Pollen: Structure, Types and Effects : Structure, Types and Effects [1 ed.] 9781617280481, 9781616686697

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Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Pollen: Structure, Types and Effects : Structure, Types and Effects, edited by Benjamin J. Kaiser, Nova Science Publishers, Incorporated, 2010.

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Pollen: Structure, Types and Effects : Structure, Types and Effects, edited by Benjamin J. Kaiser, Nova Science Publishers, Incorporated, 2010.

ENVIRONMENTAL SCIENCE, ENGINEERING AND TECHNOLOGY

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved.

POLLEN: STRUCTURE, TYPES AND EFFECTS

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Pollen: Structure, Types and Effects : Structure, Types and Effects, edited by Benjamin J. Kaiser, Nova Science Publishers, Incorporated, 2010.

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Pollen: Structure, Types and Effects : Structure, Types and Effects, edited by Benjamin J. Kaiser, Nova Science Publishers, Incorporated, 2010.

ENVIRONMENTAL SCIENCE, ENGINEERING AND TECHNOLOGY

POLLEN: STRUCTURE, TYPES AND EFFECTS

BENJAMIN J. KAISER

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved.

EDITOR

Nova Science Publishers, Inc. New York

Pollen: Structure, Types and Effects : Structure, Types and Effects, edited by Benjamin J. Kaiser, Nova Science Publishers, Incorporated, 2010.

Copyright © 2010 by Nova Science Publishers, Inc.

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Available upon request

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Pollen: Structure, Types and Effects : Structure, Types and Effects, edited by Benjamin J. Kaiser, Nova Science Publishers, Incorporated, 2010.

CONTENTS Preface

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved.

Chapter 1

vii 2n Pollen Formation: 40 Cytological Mechanisms of Nuclear Meiotic Restitution Nataliya V. Shamina

Chapter 2

Pollen Biology and Hybridization Process: Open Problem in Walnut Paola Pollegioni, Keith Woeste, Irene Olimpieri, Fulvio Ducci and Maria Emilia Malvolti

Chapter 3

Variable Sized Pollen Grains due to Impaired Male Meiosis in the Cold Desert Plants of North West Himalayas (India) Vijay Kumar Singhal and Puneet Kumar

1 65

101

Chapter 4

Capture of Male Gamete Dynamics in Pollen Tubes Tomonari Hirano and Yoichiro Hoshino

127

Chapter 5

Sunflower Pollen: Theoretical and Practical Aspects V. Popov, T. Dolgova and V.V. Dokuchaev

135

Chapter 6

Regulation of Pollen Fertility in the ‗9E‘-CMS-Inducing Cytoplasm of Sorghum: Interaction of Plant Genotype with Environment L.A. Elkonin, M.I. Tsvetova, V.V. Kozhemyakin and O.P. Kibalnik

157

Chapter 7

Pollen Development and Ubiquitin Proteasome System Sandrine Bonhomme

Chapter 8

Pollen Vigor and Seed Production in Sympatric Population of Two Orchid Species and their Hybrids Elisa Vallius

197

Microspores and their Applications in Basic and Applied Plant Sciences Mehran E. Shariatpanahi and Alisher Touraev

217

Chapter 9

Pollen: Structure, Types and Effects : Structure, Types and Effects, edited by Benjamin J. Kaiser, Nova Science Publishers, Incorporated, 2010.

179

vi Chapter 10

Chapter 11

Chapter 12

The Role of Anion Channels in Pollen Germination and Tube Growth Maria Breygina, Anna Smirnova, Natalie Matveeva and Igor Yermakov Phenotypic Plasticity in Pollen and Ovule Production of Nicotiana Longiflora and N. Plumbaginifolia (Solanaceae): Implications for Plant Mating System Variability Figueroa-Castro Dulce María, Timothy Holtsford and Angela Etcheverry A Critical Presentation of Innovative Techniques for Automated Pollen Identification in Aerobiological Monitoring Networks Rossana Dell‘Anna, Antonella Cristofori, Elena Gottardini and Francesca Monti

235

255

273

Chapter 13

Orbicules in Relation to the Pollination Modes B.G. Galati, M.M Gotelli, S. Rosenfeldt J.P. Torretta and G. Zarlavsky

Chapter 14

Hybridization Barriers between Cotton (Gossypium Hirsutum) and Species of the Malvaceae Family Stella K. Kantartzi

305

Somatic Embryogenesis in Dandelions (Taraxacum) using Anther Cultures Luděk Záveský

317

Chapter 15

Chapter 16

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Contents

Chapter 17

Chapter 18

Aerobiological Notes of Chenopodiaceae-Amaranthaceae Pollen in the Middle-West of Spain David Rodríguez de la Cruz, Estefanía Sánchez-Reyes and José Sánchez-Sánchez Pollen Viability in Hybrid Swarm Populations of Pinus Mugo × Pinus Sylvestris in Slovakia A. Kormutak, B. Vookova, T. Salaj, J. Salaj, V. Čamek,P. Boleček, P. Maňka and D. Gömöry Aperture Ontogeny in the Proteaceae Grevillea Rosmarinifolia Béatrice Albert and Sophie Nadot

Index

Pollen: Structure, Types and Effects : Structure, Types and Effects, edited by Benjamin J. Kaiser, Nova Science Publishers, Incorporated, 2010.

289

331

341

349 355

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PREFACE Pollen is a fine to coarse powder containing the microgametophytes of seed plants, which produce the male gametes (sperm cells). Pollen grains have a hard coat that protects the sperm cells during the process of their movement between the stamens to the pistil of flowering plants or from the male cone to the female cone of coniferous plants. When pollen lands on a compatible pistil of flowering plants, it germinates and produces a pollen tube that transfers the sperm to the ovule of a receptive ovary. Among other topics, this book reviews research on pollen biology and the hybridization process in walnuts, modern conception on the structure and development of the sunflower anther; the role of anion channels in pollen germination and tube growth. Chapter 1 - This chapter is an illustrated catalogue of meiotic division abnormalities, preferably cytoskeleton aberrations in karyo- and cytokinesis, leading to 2n gametes formation in plants. It includes 40 meiotic restitution mechanisms in pollen mother cells. Chapter 2 - This review focuses on the pollen biology of Juglans, and in particular Juglans nigra (Eastern Black walnut) and Juglans regia (Persian or English walnut), which are economically important species in Europe, Asia and North America. Both species are monoecious, heterodichogamous and wind –pollinated. Their mating system is predominantly outcrossing, although under particular environmental conditions self-pollination is possible. Hybrids between the two species, Juglans × intermedia (Carr) can occur naturally, although they often have reduced fecundity. Compared to the parental species, most J. × intermedia (J. nigra × J. regia) hybrids show increased vegetative vigor, distinct disease resistance, high wood quality, and greater winter-hardiness. For these reasons here is great demand for J. × intermedia for forestry, especially in Northern Europe. The authors review several aspects of Juglans pollen biology that frustrate the production of J. × intermedia and limit the progress of researchers and plant breeders who work with this genus. The authors also discuss the ways in which scientists and breeders are working to overcome problems related to pollen storage and viability testing, pistillate flower abscission (PFA), fertilization and embryogenesis in Juglans, and the use of microsatellites to monitor gene flow, ploidy, parentage, and hybridogenesis all with an eye toward practical solutions to the current shortage of J. × intermedia for research and applied forestry. Chapter 3 - The cold deserts in Western Himalayas spread over approx. 74,809 Km2, cover Leh and Kargil districts of Ladakh in Jammu & Kashmir, Uttarkashi in Uttarakhand and Lahaul- Spiti along with some parts of Chamba and Kinnaur districts of Himachal Pradesh. These cold deserts consist of rugged mountains, snow clad peaks, bare rocks, steep

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viii

Benjamin J. Kaiser

sandy slopes with rock gravels, low capacity of soil to retain moisture, and have oppressive and inhospitable conditions which include freezing temperature, dry arid weather, high velocity winds and low precipitation. As a consequence of such harsh climatic conditions prevailing in these regions, plants tend to become prostrate, thick, bushy, hardy, mat forming and spiny with long roots and small succulent or woolly leaves. Majority of these plants are perennials and survive through underground parts. Further, the plants of the area are exposed to high incidence of UV rays and are under considerable pressure of human intervention and natural disasters which include agriculture, heavy grazing, snow avalanches, windstorms, landslides and increasing entry of tourists and transport vehicles. As a consequence of such stresses, the plants of the area are expected to show considerable amount of irregularities during male meiosis and in pollen grains. Keeping above assumptions in mind the work on cytomorphological diversity in the plants of Lahaul-Spiti, a part of the Western Himalayan cold deserts, was undertaken. During the course of study spanning 5 years (2005-2009) the authors have come across several species which depict various irregularities during male meiosis resulting into the formation of variable sized apparently fertile/ stained pollen grains and considerable amount of pollen sterility. These heterogeneous sized pollen grains and pollen malformation are reported mainly in species depicting the phenomenon of cytomixis involving chromatin transfer among proximate pollen mother cells (PMCs) viz., (Anemone rivularis, 2n=16; Aquilegia fragrans, 2n=16; Astragalus bicuspis, 2n=16; A. frigidus, 2n=16; A. himalayanus, 2n=16; A. rhizanthus, 2n=16; Caltha palustris, 2n=32; Clematis grata, 2n=16; C. orientalis var. acutifolia, 2n=32; Geranium pratense, 2n=56; Hedysarum astragaloides, 2n=14; Meconopsis aculeata, 2n=56; Medicago falcata, 2n=16; M. sativa, 2n=32; Melilotus officinalis; 2n=16, Parnassia laxmanii, 2n=18; Pleurospermum candollii, 2n=22; P. govanianum, 2n=18; Potentilla atrisanguinea var. atrisanguinea, 2n=84; P. atrisanguinea var. argyrophylla, 2n=84; P. cuneifolia, 2n=28; P. fruticosa var. rigida, 2n=14; Ranunculus hirtellus, 2n=32; R. laetus, 2n=28; Rosularia alpestris, 2n=28; Silene vulgaris, 2n=24; Thalictrum foetidum, 2n=42; Trifolium pratense, 2n=14; T. repens, 2n=16 and Trigonella emodii, 2n=16). The resultant PMCs after partial or complete chromatin transfer in these species depict hyper-, hypoploid and anucleated nature which give rise to apparently fertile diploid (2n), haploid (n), and aneuploid, and sterile or micro pollen grains. Such variable sized fertile and sterile pollen grains are also resulted in species depicting irregular synapsis (Dianthus angulatus, 2n=30; Ranunculus laetus, 2n=28; Rosularia alpestris, 2n=28), spindle irregularities (Potentilla atrisanguinea var. argyrophylla, 2n=84; Ranunculus hirtellus, 2n=32; R. laetus, 2n=28; Rosularia alpestris, 2n=28), triploids (Chrysanthemum pyrethroides, 2n=27; Heracleum brunonis, 2n=33), pentaploids (Agrimonia eupatoria, 2n=70) and polyploids with irregular meiotic course (Chrysanthemum pyrethroides, 4x; Clematis orientalis var. acutifolia, 4x; Potentilla atrisanguinea var. atrisanguinea 12x; P. atrisanguinea var. argyrophylla, 12x; Ranunculus hirtellus, 4x; R. laetus, 4x). Double sized pollen grains referred as ‗2n‘ pollen grains resulted as a result of either direct fusion of two pollen grains (Caltha palustris, Potentilla atrisanguinea var. argyrophylla) or are the end products of fused PMCs (syncytes) as is the case in Meconopsis aculeata and Clematis orientalis var. acutifolia are also recorded. Though cytological status of such variable sized fertile pollen grains could not be ascertained presently, they can play an important role in the origin of intraspecific polyploids, aneuploids and taxa with B-chromosomes. Chapter 4 - The process of double fertilization involves 2 sperm cells that are delivered into female gametes via pollen tubes. To analyze this process, the authors utilized the

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Preface

ix

bicellular pollen species Alstroemeria and Cyrtanthus, which produce 2 sperm cells in the pollen tubes. Novel procedures have been developed for the in vitro observation of pollen tubes under precise control conditions, for flow cytometric analysis with single cell manipulation, and for immunocytochemical analysis to detect cytoskeletons. Using these unique methods, male gamete dynamics have been elucidated during pollen tube development. In particular, the authors have focused on male germ unit (MGU) defining that the generative cells or the pair of sperm cells in pollen tubes or pollen grains are closely associated with the vegetative nucleus. This MGU is proposed to function as a vehicle for the transmission of male gametes to the female gametes and to participate in the fusion with the target female cell during fertilization. In this chapter, recent insights into male gamete dynamics using novel procedures are discussed. Chapter 5 - Modern conception about the structure and development of sunflower anther in the normal state and under the different types of sterility are presented in the investigation. In the review different types of male sterility: nuclear male sterility, cytoplasmic male sterility and sterility which induces different factors is considered. This research is based on the scientific literature summarizing and experimental work results of the authors as for meiosis studying of culture sunflower and wild species and their interspecific hybrids. Spore formation capacity of androceum concerning practical breeding and seed production problems are also stated. The results of research in field of inheritance of nuclear mail sterility and cytoplasmic mail sterility, mapping of genes which control male sterility with use different molecular markers. The questions of relationship plasmone and genome in manifestation of cytoplasmic male sterility were shown. The importance of gametocytes for the induce of sunflower sterility was considered. A great attention is paid to meiosis violation in the process of chromosome formation rearrangement during the interspecific hybridization. The reasons of cytological instability of interspecific hybrids which have been obtained by crossing of annual wild species with culture sunflower and the ways of overcoming this cytological instability are discussed. Chapter 6 - Restoration of pollen fertility in plants with cytoplasmic male sterility (CMS) is known to be controlled by specific nuclear fertility-restoring genes that suppress functioning of aberrant mitochondrial genes, expression of which destroys pollen development. Usually, these genes are dominant and manifest in the F1 generation. In sorghum, pollen fertility restoration in some CMS-inducing cytoplasms (A4, ‗9E‘, ‗M35-1A‘) has aberrant mode of inheritance: it is stably expressed in self-pollinated progenies of F1 hybrids with restored fertility but unstably manifested in new hybrid genome (F1 or test-cross hybrids to CMS lines with the same cytoplasm type). The authors found that in the ‗9E‘ cytoplasm this phenomenon is caused, evidently, by environmental conditions during testcross hybrid plant development, namely water-availability and photoperiod. Experimental data testify to strong sensitivity of fertility-restoring genes to plant water-availability conditions at microspore- and gametogenesis: at high level of water availability these genes are dominant and can express in heterozygous state (in test-cross hybrids), while in drought conditions fertility-restoring genes are recessive and can function only in homozygous state. In addition, reduced photoperiod at photoperiodically-sensitive stage of sorghum plant ontogenesis also increased percentage of male-fertile F1 plants, perhaps, by ‗activation‘ of fertility-restoring genes, and these genes expressed in the next generation. Changes of environmental conditions during male-sterile hybrid plant ontogenesis can ‗activate‘ expression of fertility-restoring genes. Cytological analysis revealed significant

Pollen: Structure, Types and Effects : Structure, Types and Effects, edited by Benjamin J. Kaiser, Nova Science Publishers, Incorporated, 2010.

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Benjamin J. Kaiser

polymorphism of pollen grain (PG) types in CMS-lines with the ‗9E‘ cytoplasm and in the F1 hybrids with restored male fertility. Both sterile and fertile plants had ―fertile‖ (normally colored) PGs, PGs with anomalous shape, with delay of development at one- or two-nucleate gametophyte, with disturbed starch accumulation (incompletely filled with starch and PGs of waxy-type), with entirely degenerated content. Additional watering increased percentage of fertile PGs but no clear correlation between their frequency and seed set has been found. The data obtained demonstrate that in the ‗9E‘ cytoplasm pollen fertility, obviously, is epigenetically-regulated trait depending both from genotype and plant environmental conditions. Chapter 7 - The UPS (Ubiquitin Proteasome System) is a major post-translational regulation pathway in eucaryotes, allowing rapid and selective degradation of proteins. These proteins are either misfolded or damaged polypeptides (quality control), or key developmental/metabolic regulators. The latter are selectively recognized and labelled with a poly-ubiquitin chain by E3-ubiquitin ligases, prior to degradation in a large multi-subunit complex called the 26S proteasome. In plants, the UPS controls many processes, in particular most hormone signalling pathways, and several other important steps including cell cycle, shoot branching, biotic stress response and self-incompatibility. Recent data highlight that the UPS is also crucial for male gametophyte (i.e. pollen) development. Plant male gametophyte development requires several tightly regulated processes of nuclei movement, cell divisions and differentiation that are well described, but which molecular control still needs to be deciphered. Different transcriptomic data indicate that transcripts for most UPS actors are found in microspore and/or pollen cells. In the last few years, the role of several components of UPS, including proteasome subunits and proteins involved in targets recognition/ubiquitylation, has been demonstrated during male gametogenesis. In particular, the role of an F-box protein that is transiently transcribed in male germ cell and specifically required for mitosis entry, has allowed significant progress in building a model of the control of male gametophyte development. In this review, I try to state all published evidence for an implication of UPS components in male gametophyte development, and to highlight important ways that still need to be explored. Chapter 8 - Orchids (Orchidaceae) are known to frequently hybridize. Especially in deceptive taxa, pollen flow between species is caused by lack of fidelity in pollinator behaviour, as pollinators tend to move to different-looking plants after an ineffectual visit. Because many of these deceptive species have no post-pollination reproduction barrier between them, production of hybrid seed in sympatric populations is not rare. As a result of interspecific fertilization, either intermediate or non-typical, usually tall plants with large inflorescences appear. These hybrid plants may be polyploids with varying number of chromosomes and they suffer from very low fertility. Many of these hybrids also totally fail to produce pollen, but production of normally structured and totally or partially in-viable pollinia is also possible. In this chapter, the quality of pollen in hybrid plants and its effects on reproductive success of original species are discussed using the hybrids between Dactylorhiza incarnata and D. fuchsii in Irish dune population as an example. Quality of pollen in hybrid plants was examined using fluorescent staining method, and was found to vary from totally in-viable to partially viable. The effects of pollen source on seed production were examined with hand-pollination experiments in parental species and hybrids. Production of F1 seed between D. incarnata and D. fuchsii was comparative to intra-specific fertilization, but only a few seeds were produced following pollination of parental species with hybrid

Pollen: Structure, Types and Effects : Structure, Types and Effects, edited by Benjamin J. Kaiser, Nova Science Publishers, Incorporated, 2010.

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Preface

xi

pollen. Seed production in hybrid plants was very low compared to the parental species, but some seed was produced. Asexual reproduction via apomictic seed production in D. incarnata was examined experimentally stimulating stigma chemically and mechanically. Mechanical stimulation did not result in seed production but liquid containing chemicals from orchid pollen did trigger ovule maturation and production of some seed, as did non-related Ranunculus acris pollen. Hybrid pollen in the stigma did not essentially cause interference in seed production of D. incarnata either before or after pollination with intra-specific pollen. Pollen produced by hybrids thus seems not to play an important negative role in the reproduction of the parental species. Some hybrids, however, produce at least partially viable pollen which may lead to back-crossing and lower production of genetically normal progeny. This production of hybrids, possibility of apomictic seed production, and introgression at different levels may be important factors behind the high number of taxa and large intraspecific variation in deceptive terrestrial orchids. Chapter 9 - Nowadays, the use of isolated and in vitro cultured microspores is not limited for the production of doubled haploids which are important part of modern plant breeding programmes. Microspores became also a powerful tool to investigate important biological processes such as cell totipotency, differentiation, embryogenesis, cell cycle, plant reproduction and development. In the normal pathway microspores develop into mature pollen in vivo or when cultured in vitro in a rich medium with sugars, however they can be reprogrammed into the totipotent state and further induced to become embryogenic and produce embryos when subjected to various stresses, such as nutrient starvation, heat or cold shock. Microspore cultures are the most efficient method to produce doubled haploids, excellent system for in vitro mutagenesis and selection, attractive target for genetic transformation and for gene targeting in plants. This review paper describes the potential applications of plant microspores in plant breeding, genetics, cellular and molecular biology and biotechnology. Chapter 10 - The basic feature of polar growing cells is uneven distribution of organelles, forming distinct cytoplasmic zones. It is closely related to the cytosolic free ion gradients and transmembrane ion fluxes, which may cause uneven membrane potential distribution along the cell surface. Participation of Ca2+, H+ and K+ in pollen tube growth has been proved in numerous studies. Data on inorganic anions contribution to the growth process are scarce and controversial. In somatic plant cells anion channels have vital functions, including membrane voltage and turgor pressure regulation. In this chapter the authors give an overview of recent findings in ionic regulation of pollen germination and give evidence for the important role of anion channels in this process. The use of inhibitory analysis combined with fluorescent methods has allowed them to observe both temporal and spatial changes of membrane potential and reveal the involvement of anion channels in the regulation of this value. The authors‘ data on the key role of anion channels in structural and functional compartmentalization of the polarized pollen tube cytoplasm are considered. A contribution of mitochondrial anion channels to the pollen tube growth regulation is also discussed. Chapter 11 - Phenotypic plasticity in floral traits associated with mating systems has been demonstrated for many species. However, few studies have explored the existence of plasticity in pollen and ovule production. This is important because plant mating systems are often estimated as pollen:ovule ratio. In this chapter the authors determine the existence of phenotypic plasticity in gamete production and in mating system as estimated through pollen:ovule ratio in Nicotiana longiflora and N. plumbaginifolia. The authors measured

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Benjamin J. Kaiser

several morphological traits of flowers and then counted pollen and ovules directly on plants from greenhouse and natural environments. Then the authors developed regression models for both environments, using morphological traits to predict pollen and ovule number. The authors then applied both regression models to eight and six natural populations of N. longiflora and N. plumbaginifolia, respectively. The authors found that pollen production is plastic in N. longiflora but not in N. plumbaginifolia. There was a general trend towards a decrease in number of pollen grains in natural populations of N. longiflora; but it was significant in only four out of the eight populations sampled. In N. plumbaginifolia that trend was inverted, with natural populations producing more pollen grains; however this pattern was not significant for any of the six populations studied. Ovule production was not significantly different between environments in N. longiflora and data did not show any particular trend. In N. plumbaginifolia, ovule production had a general trend towards a decrease in natural populations, but this trend was significant in only three out of the six populations studied. Pollen:ovule ratio had a general trend towards a decrease in natural populations of N. longiflora, but this trend was significant in only one population, suggesting the existence of a trend towards selfing for that population. On the contrary, pollen:ovule ratio in N. plumbaginifolia tended to be higher in natural populations compared to greenhouse, however, it was not significant in any of the populations studied. The authors‘ results showed that plastic responses are population- and species- specific, possibly as a consequence of local adaptations to dominant selective pressures in each population. Chapter 12 - Pollen grains are one of the major causes of respiratory allergies. The authors briefly review the role of aerobiological monitoring centers in providing information about airborne pollen concentration for helping allergic patients to reduce exposition to allergens and to start appropriate drug treatments. Spatial and temporal resolution of this information should be increased. However, the effort required by the technique currently used to identify and count the airborne pollen grains hinders this improvement. Therefore, innovative classification approaches were investigated. In particular, the authors studied the feasibility of methodologies developed in the spectroscopic and biomolecular field, with the aim at providing rapid, accurate and possibly automated airborne pollen concentration measurements. In this chapter the state of the art in this field is outlined as well as the authors‘ obtained results; the authors discuss both the proof of principle of the applicability of such techniques for pollen quantification and, from a more practical point of view, the feasibility of implementing them in aerobiological centers as routine identification tools. Possible future improvements of developed techniques to solve current weaknesses are also examined. Chapter 13 - Orbicules or Ubisch bodies are corpuscles of sporopollenin that appear in the anther locule during pollen grain development. Their size ranges from 0.14 µm to 20 µm. They present different shapes with a smooth or ornamented surface. Orbicules often form aggregates and sometimes have a plaque-like appearance. Ultrastructurally, they may present a central core with different degree of transparency to electrons. Those that do not have a central core are observed completely solid. Orbicules are resistant to acetolisis, autofluorescent when irradiated with ultraviolet light and have the same reaction to colorants that the exine of pollen grains. Their presence is generally associated with a tapetal membrane in species with secretor type tapetum and with a peritapetal membrane in species with intermediate or plasmodial type tapetum.

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xiii

Although the shed of orbicules out of the anther along with the pollen grains is cited, they are usually attached to the inner surface of the locule when the anther opens. Investigations suggest that orbicules appear in approximately 80 families of Angiosperms and Gimnosperms. It is not certain whether orbicules are not developed in the rests of the families or are just not informed. Researches on ontogeny and ultrastructure of orbicules are rare. However, their tapetal origin and their simultaneous formation with the pollen grain wall are well established. The systematic value of orbicules is known and considered in a few families, such as Loganiaceae, Gentianaceae, Apocynaceae, Rubiaceae and Oxalidaceae. Evolutionary studies on these bodies or on its relationship with the different modes of pollination are lacking. Even though orbicules are so common among angiosperms, their function is unknown and only speculations are made. On this report a review on orbicules is made and an analysis of their presence, ontogeny and morphology is presented. The authors‘ aim is to supply information that will help understand orbicules function. Therefore, the orbicules morphology in relation with the pollination mode is studied. Chapter 14 - Cotton breeders have long recognized the importance of alien germplasm from the Malvaceae family as sources of genes for crop improvement. An understanding of the biological nature of the incompatibility systems that prevent hybridization and/or seed development is necessary for the successful hybridization and introgression between cotton and other Malvaceae species. In this review, the authors survey the reasons for reproductive isolation between cotton and its wild or cultivated Malvaceae relatives. Chapter 15 - The microspore and anther cultures have been used in plant breeding since the first reports on embryogenesis from pollen of Datura innoxia in 1960´s. Large numbers of studies have been focused on formation of homozygous double haploid plants in many important crops by both methods. Microspore embryogenesis is an elegant system for a selection for dominant and recessive traits in haploid plants and after artificial diploidization of them, the plants homozygous for all genes can be obtained. Reports on employment of the anther cultures are more frequent in contrast to isolated microspore cultures due to a more simple technical handling, despite the fact that there is generally a lower efficiency of this method for doubled haploids production than in pure microspore cultures. Moreover, the anther cultures lead to somatic tissue–derived plants and thus are used as the method for the somatic embryogenesis. I used anther and microspore cultures methods prospecting their potential also in apomixis research. Dandelions (Taraxacum sect. Ruderalia) as plants representing an model for studying a diplosporous type of apomixis and producing three ploidy levels (haploid, diploid and triploid) of pollen grains were subjects to establishing new protocols of such artificial embryogenesis. A production of microspore-derived plants could have the potential for observation of recombinations and segregations of apomictic genes within microsporogenesis. Different culture media were tested and callus production, differentiated shoots, roots, and finally whole viable and fertile plants were obtained using anther cultures. Obtained regenerants were of somatic origin. The first attempts of establishing microspore embryogenesis were not successful also in isolated microspore cultures. This is the only one report on obtaining somatic embryogenesis based plants via anther cultures of dandelions with potential use in pharmacy (production of drugs) and particularly for further research purposes. These methods have not been published for this genus yet. Chapter 16 - The aim of the present chapter was to describe the ChenopodiaceaeAmaranthaceae pollen dynamics in the atmosphere of two cities of the Middle-West of Spain

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Benjamin J. Kaiser

(Salamanca and Valladolid). Samples were collected by the volumetric method with the aid of two Burkard spore-traps located in the centre of both urban cities during years 2005 and 2006. This pollen type was mainly detected in the atmosphere between late Spring and late Summer, with a Main Pollen Season registered between late May and early October and maximum concentrations detected in August. The intra-diurnal pattern, calculated by means of three different methods, was very similar for both towns reaching a higher hourly concentration percentage in the second half of the day. The correlations obtained between daily pollen counts and different meteorological parameters showed that the airborne presence of this pollen type is positively associated with temperature and negatively with rainfall during MPS. According to known threshold (10-15 pollen/m3), ChenopodiaceaeAmaranthaceae pollen concentrations exceeded this threshold during 1 day in 2005 and during 12 days in 2006. Chapter 17 - Pollen viability in three hybrid swarm populations of Pinus mugo × P. sylvestris and in control populations of P. mugo in northern Slovakia was studied throughout two successive years. Germination percentage and pollen tube length were used as the principal parameters of pollen viability. In 2007, a slight reduction of pollen germination was recorded in hybrid swarms on the localities Habovka and Sucha Hora in comparison with the adjoining population of P. mugo from Rohače. Statistically significant differences between individual populations were revealed in pollen tube length only. In 2008, a pollen of improved quality was produced in all the populations tested. Pollen germination of hybrid swarms in Habovka, Sucha Hora and Tisovnica was comparable in the respective year with control population of P. mugo from Vratna valley. At the pollen tube level the hybrid swarms in Habovka and Sucha Hora deviated considerably from control population by their shorter pollen tubes. Obtained results are taken as an evidence supporting partial reduction of pollen viability in the respective hybrid swarms. Annual variation in pollen viability is supposed to be conditioned climatically. Chapter 18 - Grevillea rosmarinifolia presents three -aperturate pollen grain organized according to Garside‘s rule, a pattern that is common in Proteaceae but otherwise rarely observed in the eudicot clade where this family belongs. In this chapter the authors describe into details features of early pollen development (microsporogenesis) in order to explore the impact of callose deposition on aperture pattern determination in this species. The authors show that the partitioning of the microspore mother cell during microsporogenesis occurs through centripetal cell-plate formation, followed by additional deposition of callose onto the cell plates. The authors‘ observations strongly suggest that these additional callose deposits are implicated in the determination of aperture localisation in Grevillea.

Pollen: Structure, Types and Effects : Structure, Types and Effects, edited by Benjamin J. Kaiser, Nova Science Publishers, Incorporated, 2010.

In: Pollen: Structure, Types and Effects Editor: Benjamin J. Kaiser, pp. 1-64

ISBN: 978-1-61668-669-7 ©2010 Nova Science Publishers, Inc.

Chapter 1

2N POLLEN FORMATION: 40 CYTOLOGICAL MECHANISMS OF NUCLEAR MEIOTIC RESTITUTION Nataliya V. Shamina Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of RAS, Novosibirsk, Russia

To Munikote Ramanna, who encouraged me to study the meiotic restitution mechanisms in higher plants.

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ABSTRACT This is an illustrated catalogue of meiotic division abnormalities, preferably cytoskeleton aberrations in karyo- and cytokinesis, leading to 2n gametes formation in plants. It includes 40 meiotic restitution mechanisms in pollen mother cells.

Keywords: division spindle, cell plate, pollen mother cells (PMCs), nuclear restitution, phragmoplast, cytokinesis, cytoskeleton, meiosis, 2n-gametes, plant cell division.

INTRODUCTION Gametes with diploid chromosome number play a considerable role in higher plant evolution and speciation and are an important instrument in breeding (Mendiburu, Peloquin, 1976; Peloquin et al., 1999; Jauhar, 2003; Cai and Xu, 2007). In angiosperms, about 30% 80% of species were estimated to be of polyploid origin. Polyploidisation is a key evolutionary process in higher plants that lead to the formation of new species. The majority of higher plant species evolutionised this way, or by means of hybridization with more or less widely related species and further polyploidisation. 

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Such important phenomenon as gametophytic apomixis is also associated with polyploidy (Estrada-Luna et al., 2002; d‘Erfurth et al., 2009). Apomixis is a specific reproduction method that allows us to obtain absolute genetic copies of mother plants and is related with the process of 2n gametes formation. Despite impressive advancements of genetic engineering in development of transgenic plants, wide hybridization remains the most important and, so far, indispensible method to obtain breeding material. It is explained by the thing that most of the traits important for breeding have polygenic control and cannot be transferred by transformation. Overcoming hybrids F1 sterility is the key problem of plant wide hybridization (Udall and Wendel, 2006). Under complete or partial absence of homologs conjugation, regular chromosome distribution in two subsequent meiotic divisions becomes impossible. Developing microspores have their aneuploid chromosome number and are non-viable. Rapid development of fertile wide hybrids is possible due to sexual polyploidisation realized by 2n gametes. Such gametes form in parents or in hybrids (allohaploids) as a result of meiotic restitution process (Consiglio et al., 2004). Restitution is understood as absence of segregation of daughter or parent genomes during gametes formation. These aberrations may occur in each of two meiotic divisions, also in preand postmeiotic mitoses. If, as a consequence of such abnormalities, the results of the first meiotic division are exterminated, the first division restitution (FDR) with the formation of 2n nonreduced gametes proceeds. As for the second division restitution (SDR) – 2n reduced gametes develop (see review Hermsen, 1984). Non-reduced 2n gametes from taxonomically distant parents fuse and initiate fertile progenies, and integration of different species genomes is of hybrid advantage for the organism. Allopolyploid species are very viable and widely spread. Many of them are important agricultural crops. Wide hybridization combined with sexual polyploidization is the most important stage in the evolution for the majority of cultivated plants (Hutchinson, 1953). The first such example is the Raphanobrassica hybrid obtained at the beginning of the 20th century crossing 2n gamet producents (Karpechenko, 1927). Though it had a cabbage root and radish leaves, it laid ground for such wonderful synthetic agronomic forms as triticale (wheat-rye amphidiploid) and others. It is well known that, in certain plant cross variants, wide F1 hybrids set seeds both in backcrosses and self-pollination (Love, Craig, 1919; Muntzing, 1939; Mitsuoka, 1953; Tanaka, 1959). When studying chromosome behavior at meiosis in fertile wide F1 hybrids of monocotyledonous species, it was detected that certain aberrations of chromosome segregation in the first, second or both meiotic divisions is the condition of viable gametes formation in them. Frequent are the reports on viable gametes formation as a combination result of low level of homeological chromosome conjugation , univalents equational division type at anaphase I and the subsequent chromosome non-segregation in the second meiotic division (Маап, Sasakuma, 1977; Sasakuma, Kihara, 1981; Xu and Joppa, 2000). A number of phenomena that lead to the restitution nuclei formation in the first meiotic division with the following equational division in the second meiosis and development of viable dyads of microspores were described. In these cases the reasons for restitution nucleus formation may be drastically unequal univalents distribution between the poles (Sears, 1953; Avers, 1954; Stefani, 1986, Jauhar, 2003), absence of their movement in anaphase I (Rhoades, Dempsey, 1966; Wagenaar, 1968; Fukuda, Sakamoto,1992) and even the movement of segregated univalents from the poles to the cell centre again (Riley, Chapman, 1957). It was also reported on the formation of restitution nuclei as a result of

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2n Pollen Formation: 40 Cytological Mechanisms of Nuclear Meiotic Restitution

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cytokinesis absence (Bielig et al., 2003; Barba-Gonzalez et al., 2005; Risso-Pascotto et al., 2006). It is obvious that these meiotic restitution processes are the result of some aberrations in the division spindle cytoskeleton apparatus and, possibly, phragmoplast, but there was no cytological analysis carried out with the structures visualization in these contributions. Meiosis in dicotyledonous species is characterized by the so-called simultaneous cytokinesis which proceeds at telophase II and immediately autonomises 4 haploid nuclei located in the common cytoplasm by means of 6 phragmoplasts activities (Tiezzi et al., 1992). Nuclei are the products of the first meiotic division also located in the common cytoplasm, as cytokinesis does not proceed at telophase I. These peculiarities provide additional possibilities for the reunion of segregated chromosome groups and condition original meiotic restitution mechanisms in this taxonomic group (Carputo et al., 2000; Andreuzza and Siddiqi, 2008). At the same time, some restitution mechanisms observed monocots are typical of dicots. It is known that meiotic restitution in dicotyledonous plant species can be realized by means of 1) chromosome non-segregation at anaphase I or II (Lam, 1974; Gill et al., 1985); 2) premature cytokinesis after the first meiotic division and the absence of chromosome segregation in the second one (Mok, Peloquin, 1975; Gill et al., 1985; Watanabe, Peloquin, 1993); 3) univalents equational division at anaphase I in asynaptic meiosis and absence of the second meiotic division (Gustafsson, 1935; Ramanna, 1983; Jongedijk et al., 1991; Vorsa, Ortiz, 1992); 4) absence of the second meiotic division (Conicella et al., 1991; Werner, Peloquin, 1991); 5) disorientation and shift of second division spindles (parallel and ―tripolar‖ spindles configurations) (Mok, Peloquin, 1975; Ramanna, 1979; Genualdo et al., 1998; El Mokadem et al., 2002; Nelson et al., 2009); 6) fused spindles at metaphase II (Ramanna, 1979; Veilleux et al., 1982; Gill et al., 1985); 7) undetected division spindle abnormalities (Qu and Vorsa, 1999); 8) cytokinetic aberrations (Ramanna, 1974; Werner and Peloquin, 1991), 9) chromosome non-segregation in the first and second post-meiotic mitoses (Prakken, Swaminatham, 1952; Bastiaanssen et al., 1998); 10) aberrations of pre-meiotic mitosis (Prakken, Swaminatham, 1952). 2n-gametes, in most cases, form as a result of restitution nucleus formation in the first or the second meiotic division. Caryo- and cytokinetic abnormalities are the base for the restitution nucleus formation process. Separation of chromosome groups in the cell space and their further autonomisation are realized by transient cytoskeleton structures: division spindle and phragmoplast (see rev. Mathur, 2004; Wasteneys and Yang, 2004). Unfortunately, despite the key role which cytoskeleton structures play during meiotic restitution, investigations in this field are not numerous (Alfano et al., 1997; Genualdo et al., 1998). It is explained with several reasons. First, it is because the lack of knowledge about the cytoskeleton rearrangements cycle during plant cell division. The centrosome and polar spindle organizers have not been identified as morphological structures in plant cell; thus organelles that control the cytoskeleton cycle are not known for it (Mineyuki, 2007). Transient stages of cytoskeleton cycle in plant cell division have also not been sufficiently studied. Second, slow progress in studying cytoskeleton mechanisms of meiotic restitution is explained by the known methodic difficulties: immunocytochemical methods of cytoskeleton structure visualization have their own restrictions (sampling problem), whereas the percent of meiocytes in which restitution proceeds is not always sufficiently high. In the combination, these two circumstances considerably complicate investigations. Third, there exists the clear notion of interdisciplinary isolation of experts in cell biology specialized in cytoskeleton studies and those involved in breeding programs and mostly dealing with the analysis of

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chromosome behavior during restitution (Ramanna, 1974, 1979, 1983; Mok, Peloqun, 1975; Werner, Peloquin, 1991). However, there are efficient classical, but unfortunately forgotten methods for cytoskeleton structure visualization which are very suitable for the above-described tasks. They consist in the use of acetoformol fixation (according to Bouin, Chamberlain, Navashin) that preserves the cytoskeleton with further staining using common stains for protein (acetocarmine, acetoorseine, etc.). The value of these methods consists in, first, their simplicity, cheap price and low labour costs; it allows to analyse comparatively big amounts of material and to trace the division even in a low percent of abnormal cells. Second, they are not different in their expressed sampling problem, - loss of information during making up the preparation; it provides the analysis of synchronized pollen mother cells (PMCs) of only one anther or its separate chamber for the correct identification of abnormal meiotic stages. And, finally, it is very important that alongside with the cytoskeleton and chromosomes, classical visualization methods allow us also to observe the nuclear envelope, nuclear area, cell membranes, cell plate, conglomerates of membrane vesicles which are indispensible for the correct definition of abnormal cell division stage. Application of these methods in studying higher plant meiocytes is most effective due to these big cell sizes. To analyse abnormal meiosis, whose results are presented below, I used the method of Navashin modified fixation (Wada, Kusunoki, 1964). Under meiosis, buds were being fixed for 24 hours at room temperature using the modified Navashin fixative on the following protocol:

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 

Solution A: 1,1 g CdCl2, 10 ml glacial acetic acid, 65 ml distilled water; Solution B: 40 ml of 40% (or 37%) formol, 35 ml of distilled water.

The solutions are prepared separately and mixed in equal volumes. The material can be preserved in the fixative at room temperature during a year, and up to 3 years at +4oC without quality loss. Before making up the preparations, it is necessary to rinse buds with running water. Anthers are stained on slide under heating in a drop of 3% acetocarmine; then they are squashed with cover glass. The images were made in white light at magnitude 100 x 10.

1. NORMAL CYTOSKELETON DYNAMICS DURING POLLEN MOTHER CELLS MEIOTIC DIVISION Before the description of abnormal phenotypes, for better orientation in the cytoskeleton cycle course, I present an illustration of trivial meiotic division with successive cytokinesis in PMCs of wheat x wheatgrass (WWG) hybrid F1 (Shamina, 2005a, b; Shamina et al., 2007). Trivial is the phenotype in which other abnormalities are not observed, but the absence of homological chromosome synapsis. The meiotic process with simultaneous cytokinesis of wild type tomato PMCs is also illustrated. The cytoskeleton cycle and meiotic chromosome cycle do not differ from each other in PMCs of mono- and dicoteledonous plant species till the telophase stage.

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2n Pollen Formation: 40 Cytological Mechanisms of Nuclear Meiotic Restitution

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1.1. Cytoskeleton Cycle during First Meiosis with Successive Cytokinesis in Monocotyledonous Species

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At early prophase, the zygothene stage, chromosomes are in the local nuclear zone attaching with their telomeres to the limited nuclear envelope region forming the ‗‘bouquet‘‘ configuration. At this stage, the cytoskeleton fibres radiate from the nuclear envelope surface to the cytoplasm periphery (Fig. 1.1, a). During pachythene, chromosomes (in WWG F1 hybrids they are univalent) realize from the bouquet and are distributed all over the nuclear volume. The cytoskeleton begins to consolidate around the nuclear surface (Fig 1.1, b). In diplothene-diakinesis, a cytoskeleton ring forms around the nucleus in the meridional plane (Fig 1.1, c). Further on, simultaineously with the nuclear envelope breakdown (NEB) and the onset of prometaphase, the perinuclear ring also decays into constitutive elements – microtubule bundles (Fig 1.1, d), which then straighten, turn and enter the former nuclear area. As a result, a chaotic net of cytoskeleton elements (MT bundles) (Fig 1.1, e) which attach to chromosome kinetochores, developing spindle kinetochore fibers, also with each other, developing bipolar central spindle fibers is forming. At late prometaphase (Fig. 1.1, f),

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Figure 1.1. Process of the first meiotic division in PMCs of wheat x wheatgrass hybrid F1 of trivial phenotype: a) zygothene, chromosomes are in bouquet configuration; cytoskeleton represents radial MT bundles; b) pachythene, chromosomes realize from the bouquet; c) diakinesis, a perinuclear cytoskeleton ring is forming; d) nuclear envelope breakdown and perinuclear ring disintegration at the prometaphase onset; e) chaotic prometaphase stage (mid-prometaphase); f) late prometaphase I, univalents assemble on the spindle equator imitating the metaphase plate; g) conventional metaphase I – anaphase I; h) late anaphase I – early telophase I; i – k) successive stages of telophase I with phragmoplast/ cell plate centrifugal movement; l) dyad.

the developed kinetochore fibers with attached univalents and bipolar central fibers orient along the division axis, interact with each other, converge on the poles and form the division spindle (Fig. 1.1, g). Univalents, as a rule, are of reductional orientation, i.e. each carries one kinetochore and is aligned with only one of the poles. Therefore, the metaphase plate is absent; though at early metaphase, univalents randomly distributing over between the poles consolidate on the spindle equator. At anaphase, univalents are scattered over the spindle body and gradually reach the poles (Fig. 1.1, h). At early telophase, the spindle consists of a

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bundle of central fibers (early phragmoplast), which are the base of the forming phragmoplast, and aneuploid telophase chromosome groups. Then the cell plate forms on the telophase spindle equator (Fig 1.1, i); the central spindle fibers surround it having a hollow cylinder configuration (mid phragmoplast). Then its fibers progressively curve and lengthen. As a result, their central points centrifugally move to the cell periphery (late phragmoplast) (Fig. 1.1, j, k). Having reached the mother cell membrane, cell plate membrane vesicles (plastosomes) fuse forming daughter cell membranes, and cytokinesis is completed. A dyad with aneuploid members is the division product (Fig 1.1, l). The second meiotic division in the trivial phenotype proceeds normally, a tetrad of aneuploid non-viable microspores is the meiotic product.

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1.2. Cytoskeleton Cycle in Male Meiosis in Dicotyledonous Species with Simultaneous Cytokinesis Cytoskeleton and chromosome behavior at meiotic stages till telophase I in PMCs with simultaineous cytokinesis is not different from those having in PMCs with successive cytokinesis (Fig. 1.2, a – h). The images of cytoskeleton structures at prophase of PMCs of wild type tomato are placed here to illustrate meiosis of dicots, is slightly different from that described for WWG F1 hybrids. Thus, there is no visible cytoskeleton perinuclear ring and also its intermediate formation stages at prophase and its disassembly at early prometaphase found. It is explained by the dual cytoskeleton behavior type at prophase stage: radial fibers, moving in the cytoplasm during perinuclear system formation can preserve their morphology and be found during rearrangements, or can depolymerise. It occurs as their shortening towards the nucleus. Such short bundles undergo the same movements to the tangential position to the nuclear surface and form a very thin perinuclear ring invisible with classical methods and revealed using the immunostaining (Shamina, 2005a). This thin ring exists up to the nuclear envelope breakdown and the beginning of prometaphase; then the ring disintegrates, its short MT bundles get longer and enter the nuclear zone forming a chaotic prometaphase figure. Complete microtubule cytoskeleton depolymerisation and complete disappearance of ring perinuclear antitubuline staining at prophase I was not observed by us in any of the species analysed using immunostaining method (tomato, potato, tobacco, maize, wheat, rye). Our observations showed that this or that cytoskeleton behavior type at prophase (rearrangements of long or shortened fibers) is encountered with this or that frequency in PMCs of all investigated species regarding both mono- and dicotyledonous plants. Sometimes, cells with developed perinuclear rings, just as without them, can be observed among PMCs within one anther at light microscopic level. The cytoskeleton cycle without depolymerisation at prophase of 100% PMCs is realized in the wild type meiosis of Elytrigia elongatum and common wheat Triticum aestivum for cultivar Albidum 114, and also in the half of the wide cereal hybrids (WWG and wheat x rye WR F1 hybrids) we analysed. Out of 105 cross variants we analysed for F1 of WWG F1 hybrids, in 53 of them, developed perinuclear rings were frequently encountered at prophase I, i.e. their either prevailed or appeared in 100% of PMCs. In the rest of cross variants, PMCs with developed perinuclear rings were constantly present as an admixture in all the anthers. Apparently the same picture was observed in the wheat-rye F1 hybrids we analysed, also in maize and rice haploids. After the division spindle formation and anaphase process, telophase chromosome groups at early telophase I become separated by the central spindle fibers (Fig. 1.2, h). At mid

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telophase, on the poles, mass polymerization of new MT bundles directed to the equator with their (+) ends begins. At late telophase, this system of developing interzonal fibers often fills up the space between daughter nuclei (Fig. 1.2, i, j). It consists of the central spindle fibers and newly-formed opposite polar MT bundles conjuncted with their (+) ends. During the interzonal cytoskeleton system formation, the nuclear envelope forms around telophase chromosome groups, and microtuble bundles diverge from its surface. Such configuration of interzonal system connecting daughter nuclei and filling in practically the whole cytoplasm has the cytoskeleton in meiotic interkinesis of all the dicotyledonous species we investigated. To the onset of prophase II, the interzonal cytoskeleton system gets depolymerized (Fig. 1.2, k, l). MTs (+)- ends disjunct, MTs shorten becoming closer to daughter nuclei. Cytoskeleton rings develop at the end of prophase II around daughter nuclei. Spindles at metaphase II of the dicots PMCs are mutually perpendicular or located at the angle of 600; so, their polar regions are maximally distant from each other (Fig. 1.2, m, n). After the formation of 4 daughter nuclei, the formation of the so-called secondary spindles begins in the common cytoplasm at telophase II, i.e. radial MT bundles connecting non-sister daughter nuclei (VanLammeren et al., 1985; Traas et al., 1989). Setting of cell plates proceeds simultaineously in the system of six phragmoplasts (Fig. 1.2, o). Daughter cell membranes form also simultaineously, realizing the so-called simultaineous cytokinesis and developing four microspores, disposed tetrahedrically (Fig. 1.2, p).

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Figure 1.2. Cytoskeleton dynamics in the meiotic division of tomato (Lycopersicon esculentum L.) PMCs: a) diakinesis; b) early prometaphase I; c, d) mid prometaphase I (chaotic stage); e) late prometaphase; f) metaphase I; g, h) anaphase I; i, j) interzonal cytoskeleton system at telophase I – interkinesis; k, l) interzonal cytoskeleton depolymerisation at prophase II; m) prometaphase II; n) metaphase II; o) late telophase II; the 4th nucleus is out of focus; o) tetrad.

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1.3. Simultaneous and Successive Cytokinesis Compared During evolution, plants have elaborated the way of cytokinesis which is modified exocytocis. Membrane vesicles transport from Golgi apparatus to the equatorial cytoplasmic zone, form a monolayer there (cell plate), then fuse and develop daughter cell membranes (Staehelin and Hepler, 1996). Transport of membrane vesicles to the equator is realized by the temporal cytoskeleton structure – phragmoplast. In meiosis, cytokinesis can be successive, after each division (as in most monocots) or only at the telophase of the second meiosis, simultaneously autonomising four daughter nuclei by six phragmoplast activities (as in most dicots). Phragmoplasts that form in successive and simultaneous cytokinesis do not principally differ in their structure: it is a system of MT bundles diverging from the telophase chromosome groups and overlapping with their (+)-ends on the equator. However, phragmoplasts in successive and simultaneous cytokinesis differ in their fibers architecture, also in the way of cell plate formation. The phragmoplast in the successive cytokinesis, in monocots, is a system of long fibers encircling the growing cell plate edge as a palisade and centrifugally moving. In simultaneous cytokinesis of dicots, phragmoplasts are a multitude of long fibers that cut through the whole cytoplasm in the interzone between nuclei. This phragmoplast has no hollow cylinder configuration and does not make centrifugal movement. It is a solid structure similar with a metaphase spindle, but more voluminous (Shamina and Dorogova, 2006). The phragmoplast function in dicots and monocots does not principally differ: transport of membrane vesicles into the region of MTs overlap for cell plate formation. However, cell plate formation is realized in different ways. In monocots, the phragmoplast expands centrifugally together with a growing cell plate, but it is immobile in dicots. The points into which plastosomes are to move during the monolayer formation (cell plate) are determined by the points of phragmoplast MTs (+) ends overlapping. These central points in mobile phragmoplast fiber ‗draw‘ the cell plate plane during their centrifugal movement. In case of an immobile phragmoplast, the whole multitude of points into which plastosomes are to move, is set by a multitude of MT (+)-ends overlap that densely cut through the whole cytoplasm and are located on the equatorial plane. All these distinctions allow us to point out two different phragmoplast types in higher plant meiosis: mobile and immobile – in successive and simultaneous cytokinesis, respectively. To form up these two phragmoplast types, central division spindle fibers are utilized. It is not surprising, as central spindle fibers are in fact a phragmoplast – a system of MT bundles oriented to the equator with their (+)-ends and overlapping in this region. In the immobile phragmoplast formation, polar MT s play a considerable role, they complete the central spindle fibers in phragmoplasts between sister nuclei and completely make up phragmoplasts between nonsister nuclei. The role of polar MTs in the mobile phragmoplast formation and function was proposed (Shamina et al., 2007a). The most important distinction of simultaneous cytokinesis from that of successive consists in the absence of cytoplasm division after the first meiotic division. An immobile phragmoplast develops between daughter nuclei at telophase I in dicot PMCs with simultaneous cytokinesis, but it does not build a cell plate. Probably, this cytokinetic arrest is realized by means of a switch off of one of the basic cytokinetic processes – production of cell plate membrane vesicles (plastosomes). If it takes place, just as in transgenic tobacco line

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Res91, then cytokinesis is realized at telophase I of dicot meiosis by means of cell plate development and formation of daughter cell membranes (Shamina et al., 2000b).

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2. PROPHASE ABNORMALITIES AS THE REASON FOR MEIOTIC RESTITUTION In the prophase of a dividing eukaryotic cell, the cytoskeleton begins the process of transfer from the interphase system to the division spindle. In PMCs it is to proceed in the way that microtubule bundles orientation would change for the opposite in a cell. MTs (+)ends of radial interphase system are on the cell periphery, and (-)-ends are by the nucleus, as plant cell MTOCs are concentrated on the nuclear envelope surface (Vantard et al., 1990; Stoppin et al., 1994; Lambert, Lloyd, 1994; Azimzadeh et al., 2001). It is on the contrary in the division spindle: (+) MT ends are in the cell centre, on the spindle equator and kinetochores, and (-) ends are located on the spindle poles, on the cell periphery (Euteneuer et al., 1982). In animal cells such reorientation of elements of microtubule cytoskeleton performs due to the doubling of polar organizers – centrioles – and interaction of MT bundles diverging from them. Plant cell is deprived of centrioles and any other morphologically identified spindle polar organizers. Therefore, mechanisms of cytoskeleton reorientation during interphase-metaphase transition fall away from observation. Studying the cytoskeleton reorientation process at prophase is especially complicated by its depolymerization or drastic shortening of fibers for this period (Сhan, Cande, 1998; Peirson et al., 1997; Zee, Ye, 2000). But cytoskeleton rearrangement can be traced at these stages in the PMCs where cytoskeleton depolymerization does not proceed. In plant cell prophase, the cytoskeleton undergoes considerable rearrangements, the result of which is the perinuclear cytoskeleton system formation (DeMey et al., 1982; Vos et al., 2008). It is necessary to note that, in plant cell mitosis, perinuclear cytoskeleton systems were described, though their function and formation mechanisms remain obscure (Ambrose and Cyr, 2007). They are a loose cage of straight chaotic MT bundles converging with their (-)ends in several points (Schmit et al., 1983; Wick, Duniec, 1984). During prophase, such a ‗‘loose cage‘‘ can have a higher degree of structural organization, making the so-called prophase spindle or polar caps as a result of MTs bipolar orientation and even their convergence on the poles. During the nuclear envelope breakdown, the prophase spindle disappears (Wang et al., 1991), and its relation with prometaphase cytoskeleton has not been shown. At meiotic prophase, a cytoskeleton transfer from the interphase radial system to the perinuclear ring (Shamina, 2005a) is realised. As a result of our study of wild-type phenotypes with and without cytoskeleton depolymerisation at transient stages and of abnormal meiotic divisions, the cytoskeleton cycle undergoes the following stages at prophase (Shamina, 2005a): 1) transition from the reticular cytoskeleton to straight radial MT bundles located in the meridional plane, whose proximal ends are in the narrow ring zone (plane reorientation); 2) MT bundles reorientation at 90 o from the radial orientation to that of tangential to the nucleus; 3) prophase perinuclear cytoskeleton system formation – the meridional microtubular ring; the last process includes an MT bundles curvature and their co-

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orientation in the ring body. According to our data, variations of MT depolymerization/ repolymerisation (shortening) do not principally affect the cytoskeleton cycle at this stage. Due to the formation of perinuclear system at late prophase, the cytoskeleton fibers become closer to the nucleus (VanLammeren et al., 1985; Staiger, Cande, 1990; 1991; Suzuki, Tanaka, 1999; Ambrose et al., 2007) that, further on, provides – after the nuclear envelope breakdown - their contact with chromosome kinetochores. Aberrations of this important stage lead to drastic abnormalities in division spindle formation, corresponding disturbances of chromosome segregation and nuclear meiotic restitution.

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2.1. Cytoskeleton Conservation in the Interphase Radial Configuration The cytoskeleton block in the radial position was observed by us during meiosis of 10% of PMCs of WWG F1 hybrids № 9-00 (Triticum aestivum L. SP-718 x Agropyron glaucum L.). It is rather a rare abnormality of cytoskeleton cycle. If, in wild type meiosis and the trivial phenotype of WWG F1 hybrids, MT bundles become closer to the nuclear envelope at late prophase, all the processes of cytoskeleton reorganization are arrested in this abnormal phenotype. Cytoskeleton fibers, up to telophase, preserve their interphase orientation; perinuclear ring, division spindle, and the phragmoplast are not developing. Accordingly, processes of caryo- and cytokinesis do not proceed. At the same time nuclear envelope breaks down at the beginning of conventional prometaphase and its reformation at the end of conventional telophase proceeds normally. Between these events chromosomes remain immobile in the former nuclear area. The product of the first meiotic division is a monad with a restitution nucleus. In the second meiotic division, a common spindle is developing in such a cell. After the regular division in the second meiosis, such a cell forms a dyad with nonreduced 2n members. The reason for meiotic restitution in this phenotype is the impossibility of MT (+)-ends attachment to chromosome kinetochores due to their spatial distance: in the radial prophase configuration, MT (-)-ends are located in the central cytoplasm region, and (+) ends are on the periphery (Shamina et al., 2003а).

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Figure 2.1. Conservation of radial configuration of MT bundles during the first meiotic division in PMCs of WWG F1 №9-00: a) PMC before nuclear envelope breakdown; b, c) radial cytoskeleton at non-nuclear stages from prometaphase I to telophase I (nuclear envelope is absent, the former nuclear area is not different from the cytoplasm; d) monad with restitution nucleus at prophase II.

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2.2. Fused Spindle. Approachment of Nuclei at Prophase II of Meiosis with Simultaineous Cytokinesis in Dicotyledonous Species As a mechanism of meiotic restitution, the phenomenon of fused spindles has been known for a long time in dicotyledons (Jorgensen, 1928). It consists in the formation of common division spindle at metaphase II after normal first meiotic division. We observed five reasons initiating this process: approachment of prophase nuclei, fusion of perinuclear rings (p. 2.3 of the present review), underformation of interzonal cytoskeleton system in interkinesis (p. 7.9), aberration of interzonal cytoskeleton system in interkinesis (p. 7. 10), approachment and fusion of chaotic prometaphase figures in common cytoplasm (p. 4.4). In this part, behavioral aberrations of daughter nuclei are described at the prophase of the second meiotic division. In wild type meiosis with simultaineous cytokinesis in dicotyledons, daughter nuclei of the first meiotic division keep at the distance from each other in the common cytoplasm due to the developed system of interzonal cytoskeleton (VanLammeren et al., 1985; Hogan, 1987; Traas et al., 1989). At prophase II this system depolymerises (Peirson et al., 1997), interzonal MT cytoskeleton is replaced for the perinuclear MT system shaped as rings (Shamina, 2005a), but nuclei do not approach each other. After the nuclear envelope breakdown, the second meiotic division spindles form out of microtubules of perinuclear systems. Mutually perpendicular orientation of these spindles is achieved at late prometaphase II, after the chaotic stage. Spindles are located in the common cytoplasm, six phragmoplasts develop at telophase II connecting all the nuclei, and simultaineous cytokinesis autonomising four haploid nuclei proceeds. During male meiosis in two potato (Solanum tuberosum L.) clone groups 1) CD1015, CD1050 and 2) RH95-237-06, RH95-237-14; RH95-237-03; RH96-2013-03 abnormal behaviour of daughter nuclei at prophase II is observed. In 50% PMCs of the first clones group and in 90-100% PMCs of the second clones group, at this stage, migration of daughter nuclei and their abnormal approachment occur. After the nuclear envelope breakdown, a common spindle possessing chromosomes of both daughter nuclei is developing in the cell centre. After chromosomes segregation by this spindle, two unreduced 2n nuclei form in the common cytoplasm, and then a dyad forms out of such cells. Studies of MT cytoskeleton behavior at meiotic stages preceding the appearance of fused spindles (at telophase I – interkinesis – prophase II) do not reveal any deviations in all enumerated clones, both under cytoskeleton visualization using the classical method (Navashin fixation) and immunostaining. Approachment of nuclei begins after the transition from the interzonal cytoskeleton in interkinesis to the perinuclear one at prophase II. After chromosome segregation within the common spindle, two daughter nuclei with diploid chromosome number develop in the common spindle at anaphase II. At telophase II, an immobile phragmoplast develops between them, and cytokinesis with the formation of a dyad of unreduced microspores proceeds (Shamina et al., 2004).

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Figure 2.2. Approachment of daughter nuclei in PMCs of potato (Solanum tuberosumL.) clone with fused spindle phenotype at prophase II: a) normal position of daughter nuclei in interkinesis; b) approachment of nuclei in the common cytoplasm at prophase II; c) common spindle at metaphase II.

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2.3. Fused Spindle. Fusion of Cytoskeleton Perinuclear Rings at Prophase II Potato (Solanum tuberosum L.) clone СЕ10 is the producent of nonreduced gametes with fused spindle phenotype. In PMCs of this clone, the above-described (p. 2.2) approachment of daughter nuclei at prophase II is accompanied by the reorganization of perinuclear cytoskeleton system. This clone is characterized by complete asynapsis and curved C-shaped spindle formation at metaphase I. Due to the spindle curvature, its poles and, as a consequence, daughter nuclei become close at telophase I (see p. 3.2). However, their position corrects itself and becomes normal as a result of development of cytoskeletal interzonal system at late telphase I. Interzonal cytoskeleton system elongates and separates daughter nuclei, so they become maximally distant from each other, just as in normal meiosis. At prophase II, the nuclei surrounded by perinuclear cytoskeleton rings move to the cell centre and approach each other. Then perinuclear rings disjunct, fuse and form one common ring encircling both nuclei. After nuclear envelope breakdown, a common division spindle forms out of this common ring. Chromosome segregation proceeds normally, the phragmoplast develops between two daughter cells, and cytokinesis occurs. The product of meiosis is a dyad with unreduced 2n members (Conicella et al.,2003). Approachment of nuclei and fusion of perinuclear rings at prophase II occurs in 50% of PMCs of clone CЕ10. This phenomenon indicates the thing that MTs of perinuclear rings have their own active dynamics.

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Figure 2.3. Fusion of perinuclear rings (marked in arrows) of daughter nuclei at prophase II in potato clone CE10 PMCs a) disjunction of rings of approached nuclei b) the rings fusion, c) common perinuclear ring around daughter chromosome groups, d) common chaotic cytoskeleton figure at prometaphase II

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2.4. Cortical Cytoskeleton Ring and Meiotic Restitution In the meiotic division of PMCs of maize (Zea mays) haploid №1498, multiple spindle formation in the mononuclear PMCs in the first meiosis, and the deviation in the structure of cytoskeleton configuration – the perinuclear ring at prophase II - was found in 50% of cells. After nuclear envelope breakdown, in the first meiotic prometaphase, a normally appearing chaotic cytoskeleton figure was developing, out of which several spindles were developing instead of one; chromosomes were randomly distributed among them (‗‘multiple spindles‘‘ phenotype). In interkinesis, multinuclear monads were forming out of PMCs with multiple spindles. Usually, in such cells, at the second meiotic division, several spindles develop (due to multiple nuclei), and the meiotic division product of monocotyledons PMCs with multiple spindles phenotype is a multinuclear monad at tetrad stage. But in haploid №1498, unlike them, at prophase II, a cortical cytoskeleton ring was forming encircling all the micronuclei. At prometaphase II, in such cells, there developed a common chaotic cytoskeleton figure out of which one common biplolar spindle was forming. Chromosome segregation and cytokinesis in the second meiosis occurs normally, and a dyad was the meiotic division product. The cells – members of such a dyad – are predecessors of unreduced 2n-gametes (Shamina et al., 2007c).

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Figure 2.4. Cortical cytoskeleton ring at prophase II in multinuclear PMCs of maize haploid leads to chromosome integration into a common spindle at metaphase II a) multiple spindles at metaphase I; b, c) common cortical ring at prophase II (arrows), some parts of the ring may be out of focuse; d) common division spindle at metaphase II.

2.5. Autonomous Cytoskeleton Ring In WWG hybrid №14-2 (T. aestivum, cv. Novosibirskaya 67 x A. glaucum 13-1), at prophase I, PMCs are characterized by a drastically excentric position of nuclei in 7-10% of cells. Radial cytoskeleton is also asymmetrical: MT bundles radiate from the nucleus to the periphery are of different length. At late prophase, in the free cytoplasm region, where radial MT bundles had the biggest length, a ring figure of curved MT bundles is gradually forming. Under normal meiosis, an MT ring closely encircles the nucleus, but in PMCs of this hybrid, the ring is fully autonomous to the nucleus lying in the free cytoplasmic region. It is orientated also in the meridional plane within flattened tablet-shaped cereal meiocyte, just as a normal perinuclear ring. Later, a bipolar spindle without chromosomes forms out of this ring close to the nucleus.

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Figure 2.5 Formation of autonomous perinuclear ring and autonomous division spindle as a consequence of excentric position of the nucleus in PMCs WWG F1 hybrids: a) excentric nucleus at prophase I, radial cytoskeleton is asymmetrical, b, c) autonomous cytoskeleton ring at late prophase I, d) autonomous spindle in the cell with a restitution nucleus at interkinesis.

Chromosomes cannot contact spindle fibers and then form a restitution nucleus. At telophase, in the equatorial zone of the autonomous spindle, the phragmoplast/cell plate is developing which expand centrifugally. As the spindle and, respectively, phragmoplast are on the periphery, the cytoplasm incompletely divides, and daughter cell membranes form like incisions on the mother cell membrane. Sometimes there occurs cytokinesis with the separation of unnuclear cytoplast; in part of cells, cytokinesis may be arrested (Shamina et al., 2003a).

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2.6. Chromosome Arrest in the Zygothene „‟Bouquet” Configuration: Monopolar Chromosome Migration in a Bipolar Spindle At prophase, chromosome behavioral abnormalities can be the direct reason for the restitution nucleus formation at telophase on the background of normally proceeding cytoskeleton cycle. In male meiosis in WWG F1 hybrid №1-2-1, rice (Oryza sativum) № C45 and maize (Zea mays) №4479-4 haploids, we observed chromosome arrest in the ‗‘bouquet‘‘ configuration up to NEB. Occurrence frequency of PMCs with chromosome arrest in the bouquet varied from 30 to 80% in these genotypes. Chromosome compactization was normal. Stages of cytoskeleton cycle: perinuclear ring and bipolar division spindle formation – were also normal. At prometaphase, obviously due to the preserving univalents orientation, a monopolar figure consisting of reductionally oriented univalents and their kinetochore fibers, was developing. Then this monopole interacted with central spindle fibers and, as a result, a bipolar spindle was forming with all chromosomes (univalents) were attached to one pole. At anaphase, all univalents migrated to this pole forming a restitution nucleus. At telophase, on the spindle equator, a phragmoplast and a cell plate were forming, cytokinesis proceeded normally; the division result was a dyad. One cell had a 2n nucleus (or n in haploids), the other was unnuclear (Shamina et al., 2007b). Similar meiotic products – dyads with one anucleate member and the other with restitution nucleus - were reported in meiosis in a wide lily hybrid – a producent of 2n gametes (Barba-Gonzalez et al., 2005).

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Figure 2.6. Consequence of chromosomes arrest in the ‗‘bouquet‘‘configuration: monopolar segregation in the bipolar spindle in PMCs of WWG F1: a) chromosomes in the ‗‘bouquet‘‘ configuration at diakinesis; b) monopole at prometaphase I; c) monopolar segregation of univalents at anaphase I in bipolar spindle; d) telophase I with drastically unequal telophase chromosome groups.

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3. EARLY PROMETAPHASE ABNORMALITIES LEADING TO NUCLEAR RESTITUTION As a result of perinuclear cytoskeleton ring formation at prophase, (+) and (-) MT ends become to be at the equal distance from the nuclear area. It can be determined as the first stage of cytoskeleton reorientation during the interphase – metaphase transition. Prometaphase is separated from prophase by such an important morphological event as nuclear envelope breakdown (NEB). As a result, cytoskeleton can contact with chromosomes, and prometaphase - the process of direct division spindle formation – begins. Early prometaphase is determined by us as a period from the perinuclear ring breakdown (during NEB) to chaotic cytoskeleton figure formation (Shamina, 2005a). The main outcome of early prometaphase is entering the former nuclear area by microtubular bundles to contact chromosomes. (+) Mt ends become turned to the cell center having finished their turn at 180° at this stage compared to their orientation at interphase. The stages of MTs rearrangement during early prometaphase, include 1) perinuclear ring disassembly into separate fibers (MT bundles), 2) straightening of these bundles, 3) their invasion the former nuclear area. The result of these processes is the formation of the chaotic prometaphase cytoskeleton figure and a transfer to mid prometaphase. As for the process of MT bundles entering the former nuclear area after NEB, A. Bajer supposed it in his research of mitotic ultrastructure in the endosperm of African lily Haemanthus (Bajer, 1968; 1987; Bajer, Mole-Bajer, 1972). One can say that the meiotic perinuclear ring is an analog of the mitotic prophase spindle. It is interesting to compare their behavior during prophaseprometaphase transition. At mitosis, the prophase spindle develops from a perinuclear cytoskeletal cage by means of bipolarization of MT convergence centers (DeMey et al., 1982; Smirnova, Bajer, 1994). After NEB, MT bundles are chaotically oriented, i.e. the prophase spindle loses its clear organization and structure (Lambert, Bajer, 1975; DeMey et al., 1982; Wang et al., 1991). Unfortunately, the relationship of prophase – metaphase spindles is not shown because of methodic problems (low cell number in the analysis does not allow one to trace intermediate stages of the process). The meiotic perinuclear ring does not have convergence points and the traits of bipolar orientation of its constitutive bundles. Besides, it is not a loose cage, but a well organized and oriented ring structure. Probably, it is explained by differences in MT perinuclear system at mitotic and meiotic prophase. Phenotypes with

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abnormal cytoskeleton cycle at meiosis allow us to demonstrate the direct relationship between the perinuclear ring at prophase, chaotic prometaphase figure and the metaphase spindle in the sense that spindle formation proceeds by means of perinuclear ring MTs utilization.

3.1. Cytoskeleton Conservation in the Perinuclear Ring Configuration

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In WWG F1 hybrids № 9-3 (Triticum. aestivum L., cv. Novosibirskaya 67 x Agropyron glaucum L.), the perinuclear ring disintegration is arrested at the earliest stage of this process in 5 -7% of PMCs. As a result, after NEB, chromosomes remain scattered in the former nuclear area and do not contact with the microtubular cytoskeleton. The perinuclear ring with a completely preserved structure encircles the former nuclear area at the stages from prometaphase I till telophase I or interkinesis. The division spindle does not develop. No cytokinetic traits are found in such cells at the light level. At late (conventional) telophase I, a nuclear envelope re-forms around the chromosome group. In the second meiotic division, the formed mononuclear monad divides with a dyad formation having potentially viable predecessors of unreduced 2n gametes (Shamina, 2005b). Perinuclear ring conservation was also observed in WWG F1 hybrids № 2-2 и 2-5 (T. durum cv. Altaiskaya Niva х E. elongatum) with the abnormality frequency about 10%. Abnormal conservation of perinuclear cytoskeleton system during the first meiotic division was described in PMCs of mutant mel1 in rice (Oryza sativum) (Nonomura et al., 2007).

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Figure 3.1. Conservation of cytoskeleton ring configuration at prometaphase-telophase I in PMCs of WWG F1 hybrids: a) late prophase I: perinuclear ring is formed, the nuclear area is distinct, b, c) conventional metaphase-telophase I: nuclear envelope broke down, the perinuclear ring is preserved in the cytoplasm, the former nuclear area is indistinguished from the cytoplasm; d) monad with a restitution nucleus.

3.2 Aberration in Straightening of Microtubules of Perinuclear Ring: CSpindle According to our observations, this abnormality is the most widely spread and a mass spindle aberration in plants and is encountered in the meiosis of 80% WWG F1 hybrids and in all WR F1 hybrids we studied with the frequency 30-100%. It is found only in the phenotypes with univalents reductional orientation (each univalent is attached only to one

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spindle pole, i.e. carries one kinetochore fiber). Such spindles also develop in 100% of PMCs of synaptic tomato mutant as6, Brassica juncea haploids and potato clones СЕ10, ВЕ62, ВЕ1050, characterized by complete asynapsis. C-shaped spindles are also typical of the phenotype of meiotic mutation ms28 in maize with normal synapsis of homological chromosomes (Shamina et al., 1981). The spindle curvature in M1 was described for haploids (Sadasivaiah, Kasha, 1971), synaptic higher plant meiotic mutants (Iwanaga, 1984; Chan, Cande, 1998), wide hybrids (Darlington, 1965). In this phenotype, after nuclear envelope breakdown, the perinuclear ring disintegrates into individual MT bundles and disappears as a consolidated structure. The further spindle formation process proceeds the following way. At early prometaphase, univalents with attached MT bundles are chaotically scattered in the former nuclear area. One can often observe the thing that MT bundles are curved at the chaotic prometaphase stage. Later on, they develop a spindle of abnormal shape and location. The shape abnormality is caused by the fiber curvature, so the metaphase spindle is crest-shaped. The abnormal spindle location is expressed in the thing that its equatorial zone has a shift from the center to the cell periphery. Reductionally oriented univalents roughly congress on the spindle equator and imitate the metaphase plate. We defined this stage as metaphase I. Our observations show that the location of reductionally oriented univalents on the spindle equator is an obligatory stage of asynaptic meiosis. We constantly observed the formation of such a ‗‘pseudometaphase plate‘‘ in all the phenotypes with asynaptic meiosis we analysed – both in straight and curved spindles. At anaphase I univalents have their uncoordinated migration to the poles and turn scattered along the whole spindle body. This stage is very prolonged compared to the ―metaphase‖, it is more often encountered in preparations and, probably, due to this reason, it is mistakenly determined as abnormal metaphase. Finally, univalents reach the poles, and aneuploid chromosome groups develop on the poles of a curved spindle. On the curved spindle equator, a phragmoplast/cell plate develops and cytokinesis occurs. The cytokinetic process is normal, but, when the cell plate reaches the closest mother cell membrane and contacts it, the plastosomes fusion and development of daughter cell membranes, which incompletely divide the cytoplasm as an incision, proceeds. A premature stop of cytokinetic processes takes place. An incised binucleate monad is the division product in this case. Due to the spindle bending, its poles are close, and telophase chromosome groups can separate from the poles and migrate to the cell center. Based on the close daughter nuclei in the common cytoplasm in the second meiosis, a common division spindle develops most often. A dyad with unreduced members is the product of meiosis (Shamina et al., 1999). Such a phenotype is observed in a number of WWG F1, WR F1 hybrids, maize and rice haploids, in meiotic maize mutant ms28 . However, telophase figure correction occurs at this stage in many of genotypes with Cspindles we analysed. MT bundles polymerise from the curved spindle poles towards each other, connect on the equator and form a broad phragmoplast which crosses most part of the cytoplasm (Shamina et al., 2007a). In such phenotypes the cytokinesis proceeds normally. In additionl, there may be a correction of phragmoplast/cell plate centrifugal movement, which proceeds on the polarized cytokinesis type (Cutler and Ehrhardt, 2002; Cook, 2004), i.e. centrifugal movement continues also after the system reaches the closest mother cell membrane.

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Figure 3.2. С-spindle and meiotic restitution in PMCs of maize haploid № 2906. a) curved spindle at conventional metaphase – anaphase I, b) assymetrical phragmoplast position (arrows) because of the spindle curvature; c) binucleate monad with an incision (arrow) at interkinesis.

The spindle bending is also typical for the meiosis in haploids (Sadasivaiah, Kasha, 1971) and synaptic higher plant mutants (Iwanaga, 1984; Chan, Cande, 1998). It proves our supposition on this abnormality reasons at asynaptic meiosis. Besided, the curved spindle abnormality is almost exclusively typical of the first meiotic division. The spindle bending in the second metaphase was traced by us only in two phenotypes out of many dozens of forms with C-spindles we analysed. C-shaped spindles were described earlier in wide hybrid meiosis (Darlington, 1965), but their formation mechanism was not investigated.

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3.3 Arrest of Cytoskeleton Invading the Former Nuclear Area In WWG F1 hybrid № 5-5 (T. aestivum сv. ANK9 x A. glaucum 52-3), the process of cytoskeleton cycle, at early prometaphase, is arrested at later stages. The perinuclear ring loses its integrity and becomes disordered, microtubules straighten in its composition, but do not move. They do not leave the perinuclear zone, do not enter the former nuclear area and do not contact with chromosomes and each other. From prometaphase to conventional telophase, MTs encircle the former nuclear area – but not as a consolidated ring, as it is in the abovedescribed phenotype (see. p. 3.1), but they encircle it as a set of tangentially oriented straight microtubular bundles. As an outcome, the division spindle is not forming and chromosomes remain scattered in the center of the cell without attachment to the cytoskeleton. This

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Figure 3.3. Cytoskeleton configuration at conventional metaphase I - telophase I under the arrest of fibers penetration into the former nuclear area of PMCs in WWG F1 hybrids: a - c) cytoskeleton conservation in the configuration of a disorganized perinuclear ring during metaphase I – telophase I, d) monad with restitution nucleus at interkinesis.

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cytoskeleton configuration preserves till interkinesis. As an outcome of this, chromosome segregation and cytokinesis are arrested in the first meiotic division. At late telophase, the nuclear envelope reforms around the whole chromosome set, and a restitution nucleus is developing. The PMC share is – 5-7% with this abnormality. The analogous phenotype is also demonstrated by WWG F1 hybrid № 59-8 (T. durum cv. Altaika х E. elongatum) at frequency 10%. (Shamina et al., 2003c).

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4. MID PROMETAPHASE ABNORMALITIES AND MEIOTIC RESTITUTION The period of cytoskeleton chaotic configuration that forms after the penetration of MT bundles into the former nuclear area is indicated as mid prometaphase. This period makes us refer it to a separate substage due to its temporal prolongation compared to other prometaphase substages. First, it is possible to assume that, at this time, MTs attach to chromosome kinetochores, and our observations prove it. Kinetochore spindle fibers formation can normally be observed at the chaotic stage in objects with quite big PMCs, also in forms characterized by the prolonged prometaphase stage. It is a most important stage of division spindle formation, during which the bipolar spindle fibers development occurs (Shamina, 2005a). MT bundles attach to chromosome kinetochores forming bipolar kinetochore spindle fibers. If chromosomes are bivalent or they are a pair of chromatids with oppositely oriented kinetochores, then the system of ‗‘kinetochore MT bundle – chromosome – kinetochore MT bundle‖ – is a bipolar fiber, which is the bipolar spindle element. If chromosomes are univalent, and each of them has one (unsplitted) kinetochore, each element carries one kinetochore MT bundle, and, further on, it attaches only to one of the spindle poles and does not represent a bipolar spindle fiber. Free MT bundles, at the chaotic prometaphase stage, attach each other with their (+) ends and form another important spindle element – bipolar central fibers. Later they serve as the base of a developing phragmoplast. Analysis of abnormal phenotype of WWG F1 hybrid № 27-1 (T. aestivum cv. Saratovskaya 29 x A. glaucum) and WWG F1 № 5-98 (T. durum cv. Altaika x E. elongatum) with a very prolonged chaotic stage and a full arrest of kinetochore spindle fibers formation allowes us to reveal the process. The main process of midprometaphase or the chaotic stage is the formation of the basic elements in bipolar spindle development, its bipolar fibers: central and oppositely oriented kinetochore fibers (Shamina, 2005a). Aberrations of these processes lead to those of division spindle structure and phragmoplast, abnormalities in caryo- and cytokinesis and to the meiotic restitution.

4.1. Monopolar Spindle One of the key stages of plant spindle bipolarity setting is the formation of bipolar fibers out of antiparallel MT bundles. Under the aberration of this stage, and the rest prometaphase processes preservation (MT co-orientation in the spindle body, their convergence on the poles), monopolar spindles develop. As two types of bipolar spindle fibers – central and the opposite kinetochore ones – participate in the plant spindle formation, the absence of these

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both elements leads to the monopolar spindle development. Bipolarity of central spindle fibers is provided as a result of free MT bundles (+) ends junction. Double kinetochore fiber bipolarity is provided by the opposite orientation of chromosome kinetochores. Therefore, for monopolar plant spindle formation, a combination of two abnormalities is necessary: absence of mutual attachment of free MTs (+) ends, and absence, due to some reason, of oppositely oriented kinetochore fibers. A combination of two such abnormalities is a rare event. In the meiosis of WWG F1 hybrid № 30-2, chromosomes are presented by univalents with an unsplitted kinetochore (reductional univalents). Thus double kinetochore fibers formation, in this case, is impossible; only one MT bundle is attached to each univalent. During central spindle fibers formation, interaction of free MT (+)-ends is aberrated, probably by the effect of the corresponding mutation which is manifested in conditions the allohaploid F1 hybrid genotype. Instead of bipolar fibers, free MT bundles and univalents with a single kinetochore MT bundle, participate in the spindle formation. Such monopolar spindle elements interact; their (-)-ends converge with the formation of a monopolar figure. Such a monopolar cone is compact, there is no anaphase chromosome movement in it as a rule, phragmoplast is absent. At telophase, a monad with a restitution nucleus is developing (Shamina et al., 2003b). Such a phenotype was also observed by us in WR F1 hybrid and haploid of Brassica juncea. In a number of wide cereal hybrid phenotypes, the monopolar spindle has a looser structure and the shape of a non-compact cone, but that of a monoaster. At telophase, a monads with many micronuclei developes out of such cells. However, sometimes, in such spindles, anaphase univalent migration is realized to the single pole, and a restitution nucleus is forming (Shamina, 2005b). The phragmoplast does not form in such monopoles, and cytokinesis does not proceed.

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Figure 4.1. Monopolar spindles in PMCs of WWG F1 hybrids at metaphase I: a) compact monopolar spindle at conventional metaphase I – telophase I; b) one of not numerous chromosome anaphase movements in a compact monopolar spindle; c) loose radial monopolar spindle at metaphase I; d) telophase I in PMCs with a loose monopole.

Monopolar spindles are a well known and most frequently encountered abnormality of centriolar spindle in animal cells. This abnormality leads to the arrest of chromosome segregation and was described in the phenotype of homozygotes on mutations polo (Sunkel and Glover, 1988), mgr (Gonzalez et al., 1988), mast in Drosophila melanogaster (Lemos et al., 2000), in cell culture of the Chinese hamster (Wang et al., 1983). Monopolar spindles also form as a result of the influence on dividing animal cells by specific inhibitors (Mazia et al., 1981). Besides, monopolar spindles function in some types of modified cell division, e.g. in the meiosis of Sciara (Abbot et al., 1981; Fuge, 1994). The reason for monopolar spindle

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formation instead of a normal bipolar one in animal cells is segregation arrest of polar spindle organizers (centrosomal structures) (Sawin et al., 1992; Heck et al., 1993; Blangy et al., 1995). In acentriolar plant cell, the monopolar spindle has not been described in natural conditions for a long time; it was reported on its formation under the effect of a number of chemical agents (Tiwari et al., 1984; Binarova et al., 1998; Smirnova et al., 2002).

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4.2. Autonomous Spindle Among abnormal meiotic divisions, there are those in which spindle kinetochore fibers formation is completely arrested. Chromosomes remain a disoriented group in the former nuclear area, and MT bundles, with lesser of bigger success, undergo the division spindle formation process. Under the aberration of MT bundles attachment to kinetochores, the socalled autonomous spindle - bipolar cytoskeleton system deprived of chromosomes and consisting of only central spindle fibers – is developing. Immobile chromosomes lying separately form restitution nucleus. A phragmoplast and a cell plate can develop on the autonomous spindle equator, which are indicative of such spindle fibers bipolarity. A chaotic cytoskeleton configuration is also encountered. Under simultaineous aberration of MT (+)ends attachment to kinetochores, and their conjuction with each other, an autonomous monopolar spindle is forming. During male meiosis in WWG F1 № 27-1 (T. aestivum cv.Saratovskaya 29 х A. glaucum) and TA F1 № 5-98 (T. durum сv. Altaika x E. elongatum), the cytoskeleton cycle is of normal procedure till the chaotic prometaphase stage. Free MT bundles conjunct with each other by their (+)-ends forming bipolar central spindle fibers, whereas kinetochore ones are absent. Chromosomes are chaotically scattered in the former nuclear area in the cell center, close to the formed bipolar spindle, or they form a compact group close to it, but do not connect to the spindle fibers. The division in the second meiosis of obtained monad with a restitution nucleus results in the formation of a dyad with 2n members at tetrad stage.

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Figure 2. Autonomous spindles at metaphase I im PMCs of WWG F1 hybrid: a, b) bipolar autonomous spindle (arrows) at metaphase I; c) chaotic autonomous ―spindle‖ at metaphase I (arrows); d) common spindle in the monad with restitution nucleus at metaphase II.

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4.3. Chromosomes Monopolar Migration in a Bipolar Spindle. “Comet” Phenotype

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This abnormality was observed in meiosis of wide cereal F1 hybrids: WR F1 (T. aestivum cv. Saratovskaya 29 х S. cereale cv. Onokhoiskaya), WWG F1 № 27-1, (T. aestivum cv. Saratovskaya 29 х A. glaucum), № 21-1 (T. durum cv. Altaiskaya Niva х E. elongatum), also in wheat-rye alloplasmic line CYANK9 and in monosomic line 2А of wheat T. aestivum cv. Milturum 533. In this phenotype, the spindle kinetochore fibers formation is aberrated in 15% of PMCs. The metaphase spindle is developing bipolar, but all the chromosomes move to one of the poles, whereas the opposite pole remains ―empty‖. An autonomous bipolar spindle is forming in such cells, and it is composed of exclusively central spindle fibers. Chromosomes contact it with their arms and slide along its surface to one of the poles as a unified group (Seryukova et al., 2003). The telophase chromosome group on one pole and spindle fibers diverging from it looks very originally, reminding of a comet. A cell plate develops at telophase on the spindle equator (in the middle of a ―comet tail‖), and the spindle becomes barrel-shaped which is typical of the phragmoplast. Centrifugal movement of phragmoplast/cell plate and daughter cell membrane formation occur normally, and an anucleate cytoplast splits from the cell. The other cell contains a restitution nucleus. Rather often cytokinesis may be arrested. Chromatin movement along the autonomous spindle have been described in mouse oocytes (Deng et al., 2009). Migration of all chromosomes to one pole was described as a reason for restitution nucleus formation (Sears, 1953; Avers, 1954; Stefani, 1986, Jauhar, 2003). This phenomenon was also reported in animal meiosis of meiotic mutants fusolo и solofuso (Drosophyla) (Bucciarelli et al., 2003). However, the mechanisms of this phenomenon remained unknown, as there was no division spindle visualization as a cytoskeleton structure. We managed to reveal two reasons for chromosome monopolar segregation in a bipolar spindle (see also p. 2.6).

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Figure 4.3. Result of chromosome monopolar migration along the autonomous spindle (arrows) in PMCs of WWG F1 hybrid a, b) late anaphase I; c) telophase I; d) autonomous phragmoplast (arrow).

4.4. Fused Spindle. Approachment and Fusion of Cytoskeleton Chaotic Figures at Mid-Prometaphase II in PMCs with Simultaneous Cytokinesis in Dicots During normal meiosis chaotic prometaphase II figures representing randomly oriented MT bundles in a complex with chromosomes are located at the maximal distance from each

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other in common cytoplasm. Then spindle fibers orient along the axes of the future division, co-orient, converge on the poles, and two mutually perpendicular division spindles develop in the common cytoplasm (Traas et al., 1989). In potato clones – producents of 2n gametes ВЕ1050; ВЕ62 in 50% PMCs, and in tomato (Lycopersicon esculentum) meiotic mutant as6 in 100% of PMCs, prometaphase chaotic figure migration proceeds to the cell center and their fusion into a common chaotic figure takes place. All the enumerated genotypes are characterized by a complete asynapsis, curved spindles at metaphase I, approachment of daughter nuclei on the poles of a curved spindle at telophase I and their further correction by the developing cytoskeleton interzonal system. These nuclei enter prometaphase II and are located at a normal distance from each other. But, after NEB and the onset of prometaphase, chaotic figures approach each other and fuse in the cell center. Then their fibers co-orient, converge on the poles and form a common spindle. We have never observed such a fusion of cytoskeleton figures at late prometaphase II (set of roughly bipolarly oriented spindle fibers non-converged on the poles without a developed metaphase plate) or developed spindles at M II (Shamina et al., 2004). Results of our observations using the method of immunostaining showed that the MT cytoskeleton per se does not play any role in chaotic figures approachment.

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Figure 4.4. Approachment of chaotic cytoskeleton figures at prometaphase II in PMCs of tomato (Lycopersicon esculentum) meiotic mutant as6. a, b) successive migration of chaotic prometaphase figures to the cell center; c) formation of common chaotic prometaphase figure; d) common spindle at metaphase II.

5. ABNORMALITIES OF LATE PROMETAPHASE AS A MEIOTIC RESTITUTION MECHANISM The span between the chaotic stage and metaphase is indicated as late prometaphase. The basic cytoskeleton dynamic process at this period is cytoskeleton realization from the chaotic configuration and bipolar spindle formation, i.e.spindle fibers orientation along the future division axis. Research of late prometaphase with the help of abnormalities typical for this stage allows one to reveal some of its constitutive morphological processes and to determine their interrelation. At late wild type meiotic prometaphase, the formed bipolar spindle elements (chromosomes with oppositely directed spindle kinetochore fibers and bipolar central spindle fibers) orient along the future division axis and form a system of bipolar fibers – the division spindle. At the same stage, the final spindle formation processes – fibers (-)-ends convergence and spindle poles development, also development of metaphase plate (chromosome congression) - take place.

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During normal meiosis, these processes occur rapidly, so that many their details are indiscernible. Multiple observations of this stage in normal and abnormal meiosis allow us to assert that bipolar spindle fibers orientation proceeds as a result of their turn and migration in the cytoplasm; each fiber is independent from the rest (Shamina, 2005a). These peculiarities can be traced directly under the formation of (relatively) normal bipolar spindles in the meiosis of wide F1 hybrids. Due to a number of reasons (probably univalent chromosomes) their prometaphase stage is considerably prolonged compared to the norm, and its distinct events and intermediate stages become accessible for direct observation. The main result of late prometaphase is the completion of bipolar spindle formation – the pledge and condition of mother cell genome division into two parts. Aberrations of this process can lead to the formation of restitution nuclei.

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5.1. Chaotic Spindle Prometaphase I of meiosis in WWG F1 hybrid № 27-1 (T. aestivum cv. Saratovskaya 29 x A. glaucum) is drastically abnormal. First, it is expressed in the unusually longstage of ‗‘chaotic bundles net‘‘ at which spindle fibers are disorderly scattered over the cytoplasm. At this stage, the course of prometaphase is arrested in part of the cells, and the division spindle is not developing. This figure (spindle fibers chaotic net and a group of disoriented chromosomes in the center) is preserved in the metaphase, anaphase and telophase of the first meiotic division. At the end of telophase I, chromosomes are encircled by the nuclear envelope, and a restitution nucleus or a set of micronuclei is forming. Chromosomes carry MT bundles on kinetochores, i.e. spindle kinetochore fibers are developing. But there is no chromosome anaphase movement and chromosomes remain in the cell centre. It is possible to distinguish long central spindle fibers oriented chaotically. This abnormality is the result of abrerration of bipolar orientation process of the developed spindle fibers at late prometaphase. Cytokinesis does not proceed due to the absence of a developed bipolar phragmoplast (it can also be called chaotic here). In the second meiotic division, a common spindle and a dyad with 2n members (Seriukova et al., 2003) develop. The abnormality‘s frequency – up to 10%.

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Figure 5.1. Chaotic spindle and its consequences in PMCs of WWG F1 hybrid. a, b) chaotic spindle at metaphase I; c) monad with a restitution nucleus in interkinesis; d) common spindle at metaphase II.

The similar picture is observed in the abnormal meiosis in dicots, at the prometaphase of both first and second meiotic division. In PMCs of pea (Pisum sativum) meiotic mutant ms3, at metaphase I, abnormal cytoskeleton figures in a form of a network of chaotically oriented MT bundles, built instead of a bipolar spindle. Chromosomes with attached kinetochore fibers are disorderly scattered among free MT bundles; individual spindle fibers do not interact with

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each other, do not obtain bipolar orientation, do not converge with their (-)-ends. Chromosome movement is arrested at anaphase, a monad with micronuclei develops in the interkinesis. As a result of these aberrations, at telophase II, the cell divides into many fragments forming a polyad at the stage of tetrads (Shamina et al., 2000a). A net of chaotically scattered spindle fibers in the cytoplasm at metaphase II, after the normal first meiotic division, is a typical phenotypic feature of sugar beet mutant line SOAN112. The first male meiotic division proceeds without deviations. Drastic abnormalities of cytoskeleton dynamics appear at prometaphase II. After NEB, the MT cytoskeleton concentrated in the perinuclear region, begins to rearrange for the spindle formation of the second meiotic division. However, instead of two bipolar mutually perpendicular spindles, in 30% of the mutant PMCs, a chaotic net of disoriented criss-crossed MT bundles with disorderly scattered chromosomes with kinetochore fibers, is developing. A bipolar system of mutually parallel MT bundles, typical of spindles, does not develop. MTs do not converge on pole ends and it can be well seen when operating a microscope screw. At anaphase, chromosomes segregate into chromatides, but anaphase movement is arrested or, either, chromosomes move at a small distance. Abnormal daughter cell membranes sometimes divide the cell incompletely and look like incisions on the mother cell membrane. This is the result of multiple disoriented short cell plates formation on the chaotic fibers. Herewith, an incised monad with micronuclei is forming

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5.2. Spindle Disorientation at Metaphase II in the Meiosis with Simultaineous Cytokinesis in Dicots Orientation of developed bipolar spindle fibers, just as that of spindles proper, along the cell division axis is realized at late prometaphase. In the abnormal phenotype ‗‘disoriented spindle‘‘, bipolar spindles develop, but orientation of division axes changes in the cell. Herein, in the second meiotic division of dicots PMCs, poles of spindles located in the common cytoplasm, can converge, and it leads to the formation of a nucleus with a doubled chromosome set (Mok, Peloquin, 1975; Dorogova and Shamina, 2000; Taschetto and Pagliarini, 2003; Camadro et al., 2008). The AtPS1 gene and a corresponding set of mutants that produce pollen grains which are up to 65% diploid and give rise to numerous triploid plants in the next generation was described in Arabidopsis (d‘Erfurth et al., 2008).

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Figure 5.2. Spindle poles convergence under the spindles disorientation in the second meiotic division in tobacco (Nicotiana tabacum L.) PMCs. a) normal spindles position at MII in PMCs of wildtype tobacco; left spindle metaphase plate is seen from the equator, right – from the pole, b - d) spindle disorientation with poles convergence in the second meiotic division of tobacco PMCs, line Res91.

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6. ANAPHASE ABNORMALITIES THAT LEAD TO MEIOTIC RESTITUTION It is surprising, but chromosome segregation abnormalities per se (chromosome anaphase movement arrest in the normal spindle) are not so often encountered among forms with abnormal meiosis as one would expect There are literary reports on chromosome anaphase movement aberrations (Rhoades, Dempsey, 1966; Wagenaar, 1968; Fukuda, Sakamoto,1992). But, as these observations were made without spindle visualization, it is impossible to exclude the thing that these abnormalities are caused by spindle structure aberrations, but not the mechanism of anaphase movement proper. Chromosome movement can be decelerated in a normal spindle, there may be chromosome laggards. As our wide observations of hundreds of different plant forms with abnormal meiosis show, if the anaphase began and the division spindle is normally developed, then chromosomes reach the poles, sooner or later. We manage to trace univalents that do not reach the poles and remain in the body of a normal spindle till the end of telophase only in several genotypes of haploids and allohaploids. Cytokinesis is not aberrated, as a rule, and a dyad with mironuclei is the product of such a division in most cases. If cytokinesis does not proceed, monads with micronuclei or a restitution nucleus develop. In the case of micronuclei formation, univalents move at a bigger distance, and the spindle may be longer. Restitution nucleus forms when chromosomes migrate at a comparatively short distance at anaphase and remain close to the equator (p. 6.1).

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6.1. Aberration of Anaphase Chromosome Movement This phenotype is observed in PMCs of WWG F1 hybrid № 519 in 20% of cells. At metaphase I, univalents, just as in the trivial phenotype, group on the spindle equator imitating the metaphase plate. At anaphase I, univalents begin to move, but do not diverge far from the equator. The phragmoplast can develop independently from the chromosomes position, or its development or function may be arrested. Cytokinesis can be realized (incomplete, so daughter cell membranes are incision-shaped) or not. In any case, nondiverged chromosomes form a restitution nucleus out of which a common division spindle is developing at metaphase II.

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Figure 6.1. Arrest of chromosomes at early anaphase in PMCs of WWG F1 hybrid: a) PMC at early telophase I; b, c) attemption of phragmoplast (arrows) centrifugal movement in PMCs with chromosome arrest in the spindle body; d) monad with a restitution nucleus at interkinesis.

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6.2. Spindle Shortening During Anaphase The chromosome segregation is provided by two processes: kinetochore spindle fibers shortening (anaphase A) and central spindle fibers lengthening (anaphase B) (Bruss-Mascher et al., 2004; Bouck and Bloom, 2005). By means of anaphase A, chromosomes become spatially distant from each other; by means of anaphase B – spindle poles. In dividing higher plant cells, the process of anaphase B is realized alongside with anaphase A (Yu and Russell, 1993). In PMCs of maize dihaploid №4611 we observed a drastic shortening of normal division spindle at anaphase I in about 35% of cells. Chromosome synapsis is of normal procedure in the dihaploid, the division spindle has normal size and shape at metaphase I. Chromosomes start anaphase movement coordinatedly, as anaphase groups. After chromosomes pass the distance, which is about one half of half-spindles length, the spindle begins to shorten, and chromosomes move backward. Finally, they are on the equator, there where they began their movement from. At late telophase, approached chromosome groups are encircled by the common nuclear envelope forming a restitution nucleus. Two closely approached daughter nuclei may also develop. Cytokinesis is arrested: phragmoplast fibers are also abnormally shortened, centrifugal movement of phragmoplast/cell plate does not proceed. A common division spindle is forming in the monad at metaphase II. As a result of the second meiotic division, dyads with 2n members develop at the stage of tetrads. One can hypothesise that shortening of a normal metaphase spindle, under the onset of anaphase, is caused by the aberration of activity balance of motor cytoskeleton MAPs, kinesins (Lee and Liu, 2007). Some of them provide transport towards (+)-MT ends separating the spindle poles, others – towards (-)-ends closing the poles (Hamada, 2007; Bannigan et al., 2007). The first ones activity prevails at normal anaphase. Abnormally shortened spindles are described in the phenotype of Arabidopsis mor-1 and clasp-1 mutants (Kawamura et al., 2006; Ambrose et al., 2007). The coordination of spindle length is important for regular cell division, and it is a conserved feature of eukaryotic cell (McNally et al., 2006; Yang et al., 2003; 2005).

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Figure 6.2. Spindle shortening during anaphase I in PMCs of maize dihaploid a) normal chromosome segregation at the onset of anaphase I; b) spindle shortening, disappearance of its polar regions; c) dumb-bell restitution nucleus; a reduced phragmoplast is seen on the nuclear sides, d) common spindle in the monad at metaphase II.

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7. CYTOSKELETON ABNORMALITIES AT TELOPHASE AND MEIOTIC RESTITUTION Cytokinesis is the fundamental cell division process whose mechanisms remain mostly unclear in plant cell (Jurgens, 2005; Segui-Simarro et al., 2007; VanDamme et al., 2008). Cytoskeleton abnormalities and daughter genomes autonomisation failure also lead to the meiotic restitution. Daughter nuclei, being in the common cytoplasm, can approach each other, and it leads to the congression of their chromosomes in the common spindle in the further cell division – both meiotic and post-meiotic. Due to the analysis of meiotic division abnormalities, we managed to describe the process of mobile phragmoplast functioning and formation in PMCs of monocots and also to divide the telophase into substages (Shamina et al., 2007). Bipolar phragmoplast fibers (central spindle fibers) develop at mid prometaphase, and the phragmoplast proper (central spindle, bipolar system of bipolar fibers, early phragmoplast) is developing at late prometaphase. After chromosomes segregation to the opposite poles at anaphase, the spindle cytoskeleton composition changes. Kinetochore fibers depolymerise and shorten and, at early telophase, the spindle consists of only central fibers. At late anaphase – early telophase, the central spindle looks like a solid column between telophase chromosome groups. We determined this structure as an early phragmoplast. At early telophase, on the spindle equator, a cell plate begins to develop as a monolayer of membrane vesicles (plastosomes). These vesicles are Golgi-derived, and are transported here by the central spindle fibers. After the cell plate crosses the whole spindle, its central fibers redistribute so that they surround the cell plate growing edge and obtain a form of a hollow cylinder – mid phragmoplast. The fibers encircle the growing cell plate as a palisade. Probably, at this stage, central spindle fibers lose their lateral connections with each other, which they had in the anaphase spindle. The phragmoplast in PMCs is not a set of short fibers around the cell plate, but a system of long pole-to pole fibers linking polar regions with the equator and surrounding the growing cell plate edge. At mid telophase, phragmoplast fibers become longer, curve and, due to this, their central points (regions of MTs (+) ends overlapping) make centrifugal movement (Shamina et al., 2007a). We called this expanding barrel-shaped structure with progressively curving fibers late phragmoplast. As the phragmoplast moves to the periphery, its fibers become more and more curved. The cell plate expands by means of new membrane vesicles attachment to its growing edge, and phragmoplast fibers continue to encircle its growing edge providing vesicles transport to this region. During centrifugal movement the number of phragmoplast fibers increases, probably by means of polar synthesis of new MTs (Shamina et al., 2007a). When the cell plate reaches the mother cell membrane and contact it, plastosomes fuse in its composition with the formation of daughter cell membranes. After this, the formation of daughter nuclei envelopes proceeds. The developed membranes cut the phragmoplast fibers on the equator in the region of MT (+)-ends overlapping. Separated phragmoplast fibers, after this, become part of radial interphase cytoskeleton of daughter cells. At late telophase, when the phragmoplast/cell plate practically completely cross the mother cell cytoplasm, MT bundles begin to polymerise from telophase chromosome groups to the direction opposite to the equator. They do not participate in cytokinesis, but they are part of the radial cytoskeleton typical of interkinesis. The cytoskeleton cycle returns to the initial point here.

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It is shown, that the process of phragmoplast formation and operation during mitosis in meristematic cells can also be divided into phases: phragmoplast initials, solid phragmoplast, transitional phragmoplast, and ring-shaped phragmoplast (Seguí-Simarro et al., 2004). Not numerous phragmoplast abnormalities were described in a number of phenotypes: embryonic mutants pilz and hinkel (Arabidopsis thaliana) (Mayer et al., 1999; Strompen et al., 2002), in the meiosis of mutant aph (Zea mays) (Staiger, Cande, 1993), in PMCs in wild populations of the grass Paspalum (Pagliarini et al., 1999) and wide cereal hybrids F1 (Shamina et al., 2007), also under physical and chemical influences on mitotic cells (MoleBajer, 1969; Smirnova and Bajer, 1998). A number of embryonic mutations that disturb Arabidopsis cytokinesis (Sollner et al., 2002) are known, including those arresting membrane vesicles fusion and daughter cell membrane formation. Mutants with cytokinetic aberrations in male meiosis were described in Arabidopsis (Chen and McCormick, 1996; Hulskamp et al., 1997; Spielman et al., 1997; Hauser et al., 2000; Magnard et al., 2001).

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7.1. Arrest of Basic Telophase Processes In male meiosis in WWG F1 hybrid № 9-02, about 20% dyads form at the stage of tetrads. It is explained by a complete arrest of the basic processes of telophase I: phragmoplast centrifugal movement, cell plate formation and nuclear envelope restoration around daughter nuclei. Only the amplification of phragmoplast fibers occurs, and it becomes wider, but it does not make any centrifugal movement and does not approach the mother cell membrane. At conventional interkinesis, the radial cytoskeleton is developing in the cell existing alongside with the telophase spindle (early phragmoplast). At prophase II, a cytoskeleton ring that encircles both chromosome telophase groups develops out of radial cytoskeleton elements. A common division spindle is developing at prometaphase II out of this common ring, and a dyad forms at the stage of tetrads. Cells – the dyad members – have unreduced 2n chromosome number. Aberration of regulation of the corresponding cell cycle stages is, probably, the reason for this phenotype‘s abnormalities. Experiments with the expression of a nondegradable cyclin in plant mitotic cells resulted in a phenocopy of the described abnormality (Weingartner et al., 2004). Arrest of daughter nuclei envelopes reformation and arrest of cytokinesis at telophase were described in experiments on kinase CDK1 inhibition (Wheatley et al., 1997), in dividing rat kidney cells. In S. cerevisiae (budding yeast), high levels of nondegradable cyclin CLB2 arrest cells late in mitosis, with segregated chromosomes and the presence of an elongated mitotic spindle (Surana et al., 1993). Indestructible cyclin Cdc13 arrests Schizosaccharomyces pombe cells in anaphase with separated and condensed chromosomes and no septa (Yamano et al., 1996).

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Figure 7.1. Meiotic process under the complete arrest of telophase I processes in PMCs of WWG F1 hybrid: a) telophase I, b) interkinesis, c) prophase II with a common perinuclear ring (arrows), d) common spindle at metaphase II.

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7.2. Complete Arrest of Cytokinetic Processes at Early Phragmoplast Stage This phenotype observed in a rice haploid № 207 and WWG F1 hybrid № 738 differs from the pervious one in the thing that not all telophase processes are arrested in it, but only cytokinetic processes. At telophase I – interkinesis, the cytoskeleton preserves its early phragmoplast configuration, i.e. it is the central spindle with telophase chromosome groups (then with the interphase nucleus) on the poles. Mid-phragmoplast formation as a hollow cylinder, also barrel-shaped late phragmoplast with curved fibers expanding centrifugally, does not occur. Phragmoplast fibers do not amplify. The cell plate does not develop. Unlike the previous abnormal phenotype, nuclear envelope formation proceeds here around daughter nuclei, so they co-exist with the cytoskeleton which remains in the anaphase spindle configuration. As a result, a binucleate monade is forming. During interkinesis, daughter nuclei often migrated from telophase spindle poles and approach in the common cytoplasm. At the second meiotic division, in monads with approached daughter nuclei, a common division spindle is forming, and then – a dyad of unreduced microspores at tetrad stage. The fbnormality frequency is up to 40%. Arrest on the solid phragmoplast stage is described in postmeiotic mitosis in double kinesin mutant in Arabidopsis (Lee et al., 2007).

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Figure 7.2. Arrest of all cytokinetic processes at telophase I in PMCs of rice haploid. a) metaphase I; b) late anaphase I; c) monad with an anaphase spindle and interphase nuclei at interkinesis; d) common spindle at metaphase II.

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7.3. Arrest at Early Phragmoplast Stage with Element of Centrifugal Movement: Gamma-Phenotype In a number of phenotypes of WWG hybrids F1 (Triticum aestivum cv Novosibirskaya 67 x Agropyron glaucum) and WR F1 hybrids (Triticum aestivum cv Yubileinaya x Secale cereale v Hariuchiban), after chromosomes reach the poles, a weakly curved or a straight spindle curves more and more during telophase, so that the polar regions approach. Sometimes, the spindle curves so strongly that telophase chromosome groups interchange their positions, and the whole figure looks like the Greek letter gamma. That is why we called this abnormality gamma-phenotype. The cell plate is not forming; but in many PMCs, it is possible to observe the appearance of big vacuole-like structures (not shown) as we interpreted as plastosomes conglomerates. Phragmoplast fibers lengthening and curvature are realized, but they do not amplificate. Approached chromosome telophase groups then either are encircled by the common nuclear envelope and form the restitution nucleus, or they are encircled by the nuclear envelope individually. Mono- and binucleate monads enter their second meiotic division. The restitution nucleus or approached daughter nuclei form the common division spindle at prometaphase II (Shamina et al., 2009). In this case, the meiotic products are dyads with nonreduced members at the stage of tetrads. The number of cells with this abnormality in the anther may be high (up to 80%). Unlike the previous abnormality (p. 7.2), where not only mid-phragmoplast formation is arrested, but also further cytokinetic processes, in this phenotype, the early phragmoplast structure is affected by the processes of phragmoplast centrifugal movement (fibers curvature and elongation) at mid-late telophase. Normal mid-phragmoplast (hollow cylinder) fibers perform their centrifugal movement autonomously. Normally, the central point of each fiber (MTs (+)-ends overlap) moves centrifugally, indicating its own line along the radius from the center to the periphery. Movements of all fibers accompanied by plastosome transport to their central points lead to the centrifugal cell plate growth. Probably, in the gamma-phenotype, anaphase spindle central fibers do not lose their lateral connections, cannot autonomies, and the early phragmoplast behaves then like a single fiber. It is necessary to point out the principle distinction of curved spindles in the gammaphenotype and C-shaped spindles that appear because of aberrations in straightening of perinuclear ring MTs at early prometaphase (p. 3.2). The reasons for the formation of meiotic restitution nuclei and its mechanisms are completely different in these two cases.

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Figure 7.3. Progressive spindle curvature during telophase I in PMCs of WWG F1 hybrid a) anaphase I; b - d) telophase chromosome groups approacment caused during telophase I.

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7.4. Consolidation of Laggard Chromosomes into the Restitution Nucleus The following process of restitution nucleus formation was described in a number of contributions on cytological research of meiosis in allohaploids: univalents do not reach the poles at anaphase being scattered in the spindle body, and then consolidate into a restitution nucleus at telophase. As a rule, cytokinesis does not occur. This consolidation mechanism is unknown. A mononuclear monad, but not a monad with micronuclei, is developing; it would not occur without this consolidation. Restitution nuclei have a very original torus shape with a cytoplasm piece in the center (Chistyakova, 1974; Xu and Joppa, 2000, Fig. 1a). We believe that this variant of gamma-phenotype is the mechanism for restitution nucleus formation: an arrest at the stage of hollow cylinder formation and a progressive curvature of early phragmoplast on the background of univalents anaphase movement arrest. Chromosomes do not reach the poles, remain scattered in the spindle body and, as it curves, consolidate into a spiral or ring figure. The cell plate is absent, cytokinesis is completely arrested. At the end of the conventional telophase, chromosomes, which are located as a ring (or even spiral), are encircled by the nuclear envelope. A restitution nucleus is forming often ring-shaped: as a torus with a cytoplasmic ‗‘cork‘‘ in the center. Nevertheless, at metaphase II, in such cells, a common division spindle is forming, cytokinesis occurs normally, and a dyad of nonreduced microspores is the product of meiosis.

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Figure 7.4. Consolidation of laggard chromosomes scattering within the spindle body into the restitution nucleus at telophase I in PMCs WWG F1 hybrid by means of progressive curvature of telophase spindle; a) late anaphase I with chromosome laggards, b) spindle curvature with scattered chromosomes in it at mid-telophase I; c) ring spindle at late telophase I, d) spiral spindle at late T1.

7.5. Arrest of Phragmoplast Development at the Stage of Hollow Cylinder In this abnormal phenotype, the stage of transition from mid-phragmoplast (hollow cylinder) to configuration of late phragmoplast and centrifugal movement is arrested: no progressive fiber elongation, curvature and amplification. The cell plate is not forming. The cytoskeleton is at ‗‘hollow cylinder‘‘ stage till late telophase, when a nuclear envelope develops around telophase chromosome groups. Cytokinesis does not occur. Daughter nuclei approach each other at the equator in the common cytoplasm, often penetrating the cylinder. As a result, binucleate monads with closely approached daughter nuclei are developing. In the second meiotic division, a common spindle at metaphase II and a dyad with 2n cells at the stage of tetrads are forming. The abnormality frequency is up to 35%.

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Figure 7.5. Arrest of phragmoplast development at the stage of hollow cylinder in PMCs of WWG F1 hybrid № 30-546. a, b) phragmoplast conservation at hollow cylinder stage in interkinesis; daughter nuclei migrate inside the phragmoplast and approach each other c) binucleate monads in interkinesis; d) common division spindle at metaphase II.

7.6. Excessive Curvature of Phragmoplast Fibers during Centrifugal Movement

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One of the models of phragmoplast centrifugal movement at meiosis presupposes the thing that this process is a modification of B-anaphase (Shamina et al., 2007a). Phragmoplast fibers not only elongate, as in B-anaphase, but also curve; due to this their central points move centrifugally, and the poles remain fixed. Aberrations in phragmoplast fibers curvature contribute to the restitution process.

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Figure 7.6. Excessive curvature of phragmoplast fibers as a reason for telophase chromosome groups approachment and their congression into the restitution nucleus in PMCs of WWG F1 hybrid: a) midtelophase I; cell plate formation is aberrated; b, c) abnormally drastic phragmoplast fibers curvature at late telophase I; telophase chromosome groups approach the equator following fibers polar ends; d) telophase chromosome groups congression into a restitution nucleus.

In PMCs of WWG F1 hybrid № 328, at mid – late telophase I, the phragmoplast morphology considerably changes. Telophase chromosome groups move to the cell equator and closely approach each other. This shift takes place because of an excessive phragmoplast fibers bending whose polar ends abnormally approach each other on the equator. Because of the thing that the cell plate does not develop in 30% of PMCs, approached chromosome telophase groups congress forming a restitution nucleus. The mononuclear monad, in the second meiotic division, results in a dyad with non-reduced 2n members. The percentage of cells with abnormal phragmoplast fibers curvature is up to 50%.

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7.7. Aberration of Phragmoplast Centrifugal Movement

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In some phenotypes, aberration of cytokinesis occurs because of abnormalities in centrifugal phragmoplast movement and consequent secondary aberrations of cell plate formation. It is expressed in the thing that phragmoplast expansion decelerates, or stops halfway and does not reach the mother cell membrane. The cell plate, in such a phragmoplast, is always abnormal: it is not a monolayer of vesicles, but cloud-shaped or is strongly meandered. Sometimes, in such case, the phragmoplast/cell plate has assymetrical expansion, i.e. one edge irregurally moves and the other does not. As a result, the system reaches the mother cell membrane and contacts it with its only one part; the cell plate incompletely divides the cytoplasm, and daughter cell membranes are incision-shaped on the mother cell membrane or invaginations or caves inside the mother cell cytoplasm. If the phragmoplast/cell plate does not reach the mother cell membrane, then membrane vesicles do not fuse, and there is no daughter cell membranes formation. In such cases, the cell plate then disappears dispersing into its constitutive membrane vesicles, or plastosomes, and the daughter nuclei approach each other. In such phenotypes, at late telophase, the cytoplasm turns to be completely filled with plastosomes conglomerates whose formation also continues at interkinesis. This cytokinetic abnormality was observed by us in the phenotype of meiotic mutant ms43 (Shamina and Dorogova, 1995) in maize, also in WWG F1 hybrid № 13-2 and № 13-4 (T. aestivum ANK9 x A. glaucum 52-3). The abnormality frequency is from 15 to 60%. It is shown that failure of kinesins function leads to aberration of phragmoplast centrifugal movement, and cell plates formed by these phragmoplasts did not reach the mother cell membrane (Hiwatshi et al., 2008).

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Figure 7.7. Phenotypic manifestation of aberration in phragmoplast centrifugal movement of PMCs of WWG F1 hybrid; a - c) abnormal cell plate (marked in arrow) does not reach the mother cell membrane, its shape is drastically aberrated; d) daughter chromosome groups from previously approached nuclei encircled by the common perinuclear cytoskeleton ring at prophase-early prometaphase II; such common ring is a predecessor for common spindle in M II.

7.8. Arrest of Radial Cytoskeleton System Formation at TII (in „‟Parallel Spindles‟‟ Phenotype) in the Simultaineous Cytokinesis in the Dicot PMCs This mechanism of nuclear meiotic restitution is one of the most important in potato breeding (Alfano et al., 1999; Carputo et al., 2003). Simultaineous cytokinesis at telophase II of wild-type dicot male meiosis is realized due to the formation of six immobile

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phragmoplasts that conjunct four nuclei lying on the tetrahedron tops. Such location is achieved by means of the corresponding spindles orientation at metaphase II. In this case, the tetrad microspores also have their tetrahedral location. Four phragmoplasts form at metaphase II under parallel spindle co-orientation, and a coplanar microspores tetrad is the division product.

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Figure 7.8. Consequences of arrest of radial MT systems at TII in sugar beet (Beta vulgaris L.) PMCs. a) metaphase I; b) prophase II; c) metaphase II with parallel spindles; b) dyad with binucleate members at tetrad stage.

Binucleate dyads are the meiotic product at telophase II under parallel spindles location and simultaineous arrest of radial MTs (additional spindles, phragmoplsts) formation. It leads to the meiotic restitution, i.e. reunion of chromosomes of these nuclei in the first postmeiotic mitosis. This mechanism was described in the phenotype of potato mutant clone with parallel spindles and aberration of radial MT bundles (Alfano et al, 1997; Genualdo et al., 1998).

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7.9. Fused Spindles. Underformation of Cytoskeleton Interzonal System at Telophase I Approachment of daughter nuclei is observed at telophase I in sugar beet (Beta vulgaris L.) mutant lines В-24 and SOAN 5-10, which demonstrate high percentage of nonreduced gametes. Analysing cytoskeleton dynamics in the meiosis of this mutant line, we found that cytoskeleton aberrations at telophase I – interkinesis lead to the backward movement of daughter nuclei to the cell center and the restitution process. Normally, at telophase I, the system of central spindle fibers separating daughter chromosome groups and, later, interphase nuclei, symmetrically widen due to new polar MTs polymerization, and interzonal fibers amplificate because of this process. Thus, the so-called immobile phragmoplast develops between daughter nuclei. It disappears only at prophase II. Unlike this, the system of interzonal MTs is aberrated in mutant meiocytes at telophase I. Its fibers do not amplify, most part of MT bundles disappears and, as a result, the system looks abnormal: daughter nuclei not supported by MTs move to the center and abnormally approach each other. Approachment of daughter nuclei is preserved at interkinesis and prophase II, which has the major influence on the process of the second meiotic division. At prometaphase II and metaphase II, a common spindle is developing in such cells. At telophase II, on the common spindle equator, a phragmoplast/cell plate is forming, and cytokinesis takes place. Instead of a tetrad, a dyad forms; it initiates the formation of nonreduced gametes (Dorogova et al.,1999). The abnormality frequency is about 60%.

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Figure 7.9. Approachment of daughter nuclei at interkinesis at underformation of intezonal cytoskeleton system in PMCs of sugar beet (Beta vulgaris) line B-24: a) underformation of interzonal cytoskeleton system, b, c) approachment of nuclei at interkinesis; d) common spindle at MII.

Approachment of daughter nuclei, as a result of abnormal MT dynamics in the mutant, reveals the framework function of interzonal fibers at TI of dicots. This abnormality differs in its mechanism from that described in p. 3.2., which also causes fused spindles formation. In phenotype 3.2, approachment of daughter nuclei occurs later, at prophase II, because of an unknown mechanism on the background of normal MT cytoskeleton behavior. The interzonal system of polar MT bundles forming de novo can perform an unexpected function in the correction of daughter nuclear position. If a division spindle forms as a curvature (C-shaped) – it practically always happens under asynaptic meiosis, - telophase chromosome groups and daughter nuclei become abnormally approached. Approachment of daughter nuclei would inevitably lead to their restitution with the absence of cytokinesis after the first meiotic division in dicots. Such a mechanism of restitution nuclei formation was described by us in the meiosis of monocots: development of C-spindle at metaphase I and arrest of cytokinesis at telophase I lead to the approachment of daughter nuclei and their restitution (p. 3.2). However, approachment of nuclei on the poles of a curved spindle in the common cytoplasm in PMCs at telophase I has never led to restitution in any of the numerous cases of asynaptic meiosis with C-spindle of different dicot species we analysed. The thing is that the system of straight interzonal microtubules forming at telophase from the spindle poles inevitably separted abnormally closed telophase nuclei. As a result, at interkinesis, nuclei were always diametrically opposite to each other in the cortical cytoplasm region, just as in wild type meiosis, i.e. the position of nuclei was completely corrected. We observed this phenomenon under the spindle curvature in the asynaptic meiosis of potato clones (Solanum tuberosum L.) CE10, BE1050, ВЕ62, tomato mutant as6 (Lycopersicon esculentum L.), haploids of Brassica juncea Rajat.

7.10. Fused Spindles. Aberration of Fibers of Interzonal Cytoskeleton System Brassica juncea is a natural tetraploid. It is believed that it originated from the cross of diploid species Brassica rapa and Brassica nigra (Prakash, 1980). The tetraploid progeny from such a cross could appear only under the formation of nonreduced gametes by both parents. In this case, it is possible to expect this trait manifestation in the meiosis of haploids B. juncea. True, the haploids meiotic products at the stage of tetrads have an admixture of dyads up to 20%. Analysis of meiotic process in PMCs with cytoskeleton visualization revealed two mechanisms of meiotic restitution that lead to dyads formation.

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1st mechanism: aberration of interzonal cytoskeleton system formation at telophase Iinterkinesis. This abnormality represents a failure of polar MTs (+)-ends overlapping on the equator during the interzonal system formation. Under normal polymerization of polar MTs, but with aberration of their (+)-ends overlapping on the equator, the structure of interzonal cytoskeleton system becomes aberrated. Instead of an immobile phragmoplast, it is radial MT bundles diverging from daughter nuclei arise. Distal ends of these MTs criss-cross with each other in the cell equatorial region, and this is a good diagnostic trait of this abnormality. As a result, the interzonal MT system is not consolidated, and leads to a shift of daughter nuclei, their approachment and to the common spindle formation in the second meiotic division. 2nd mechanism – monopolar spindle formation at metaphase I in about 7% of cells. This abnormality was described above (p. 4.1). It leads to a complete arrest of chromosome segregation at anaphase I and the restitution nucleus formation at telophase I. At metaphase II, a common division spindle is forming and a dyad instead a tetrad at telophase II. Monopolar spindle formation was described by us also in the meiosis of monocot plant forms: WWG and WR F1 hybrids, where they are also the reason for meiotic restitution. The both mehanisms of nuclear restitution in the meiosis of Brassica juncea are based on one abnormality of cytoskeleton dynamics: aberration of connection of (+)-ends of antiparallel polar MTs that proceeds either at prometaphase I which, in its turn, leads to the monopolar spindle formation, or at telophase I, and it aberrates the structure of interzonal cytoskeleton system.

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Figure 7.10. Meiosis in PMCs of Brassica juncea haploid №6. Aberration of MTs (+)-ends overlapping of interzonal cytoskeleton system and the shift of daughter nuclei caused by it (disconnected fibers are indicated by arrows); a - c) disconnection of interzonal MT bundles at telophase I (arrows); c) displacement of daughter nuclei at interkinesis; d) dyad at tetrad stage.

8. CELL PLATE ABNORMALITIES LEADING TO MEIOTIC RESTITUTION The cytoskeleton play a key role in the cell plate formation: membrane vesicles (plastosomes) transport occurs along phragmoplast fibers from Golgi apparatus into the cell equatorial zone (Lee et al., 2001). Nevertheless, many aspects of cell plate and daughter cell membranes formation are realized independently from the cytoskeleton function (Otegui and Staehelin, 2000). Aberration of these processes leads to that in daughter genomes autonomisation and, as a consequence, it can be the reason for meiotic restitution. Formation of daughter cell membranes is a very important terminal cytokinetic stage. It occurs when the cell plate reaches the mother cell membrane and contacts it (Ehrhardt and Cutler, 2002). After this, plastosomes fuse and daughter cell membranes develop (Esseling-Ozdoba et al., 2008).

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8.1. Absence of Cell Plate This aberration is quite widely spread in abnormal cereal meiosis. We observed it at the frequency from 3 to 30% in several dozens of haploid and allohaploid genotypes of maize, rice, wheat, rye, wheat-grass and their wide hybrids. Under the presence of a developed phragmoplast, no membrane vesicles observed, - either as a cell plate (monolayer) or other associations. The phragmoplast fibers move centrifugally and can reach the mother cell membrane, but the formation of daughter cell membranes is impossible. Daughter nuclei, in rather a high cell percentage, approach in the common cytoplasm, and their chromosomes can congress into the common division spindle at metaphase II (Shamina et al., 1999). A dyad of unreduced microspores is the meiotic product in PMCs with such a phenotype. Investigations carried out at the light level, cannot reveal the thing if the cell plate absence is the consequence of arrest in the synthesis of membrane vesicules by Golgi apparatus or an aberration of phragmoplast transport. Indirect data (absence of plastosome conglomerates in the cytoplasm) are in favour of the first supposition. Abnormal cytokinesis without cell plate in mobile phragmoplast was also described in meiosis of Magnolia (Brown and Lemmon, 1992; Dinis and Mesquita, 1993) and Conocephalum (Bryophyta) (Brown and Lemmon, 1998).

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Figure 8.1. Approachment of telophase chromosome groups under the absence of cell plate on the background of the realization of phragmoplast (marked in arrows) centrifugal movement in PMCs of WWG F1 hybrids: a, b) telophase process with the absence of cell plate, c) binucleate monade at prophase II, nucleiare encircled by the common perinuclear cytoskeleton ring (arrows), d) common spindle at metaphase II.

8.2. Rotation of Phragmoplast on the Background of Cell Plate Absence A variant of the previous phenotype is continuation of ‗‘empty‘‘ phragmoplast centrifugal movement also after it reaches the mother cell membrane. At early - mid telophase, the cell plate is absent, and then an ‗‘empty‘‘ phragmoplast – as a hollow cylinder, and, later, as the hollow barrel (as its fibers become curved), begins the centrifugal movement to the mother cell membrane. Having reached it, the phragmoplast does not stop moving and continues to expand. Daughter nuclei are formed in interphase, but centrifugal movement of the phragmoplast still continues. As a result, it turns within the PMC - which is tablet-shaped in cereals – in complex with telophase chromosome groups or daughter nuclei. The telophase spindle axis becomes to be located along the short axis of a tablet-shaped PMC as a consequence of this turn. Daughter nuclei or chromosome groups approach each other in the common cytoplasm, which is the reason for the restitution process. The percentage of cells

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with a common spindle is much higher at the phragmoplast turn than without it at metaphase II. Such excessive phragmoplast movement without a cell plate is observed in most of WWG F1 and WR F1 hybrids with the ―cell plate absence‘‘ phenotype. In the WWG hybrid F1 № 199 (Е. еlongatum x T. aestivum cv. Lutescence 132), this abnormality was mass (in 30% PMCs).

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Figure 8.2. Approachment and abnormal co-orientation of daughter nuclei within the cell under the cell plate absence in PMCs of WWG F1 hybrid: a) late telophase without cell plate; b, c) two optical sections of the same PMC with displaced daughter nuclei; c) abnormal position of nuclei at telophaseinterkinesis.

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8.3. Aberration of Cell Plate Shape and Structure In male meiosis in several wide cereal hybrids F1, the cell plate abnormalities are observed, whereas the phragmoplast centrifugal movement proceeds regularly. The abnormal cell plate is either fragmental or it looks like an amorphous conglomerate of vesicles or cisternaea on the cell equator, or it has an abnormal wide diffusional shape. Concomitantly, the daughter cell membranes do not form or form abnormal. In such cases, the daughter cell membranes do not divide the cytoplasm completely and are an incision or a cave on the mother cell membrane. As a result of cell division with such a phenotype, binucleate monads are developing. In the second meiotic division, chromosomes of both nuclei sometimes congress in the common spindle, and a dyad at tetrad stage is forming. The percentage of such abnormalities is, as a rule, not so high. Aberrant cell plate formation is described in the phenotype of Arabidopsis mutant mor1 (Eleftheriou et al., 2005).

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Figure 8.3. Abnormalities of the cell plate formation during the first meiotic division in cereal PMCs: a) phragmental cell plate as a chain of lacoons; b) amorphous congression of vesicles on the cell equator; c) wide diffuse cell plate not forming daughter cell membranes; d) binucleate monad.

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8.4. Arrest of Plastosomes (Cell Plate Membrane Vesicles) Fusion In the phenotype of meiotic mutant pam1 (Zea mays), at the stage of dyads (interkinesis), the appearance of some amount – about 10% - of binucleate monads with drastically changed morphology is observed. Daughter nuclei are located one above the other in them. Nevertheless, all telophase PMCs of the mutant have no deviations from the norm; in particular, the phragmoplast and cell plate are normally developed. Telophase chromosome groups in such PMCs are of normal position, i.e. they are always in the plane parallel to the slide. It is necessary to note that in normal maize dyads and tetrads the nuclei are also always in one plane parallel to the slide. Normally, in cereals, the division axis of PMCs on squashed preparations is also oriented this way, as cells have ratios 3:3:1 counting from the division axis. Thus, in the mutant, after telophase I, during interkinesis, the cells – precursors of deformed monads – considerable change their shape, turn flattened from the poles and, as a consequence of this, they have an abnomal position on the preparation. Such cells ratios become 1:3:3: counting from the division axis. The analysis of cytokinesis in them showed that it proceeds normally, but the final stage – fusion of membrane vesicles and daughter cell membranes formation - do not occur in such cells. Having reached the mother cell membrane, the phragmoplast/cell plate does not stop and continue its centrifugal movement. Herein, the cells expand laterally and, hence, they flatten from the poles. As a result of the phragmoplast/cell plate excessive expansion, the cell deforms and changes its positon on the squashed preparation: division axis is perpendicular to the slide. Telophase chromosome groups turn into interphase daughter nuclei, so that cytokinesis continues in the conventional interkinesis. Simultaineous presence of formed interphase nuclei and cell plate (but not daughter cell membranes) in cereal PMC is not encountered in wild type meiosis and is a typical phenotypic trait of mutant pam1. During ‗‘excessive‘‘ cytokinetic process, the formed cell plate gradually disperses. Plastosome dispersion begins from the center: edges of semidispersed cell plate sometimes turn into incomplete daughter cell membranes as ring incisions around the mother cell membrane. At metaphase II, in common cytoplasm, a common division spindle often developes, as daughter nuclei close approach each other. Dyads with 2n members at the stage of tetrads are the meiotic product (Dorogova and Shamina, 2001). Such a phenotype is also observed in number of maize haploids at a high PMC percentage (25%). In those cases the phragmoplast turn during excessive centrifugal movement is also observed (see also p.8.2).

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Figure 8.4. Aberration of daughter cel membranes formation in PMCs of meiotic mutant pam1 (Zea mays L.). a) interphase nuclei and cell plate at early interkinesis; daughter cell membranes do not form, b) turn of telophase figure inside PMCs, as a result of continuation of phragmoplast/cell plate centrifugal movement after they reach the mother cell membrane; c) approachment of daughter nuclei in the common cytoplasm; the cell plate is dispersed; d) common division spindle at MII.

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Gu and Verma (1997) showed that in BY-2 cells, under arrest of membrane vesicles fusion cased by the phragmoplastin overexpression, the cell plate gradually becomes abnormally S-shaped and even shifts into the diagonal or longtitudinal (along the long cell axis) position. We believe that it is consequence of the cell plate excessive growth under nonstop cytokinesis. As BY-2 cells are enveloped by a rigid cell wall, the cell plate excessive growth leads not to the cell expansion in the equatorial region, but to the deformation and shift of the cell plate per se. This shift is quite possible: rotation of the phragmoplast/cell plate was described in some plant cell types under their differentiation (Palevitz, Hepler, 1974). Peculiarities of knolle mutant phenotype with failure of plastosomes fusion (Lukowitz, Mayer, 1996; Lauber et al., 1997; Batoko and Moore, 2001) also indicate the processes of ―non-stop cytokinesis‖. First, it is the disorientation of daughter cell wall stubs that indicates their shift mentioned by the authors. Second, it is excessive thickness of these stubs which is indicative of excessive amount of membrane vesicles that moved into this region. Phenotypes of mutants tangled and discordia characterized with division plane disorientation (Walker et al., 2007; Wright et al., 2009) could also be interpreted as examples of ―non-stop‖ cytokinesis. Vivid traits of excessive cytokinesis are observed in dividing plant cells under caffeine action. This agent specificaly inhibits the fusion of plastosomes and daughter cell membranes formation. In studying the ultrastructure of dividing cell of stamen hairs in spiderwort under the action of caffeine, Helper and Bonsignore (1990) pointed out considerable plastosome conglomerates on the cell plate edges adjoining the mother cell membrane. In our opinion, this is the result of excessive, non-stop cytokinesis when the phragmoplast/cell plate is deprived of its ability to continue centrifugal movement within the walled cell. In the present review, the pragmoplast non-stop movement is described in p. 8.2. for the phenotype with cell plate absence. In this case – because of the impossibility of daughter cell membranes formation – the ‗‘empty‘‘ phragmoplast continues its centrifugal movement after it has reached the mother cell membrane and, as a result, it turns within it.

8.5. Longtitudinal Orientation of Cell Plate and Daughter Cell Membranes According to Division Axis In PMCs of WWG F1 hybrid № 27 and № 734, at frequency 7-10%, a considerable aberration in cell plate formation, which proceeds because of aberrations in cell plate membrane vesicles (plastosomes) transport to the equator and, to be more precise, their distribution over the phragmoplast. Membrane vesicles are not transported along all the phragmoplast fibers towards the cell equator, but are distributed along some closely located fibers and fill in the space among them – from pole to pole as a stretched conglomerate. If this plastosome congression adjoins the mother cell membrane, daughter cell membranes develop as a narrow cistern, the incision. Abnormal cytokinesis, thus, occurs not perpendicularly to the division spindle axis, but along to it. An unnuclear cytoplast separates from the cell, or an incision is developing on the mother cell membrane, and daughter nuclei turn to be in the common cytoplasm. Their chromosomes can congress in the common spindle in the following division which is, as a rule, of normal procedure.

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Figure 8.5. Abnormal cell plate formation under aberration of phragmoplast fibers transport function in PMCs of WWG F1 hybrid: a - c) longtitudinal location of a mass of cell plate membrane vesicles (arrows) among the phragmoplast fibers in local part of the phragmoplast, d) abnormal daughter cell membranes located along the cell division axis.

This phenotype adds to the group of plant cell division abnormalities which are characterized by an incomplete arrest of some process that refers to the whole multitude of cytoskeleton elements (fibers, MT bundles) in a cell. In these abnormalities, part of fibers population continues to follow the cytoskeleton cycle, and part of it stops at the previous stage. Such are i) the ―combined spindle‖ phenotypes that consist of a mixture of straight and curved fibers (Shamina, 2005 b), ii) part of centrifugally moving phragmoplast fibers (p. 9.5), iii) triple phragmoplast in a cell with tripolar spindle in which, this or that cytokinetic process sometimes ‗‘switches off‘‘ in one or two of its three constituents. Thus, the cell plate formation or phragmoplast expansion may occur in only one or two of the three phragmoplasts (Shamina, paper in preparation).

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9. INCOMPLETE CYTOKINESIS AS INCISIONS ON THE MOTHER CELL MEMBRANE AND ITS CONSEQUENCES In all cases, when the phragmoplast/cell plate contact the mother cell membrane – due to this or that reason – not simultaneously over its whole circumpherence, daughter cell membranes separate the cytoplasm incompletely, and look like more or less deep incisions on the mother cell membrane. It happens as a consequence of the thing that an assymetrically located cell plate, at the moment of contacting the mother cell membrane, do not completely cross the cytoplasm. A binucleate incised monad formed as a result of such a division is often the reason for 2n cells formation in the following division. Approachment of daughter nuclei in the common cytoplasm can often be the reason for the development of 2n gametes out of incised binucleate monads. In many cases, two division spindles form in the common cytoplasm at metaphase II of such a monad; the meiotic product is a triad with two haploid or aneuploid members and one binucleate cell. This last one may congress chromosomes of the two nuclei in the common spindle at postmeiotic mitosis and results in a cell – the precursory of a 2n gamete. Sometimes, at metaphase II, spindles are not in parallel, but located at an angle to each other. As a result, the polar regions of two spindles approach, often in one point, and a restitution nucleus forms here.

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Figure 9. Restituion process in binucleate incised monads formed as a result of incomplete cytokinesis in cereal PMCs: a) binucleate monad with abnormal daughter cell membranes as an incision on the mother cell membrane, b) parallel spindles at metaphase II, c) displaced spindles at metaphase II with a converged pair of poles (arrow), d) triad with a 2n member.

According to our observations of simultaneous cytokinesis in PMCs of monocots, the reasons for asymmetrical cell plate position and incisions development can be the following:

9.1. Assymmetrical Position of the Division Spindle and Phragmoplast

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For regular cytokinesis, a spindle should be positioned right in the cell center. If it becomes approached to some mother cell membrane area, the first cell plate contact with mother cell membrane leads to fusion of plastosomes and the stop of cytokinesis. We observed this phenomenon in the meiosis of WWG hybrids F1 № 86-2 (T. aestivum ANK26А х A. glaucum), № 97-13 (T. aestivum AHK9 x A. glaucum) in multinucleate meiocyte division, where some spindles were located on the cytoplasm periphery (also the second meiotic division in PMCs on mutant ms43 in maize), in the abnormal phenotype of a multiple spindle in WR hybrids F1.

9.2. Spindle Curvature When the spindle is curved, its equatorial region, as a consequence, is displaced to the mother cell membrane. Incomplete cytokinesis is observed in the phenotype of maize meiotic mutant ms28, also in curved spindles under asynaptic meiosis (wide cereal hybrids F1, haploids, synaptic mutants) (see p. 3.2).

9.3. Cytoskeleton Disorganization Under spindle disorganization, when it represents a chaotic network of fibers, multiple cell plates may develop on random MT bundles. Only those cell plates that met the mother cell membrane turn into abnormal daughter cell membranes in a form of short incisions. Cell plates, located deep in the cytoplasm, are unable to reach the mother cell membrane, disperse, because the fusion of vesicles and formation of daughter cell membranes do not proceed (p. 5.1).

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9.4. Abnormal Phragmoplast/Cell Plate Expansion Under the pragmoplast aberrated centrifugal movement, the cell plate expands asymmetrically and often reaches the mother cell membrane not simultaneously over the whole circumpherence. After the contact, there is a fusion of plastosomes, and daughter cell membranes look like incisions. This abnormality is typical of the phenotype of maize meiotic mutant ms43 (p. 7.7).

9.5. Under Asymmetrical Phragmoplast Movement in the Lateral Direction In PMCs of WWG hybrid F1 no. 625 (T. aestivum cv. Novosibirskaya 67 × A. glaucum) and wheat–rye hybrid F1 no. 8245 (T. aestivum cv. Lutescens x Secale cereale cv. Onokhoiskaya), a curious phenomenon is observed at TI with 15% frequency.

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Figure 9.5. Fragmentary phragmoplast function and incomplete asymmetrical cytokinesis in WR F1: a, b) centrifugal movement of lateral phragmoplast part (arrow) and arrest of its rest parts movement at telophase I, c) result of cell division with this phenotype: binucleate incised monad, d) approachment of spindle poles at metaphase II

The phragmoplast, normal and completely developed out of the straight spindle, moves centrifugally and forms the cell plate only with its some part, and the rest parts remain immobile. As a result, the cell plate incompletely crosses the cytoplasm and, having contacted the local part of mother cell membrane, performs into abnormal daughter cell membranes in a form of incision.

9.6. Cell Plate Dispersion Incomplete daughter cell membranes in a shape of an incision arises under the break of a normal cell plate as a consequence of plastosomes dispersion with the aberration of daughter cell membranes formation (maize mutant pam1 phenotype). Here, in part of cells, a full ring incision that encircles the cell equatorial area arise (p. 8.4).

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9.7. In Phenotypes with Cell Plate Structure Aberrations (P. 8.3), Incomplete Cytokinesis as Incisions is a Common Phenomenon Such a mechanism of restitution nuclei formation was described in microsporogenesis in Brachiaria (Gallo et al., 2007).

9.8. Cytokinesis Correction in the Gamma-Phenotype In many of WWG F1 and WR F1 hybrids with an arrest of middle phragmoplast (hollow cylinder) formation and its progressive curvature during telophase (p. 7.3), an addditonal phragmoplast that consists of polar MT bundles, overlapping on the equator, forms berween approached telophase groups (Shamina et al., 2009). This phragmoplast forms a cell plate and carries out cytokinesis, which is successful in many cases, but it is incomplete in some PMCs percentage with daughter cell membranes as an incision.

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Figure 9.8. Correction of cytokinesis in gamma-phenotype WR hybrid F1 with the formation of binucleate incised monads or dyads: a) approachment of telophase chromosome groups at late telophase I as a result of an early phragmoplast progressive curvature, b, c) additional phragmoplast (arrow) formation between approached telophase chromosome groups, d) binucleate incised monad at prophase II – the result of incomplete cytokinetic correction.

10. GENERAL MEIOTIC ABNORMALITIES LEADING TO RESTITUTION Most of meiotic restitution mechansms consists in the aberration of chromosome spatial separation during karyokinesis or/and autonomisation of diverged chromosome groups (or daughter nuclei) during cytokinesis. Intracellular distant transport, including chromosome segregation and cell plate formation, is realized by the cytoskeleton. However, in this part of the catalogue, meiotic abnormalities are considered, that are not related to cytoskeleton cycle abnormalities, but conditioned by the aberration of the elements of the whole set of meiotic division processes.

10.1. Omission of one or Both of Meiotic Divisions Haploid microspores are the result of two successive meiotic divisions. If meiosis is omitted (apomeiosis), 2n gametes genetically identical to parental genotype are formed

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(Tavoletti, 1994). If only one of the meiotic divisions proceeds, then microspores are also diploid (Carputo et al., 2003; Hayashi et al., 2009). It was reported on the equational division of univalents (into chromatids) at anaphase I in the asynaptic meiosis and the absence of the second meiotic division (Gustafsson, 1935; Ramanna, 1983; Jongedijk et al., 1991; Vorsa, Ortiz, 1992; Ramanna et al., 2003; Barrel and Grossniklaus, 2005), Our observations of male gametogenesis in the natural apomict Arabis holboelli and maize haploid №4607 showed that, under complete asynapsis, univalents equational orientation, normal chromatids segregation and cytokinesis, pollen grains develop out of the only dyad members formed as a result of this process. The cytoskeleton rearrangements were not aberrated. We believe that this phenotype, just as those described in the above cited contributions, is an ‗‘omission‘‘ of not the second one, but the first meiotic division, as the processes are exclusively typical of the second meiotic division. Such phenotype represents first division restitution mechanism. In literary sources, it was also reported on the absence of the second meiotic division in the synaptic forms (Conicella et al., 1991; Werner, Peloquin, 1991; Park et al., 2002). Omission of the second meiotic division in forms with normal chromosome synapsis and bivalent formation is genetically equivalent to the second division restitution mechanism.

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Figure 10.1. First meiotic division in maize haploid with univalents equational division and the absence of the second meiotic division: a) PMC at diakinesis, b) MI with the metaphase plate organized by equationally oriented univalents, c) anaphase I, d) dyad of haploid microspores.

10.2. Equational Division of Univalents in the First Meiotic Division and Failure of Chromosomes Segregation at the Second Meiosis This abnormality leads to the restitution nucleus formation at the first meiotic division. In most cases, further random segregation of chromatides at the second meiosis leads to the formation of nonviable aneuploid gametes. However, in a number of wide F1 cereal hybrid genotypes, undetected aberrations of division spindle formation at prometaphase II lead to chromosome nondivergence, formation of restitution nuclei and potentially viable 2n gametes. Such phenotypes were described in literature (Маап, Sasakuma, 1977; Sasakuma, Kihara, 1981).

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Figure 10.2. Equational division of univalents at MI with a furthet arrest of division spindle formation at MII in PMCs of WWG hybrid F1: a) metaphase plate in the division spindle at MI consisting of equationally oriented univalents, b) dyad with nonreduced members, c) dyad at MII without traits of division spindle formation: cytoskeleton fibers are not revealed, d) dyad at the stage of tetrads: nuclear envelope encircled the nondiverged chromosomes.

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10.3. Cytomixis The known phenomenon of migration of nuclei from one PMC into the other through cytomictic channels (Caetano-Pereira and Pagliarini, 1977; DeSousa and Pagliarini, 1977; Utsunomiya et al., 2004) can be the reason for 2n gametes formation. This process was described as mass in the wild type meiosis of Dactylis glomerata, where it causes gametes formation with a doubled chromosome number out of recipient cells (Falistocco et al., 1995). In a number of forms with abnormal meiosis we analysed, cytomixis was observed. We found out that the transfer of nuclear material is realized together with the nuclear envelope (Shamina et al., 2000b), and the common perinuclear cytoskeleton ring develops at late prophase around the basic and additional nuclei. This ring functions then as a base for common division spindle formation and congression of chromosomes of both nuclei. In some transgenic tobacco lines, 10% of nucleus cells transferred completely to the neighbouring meiocyte at early prophase I (Shamina et al., 2000b), and that was the base for meiotic restitution. It is necessary to note viable 2n gametes, by means of cytomixis can develop only in diploids with a normal homological chromosome synapsis. Formation of 2n gametes as a result of meiocytes fusion was described in cereal meiosis (Risso-Pascotto et al., 2006).

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Figure 10.3. Congression of neighbouring PMCs chromosomes as a result of cytomixis in WWG F1 hybrid: a) donor cell at initial stages of cytomixis: the nucleus stretches towards cytomictic channels (contact point of two PMCs is marked with arrow), b) recipient cell with an additional nucleus that migrated into it as a result of cytomixis, c) double division spindle in the recipient cell at MI, d) common cytoskeleton ring in the binucleate recipient cell at prophase I, the condition for further spindle formation with a doubled chromosome number.

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10.4. Cytokinesis Aberrations in the Last Premeiotic Mitosis The phenotype of meiotic mutant pam1 and haploid № 2905 in maize is characterized by cytokinetic arrest in the meiosis and part of cells of the last premeiotic mitosis. It leads to the thing that some cells turn binucleate at prophase I.

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Figure 10.4. Meiotic restitution as a result of the absence of cytokinesis in the last premeiotic mitosis. Binucleate PMCs at prophase I, common cytoskeleton perinuclear ring (marked with arrows) in binucleate PMCs at prophase I of meiotic mutant pam1, (Zea mays) and WWG F1 hybrid: a) binucleate PMC at prophase I in the maize haploid, b) common perinuclear ring around two nuclei at prophase I in PMCs of mutant pam1 c) common perinuclear ring around two nuclei at prophase I in PMCs of WWG F1 hybrid, d) common spindle with a double chromosome set at metaphase I.

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At late prophase, a common perinuclear ring initiating the common diision spindle at MI is forming round these nuclei. As this spindle includes a double chromosome number, diploid microspores will be the product of such cell (Dorogova and Shamina, 2001). Such phenotype is also encountered in WWG F1hybrids with frequency of 10-15%. 2n gametes formation as a result of failure of the last premeiotic cytokinesis is reported for some forms of oat Avena vaviloviana (Katsiotis and Forsberg, 1995).

10.5. Backward Movement of Daughter Chromosome Groups from the Spindle Poles into the Cell Center This phenomenon was earlier described under the formation of restitution nuclei (Sears, 1953; Avers, 1954; Stefani, 1986, Jauhar, 2003). However, as there was no cytoskeleton visualization made in these contributions, its mechanisms remained unknown. Abovedescribed are some cytoskeleton abnormalities that lead to the congression of chromosome groups diverged to the poles: anaphase spindle shortening (p. 6.2), early phragmoplast curvature or gamma-phenotype (p. 7.3), excessive fibers curvature of late phragmoplast (p. 6.6), absence of cell plate (pp. 8.1, 8.2). An original process of telophase chromosome groups congression in the cell center is observed in WWG F1 hybrid № 27-645 (T.aestivum c Алтайская Нива x E. elongatum), Univalents that constitute aneuploid daughter chromosome groups move at telophase I from poles to the equator sliding with their arms along the telophase spindle surface. They can move both as a group, or individually. Congressed on the equator, chromosomes form a unified group which is surrounded by the nuclear envelope and forms a restitution nucleus. Cytokinesis is arrested here: there is no phragmoplast centrifugal movement and cell plate

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formation. In the second meiotic division, a common division spindle and a dyad with nonreduced members are developing.

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Figure 10.5. Backward movement of telophase chromosomes and telophase chromosome groups to the spindle equator in PMCs of WWG F1 hybrid № 27-645: a) backward movement of telophase chromosome groups to the cell center, b) formation of restitution nucleus out of chromosomes approached on the equator, c, d) returning of telophase chromosomes to the equator from the poles.

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One can hypothesise that this abnormality is a result of untimely activity of chromokinesines. These transport proteins are localized on chromosome arms and transport them to MT (+)-ends in the process of metaphase plate formation during late prometaphase (Mazumdar and Misteli, 2005). It is possible, that this process additionally acts at telophase I in WWG hybrid F1 №.27-645. Such a phenomenon of untimely onset of intracell process is known in cytokinesis in Schizosaccharomyces pombe, when the cell forms several septs one by one (Fankhausher and Simanis, 1994; Song et al., 1996). The return of chromosomes completely diverged to the poles back to the cell equator was reported on in animal cells under division with the expression of nondestructible form of cyclin B (Wheatley et al., 1997).

DISCUSSION Cell meiotic division is realized by a set of many complicated multistage processes aimed at one result: obtaining haploid spores. Thus, it is not surprising that, in detailed research of abnormal meiosis, there are so many different ways leading to meiotic restitution and 2n gametes formation. Besides this, abnormal processes of pre- and postmeiotic divisions can also affect the ploidy of gametes.

Meiotic Restituion Mechanisms Typical of only Species with Successive Cytokinesis Formation of restitution nuclei in meiosis with successive cytokinesis has its peculiarities. They consist both in the fact of successive autonomisation of two meiotic division products and in the aberrations of specific mechanisms of mobile phragmoplast formation and operation. Thus, the central position of the division spindle is very important in the cell. If it is abnormal, the cytokinetic abnormalities lead to the formation of incomplete cell membranes as incisions which, in its turn, often results in meiotic restitution. On the contrary,

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in PMCs of wild type dicots with simultaneous cytokinesis, the division spindle is very often peripheric or excentric in the first meiosis; it does not cause any abnormalities, as cytokinesis is not realized at telophase I. Incomplete cytokinesis after the first meiotic division is one of the restitution mechanisms typical of only monocots with simultaneous cytokinesis. Out of p. 9 it is seen how various the reasons for incomplete cytokinesis are in male meiosis in monocots. Incomplete daughter cell membranes can be a consequence of cytoskeleton and other abnormalities during a long period from prometaphase to telophase completion. It is obvious that abnormal daughter cell membranes as an incision on the mother cell membrane in walless cell division are stubs analogs in the mitosis of walled cells (Nacry et al., 2000; Assaad et al., 2001; Muller et al., 2002). Thus, when analyzing abnormal mitosis, it is also necessary to keep in mind that the appearance of stubs proper in meiotic division products of walled cells does not say anything about the reasons of their development and requires a detailed analysis of the whole mitotic process. Approachment of daughter nuclei on the poles of a curved spindle occurring with parallel cytokinetic aberrations (Shamina et al., 1999) refers to monocots meiotic restitution mechanisms. Restitution process can occur under cytokinesis which, here, looks like a not deep incision on the mother cell membrane. An important feature of this restitution mechanism in monocots is the absence of the process that corrects abnormal approachment of daughter nuclei or chromosome groups. On the contrary, in the male meiosis with simultaneous cytokinesis of dicots, such a mechanism functions, and it is based on the thing that, unlike monocots, formation of daughter nuclei proceeds in them also at early telophase. Further on, from daughter nuclear envelopes to the equator, polar MTs polymerise, and they form a developed system of interzonal fibers (immobile phragmoplast). These fibers separate daughter nuclei if they are abnormally approached due to some reason. This correction mechanism is absent in PMCs of monocots with simultaneous cytokinesis, as their phragmoplast is a derivative of the central spindle, and the role of polar MTs is secondary. It is obvious that the described correction mechanism will function in dicots under daughter chromosome pretelophase approachment caused not only by the spindle curvature, but other reasons. Thus, the approachment of chromosome groups on a curved spindle poles (p. 3.2), as a result of spindle shortening at anaphase (p. 6.2), backward movement of telophase groups to the equator (p. 10.5), are the restitution mechanisms typical of meiosis with successive cytokinesis. They correct in dicots meiosis with simultaneous cytokinesis. Cytokinesis abnormalities in the first meiosis are a typical mechanism of meiotic restitution in monocot plant species. In this case, daughter nuclei easily approach in the cytoplasm, and it leads to the congression of their chromosomes in the common spindle of the following cell division. The original aberrations of successive cytokinesis that are a reason for meiotic restitution, are numerous and diverse (points 7-1 – 7.7). They are a valuable source of information to study temporal and spatial regulation of plant cytokinesis, also the mechanisms of centrifugal movement of phragmoplast/cell plate at normal meiosis (Shamina et al., 2007a).

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Meiotic Restitution Mechanisms Typical of only Species with Simultaneous Cytokinesis As in male meiosis of the majority of dicots, cytokinesis is absent after the first meiotic division, they often have occasions in genetic material congression in the common cytoplasm before and during the second meiotic division and common division spindle formation at metaphase II. This abnormality is called ―fused spindles‖ and it is the basic source of nonreduced gametes formation in dicots (see review Ramanna, 1979). In the present review, there are 5 mechanisms that lead to fused spindles and they are described in more or less detail. Two of these mechanisms are abnormalities of interkinetic interzonal cytoskeleton system that corrects the position of daughter nuclei in the common cytoplasm, the gist of rest 3 is so far unclear (Dorogova et al., 1999; Shamina et al., 2001).

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1st mechanism fs: aberration of polar MT polymerization from the polar regions of telophase spindles and from the envelope of daughter nuclei in sugar beet line phenotype В-24 (p .7.9), 2nd mechanism fs: aberration of opposite polar MT (+)- ends attachment under the formation of interzonal cytoskeleton system, as in haploid Brassica juncea (p. 7-10), 3rd mechanism fs: approachment of daughter nuclei at prophase II unaccompanied by visible abnormalities of cytoskeleton structural behaviour (p. 2.2), 4th mechanism fs: fusion of perinuclear cytoskeleton rings of approached prophase nuclei and the common perinuclear ring formation, out of which a common fused spindle forms (p. 2.3). Possibly, it is a necessary condition for a fused spindle formation at the approachment of daughter nuclei. 5th mechanism fs: approachment, due to an unknown reason, and fusion of chaotic prometaphase figures (mid-prometaphase) in the second meiotic division (p. 4.4). Partial or complete arrest of polar MTs polymerization at telophase I that leads to fused spindles, is interesting to be compared to the similar cytoskeleton abnormality that proceeds in the second meiotic division on the background of parallel spindles (ps) (p. 7.8). This abnormality also leads to the formation of unreduced 2n gametes and consists in polar MTs polymerization arrest and, thus, the impossibility of phragmoplasts formation between nonsister daughter nuclei. Despite the identity of aberrations proper, the restitution mechanisms turn different in these two cases (fused spindles and parallel spindles). Approachment of daughter nuclei at prophase II is the reason for fused spindles phenotype, typical of many different dicot forms producing 2n gametes. Normally, at this stage, the interzonal cytoskeleton depolymerises, nuclei become deprived of its support, but do not approach each other in wild type meiosis. According to our data obtained both with the classical method and immunostaining, approachment of prophase nuclei is not accompanied by any aberrations in the microtubule skeleton behaviour (Conicella et al.,2003). Thus, the reason for the approachment of nuclei at prophase II remains unknown in these phenotypes. An interesting phenomenon is the fusuion of perinuclear cytoskeleton rings in approached prophase nuclei. It is a necessary stage of daughter cell genomes congression at prometaphase II and the development of common division spindle. It is unknown whether this phenomenon is cytoskeleton behavioural abnormalities or it is part of a special mechanism that provides genome consolidation in the division and ‘‘switches on‘‘ untimely. The

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importance of common cortical cytoskeleton ring for genome consolidation by means of a common spindle formation also demonstrates the restitution mechanism described in p. 2.4 of maize haploids. In this case, many micronuclei in a monad – the first meiotic abnormality product – are encircled by a cortical cytoskeleton ring at prophase II. As a result, a common division spindle at metaphase II and a dyad of normal microspores at the stae of tetrads are forming. Approachment and fusion of mid-prometaphase figures are not accompanied by any visible aberrations in MT cytoskeleton structures, and the reason for this phenomenon is not yet known. Processes that occur in the second meiotic division in PMCs with ‗‘fused spindles‘‘ phenotype can be compared to the process of genetic material congression at prophaseprometaphase of the first meiotic division in multinucleate PMCs of both mono - and dicot plant species. PMCs can be multinucleate when entering meiosis as a result of cytoskeleton abnormalities of the last premeiotic mitosis, also as a result of cytomixis. We observed the first meiotic division in binucleate PMCs of maize meiotic mutant pam1 (Dorogova, Shamina, 2001), and also phenotypes with cytomixis in different mono- and dicot species (Shamina et al., 2000). In all these numerous cases, the first meiotic division had the same procedure in multinucleate cells: all nuclei approached in the center, were encircled by the common perinuclear MT ring and formed their unified division spindle at metaphase I. If multinucleate cells formed as a result of chromosome segregation abnormalities and/or cytokinesis in the first meiotic division, in the second one several division spindles developed out of multiple nuclei (Shamina et al., 1981). In other words, in the second meiotic division, the process of genetic material consolidation is not observed, which is obligatory in the first meiotic division. Obviously, nuclear - cytoplasmic interactions proceed in a different way in dicots: active genetic material consolidation is realized at the first meiosis, its autonomisation – at meiosis II. One can hypothesise that consolidation of genetic material of daughter nuclei in the second meiotic division of dicots with ‗‘fused spindles‘‘ phenotype (points 2.2. 2.3. 4.4), and ‗‘common cortical ring‘‘ (p. 2.4), there is some aberration of signal mechanism that makes the cell divide on the ‗‘scenario‘‘ of the first meiotic division. Spindle disorientation at metaphase II in the common cytoplasm refers to specific restitution mechanisms of dicot plant species: parallel (p. 7.8) and the so-called ‗‘tripolar‘‘ configurations (p. 5.2) (Mok, Peloquin, 1975). As it was noted above, parallel spindle coorientation leads to the restitution only in combination with an additional abnormality: arrest in polar MT bundles formation at telophase II (Genualdo et al., 1998). Tripolar configurations are disoriented spindles whose two poles are in one point, i.e. considerably approached (p. 5.2) (Mok, Peloquin, 1975; Ramanna, 1979).

Meiotic Restitution Mechanisms Common for Mono- and Dicotyledonous Species Cytoskeleton reorganization from prophase till the onset of telophase occurs in the meiosis of mono- and dicotyledonous plants the same way. Thus, cytoskeleton abnormalities observed at these stages and leading to the formation of restitution nuclei can be considered universal. And some of such abnormalities were really found by us both mono- and dicot plant species, and we are convinced that further wide investigations will find out the same for

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the rest of abnormalities. Aberration in chromosome segregation that leads to the formation of restitution nuclei in meiosis of monocots was described also in the meiosis of dicots (without division spindle visualization) (Lam, 1974). It is possible to consider the following common restitution mechanisms for mono- and dicotyledonous plant species: 1) chromosome arrest in the ‗‘bouquet‘‘ and drastically unequal chromosome segregation to the poles related with this thing (p. 2.6), 2) arrest of cytoskeleton cycle at the stage of radial bundles (p. 2.1), 3) autonomous cytoskeleton ring formation under accentric nucleus position (p. 2.5), 4) cortical cytoskeleton ring formation at prophase of the second meiosis (p. 2.4), 5) arrest of perinuclear cytoskeleton ring disintegration at early prometaphase (p. 3.1), 6) arrest of cytoskeleton penetrating the former nuclear area at early prometaphase (p. 3.3) (found in dicot lines Res91, Res79 transgenic tobacco Nicotiana tabacum (Sidorchuk, personal communication); 7) monopolar spindle (p. 4.1) (in dicots - haploid Brassica juncea), 8) chromosome monopolar movement in a bipolar spindle under the absence of kinetochore fibers (p. 4.3 ‗‘comet‘‘ phenotype), 9) arrest of kinetochore fibers formation at mid-prometaphase (p. 4.2), 10) arrest of cytoskeleton realisation from the chaotic configuration at late prometaphase (p. 5.1, chaotic spindle) (it dicots it is observed in the phenotype of pea meimutant ms3) 11) abnormalities of anaphase chromosome movement (p. 6.1), 12) spindle shortening at anaphase (p. 6.2), 13) cell plate absence (p. 8.1) (in dicots – meimutant tetraspore in Arabidopsis) (Spielman et al., 1997), 14) cell plate abnormalities (p. 8.3), 15) arrest of daughter cell membranes formation (p. 8.4) and 16) the so-called general abnormalities (p. 10), but p. 10.5. Absence of one of meiotic divisions (Ramanna, 1983; Gill et al., 1985; Conicella et al., 1991) and ‗‘premature cytokinesis‘‘ – when the cytoplasm divides at telophase I and the second meiotic division does not occurs (p. 10.1) (Watanabe, Peloquin, 1993) was described in dicots among them.

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General Review of Meiotic Restitution Cytoskeleton Mechanisms According to their mechanisms, the abnormalities we described, that lead to meiotic restitution, are divided into three groups;1) formation of restitution nuclei, 2) congression of daughter genomes, 3) consolidation of cytoskeleton structures, 4) regulatory aberrations. Formation of restitution nuclei is a result of a complete arrest in spatial separation of daughter genomes. We detected several cytoskeleton abnormalities that lead to the formation of restitution nuclei. These are the above-described arrest of radial cytoskeleton reorientation, perinuclear ring conservation, autonomous ring, ring spindle, arrest of MT entering the former nuclear area, arrest of kinetochore MT formation, arrest in bipolar spindle fibers development, arrest of bipolar spindle formation (chaotic nonpolar spindles). Thus development of restitution nuclei occurs due to drastical aberrations of segregation apparatus, - the division spindle. In asynaptic meiosis the restitution nuclei form as a result of equational univalents segregation in the first meiotic division. Congression of daughter genomes, as a restitution process, occurs after chromosomes segregation, and the formation of the nuclear envelope around them. After this, as a result of cytoskeleton abnormalities, a recurrent approachment of daughter nuclei in the common cytoplasm can proceed. The basic reason for this is aberrations in structures that realize daughter genomes autonomisation, i.e. cytokinetic structures. As a consequence, daughter nuclei approach in the common cytoplasm and, in the next division, after NEBs, both

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daughter genomes become involved in one common division spindle. As a result of segregation, nuclei with a doubled chromosome number are developing. We have never observed the fusion of envelopes of approached daughter nuclei. According to our data, approachment of nuclei in the common cytoplasm can occur as a consequence of abnormalities of cytoskeleton-maintaining structures: cenral spindle fibers and interzonal MT system, also due to the absence of cell plate or daughter cell membranes. Approachment of daughter nuclei can be spontaneous in the common cytoplasm, just as in phenotype fs in dicots. Consolidation of cytoskeleton structures, as our data testify, also lead to the congression of segregated daughter genomes and, hence, to the meiotic restitution. Spontaneous approachment of daughter nuclei with developed perinuclear cytoskeleton rings occurs in 50% of PMC potato clone CE10 at prophase II. After complete approachment of nuclei, rings disjunct and fuse forming one common ring around both nuclei. After NEB, a common division spindle forms out of a common perinuclear cytoskeleton system. In 100% of PMCs of tomato meiotic mutant, as6, at mid prometaphase II, approachment and fusion of cytoskeleton chaotic figures proceed. No other MT cytoskeleton aberrations are observed in this phenotype. Regulatory aberrations make up a considerably big group of meiotic restitution mechanisms. It is the omission of one or both meiotic divisions (pp. 10.1, 10.2) and also the manifestation of processes typical of other studies at certain meiotic stages (p. 10.5, fused spindle phenotype, pp. 2.2, 2.3, 4.4).

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ACKNOWLEDGMENTS The author would like to express her gratitude to G.M Seriukov and E.G. Seriukova for their kindly provided material of wheat-wheat-grass F1, L.F. Dudka and V.Ya. Kovtunenko for their wheat-rye F1 hybrids, O.A. Shatskaya for maize haploids, Zh.M Mukhina for rice haploids and Alexander V. Zhuravlev for the English version of this big contribution.

REFERENCES Abbot AG, Hess JE, Gerbi SA. 1981. Spermatogenesis in Sciara coprophila. 1. Chromosome orientation on the monopolar spindle of meiosis 1. Chromosoma 83: 1-18. Alfano F, Cammareri M, Errico A, Frusciante L, Conicella C. 1999. 2n Gametes in Solanum tuberosum dihaploids. Amer J Potato Res 76: 281-285. Alfano F, Errico A, Conicella C. 1997. Potato meiotic mutants and importance of changes in the cytoskeleton. Cell Biol Internat. 21: 3-5. Amanda J. Wright,1 Kimberly Gallagher,2 and Laurie G. Smith. 2009. Discordia1 and alternative discordia1 function redundantly at the cortical division site to promote preprophase band formation and orient division planes in maize. Plant Cell 21: 234-247 Ambrose JC, Cyr R. 2007. The kinesin ATK5 functions in early spindle assembly in Arabidopsis . Plant Cell 19: 226–236.

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In: Pollen: Structure, Types and Effects Editor: Benjamin J. Kaiser, pp. 65-99

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Chapter 2

POLLEN BIOLOGY AND HYBRIDIZATION PROCESS: OPEN PROBLEM IN WALNUT Paola Pollegioni,1, Keith Woeste,2,† Irene Olimpieri,1 Fulvio Ducci 3,‡ and Maria Emilia Malvolti,1, 1

C.N.R. Institute of Agro-environmental and Forest Biology, Porano, Terni, Italy 2 U.S.D.A. Forest Service, Hardwood Tree Improvement and Regeneration Center, Department of Forestry and Natural Resources, Purdue University, Lafayette IN, USA 3 C.R.A. Research Centre for Silviculture, Arezzo, Italy

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ABSTRACT This review focuses on the pollen biology of Juglans, and in particular Juglans nigra (Eastern Black walnut) and Juglans regia (Persian or English walnut), which are economically important species in Europe, Asia and North America. Both species are monoecious, heterodichogamous and wind –pollinated. Their mating system is predominantly outcrossing, although under particular environmental conditions selfpollination is possible. Hybrids between the two species, Juglans × intermedia (Carr) can occur naturally, although they often have reduced fecundity. Compared to the parental species, most J. × intermedia (J. nigra × J. regia) hybrids show increased vegetative vigor, distinct disease resistance, high wood quality, and greater winter-hardiness. For these reasons here is great demand for J. × intermedia for forestry, especially in Northern Europe. We review several aspects of Juglans pollen biology that frustrate the production of J. × intermedia and limit the progress of researchers and plant breeders who work with this genus. We also discuss the ways in which scientists and breeders are working to overcome problems related to pollen storage and viability testing, pistillate flower abscission (PFA), fertilization and embryogenesis in Juglans, and the use of microsatellites to monitor gene flow, ploidy, parentage, and hybridogenesis all with an 

E-mail: [email protected] E.mail: [email protected] ‡ E-mail: [email protected] † 

[email protected]

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Paola Pollegioni, Keith Woeste, Irene Olimpieri et al eye toward practical solutions to the current shortage of J. × intermedia for research and applied forestry.

Keywords: J. × intermedia, Persian walnut, black walnut, hybrid

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INTRODUCTION Juglans is one of eight genera composing the family Juglandaceae and consists of 21 species of deciduous, monoecious trees distributed in North and South America, Southeastern Europe, Eastern Asia and Japan (Manning, 1978). Juglans species are traditionally divided into four distinct sections mainly based on leaf architecture, wood anatomy, pollen and fruit morphology: Dioscaryon Dode (traditionally Juglans), Rhysocaryon Dode (black walnuts), Cardiocaryon Dode (Asian butternuts) and Trachycaryon (American butternut) (Dode, 1909). Dioscaryon contains just one species, Juglans regia L. (Persian or English walnut) which is native to Eurasia from the Balkans to southwest China. Persian walnut bears four-celled nuts singly or in pairs, with smooth, thin shell and a dehiscent husk that separates easy from the nut at maturity. Section Juglans also includes the iron walnut, Juglans sigillata Dode, a type from Southern China and Tibet, with thick, rough-shelled nuts and very dark-colored kernels. The iron walnut has been considered as an ecotype of J. regia for long time, but it is also accepted as a separate species by some botanists (Manning, 1978). The Rhysocaryon section is endemic to the Americas and includes approximately 16 species: seven North American species, Juglans californica S. Wats. (Southern California black walnut), Juglans hindsii (Jeps) Rehder, Juglans major (Torr. Ex Sitsgr.) Heller, Juglans microcarpa (Texas black walnut) Berl., Juglans jamaicensis C.DC (West Indies black walnut), Juglans mollis Engelm., and Juglans nigra L. (Eastern Black walnut); four Central America species, Juglans olanchana Standl. & L.O. Willimas, Juglans steyermarkii Mann., and Juglans guatemalensis Mann, Juglans pyriformis Liebm.; and five South American species, Juglans australis Griesb., Juglans boliviana (C.DC.) Juglans soratensis Mann., Dode, Juglans neotropica Diels, and Juglans venezuelensis Mann. All members of Rhysocaryon section exhibit fourchambered nuts with thick, ridged or striate, not completely smooth shells and indehiscent and persistent husks. These species are so closely related that their discrimination is often difficult. Section Cardiocaryon (Oriental butternuts) includes three species all native to East Asia: Juglans ailantifolia Carr., Juglans cathayensis Dode, and Juglans mandshurica Mahim. Asian butternuts produce two chambered nuts with 4-8 prominent ridges and indehiscent husks, and are borne in long racemes of up to 20 nuts. Their susceptibility to walnut bunch disease has limited their horticultural diffusion in the eastern U.S. Section Trachycaryon consists only of J. cinerea L. butternut, a North America species, characterized by twochambered nut with high prominent ridges on the shell and an indehiscent husk. A Complete description of ecological distribution and the morphological variation in Juglans genus are found in two extended reviews, Manning, (1978) and McGranahan & Leslie, (2009). Earlier molecular studies based on nuclear RFLPs (Fjellstrom & Parfitt, 1995) and matK and ITS sequences (Stanford et al., 2000) confirmed the traditional taxonomic classification of Juglans and are consistent with biogeography and fossil history. Fossil evidence supported the ancient divergence of sections Cardiocaryon and Rhysocaryon almost simultaneously

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with the origin of the genus in the middle Eocene (~ 45 Ma) in North America (Manchester, 1987). Black walnut spanned from the West to East coast of North America extending into the Southern Hemisphere as far as Ecuador, whereas members of Cardiocaryon section crossed the Bering land bridge, existed from the early Eocene (55 million years before present) until the late Miocene, and spread into Eurasia; this theory implied that Dioscaryon section evolved from a common ancestor with Cardiocaryon. Nevertheless, recent study based on non-coding intergenic spacer (NCS) regions of chloroplast DNA supported section Juglans as the oldest lineage within the genus Juglans and the section Rhysocaryon as the youngest, in contrast to fossil evidence (Aradhya et al., 2007): Juglans section may be an independent, monophyletic clade sister to sections Cardiocaryon and Rhysocaryon. However the authors also postulated that the evolutionary history of Juglans section may have been confounded by geographic isolation, bottleneck events, human selection and introgression among isolated population during the post Pleistocene glaciations. Walnuts are among the most important trees in the world for nut and wood production. In particular two species, J. nigra L. (Eastern black walnut) and J. regia L. (Persian or English walnut) are widely cultivated. Most of the member of Juglans are of low economic value and are used only occasionally as timber or in the brown dye industry.

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JUGLANS REGIA L. (PERSIAN OR ENGLISH WALNUT) Juglans regia, the Persian or English walnut, is one of the most economically important member of the genus Juglans. Persian walnut is widely cultivated throughout the temperate regions of the world for its high quality wood and edible nuts. Persian walnut wood has a light yellow color and is characterized by a hard and homogenous grain. It is used for the production of furniture, panels and other manufactured products. Its non-edible parts, such as leaves and husks, find broad application in cosmetic and dye industries, and in traditional medicine (Amaral et al., 2008). For example, leaf extracts have a remarkable capacity to scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can at least partially justify the therapeutic use of J. regia leaves in folk medicine (Almeida et al., 2008). In addition, during the last decade several studies described the biochemical composition of walnut nutmeats, mainly with respect to their nutritional and health benefits. Walnuts are rich in -6 (linoleic acid) and -3 (linolenic acid) essential polyunsaturated fatty acids which cannot be produced in the human body and must be taken up through food (Caglarirmak ,2003; Amaral et al., 2003; Pollegioni et al., 2006). An inverse relationship between the relative risk of coronary heart disease and the frequent daily consumption of small amounts of walnut nuts was found. Feldman (2002) reported that: ―Compared to most other nuts, which contain monounsaturated fatty acids, walnut are unique because they are rich in ω6 and ω3 acids‖.

Walnuts also contain significant amounts of tocopherols, in particular - tocopherol, which protects storage lipids and proteins from oxidation (Verardo et al. 2009). Persian walnut is considered native from South-Eastern Europe to North-Western China (Xinjiang province) through Turkey, Caucasus, Iran, Pakistan, Northern India, Pakistan,

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Nepal and Tibet (Huntley & Birks, 1983). In recent decades, the origin of the European walnut has been a debated subject among foresters, botanists and bio-geographers. According to a traditional theory, the diffusion of the walnut species in Europe followed the ancient trade routes, passing from China into India, Persia and Greece (Forte, 1993). Nevertheless, it is still debated if the species was extinguished during the Pleistocene glaciations or if it survived the rigours of the cold, dry glacial intervals in refugia in Southern Europe and the Balkans, as suggested by some paleopalynologic studies (Huntley & Birks, 1983; Carrión & SànchezGomez, 1992; Fornari et al., 1999). Without regard to this debate, the first post-glacial appearances of Persian walnut pollen in Europe occurred around 1500-2500 yr BP and corresponded to the establishment of the Greek and Roman settlements (Huntley & Birks, 1983; Beer et al., 2008; Chester, 2009). From Greece, the cultivation of walnuts spread to Rome where walnuts were called Jovis Glans (Jupiter‘s acorn), from which comes the name of Juglans genus. From Italy, J. regia was exported to France, Spain, Portugal and Southern Germany (McGranahan & Leslie, 2009). Although there is evidence that environmental change could influence its expansion (Winter et al., 2009), J. regia generally grows wherever the climate is temperate from the 10th to about 50th parallel Northern latitude. J. regia grows best where the mean annual temperature is in the range of 10.5-15°C and annual precipitation is up to 700 mm. Persian walnut grows at altitudes from sea level to 1000-1200 m. a. s. l. (Forte et al., 1993). It is considered a frost-sensitive species because it is threatened by the occurrence of both early and late-season frost. As observed by Fady et al., (2003), late spring frosts have a negative impact on architectural traits and thus on wood quality. Early budbreak leads to loss of apical dominance and defective stem form when late spring frosts occur. Recently, Loacker et al., (2006) found a striking positive correspondence in alpine meadows between the number of germinated Persian walnut seedlings and higher average minimum temperatures during winter; conversely, germination rate was negatively associated with the number of days with severe frost They reported that in the last thirty years, climate warming promoted the expansion of J. regia in the South- and South-West-facing forests of inner Alpine valleys (Tyrol, Austria) which are often dominated by Scots pine (Pinus sylvestris L.). Persian walnut is also sensitive to soil conditions, developing best on deep, well-drained, moist and fertile soils rich in Calcium with a pH range from 6 to 7.5 (McGranahan and Leslie, 2009). Walnut is known to have very low tolerance for drought and flooding, which cause root system anaerobiosis (Mapelli et al., 1997) and enhance susceptibility to several Juglans diseases, including walnut blight (Belisario et al., 1997), anthracnose (Belisario et al., 2008) and root/collar infection by Phytophthora cinnamomi (Belisario et al., 2009). Persian walnut is cultivated in Southern and Western Europe, but also in Central Asia, Northern India, China, South Africa, Argentina, Chile, USA, Australia, New Zealand and Japan (McGranahan & Leslie, 1991). China leads world production, followed by the USA, Iran, Turkey, Ukraine, Romania, France and India. (FAOSTAT data, 2004). The major exporters are the USA, which exports 115.000 Metric Tons, followed by France (23.000 MT), China (22.000 MT) and India (17.000 MT). In the United States, 99% of the walnut crop is produced in California, where the crop has been grown since the 18th century when plants were imported from South America by Spanish missionaries (Potter et al., 2002). China has encouraged J. regia production and expects to have over 1 million hectares of walnut by 2012 (McGranahan and Leslie, 2009). In Europe J. regia is considered one of the most valuable broadleaved tree species. For example, in Italy, Campania is traditionally the most important

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region for walnut cultivation.Walnut cultivation in Italy decreased after the Second Word War (from 80 to 10 MT) because of land abandonment and the mechanization of agricultural lands (Di Vaio & Minotta, 2005). Local varieties/accessions were increasingly neglected because of their irregular fruit size and limited market demand. Never the less, in the last twenty years almost 100,000 ha of forest tree plantations were established on former agricultural lands with grants from the European Union. In a large percentage of these plantations (40–50%), Persian walnut (Juglans regia L.) was planted as the main species, due to the high value of walnut wood in the European market (Paris et al., 2005).

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JUGLANS NIGRA L. (EASTERN BLACK WALNUT) Juglans nigra L. (Eastern black walnut) is one of the most valuable hardwood species. It grows as scattered individual trees or in small, spatially distinct groves throughout the deciduous forests of eastern North America. It is a fast growing species, producing high quality timber on a relatively short rotation of about 60 years (Beineke, 1983). Eastern black walnut is native to most of the eastern U.S. from New Hampshire south to Georgia and west to Texas. Its western border includes parts of the states of Oklahoma, Kansas and Nebraska, with the northern limits crossing Minnesota, Wisconsin, Michigan and Ontario, Canada. On the western border in Kansas, in locations where environmental conditions are favorable for black walnut cultivation, it is abundant and occupies 50 percent or more of the basal area in stands of several hectares (Grey & Naughton, 1971). According to Williams et al., (2004), black walnut probably re-colonized the Midwest as a single, large population from a glacial refugium in the Lower Mississippi valley between 14,000 yr BP and 12,000 yr BP. Beginning in the 17th century, J. nigra was imported from the Eastern and Central hardwood forests of the United States to the European continent for ornamental purposes, and subsequently for its rapid growth, which led to its use for wood and as rootstocks. It is cultivated in Central Europe, the Balkans, Caucasus, Russia and Eastern-Central Asia. For example in Italy, black walnut is usually found in private and public parks of Pianura Padana, where is also used for reforestation and recovering degradated areas (Fenaroli & Gambi, 1975). In its native range, the vast majority of black walnut occurs in natural stands. Walnut plantations only cover about 13,800 acres in the United States, which represents about 1 % of all black walnut volume in U.S. (Shifley, 2004). According to Rink et al., (1994), intense harvesting pressure in the first part of the 20th century resulted in severely fragmented black walnut populations and consequently in significant losses of genetic diversity. Recently, a broad-scale study of the genetic structure of 43 indigenous populations of J. nigra, using (neutral) microsatellite markers indicated that the large deforestation and fragmentation that occurred across the range of black walnut after European settlement had little effect on the neutral genetic diversity of the species (Victory et al., 2006). In spite of differences in adaptative traits observed in provenance tests, high genetic homogeneity was found among American walnut populations. The authors postulated that because strong adaptative differences can persist in the face of high levels of gene flow, the use of functional markers, tightly linked to trait of interest, could be more useful for detecting regional adaptation in black walnut. Furthermore, because walnut trees can live to

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greater than 200 years of age, it is possible that an insufficient number of generations have passed to detect the effects of recent forest fragmentation. J. nigra is most valued for its lumber and veneer. The wood is used for multiple purposes, including the production of fine furniture, interior panelling, plaques and gunstocks. The wood machines easily, though it is hard, and when finished it has a dark luminous beauty. Uniformity of color is an extremely important factor in wood quality that is not present in black walnut because of the contrast in color between the sapwood and the darklycolored heartwood (Cassens, 2004). The heartwood of J. nigra is markedly darker than J. regia heartwood. Beritognolo et al., (2002) studied the role of transition zone (innermost sapwood) in the transformation of sapwood to heartwood and in the accumulation of phenolic substances in J. nigra heartwood. Although the mechanisms underlying heartwood formation are not completely elucidated, their results supported the hypothesis that flavonols are synthesized de novo in J.nigra in aged xylem tissues during the transformation of sapwood to heartwood. In addition flavonol accumulation appeared to be regulated mainly at the transcription level by the expression of chalcone synthase (CHS), flavanone 3-hydroxylase (F3H) and dihdroflavonol 4-reductase (DFR) enzymes. The nut produced by the black walnut has a furrowed, hard and thick shell that protects the edible seed. Well-managed seedling black walnuts produce nuts averaging 20% kernel but after shelling only 6 to 10% usable kernel is recovered. Nevertheless each year, American consumers use 2 million pounds of black walnut kernels in cookies, cakes and ice cream products (Reid et al., 2004). In addition, ground black walnut shell is extremely valuable for industrial applications such as metal cleaning and polishing and oil well drilling (Cavender, 1973). More than 400 black walnut cultivars have been named and released during the past century (Woeste 2004). Twenty of the most popular have been analysed and showed considerable genetic variation in nut quality, blooming date, leafing date, age of first bearing and growth rate (Reid et al., 2004). Despite its wide and geographically diverse native range, J. nigra is generally considered by silviculturists to be site sensitive; it only competes well against other temperate forest species on a limited number of site types. The growing season of J. nigra ranges from 140 to 280 days. Black walnut is tolerant of annual precipitation and temperature variations. For example, annual precipitation is less than 640 mm in northern Nebraska and about 1780 mm in the Appalachians of Tennessee and North Carolina. Mean annual temperatures range from about 7°C at the north of J. nigra‘s range, to 19°C at the south (Schlesinger & Funk, 1977). Black walnut generally requires moist, well drained, loamy, deep, nearly neutral soils; it grows best on sandy loam, loam or silt loam soils that hold a large amount of water that can be used by the tree during dry periods of the vegetative season (Beineke, 1983). J. nigra plants can reach a height of 45 m and a trunk diameter of 2 m; the root system is deep and wide spreading, with a definite taproot, at least in early life. As reported by Burke & Williams (1973), the taproot of 9-year-old black walnut trees, excavated from an Indiana plantation, was 2,3 m long, with lateral roots extended more than 2.4 m. In comparison with J. regia, J. nigra appeared to be more tolerant to water logging (Mapelli et al., 1997) and resistant to some walnut diseases, including, bacteriosis and infection by Phytophthora cinnamomi (Belisario et al., 1997; 2009; 2008). The most serious foliar disease of black walnut is anthracnose, caused by Gnomonia leptostyla (Fr.) Ces. Symptoms of walnut anthracnose develop on leaves, stem and fruit as irregular necrotic areas that are often surrounded by small chlorotic halos. In severe cases, these lesions may cause premature

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defoliation, fruit drop, or poorly filled nuts (Funk et al., 1981). The selection of resistant genotypes toward the anthracnose disease could represent a valid alternative to cultural and chemical (fungicides) management (Woeste and Beineke, 2001). A wide range in susceptibility to walnut anthracnose appears to exist in J. nigra, but. no specific genotype has been reported to be immune. Two black walnut cultivars, ―Thomas‖ and ―Ohio‖, have been noted for their anthracnose resistance, although both cultivars could contract the disease under condition of high pressure (Berry, 1960). As reported by Mielke et al., (2004), trees in adjacent J. nigra plantations located in North America frequently exhibit different levels of disease incidence. Genotypes derived from the western edge of the natural range of black walnut (Kansa and Oklahoma) appeared most susceptible, perhaps because of low selective pressure for anthracnose resistance in this relatively arid region. These observations clearly suggested the existence of natural resistance to anthracnose. Studies also indicated that the natural resistance to G. leptostyla is highly heritable (Beineke & Masters, 1973), encouraging genetic breeding programs in walnut. The genetics of the pathogen have never been researched, and it is possible that the fungus has multiple races and local or regional variation in virulence.

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INTERSPECIFIC HYBRID (JUGLANS × INTERMEDIA CARR) Although phylogenetic analysis based on nuclear RFLP, matK and ITS sequence has demonstrated that black walnut and Persian walnut belong to different sections of genus Juglans, Rhysocaryon and Dioscaryon respectively (Stanford et al., 2000), a hybrid between them, Juglans × intermedia (Carr), can occur naturally. Generally, the female parent of J. × intermedia is J. nigra and the male parent is J. regia (J. nigra × J. regia). In fact, because J. nigra pistillate flowers usually mature at least two to three weeks later then J. regia catkins, there is a considerable phenological barrier to hybridization which is overcome only rarely in nature. The percentage of hybrid progeny in a mixed population is usually less than 10 % (Funk, 1970). The difficulty obtaining hybrids of the two species could be the result of an incompatibility in flowering phenology or some mechanism(s) of genetic incompatibility (Sartorius 1990), failure of fertilization (pre-zygotic factors), or embryo abortion (postzygotic factors). In addition to synchrony of flowering, hybridization rate may be affected by air temperature, which influences pollen germination and penetration through the stigma and the style to the J. nigra ovary. As described in the next section, Luza et al. (1987) found clear differences in temperature optima for pollen germination and tube grow in J. nigra and J. regia. Compared to the parental species, most J. × intermedia hybrids show increased vegetative vigour, distinct disease resistance, good wood quality, and greater winter-hardiness than Juglans regia (Fady et al., 2003). In particular they showed strong apical dominance, late budbreak and resistance to spring frost damages. They were superior to the parents in growth at sites with medium to low fertility and were moderately tolerant of flooding. As reported by Mapelli et al., (1997), some walnut hybrid genotypes could be exposed to anoxia stress for 10 to 12 days before they showed visible signs of injury. For these reasons there is a great demand for J. x intermedia for forestry, especially in Northern Europe. Recent investigation on the resistance to anthracnose infections of J. regia, J. nigra and inter-specific hybrids

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(J. nigra × J. regia) plants proved that J. regia is susceptible, J. nigra is relatively resistant, while hybrids showed an intermediate behavior toward Gnomonia leptostyla infection (Anselmi et al 2005). In general, J. × intermedia hybrids flower profusely but never bear much seed; inadequate chromosome pairing in megaspore and microspore mother cells can frequently occur (McKay 1941) Often the seed produced is not able to germinate well, averaging only 27 percent (Funk, 1970). Walnut trees that show a particular aptitude for producing hybrids are defined as ―hybridogenic‖ plants. The identification and selection of hybridogenic parents is the first step toward obtaining hybrid progeny in walnut. In addition, although some trees appear to be hybridogenic under natural conditions, it has been difficult to produce hybrids using controlled crosses (McKay 1965; Scheeder 1990). As reported below, breeders have encountered difficulties obtaining sufficient Persian walnut pollen at the time J. nigra pistillate flowers are receptive. Suitable and relative simple method for pollen storage and viability testing is now available for Juglans (Luza & Polito, 1985, 1988b). In addition, pistillate flower abscission (PFA), caused by excessive pollen load, has been reported in Persian (Catlin et al. 1987) and black walnut (Beineke and Masters 1976). PFA may decrease the final nut set (Figure 1).

Figure 1. Summary of the hallmark events (the low pollen viability, the pistillate flower abortion, fertilization and embryogenesis) of inter-specific hybridization between Eastern black walnut and Persian walnut.

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Thus, the production of hybrid plants depends mostly on successful natural hybridization. In practice, forest nurseries commonly collect seeds from J. nigra trees that are expected to be pollinated by J. regia. After one or two years of cultivation, the hybrid genotypes are distinguished mainly by phenotypic traits such as leaf and bud shape. As suggested by Hussendorfer (1999), ―the natural variation of phenotypic traits sometimes leads to the problem of miss-identification of hybrids‖. In the past, several methodologies have been developed to distinguish between J. nigra and J regia and to identify French and German inter-specific walnut hybrids. They were based on morphological traits (Jay-Allemand et al., 1990), biochemical markers such as isozymes (Germain et al., 1993; Hussendorfer, 1999), PCR-markers as Restriction Amplified Polymorphisms (RFLPs) (Tanzarella & Simeone, 1996) and Random Amplified Polymorphic DNA (RAPDs) (Malvolti et al., 1997). Although isozymes and RFLPs are codominant markers, they are not frequently used for hybrid identification because they are time-consuming, expensive and characterized by low levels of polymorphism. Malvolti et al., (1997) reported that a subset of twenty selected RAPDs markers were a powerful tool to discriminate between J. x intermedia genotypes and backcross plants ((J. nigra x J. regia) x J. regia ) and ((J. nigra x J. regia) x J. nigra). Nevertheless, RAPDs (dominant markers) can show a low reproducibility and are not useful for pedigree and parentage analysis. Vegetative propagation of the identified walnut hybrids, by cutting or micropropagation, has proved difficult. Numerous juvenile and mature clones cannot be propagated at a commercial scale because of their limited ability to form adventitious roots. Claudot et al., (1993) detected a strong accumulation of hydrojuglone glucoside (precursor of juglone) in phloem and parenchymal cells in seedling and rejuvenated material, whereas a high content of flavonol glycosides (myricitrin and quercitrin) in the peripheral zone of mature shoots. It was postulated that these polyphenols may inhibit adventitious root generation in microcuttings. The expression of antisense chalcone synthase RNA (key enzyme in flavonoid biosynthesis) in transgenic hybrid walnut microcuttings confirmed the previous results: decreased flavonoid content in stems of antisense chs transformed lines was associated with enhanced adventitious root formation (Euch et al., 1998). In addition the widespread use of micropropagation in order to produce hybrid walnuts has been limited by the low survival of shoots cultured in vitro during acclimatization. An antagonism between the number of roots and the number of leaves in the walnut plantlet was observed (Cheneval et al., 1995; 1997). They noted that a low sucrose concentration in the propagation medium promotes photosynthetic activities of shoots and consequently the establishment of photoautrophy but also reduces the development rate of root system. Somatic embryogenesis was developed from cotyledons of immature nuts of J. × intermedia hybrids (Cornu, 1988). Unfortunately only few propagated somatic embryos completed their growth and produced whole plants. Nevertheless recent increases in industrial demand for wood have led to expanded planting areas and the establishment of new seed orchards for production of J. × intermedia trees.

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FLORAL AND POLLINATION BIOLOGY IN WALNUT Black and Persian walnut are wind-pollinated, monoecious, dichogamous and hypothetically entirely self-compatible species, with the same number of chromosomes (2n = 32). Male (staminate) and female (pistillate) flowers are on the same tree but separated from each other. Both species are characterized by a dichogamous bloom habit: the period of the female flower receptivity does not overlap the period when male flowers shed pollen. As discussed by Bertin & Newman (1993), dichogamy represents an evolutionary mechanism to encourage an outcrossing mating system, reducing or preventing self-pollination. Nevertheless this bloom habit does not eliminate the possibility of self-fertilization in walnut because the temporal separation of female and male flower bloom is sometimes incomplete (Forde & Griggs, 1975). The mating system of walnuts exhibits a phenotypic dimorphism defined as ―heterodichogamy‖: if the male flower shed their pollen before the pistillate flowers are receptive, the genotypes are classified as ―protandrous‖, whereas if the mature pollen is released after the period of the female flower receptivity, the genotypes are classified as ―protogynous‖. Most J. regia trees are protrandrous, only a few cultivars, such as ―Chico‖ and ―Amigo‖, are protogynous. On the contrary, a high incidence of protogyny is detected in J. nigra species (Funk et al., 1970). According to Gleeson (1982), heterodichogamy in Persian walnut is regulated by two dominant-recessive alleles at a single locus, with protogyny as a dominant phenotype. In addition, the mode of dichogamy in Juglans seems to be correlated with the extent of both staminate and pistillate flower differentiation that occurs prior to the onset of the dormant season. As is typical for many winter-deciduous tree species, floral organogenesis and differentiation in Juglans begins in the growing season prior to dormancy and ends in the spring during the weeks before bloom. Luza & Polito (1988a) showed that in each protandrous tree, the staminate flower primordia entered the dormant season with anthers having all wall layers and four microsporgia fully differentiated; in the protogynous tree, anthers presented only as undifferentiated structures. Subsequently, Polito & Pinney (1997) observed that pistillate floral primordia in protogynous individuals progressed to the initiation of a perianth (four sepal primordia), whereas in protandrous individuals development stopped at an early stage corresponding to initiation of the involucral ring.

STAMINATE FLOWER AND POLLEN STRUCTURE In walnut the staminate (male) flowers are small and densely grouped in catkins, 10-15 cm long, borne laterally on 1-year-old wood. Each catkin includes up to 40 sessile petaless florets surrounded by green sepals. The individual flowers lack petals and are characterized by numerous stamens. Each stamen terminates in a pollen-bearing anther (Figure 2). At maturity, each catkin is able to release two million pollen grains that are subsequently dispersed by wind over long distances (Impiumi, & Ramina, 1967). Emergence of the staminate inflorescence and shedding of pollen increase with rising temperatures and are associated with lower relative humidity: cold weather has the opposite effect and reduces pollen dispersal. In addition, the pollen mother cells of anthers are usually very sensitive to

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spring frost. Frost frequently causes partial or full abortion of normal meiosis, causing catkins to shed sterile pollen (Kvaliashvili et al., 2006). Pollen grains consist of three distinct portions (Polito et al., 1998a). The central, living, cytoplasm in which is found the nuclei responsible for fertilization, is surrounded by two distinct layers that compose the pollen wall: the inner layer, the ―intine‖, and the outer layer, the ―exine‖.

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Figure 2. Male walnut catkins borne laterally on 1-year-old wood. Each catkin includes up to 40 sessile, petaless florets surrounded by green sepals. The individual flowers lack petals and are characterized by numerous stamens. Each stamen terminates in a pollen-bearing anther.

The intine is a thin inner wall made of mostly pectin and cellulose. The exine is composed of ―nexine‖ and ―sexine‖, and is perforated by numerous pores (germination apertures). The pollen wall is resistant to degradation and treatment with intense heat; strong acids and bases usually have little effect upon the pollen wall. In particular, the walls of the pollen grains of J. nigra (the exine) include a sexine three times thicker than the underlying nexine (Calzoni et al., 1990). Of the structural elements of the sexine, the ―tectum‖ appears strongest, crossed by thin channels and decorated by spinulose extroflections. Bacula are differently shaped and irregularly distributed; thin lamellar structures are rarely present. In J. regia the exine is not as thick as in J. nigra, although the sexine/nexine ratio remains unvaried. In both species, the intine is widely spread through the oncus and nexine is homogeneous and broken at the pores without opercula. Meiosis in the pollen mother cells, and maturation of pollen grains, occur before (protandrous) or after (protogynous) pistillate flowers bloom. The pollen grains, which contain the male gametes, are transferred to the sticky stigmatic surface of receptive female flowers by wind. After 7 to 8 hours, in warm and sunny conditions, or 24 to 36 hours in cold and humid weather, the pollen grain germinates (Kvaliashvili et al., 2006). Pollen germination requires hydration of the dry cytoplasm followed by expansion of the inner wall through one of the pores in the outer wall. As described by Polito et al., (1998a), the cytoplasm of pollen grain moves into the long pollen tube defined by the growing wall. After pollen germination on the surface of stigma, multiple pollen tubes grow through the style; some of them penetrate the ovary but only one reaches the embryo-sac and fertilizes the egg cell. Within the pollen tube, two non-motile sperm cells are ultimately formed and are conveyed through the tube,

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keeping pace with tip growth. Fertilization occurs one week from the time of pollen germination on the stigmatic surface. The ability of walnut pollen to germinate can vary among years and during the same vegetative season. Understanding this critical process is not only important for deciphering the basic mechanism of sexual reproduction in walnut but also has value for the potential manipulation of nut production. In particular the barriers underlying the partial incompatibility between J. nigra and J. regia are still unclear. In order to prevent the ―wrong‖ cross, many plants have developed barriers that operate in the pistil either before fertilization, inhibiting pollen tube germination and elongation, or after fertilization, causing abortion of the illegitimate embryo. The barriers in interspecific-crosses are mostly referred to as ―incongruity‖, indicating the lack of communication due to the absence of co-evolution of two species (Hogenboom, 1984). Detecting the signals that regulate the compatible interaction between a pollen tube and all the female cells in its path is crucial for breeders to break species barriers and produce J x intermedia hybrids. Pollen germination and pollen tube growth involve a high number of signalling events, including cell-environment interaction, intercellular and intracellular communications. It‘s very well known that pollen germination and tube growth are significantly regulated by the temperature, the transport of inorganic ions such as Ca+2 and K+ across the plasma membranes of pollen, and by the synthesis of signal molecules such as gametophyte-specific flavonol diglycosides (Taylor & Hepler, 1997). Clear differences in temperature optima for pollen germination and tube grow were found in J. nigra and J. regia: maximum germination occurred at 32°C and 28°C respectively (Luza et al., 1987). Pollen germination percentage increased with temperature in both species but declined abruptly and approached zero at approximately 40°C; no germination of pollen occurred below 14°C. In addition, a positive linear correlation between staminate bloom date and optimum temperatures for pollen germination was detected; higher optimum temperatures were associated with late blooming dates. No differences in optimum temperature (33°C) for pollen tube elongation in vitro were detected between black and Persian walnut. Nevertheless the minimum temperature that would support pollen tub elongation in J. regia was lower than in J. nigra. According to Luza et al., (1987), although some degree of phenotype plasticity may influence the responses to the temperature, differences in the ability of pollen to germinate at various temperatures could be genetically fixed. Significant variations in the mineral ion composition of pollen were also identified between black and Persian walnut. Notable differences were observed in P, N, Mg+2, Ni, and K+ content. As proposed by Calzoni et al., (1990), the capacity of the sporophyte parent to accumulate mineral elements into pollen grains during dehydration can be considered as species-specific. In particular, studies in Arabidopsis demonstrated that an inward K+ current across the plasma membrane may play a role in the activation of the osmotic water influx required for pollen germination and the regulation of pollen turgor pressure during tube elongation (Fan et al., 2001). Extracellular acidification induced by a H+-ATPase pump and high concentration of external Ca+2, typical of the micropylar apparatus and the receptive synergid cell, may negatively regulate the pollen inward K+ channels, inhibiting tube growth. Significant variation in macro- and microelements found in walnut pollen could represent a discriminating factor between J. nigra and J. regia and negatively affect the ability of pollen tube to grow through the style and ovary tissue when interspecific pollinations occur.

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The low probability of fertilization between Persian and black walnut also may be caused by inefficient pollen –pistil recognition during germination and pollen tube elongation. Successful fertilization depends on specific pollen –pistil interactions and only ―compatible‖ pollen grains are able to complete the passage through stigma, style and ovary (Geitmann & Palanivelu, 2007). Pollen tube growth takes place in the extracellular matrix (ECM) of the stigmatic and stylar transmitting tissues (TT) and along the ovule surface. Pollen tube growth has been described as a specialised form of plant cell movement in which the pollen cytoplasm moves forward, leaving behind cell wall materials connecting the tube to the empty pollen grain that remains anchored on the stigmatic surface. This process involves cytoskeletal elements such as actina, myosin, microtubules and the synthesis of wall degradating enzymes (Taylor & Hepler, 1997). The pistil ECM provides chemical and physical support as well as directional cues for pollen tube elongation toward the ovules. The ECM is enriched with secretory materials such as free sugars, polysaccharides, glycoproteins and glycolipids. Arabinogalactan proteins (AGPs), which are ubiquitous to plants, represent the major class of proteins in the ECM of the transmitting tissue and in the stigmatic exudates. AGPs are a class of hydroxyl-proline-rich glycoptoteins characterized by a high carbohydrate content that include arabinose and galactose residues (Bacic et al., 1988). In the last fifteen years, numerous studies demonstrated that AGPs of the transmitting tissues play a major role in pollen recognition and adhesion on the stigma: they serve as nutrients and adhesive substrates for the tube pollen elongation (Cheung et al., 1995; Taylor & Hepler, 1997; Sanchez et al., 2004; Geitmann & Palanivelu, 2007). Within the receptive female flower, TTS proteins display a gradient of increasing concentration and glycosylation from the stigmatic surface to the ovarian transmitting tissue. The increase in acidity associated with increased TTS protein glycosylation may have a chemotropic effect, guiding pollen tube from the stigma to the ovary (Wu et al., 1995). TTS proteins are also deglycosylated and then incorporated into the pollen tube wall, providing nutrient and energy for tube elongation process. Recently Sanchez et al., (2004) reported that new, interesting signalling systems are involved in pollen tube growth, including ethylene and GABA. Furthermore, Geitmann & Palanivelu, (2007) suggested a putative ovule—based pollen repulsion mechanism during inter-specific crosses. This short-range repulsion of the pollen tube is used to inhibit the access of multiple pollen tubes to an ovule, but it also prevents intra genomic conflicts that would rise from the egg cell being fertilized by genetically distinct sperm. As demonstrated by Palanivelu & Preuss (2006), the repulsion initiated prior to tube reception in the female gametophyte maybe mediated by synthesis of nitric oxide (NO). This study also showed that in Arabidopsis the repellent signal from ovule was less effective than in closely related species. According to Calzoni et al., (1990), the pattern of soluble cytoplasm, membrane and cellwall proteins of J. nigra and J. regia pollen vary quantitatively and qualitatively. In particular the affinity chromatography of salt-soluble proteins of the pollen wall revealed a glycoprotein fraction eluted with 300 mM of methyl--D-mannopyranoside present in J. regia and completely absent in J. nigra. We can postulate that these differences in the chromatography profiles may reflect differences in enzymatic activities critical for hydration during pollen germination, for adhesion and penetration through the stigmatic and stylar transmitting tissue, and for proper pollen tube guidance. Which molecules, structures and interactions are relevant for the expression of the incongruity in Juglans during inter-specific pollination are not yet

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understood. Therefore functional and genetic redundancy among molecules involved in the pollen-pistil recognition / fertilization permits rare crosses among hybridogenic plants.

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POLLEN VIABILITY AND CONSERVATION Taking into consideration the wide variability associated with walnut flower phenology, a suitable method of pollen storage is essential. One of the main problems in black and Persian walnut production is that it is difficult for breeders to obtain sufficient amounts of desired pollen at the time pistillate flowers are receptive because of the dichogamous nature of the species. For example, J. regia pollen matures and is usually shed about one month in advance of J. nigra pistillate receptivity. In this case, short-term storage of Persian walnut pollen is needed in order to carry out the inter-specific cross. In black walnut the situation is frequently reversed, and the pistillate flowers are receptive a week in advance of pollen maturation, requiring a pollen storage for one year (Griggs et al., 1971). As reported by Luza & Polito (1985), the life span of walnut pollen appears to be very short under natural conditions and its vitality can be affected by temperature, relative humidity and maturity. Studies of pollen germination in vitro and of tube growth revealed differences among 21 Persian walnut genotypes for ability to germinate. The samples were collected in the experimental fields of the University of California, Davis (Luza & Polito, 1985). In a different study, similar results were observed between 32 walnut cultivars from different sites of Turkey (Sütyemez, 2007). The most remarkable indication of low vitality of Persian walnut pollen was given by observations after 24 hours of incubation at 24°C. Under these conditions, pollen lost the ability to germinate in vitro within two days for all cultivars tested. Black walnut pollen seems to be viable at least 24 hours at 24°C, with an average of 21% of pollen germination (Beineke & Masters, 1983). Polito et al., (1998a) concluded that rapid desiccation was the probable cause of pollen death, and this factor may be a serious problem for breeders who wish to store pollen for an extended period and for production of J. × intermedia hybrid progeny. Few methods have been developed for storing walnut pollen, and none of them are easy to apply. In black walnut, refrigeration (14°C), without desiccation provided satisfactory short-term storage for one to three weeks. According to Beineke & Masters (1983), freezer storage and treatment in desiccators were inconsistent and for the most part damaging. Nevertheless in this study the maximum pollen germination was 36.2 % after only one week of storage. Persian walnut pollen can also be stored at typical freezer temperatures (-20°C) but only with careful control of the relative humidity (RH) of the storage environment. As described by Luza & Polito (1995), most of the Persian walnut pollen did not germinate after three months of storage at -20°C when RH was not controlled. Pollen storage for three months up to one year at -20°C is possible if the RH remains near 33 percent. This can be achieved by storing pollen over a saturated solution of magnesium chloride (MgCl·6H2O), although under these conditions the germination ability may vary and be near zero for some cultivars. Hall and Farmer (1971) proved that liquid nitrogen storage (-196°C) of black walnut pollen was effective and suggested the possibility of long-term viability retention. Results of Luza & Polito (1988b) study indicated that Persian walnut pollen may be stored at -196°C and pollen germinability can be maintained if the pollen grain moisture content is

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controlled and reduced to a value between 7.50% and 3.20% by gentle drying for 24 hours after collection. They concluded that excessive moisture (more than 30%) may be lethal, inducing ice-crystal formation in the pollen cell during freezing. Intracellular ice formation can induce fractionation of organelles and disruption of membranes. As described in the previous section, the viability and germinability of pollen depends strongly on the state of the vegetative cell membranes. Obviously the apparatus for liquid nitrogen long-term storage are not widely available and this method is not easy to apply in many instances.

PISTILLATE FLOWER STRUCTURE AND RECEPTIVITY

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Pistillate flowers of both, J. regia and J. nigra, are borne at the tips of terminal shoots on current season‘s wood, in spikes of typically two to three flowers. McGranahan and Leslie (2009) reported that female flowers are also produced on the tips of lateral shoots in some cultivars. This type of flowering is called ―lateral bud fruitfulness‖ and is often correlated with high nut yield in young trees. Pistillate flowers lack visible sepals and petals, are pubescent, small and green. In particular, the entire basal portion of the flower is enclosed with a hairy sticky involucre fused to four sepals. The husk of the mature walnut fruit is derived mainly from the tissues of the involucre and sepals (Figure 3).

Figure 3. Longitudinal section of walnut pistillate flower. Pistillate flowers lack visible sepals and petals, are pubescent, small and green. In particular entire basal portion of the flower is enclosed with a hairy sticky involucre fused to four sepals. The ovary is the enlarged basal portion of the pistil which also includes a short style and bilobular stigma. At the base of the locule is a one ovule which is surrounded by a single integument. Surrounded by the integument is a region called nucellus in which is present the embryo-sac that contains an egg cell and two synergid cells at micropylar pole, two polar nuclei in the centre, and three antipodal cells at chalazal pole.

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The ovary is the enlarged basal portion of the pistil which also includes a short style and a large feathery bilobular stigma. The structure of the stigma facilitates the interception of wind-borne pollen, the recognition of walnut pollen and the exclusion of pollen of other species. The surface of the stigma secretes a thin layer of exudates providing a suitable medium for pollen germination and the initial growth of the pollen tube (Polito et al., 1998a). The shell of fully matured fruit is derived from the ovary wall. At the base of the locule is one ovule which is surrounded by a single integument. At the end of fruit development, the integument becomes the seed coat, the brownish pellicle that enclosed the kernel at maturity. A region called nucellus is surrounded by the integument. The nucellus constitutes the bulk of the ovule during fertilization. The cells of the nucellus degrade as the fruit develops. Within the nucellus is present the embryo-sac (seven-celled structure) containing the female germ cell (egg cell). At the time of pollination most of the embryo-sac structure contain an egg cell and two synergid cells at the micropylar pole, two polar nuclei in the center, and three antipodal cells at chalazal pole (Figure 3). When pollen enters through the embryo-sac, one sperm cell is discharged and fuses with the egg cell to form the zygote and subsequent embryo. The second sperm cell fuses with two polar nuclei to provide a nutritive tissue called endosperm (triploid tissue). In the early stages of fruit growth the endosperm is consumed and disappears in mature fruit. One week after fertilization, the zygote has already started cell division and the proembryo is composed of a maximum of eight cells. Polito et al., (1998a) reported that fertilization in walnut usually occurs five to seven days after pollination. Luza and Polito (1991) showed that porogamy and chalazogamy are alternate pathways in walnut flowers. In angiosperms, pollen tubes typically enter the ovule through the micropyle, a phenomenon referred as porogamy. Chalazogamy refers to pollen tube entry through funiculus and chalaze tissues rather than though the micropyle. According to Luza and Polito (1991), in J. nigra and J. regia the occurrence of chalazogamy / porogamy is correlated with the developmental stage of pistillate flower. They observed that during the earlier stages of anthesis, pollen tubes arrive at the ovary when the integument is less developed and a considerable space is present between the apex of the nucellus and the base of the stylar canal. In this case the pollen tubes may be unable to cross this open space and bypass the micropyle. They grow along the surface of winged outgrowths to the chalazal end of the ovule. When the development of ovary progress to the point that integument is close to the bottom of the style, then porogamy occurs. Nevertheless in J. regia cv. Franquette, Tadeo et al., (1994) observed that five days after the time of pollination one of the synergid cells had a normal structure whereas the other usually was degenerated. In porogamy, the pollen tube contents are discharged into one of the synergids prior to fertilization, causing the breakdown of this cell. Since only one of the synergid cell survived in all embryo-sacs analysed in this study, Tadeo and colleagues (1994) suggested that pollen tubes might have entered the ovule mainly via the micropyle. Pistillate flowers that are not fertilized continue to grow for the next three weeks, at which point they drop. In the embryo-sac of unpollinated ovaries, fusion of the two polar nuclei occurs in the early development stages, leading to a 2n endosperm tissue. The absence of pollination accelerates cellularization of the 2n endosperm, causing degeneration of embryo-sac. Tadeo et al., (1994) evaluated the putative role of endogenous gibberellins (GAs) in walnut fruit development. A wide body of evidence suggested that pollination process may be particularly dependent on GAs. They observed different patterns of GA change in pollinated

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and unpollinated ovaries of ‘Franquette‘. In particular, gibberellin A1 (GA1), which is thought to be an active GA controlling vegetative growth in higher plants, showed a transitory increase prior to and immediately after fertilization, and a gradual decrease subsequently in pollinated ovaries. In unpollinated ovaries, the transitory GA1 peak occurred at the same moment but was higher (2-fold) than in pollinated ovaries. Thirteen days after pollination, GA1 levels were much lower in unpollinated flowers than in pollinated flowers. It has been postulated that GAs may preserve embryo-sac viability and extend the period of maximum pollen receptivity. GA1 may also postpone the beginning of senescence in unpollinated ovaries and protect the reproductive structures of the ovary before fertilization. According to Tadeo et al. (1994), fertilization induced immediately the gradual reduction of GA1 at the beginning of embryo cell division. They proposed that GA1 may be a critical component for embryogenesis. Growth arrest and flower abscission coincided with very low amounts of gibberellins. For a long time the design of walnut orchards has been focused on maximizing pollen density during pistillate flower bloom to improve nut yield. Moreover there were major breeding efforts to modify the quality and quantity of walnut production by selecting suitable genotypes and/or carrying out controlled crosses between useful parental trees with a handpollination into receptive female flowers. The pistillate flower is usually receptive to pollen for a short time, seven days at most, if conditions are ideal. Generally hot and dry environmental conditions reduce the period of optimal receptivity. Before the expansion of the stigma, female flowers are not able to retain wind-borne pollen and to produce the layer of exudates in which pollen can germinate and tube growth occurs. Considering the low pollen viability observed at room temperature, pollen that lands on stigmas does not have a good chance to survive until the female flowers become receptive. Polito et al., (1998a) reported that pistillate flowers were highly receptive when the two stigmatic lobes were separated from one another to form a V-shape. Once the stigmatic lobes were orientated at more than 45 degree angle to the longitudinal axis of the ovary, the surface began to dry out and the female flowers were not longer receptive. Nevertheless, as described in the next section, some evidence suggests that a large and uncontrolled amount of pollen can adversely affect the final nut set by inducing pistillate flower abscission.

PISTILLATE FLOWER ABSCISSION (PFA) Pistillate flower abscission (PFA), was reported for the first time in Persian and black walnut by Catlin et al., (1987) and Beineke and Masters (1976) respectively. Pistillate flower abscission is the loss of the pistillate flowers early in the season, typically two to three weeks after bloom and prior to fruit drop due to lack of pollination. According to Catlin et al., (1987), the ovary enlargement in PFA-type flowers stops at a diameter of 3 to 4 mm, leading to abscission 10 to 14 days later. The abscission of un-pollinated flowers occurs three to four weeks after bloom, and their ovaries have been enlarged to more than 7 mm in diameter. Two different areas of separation were also detected: the distal and proximal area of the peduncle. PFA-type abscission of flowers occurs at the zone between the peduncle and vegetative apex, causing drop of flowers still attached to the peduncle, in contrast to separation between the

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ovary and peduncle, which was typical of non-fertilized flowers. In addition PFA-type flowers showed cell and tissue necrosis at the tip of the stigma, in the inner wall of the ovary, the integuments and throughout the placental evaginations (Catlin & Polito, 1989). Pistillate flower abscission has emerged rapidly as a major non-pathogenic problem of walnut production, reducing final yield. In particular, Catlin et al. (1987) recorded levels of PFA in excess of 90 percent in ‗Serr‘ orchards planted in the Sacramento Valley of California. ‗Serr‘ appears to be the most susceptible cultivar. Other J. regia cultivars, such as ‘Chandler‘, ‘Howard‘ and ‘Vina‘, are affected by PFA but usually with less loss (Catlin & Olsson, 1990; Rovira & Aletà, 1997; Polito et al., 1998b). Moreover this phenomenon may vary among varieties and sites, and it is not consistent between years. Rovira & Aletà (2001) evaluated the incidence of PFA in 19 different cultivars and selections of J. regia: five California cultivars (‗Chandler‘, ‗Chico‘, ‗ Hartley‘, ‗Serr‘, and ‗Vina‘), four Chilean selections (‗AS-0‘, ‗AS-1‘, ‗AS-5‘ and ‗AS-7‘) five French cultivars (‗Franquette‘, ‗Lara‘, ‗Mayette‘, ‗Marbot‘ and ‗Parisienne‘) and five selections from Spain (‗MBT-49‘, ‗MBT-31‘, ‗MBT-247‘, ‗MBT-119‘, and ‗MBT-122‘) located at IRTA-Mas Bovè (Spain). Significant differences were observed between years, among groups of cultivars of different geographic origin and within cultivars. The Spanish selections were the most affected group with 73.4% PFA, compared to Chilean selections that showed only 6.8%. Unexpectedly, French and Californian cultivars presented an intermediate behaviour, showing lower mean values of PFA in ‗Chandler‘, ‗Chico‘, ‗Franquette‘, ‗Hartley‘ and ‗Serr‘ cultivars than those observed in California. Nevertheless in all cases the number of dropped flowers due to PFA was negatively correlated with final nut set. In addition, although PFA incidence is difficult to predict and control, the heritability of this trait seems high (narrow sense heritability = 0.61); selection of parents with low abortion could produce offspring with lower levels of PFA (Hassani et al., 2006). Finally a bias in the measures of PFA incidence could have occurred in the previous studies. PFA level was usually quantified as a percentage of the necrotic pistillate flowers 3-4 mm in diameter dropped while attached to the peduncle. In a recent study, Gonzàlez et al. (2008) reported two separation areas in PFA-type flowers of ‗Serr‘ walnut. The distal separation area of the peduncle was present in 36 % of the cases, causing flower drop without the peduncle; the remaining 64% showed an attached peduncle. The absence of the peduncle may be attributed to abscission from lack of pollination and may have misled the researchers. They also noted a new and interesting symptom useful for discriminating between these two types of abscission. The scar caused by PFA presented an irregular surface, was brown, and 1-2 mm in diameter, versus the scar caused by lack of pollination, which had a smooth surface, was chalky and 4 to 5 mm in diameter. During the late 1980s several studies were conducted to determine the cause of this disorder. Mineral nutrient deficiency, the phytotoxic effect of copper sprays used for control of blight/anthracnose disease, unmet chilling requirement, tree age, water stress, environmental conditions, defective ovarian development (Catlin et al., 1987), low nitrogen content and competition for carbohydrate (Deng et al., 1991) were excluded as plausible causes of PFA. In the walnut orchards of Balatonboglàs Winery (Hungary), Pór & Pór (1990) observed that the nut yield decreased significantly as distance from the pollenizer decreased. After noting PFA-type drop when large amounts of pollen were applied to flowers in the course of making crosses for breeding, McGranahan et al. (1994) proved that pistillate flower abscission was caused by the presence of excess pollen on the stigma. They discarded the hypothesis that the high number of pollen tubes growing through the stigma and the style to

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the ovary may influence the fertilization rate. Dead pollen induced the same amount of PFA as live pollen. Polito et al., (1998b) confirmed the previous findings, detecting a positive correlation between PFA and pollen load. High PFA was always associated with high numbers of pollen grains on stigmas. They combined data from walnut orchards at different sites in California and deduced that 50 % PFA occurred in ‗Serr‘ when an average of 85 pollen grains per flower were present. Polito et al., (2006) evaluated the putative involvement of dichogamy in pollen load. Analysis of pollination dynamics in a California ‗Chandler‘ walnut orchard, using microsatellite markers, permitted them to discriminate the effective sources of pollen during the dichogamous bloom cycle of the trees. The most likely source of excess pollen necessary for PFA induction was the self-pollen shed from catkins at the beginning of female flower receptivity. Therefore the extent of bloom overlap (self-pollination) may also have a role in the evolution of walnut dichogamy and PFA may have been a mechanism to improve progeny fitness. As reported by McGranahan et al., (1994):

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―Trees with overlapping male and female blooms would be at a reproductive disadvantage and thus dichogamy would be favored. In forest tree competition, this phenomenon would tend to discourage walnut trees crowded by other walnut from producing a heavy nut set, instead they could put their energy into vegetative growth ‖.

They suggested that comparing ―bloom overlap‖ and PFA levels in some cultivars might prove meaningful. In the last decade, data supporting the theory that excess pollination causes PFA were collected in central Chile where ‗Serr‘ pollination was insufficient due to poor overlap of male and female flowers. The consequent reduction in self-pollination led to a correspondingly lower percentage of PFA (Gratacòs et al., 2006). On the other hand no significant correlation was detected between the incidence of pistillate flower abscission and bloom overlap for 19 walnut cultivars and selections planted in Spain (Rovita et al., 2001). As indicated by Kruger (2000), a reduction of pollen density in the orchard and minimization of the losses caused by pollen-induced pistillate flower abscission may be best achieved by removing pollinizer trees from the site and/or mechanical shaking of the trees with the objective of removing some of the catkins. This suggestion requires careful consideration of how many pollinizers are adequate and which orchard configuration is suitable to provide sufficient but not excessive pollen loads. Preliminary experiments carried out by Polito et al., (1998b) demonstrated that removing some pollinizers from a ‗Serr‘ orchard improved walnut yield from 20 to 86 percent. An alternative approach implies the use of tree shakers at the beginning of the male bloom when the first catkins fall from the trees. At that time, most of the catkins are half size or longer, and easy to remove without damaging the trees by injuring shoot tips. Considering that the density of walnut pollen is constant for 160m around a pollenizer, even in the absence of wind (Impiumi & Ramina, 1967), Polito et al., (2006) suggested that entire rows of trees be shaken if they are within 47m of the cultivar affected by PFA. Lemus (2005) and Gratacòs et al., (2006) have successfully applied mechanical shaking treatments in Chilean ‗Serr‘ orchards. By shaking walnut trees when 15% and 50% of female flowers were receptive, they produced an increase in nut yield of 25-30%. Both of these techniques are time-consuming and require information about phenology and the history of the orchard. These methods can fall short for practical reasons as well. Many ‗Serr‘ growers do not own shakers and find it difficult to coordinate the required activities in

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the necessary time frame. Other growers have obtained mixed results from these methods because their proximity to other orchards increases their pollen load (Beede et al., 2008). Recent research has focused on the physiology and efficacy of ethylene antagonists as management tools for controlling PFA in walnut orchards, especially in ‗Serr‘ orchards. It is well known that the final stages of fruit development are controlled by hormones such as ABA and ethylene. In particular ethylene is a natural hormone associated with organ senescence and dehiscence of flowers and fruit. Although ethylene has been extracted and identified in walnut fruit, its precise role is unclear. Polito et al. (2005), has postulated that the overloading of pollen on stigmas may increase ethylene biosynthesis, inducing pistillate flower abscission. As proved by Johnson (2008), a peak in ethylene production was detected approximately 12-30 hours after pollination in excised pistillate flowers, with pollinated flowers producing more ethylene than non-pollinated ones. It‘s also interesting to note that ethylene production was inducted by both live and dead pollen. An increasing number of researchers have focused their attention on the potential for reducing the effect of pollinationinduced ethylene by applying two inhibitors, aminoethoxyvinylglycine hydrochloride (AVG) and 1-methylcycloproane (1-MCP). The modes of action for these two molecules are distinct. AVG, as Retain® (Valent Bioscience), inhibits ethylene synthesis, while 1-MCP, as an isopropanol-based adjuvant or as a gas, is a competitive inhibitor of ethylene action. Johnson (2008) observed that AVG and 1-MCP produced a significant decrease and increase, respectively, in ethylene biosynthesis by pollinated flowers. In the latter case, the observed increase in ethylene production may have been due to a feedback mechanisms triggered when 1-MCP blocked the ethylene receptors. In recent studies, the effect of AVG and 1-MCP application was tested with mixed results. Early application of AVG (125ppm) consistently reduced PFA in ‗Serr‘ orchards located in different Chilean walnut production areas (Lemus et al., 2007) and in San Joaquin County, California (Beede et al., 2008; Johnson, 2008). In particular, AVG-treated trees showed a 57 to 70% yield increase over the untreated controls. Flowers treated in the pre-receptive and early stages of stigma development performed better than flowers at peak receptivity. The AVG residual must be sufficient to inhibit ethylene production caused by excessive pollen load during the 5 to7 day receptivity period. Surprisingly, a field experiment in a ‗Chandler‘ orchard showed no reduction of PFA using AVG, but the effectiveness of 1-MCP (1-10ppm) against pistillate flower abscission was verified (Johnson, 2008). Although these studies are not conclusive, the role of ethylene in the regulation of fertilization and fruit development deserves thorough investigation. The use of AVG and 1MCP could represent powerful tools to overcome PFA in the orchard management but also in breeding programs in order to carry out controlled crosses. Rovita and Aletà (1997) reported that an artificial load of pollen applied to female flowers raised significantly the percentage of PFA, compared to open-pollinated reference flowers. Gonzàlez et al., (2008) observed that only very low concentration of ‗Serr‘ pollen (maximum 5 grains per mm-2 of applied surface) could prevent PFA in controlled crosses. The theory that hand–pollinations using pollen from a single source may influence the rate of pistillate flower abscission and fertilization in walnut has not been evaluated until recently. In all experiments previous described, a single pollen source was used, either self pollen or pollen from a single donor. McGranahan et al., (1994) also noted that PFA was first discovered in commercial walnut orchards where low pollen diversity is expected. In addition, investigation of self pollinated flowers proved that some Georgian walnut varieties

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were self-sterile. Kvaliashvili et al.,(2006) proposed that self pollen promoted pistillate flower abscission. There is a growing but conflicting body of evidence that high pollen diversity can enhance plant fecundity, although the mechanisms underlying such results are still poorly understood (Kron and Husband, 2006). For many plants, the number of pollen genotypes deposited on a stigma is positively correlated with reproductive success. In controlled crosses, increasing the diversity of the pollen source increases the probability that a female flower will receive pollen from a genetically compatible donor, it enhances the number of ovules fertilized per tree (pre-zygotic factors) and/or reduces embryo abortion (post-zygotic factors).

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INVESTIGATION OF GENETIC HYBRIDIZATION IN WALNUT The importance of intra- and inter-specific hybridization for the genetic improvement of forest trees has been evident for at least 50 years (Schreiner 1960). Nevertheless, tree improvement often has been narrowly focused on selection and breeding within a single native species. As suggested by Schreiner (1963), inter-specific hybridization also provides the maximum genetic diversity needed for greatest genetic improvement. Sometimes interspecific hybrids may be difficult to obtain, however, even with the use of controlled pollination. This is the case for hybridization between Juglans nigra L. and Juglans regia L. that produces Juglans × intermedia Carr. As reported in the previous sections, the hybridization between black and Persian walnut species is rare under natural conditions and difficult using controlled pollination because of phenological and genetic incompatibilities. It requires the overlapping of the bloom time for the two parental trees, an appropriate temperature for pollen germination and penetration though the stigma and the style to the J. nigra ovary (Luza & Polito, 1987), and genetic compatibility pre- and post-pollination (Sartorius, 1990). In the last thirty years, seed orchards for hybrid production have been designed; generally one plus tree as a female parent and several plus trees as fathers were deployed to ensure enough pollen pressure. The oldest and best known European J. × intermedia (NG23 × RA) was obtained in France by the open-pollination of the mother J. nigra NG23 with four J. regia plus trees RA984, RA996, RA331, RA295 as male parents (Becquey 1990). However, selection by phenological observations and clonal (graft) propagation of hybridogenic parent trees required more than ten years (Jay-Allemand et al. 1990). In these studies (Pollegioni et al, 2009a, b), we reported a new method based on (neutral) microsatellite markers which permitted the identification of new interspecific hybrids and, at the same time provided a rough idea of which walnut genotypes might be useful for establishing new seed orchards for inter-specific F1 hybrid production. The use of genotypes with demonstrated compatibility may increase the efficiency of F1 production. This method should also provide a powerful tool to evaluate the barriers to hybridization between Juglans species and to detect the factors that reduce hybrid fertility

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RETROSPECTIVE IDENTIFICATION OF HYBRIDOGENIC WALNUT TREES The identification and selection of genotypes with a spontaneous ability to cross (hybridogenic parent trees) is a simple and efficient method for obtaining hybrid progeny. Pollegioni et al. (2009b) reported a new method for retrospective identification of hybridogenic walnut trees based on microsatellite (SSR) fingerprinting and parentage analysis in order to establish new seed orchards for hybrid production. Woeste et al. (2002) developed a panel of thirty nuclear microsatellites in J. nigra L. as markers for a wide range of genetic investigations. A subset of these markers has been successfully used for clonal identification (Robichaud et al., 2006) and a broad-scale study of the genetic structure of J. nigra populations in the Central Hardwood Region of the United States (Victory et al., 2006). At the same time, a subset of microsatellites were also selected and screened in J. regia L. as a starting point for the genetic characterization of walnut cultivars (Dangl et al., 2005) and the variety ‗Sorrento‗ (Foroni et al.,2005). Microsatellites, known as simple sequence repeats (SSRs), are short (1-6 bp long), tandemly repeated DNA sequences widely dispersed throughout eukaryotic genomes. These markers require the design of primers for the conserved flanking regions of the microsatellite and the PCR amplification of the repeat region. The single-locus markers are characterized by hypervariability, abundance, high reproducibility, Mendelian inheritance, and co-dominant expression. These positive features make them suitable tools for parentage analysis (Streiff et al., 1999) and molecular fingerprinting of hybrids (Nandakumar et al., 2004). Nevertheless, a detailed study of the inter-species transportability of the microsatellite markers in walnut was not yet available. Peakall et al. (1998) demonstrated that the successful cross-species amplification of SSRs does not prove the maintenance of the repeat motif in the non-source species. Studies employing cross-species amplification should therefore be accompanied by knowledge of the underlying DNA sequence. Over the last six years under the framework of the national Project RI.SEL.ITALIA (financially supported by the Italian Ministry of Agricultural Policy, Sottoprogetto 1.1 ―Biodiversità e Produzione di Materiale Forestale di Propagazione‖, coordinator Dr. Fulvio Ducci CRA-Arezzo), the C.N.R. Institute of Agro-environmental and Forest Biology (Porano) has been intensively evaluating walnut germplasm in Italy. As a result of these efforts, a promising mixed population, including J. nigra, J. regia and some J. × intermedia hybrids, was discovered in Northern Italy, Veneto region, Villa Mezzalira Park, Bressanvido (Pollegioni et al. 2009a). Ten microsatellites tested in the mixed walnut population collected in Villa Mezzalira‘s Park amplified in both species, producing fragments of variable size; eight (7.14 %) were common, 68 (60.7 %) amplified in J. nigra and 36 (32.1 %) in J. regia only (private alleles). Indices of genetic diversity revealed a high level of variability. DNA fingerprinting analysis divided the total sample set (138 plants) into three main groups: J. nigra (82), J. regia (49) and diploid (2n = 32) J. x intermedia hybrids (7). Forty-nine J. regia, 8 J. nigra, 3 diploid hybrids, are adult trees growing in the Park (Table1); 15 J. nigra adults plants (J. nigra NC) were located outside the park; 59 J. nigra and 4 diploid hybrids were sixyear old plants grown at the Veneto regional nursery (Montecchio Precalcino, Vicenza) from seeds collected in the Park.

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Table 1. Characteristics of 139 plants sampled in Villa Mezzalira Park, Bressanvido (Northern Italy 45° 39′ 0′ ′ N, 11° 38′ 0′ ′ E) genotyped using SSR markers (Pollegioni et al., 2009a). Species

Group

Adult Trees (N)

Genotype label

Six year old Treesb (N)

Genotype Label

J. nigra N

8

N3, N4, N5, N17, N18, N22, N23, N24

59

N25-N83

67

J. nigra NC a

15

NC1-NC15

-

-

15

J. regia

49

R6-R16, A.E., B2-B20, V1-V17

-

-

49

Diploid hybrid

3

H1, H2, H19

4

IMP3, IMP4, IMP9, IMP18

7

Triploid hybrid

1

N21

-

-

1

Total

J. nigra L.

J. regia L.

J. × intermedia Carr. Total a

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b

76

63

139

Fifteen black walnut adults plants located outside the park were labelled J. nigra-NC. Six-year old plants growing at the Veneto regional nursery (Montecchio Precalcino, Vicenza), Italy, from seeds collected inside the park.

By genotyping the adult trees in the population with microsatellites, a triploid hybrid plant with two genome parts from J. nigra and one part from J. regia was identified (N21 tree). Cytological analysis proved that the N21 tree is triploid and that it contains 48 somatic chromosomes (Figure 4). The analysis to identify the maternal parents of the seedling trees from the population in the park (exclusion method) indicated that J. nigra N17 was the ―putative‖ hybridogenic mother plant of the seven diploid hybrids. Analysis of the sequence of the amplified fragments confirmed the cross-species amplification of the SSRs, but inter-specific differences in allele sizes were due not only to simple changes in the number of repeats but also to mutations in the flanking regions: insertion and deletion events in the flanking regions contributed to the variation in allelic size among and within Juglans species. The same battery of 10 SSR primer pairs was used to perform the DNA fingerprinting and parentage tests of eight half-sib families collected in the Villa Mezzalira‘s Park with the specific objectives of 1) detecting the presence of J. × intermedia in these progenies, 2) identifying J. nigra mother trees that spontaneously crossed with J. regia (hybridogenic mothers), and 3) verifying the differential reproductive success (DRS) of J. regia male parents (hybridogenic fathers) for production of hybrid offspring genotypes. (Pollegioni et al., 2009b). Seeds were collected from seven adult J. nigra trees and the triploid hybrid plant in Villa Mezzalira Park. The seeds were planted in a field at the CRA Institute for Silviculture (Arezzo), and eight open-pollinated progenies (461 total seedlings) were obtained: forty-one

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seedlings from plant N3; 29 from N24; 88 from N17; 24 from N18; 76 from N22; 71 from N23; 114 from N24, and 18 from the triploid N21 (Table 2).

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Figure 4. Somatic chromosome number (3n = 48) of developing (premeiotic mitosis) pollen mother cells of N21 hybrid genotype was evaluated microscopically after traditional aceto-carmine staining.

The high levels of polymorphism (129 alleles) detected positively influenced the exclusion and identity probabilities described in the study. The allelic richness and the observed heterozygosity measured for each locus in the tested samples provided high combined power of exclusion and low probability of identity. The study clearly demonstrated the power of SSR markers for DNA fingerprinting and parentage analysis. Principal Coordinate Analysis, which was performed on the Simple Match‘s similarity coefficient was computed using 129 alleles. It revealed distinct J. nigra and J. regia clusters and the presence of several intermediate individuals. Three main groups were detected (Figure 5); two that included 49 J. regia and 82 J. nigra trees were clearly separated by the first principle coordinate. The second principal coordinate divided the J. nigra trees in two subgroups. J. nigra-NC plants, located outside the site area, were found to be genetically distinct from the other eastern black walnut trees planted inside the Park. Seven diploid hybrids (H1, H2, H19, IMP3, IMP4, IMP9 IMP18) were incorporated in the third main group, located in an intermediate position between black and common walnut. As expected, the triploid hybrid plant (N21), with two genome parts of J. nigra and one part of J. regia, was placed between black walnut and the hybrid groups. Cluster analysis showed that the third group was composed of genotypes genetically distinct from individuals of the two parental species, but this placement does not prove the trees in this group are all interspecific hybrids. The identification of diploid hybrids was definitively performed by assigning the459 offspring genotypes to four putative classes: two black walnut (J. nigra N, J. nigra NC), one J .regia, and one J. × intermedia. The assignment analysis by the Paetkau et al. (1995) frequency method and Rannala & Mountain (1997) partial Bayesian method, combined with the exclusion-simulation significance test of Cournet et al. (1999), revealed the presence of 198 diploid J. x intermedia hybrids among the total of the progeny seedlings (42.9%). Maternity checks were performed on all individuals. A few errors of sampling (0.06 %) were found. These probably resulted from accidental mixing of seeds during collection of

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progenies (Table 2). Four distinct hybridogenic J. nigra mother trees were identified, including N17 as expected, but also N23, N24 and the triploid hybrid plant N21. The three hybridogenic black walnut plants had different reproductive success rates.

Figure 5. Pollegioni et al., 2009b. Principal Coordinate Analysis of 600 Juglans individuals based on genotypic similarity as determined by simple match coefficients based on 10 SSR loci. J. regia (N = 49), J. nigra N (N = 67), J. nigra NC (N = 15), N21 triploid hybrid, ◊ diploid hybrids (N = 7) and J. nigra offspring (N = 461).

The identification of three distinct hybridogenic J. nigra mothers was an important practical result. Indeed, genetic improvement, especially of long living plants, requires the availability of selected ―plus‖ genotypes able to produce a consistent quantity of hybrid progeny. The authors‘ approach also permitted the quantification of the differential reproductive success of each mother. Thus, even though these results should be confirmed by observations over additional years, it should be possible to focus breeding research on two plants with a relatively high rate of hybrid production: N24 (87%) and N17 (70%). The authors also showed that the triploid hybrid plant N21 produced fertile female flowers, although the number of progeny was limited (18 total seedlings: 15 hybrid and 3 J. nigra genotypes). As described by Funk (1970) some J. × intermedia trees flower profusely but never bear much seed. In addition two hybrid plants out of 15 displayed an unusual and fatal karyotype. The most likely explanation for the unusual microsatellite profiles in some of the progeny is irregular meiosis in the original triploid hybrid parent and subsequent elimination/ addition of chromosomes.

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Table 2. Maternity analysis and identification of hybridogenic mother trees (Pollegioni et al., 2009b).

Maternal tree

Number of putative offspring

Non-maternity (seed mixture)

Maternity assignment b

Total number of offspring

Hybrid progeny J. ×intermedia Carr c (N)

N3

41

0

-

41

0

N4

29

0

-

29

0

N17

88

0

-

97

68 (70%)

N18

24

9

N17 (9)

15

0

N21

18

0

-

18

15 (83.3%) a

N22

76

3

N23 (3)

73

0

N23

71

0

-

74

17 (22.9%)

N24

114

0

-

114

100 (87.7%)

a

Two hybrids offspring, N21- 14 and N21-15, triploid for one locus, were included. b The maternity was re-assigned combining the exclusion method based on Mendelian segregation rules with maximum-likelihood approach (Marshall et al. 1998). c Based on genotyping eight half-sib progenies using ten microsatellite loci.

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Paternity of 198 diploid hybrids detected in four open-pollinated families was inferred by using a likelihood-based approach (Marshall et al. 1998) based on nine microsatellite loci. Differential male reproductive success was observed among pollen donors within the research site (Figure 6).

Figure 6. Pollegioni et al., 2009b. Number of hybrid offspring produced by each J. regia male that pollinated J. nigra females, N17, N21, N23, N24, and ---- total. Assignment was based on greatest likelihood. The successful pollinations corresponded to the number of times a pollen donor (J. regia) pollinated a mother tree (J. nigra).

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In the production of hybrid progeny male reproductive success was unevenly distributed both in amount and in space. In particular 49 (47.5 %) of the total diploid hybrids detected in four half-sib families were sired by only three J. regia genotypes (B6, V15 and B7). Although phenological data was not recorded for the individuals at the research site and the authors‘ experimental design could not differentiate among all possible reasons for unequal paternal success, the results do guide speculation. Juglans nigra generally blooms later than J. regia, so the amount and timing of pollen shed, distance of pollen donor from seed trees, plant size, and weather conditions, may have had a profound effect on the distribution of male reproductive success.The authors did not find a significant correlation between reproductive success of Persian walnut trees and the distance from black walnut mother plants. Spatial factors may have influenced pollination in their study, but they were probably not a major determinant of male success. The timing of pollen release and the presence of some mechanisms of genetic incompatibility could be plausible explanations for the observed fertilization pattern. The paternal plants may have been the only trees releasing pollen when the maternal trees had receptive stigma (synchronous flowering). On the other hand, as reported previously, pre-zygotic factors, such as pollen germination and tube growth rate, or post-zygotic factors, such as genetic complementation, could have affected male reproductive rate and may have been particularly relevant in this case where inter-specific crosses were made (Wheeler et al. 2006). In conclusion, although fluctuations in pollen production can occur among years, and the experiment was carried out on a relatively small sample of parent trees, parentage analysis of half-sib families based on microsatellite markers permitted the identification of new interspecific hybrids and, at the same time provided a rough idea of which walnut genotypes might be useful for establishing new seed orchards for inter-specific F1 hybrid production. The use of genotypes with demonstrated compatibility may increase the efficiency of F1 production. This method should also provide a powerful tool to evaluate the barriers to hybridization between Juglans species and to detect the factors that reduce hybrid fertility. Finally the retrospective selection of hybridogenic trees is a valid approach for the identification of new parental combinations when no phenological and morphological data of the trees are available.

ACKNOWLEDGMENTS The authors thank Dr. Agnes Major, Susanna Bartoli, Giovanni De Simoni, Claudia Mattioni, Marcello Cherubini and Daniela Taurchini for their support in statistical and laboratory analysis. A warm thank to Prof. Chuck Leslie (Walnut Breeding Department,University of California, Davis) for the critical review of the manuscript. The use of trade names is for the information and convenience of the reader and does not imply official endorsement or approval by the United States Department of Agriculture or the Forest Service of any product to the exclusion of others that may be suitable.

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Sütyemez, M. (2007). Determination of pollen production and quality of some local and foreign walnut genotypes in Turkey. Turkish Journal of Agriculture and Forestry, 31, 109-114. Tadeo, F.R., Talon, M., Germain, E., Dosba, F. (1994). Embryo sac development and endogenous gibberellins in pollinated and unpollinated ovaries of walnut (Juglans regia). Physiologia Plantarum, 91, 37-44Tanzarella, O.A. & Simeone, M. (1996). Final Report Project UE ―W –Brains‖ AIR3 – CT92-01429. Taylor, L.P. & Hepler, P.K. (1997). Pollen germination and tube growth. Annual Review of Plant Physiology and Plant Molecular Biology, 48, 461-491. Verardo, V., Bendini, A., Cerretani, L., Malaguti, D., Cozzolino, E., Caboni, M.F. (2009). Capillary gas chromatography analysis of lipid composition and evaluation of phenolic compounds by micellar electrokinetic chromatography in Italian walnut (Juglnas regia L.): irrigation and fertilization influence. Journal of Food Quality 32, 262-281. Victory, E.,. Glaubitz, J.C, Rhodes, Jr O.E., Woeste, K., (2006): Genetic homogeneity in Juglans nigra (Juglandaceae) at nuclear microsatellites. American Journal of Botany, 93, 118- 126. Wheeler, N., Payne, P., Hipkins, V., Saich, R., Kenny, S., Tuskan, G. (2006). Polymix breeding with paternity analysis in Populus: a test for differentiation reproductive success (DRS) among pollen donors Tree Genetics & Genomes, 2, 56-60. Williams, J.W., Shuman, B.N., Webb III, T., Bartlein, P.J., Leduc, P.L. (2004). LateQuaternary vegetation dynamics in North America: scaling from taxa to biomes. Ecological Monographs, 74, 309-334Winter, M.B., Wolff, B., Gottschling, H., Cherubini P. (2009). The impact of climate on radial growth and nut production of Persian walnut (Juglans regia L.) in Southern Kyrgyzstan. European Journal of Forest Research, 128, 531-542. Woeste, K. 2004. An online database of Juglans names and origins. HortScience 39:1771. Woeste, K.E., & Beineke W.F., (2001). An efficient method for evaluating black walnut for resistance to walnut anthracnose in field plots and the identification of resistant genotypes. Plant Breeding 120, 454-456. Woeste, K., Burns, R., Rhodes, O., Michler C. (2002). Thirty polymorphic nuclear microsatellite loci from black walnut. Journal of Heredity, 93, 58-60. Wu, H.M., Wang, H., Cheung, A.Y. (1995). A pollen tube growth stimulatory glycoprotein is deglycosylated by pollen tubes and displys a glycosylation gradient in the flower. Cell, 82, 395-403.

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Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Pollen: Structure, Types and Effects : Structure, Types and Effects, edited by Benjamin J. Kaiser, Nova Science Publishers, Incorporated, 2010.

In: Pollen: Structure, Types and Effects Editor: Benjamin J. Kaiser, pp. 101-126

ISBN: 978-1-61668-669-7 ©2010 Nova Science Publishers, Inc.

Chapter 3

VARIABLE SIZED POLLEN GRAINS DUE TO IMPAIRED MALE MEIOSIS IN THE COLD DESERT PLANTS OF NORTH WEST HIMALAYAS (INDIA) Vijay Kumar Singhal* and Puneet Kumar Department of Botany, Punjabi University, Patiala -147002, Punjab (India)

ABSTRACT

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The cold deserts in Western Himalayas spread over approx. 74,809 Km2, cover Leh and Kargil districts of Ladakh in Jammu & Kashmir, Uttarkashi in Uttarakhand and Lahaul- Spiti along with some parts of Chamba and Kinnaur districts of Himachal Pradesh. These cold deserts consist of rugged mountains, snow clad peaks, bare rocks, steep sandy slopes with rock gravels, low capacity of soil to retain moisture, and have oppressive and inhospitable conditions which include freezing temperature, dry arid weather, high velocity winds and low precipitation. As a consequence of such harsh climatic conditions prevailing in these regions, plants tend to become prostrate, thick, bushy, hardy, mat forming and spiny with long roots and small succulent or woolly leaves. Majority of these plants are perennials and survive through underground parts. Further, the plants of the area are exposed to high incidence of UV rays and are under considerable pressure of human intervention and natural disasters which include agriculture, heavy grazing, snow avalanches, windstorms, landslides and increasing entry of tourists and transport vehicles. As a consequence of such stresses, the plants of the area are expected to show considerable amount of irregularities during male meiosis and in pollen grains. Keeping above assumptions in mind the work on cytomorphological diversity in the plants of Lahaul-Spiti, a part of the Western Himalayan cold deserts, was undertaken. During the course of study spanning 5 years (2005-2009) we have come across several species which depict various irregularities during male meiosis resulting into the formation of variable sized apparently fertile/ stained pollen grains and considerable amount of pollen sterility. These heterogeneous sized pollen grains and pollen malformation are reported mainly in species depicting the phenomenon of cytomixis involving chromatin transfer among proximate pollen mother cells (PMCs) viz., (Anemone rivularis, 2n=16; Aquilegia fragrans, 2n=16; Astragalus bicuspis, 2n=16; *

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Vijay Kumar Singhal and Puneet Kumar A. frigidus, 2n=16; A. himalayanus, 2n=16; A. rhizanthus, 2n=16; Caltha palustris, 2n=32; Clematis grata, 2n=16; C. orientalis var. acutifolia, 2n=32; Geranium pratense, 2n=56; Hedysarum astragaloides, 2n=14; Meconopsis aculeata, 2n=56; Medicago falcata, 2n=16; M. sativa, 2n=32; Melilotus officinalis; 2n=16, Parnassia laxmanii, 2n=18; Pleurospermum candollii, 2n=22; P. govanianum, 2n=18; Potentilla atrisanguinea var. atrisanguinea, 2n=84; P. atrisanguinea var. argyrophylla, 2n=84; P. cuneifolia, 2n=28; P. fruticosa var. rigida, 2n=14; Ranunculus hirtellus, 2n=32; R. laetus, 2n=28; Rosularia alpestris, 2n=28; Silene vulgaris, 2n=24; Thalictrum foetidum, 2n=42; Trifolium pratense, 2n=14; T. repens, 2n=16 and Trigonella emodii, 2n=16). The resultant PMCs after partial or complete chromatin transfer in these species depict hyper-, hypoploid and anucleated nature which give rise to apparently fertile diploid (2n), haploid (n), and aneuploid, and sterile or micro pollen grains. Such variable sized fertile and sterile pollen grains are also resulted in species depicting irregular synapsis (Dianthus angulatus, 2n=30; Ranunculus laetus, 2n=28; Rosularia alpestris, 2n=28), spindle irregularities (Potentilla atrisanguinea var. argyrophylla, 2n=84; Ranunculus hirtellus, 2n=32; R. laetus, 2n=28; Rosularia alpestris, 2n=28), triploids (Chrysanthemum pyrethroides, 2n=27; Heracleum brunonis, 2n=33), pentaploids (Agrimonia eupatoria, 2n=70) and polyploids with irregular meiotic course (Chrysanthemum pyrethroides, 4x; Clematis orientalis var. acutifolia, 4x; Potentilla atrisanguinea var. atrisanguinea 12x; P. atrisanguinea var. argyrophylla, 12x; Ranunculus hirtellus, 4x; R. laetus, 4x). Double sized pollen grains referred as ‗2n‘ pollen grains resulted as a result of either direct fusion of two pollen grains (Caltha palustris, Potentilla atrisanguinea var. argyrophylla) or are the end products of fused PMCs (syncytes) as is the case in Meconopsis aculeata and Clematis orientalis var. acutifolia are also recorded. Though cytological status of such variable sized fertile pollen grains could not be ascertained presently, they can play an important role in the origin of intraspecific polyploids, aneuploids and taxa with B-chromosomes.

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Keywords: Aberrant cytokinesis, cytomixis, heterogeneous pollen size, imbalanced polyploids, synaptic mutants, Lahaul-Spiti, irregular male meiosis, pollen sterility, multiple spindles, syncyte.

INTRODUCTION The cold deserts in Western Himalayas are spread over approx. 74,809 Km2, cover Lahaul-Spiti, parts of Kinnaur and Chamba districts of Himachal Pradesh along with Leh Ladakh region of Jammu and Kashmir and Uttarkashi area of Uttarakhand together constitute the cold deserts of India. Lahaul-Spiti, the presently studied area is the largest district of Himachal Pradesh with an area of ca.13, 835 km2, situated between 310 44 57 and 320 59 57 N latitude and between 760 29 46 and 780 41 34 E longitude. It has Chamba on its west, and Kangra, Kullu and Kinnaur districts on its south. In the north is Ladakh (Jammu and Kashmir), while on its east is Tibet (Fig. 1). These cold deserts consist of rugged mountains, snow clad peaks, bare rocks, steep sandy slopes with rock gravels and soil with low capacity to retain moisture. On the basis of geographical conditions, Lahaul - Spiti district is divided into two main regions, i.e., Lahaul Valley and Spiti Valley which are different in many aspects. Kunzum Pass (4,550 m) keeps these two sub-divisions isolated from each other for more than six months in a year due to heavy snowfall.

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Climatic conditions prevailing in the Lahaul and Spiti are oppressive and inhospitable which include freezing temperature (>-300 C), dry arid weather, high velocity winds and low precipitation. The flora of the area exhibits a number of ecological, morphological, physiological and reproductive adaptations, which help them to withstand the impact of harsh climatic conditions prevailing in the region. Due to such specific ecological conditions plants tend to become prostrate, thick, hairy, bushy, hardy, sturdy, mat and cushion forming and spiny with long roots and small succulent or woolly leaves. Another remarkable feature of the plants is their adaptability to survive through different means of vegetative propagation. Majority of these plants are observed to be perennial in nature and perennate by means of rootstocks, runners, bulbs, rhizomes, tubers, etc. Inspite of extreme xeric and inhospitable conditions, the area is quite rich in plant wealth, and is represented by a variety of plants which include majority of angiosperms of dry alpine and temperate zones, and few gymnosperms and pteridophytes. Further, the plants of the area are exposed to high incidence of UV rays and are under considerable pressure of human intervention and natural disasters which include agriculture, heavy grazing, snow avalanches, windstorms, landslides and increasing entry of tourists and transport vehicles.

Figure 1. Map depicting collection sites in the cold deserts of Lahaul-Spiti in the North western Himalayas.

The cold deserts of Lahaul –Spiti are quite rich floristically with as many as 985 species belonging to 353 genera and 79 families of flowering plants (Aswal & Mehrotra, 1994). Also due to geographical isolation and specific ecological conditions the flora of the region shows plenty of endemics. A cursory look at the work carried out on the plants of this region reveals that most of the earlier attempts pertain to studies on floristic diversity and gathering of

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ethnobotanical information. Barring a few studies carried out by the researchers at IHBT, Palampur (Himachal Pradesh) on some specific medicinal herbs viz., Aconitum heterophyllum, Acorus calamus, Curcuma aromatica, Dracocephalum heterophyllum, Hedychium spicatum, Podophyllum hexandrum, Salvia sclera and Valeriana jatamansi, no systematic attempt has been made so far to explore the existence of cytomorphological diversity in the plant resources of Lahaul-Spiti. Besides, no satisfactory information is available on the ecological preferences and distribution pattern, flowering and fruiting period, reproductive potentiality and regeneration capacity of the plants of the region. Even the basic information on the species about the cytological status, meiotic behaviour particularly on male side and pollen/seed viability is lacking. Keeping in view the above lacunae and importance of male meiotic studies and consequent pollen grain formation, an attempt has been made by the authors to study the cytomorphological diversity including detailed meiotic course and microsporogenesis in the plants of Lahaul-Spiti. Extensive and intensive surveys were conducted in the cold desert regions of Lahaul-Spiti (Himachal Pradesh) in the North West Himalayas consecutively for five years (2005-2009). Various localities in the Lahaul Valley and Spiti Valley falling in the altitudinal range of 2400 m to 5020 m (Fig.1) were visited during the months of May-September when the area is open for traffic and majority of the plants are in full bloom. The materials for male meiotic studies were collected from the wild plants growing in the cold desert regions of Lahaul-Spiti. Voucher specimens of the cytologically worked out plants were deposited in the Herbarium, Department of Botany, Punjabi University, Patiala (PUN1). Floral buds of suitable sizes were collected from healthy plants and fixed (temperature in the field at the time of fixation of floral buds was 10-30˚ C) in freshly prepared Carnoy‘s fixative (6 Alcohol: 3 Chloroform: 1 Acetic acid, v: v: v). These materials were subsequently transferred to 70% alcohol and stored in refrigerator at 4˚C until use. Pollen mother cells were prepared by squashing the young and developing anthers in 1% acetocarmine. A number of freshly prepared slides were carefully examined to determine the chromosome counts at diakinesis, metaphase-I, anaphase-I, metaphase-II and anaphase-II, and detailed meiotic course including microsporogenesis. On an average, 50 meiocytes were analyzed at the earlier stages of prophase-I, and metaphase-I for recording the chromosomal associations. About 100 PMCs were observed at A-I/T-I and A-II/T-II for analyzing the segregation of chromosomes. For sporad analysis, 100-200 PMCs were observed to study the products of meiosis. Pollen fertility was estimated through stainability test for which mature anthers from opened flowers were squashed in glyceroacetocarmine (1:1) mixture and 1% aniline blue dye. In each case 500-2000 pollen grains were analyzed for estimating pollen fertility and pollen size. Well filled pollen grains with stained nuclei were taken as apparently fertile while shrivelled and with faintly and unstained cytoplasm were counted as sterile. Pollen grain size was measured using Ocular micrometer. Photomicrographs of pollen mother cells, sporads and pollen grains were made from the freshly prepared and temporary mounts using Leica Qwin Digital microscope and Nikon 80i Eclipse microscope. Mode of natural regeneration/propagation (seeds, rootstocks, runners, bulbs, rhizomes or tubers) in the species was also noticed from the plants growing wild in the natural habitat.

1

Herbarium code of Botany Department, Punjabi University, Patiala as per ―Index Herbariorum‖ by Holmgren & Holmgren (1998).

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Meiosis is one of the most sensitive stages in the life cycle of seed plants (Namuco & O‘Toole, 1986; Saini, 1997; Porch & Jahn, 2001; Erickson & Markhart, 2002; Romanova & Tret‘yakova, 2005; Fuzinatto et al., 2008) and any disturbance during the meiotic course results into reduced viability of gametes. Meiosis is a highly specified and genetically programmed process which comprises pairing of homologous chromosomes, crossing over, and the reduction in chromosome number. And like many other biological processes, meiosis is also controlled by a large array of genes (Ramanna, 1974; Mok & Peloquin, 1975; Baker et al., 1976; Mok et al., 1976; Koduru & Rao, 1981; Kaul & Murthy, 1985; Falistocco et al., 1994; Pagliarini, 2000; Villeneuve & Hillers, 2001). Mutation in any of these genes which govern micro- or megasporogenesis from pre-meiotic to post meiotic events may results into serious irregularities, changed rhythm of cell cycle and ultimately leading in the genetically aberrant end products having adverse impact on fertility and overall reproductive efficiency of the species. During the course of this study cytological investigations have been made in 305 accessions of 140 species of dicots from the cold deserts of Lahaul-Spiti. Out of these, 43 species (Agrimonia eupatoria L., 2n=5x=70; Anemone rivularis Buch.-Ham. ex DC. 2n=2x=16; Aquilegia fragrans Benth., 2n=2x=16; Astragalus bicuspis Fisch., 2n=2x=16; A. chlorostachys Lindl., 2n=2x=16; A. frigidus (L.) A.Gray, 2n=16; A. himalayanus Klotz., 2n=2x=16; A. rhizanthus Royle ex Benth., 2n=2x=16; Caltha palustris L., 2n=4x=32; Chrysanthemum pyrethroides (Kar. et Kir.) Fedtsch., 2n=3x=27, 2n=4x=36; Clematis grata Wall., 2n=2x=16; C. orientalis L. var. acutifolia Hook. f. et Thoms., 2n=4x=32; Delphinium brunonianum Royle, 2n=16; D. denudatum Wall. ex Hook. f. et Thoms., 2n=16; D.vestitum Wall. ex Royle, 2n=16; Dianthus angulatus Royle ex Benth., 2n=2x=30; Geranium pratense L., 2n=4x=56; Hedysarum astragaloides Benth., 2n=14; H. microcalyx Baker, 2n=14; Heracleum brunonis (DC.) Benth., 2n=33; H. thomsonii C. B. Clarke, 2n=22; Ligusticopsis wallichianum (DC.) Pimenov Kljuykov, 2n=22; Meconopsis aculeata Royle, 2n=4x=56; Medicago falcata L., 2n=16; M. sativa L., 2n=4x=32; Melilotus officinalis (L.) Pallas, 2n=2x=16; Parnassia laxmanii Pall.ex Schult., 2n=18; Pleurospermum candollii (DC.) Benth. ex C. B. Clarke, 2n=2x=22; P. govanianum (Wall. ex DC.) Benth. ex C. B. Clarke, 2n=18; Potentilla atrisanguinea Lodd. var. atrisanguinea, 2n=12x=84; P. atrisanguinea Lodd. var. argyrophylla (Wall. ex Lehm.) Griers., 2n=6x=42, 2n=12x=84; P. cuneifolia Bertol, 2n=4x=28; P. fruticosa L. var. rigida (Wall. ex Lehm.) Th. Wolf., 2n=2x=14; Ranunculus hirtellus Royle, 2n=4x=32; R. laetus Wall. ex Royle, 2n=4x=28; Rosularia alpestris (Kar. et Kir.) Boriss, 2n=2x=28; Saxifraga hirculus L., 2n=32; Sedum oreades (Decne.) R.Hamet, 2n=22; Silene vulgaris (Moench) Garcke, 2n=2x=24; Thalictrum foetidum L., 2n=6x=42; Trifolium pratense L., 2n=2x=14; T. repens L., 2n=4x=16 ; Trigonella emodii Benth., 2n=2x=16) depict the abnormal meiotic course comprising cytomixis, syncyte PMCs, asynapsis, structural heterozygosity, sticky and pycnotic chromatin, interbivalent connections, supernumerary nucleolei, out of plate bivalents, multiple spindles, non-synchronous separation of bivalents, laggards and chromatin bridges, aberrant cytokinesis, micronuclei/ included micronuclei, monads, dyads, triads, and polyads during microsporogenesis and consequently variable sized apparently fertile pollen grains and considerable amount of pollen sterility. A number of meiotically irregular phenomena observed in the presently investigated plants resulting into pollen malformation are discussed separately.

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I. CYTOMIXIS The phenomenon of cytomixis involving transfer of chromatin material between proximate meiocytes was discovered long ago in Crocus sativus by Kornicke (1901). The phenomenon is widely spread in the flowering plants and reported to occur in numerous plants (Singhal & Gill, 1985; Falistocco et al., 1995; Ghanima & Talaat, 2003; Ghaffari, 2006; Lattoo et al., 2006; Kumar & Singhal, 2008; Singhal & Kumar, 2008a, 2008b; Gupta et al., 2009; Singhal et al., 2007, 2008, 2009a, b; Kumar et al., 2008a, b, 2010). Cytomixis is better known to exist in genetically imbalanced plants like, hybrids, mutants, and aneuploids, and polyploids (de Nettancourt & Grant, 1964; Basavaiah & Murthy, 1987; Premachandran et al., 1988; Peng et al., 2003; Zhou, 2003; Sheidai & Attaei, 2005; Li et al., 2009). Predominantly, cytomixis is observed in meiotic cells (Romanov & Orlova, 1971, Bauchan, 1987, Dagne, 1994), however it has also been reported to occur in the somatic cells (Wang et al., 2004, Guzicka & Wozny, 2005). Although transfer of chromatin material has been reported in numerous species, there are conflicting opinions and explanations regarding the causes and significance of cytomixis. Possible causes suggested earlier include the effect of fixation, pathological changes, physiological control, chemicals and herbicides, pollution, temperature, stress factors and genetic control. Pressure differences and clumped chromatin bridges during premeiotic anaphase are the other explanations put forth by some authors. In the present case, cytomixis seems to be a natural phenomenon under direct genetic control as mentioned earlier (Singhal & Gill, 1985; Haroun, 1995; Bellucci et al., 2003; Ghanima & Talaat, 2003; Haroun et al., 2004; Lattoo et al., 2006; Singhal et al., 2007, 2008, 2009a,b, 2010; Kumar & Singhal, 2008; Singhal & Kumar, 2008a, b). Presently cytomixis involving the inter PMC (pollen mother cell) transfer of chromatin material has been reported to occur in 30 species (Anemone rivularis, 2n=16; Aquilegia fragrans, 2n=16; Astragalus bicuspis, 2n=16; A. frigidus, 2n=16; A. himalayanus, 2n=16; A. rhizanthus, 2n=16; Caltha palustris, 2n=32; Clematis grata, 2n=16; C. orientalis var. acutifolia, 2n=32; Geranium pratense, 2n=56; Hedysarum astragaloides, 2n=14; Meconopsis aculeata, 2n=56; Medicago falcata, 2n=16; M. sativa, 2n=32; Melilotus officinalis; 2n=16, Parnassia laxmanii, 2n=18; Pleurospermum candollii, 2n=22; P. govanianum, 2n=18; Potentilla atrisanguinea var. atrisanguinea, 2n=84; P. atrisanguinea var. argyrophylla, 2n=84; P. cuneifolia, 2n=28; P. fruticosa var. rigida, 2n=14; Ranunculus hirtellus, 2n=32; R. laetus, 2n=28; Rosularia alpestris, 2n=28; Silene vulgaris, 2n=24; Thalictrum foetidum, 2n=42; Trifolium pratense, 2n=14; T. repens, 2n=16 and Trigonella emodii, 2n=16). Transfer of chromatin in these species occurred from early prophase up to sporad stage involving two and more PMCs (Figs.2a-m.). In majority of the species the frequency of PMCs involved in cytomixis during the meiosis-I was more compared to meiosis-II. The transfer of chromatin material occurs through narrow (Figs.2a, d, f, g) and broad (Figs. 2b, h, i) cytoplasmic channels by the formation of chromatin bridges between the proximate PMCs. The number of chromatin bridges varied from one to many (Figs.2a, g, h, i). The transfer of chromatin material observed to be unidirectional (Figs. 2a, c, d, e, f, g, h, i) as well as bidirectional (Fig.2b) in the PMCs. In some cases along with chromatin material, nucleolus is also get transferred from donor PMC to that into recipient and as a result the recipient PMCs with two nucleoli are often seen (Fig. 2a). The chromatin transfer was either partial or complete.

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Figures (2a-o). PMCs showing cytomixis and pollen grains, a) In Geranium pratense, two PMCs at early prophase stage of meiosis showing chromatin transfer (arrowed) and recipient PMC with two nucleoli (arrow head). b) In Ranunculus laetus, PMCs at early prophase stage showing bidirectional chromatin material transfer (arrows). c-g) In Thalictrum foetidum, PMCs showing chromatin material transfer at various stages of meiosis, c) early prophase-I; d) metaphase-I; e) anaphase-I; f) telophase-I; g) telophase-II. h-i) In Meconopsis aculeata, PMCs showing chromatin material transfer through two and multiple chromatin strands (arrow). j) In Ranunculus laetus, A group of four PMCs involved in chromatin material transfer (arrow). k) In Potentilla cuneifolia, an anucleated PMC (arrow head) and other proximate hyperploid PMC with two chromosome complements. l) In Caltha palustris, Two PMCs showing chromatin material transfer at earlier stages of meiosis-I (arrow head) and other nearby anucleated PMC with narrow chromatin strand on periphery. m) In Potentilla cuneifolia, an anucleated PMC (arrow) and adjacent PMC showing 21 bivalents and extra bivalents on the margin (arrow head). n) In Thalictrum foetidum, stained fertile two different sized pollen grains and an unstained sterile micro-, pollen grain (arrow). o) In Potentilla var. argyrophylla, stained fertile heterogeneous sizes and unstained sterile pollen grains (arrows).

In partial transfer only some part of the chromosome complement was transferred while in complete transfer whole of the chromatin material moved from the donor to the recipient PMC. In a number of PMCs extra chromatin masses other than their normal chromatin

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material were also observed which might have resulted due to partial transfer of chromatin. Complete transfer of chromatin lead to the formation of anucleated (donor) and hyperploid (recipient) PMCs (Figs. 2k, l, m). The donor PMCs were left with hypoploid chromosome number. The resultant pollen mother cells after partial or complete chromatin transfer in majority of the species leads to the formation of apparently fertile diploid (2n), haploid, aneuploid and sterile or micro-pollen grains (Figs. 2n, o). The phenomenon of cytomixis causing high degree of pollen malformation in the form of sterile and heterogeneous size fertile pollen grains are frequently noticed in Anemone rivularis, Aquilegia fragrans, Astragalus bicuspis, A. himalayanus, A. rhizanthus, Caltha palustris, Clematis grata, C. orientalis var. acutifolia, Meconopsis aculeata, Medicago sativa, Melilotus officinalis, Pleurospermum candollii, Potentilla atrisanguinea var. atrisanguinea, P. atrisanguinea var. argyrophylla, P. cuneifolia, P. fruticosa var. rigida, Ranunculus hirtellus, R. laetus, Rosularia alpestris, Silene vulgaris, Thalictrum foetidum, Trifolium pratense, T. repens, and Trigonella emodii. Although the cytological status and chromosome constitution of such apparently fertile pollen grains could not be ascertained presently but their role in the origin of intraspecific polyploids, aneuploids, B-chromosomes and thereby in the evolution of these plant species cannot be ruled out. The phenomenon of cytomixis in the presently investigated species is also observed to be associated with several other meiotic abnormalities which include sticky and pycnotic chromatin, interbivalent connections, spindle irregularities, non-synchronous separation of bivalents, laggards and bridges, and consequently abnormal microsporogenesis.

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II. CHROMOSOME STICKINESS Stickiness in chromosomes is the other meiotic abnormality encountered quite frequently in Anemone rivularis, 2n=16; Astragalus frigidus, 2n=16; Caltha palustris, 2n=32; Clematis grata, 2n=16; C. orientalis, 2n=32; Dianthus angulatus, 2n=30; Hedysarum microcalyx Baker, 2n=14; Meconopsis aculeata, 2n=56; Medicago sativa, 2n=32; Ranunculus hirtellus, 2n=32; R. laetus, 2n=28) at the different stages of meiosis (Figs. 3a-f) from early prophase-I and persisted till the second meiotic division. However, it was more frequently observed in the PMCs during MI. Due to this, the individual chromosomes/bivalents lose their shape completely and form a clump of chromatin mass and in most of the cases it is even difficult to determine the exact chromosome number. Extent of chromatin stickiness varies in different species. Chromosome stickiness either involves a few bivalents, where it was possible to identify the meiotic stage or the entire chromosome complement (Figs. 3a, b). In such cases the chromosome stickiness impaired chromosome segregation and led to the formation of pycnotic nuclei (Fig. 3c). In severe cases, chromosome stickiness resulted in very thick chromatin bridges at telophase-II (Figs. 3d, e, f). Chromosome stickiness also resulted in the delayed separation of bivalents/chromatids at anaphase-I/II. The sticky bivalents/chromatids sometime lagged behind at anaphases/telophases and formed micronuclei at sporad stages. In Meconopsis aculeata and Ranunculus laetus, chromatin stickiness is very severe at telophases (Figs. 3d, e,) and chromatin material fail to separate during anaphases/telophases and resulted into the formation of unreduced ‗2n‘ pollen grains.

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The phenomenon of chromatin stickiness was first reported in Zea mays by Beadle (1932) and identified the affect associated with a recessive allele. Primary cause and biochemical basis of chromosome stickiness are still unknown, and genetic as well as environmental factors have been suggested to cause chromosome stickiness. Genetically controlled chromosome stickiness has been observed in crop plants like Zeya mays (Beadle, 1932; Golubovskaya, 1989; Caetano-Pereira et al., 1995), Alopecurus myosuroides (Johnsson 1944), pearl millet (Rao et al., 1990), wheat (Zanella et al., 1991) and soybean (Bione et al., 2000). Several agents have been reported to cause chromosome stickiness, including X-rays (Steffensen, 1956), gamma rays (Rao and Rao, 1977; Al Achkar et al., 1989), low temperature (Eriksson, 1968), herbicides (Badr & Ibrahim, 1987) and some chemicals present in the soil (Levan, 1945; Steffensen, 1955; Caetano-Pereira et al., 1995). Gaulden (1987) postulated that sticky chromosomes may result due to changes in specific non-histone proteins (topoisomerase II and the peripheral proteins) that are integral components of the chromosome and whose function is necessary for separation and segregation of chromatids, the changes being caused either by mutation in structural genes for the proteins (heritable stickiness) or by direct action of mutagens on the proteins (induced stickiness). The stickiness in chromosomes in majority of the presently observed species seems to be associated with the phenomenon of cytomixis and other meiotic irregularities as has been observed earlier by other workers (Chauhan, 1981; Mary & Suvarnalatha, 1981; Singhal & Gill, 1985; Kumar & Singhal, 2008; Kumar et al., 2008a, b, 2010; Singhal & Kumar, 2008a).

Figures (3a-f). Chromosome stickiness, a) In Clematis grata, a group of PMCs at metaphase-I showing complete chromosome stickiness. b) In Meconopsis aculeata, PMCs at metaphase-I showing partial chromosome stickiness. c) In Caltha palustris, a PMC at early prophase –I showing pycnotic chromatin masses (arrows). d) In Meconopsis aculeata, a group of PMCs at telophase-II showing severe chromosome stickiness (arrows). e) In Ranunculus laetus, Normal PMCs at telophase-I showing two poles (arrow head) and PMCs showing complete chromosome stickiness with restitution nuclei (arrow) f) In Ranunculus laetus, Two sticky chromatin bridges formed, one involving two poles and other between the micronucleus and one pole at telophase-II.

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III. SYNAPTIC MUTANTS Ranunculus laetus (2n=28) and Rosularia alpestris (2n=28) the presently investigated species show meiotic irregularities in the form of irregular or impaired synapsis (univalents) resulting into abnormal segregation of chromosomes during anaphases/telophases, irregular microsporogenesis, high pollen sterility and, pollen grains of variable sizes (Figs. 4a-k). Such plants showing aberrant synaptic behaviour are generally classified into two categories on the basis of their effects on homologous chromosome pairing. Plants showing partial or complete failure in pairing of homologous chromosome are classified as asynaptic mutants, while those with premature separation of homologous chromosomes are classified as desynaptic mutants (Cai & Makaroff, 2001). Asynaptic mutants are resulted due to lack of chromosome pairing during the earlier stages of prophase –I (Randolph, 1928) and the desynaptic mutants are resulted because of the falling apart of the synapsed homologous chromosomes due to their inability to generate or retain chaismata (Li et al., 1945; Rieger et al., 1976; Koduru & Rao, 1981). At present it is difficult to pinpoint the asynaptic or desynaptic behaviour of chromosomes because both the species are not amenable for the analysis of chromosomal configurations during pachytene stages of meiosis (Fig. 4a). On the basis of present observations we safely refer these plants as synaptic mutants as has also been suggested by Riley and Law (1965), and Kaul and Murthy (1985). In both the species all the chromosomes remained as univalents during the first meiotic division (Figs.4b, f). The univalents at the earlier prophase stages and metaphase-I remain randomly dispersed in the PMCs. The metaphase –I and anaphase-I stages in these cases are indistinguishable and the term metaanaphase stage is used here as has already been coined by Person (1955). The univalents in PMCs are present in unorganized/multipolar groups (Fig. 4g). During the late meiotic stages (anaphases/telophases) the univalents remained as laggards (Fig. 4h). These laggards in majority of the meiocytes fail to be included in the daughter nuclei and often formed micronuclei (Fig. 4i). Another irregularity frequently observed in meiocytes is the failure of first meiotic division resulting to the formation of restitution nuclei. Consequent to these irregularities PMCs during microsporogenesis revealed the formation of abnormal sporads such as, monads, dyads, triads, polyads, and tetrads with micronuclei (Figs. 4i). As a result of impaired meiotic course due to synaptic irregularity in these plants total pollen sterility in Rosularia alpestris (Fig. 4k) and heterogeneous size fertile pollen grains have been observed in Ranunculus laetus (Fig. 4j). A large number of synaptic mutants of spontaneous origin are already known in several plant species (Kaul & Murthy, 1985; Singh, 2002). Various causes have been assigned which influence chromosome pairing in synaptic mutants, like temperature, humidity and chemicals (Prakken, 1943; Ahloowalia, 1969; Koduru & Rao, 1981). The asynaptic nature of such individuals could be attributed to their intraspecific hybrid status where chromosomes of two probable and distantly related parents remain unpaired. Similar type of univalent formation with high pollen sterility and pollen grains of variable sizes has already been reported in the interspecific hybrids of Nicotiana (Palakarcheva & Dorossiev, 1992; Krusteva, 1995; TrojakGoluch & Berbeć, 2003) and in Dianthus angulatus from this laboratory (Kumar et al., unpublished data). Also the freezing temperature causing mutations in certain genes controlling the synaptic behaviour of homologous chromosomes during pairing/separation is not ruled out here.

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Figures (4a-e) PMC with synaptic irregularities in Rosularia alpestris, a) A PMC at early prophase-I, b) A PMC at diakinesis showing 28 univalents. c) Univalents randomly spread in two PMCs. (d, e) Sporads with micronuclei and different sized microspores. (3f-k) PMCs with synaptic irregularities in Ranunculus laetus, f) A PMC showing 28 univalents. g) Chromosomes randomly spread in a PMC. h) Multipolar PMC with some laggards/unoriented chromosomes. i) Dyads (with and without micronuclei) and triad. j) Heterogeneous sizes stained pollen grains of Ranunculus laetus. k) Unstained and variable sized sterile pollen grains of Rosularia alpestris.

IV. MULTIPLE SPINDLES Multiple spindle formation during the first and second divisions of meiosis has been reported in several plant species (Clark, 1940; Vasek, 1962; Tai, 1970; Tilquin et al., 1984; Staiger & Cande, 1990; Shamina et al., 2000; Caetano-Pereira & Pagliarini 2001; RissoPascotto et al., 2005; Calisto et al., 2008; Felismino et al., 2008; Wang et al., 2009). In Brachiaria ruziziensis (Risso-Pascotto et al., 2005), and Zea mays (Caetano-Pereira & Pagliarini, 2001), the production of polyads have been strongly correlated to multiple spindles. Investigations in Rubus (Thompson, 1962) and Fuchsia (Tilquin et al., 1984) have shown that multiple spindles are probably a consequence of high ploidy level. Multiple spindles result in a multipolar cell division and provide a mechanism to decrease the ploidy level of polyploids (Tai, 1970). High pollen sterility is a common consequence of this anomaly (Mendes-Bonato et al., 2002) and also the formation of heterogeneous sizes pollen grains. Recently, d'Erfurth et al. (2008) have isolated and characterized AtPS1 (Arabidopsis thaliana Parallel Spindle 1) gene involved in controlling the diploid (2n) gamete formation in

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Arabidopsis thaliana due to abnormal spindle orientation at male meiosis II. Consequently, Atps1 mutants produce diploid pollen grains and spontaneous triploid plants in the next generation. Cytological analysis carried out during the present investigations reveal that majority of the species depict normal spindle formation where bivalents segregated regularly to opposite poles during anaphase I and telophase I. However, in some of the presently studied species (Agrimonia eupatoria, 2n=70; Chrysanthemum pyrethroides, 2n=36; Heracleum brunonis, 2n=33; Potentilla atrisanguinea var. atrisanguinea, 2n=84; P. atrisanguinea var. argyrophylla, 2n=84; Ranunculus hirtellus, 2n=32; R. laetus, 2n=28; Rosularia alpestris, 2n=32; Thalictrum foetidum, 2n=42) spindle fibers did not converge into focused poles during anaphases and segregated chromosomes failed to converge at the poles (Figs. 5a-d). In such cases one or more supernumerary spindles per PMCs are formed, apart from the two regular/primary bipolar spindles. These supernumerary spindles consisting variable number of chromosomes and ranging from one chromosome per spindle to many chromosomes. The presence of multiple spindles, however, did not affect the function of regular spindles. Meiosis proceeded through the telophase II and pollen grain formation, regardless of the chromosome numbers present per pole and orientation. Consequent to these spindle irregularities, abnormal sporads with triads and polyads with unbalanced microspores/sporads with micronuclei were resulted (Figs 5e, f, g). The products of such irregular sporads lead to low pollen fertility and formation of fertile pollen grains of heterogeneous sizes (Fig. 5h).

Figures (5a-h). PMCs showing spindle abnormalities, a) A PMC at metaphase –I showing out of plate bivalents (arrows) in Clematis orientalis var. acutifolia. (b-h) In Ranunculus laetus, b) Four micronuclei at telophase -I. c) A PMC showing multipolar distribution of chromosomes at metaphase – II. d, e, f) PMCs at telophase-II showing multiple poles and micronuclei. g) Polyad with micronuclei (arrow). h) Fully stained fertile heterogeneous sizes and faintly stained sterile pollen grains (arrow).

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V. ABERRANT CYTOKINESIS

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Unreduced gametes are also known to result from aberrant cytokinesis (Pfeiffer & Bingham, 1983; Tavoletti et al., 1991; Ray & Tokach, 1992). The origin of unreduced microspores due to aberrant or absence of cytokinesis has been reported in several plants such as potato (Ramanna, 1974), alfalfa (Pfeiffer & Bingham, 1983), Arabidopsis (Spielman et al., 1997), Brachiaria brizantha (Risso-Pascotto et al., 2003), B. nigropedata (Utsunomiya et al., 2005), B. humidicola ( Adamowski et al., 2007) and (P. alba × P. glandulosa) × P. tomentosa (Wang et al., 2009) which produce big and/or giant pollen. Mutants in Arabidopsis (Hülskamp et al., 1997; Spielman et al., 1997) have been identified that have failed in cytokinesis during male meiotic course, resulting in a single microspore with four nuclei. Generally two rounds of chromosome segregation and one simultaneous or two successive cytoplasmic divisions (cytokinesis) occur and the final product of male meiosis in flowering plants emerges as a sporad of haploid microspores. The timing of cytokinesis varies among angiosperms. In majority of the monocot plants, cytokinesis is successive, so that there is a distinct dyad stage (Boldrini et al., 2006). However, in most of the dicots, it is simultaneous and occurs after telophase II (Peirson et al., 1996). The genes responsible for the partitioning of the cytoplasm after nuclear division play a very important role in the formation of viable gametes. Any mutation in such genes may results in the formation of abnormal spindles and consequently irregular cytokinesis. Aberrant cytokinesis in Potentilla atrisanguinea var. argyrophylla (2n=12x=84) resulted in the production of polynucleated and dumble shape pollen grains (Figs. 6a, b). In Ranunculus hirtellus (2n=4x=32) additional cytokinesis in 1-2 microspores of a sporad (Fig. 6c) lead to the formation of polyads with smaller microcytes which subsequently resulted into the formation of heterogeneous sizes pollen grains and pollen sterility (Fig. 6d).

Figures (6a-d). a) Potentilla var. argyrophylla, a polynucleated pollen grain. b) In Potentilla var. argyrophylla, a dumble shaped pollen grain. c) In Ranunculus hirtellus, two microspores in a sporad showing additional cytokinesis (arrow). d) Fully stained fertile heterogeneous sizes and unstained sterile pollen grains.

VI. SYNCYTE FORMATION Gaines and Aase (1926) were the first to notice the formation of syncytes in the haploid Triticum compactum. Later, Levan (1939) defined syncyte formation as the fusion of PMCs or nuclei. Syncyte formation has been recorded presently during the study of male meiosis in

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Meconopsis aculeata (2n=56) and Clematis orientalis var. acutifolia (2n=32). In both the cases, the syncytes are formed as a result of direct fusion of PMCs during the earlier stages of meiosis (Figs. 7a-c). Syncytes are resulted either by the passage of a nucleus from one PMC into another during cytomixis (Price, 1956) or through the fusion of two PMCs as suggested by other workers (Mehra & Kalia, 1973; Rao & Koduru ,1978; Kim et al., 2009). In Meconopsis aculeata and C. orientalis var. acutifolia such syncytes are observed to undergo the normal meiotic course of bivalent formation, chromosome segregation and microsporogenesis but resulted into the formation of almost double sized pollen grains (Fig. 7d). Such pollen grains have certainly unreduced or ‗2n‘ chromosome constitution. The syncytes formed due to direct fusion of PMCs and lead to the formation of large sized pollen grains have also been reported earlier in Phleum pratense (Levan, 1941), rice (Katayama, 1964), Cyamopsis tetragonoloba (Sarbhoy, 1980) and Brachiaria decumbens (MendesBonato et al., 2001). The formation of such unreduced or ‗2n‘ gametes through syncyte PMCs seems to be one of the possible factors for the origin of polyploids in different plants as has been advocated by Price (1956) in Erianthus arundinaceus, Saccharum rubustum and S. sinense × spontaneum, and Kim et al. (2009) in Chrysanthemum.

Figures (7a-d). Syncytes and pollen grains, a, b) Clematis orientalis var. acutifolia Direct fusion of two PMCs at early prophase-I (Clematis orientalis var. acutifolia) and metaphase-I (Meconopsis aculeata). c) In Meconopsis aculeata, syncyte formation at metaphase-I (arrow). d) In Meconopsis aculeata, fully stained fertile two different sized pollen grains.

VII. IMBALANCED POLYPLOIDS Generally imbalanced polyploids are problematic for the normal completion of meiosis and are characterized by having highly irregular meiotic course. Of the 53 polyploid species

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covered presently, Agrimonia eupatoria, 2n=5x=70; Chrysanthemum pyrethroides, 2n=3x=27; Heracleum brunonis, 2n=3x=33; Potentilla atrisanguinea var. atrisanguinea, 2n=12x=84; P. atrisanguinea var. argyrophylla, 2n=12x=84 are observed to be genetically imbalanced taxa. In these species, the meiosis is noticed to be highly abnormal characterized by irregular chromosome pairing (univalents and multivalents), irregular distribution of chromosomes, laggards and chromatin bridges, spindle irregularities, aberrant cytokinesis and abnormal microsporogenesis leading to abnormal sporad formation (polyads, monads, dyads, triads, tetrads with micronuclei) coupled with heterogeneous sizes pollen grains and high pollen sterility (Figs 8a-f.).

Figures (8a-f). Irregular meiosis in the PMCs of imbalanced polyploid, Agrimonia eupatoria (n=35), a) PMCs at metaphase-I showing univalents (arrows) and bivalents. b) A PMCs showing chromosomes arranged in three poles at anaphase-II and many laggards. c) A PMCs showing unoriented chromosomes at anaphase-II. d) Laggards in the tripolar PMCs at teleophase –II. e) Polyads with micronuclei. f) Fully stained fertile heterogeneous sizes and unstained sterile pollen grains.

Besides, Geranium pratense (2n=4x=56) which exists at tetraploid level, depicts some meiotic abnormalities in the form of univalents and multivalents during late prophase-I or metaphase-I. However, further meiotic course in such cases is not affected significantly and only some pollen sterility is caused.

VIII. FUSION OF POLLEN GRAINS Cytoplasmic connections and fusion among pollen grains which have been observed in Caltha palustris (2n=32), Meconopsis aculeata (2n=56), and Potentilla atrisanguinea var. argyrophylla (2n=84) results into large/giant sized pollen grains (Figs. 9a-d). Occurrence of

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such connections among pollen grains and subsequent transfer of chromatin have also been noticed earlier in the intergeneric hybrids of Roegneria tsukushiensis x Psathyrostachys huashanica and Triticum aestivum x Psathyrostachys huashanica (Sun et al., 1993, 1994). Formation of big or giant pollen grains from such fusion which are certainly of polyploid nature and their further evolutionary importance in the origin polyploids cannot be ruled out.

Figures (9a-d). a, b, c) In Potentilla atrisanguinea var. argyrophylla, photomicrographs showing the process of pollen grain fusion through the dissolution of pollen grain walls. d) Two normal pollen grains showing fusion (arrow head) along with one large sized (arrow) stained fertile pollen grain.

IX. MULTIPLE ASSOCIATIONS IN DIPLOIDS Associations of more than two chromosomes in a diploid taxon might indicate that at least partial homology of chromosomes extends to some non-homologous pairs that is possible either due to hybrid nature or heterozygosity for reciprocal translocations (Singhal, 1982). Reciprocal translocations have been considered as an important source of intraspecific chromosomal structural polymorphism (Müntzing & Prakken, 1941; Hrishi et al., 1969; Candela et al., 1979). Presently, multiple associations of chromosomes due to reciprocal translocations have been reported in Astragalus chlorostachys (2n=16) where four chromosomes are associated to form a quadrivalent (rings and chains) (Figs. 10a, b). Pollen fertility in a structural heterozygote is reduced to a considerable extent (40%) (Fig.10c). Disjunctional orientation of multivalents in this species is preferentially seems to be of adjacent nature in contrast to the alternate orientation which normally results into high pollen fertility.

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Figures (10a-h). (a-c) In Astragalus chlorostachys, a) PMCs at metaphase-I showing a quadrivalent in the form of ring (arrow). b) A PMCs at metaphase-I showing a quadrivalent in the form of chain (arrow). c) Fully stained fertile and unstained sterile pollen grains. (d-f) In Delphinium vestitum, e) a PMC showing precocious disjunction of bivalents at metaphase-I (arrows). e) Two large sized bivalents lag at equatorial plate (arrows). f) A PMC showing late disjunction of chromosomes at anaphase-I (arrow). g) In Meconopsis aculeata, a PMC showing interbivalent connections at anaphase-I (arrows). h) In Clematis orientalis var. acutifolia, a PMC showing chromatin bridges at anaphase-I (arrow). h) In Heracleum brunonis, a PMC showing laggards at anaphase-I (arrows).

X. NON SYNCHRONOUS DISJUNCTION OF BIVALENTS Non synchronous disjunction of bivalents is either found in the hybrid taxa or the species having different sized chromosomes and the rate of terminalization. Delphinium brunonianum, 2n=16; D. denudatum, 2n=16; and D.vestitum, 2n=16 depict both delayed and precocious disjunction of some bivalents during male meiosis (Figs. 10d-f). Early disjunction of some bivalents normally does not affect the normal distribution of chromosomes at A-I but delayed separation of bivalents which normally exists in hybrids and cytologically abnormal diploids causes some meiotic disturbances (chromatin bridges and laggards) and consequently pollen malformation as has been the case in D.vestitum. In Caltha palustris, Delphinium brunonianum, D. denudatum and D.vestitum, large size of the bivalents seem to be responsible for delay in their segregation during anaphases as has been the case in

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Cyathocline purpurea and Blumea spp. (Gupta,1981) and Vicia tenera (Kaur & Singhal, 2010). In Delphinium brunonianum and D.vestitum there is a tendency of some bivalents to lag at the equatorial plate (Fig. 10e) and variation in chromosome number is caused which ultimately affect the products of PMCs leading to reduced pollen fertility and pollen grains of variable sizes.

XI. INTERBIVALENT CONNECTIONS Interchromosomal connections are known to exist in mitotic chromosomes of plants as well as animals (Chiarelli et al., 1977). Interbivalent connections are reported presently during meiosis in Astragalus bicuspis (2n=16), Clematis orientalis var. acutifolia (2n=32), Heracleum brunonis (2n=33), Meconopsis aculeata (2n=56) and Pleurospermum candollii (2n=22). Such interchromosomal connections are observed to be more prominent in the meiocytes during late prophase-I, metaphase-I and A-I stages (Fig. 10g). Interbivalent connections have been observed during the diplotene and diakinesis stages in Crotalaria (Akpabio, 1990) and Capsicum (Falusi, 2006). These workers are of the opinion that with the advancement of meiosis there is a reduction in the frequency of interbivalent/chromosomal connections. However, in the presently studied species, these connections continue up to A-I stage. Interbivalent/chromosomal connections reported presently in all the species are associated with cytomixis and other meiotic abnormalities as has also been suggested earlier by Habib and Chennaveeraiah (1976) in Capsicum annuum and woody species by Singhal and Gill (1985).

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XII. OTHER MEIOTIC ABNORMALITIES Besides the presence of above mentioned meiotic irregularities, lagging of chromatin material in the form of one to many laggards and chromatin bridges during AI/TI and AII/TII (Figs. 10h, i) are also observed in PMCs of many species such as Caltha palustris (2n=32), Clematis grata (2n=16), C. orientalis var. acutifolia (2n=32), Delphinium brunonianum (2n=16), D.vestitum (2n=16), Heracleum brunonis (2n=33), Meconopsis aculeata (2n=56), Potentilla atrisanguinea var. atrisanguinea (2n=84), P. atrisanguinea var. argyrophylla (2n=84), Ranunculus hirtellus (2n=32), R. laetus (2n=28), Rosularia alpestris (2n=28), Saxifraga hirculus (2n=32), Sedum oreades (2n=22) and Thalictrum foetidum (2n=42). In all these cases, the laggard/s failed to be included in the telophases nuclei resulted into the formation of micronuclei in the sporads. Such micronuclei consequently form the micro/sterile pollen grains. Other meiotic abnormalities such as pycnotic and fragmented chromatin material in the cytoplasm during early prophase-I and out of plate few bivalents which ultimately affect the pollen fertility and pollen size are also noticed quite frequently in Caltha palustris (2n=32), Clematis grata (2n=16), C. orientalis var. acutifolia (2n=32),, Heracleum brunonis (2n=33), Ranunculus hirtellus (2n=32) and R. laetus (2n=28)).

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CONCLUSION Meiotic diploidization, somatic chromosome doubling during mitosis in a zygote or meristematic cells or non-reduction in meiosis leading to the production of 2n gametes are the different mechanisms through which origin of polyploids can take place (Kim et al., 2009). In addition to these processes, 2n gametes may be formed by cytomixis (Falistocco et al., 1995; Kumar et al., 2008a, b, 2010; Sheidai et al., 2009 a, b; Singhal et al., 2010) or syncyte formation (Price, 1956; Kim et al., 2009). Both the processes, cytomixis and syncytes have been observed presently to produce such unreduced gametes. Cytomixis and associated meiotic abnormalities during male meiosis seem to be directly responsible for pollen malformation and variation in pollen grain size as has been reported in several plant species by authors (Kumar and Singhal, 2008; Kumar et al., 2008a, b, 2010; Singhal and Kumar, 2008a, b; Singhal et al., 2008, 2009a, b, 2010) and other workers (Chauhan, 1981; Koul, 1990; Ghanima & Talaat, 2003; Haroun et al., 2004; Ghaffari, 2006; Lattoo et al., 2006; Sheidai et al., 2009 a, b). The present findings further strengthen the view that the abnormalities during male meiosis affect the viability of pollen grains and reproductive success. On the other hand the unreduced pollen grains formed as a result of these meiotic abnormalities may help the species to produce polyploid plants that can better adapt to adverse conditions than their diploid counterparts. Present studies indicate that pollen malformation and pollen grains of heterogeneous sizes in the investigated species are the outcome of cytomixis and other associated abnormalities during male meiosis.

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ACKNOWLEDGMENTS The authors are grateful to the University Grants Commission, New Delhi for providing financial assistance under the DRS SAP I and II and ASIST programme and also to CSIR for providing Senior Research Fellowship to Mr. Puneet Kumar. Thanks are also due to the Head, Department of Botany for necessary laboratory and library facilities.

REFERENCES Adamowski, E. V., Boldrini, C. B., Pagliarini, M. S. & Valle, C. B. 2007. Abnormal cytokinesis in microsporogenesis of Brachiaria humidicola (Poaceae: Paniceae). Genetics and Molecular Research, 6, 616-621. Ahloowalia, B. S. 1969. Effect of temperature and barbiturates on a desynaptic mutant of rye grass. Mutation Research, 7, 205–213. Akpabio, K. E. 1990. Chromosomal interconnections and metaphase 1 clumping in meiosis of four species of Crotalaria L. Nigerian Journal of Botany, 3, 191–195. Al Achkar, W., Sabatier, L. & Dutrillaux, B. 1989. How are sticky chromosomes formed? Annu. Genet., 32,10-15. Aswal, B. S. & Mehrotra, B. N. 1994. Flora of Lahaul-Spiti (A Cold Desert in North West Himalaya). Dehra Dun, India: Bishen Singh Mahendra Pal Singh.

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Chapter 4

CAPTURE OF MALE GAMETE DYNAMICS IN POLLEN TUBES Tomonari Hirano1, 2 and Yoichiro Hoshino1,* 1

Field Science Center for Northern Biosphere, Hokkaido University, Kita-Ku, Sapporo, Japan 2 RIKEN Nishina Center, 2-1 Hirosawa, Wako, Saitama, Japan

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ABSTRACT The process of double fertilization involves 2 sperm cells that are delivered into female gametes via pollen tubes. To analyze this process, we utilized the bicellular pollen species Alstroemeria and Cyrtanthus, which produce 2 sperm cells in the pollen tubes. Novel procedures have been developed for the in vitro observation of pollen tubes under precise control conditions, for flow cytometric analysis with single cell manipulation, and for immunocytochemical analysis to detect cytoskeletons. Using these unique methods, male gamete dynamics have been elucidated during pollen tube development. In particular, we have focused on male germ unit (MGU) defining that the generative cells or the pair of sperm cells in pollen tubes or pollen grains are closely associated with the vegetative nucleus. This MGU is proposed to function as a vehicle for the transmission of male gametes to the female gametes and to participate in the fusion with the target female cell during fertilization. In this chapter, recent insights into male gamete dynamics using novel procedures are discussed.

INTRODUCTION Different types of pollen are characterized by the number of cells in the microspore at the mature stage. As a consequence of mitosis of the microspore nucleus, a large vegetative cell and a smaller generative cell are produced. In approximately 30% of plant families, further *

Correspondence: Yoichiro Hoshino, Field Science Center for Northern Biosphere, Hokkaido University, Kita 11, Nishi 10, Kita-Ku, Sapporo 060-0811, JAPAN, Telephone number: +81-11-706-2857, FAX number: +81-11706-2857, e-mail: [email protected],

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mitosis of the generative cell results in the formation of 2 sperm cells in the pollen. This type of pollen is called a tricellular pollen. In most other plant families, a generative cell and a vegetative cell are maintained in mature pollen and additional mitosis of the generative cell takes place in the elongating pollen tube. Based on the number of cells in mature pollen, the latter type of pollen is called bicellular pollen. Two significant plant families, Gramineaceae, which includes rice, maize, and wheat, and Brassicaceae, which includes Arabidopsis, are categorized into the tricellular pollen group. Therefore, considerable research has conducted on tricellular pollen at the molecular level, including the transcriptome analysis of isolated sperm cells in Arabidopsis (Borges et al. 2008), characterization of polypyrimidine tractbinding protein for pollen germination in Arabidopsis (Wang and Okamoto 2009), establishment of expressed sequence tag libraries from sperm cells in maize (Engel et al. 2003), proteomic analyses of mature pollen in rice (Dai et al. 2006), and transcript profiling compared between distinctive sperm cells of Plumbago zeylanica (Gou et al. 2009). In contrast, studies concerning bicellular pollen are rare; however, several significant works have been performed, including cDNA libraries constructed from generative cells (Xu et al. 1999; Okada et al. 2006) and characterization of the GCS1 protein involved in fertilization in generative cells of the lily (Mori et al. 2006), and isolation of Alstroemeria glsA expressed in generative cell development (Igawa et al. 2009). In this chapter, we focus on bicellular pollen. The defining characteristic of bicellular pollen is that sperm cells are formed by cell division from the generative cell in the germinating pollen tube. In the process of sperm cell formation in the pollen tube, it is predicted that there is cooperation between vegetative and generative cells. The complex of the male gametes in the pollen or pollen tube is known as male germ unit (MGU) (Dumas et al. 1984), which appears to be associated with the process of double fertilization. In bicellular pollen, sperm cell formation is coordinated with pollen germination following pollen tube elongation. Furthermore, the maturation of sperm cells results in the preparation for fertilization. The advantage of bicellular pollen for practical experiments is the direct observation of the processes of mitotic division and maturation in vitro. During these processes, pollen germination and/or pollen tube elongation could trigger mitosis of a generative cell. Thus, analyses of bicellular pollen play an important role in further understanding male gamete development. Moreover, data collected on bicellular pollen can be extrapolated to many plant species, as 70% of plant families produce bicellular pollen. Here, we introduce novel procedures for the analysis of male gamete dynamics in bicellular pollen. The specific futures of our study are to use liquid in vitro culture condition for pollen germination and to develop flow cytometry (FCM) analysis with selected male gametes. This in vitro strategy could contribute extensively to pollen research. Furthermore, this method will be useful for isolating sperm cells of bicellular pollen, making them available for in vitro fertilization research (Kranz and Lörz 1993; Kranz et al. 2008) by the artificial fusion of an isolated egg cell and an isolated sperm cell.

MALE GAMETE DYNAMICS IN THE POLLEN TUBE The molecular processes underlying the development of the male gametophyte have been identified by the characterization of some male gametophytic mutants in the tricellular pollen

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of A. thaliana. In the mutants of duo1 and duo2 (Durbary et al. 2005; Rotman et al. 2005), cdka;1 (Iwakawa et al. 2006; Nowack et al. 2006), fbl17 (Kim et al. 2008; Gusti et al. 2009), and duo3 (Brownfield et al. 2009a), bicellular pollen grains were formed at anthesis by the blockage of generative cell division. The genes responsible for mutant formation controlled the cell cycle after pollen mitosis I and gamete differentiation (Borg et al. 2009; Brownfield et al. 2009b). On the other hand, the molecular processes involved in bicellular pollen, from pollen grain to pollen tube, have not yet been analyzed. Improvements in our understanding of the molecular mechanisms of bicellular pollen and comparisons between bicellular and tricellular pollen are expected to lead to the identification of the genes responsible for the determination of bicellular or tricellular pollen. Several phenomena of male gamete maturation during pollen tube growth have been reported. In A. thaliana, sperm cells in the pollen grains synthesized DNA and contained approximately 1.5C DNA at anthesis (Friedman 1999). After pollination, the sperm cells continued through the S phase of the cell cycle during pollen tube growth and those deposited within the embryo sacs contained 2C DNA (Friedman 1999). Sperm cells in Nicotiana tabacum were formed in the pollen tubes and contained 1C DNA during pollen tube growth; these sperm cells were in the S phase of the cell cycle in the synergid (Tian et al. 2005). Egg cells in N. tabacum also increased DNA content from 1C at anthesis to 1C–2C at 48 h after pollination and newly formed zygotes contained 4C DNA until the first division (Tian et al. 2005). Therefore, cell fusion in N. tabacum occurred at the G2 phase in the cell cycle of sperm and egg cells, and there was cell-cycle synchronization between male and female gametes. The generative cell or the pair of sperm cells in pollen tubes or pollen grains is reported to be closely associated with the vegetative nucleus in many species, including those with bicellular and tricellular pollen (Mogensen 1992). This association produces an entity known as the MGU, which is proposed to function as a vehicle for the transmission of male gametes to the female gametes and to participate in the fusion with the target female cell during fertilization (Dumas et al. 1984). In the MGU of several species, sperm cells that are associated with the vegetative nucleus (Svn) and those that are not associated with the vegetative nucleus (unassociated sperm cells; Sua) differ in size, shape, and organelle content (reviewed in Mogensen 1992; Weterings and Russell 2004). In P. zeylanica, Sua containing numerous plastids preferentially fuse with the egg cell, whereas Svn containing numerous mitochondria fuse with the central cell (Russell 1984; Russell 1985). Furthermore, it was recently reported that numerous genes were highly upregulated in dimorphic sperm cells in P. zeylanica and both sperm cells showed different gene-expression profiles (Gou et al. 2009). This evidence of preferential fertilization and the differences in the expression of sperm cells suggest the possibility of a system for controlling male gamete maturation and specificity. It has been suggested that in A. thaliana, the position or organization of the MGU in mature pollen may influence the efficiency of male gamete delivery (Lalanne and Twell 2002). However, the influence of the vegetative nucleus in the MGU on the differentiation of the generative cell or the development of sperm dimorphism is unclear.

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NOVEL APPROACHES FOR MALE GAMETE ANALYSIS IN THE POLLEN TUBE Efficient isolation of generative cells or sperm cells from the pollen grain or pollen tube could be used for detailed male gamete analysis, such as the comparison of gene-expression profiles between the pair of sperm cells (Gou et al. 2009), and for in vitro fertilization (Kranz and Lörz 1993; Kranz et al. 1998; Uchiumi et al. 2007). In particular, male gamete isolation is indispensable for the analysis of male gamete dynamics during pollen tube growth in bicellular pollen species. Methods of male gamete isolation from pollen tubes have been developed using pollen tubes cultured in vitro or by using a in vivo/in vitro technique (Shivanna et al. 1988; Tylor et al. 1989). In one of the methods, the pollen tubes were immersed into solutions for osmotic bursting with or without enzyme pretreatment, and a high yield of generative cells or sperm cells could be obtained using this osmotic bursting method (Theunis et al. 1991). A novel method for male gamete isolation from pollen tubes grown in vitro was recently established in Alstoemeria aurea (Fig. 1; Hirano and Hoshino 2009). The isolation method was designed for DNA analysis of male gametes during pollen tube growth with FCM. The pollen tubes collected from the liquid medium were chopped with a sharp razor blade in the cell extraction buffer and the isolated male gametes in the buffer could then be used for FCM analysis. This chopping method is very simple and quick, without the need for pretreatment, but is not suitable for pollen tubes immediately after their emergence from the cut end of the style in the in vivo/in vitro method, because there is a possibility that cells from the style are chopped together with the pollen tubes. Therefore, the osmotic bursting and chopping methods should be chosen depending on the specific sample conditions. FCM analysis is widely used to estimate the DNA content in plant cell nuclei, as it is a very simple procedure that can rapidly measure DNA content in large cell populations (Doležel and Bartoš 2005). As a method for male gamete analysis, FCM has been used to assess the pollen of several species (Suda et al. 2007) and the germinated gymnosperm pollen of Cupressus dupreziana (Pichot and El Maâtaoui 2000). Recently, FCM analysis has been used for in vitro cultured pollen tubes in A. aurea and Cyrtanthus mackenii (Hirano and Hoshino 2009, 2010). When the male gametes were isolated from the pollen tubes using the chopping method described above, the timing of sperm cell formation could be detected during pollen tube growth. Moreover, MGU formation and the occurrence of DNA synthesis in sperm cells were also confirmed by the FCM-based method combined with single cell manipulation using a microcapillary controlled by a micropump (Fig. 1). In A. aurea, the proportion of MGU formation in pollen tubes cultured in vitro increased after sperm cell formation, and the change was detected by FCM analysis (Hirano and Hoshino 2009). On the other hand, the proportion of MGU formation in C. mackenii tended to decrease with extended culture after sperm cell formation (Hirano and Hoshino 2010). These results indicate that the FCM-based method has potential as a simple and quick tool for assessing male gamete behavior, such as sperm cell formation and MGU formation, during pollen tube growth. Moreover, FCM may be applicable as a high throughput technique for preparing MGU material for molecular approaches in combination with a cell-sorting system.

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Figure 1. Summary of a newly developed procedure for the analysis of male gamete behavior in the pollen tube. Pollen grains are cultured in a liquid medium, and then the elongated pollen tubes are used directly for the cytological assessment of male gametes in pollen tubes or for the isolation of male gametic cells and nuclei. Male gamete behavior in the pollen tube, such as the timing of sperm formation and MGU formation, can be detected by flow cytometry with the population of cells and nuclei isolated by means of the chopping method. In addition to the large scale analysis, DNA analysis and cytoskeleton observation in a small number of targeted cells can also be performed using single cell manipulation.

The microtubular cytoskeleton of generative cells and sperm cells differs from that of somatic cells: in the former 2 cell types, the microtubules are arranged in prominent bundles that are aligned helically or longitudinally relative to the long axis of the cell (Palevitz and Tiezzi 1992; Southworth and Cresti 1997). It has been reported that in generative cells and sperm cells isolated from pollen grains or pollen tubes, the microtubules are lost or exhibit a mesh pattern and that the cells acquire a spherical shape (Russell 1991; Thunis et al. 1991). In N. tabacum, when pollen grains were squashed in a fixation medium, the isolated generative cell was found to retain its spindle shape and the basket-like cytoskeleton of the microtubule bundle (Theunis et al. 1992). Therefore, the buffer used for the isolation of male germ cells has a great influence on the microtubular cytoskeleton of the isolated cells. For the isolation of male gametes from pollen tubes in C. mackenii, a fixative supplemented with surface active agent was useful for the protection of the microtubule bundle and for the maintenance of the cell shape (Hirano and Hoshino 2010). The male gametes isolated in the fixative could be therefore used for detailed analysis of the cell shape and cytoskeleton without interference from the cytoskeleton of the pollen tubes. The isolated MGUs from pollen tubes in C. mackenii showed sperm dimorphism in terms of nuclear shape between sperm cells and the Sua nuclei were longer than the Svn nuclei. The Sua also showed higher microtubule accumulation than the Svn and there was sperm dimorphism in terms of microtubule accumulation. These results suggest that the differences in the microtubule accumulation

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between sperm cells in MGU are possibly related to the differences in the nuclear shape (Hirano and Hoshino 2010).

CONCLUSION Various techniques, including single cell manipulation, have been developed to date and have led to improvements in the analysis of male and female gametes. As a result of these advances, unique features of double fertilization have been discovered. However, our understanding of cellular and molecular mechanisms involved in double fertilization is still limited and, in particular, the molecular processes involved in sperm cell development in bicellular pollen remain totally unknown. In this chapter, we introduced novel approaches for the analysis of male gamete development during pollen tube growth, which may be utilized for the detection of sperm cell-gene expression in bicellular pollen species, as well as cytological analysis. Differences in gene expression between pairs of sperm cells in the MGU, which was observed in P. zeylanica (Gou et al. 2009), may also be analyzed during pollen tube growth using single cell manipulation combined with a microdissection technique. These challenging studies will contribute to our understanding of the mechanisms of the MGU and the processes involved in double fertilization from the viewpoint of male gametes.

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REFERENCES Borg, M.; Brownfield, L.; Twell, D. (2009). Male gametophyte development: a molecular perspective. Journal of Experimental Botany, 60, 1465–1478. Brownfield, L.; Hafidh, S.; Durbarry, A.; Khatab, H.; Sidorova, A.; Doermer, P.; Twell, D. (2009a). Arabidopsis DUO POLLEN3 is a key regulator of male germline development and embryogenesis. Plant Cell, 21, 1940–1956. Brownfield, L.; Hafidh, S.; Borg, M.; Sidorova, A.; Mori, T.; Twell, D. (2009b). A plant germline-specific integrator of sperm specification and cell cycle progression. PLoS Genetics, 5, e10000430. Dai, S.; Li, L.; Chen T.; Chong, K.; Xue Y.; Wang, T. (2006). Proteomic analyses of Oryza sativa mature pollen reveal novel proteins associated with pollen germination and tube growth. Proteomics, 6, 2504–2529. Doležel, J. & Bartoš, J. (2005). Plant DNA flow cytometry and estimation of nuclear genome size. Annals of Botany, 95, 99–110. Dumas, C.; Knox, R. B.; McConchie, C. A.; Russell, S. D. (1984). Emerging physiological concepts in fertilization. What‘s New in Plant Physiology, 15, 17–20. Durbarry, A.; Vizir, I.; Twell, D. (2005). Male germ line development in Arabidopsis. duo pollen mutants reveal gametophytic regulators of generative cell cycle progression. Plant Physiology, 137, 297–307. Friedman, W. E. (1999). Expression of the cell cycle in sperm of Arabidopsis: implications for understanding patterns of gametogenesis and fertilization in plants and other eukaryotes. Development, 126, 1065–1075.

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Gou, X.; Yuan, T.; Wei, X.; Russell, S. D. (2009). Gene expression in the dimorphic sperm cells of Plumbago zeylanica: transcript profiling, diversity, and relationship to cell type. Plant Journal, 60, 33–47. Gusti, A.; Baumberger, N.; Nowack, M.; Pusch, S.; Eisler, H.; Potuschak, T.; Veylder, L. D.; Schnittger, A.; Genschik, P. (2009). The Arabidopsis thaliana F-box protein FBL17 is essential for progression through the second mitosis during pollen development. PLoS ONE, 4, e4780. Hirano, T. & Hoshino, Y. (2009). Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in Alstroemeria aurea. Journal of Plant Research, 122, 225–234. Hirano, T. & Hoshino, Y. (2010). Sperm dimorphism in terms of nuclear shape and microtubule accumulation in Cyrtanthus mackenii. Sexual Plant Reproduction, 23, 153162. Palevitz, B. A. & Tiezzi, A. (1992). Organization, composition, and function of the generative cell and sperm cytoskeleton. International Review of Cytology, 140,149–185. Igawa, T.; Hoshino, Y.; Yanagawa, Y. (2009). Isolation and characterization of the plant glsA promoter from Alstroemeria. Plant Biology, 11, 878–885. Iwakawa, H.; Shinmyo, A.; Sekine, M. (2006). Arabidopsis CDKA;1, a cdc2 homologue, controls proliferation of generative cells in male gametogenesis. Plant Journal, 45, 819– 831. Kim, H. J.; Oh, S. A.; Brownfield, L.; Hong, S. H.; Ryu, H.; Hwang, I.; Twell, D. (2008). Control of plant germline proliferation by SCFFBL17 degradation of cell cycle inhibitors. Nature, 455, 1134–1138. Kranz, E. & Lörz, H. (1993). In vitro fertilization with isolated, single gametes results in zygotic embryogenesis and fertile maize plant. Plant Cell, 5, 739–746. Kranz, E.; Hoshino, Y.; Okamoto, T. (2008). In vitro fertilization with isolated higher plant gametes. Methods in Molecular Biology, The Humana press Inc. 427, 51–69. Kranz, E.; von Wiegen, P.; Quader, H.; Lörz, H. (1998). Endosperm development after fusion of isolated, single maize sperm and central cells in vitro. Plant Cell, 10, 511–524. Lalanne, E. & Twell, D. (2002). Genetic control of male germ unit organization in Arabidopsis. Plant Physiology, 129, 865–875. Mogensen, H. L. (1992). The male germ unit: concept, composition, and significance. International Review of Cytology, 140, 129–147. Mori, T.; Kuroiwa, H.; Higashiyama, T.; Kuroiwa, T. (2006). GENERATIVE CELL SPECIFIC 1 is essential for angiosperm fertilization. Nature Cell Biology 8, 64–71. Nowack, M. K.; Grini, P. E.; Jakoby, M. J.; Lafos, M.; Koncz, C.; Schnittger, A. (2006). A positive signal from the fertilization of the egg cell sets off endosperm proliferation in angiosperm embryogenesis. Nature Genetics, 38, 63–67. Okada, T., Bhalla, P. L., Singh, M. B. (2006) Expressed sequence tag analysis of Lilium longiflorum generative cells. Plant and Cell Physiology, 47, 698–705. Pichot, C. & El Maâtaoui, M. (2000). Unreduced diploid nuclei in Cupressus dupreziana A. Camus pollen. Theoretical and Applied Genetics, 101, 574–579. Rotman, N.; Durbarry, A.; Wardle, A.; Yang, W. C.; Chaboud, A.; Faure, J. E.; Berger, F.; Twell, D. (2005). A novel class of MYB factors control sperm-cell formation in plants. Current Biology, 15, 244–248.

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Russell, S. D. (1984). Ultrastructure of the sperm of Plumbago zeylanica. II. Quantitative cytology and three-dimensional organization. Planta, 162, 385–391. Russell, S. D. (1985). Preferential fertilization in Plumbago: ultrastructural evidence for gamete-level recognition in an angiosperm. Proceeding of National Academy Sciences of the United States of America, 82, 6129–6132. Russell, S. D. (1991). Isolation and characterization of sperm cells in flowering plants. Annual Review of Plant Physiology and Plant Molecular Biology, 42,189–204. Shivanna, K. R.; Xu, H.; Taylor, P.; Knox, R. B. (1988). Isolation of sperm cells from the pollen tubes of flowering plants during fertilization. Plant Physiology, 87, 647–650. Southworth, D. & Cresti, M. (1997). Comparison of flagellated and nonflagellated sperm in plants. American1 Journal of Botany, 84, 1301–1311. Suda, J.; Kron, P.; Husband, B. C.; Travnicek, P. (2007). Flow Cytometry and Ploidy: Application in plant systematic, ecology and evolutionary biology. In: J. Doležel, J. Greilhuber, J. Suda (Eds.) Flow cytometry with plant cells (pp. 103–130). KGaA, Weinheim: WILEY-VCH Verlag GmbH & Co. Taylor, P.; Kenrick, J.; Li, Y.; Kaul, V.; Gunning, B. E. S.; Knox, R. B. (1989). The male germ unit of Rhododendron: quantitative cytology, three-dimensional reconstruction, isolation and detection using fluorescent probes. Sexual Plant Reproduction, 2, 254–264. Theunis, C. H.; Pierson, E. S.; Cresti, M. (1991). Isolation of male and female gametes in higher plants. Sexual Plant Reproduction, 4, 145–154. Theunis, C. H.; Pierson, E. S.; Cresti, M. (1992). The microtubule cytoskeleton and the rounding of isolated generative cells of Nicotiana tabacum. Sexual Plant Reproduction, 5, 64–71. Tian, H. Q.; Yuan, T.; Russell, S. D. (2005). Relationship between fertilization and the cell cycle in male and female gametes of tobacco. Sexual Plant Reproduction, 17, 243–252. Uchiumi, T.; Uemura, I.; Okamoto, T. (2007). Establishment of an in vitro fertilization system in rice (Oryza sativa L.). Planta, 226, 581–589. Wang, S. & Okamoto, T. (2009). Involvement of polypyrimidine tract-binding protein (PTB)related proteins in pollen germination in Arabidopsis. Plant and Cell Physiology, 50, 179–190. Weterings, K. & Russell, S. D. (2004). Experimental analysis of the fertilization process. Plant Cell, 16, 107–118. Xu, H.; Swoboda, I.; Bhalla, L.; Singh M. B. (1999). Male gamete cell-specific expression of H2A and H3 histone genes. Plant Molecular Biology 39, 607–614.

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Chapter 5

SUNFLOWER POLLEN: THEORETICAL AND PRACTICAL ASPECTS V. Popov*, T. Dolgova and V.V. Dokuchaev Kharkiv National Agrarian University, Department of Ecology and Biotechnology, Kharkiv Region, 62483, Ukraine

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ABSTRACT Modern conception about the structure and development of sunflower anther in the normal state and under the different types of sterility are presented in this investigation. In this review different types of male sterility are discussed: nuclear male sterility, cytoplasmic male sterility and sterility which induces different factors. This research is based on the summary of scientific literature and the experimental work results of the authors, in addition to the meiosis studying of the sunflower, wild species and their interspecific hybrids. Spore formation capacity of androceum concerning practical breeding and seed production problems are also studied. The results of research in the field of inheritance of nuclear mail sterility and cytoplasmic mail sterility and the mapping of genes which control male sterility using different molecular markers are presented. The questions of relationship plasmone and genome in manifestation of cytoplasmic male sterility and the importance of gametocytes for the induction of sunflower sterility has been considered. Great attention was paid to meiosis violation in the process of chromosome formation rearrangement during the interspecific hybridization. The reasons of cytological instability of interspecific hybrids which have been obtained by crossing of annual wild species with culture sunflower and the ways of overcoming this cytological instability are also discussed.

INTRODUCTION Sunflower (Helianthus annuus L.) is one of the basic oilseed crops in the agriculture world-wide, its productivity and sowing areas have a stable tendency to growth. The use of *

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heterotic effect in the breeding is to a great extent instrumental in wide distribution of cultivated sunflower. The heterosis provide high positive of productivity, collection of oil, resistance to disease and pests, as well as, uniformity according to the agronomical and commodity standards. It is obvious that success of heterosis breeding and effective industrial seed production of hybrid sunflower had been not possible without the use of the genetically determined systems of the controlled pollination, such as the phenomena of cytoplasmatic male sterility (CMS) and nuclear male sterility (NMS) [31]. More than 30 years have passed since the phenomenon discovery of sunflower sterility. Initial experiments on this study stalled until the first economic expedient results ( Leclerc‘s works of 1960-ies); since then scientific interest of the researchers about this problem has not weakened [49, 52]. An important stimulus for a study of the problem of sunflower pollen sterility was investigations on the interspecific hybridization within the limits of the genus Helianthus. The total complex of cytological abnormalities known as incompatibility manifested on the interspecific hybridization. Species interrelation and interspecific incompatibility was of particular importance for elucidating the type of the isolating mechanism [73]. The investigations show that one of the mechanisms, which lead to the speciation of sunflower species, is related to the chromosomal rearrangements [11, 14]. These chromosomal rearrangements are generally well correlated with pollen fertility of F1 hybrids obtained by crossed differ Helianthus spp. [4]. At present, researchers interest has been increasing about the benefits of the wild species of sunflowers becoming the potential donors of important economic and biological characters: disease and pest resistance, early maturing, high content of protein and oil etc. [8, 47]. Besides, general distribution of only one type of cytoplasm (PET1) in the industrial seed growing forwards the definite narrowing of genetic variety and growth of susceptible to action of disease and pests. Numerous wild Helianthus species can be of use not only for the improvement of some agronomical characteristics, but also as sources of CMS and Rf-genes. Besides, data about crossability species of sunflower, character of micro- and macrosporogenesis, features of fertilization and zygote development are used in the phylogenetic analysis [4, 34, 43]. In this connection the purpose of the present review was an analysis of accumulated data related to the questions of development and forming of sunflower fertile pollen and as well as generalization of modern information about the mechanisms of origin of male sterility and its use for the decision of fundamental and applied aspects of breeding and genetics.

FLOWERING BIOLOGY, ANTHER DEVELOPMENT AND POLLEN FORMATION IN A FERTILE SUNFLOWER The sunflower inflorescence is a head, consisting of a disc-like receptacle and florets. Sunflower florets are of two types: semifloret and tubulous. The semiflorets are disposed on the perimeter of a floral disk, and the tubulous ones cover its entire surface. The semiflorets are sterile; they consist of an ovary and a single-petal corolla. A style, a stigma and anthers are absent. The sunflower tubulous florets are monoecious. There are five stamens; anthers are adnate as a tube. A pistil consists of two carpels, a stigma is bilobate.

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Pirev [68], Ryabota [74], Horner [40], Simonenko [81] and other researchers studied the development of anthers and the forming of sunflower pollen using both light and electronic microscopy. Horner [40] described 11 stages of pollen grains development. According to his classification the first stage is characterized by the forming of sporogenous tissue – secondary archesporium. Stages 2-5 are directly related to passage of meiosis, beginning from a prophase I and concluding by the tetrad forming. Stages 6-11 are characterized by the forming of sunflower pollen grains. In sunflower microsporangium development starts in a general way as angiosperm plants. In the homogeneous meristematic tissue of stamen tubercles (cone, protuberance) four isolated hearths of primary archesporium cells begin forming when the diameter of the inflorescence mounts to 1,5-2,0 cm. The anther walls arise after the series of the divisions of a parietal layer. Parietal layers laying an epidermis are ontogenetic family to sporogenic tissue. The epidermis cells, being the most outward layer of the anther, are divided only along, lengthen and have a dense membrane. Endothecium is located directly under the epidermis. It reaches its complete development when pollen grains have been ready to go out of the anther, owing to the presence of fibrous layer, which gets dry and promotes the anther dissection. One row of the middle cells (or vanishing) of layer is disposed under endothecium. The characteristic of the cells of the layer is their flattening as far as tapetum development. Then they fully disappear. The internal layer of the anther is tapetum. In the sunflower anther it is presented by one row of cells with large nuclei. Tapetum or a covering layer of the anther has been formed to the moment of preparation of pollen mother cells (PMC) to meiosis passage. After the formation of all tapetum cells their nuclei continue to divide without cytokinesis. Therefore they become binuclear from uninuclear or have one polyploid nucleus. At first tapetum is located on the periphery of the anther sac. A layer of the internal tapetum is selected connective outside. The cells of the internal tapetum are of another origin than are the external cells, and in this phase of the anther development these are morphologically substantially differentiated from the latter. A tapetal layer reaches its maximum development to the time of microspores tetrad formation. Thus the wall of the sunflower anther consists of epidermis, endothecium, middle (or intermediate) layer and tapetum to the moment of preparation of PMC to pass meiosis as well as all angiosperms (Fig. A). Each meiocyte is surrounded by callose and becomes the PMC. The process of meiosis begins in PMC approximately 16-18 days before flowering when a heads have reached 2,5-3,0 cm in the diameter. Meiotic studies of the sunflower showed that its stages pass asynchronously in the different sacs of one anther. Even within the limits of one sac the synchronism of division is ambiguously expressed. After the completion of the first division of meiosis in the PMC cell partition does not appear between two daughter‘s nuclei. After the second division all four cells appear simultaneously by the initiation of furrows from the periphery to the centre and divide the protoplast of PMC into separate microspores. So the formation of tetrads takes place on typically simultaneously with the tetrahedron location of microspores in tetrads (Fig. H). In that period the binuclear cells of tapetum are located on the periphery of the pollen sac. The lysis of callose and forming of membranes of young microspores begins (each microspore is surrounded by primexine). Tetrads are disintegrated. The tapetum cells and their walls are made loose and begin to separate away from the other wall layers, but tapetum is still located

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on the periphery of the pollen sac. A microsporogenesis in anther grains is completed 2-3 days before flowering.

Figure A. Micrographs of anther development and forming of pollen in sunflower: A – anther wall before meiosis in PMC of fertile plants (1- epidermis, 2- endothecium, 3- middle layer, 4- tapetum); B – normal diakinesis with 17II; C – polyvalent in diakinesis; D – lagging chromosomes in metaphase I; E – bridges in anaphase I; F – bridge and lagging chromosomes in metaphase II; G – lagging chromosomes in anaphase II; H – a normal tetrad; I – a tetrad with irregular nuclei; J – a tetrad with 2 reduced cells; K – a diad with micronuclei; L – a triad; M – a pentad; N – a hexad; O and P – the cells with the picnotic nuclei; Q – the abnormality of the cytokinesis; R – a mature pollen grain with two falcate-like sperm and one vegetative nucleus; S – the fertile pollen grains with 3 and 4 apertures; T-V – different types of a sterile pollen; W – the pollen of sterile plant before the beginning of flowering with abnormal meiosis; X – anther cross section of a fertile plant before the beginning of flowering (see epidermis and endothecium). Fig. A, R-X from Ryabota [74].

With the release of microspores from tetrads, the cell membranes of tapetum are destroyed, protoplasts are protruded and introduced into the cavity of the pollen sac, forming a general mass of tapetal periplasmodium. With the development of microspores periplasmodium is intensively used for their nutrition and to the moment of pollen maturation fully disappears together with the middle layer. Therefore the walls of mature sunflower anthers are presented by two well developed layers: by epidermis and endothecium (Fig. X). Uninuclear microspores are quickly being increased in their volume, their membrane is intensively being formed, the rudiments of spiny growths appear on an exine. Before the first differential mitosis of the primary nucleus at the beginning small, then larger vacuoles appear which fusing together, create a large one. Cytoplasm together with the nucleus are driven back by the vacuole to the membrane of the pollen grain, forms a parietal layer. A nucleus having an indefinite form after a while is divided forming vegetative and generative nuclei.

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Later on there is ―overgrowing‖ of the central vacuole and a generative nucleus moves from the membrane to the centre of the anther grain. 2-3 days before the beginning of flowering two sperms appear as a result of the generative nucleus division. The mature pollen grain of sunflower contains two falcate-like sperm and the vegetative nucleus with a clear large nucleole (Fig. R). In microspores soon after their release from tetrads (at the stage of uninuclear pollen grains) the exine begins forming, noticeable by the appearance of the rudiments of spiny growths on their surface. The exine of fertile sunflower pollen grains belongs to a compound type and consists of three layers: sexine, mexine and nexine [69]. At this stage the sporoderma of the pollen grain are fully formed and includes ectexine, endexine and intine. The ectexine is cellular, during pollen maturation of its cells are filled with the matters synthesized by organoids and tapetum protoplast and then by the products of its lysis and final destruction. The endexine has a lamellar structure and is built from the layers parallel to the protoplast surface of pollen grain. The intine is laid under the endexine since the binuclear stage. It has been fully developed to the moment of filling of the whole pollen grain by cytoplasm and forming of two sperms. Intine has not the same thickness and its internal surface reflects unevenness and protrusion out of the protoplast relief, therefore varies in all thickness and configuration in the different pollen grain areas. In the region of apertures it is sharply thickened probably executing protective functions and covering the protoplast of pollen grain from external influence as the exine is absent here. A sunflower protoplast is covered by the incrassate layer of intine, which is protruded in aperture here (Fig. S). Tailings of the finally lysed tapetal plasmodium penetrate into the exine through the outward openings, filling the cavities in the ectexine [79]. At maturity, the pollen grain of the sunflower is spherical covered with spines. The colouring of sunflower pollen is more often yellow-orange, as well as, it can be white-cream and white. This trait is controlled by one or two genes depending on the genotypes involved in crossing [25, 31]. The diameter of a mature pollen grain depends on a genotype and varies from 33 to 39 μ [35]. In the majority of pollen grains one can reveal three, seldom four apertures (Fig. S). During development and germination of plant pollen a large number of genes are displayed, among which one part is being expressed at the early stages of maturity of microspore and the other-after mitosis in microspores. According to the estimations of researchers, the mature plant pollen grains contain mRNA produced by approximately 20 thousands of different genes out of these over 4 thousands, probably, are specific for pollen [59]. Up to present a few specific genes of pollen – SF3 [7], SF16 [22], SF21 [48] and new isoform α-tubulin [24] has been revealed in the sunflower. SF3 and SF16 genes are expressed at the late stages of the development of sunflower pollen, while transcripts of SF21 gene appear in the stigma and conducting tissues of the pestile. Probably, these genes take part in the processes of maturation, germination and growth of pollen tubes. The data accumulated on the genetics of pollen allow a conclusion that the significant number of genes of the angiosperm plants is expressed at both diploid and haploid stages of life cycle [64]. In this connection, pollen selection is recently considered as an effective breeding technique, capable of readily causing essential changes in the structure of sporophyte populations [56, 65]. Influence of heating a heterogeneous pollen population on some quantitative characters of the resulting sporophytic generation was studied in the

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sunflower. Heating pollen at 600C for 3 hours was most effective. This treatment significantly increased the frequency of taller plants as well as the portion of genotypes with longer emergence-flowering stage and minimum number of branches [57].

DEVELOPMENT OF ANTHERS AND FORMING OF POLLEN DURING DIFFERENT TYPES OF SUNFLOWER MALE STERILITY There are a several types of sunflower male sterility, depending on the factors determining its manifestation: CMS, NMS, induced male sterility and sterility arising from because of different chromosomal mutations (Table 1). Each of the above-stated types of sterility has not only a theoretical value, for example, for finding out of interaction of genome and plasmon (at CMS), a mechanism of action of nuclear genes (at NMS and CMS), influence of different substances on microsporogenesis, but also a practical value for the production of highly productive hybrids of different direction. Table 1. Basic types of sunflower male sterility Type of sterility Nuclear

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Cytoplasmic

Reason for occurrence of male sterility Nuclear genes ms and msk Interaction plasmagenes and Rf genes nuclei

Modified

Biotic and abiotic environmental factors and gametocytes

Chromosomal

Different types of abnormalities at meiosis

Note Known 11 ms genes and 1 msk gene 4 Rf genes identified Induced downy mildew, high temperature, 24-hours photoperiod, as a gametocyte use gibberellin Often observed at interspecific hybridization

Currently in literature there is sufficient information about the cytological changes of sunflower anthers at nuclear and cytoplasm male sterility [1, 46, 69, 74, 80, 81]. A distinction between these two types of sterility is in that there is nuclear control in first case and, accordingly, sterility manifestation will depend only on the state of nuclear genes. In the second case the sterility display depends on the interaction between plasmon and genome. 1) Nuclear male sterility The first description of sunflower male sterility can be found in the research paper of Kuptcov [49]. A genetic analysis conducted by him showed monogenic recessive control over this trait. F1 hybrids are fully fertile at crossing NMS lines with any fertile genotype. Subsequent genetic researches exposed controlling of NMS in sunflower by 12 genes, which were designated as ms1-ms11[42, 43, 53, 70, 71] and msk [88]. Some of the genes, controlling NMS was mapped with the use of DNA-markers [16, 66]. The cytoembryological study of sunflower NMS has shown that the development of the anthers being in fertile and sterile on pollen samples run identically from the earliest phases to the formation of microspores and their release them from tetrads [69]. Morphologically

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.

noticeable abnormalities appear in the development of sterile anthers, as a rule, at the moment of release microspores from tetrads and exine formation beginning. In the sterile pollen there is no sexine with spines, specific for normal sunflower pollen. A further development of the sterile pollen grains notably falls behind normal and then ceases totally. As a result sterile pollen grains in comparison to fertile ones are considerably poorer by nuclear and cytoplasmic content, nucleic acids, proteins, and also storage, physiologically active proteins and other matters. Obscure differentiation of vegetative and generative nuclei, low viability of the latter are noted as well. The membranes of sterile pollen grains, lacking sexine with spines, are distinct of less thickness, absence of pores and, accordingly, other physical and chemical and physiological properties. In sterile as to the pollen anthers interaction of microspores with surrounding tissues changes (i.e. sporophyte with gametophyte), that is manifested in the slowed process of disintegration of the middle and then also tapetal tissues. As a rule in the endothecial cells fibrous incrassation is absent and their function is destroyed. The listed deviations from normal development result in deceleration, and then cassation of sterile pollen grains development at uninuclear, and more frequently at the binuclear stage. Future on goes degeneration of cytoplasm, deformation and disintegration of nuclei, simultaneously their membranes become deformed. Sterile pollen compresses in lumps and during flowering the pollen does not shatter from the anther. The features of anthers and pollen grains development depending on the type of sterility are described by Vol'f [89] and Ryabota [74]. They have shown that meiosis at some samples passes without visible anomalies and development of tapetum for them takes place normally up to the formation of uninuclear pollen. Then periplasmodium appears from the tapetal tissue, which is deeply introduced in the cavity of the pollen sac, driving back pollen grains to one of anther sides or to its centre. Such a location of periplasmodium is the result that pollen grains of sterile plants are not able to use its content for their growth and development. Thus, the stronger pollen sterilization is expressed, the deeper it deeper gets to the cavity of the pollen sac. With the beginning of sterile pollen degeneration also the plasmodium lyses. Epidermis and endotecium of the walls of mature anther of sterile plants are developed weaker, than in fertile ones. Pollen development at different types of NMS is ceased at the uninuclear or binuclear stages, but sometimes there are separate pollen grains, which reach at the third-nuclear stage. Then the degeneration of sterile pollen begins, which in broad sense results in separating cytoplasm of pollen grain from its membrane and its condensing round the nuclei, deformation of membrane, causing the disturbance of the normal form of pollen grain, comes then: lysis of cytoplasm and nuclei (Fig. T-V). The creation of sunflower heterotic hybrids on the basis of NMS comes across with a series of difficulties. Therefore their production has become simpler with the use of CMS. 2) Cytoplasmic male sterility The first sunflower CMS was observed by Leclercq in the 1960s from the crosses between the wild type of H. petiolaris with the cultural one – H. annuus [52] and the active study of the genetic system of sunflower CMS-Rf began from that time. Today, over 70 sources of CMS are known, out of these wild species of genus Helianthus are basic [77]. Most CMS sources are identified in the annual species of sunflower. Also sunflower CMS can be also induced by the chemical matters of different nature [42].

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Naturally, the involvement of different accessions, which have a sterile cytoplasm in the breeding process is impossible and unthinkable without the restorers of pollen fertility, which serve as testers for a certain type of cytoplasm. The larger is identified for a classic cytoplasm – PET1 and the creation of new inbred lines carrying the Rf genes to this cytoplasm is not problematic as compared to the other CMS types. The known commercial lines are restorers of pollen fertility. At present, the study of inheritance and alleles interaction of genes fertility restoration is being intensively conducted to determine the solution of certain breeding problems [44, 78]. The results of the study on inheritance of Rf genes show that one gene (Rf1) with its possible interaction at least with 2-3 genes play a key role in fertility restoration of pollens [44]. Undoubtedly it is of interest to search Rf genes-donors for different cytoplasm in wild species, interspecific hybrids, cultivars and other initial materials. In our research, the ability of sunflower accessions produced by interspecific hybridization to restore fertility of pollen in the accessions having cytoplasm of PET1, MAX1 and GIG1 was not identical. It was shown that the level of restoration fertility varied from 0 to 100 %, so there was a partial restoration of pollen fertility in some hybrid combinations. A comparison of the restoration ability of some accessions with three different cytoplasm have revealed the interaction between nuclear genes and plasmagenes that manifested up in a different ability of samples to restore fertility. The multidirectional effects were also found out; for example, one of the accessions fully restored fertility in the sterile analogue on the basis of PET1 and partly for GIG1 and MAX1 [84]. A number of the actual research aspects related to the genetics of pollen fertility restoration belongs to the mapping of genes using markers based on RFLP and PCR analyses. Molecular mapping of Rf1 [32, 39, 45, 50, 85] and Rf4 [27] genes has been made with their use that will allow the involvement of markers linked with these genes in a practical breeding, primarily using MAS (marker-assisted selection). In a general way, the changes that occur during forming of anther walls are similar to NMS of the sunflower [62] and CMS [40]. Common in these types of male sterility, overgrowths of cells of external and internal tapetum without its transformation into plasmodial take place. The comparison of the cytological features of pollen development in the sunflower accessions with NMS and CMS was attempted by Simonenko [81]. He determined the presence of clear cytological distinctions between NMS and CMS. These differences involve the disturbances at NMS to be revealed at the stage of pollen grain forming and be manifested in absence of spines on the exine due to disturbances in supply of nutritive synthesized by the tapetal tissue. In the case of CMS there is anomal development of the anther already at the early stages of its development. It involves a process of the degeneration of cellular walls of tapetum resulting in drainage of their content and gradual incrassation and occupying of anther space. In some cases the meiosis can pass with tetrad formation but an overgrowing tapetum squeezes uninuclear pollen grains that bring about their following degeneration. All contents of anther are destroyed at the last stage. While studying the histological aspects on the development of anther walls in the sunflower Meric [60] has shown that the distinctions between fertility and CMS plants aren‘t revealed to the tetrad stage. In a sterile line НА89А has a vacuolated and widened middle layer at the late tetrad stage, tapetum is also enlarged and does not form the typical fertile form periplasmodium. During the further development of the anther wall the middle layer and

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tapetum do not disappear. The safe tapetum hinders the development of endotecium (endothelial cells do not have fibrous incrassation). In plants, most anthers at CMS (as well as at NMS) endothelial cells do not broaden and not have fibrous incrassation. The maintained tapetal cells continue to produce an inhibitor, which hinders endotecium differentiation [15]. Simonenko used electron microscopy for the study of the sunflower CMS [80]. The anomalous structure of cytoplasm organelles and tapetum nuclei were observed. As a result, tapetum had an unnormally active development and remained near the wall, additionally, did not transform into periplasmodium and the microspore tetrads degenerated. For example, degeneration of microspores of tetrads and microspores in the CMS of the sunflower is exposed during the result of violation disturbances in the normal development of tapetal layer. Direct evidence of the critical role of tapetum in pollen development is in the example of transgenic plants. It has been shown that a foreign ribonuclease gene expressing a toxic product under the effect of a specific promoter for tapetal tissue initiates forming of sterile anthers [58]. The change of mitochondrial genome, which is carried by all cells of CMSplant, takes place exactly in the tapetal tissue. The molecular mechanisms of the interaction of nuclear and mitochondrial genomes are studied for the majority of crops. It has been shown that CMS-forms have chimera genes, which appear as a result of the reorganization of the mitochondrial genome. The differences in the structure of mitochondrial DNA (mtDNA) of sterile and fertile forms allow identification and localization of CMS-loci in the mitochondrial genome and allow study of their molecular structure [23]. The reorganization of the mitochondrial genome related to disturbances of pollen formation was shown in higher plants of different systematic groups. At present CMS-loci are located in the mitochondrial DNA of plants of different species: corn, petunia, rice, kidney bean, sorghum, garden radish and sunflower [19]. However, it was shown by different researchers that on the different CMS-systems the nature of disturbances resulting in the formation of nonviable pollen can be substantially different. The field measuring 17 kb, which is characterized by the presence of inversion (12 kb) and the insertion of 5 kb flanked by the inverted repetitions measuring 261 bp was observed during the research of the mitochondrial DNA of sunflower sterile forms. Such structure of mtDNA of sterile analogues determines their distinction from the fertile sunflower accessions. In turn, the insertion size of 5 kb consists of the open reading frame – orf522, which the atpA gene joins. Transcription of these areas takes place together and the product of these genes is protein with a molecular weight of 16 kDa. It is suggested that this protein blocks the chain of electrons transport by insertion into the membranes of mitochondria [38, 61]. Subsequently it was shown that many sources of CMS of sunflower differ in the certain areas of mtDNA. Ten basic mitochondrial types were selected from the data of blothybridization of nine mitochondrial genes of atp6, atp9, cob, cox1, cox2, cox3, 18s, 5s, nd5 and 3 open reading frames – orf522, orf708, orf873 [37]. In each selected type they include from 2 to 4 CMS sources. Some CMS sources were not included in any of the identified groups showing unique hybridized signals on certain mitochondrial loci. All these results show a unique structure of some sunflower cytoplasm and complexity of their organization. This research also exposes a molecular organization of the system of CMS-Rf in sunflower and brings us closer to understanding of mechanisms of nuclear - mitochondrial interactions, that in turn allows the conduct of the purposeful selection of pairs in the system ―cytoplasm – Rf genes‖ in a practical breeding.

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The investigations of the last several years prove that one of the mechanisms, which lead to the formation of abortive pollen of the sunflower, is also related to the phenomenon of apoptosis. The premature apoptosis of tapetum cells, which spreads on the other tissues of the anther, was observed when studying CMS - PET1 in the sunflower. It is related to the release cytohrome c from mitochondria, which activates a proteolytic enzyme cascade that results in the degradation of nuclear DNA and cell death. In this example the key role of plant mitochondria in the programmed cell death is shown [6]. It should be noted that the information available in literature about the mechanisms of genome - plasmon interactions in the sunflower does not illustrate a complete picture of the origin of sterile pollen. Foremost, it is stipulated by the presence of a large number of different CMS types in the sunflower. The identical phenotypical manifestation of sterility is probably related to various disturbances in their mitochondrial genome. For the final elucidation of the processes, which result in the formation of abortive pollen, it is necessary to establish how expression of CMS-genes and also the expression of Rf genes in time and space are controlled.

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3) Induced male sterility in sunflower For most plant species the influence of different environment factors on manifestation of male sterility was shown. Morphological changes are especially expressively noticeable in sunflower as a result of its affection by a very harmful disease downy mildew, substantially reducing the plants productivity. These changes at a strong degree of affection of plants are observed during the change of the flowering date (the affected plants burst into blossom 3-10 days before) and increase of flowering duration (to 20 days), the change of male and female reproductive mechanisms and a reduction in the degree of growth (a difference can arrive several times as compared to normal plants). The abnormalities are revealed by change of in anther colouring from dark to light, a greater part of pollen grains are shrunk. Such badly infected plants do not form seeds unlike plants which have a low degree of affection. They possess male sterility, but able to set a small number of seed (to 30 seeds per head), thus they a bit differ from healthy plants morphologically [2, 67]. Demurin with the coauthors [20] described sunflower sterility, which was induced by the change in the duration of photoperiod, and named it as photoperiodical male sterility. As a result of 24-hours photoperiod during 5 days, the authors‘ research work observed some changes in flowering of sunflower tubular flowers. These violations showed in complete absence of stamen filaments growth and, as a result, the anther tube with pollen didn‘t go out from a corolla, although the development of a corolla and anthers was normal. Thus selfpollination did not take place, i.e. plants were sterile. The obtained male sterility plants have been used as a maternal component of crossing for the production of the hybrid seed by the authors. The researchers succeeded producing hybrid seeds by crossing photoperiodic male sterility accessions with normal fertile plants. It points to the normal production of female gametes. The uses of chemical castration with the purpose of elucidation is a mechanism of the gametocytes action and involve male sterility in plants. This method of selection for the production of high-yielding hybrids of basic crops date back to between the 1960s to the 1980s [30]. The gametocytes used for sterilization must primarily induce 100% sterility of

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male reproductive sphere with a clear morphological display on anthers and possess high efficiency in different climatic areas and at the different stages of plant development [10]. A present interest relating to the synthesis of new chemical matters of different natures is at the forefront of research. [41]. As a result, it has been shown that androecia is more sensitive to the gametocytes action of chemical nature than is gynaecium. This is because gametocytes use various chemical matters related to the different groups of compounds (retardants, growth regulators). According to the research of Campos [12], it is shown that ethrel (2-chloroethylphosphonic acid) induces male sterility. Triphathi [87] observed anomalous development of anthers after treatment of the sunflower leaves by 1.5 % benzotriazole (C6H5N3), which induced 100 % pollen sterility. In the treated plants the pollen underdevelopment was generally related to anomalous tapetum development. The sterility was conditioned by early tapetum degeneration and their cells had degenerative and non-functioning (with a changed structure) organoids (mitochondria and plastids). Thereby the tapetum did not provide the proper feeding for developing microspores in the treated sunflower plants by benzotriazole. Pollen grains formed from such microspores were deprived of nuclei, cellular organoids and were fully sterile. However, the growth regulator gibberellin appeared to be most effective for sterilization of sunflower pollen [2, 17, 18]. The choice of an optimum gibberellin concentration for male sterility induction has a decisive value. It depends on whether it will be able to get the male sterile sunflower forms and whether the sterility of female reproductive sphere will not be caused. The essence of this approach is in the individual sprinkling of the solution of gibberellin on sunflower plants, while they are in the phase of wall outlet (the formation of generative organs) at 6-8 pairs of real leaves. Today the unequal results of gibberellin influence have been obtained on the sunflower generative vehicle. In the experiments of Anaschenko [2] and Chiryaev [18] it is shown that the optimum concentration of gibberellins is its 0,005% solution inducing 100% sterility of sunflower pollen. It is recommended to use such concentration for cultivars and to increase it to 0,01% with the purpose of the production of induced male sterility in inbred lines. However, from the point of view of some authors, who have conducted detailed research on the influence of gibberellin on the generative organs of sunflower, consider that the plants production with 100% sterility is contiguous to a series of difficulties, related to the control complication of the beginning of microsporogenesis, also the influence of environmental conditions and the sterilization of female generative sphere. So, during the investigations a different gametocyte effect of gibberellin was exposed that allowed authors to select three forms of male sterility, differentiating some changes at the late stages of meiosis and morphologically as well [26]. Using gibberellin during gametocyte sterility, it is significant to note when sterility begins: while the cells of internal tapetum are spread out; thus, the sterility has begun in the period during the forming of secondary archesporium (stage 1 by Horner); or if the treatment of plants by gibberellin is made in the period of the forming of primary archesporium in the anthers; or still earlier by gibberellin. The stage of pollen grains development, at which their final sterilization takes place during a plants treatment of gibberellin (stage 6) coincides with that at CMS [40]. The differentiation of tapetum and anther wall layers in the absence of sporogenic tissue forming is marked during the treating of gibberellin (0,01%). Pollen sacs surrounded by tapetum, the middle layer and epidermis are empty [46]. During the treatment of sunflower anthers in the same phenophase by the solution of gibberellin in a smaller

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concentration (0,005%) the degeneration of pollen grains takes place only on the stage of binuclear pollengrains (stage 9) [46]. Cytological disturbances of anthers development were studied at gametocyte male sterility of sunflower by the authors‘ research [81, 46]. The distinctions were exposed only at the late stages of pollens grains forming (before the forming of bicellular pollen) by Simonenko and Karpovich. Their research noted that development ceased and there was a gradual degeneration of tapetum. Konstantinova notices more early effects of the influence of gibberellin on anther development, which have been already noticeable since the stage 1 (acc. to Horner). Probably, such a different effect of gametocyte is related to its different concentration, which the authors used in the research. Konstantinova used a large concentration of solution of gibberellin, twice as much – 0,01% ,as compared to the research of the other author (0,005%). A specificity of the action of gibberellin in different concentrations on the different stages of microsporogenesis was shown by Konstantinova.

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4) Sunflower sterility arising at interspecific hybridization At present, interest has been growing about the wild species of sunflowers‘ potential sources of resistance genes to the biotic and abiotic factors of the environment, high-quality composition of oil, and the sources of new types of CMS etc. [29, 47]. Recently a number of wild species of the genus Helianthus have been explored while being crossed with a cultivated sunflower [72]. The genus Helianthus is intensively studied for the purpose of the species origin, speeds evolution progress and was, in a wider sense, a model object for understanding of mechanisms of speciation in plants [11, 36, 83, 86]. The crossability between sunflower species may suggest their phylogenetic relation in the genus Helianthus [34, 73]. Nowadays such results are corrected by using molecular data [33, 51, 54, 75, 76, 82, 90]. In the case of a successful production of sunflower interspecific hybrids, their further use in breeding will depend on the features of development of such hybrids and, foremost, normal forming of female and male generative sphere. However, it has been shown that the microsporogenesis passes with considerable violations by numerous cytological investigations of interspecific hybrids obtained within the limits of different of plants‘ genera, including the genus of Helianthus, is a reason for hybrids sterility [4, 9, 34, 43]. The plants‘ sterility can be manifested at interspecific hybridization in crosses of not only different genomic species but also species having identical number of chromosomes. Foremost, it is related to a violation of recombination of the inherited material that results in most cases of the hybrids sterility of the first generation. For most plants species a direct relationship between pollen sterility and disturbance arising in meiosis has been determined. The same peculiarity is relevant to the genus Helianthus. During interspecific hybridization there can be the situations when chromosomes of the crossed accessions are fully homologous or their partial or complete unhomology is observed. In these cases the question is about congruent and incongruent crosses, accordingly, at which, there is a fully fertile offspring in the first case and in the second or other degree a sterile one. The annual species of sunflower have a high degree of compatibility at crossing between themselves and are able to form a fertile offspring, while, crossing between annual and perennial species is hardly managed. The research of Georgieva-Todorova [34], Atlagic [3-5], Jan [43] and others have shown that there are substantial deviations in meiosis in the

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Table 2. The level of chromosomal conjugation and frequency of chromosomal associations at the prophase of the meiosis I in sunflower

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Hybrid combination

H. neglectus × H. annuus Х1006-B H. annuus ANN-1064 × H. annuus Х1006-B H. praecox × H. annuus Х1006-B H. annuus ANN1366 × H. annuus Х1012-B H. annuus H-151 × H. annuus Х908-B H. argophyllus × H. annuus Х908-B H. annuus ANN1064 × H. annuus Х908-B H. praecox × H. annuus Х908-B BC1 (H. praecox × H. annuus Х908-B) × H. annuus Х908-B BC2 (BC1 × H. annuus Х908-B

Meiocytes studied

Number of chiasmata

Number of closed bivalents per meiocyte

per meiocyte 22.15 ±4.86 22.02 ±0.12 19.72 ±0.11 21.92 ±0.11 23.80 ±0.14 20.59 ±0.13 22.62 ±0.14 19.94 ±0.13

per bivalent 1.30 ±1.30 1.30 ±0.01 1.16 ±0.01 1.29 ±0.02 1.40 ±0.01 1.21 ±0.01 1.33 ±0.01 1.17 ±0.01

206

19.77 ±1.12

1.16 ±0.07

2.70 ±1.09

102

20.00 ±1.13

1.18 ±0.07

2.92 ±1.14

73 287 192 260 233 154 188 206

5.03 ±2.51 4.99 ±0.12 2.67 ±0.10 4.83 ±0.10 6.59 ±0.12 3.53 ±0.13 5.55 ±0.13 2.88 ±0.13

Univalents, %

Tetravalents, %

Hexavalents, %

17II , %

Other abnormalities, %

16II + 2I

15II + 4I

15II + 1IV

13II + 2IV

14II + 1V I

0

0

1.4±1.4

0

0

1.4±1.4

1.0±0.6

0

0.3±0.3

0

0

11.5±1.9

9.4±2.1

2.1±1.0

4.7±1.5

0.5±0.5

1.0±0.9

3.1±1.3

3.1±1.1

0.4±0.4

0

0

0

2.3±0.9

0.4±0.4

0

2.6±1.0

0.9±0.6

0.9±0.6

4.2±1.3

2.6±1.3

0

9.1±2.3

1.3±0.9

0

3.2±1.4

1.1±0.8

0

0

0

0

0

1.9±1.0

1.5±0.8

5.3±1.6

0

1.9±0.5

15.0±2.5

76.7 ±3.0

13.6±2.4

2.9±1.2

2.4±1.1

0

0.5±0.5

3.9±1.4

59.8 ±4.9

8.8±2.8

3.9±1.9

1.0±0.9

0

0

26.5±1.3

97.3 ±1.9 87.1 ±2.0 79.2 ±2.9 94.6 ±1.4 91.0 ±1.9 83.8 ±3.0 99.0 ±0.7 76.2 ±3.0

ion?docID=3018106.

Table 3. The frequency of pollen mother cells with abnormalities in meiosis in the interspecific hybrids of the genus Helianthus

H. praecox × H. annuus Х1006-B

2811

29.6 ±0.9

401

4460

8.6 ±0.4

588

3760

7.3 ±0.4

623

4041

6.5 ±0.4

703

H. argophyllus × H. annuus Х908-B

3433

6.7 ±0.4

469

H. praecox × H. annuus Х908-B

3962

BC1(H. praecox × H. annuus Х908B) × H. annuus Х908-B

4495

BC2 (BC1 × H. annuus Х908-B

3072

50.0 ±0.8 12.8 ±0.2 9.6 ±1.7

556 660 464

533 256 585 278 578 372 261 565 376

with abnormalities, %

603

Telophase II

total

8.9 ±0.4

with abnormalities, %

4343

84

Anaphase II

total

H. annuus ANN-1064 × H. annuus Х1006-B

24.8 ±4.7 17.1 ±1.5 25.2 ±2.2 15.0 ±1.5 17.7 ±1.5 15.2 ±1.4 14.7 ±1.6 47.1 ±4.5 15.2 ±1.4 13.8 ±1.6

with abnormalities, %

101

Metaphase II

total

38.6 ±1.5

with abnormalities, %

1083

Anaphase I

total

H. neglectus × H. annuus Х1006-B

with abnormalities, %

Hybrid combination

H. annuus ANN-1366 × H. annuus Х1012-B H. annuus H-151 × H. annuus Х908-B H. annuus ANN-1064 × H. annuus Х908-B

total

with abnormalities, %

Metaphase I

total

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Meiocytes studied

78.6 ±4.5 11.6 ±1.4 41.1 ±3.1

73

78.1 ±4.8

91

94.5 ±2.4

202

28.7 ±3.2

432

8.1 ±1.3

399

6.8 ±1.3

493

2.6 ±0.7

327

63.0 ±2.7

370

19.46±2. 1

7.7 ±1.1

562

481

13.9 ±2.5

535

2.6 ±0.7

14.0 ±2.1 12.6 ±1.4 7.5 ±1.4 66.7 ±2.9 10.8 ±0.4 7.55 ±1.4

237

44.35±3. 2 10.1 ±1.3

524

2.7 ±0.7

288

5.9 ±1.4

504

0.8 ±0.4

508

1.6 ±0.6

499

1.8 ±0.6

504

0.2 ±0.2

456

9.0 ±1.3

414

10.9 ±1.5

442

1.8 ±1.6

371

70.1 ±2.4

603

19.5 ±1.6

536

11.0 ±1.4

586

8.2 ±1.1

434

7.8 ±1.3

375

9.3 ±1.5

442 499 357

51.4 ±2.4 16.6 ±1.7 5.9 ±1.3

ion?docID=3018106.

Table 4. The frequency of the abnormalities in meiosis at the tetrad stage in the interspecific hybrids of the genus Helianthus

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Hybrid combination

Total number studied cells

Average frequency of tetrads, % normal

with micronuclei

Monads, %

Diads, %

Triads, %

Polyads, %

Other abnormalities, %

H. neglectus × H. annuus Х1006-B

368

76.1±2.2

13.6±1.8

0

1.9±0.7

6.0±1.2

0.8±0.5

1.6±0.7

H. annuus ANN1064 × H. annuus Х1006-B

1137

91.5±0.8

0.4±0.2

0.3±0.2

0.1±0.1

1.0±0.3

6.0±0.7

0.7±0.2

H. praecox × H. annuus Х1006-B

671

89.4±1.2

6.8±1.0

0

0.9±0.4

1.0±0.4

1.2±0.4

0.6±0.3

H. annuus ANN1366 × H. annuus Х1012-B

938

92.8±0.8

0.9±0.3

0.4±0.2

0.3±0.2

2.2±0.5

1.3±0.1

2.1±0.5

H. annuus H-151 × H. annuus Х908-B

919

94.7±0.7

0.1±0.1

0

3.5±0.6

1.4±0.4

0.1±0.1

0.2±0.1

H. annuus ANN1064 × H. annuus Х908-B

560

89.3±1.3

1.8±0.6

0.2±0.2

1.8±0.6

0.9±0.4

0.7±0.4

5.3±0.9

H. argophyllus × H. annuus Х908-B

862

99.0±0.3

0.3±0.2

0.1±0.1

0

0.3±0.2

0

0.2±0.2

H. praecox × H. annuus Х908-B

1134

44.8±1.5

9.0±0.8

0.5±0.2

15.5±1.1

18.3±1.3

3.3±0.5

8.5±0.8

BC1 (H. praecox × H. annuus Х908-B) × H. annuus Х908-B

916

86.3±1.1

4.26±0.6

0

0.4±0.2

8.3±0.9

0.3±0.2

0

BC2 (BC1 × H. annuus Х908-B)

539

93.7±1.0

3.3±0.8

0

0.2±0.2

2.6±0.7

0.2±0.2

0

ion?docID=3018106.

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150

V. Popov, T. Dolgova and V.V. Dokuchaev

interspecific hybrids in the system of crossings between annual and perennial species. Such hybrids have abnormality in passing the meiosis and as a result sterility was observed. The interspecific hybrids obtained within the limits of the section of Helianthus (annual type of development) are characterized by the stable passage of meiosis and high fertility (Table 2-4) [21, 34, 43]. However, there can be serious issues at the recombination of chromosomes from their partial or complete unhomology in interspecific hybrids, in spite of relatively good compatibility between annual species. So, the study of meiosis in the interspecific hybrids obtained from the cross of H. praecox with the cultural sunflower species H. annuus showed the presence of various distortions in 50 % PMC (Table 3). Thus, in the crosses of wild species of H. annuus with different inbred lines, an amount of cells with distortions was 6,58,9 % (Table 3). The formation of tetravalents and hexavalents was typical of main disturbances (Fig. C) in diakinesis and on the subsequent stages of meiosis were bridges (Fig. E) and lagging chromosomes (Fig. D, G) in the hybrid of H. praecox × H. annuus X908-B. The monads, dyads, triads, pentads, hexads as well as cells with irregular nuclei, pycnotic nuclei, and abnormality of the cytokinesis or 1-4 micronuclei were observed on the stage of tetrads forming (Fig. I-Q). The obtained results allowed us to suppose that the studied species H. annuus (cultural) and H. praecox, though belonging to one taxonomical unit, probably have a considerable number of distinctions in the structure of chromosomes that requires additional research involving methods related to a differential colouring of the chromosomes, and, as well as, hybridization in situ. Currently the last method has already found its application in cytogenetically research of sunflower [13, 28, 55, 63]. Backcrossing of F1 interspecific hybrid by the sunflower cultural form (H. praecox × H. annuus) × H. annuus led to a reduction of number of meiocytes with disturbances in 4 times (Table 3). A meiotic index has been determined for the estimation of meiosis stability in hybrids which is a percent of normal tetrads from a total amount of the studied. A plant is considered to be cytologically stable at a meiotic index 90 % and higher, and so able to reproduce a stable number of chromosomes in offspring. A value of meiotic index was 86,3% in BC1 plants that is indicative of a cytological instability of this hybrid. At repeated backcrossing by the sunflower cultural form, the amount of the anomalous final meiosis products decreased as compared to BC1 2 times, and a meiotic index at BC2 reaches at 93,7%; that is indicative of a tendency to cytological stability of BC2 [91].

CONCLUSION Since the moment of the first description of anther development and sunflower pollen, more than 40 years have passed and during this period definite accomplishments have been achieved. Substantial results on the comparative study of morphological, anatomic, cytological, genetic and biochemical features of sterile and fertile accessions of sunflower have been accumulated. In addition, interesting data in the area of artificial induction of sterility under the influence of exogenous physical and chemical factors have been achieved. Leclerc‘s discovery of CMS in the sunflower gave an impetus for this biological phenomenon to be studied at different levels: beginning from morphological features of androecium and concluding with plasmon and genome interaction.

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Sunflower Pollen: Theoretical and Practical Aspects

151

Today the genes controlling NMS and CMS have been already identified, moreover, the researches on their mapping and establishment of linkage group according to different DNAmarkers are actively conducted, and that in turn will allow an optimization of a laborious selection process. The exposed cytoplasm polymorphism in the genus Helianthus has revealed new possibilities in sunflower breeding; thus, it has extended a genetic variety of the initial material. On the basis of the identified CMS sources it is possible to produce new breeding material and practical results have been already achieved in this direction. One of the causes of pollen sterility is interspecific hybridization that widely used for introgressing genetic material from wild species into the genome cultivated species for improving important agronomical characteristics. Numerous cytogenetic investigations have shown microsporogenesis in the interspecific hybrids passes with various abnormalities and that it is a principal reason of hybrids sterility. Thus, if annual species of sunflower have, as a rule, high compatibility while crossing between itself and are able to form the fertile offspring, then crossing of annual species with perennial result in the complete sterility of hybrids. Methods of molecular genetics have taken recently a central place in the study of mechanism of forming sterility, differential expression of genes in the process of microsporogenesis and mechanism of incompatibility in the interspecific crossing. Nowadays with the use of molecular methods the following scientific directions are actively developed: study of tissue-specific expression of genes, Rf-genes mapping, phylogenetic analysis of the genus Helianthus.

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[65] Ottaviano, E., Sari Gorla, M., & Mulcahy, D. L. (1990). Pollen selection: efficiency and monitoring. Isozymes: structure, function and use in biology and medicine (pp. 575588). Wiley-Liss. Inc. [66] Perez-Vich, B., Berry, S. T., Velasco, L., et al. (2005). Molecular mapping of nuclear male sterility gene in sunflower. Crop science, 45, 1851-1857. [67] Petrenkova, V. P., Krivosheeva, O. V., Markova, T. Yu. et al. (2005). Diseases and pests of sunflower. Kharkiv: Yurjev Plant Production Institute (in Ukrainian). [68] Pirev, M. N. (1968). A study of the male reproductive organs of sunflower with normal and sterile pollen. Bull. Acad. Sci. Moldav. SSR, 14, 62-71 (in Russian). [69] Pirev, M. N. (1969). Cytoembriological researches of the phenomenon to male sterility of sunflower. A study of nature of pollen sterility and use of it in the breeding of agricultural plants (pp. 85-96). Kishinev: Kartya Moldovenyaske (in Russian). [70] Pogorleckiy, B. K. (1973). Genetic marking of male sterility of sunflower, Genetics, 9 5, 23-29 (in Russian). [71] Pogorleckiy, B. K., & Burlov, V. V. (1971). About the inheritance of male sterility in sunflower, Genetics, 7 8, 59 (in Russian). [72] Popov, V. N., Yushkina, L. L., Sharypina, Ya. Yu., et al. (2005). Genotypic features of combining ability of cultural sunflower with wild species and use of embrioculture during remote hybridization. Cytology and genetics, 39, 3-8 (in Russian). [73] Rieseberg, L. H., Kim, S. C., Randell, R. A., et al. (2007). Hybridization and the colonization of novel habitats by annual sunflower. Genetica, 129, 149-165. [74] Ryabota, A. N. (1971). Cytogenetic researches of male sterility of sunflower (Helianthus annuus L.). Thesis of candidate of biological sciences. Kharkiv (in Russian). [75] Schilling, E. E. (1997). Phylogenetic analysis of Helianthus (Asteraceae) based on chloroplast DNA restriction site data. Theor. Appl. Genet., 94, 925-933. [76] Schilling, E. E., Linder, C. R., Noyes, R. D., et al. (1998). Phylogenetic relationships in Helianthus (Asteraceae) based on nuclear ribosomal DNA internal transcribed spacer region sequence data. Systematic botany, 23 2, 177-187. [77] Serieys, H. (2005). Identification study and utilization in breeding programs of new CMS sources. Proc. 2005 sunflower subnetwork progress report (pp. 47-53). FAO, Rome, Italy. [78] Sharypina, Ya. Yu., Popov, V. N., Dolgova, T. A., et al. (2008). Study of inheritance of morfological traits in sunflower. 1. Genetic control of sunflower flowers, branchiness and restoration of pollen fertility. Cytology and genetics, 5, 47-53. [79] Simonenko, V. K. (1977). Ultrastructure of developing microspore of sunflower. Cytology and genetics, 11 5, 395-397 (in Russian). [80] Simonenko, V. K. (1982). Development of anther and microspores at fertility and CMS- lines of sunflower. Cytology and genetics, 16 5, 34-41 (in Russian). [81] Simonenko, V. K., & Karpovich, E. V. (1978). Cytological display of different types of male sterility at a sunflower. Scientific and technical bulletin SGI, 31, 32-38 (in Russian). [82] Sossey-Alaoui, K., Serieys, H., Tersac, M., et al. (1998). Evidence for several genomes in Helianthus. Theor. Appl. Genet., 97, 422-430.

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[83] Strasburg, J. L., & Rieseberg, L. H. (2008). Molecular demographic history of the annual sunflower Helianthus annuus and H. petiolaris – large effective population sizes and rates of long-term gene flow. Evolution, 62 8, 1936-1950. [84] Sytnik, D. V., Popov, V. M., & Kirichenko, V. V. (2006). Interaction of different types of cytoplasm with Rf genes wild species and interspecific hybrids of sunflower. Breeding and seed production, 92, 116-121 (in Russian). [85] Tang, S., Yu, J. K., Slabaugh, M. B., et al. (2002). Simple sequence repeat map of the sunflower genome. Theor. Appl. Genet., 105, 1124-1136. [86] Timme, R.E., Simpson, B. B., & Linder, C. R. (2007). High-resilution phylogeny for Helianthus (Asteraceae) using the 18S-26S ribosomal DNA external transcribed spacer. American journal of botany, 94 11, 1837-1852. [87] Triphathi, S. M., & Singh, K. P. (2008). Abnormal anther development and high sporopollenin synthesis in benzotriazole treated male sterile Helianthus annuus L. Indian J. Exp. Biol., 46, 71-78. [88] Vilichku, F. K. (1989). Genetic study of new type of gene male sterility at the sunflower. Proceedings on the applied botany, genetics and selection, 125, 79-81 (in Russian). [89] Vol'f, V. G. (1966). Use of male sterility in the sunflower breeding / Plant breeding of with the use of cytoplasm male sterility. Proceedings of conference on the use of cytoplasm male sterility in a breeding and seed production of cultural plants (pp. 423433). Kiev (in Russian). [90] Wills, D. M., & Burke, J. M. (2006). Chloroplast DNA variation confirms a single origin of domesticated sunflower (Helianthus annuus L.). Journal of heredity,97 4, 403408. [91] Yushkina, L. L., Nesterova, E. V., Kirichenko, V. V., et al. (2009). Cytogenetic study of interspecific hybrid Helianthus praecox × H. annuus, his paternal forms and two backcrosses. Cytology and genetics, 43 1, 42-47 (in Russian).

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In: Pollen: Structure, Types and Effects Editor: Benjamin J. Kaiser, pp. 157-177

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Chapter 6

REGULATION OF POLLEN FERTILITY IN THE „9E‟CMS-INDUCING CYTOPLASM OF SORGHUM: INTERACTION OF PLANT GENOTYPE WITH ENVIRONMENT L.A. Elkonin, M.I. Tsvetova, V.V. Kozhemyakin and O.P. Kibalnik† Agricultural Research Institute for South-East Region, Saratov, 410010, Russia

ABSTRACT

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Restoration of pollen fertility in plants with cytoplasmic male sterility (CMS) is known to be controlled by specific nuclear fertility-restoring genes that suppress functioning of aberrant mitochondrial genes, expression of which destroys pollen development. Usually, these genes are dominant and manifest in the F1 generation. In sorghum, pollen fertility restoration in some CMS-inducing cytoplasms (A4, ‗9E‘, ‗M351A‘) has aberrant mode of inheritance: it is stably expressed in self-pollinated progenies of F1 hybrids with restored fertility but unstably manifested in new hybrid genome (F1 or test-cross hybrids to CMS lines with the same cytoplasm type). We found that in the ‗9E‘ cytoplasm this phenomenon is caused, evidently, by environmental conditions during test-cross hybrid plant development, namely water-availability and photoperiod. Experimental data testify to strong sensitivity of fertility-restoring genes to plant wateravailability conditions at microspore- and gametogenesis: at high level of water availability these genes are dominant and can express in heterozygous state (in test-cross hybrids), while in drought conditions fertility-restoring genes are recessive and can function only in homozygous state. In addition, reduced photoperiod at photoperiodically-sensitive stage of sorghum plant ontogenesis also increased percentage of male-fertile F1 plants, perhaps, by ‗activation‘ of fertility-restoring genes, and these genes expressed in the next generation. Changes of environmental conditions during male-sterile hybrid plant ontogenesis can ‗activate‘ expression of fertility-restoring genes. Cytological analysis revealed significant polymorphism of pollen grain (PG) types in CMS-lines with the ‗9E‘ cytoplasm and in the F1 hybrids with restored male fertility.  †

[email protected] All-Russian Research Institute for Sorghum and Maize ―Rossorgo‖, 410050, Russia, P.O.Box Zonalnoye

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L.A. Elkonin, M.I. Tsvetova, V.V. Kozhemyakin et al. Both sterile and fertile plants had ―fertile‖ (normally colored) PGs, PGs with anomalous shape, with delay of development at one- or two-nucleate gametophyte, with disturbed starch accumulation (incompletely filled with starch and PGs of waxy-type), with entirely degenerated content. Additional watering increased percentage of fertile PGs but no clear correlation between their frequency and seed set has been found. The data obtained demonstrate that in the ‗9E‘ cytoplasm pollen fertility, obviously, is epigeneticallyregulated trait depending both from genotype and plant environmental conditions.

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INTRODUCTION Development of fertile pollen grain – plant male gametophyte – is a key step of plant sexual reproduction. This is a complex process that includes sequential stages of anther formation, differentiation of sporogenic tissue, meiosis, and maturation of pollen grain. These stages are controlled by numerous closely interacting nuclear and cytoplasmic genes. Distortion of this interaction by mutations, or by remote hybridization results in nuclear (NMS) or cytoplasmic (CMS) male sterility. At present time, different types of male sterility mutations have been described in more than 300 plant species (Kaul, 1988). These mutations are used for investigation of genetic control of pollen development as well as for obtaining of F1 hybrid seeds in plant breeding and in industrial hybrid seed production. In this connection, CMS systems have much greater importance and have been intensively investigated in the past years. Nowadays, it was clearly established that CMS arises from disturbed interaction of nuclear and mitochondrial genomes. At the molecular level this disturbed interaction results in expression of specific CMS-associated mitochondrial genes originating from high recombination activity peculiar to mitochondrial genome (Chase & Gabay-Laughnan, 2004; Hanson & Bentolila, 2004; Fujii & Toriyama, 2008). These genes encode proteins impairing mitochondrial functions at the stage of microsporogenesis and/or microgametogenesis. However, expression of these genes takes place only in hybrids, when they interact with alien nuclear genomes. In fertile lines-donors of CMS-inducing cytoplasms, functioning of CMSinducing genes is inhibited by certain nuclear fertility-restoring genes, which suppress expression of these genes at the transcriptional or post-transcriptional level. Such fertilityrestoring genes (Rf) are specific for a definite type of cytoplasm and usually have dominant mode of expression. A large number of genetically different types of CMS-inducing cytoplasms have been revealed in sorghum using intra- and interspecific hybridization (Schertz & Pring, 1982; Pring et al., 1995). These cytoplasms differ in their response to different fertility restorer lines, with respect to mitochondrial (mt) DNA restriction patterns, morphology and histological structure of anthers and by the stage of pollen degeneration. Based on anther morphology, different CMS-inducing cytoplasms have been subdivided into two distinct groups: those with small anthers without fertile pollen which degenerates during microsporogenesis (A1, A2, A5, A6), and those with large non-dehiscent anthers that may contain some stainable pollen (A3, A4, 9E) (Schertz et al., 1989). These groups have different restriction fragment length polymorphism (RFLP) patterns and can be clearly distinguished using specific mtDNA sequences (Xu et al., 1995). In addition, the cytoplasms belonging to the small-anthered group have a deletion in the rpoC2 chloroplast gene which encodes the RNA polymerase

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subunit (Chen et al., 1995). Restoration of male fertility in the A1 (review: Kaul, 1988) and A2 (Murty & Gangadhar, 1990) cytoplasms is of the sporophytic mode and is controlled by one or few dominant genes. The A3 cytoplasm is characterized by a gametophytic mode of restoration (Tang et al., 1996). The CMS-inducing effects of the ‗9E‘ and A4 (IS7920C) cytoplasms have been described by O. Webster and S.P. Singh (1964) and by J. Worstell with colleagues (1984). According to mtDNA analyses (Pring et al. 1982; Xu et al. 1995) these cytoplasms are related but not identical. In contrast to other cytoplasms they are characterized by a rearrangement in the coxI mitochondrial gene, which results in the synthesis of a variant 42-kDa cytochrome c oxidase subunit I (instead of the 38-kDa fragment in the A1 and A2 cytoplasms) (Bailey-Serres et al. 1986). In some types of sorghum CMS (A3, A4, ‗9E‘), restoration of male fertility is characterized by an unusual inheritance pattern: the Rf-genes function in the self-pollinated progenies of F1 hybrids but are not expressed or poorly expressed in backcrosses of these hybrids to parental CMS-lines or in testcrosses to CMS-lines with the same cytoplasm type (Elkonin et al., 1998, 2005; Tang et al., 2007). For the A3 cytoplasm, paramutation was suggested as the mechanism leading to silencing of restoring alleles (Tang et al., 2007). To explain the results with ‗9E‘ cytoplasm we suggested that cytoplasmic reversions from male sterility to male fertility may occur under the influence of fertility-restoring genes (Elkonin & Kozhemyakin, 2000). In addition, we observed that the level of male fertility of the F1 hybrids in the ‗9E‘ and ‗M-35-1A‘ cytoplasms correlates with the level of plant water availability during panicle development stage (Elkonin et al., 2005). On the basis of these data we suggested epigenetic mechanism causing heritable activation of fertility-restoring genes for the ‗9E‘ cytoplasm (Elkonin et al., 2006). In this chapter, we describe in detail the results of our investigations on fertility restoration in the ‗9E‘ cytoplasm and observations of pollen development in CMS-lines and in F1 hybrids in this cytoplasm with complete or partial restoration of pollen fertility.

POLLEN DEVELOPMENT IN CMS-LINES WITH „9E‟ CYTOPLASM We have studied pollen development in male-sterile plants of the line [9E] T×398 (Sorghum bicolor (L.) Moench). The seeds of this line were generously supplied by the late Dr. K. Schertz (Texas Agricultural Experimental Station, USA). This line has cytoplasm of the IS17218 accession; it was used as a source of the ‗9E‘ cytoplasm. Using this line another CMS lines in this cytoplasm, [9E] Milo-10 and [9E] Pishchevoye-614, were obtained by substitution backcrosses (BC) with the lines Milo-10 and Pishchevoye-614 (P-614; taken from the collection of the All-Russian Research Institute for sorghum and maize ―Rossorgo‖, Saratov, Russia). Anthers of these male-sterile lines have the size equal to the anthers of their isonuclear analogues in their own, ‗fertile‘, cytoplasm, although often they are asymmetric and have unequal and/or curved lobes with pointed tips (Fig. 1). These anthers do not dehisce, and the bagged panicles remain sterile. Under open-pollinated conditions they are able for normal seed set demonstrating the absence of male-sterile cytoplasm effect on female reproductive structures.

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Figure 1. Anthers of CMS-line [9E] Milo-10 and its fertile analogue (left).

For cytological analysis of pollen development the spikelets from different parts of the panicle taken at the microsporogenesis and at the flowering stages were fixed in acetic alcohol (1:3), and were stored in 75% alcohol in refrigerator. For analysis of meiosis anthers were stained with 2% acetocarmin, and macerated on a glass slide in the mixture of 45% acetic acid with 70% chloral-hydrate (1:1) colored with acetocarmin. For pollen analysis, the anthers from 10 to 15 spikelets were placed on a glass slide and macerated. Pollen grains were stained with 1% iodine-potassium iodide stain for the estimation of starch accumulation. A minimum of 400 pollen grains were studied per plant. Cytological analysis of the meiotic behavior of two CMS-lines in the ‗9E‘ cytoplasm, [9E] Milo-10 and [9E] P-614, demonstrated the higher frequency of abnormalities in CMSlines in comparison with their fertile analogues, Milo-10 and P-614 (Table 1). In the diakinesis, unpaired chromosomes were observed. During metaphase I one to three chromosomes placed out the metaphase plate or the spindle (Fig. 2 A,B). There were laggards in some microspore mother cells during telophase I and/or bivalents failed to separate (Fig.2 C). These aberrations resulted in irregular segregation accompanied by the formation of micronuclei in dyads. Similar abnormalities were observed in the second meiotic division that resulted by the formation of micronuclei in the microspores (Table 1). Table 1. Effect of the ‘9E’ cytoplasm on the frequency and the types of meiotic abnormalities in two lines of sorghum The frequency of disturbances, % Stage of meiosis Diakinesis

Milo10 8.6

Metaphase I

2.2

-

3.1

6.2

Ana-telophase I

3.2

6.6

0.0

10.5*

Diad

4.8

15.7*

7.0

11.0

Metaphase II

2.9

8.9

4.6

14.0*

Ana-telophase II

8.8

8.5

-

-

Microspore

2.1

6.6**

2.4

5.0*

[9E] Milo-10

P-614

[9E] P-614

0.0

-

6.9

The types of disturbances 2-4 univalents Chromosomes out of a spindle or an equatorial plate One or several laggards; non-separated bivalents Micronuclei; abnormal disposition of spindles Chromosomes out of a spindle or an equatorial plate; micronuclei; chromosome scattering over the spindle One or several laggards; micronuclei; absence of cytokinesis Micronuclei; degeneration; abnormal cenocyte-like structures

*, ** Significantly differed from fertile analogue at p≤ 0.05 and p≤ 0,01, respectively.

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In the tapetal tissue of CMS-plant anthers, cell vacuolization was more prominent in comparison with the fertile analogues, it being observed already at the early stages of meiosis (Fig. 2 D,E). This phenomenon is typical for many CMS-lines, and has been described in different plant species (Kaul, 1988).

Figure 2. Disturbances in microspore mother cells and in tapetal cells during microsporogenesis in ‗9E‘ CMS-plants of sorghum: A – chromosome out an equatorial plate; B – premature disjunction of bivalent; C – delayed disjunction of bivalent; D – vacuolization of tapetal cells during diakinesis in male-sterile plant; E – tapetal cells during prophase I in fertile plant; M – microspore mother cell; t – tapetal cells. Scale bar 50 µm.

However, in spite of the above-mentioned abnormalities, more than 90% microspores up to the late vacuolated stage in all the CMS-lines under study had normal appearance. Disturbances in pollen development were observed mainly at post meiotic stages that resulted in formation of anomalous pollen grains. They were as follows (Table 2): (1) dark-colored PGs filled with starch but having abnormal form (Fig. 3 B); (2) tan or light brown pollen grains (PGs) that were scored as waxy-type (Fig. 3 C); it is known that such coloration is connected with the low content of amylose, which is substituted by amylopectin (Itoh et al., 1997; Hirano, Sano, 2000); (3) PGs incompletely filled with starch (Fig. 3 D); (4) PGs with the other disturbances in starch accumulation (incompletely filled waxy-type PGs and the others) (Fig. 3 E,F); (5) PGs delayed at one or two-nucleate stage, some of them also have disturbed starch accumulation (Fig. 3 G); (6) PGs with partially or completely degenerated contents (Fig. 3 H). In addition, some other types of abnormalities were found rarely (< 0.1%): the abnormal structures or degenerated giant cenocyte microspores (Fig. 3 I,J), which were formed as a result of the absence of cytokinesis in telophase II, chromatide bridges, 3-polar spindles, etc.

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L.A. Elkonin, M.I. Tsvetova, V.V. Kozhemyakin et al. Table 2. Distribution of different types of pollen grains in CMS-lines with ‘9E’ cytoplasm and their fertile analogues

Line

Proportion of pollen grains.% Disturbances in starch accumulation Abnormal Normal WaxyIncompletely Other shape type filled PGs types

T×398 [9E] T×398 Milo-10 [9E] Milo-10 P-614 [9E] P-614

48.3 10.0*** 59.0 7.2*** 56.8 8.2***

44.3 32.3 7.2 15.9 15.0 3.4

0.0 26.0 0.0 12.2 0.0 14.2

2.7 6.7 9.5 8.3 12.7 19.4

0.3 16.3 5.0 23.7 1.8 19.0

Delay in development

Degeneration of contents

0.0 0.0 11.1 9.0 0.2 7.2

4.3 9.0 8.1 23.7 13.5 28.6

*, ** - Significantly differed from fertile analogue at p≤ 0.05 and p≤ 0,01, respectively.

A

B

C

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D

E

G

F

H

I

J

Figure 3. Pollen grain types in plants of CMS-lines with the ‗9E‘ cytoplasm and F1 hybrids with fertility-restoring lines: A – ‗fertile‘ pollen grain; B – pollen grain with anomalous shape; C – pollen grains of waxy-type; D,E,F – pollen grains with disturbed starch accumulation; G – pollen grains with delay in development and disturbed starch accumulation; H – pollen grains with degenerated contents; I,J – structures, developing as a result of disturbed cytokinesis in second meiotic division. Scale bar 50 µm.

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Regulation of Pollen Fertility in the ‗9E‘-CMS-Inducing Cytoplasm …

163

Plants of fertile analogues of CMS-lines also contained above-mentioned pollen types but their frequency was significantly lower, in comparison with CMS-lines (Table 1). Remarkably, the dark-colored PGs filled with starch which looked fertile have been discovered in pollen of sterile plants (in some plants, their frequency was up to 22.0%). But their capacity for fertilization is unknown nowadays and needs further investigation. These data demonstrate that in the sorghum ‗9E‘ cytoplasm pollen degeneration results mainly from the failure of normal starch accumulation at different stages of PGs maturation. In maturing pollen, expression of several metabolic and regulatory genes of sucrose → starch metabolism has been revealed (Datta et al., 2001). In the ‗9E‘ CMS-lines significant variability of PGs phenotypes suggests the different disturbances of starch synthesis pathway. As for waxy-phenotype of PGs, it also may be caused by different disturbances of starch accumulation pathways. The mutations conditioning such phenotype are well-known in a number of cereals (Bashkirov et al., 1987; Hirano, Sano, 2000; Pedersen et al., 2004, etc.). Our data demonstrate that PGs with similar phenotype may develop not only as a result of action of specific mutation but also as a result of metabolic distortion specific to different male-sterility-inducing factor(s); similar waxy-type pollen phenotypes we observed in malesterile nuclear mutants induced by streptomycin and ethidium bromide and in autotetraploids (unpublished). In the majority of investigated CMS-plants, anthers with completely degenerated contents or PGs that are devoid of any detectable starch are usually observed. Starch accumulation in developing PGs of CMS-lines is a rather rare phenomenon (Laser, Lersten, 1972; Chhabra, 1997; Itabashi et al., 2009). Thus, the ‗9E‘ CMS-inducing cytoplasm represents the relatively rare phenotypic manifestation of CMS.

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POLLEN DEVELOPMENT IN F1 HYBRIDS WITH RESTORED MALE FERTILITY To study genetic control of pollen abortion induced by the ‗9E‘ cytoplasm, we made crosses of male-sterile plants of the lines [9E]T×398, [9E]Milo-10 and [9E]P-614 with euplasmic line-donor of the ‗9E‘ cytoplasm, IS12603, and with a number of fertile lines of the grain sorghum, both fertility-restorers and sterility-maintainers of the other types of sorghum CMS. In addition, in these crosses we also used fertile lines in the ‗9E‘ cytoplasm, KVV-263 and KVV-34, which were obtained by self-pollination of the fertile F1 hybrids, [9E] T×398/KVV-112 and [9Е] P-614/IS12603. Analysis of fertility of the F1 hybrids obtained from these crosses revealed that a number of lines are able to restore pollen fertility in the ‗9E‘ cytoplasm and, therefore, contain nuclear fertility-restoring gene(s) (Rf-9E) (Table 3). Among these lines are Perspectivnyi-1 (Pers-1), KVV-114, Guineyskoe k-2974, Feterita-14. However, expression of male fertility was highly unstable and varied in different seasons suggesting significant influences of environmental factors.

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L.A. Elkonin, M.I. Tsvetova, V.V. Kozhemyakin et al.

Table 3. Characterization of male fertility of F1 hybrid combinations in the ‘9E’ CMSinducing cytoplasm of sorghum Number of plants1 Hybrid combination

Year

[9E] T398 / Pers-1

1999 2000 2007 2008 2009 2002 2003 2009 2002 2008 2009 2004 2006 2004 2006 1996 1996 1996 2008 2008 2009 2003 2002

[9E] T398 / KVV-263 [9Е]Milo-10 / Pers-1

[9E]Milo-10 / KVV-263

[9E]Milo-10 / Volzhskoye-615 [9E]Rannee-7/Volzhskoye-615 [9E]Milo-10 / Guineyskoe k-2974 [9E]Milo-10 / KVV-114 [9E]P-614 / Pers-1 [9E]P-614 / KVV-263 [9E] P-614 / IS12603 [9E] KVV-263 / Milo-10

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1

f

ps

s

11 19 16 16 20 13 12 23 8 8 6 2 9 20 2 2 -

8 7 3 1 1 8 3 2 2 13 9 6 4 6 3 -

4 11 16 12 8 5 1 1 30 7 10 16 6

f – fertile (seed set >40%); ps – partially sterile (40%); ps – partially sterile (40%); ps – partially sterile (40%); ps – partially sterile (40%); ps – partially serile (40%); ps – partially sterile (40%); ps – partially sterile ( 0.14 for all comparisons). In capsule length, interaction between plant species and pollen source was non-significant (F = 0.579, df = 4, P = 0.68). There was a significant difference in width of capsules produced between the pollen sources (F = 17.83, dfTREATMENT = 4, dfERROR = 56, P < 0.001). Because the interaction between the study species and pollen source was significant (F = 3.38, dfTREATMENT = 4, dfERROR = 56, P = 0.015), the effects were tested separately for both species. A significant effect of pollen source was found in both species (one-way ANOVA: F = 4.27, df = 4, P = 0.006 and F = 26.11, df = 4, P 0.694 for all pairs).

Seed capsule width Self

Within-species

Between-species

Hybrid

Open

7 6

mm

5 4 3 2 1 0

D. incarnata

D. fuchsii

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Chart 3. Mean (+SD) seed capsule width in flowers of D. incarnata and D. fuchsii pollinated with selfpollen, cross-pollen within species, cross-pollen from the other species, pollen from a hybrid plant and natural pollination.

The proportion of embryonic seed and proportion of staining and thus viable seed of all seed differed among the hand-pollination treatments in D. incarnata (Friedman: 2 = 13.4, df = 3, P = 0.004 and 2 = 9.9, df = 3, P = 0.029, respectively, Chart 4). Proportion of embryonic seed and viable seed was lower in flowers pollinated with hybrid pollen compared to flowers with any other treatment (Wilcoxon: P > 0.29 for all pairs, respectively). The proportion of embryonic seed was also lower in capsules produced following pollination with D. fuchsii pollen than in those resulting from cross-pollination (Z = -2.55, P = 0.011), but this effect was not found in the proportion of viable seed (Z = -2.37, P = 0.314). In D. fuchsii, the data were smaller and pollination with hybrid pollen seldom resulted in seed production, and therefore this treatment was excluded from the analyses. There were differences among the treatments in the proportion of embryonic seed produced (Friedman: 2 = 6.5, df = 2, P = 0.039) but not in the proportion of alive seed (2 = 2, df = 2, P = 0.368). Pair-wise comparisons revealed that there was a trend that capsules produced after pollination with self-pollen produced lower proportion of viable seed than after pollination with crosspollen from another D. fuchsii plant or from D. incarnata (Z = -1.83, P = 0.068 for both pairs).

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Pollen Vigor and Seed Production in Sympatric Population …

A

205

Proportion of embryonic seed Self

Within-species

Between-species

Hybrid

100 90 80 70 60 % 50 40 30 20 10 0

D. incarnata

D. fuchsii

Proportion of viable seed

B Self

Within-species

Between-species

Hybrid

100 90 80 70 60 % 50 40 30 20 10 0

D. incarnata

D. fuchsii

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Chart 4. Proportions (mean+SD) of A) embryonic seed and B) viable seed of all seeds in a capsule following pollination with self-pollen, cross-pollen, inter-specific pollen on D. incarnata and D. fuchsii. The effects of pollination with hybrid påollen was examined only in D. incarnata.

Seed Germination The other part of the seed produced in hand-pollination experiment was prepared for in situ germination experiment using procedure by Rasmussen and Whigham (1993). Seeds from each inflorescence and pollination treatment were placed in different germination units marked with information of the origin. These units consist of glassless 24 × 36 mm slide mount and piece (40 × 60 mm) of plankton netting (SefarNitex 03-36/28). Seeds were placed on the netting which was folded once and closed from the open sides using the slide mount. The germination units were divided to ten groups consisting seeds from different female plants and hand-pollination treatments. The units of the same group were tied together (with 10cm spaces in between) using nylon line. In 6th of September 2005 germination units were placed to five different locations of the North Bull Island. In these sites flowering plants of D. incarnata, D. fuchsii and putative hybrids were found during field work in the summer 2005. Two groups of germination units were placed on each site: one on the lower area between the dunes, and other higher, approximately half of the height of the closest dune slack. The line keeping the units together was anchored to the ground from both ends using metal hooks, and the units were arranged in a way that the netting touched the ground. The units were then covered lightly with debris and left intact for a year.

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Twelve months later, in September 2006, the germination units were collected from the field. Three lines could not be found in the field, which decreased the number of germination areas to seven, and made it impossible to compare lower and higher positions of germination unit groups. Fungi growing hyphae inside the units appeared, however, not to be dependent of their position in the field. Seeds in germination units were examined under light microscope for protocorms or germinating seeds. There was no clear sign of germination of seeds after this 12 month period. Some seed had hyphae growing inside the testa, and this was regarded as possible starting point of germination. Unfortunately, fungi could not be identified in this study. Seeds produced by D. incarnata and D. fuchsii manage to produce seed, that was infected by fungus during 12 months. Proportions of germinating units with infected seed were between 27.3 and 50% in seeds originating from self-pollination, intra-specific pollination and inter-specific pollination. There were only a few units with seed originating from D. incarnata or D. fuchsii flowers pollinated with hybrid pollen, and none of these had infected seed.

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Seed Production in Hybrid Plants Stigma receptivity of the study species and hybrids was tested using Macherey-Nagel Peroxtesmo Ko peroxidase test paper, which has been shown to be both accurate and easy-touse (Dafni & Maués 1998). Stigma receptivity was checked from about 30 hybrid plants used in hand-pollination experiments. In all individuals examined, fresh flowers were found to have receptive stigmas. A total of 20 hybrid plants were randomly chosen for hand-pollination experiment in which two flowers were hand-pollinated with one pollinium from i) the same plant (selfpollination, if pollinia were present), ii) another hybrid plant (cross-pollination), iii) D. incarnata or iv) D. fuchsii. Mature capsules were examined as presented previously in this chapter. As hybrid plant was used as a mother plant, pollen source affected both the length and width of capsules produced in hand-pollinated hybrid flowers (Friedman: 2 = 13, N = 14, P = 0.002 and 2 = 13.3, N = 14, P = 0.001, respectively, Chart 5). Pollen originating from other hybrid plant resulted in shorter and thinner capsules than pollination with incarnata or fuchsii pollen (Wilcoxon: Z = -2.67, P = 0.008 and Z = -3.23, P = 0.001 for length, Z = -2.86, P = 0.004 and Z = -3.20, P = 0.001 for width, respectively). There were no differences in capsule length or width between pollination treatment with D. incarnata and D. fuchsii (Z = -1.32, P = 0.187 and Z = -39, P = 0.700, respectively). The number of hybrid plants producing seed capsules after pollination with own pollen was low mainly due to the lack of well-developed pollinia, and was excluded from the analyses. Likewise only a few hybrid plants produced capsules naturally, because of the presence of mesh bags most of the flowering time. Dimensions of the capsules from these two treatments are, however, shown in Chart 5.

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Between-hybrids

D. incarnata

D. fuchsii

207

Open

16 14 12 mm

10 8 6 4 2 0

Length

Width

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Chart 5. Capsule length and width (mean + SD) in hybrid flowers following pollination with selfpollen, pollen from other hybrid plant, D. incarnata or D. fuchsii, and capsule dimensions in naturally pollinated hybrid flowers.

Capsules produced in hybrid plants were swollen and contained seed, but production of embryonic and viable seed was extremely low and the effect of pollen source could only be tested between D. incarnata and D. fuchsii, which resulted in the highest viable seed sets (>50% in one plant). Pollination with D. fuchsii pollen resulted in higher proportion of embryonic seed than pollination with D. incarnata pollen (Z = -2.06, P = 0.039), but there was no significant difference in the proportion of viable seed (Z = -1.10, P = 0.273). Selfpollination resulted in viable seed production in two plants (0.25% and 1.52%, respectively). Crossing between different hybrid plants resulted in seed production in five plants out of 13, and embryonic seed was only present in two plants (proportion of viable seed 7.7% and 16.8%, respectively, Chart 6). These two plants produced seed also after pollination with D. incarnata and D. fuchsii pollen. Germination of seeds produced by hybrid plants was examined using in situ germination procedure described previously in this chapter. Proportions of germination units with infected seed were lower when hybrid plant was used as a mother plant (about 10%) than if D. incarnata or D. fuchsii was used. This may also be a result of low seed number produced in hybrids causing differences in seed mass in germination units. However, seeds originating from pollinations with self-pollen, cross-pollen (another hybrid plant), D. incarnata or D. fuchsii pollen were infected by fungi. All these infected seeds were produced by two hybrid individuals, one of which is the plant shown in Chart 6 (left).

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Chart 6. Above: Two hybrid plants that produced viable seed as pollinated with pollen from parental species. Below: Fluorescent microscopy photo of stained seeds produced by specimen shown above.

Production of Apomictic Seed Following Stigma Stimulation Treatments Twenty experimental plants were covered with light mesh bags to prevent insect visits. Plants were bagged prior to opening of first flowers and kept for eight days after treatments to make sure that stigmas had no other stimulations. During the experiment eight plants were totally wilted because of drought or partially destroyed, and were excluded from the analyses. Treatments of all flowers were conducted during the same day. Flowers were first emasculated by carefully removing the pollinia using tweezers. Experimental flowers were then marked with light plastic rings placed around the ovary. Plastic rings were of different color referring to different treatments. Each plant had at least one flower with one of the five treatments: i) cross-pollination, ii) control (no treatment or any kind of handling of flower),

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iii) D. incarnata pollinium extract, iv) hand-pollination with non-orchid Ranunculus acris pollen, and V) mechanical stimulation of stigma with clean brush. Flower within the plant receiving a certain treatment was randomly chosen between the freshly opened flowers as the location of the flower in the inflorescence may affect its seed production (Vallius 2000). For cross-pollination, pollinia of D. incarnata were collected from other plants in the same population using sharp wooden sticks on which the viscidia of pollinium easily adhere. One whole pollinium was placed to the stigma of receptive flower using tweezers. To produce infertile but chemically active pollinium extract, a total of 20 D. incarnata pollinia collected from the same population were first kept in a freezer for 24 hours and then crushed using a cooled mortar (+4°C) with 1 ml of distilled water. One (1) ml of distilled water was then added to make a solution, which was stored in a freezer. In the field solution was stored in a cooler bag in which it melted down, but did not warm too much. A drop of extract was placed to the stigmas of the treated flowers using single-use pipet. After the withering of treated flowers, bags were removed and capsules were let to develop. Stigma stimulations treatments resulted in a large variation in seed production. Flowers hand-pollinated with cross-pollen produced larger capsules than flowers other treatment groups when tested as a dry weight of seed capsules (T-test, P < 0.001, respectively). Proportion of seeds with normal-sized embryo of all seeds was 18-97%, in most plants more than 90%. Five of the individual plant studied produced seed only after hand-pollination with cross-pollen (one pollinium). The rest seven plants produced some seed especially following pollinium extract treatment, pollination with R. acris pollen, or both (Chart 7). No flowers any seed after mechanic stimulation of stigma. Polyembryony was not found in any of the seeds produced in this experiment or other studies described in this chapter.

Chart 7. Seed production of D. incarnata flowers after different stigma stimulation treatments. Numbers refer to number of study plants with the found effect.

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Interference of Fertilization in the Parental Plant (D. incarnata) by Hybrid Pollen Interference effect of hybrid pollen on fertilization of D. incarnata flowers was examined with hand-pollination experiment. A total of 15 D. incarnata plants were bagged prior to anthesis. As the flowers opened, flowers were emasculated. Pollinia used in the handpollinations were collected from non-experimental plants in the population. Hybrid pollinia were collected from plants that had sectile pollinia but showed none or weak fertility in staining test described previously in this chapter. One or two freshly open flowers were pollinated in two consecutive days with one pollinium of i) cross-pollen (Day 1) and left intact (Day 2), ii) cross-pollen (Day 1 and Day 2), iii) hybrid pollen (Day 1) and cross-pollen (Day 2) or iv) cross-pollen (Day 1) and hybrid pollen (Day 2). As capsules matured, they were collected and taken to the laboratory. Capsules were stored in small glass tubes, ovendried (60°C) for 48 hours, and weighed. There were no differences in dry weight of capsules produced following pollination with one pollinium from another D. incarnata plant, with cross-pollen on two consecutive days or with one pollinium from a hybrid plant either day before or after cross-pollination with crosspollen (Friedman: 2 = 4.87, df = 3, P = 0.182, Chart 8). Dry weight of capsules has been shown to be a reliable estimate for the number of embryonic seed in a capsule (Vallius 2001). On the basis of this result, hybrid pollen does not cause interference in the fertilization of D. incarnata. This is probably because hybrid pollen grains are not viable enough to block ovary with in-growing pollen tubes.

Dry weight of capsules

50 40 mg

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60

30 20 10 0

Cross+0

Cross+Cross

Hybr+Cross

Cross+Hybr

Pollen source in hand-pollination Chart 8. Dry weight of capsules (mean+SD) produced in D. incarnata flowers following pollination in two consecutive days with cross-pollen only Day 1, cross-pollen both days or cross-pollen either Day 1 or Day 2 and hybrid pollen on the other day.

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CONCLUSION Production of F1 Hybrid Seed Following Controlled Crossing of D. incarnata and D. fuchsii In hand-pollination experiment with D. incarnata, self-pollination and pollination with D. fuchsii pollen resulted in capsules with similar size than cross-pollination. There is no effective reproductive barrier acting between D. incarnata ssp. coccinea and D. fuchsii in the North Bull Island. Flowering times of D. incarnata plants are very variable from mid-June to mid-July as the first D. fuchsii flowers opened in the end of June. There was thus considerable overlap in flowering times. Both species occur in the dune area often within a few meters from each other. Both species are known to be pollinated by solitary and social bees (e.g., Nilsson 1981, Neiland & Wilcock 2000), and in lack of pollinator reward, flower constancy can not develop for either of the species. Pollen movement can thus be predicted to be frequent between species, if there are no morphological differences between the flowers of these species leading to pre-pollination reproductive barriers. Neiland and Wilcock (2000) reported uni-directional pollen flow from D. maculata to D. incarnata flowers via syrphid flies actively visiting both species.

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Male and Female Fitness in Hybrid Plants The viability of pollen in hybrid plants was found to be very variable. Hybrid plants with 40 chromosomes (2n), and some of those having about 60 chromosomes (3n) had no fullyformed pollinia. Most hybrids, however, had pollinia with partially or wholly staining massulae showing the viability. Bertolini et al. (2000) found high variation in chromosome numbers of Dactylorhiza species and also peculiar ploidy levels. Unfortunately information about presence of pollinia and pollen viability is was not available on orchids, and results can thus not be compared. Female reproductive success of hybrid plants is much lower than that of the parental species, D. incarnata and D. fuchsii. Reproduction of hybrids did probably not suffer from low visitation rate, because natural capsule production of hybrids was comparable to the parent species. Capsule were, however, thin and contained no or only a few embryonic seeds. Results from the hand-pollination experiment show that hybrids suffered from at least partial sterility probably due to problems in meiosis during development of reproductive cells. This can be seen in poor viability of pollen leading to low seed production in flowers pollinated with hybrid pollen, and in low seed production in all hybrid flowers regardless of pollen source. Hybrids, however, produced more seeds after pollination with pollen from the parental species than after pollination with hybrid pollen. Despite very low reproductive success in hybrid plants compared to that of the parent species, hybrids can not be regarded as totally sterile. If seeds produced by hybrids of after pollination with hybrid pollen in either of the species are able to germinate, there is a possibility of introgression and even sexual reproduction of hybrids. Even though this progress is very slow due to rareness of the events, it makes the production of new genetic compositions enabling formation of a new species in

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favourable conditions (Rieseberg 1997). As Ellstrand et al. (1996) stated, even rare hybridization events producing single, partially fertile, individual may lead to a new evolutionary lineage. Stebbins (1957) found that even >99.99% florets of hybrids between Elymus glaucus and two microspecies of Sitanion produced no seed, a single seed produced after backcross with Elymus was vigorous, fully self-fertile and almost completely isolated from the parent species. Also van der Pijl and Dodson (1966) argued that the high number of ovules and pollen grains produced by orchids increases the possibility of an almost infertile hybrid to produce at least a few fertile seeds. Furthermore, orchids being usually long-lived perennials, reproduction of nearly sterile hybrids becomes more possible (van der Pijl & Dodson 1966). The authors then argued that ―hybrid incompatibility and sterility can be only relative terms‖. Rieseberg (2001) argued that chromosomal rearrangements can lead to decreased gene flow via lower recombination rates than via low fitness of hybrids. In plants, sterility caused by chromosomal rearrangements may disappear and full fertility may be recovered upon chromosomal doubling (e.g. Rieseberg 2001). However, if new hybrid lineage has lower fitness than the parental species, it can not be maintained in sympatry, speciation is thus more probable in hybrid populations isolated from the parent species (Rieseberg 1997). The small size of orchid seed enables long-distance dispersal to new sites where hybrids may occur without interference of the parent species (van der Pijl & Dodson 1966).

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Apomixia in Dactylorhiza The number of seeds produced following chemical treatment of stigma (pollinium extract or R. acris pollen) were very low compared to that after normal cross-pollination. There is always a possibility of contamination of stigmas during treatment, but because Dactylorhiza pollinia are very compact and hidden inside the bursicle, it is not probable that in all cases some pollen grains would have ended to the stigmas of treated flowers. Because mechanical stimulation did not result in any seed production, it seems that chemical stimulation may play a part in agamospermous seed production of D. incarnata. Lupton (2006) argued that presence of a few viable seed in Spiranthes romanzoffiana capsules may be a result of failed fertilization following weak pollen tube growth. This can not explain the findings in this study, because in D. incarnata a few seed were produced following chemical stimulation of the stigma. In orchids, the development of ovules begins only after pollination being regulated by presence of ethylene and pollen-borne auxin (Zhang & O‘Neill 1993). Ovule development can be induced also with other stimulus than conspecific pollen. As ovules develop, most of them produce empty testae, but some may produce a normal embryonic seed via apomixis. Uniparental reproduction enables colonization of areas via single individuals (Hörandl et al. 2008), or in the case of hybrid plants may increase the number of hybrid individuals in a population, even though the number of seeds produced in very small compared to sexually reproducing plants. In case of Dactylorhiza, apomixia may play an important role in stabilization of allopolyploids, as vegetative reproduction is either rare or absent. Kropf and Renner (2005) argued that D. sambucina is non-agamospermous species, because there were no capsules produced in emasculated and bagged flowers. This result may be explained by lack of stimulation of stigmas in bagged flowers. Production of apomictic seed in non-cleistogamous orchids is

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probably possible only after stigma stimulation which is needed to trigger the maturation of ovules. Bullini et al. (2001) showed that Dactylorhiza insularis was allotriploid, apomictic species originating from single hybridization event between D. romana and D. sambucina. Because of non-functional pollinia, this species reproduces solely via production of agamospermous seed produced both in open and cleistogamous flowers (Diana 1997).

Interference of Fertilization in the Parental Species by Hybrid Pollen Deposition of non-viable or weakly viable hybrid pollen did not affect the seed production in D. incarnata. This is probably, because the hybrid pollinia stayed inactive and there was space available for cross-pollen to germinate on the stigma. It may even be, that hybrid pollen deposited on stigma one day before cross-pollen advanced the ovule maturation, and could even have slight positive effect on fertilization of D. incarnata. In contrast to this result, Neiland and Wilcock (1999) found that interspecific, non-orchid pollen load depressed the production of seeds in D. purpurella. Flanagan et al. (2009) found that seed production in Mimulus ringens (Phrymaceae) was lower if pollinating insects also visited flowers of competing species. However, they found that decreased seed set was a result of lower pollen deposition, and presence of interspecific pollen on stigma did not cause interference reproduction of this species. Variation in these results may be caused by the amount of interspecific pollen on stigmas of experimental plants, as large number of pollen grains may physically block the receptive area.

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Speciation and Conservation of Dactylorhiza If polyploid hybrid plants manage to somewhat reproduce sexually or asexually, development of new species is more probable than usually considered on the basis of low fertility common in hybrid plants. However, as Arnold and Hodges (1995) argued, hybrids may have an equal fitness compared to parental species. Polyploidy has also advantages, as shown especially in crop plants (e.g. Gustafsson 1968), even though also negative consequences of polyploidy have been reported (Thompson et al. 1997) Hybrids between D. incarnata and D. fuchsii are robust and conspicuous plants compared to parental species and other surrounding vegetation. These plants produce a vast number of flowers and are frequently visited by insects. Frequent visitation probably results in pollen deposition at least from parental species, and thus production of backcrosses. Furthermore, frequent visits may trigger ovule production and also production of apomictic seed in hybrid plants, as was shown with stigma stimulation experiment in D. incarnata. Dactylorhiza, especially the allopolyploid species, has high levels of genetic variation, which has been accumulated over a long time (Hedrén et al. 2007). Hedrén (2001) and Pillon et al. (2006) argued that even if the many allotetraploid taxa in Dactylorhiza are endemic and therefore of high interest of conservation, it is at least equally important to protect the diploid species. Hybridization events do happen in nature also today, and new allopolyploids may still arise from these parental species. Conserving still non-existing species by ensuring the viability of sympatric populations is definitively worth consideration.

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ACKNOWLEDGMENTS The study presented in this chapter was financially supported by the Academy of Finland. I am grateful to my colleagues at the Botany Department of the Trinity College Dublin. I also want to thank Gaia Franchini for essential help with an article in Italian.

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Flood, D.T. (1977): Historical evidence for the growth of North Bull Island. In: D. W. Jeffrey, R. N. Goodwillie, B. Healy, C. H. Holland, J. S. Jackson & J. J. Moore (Eds). North Bull Island Dublin Bay –a modern coastal natural history (pp. 9-12). Dublin: The Royal Dublin Society. Flanagan, R. J., Mitchell, R. J., Knutowski D. & Karron, J. D. (2009) Interspecific pollinator movements reduce pollen deposition and seed production in Mimulus ringens (Phrymaceae). American Journal of Botany, 96, 809-15. Foley, M. & Clarke, S. (2005) Orchids of the British Isles. Cheltenham, UK: Griffin Press Publishing Limited. Gustafsson, Å. (1968) Reproduction mode and crop improvement. Theoretical and Applied Genetics, 38, 109-17. Gutowski, J. M. (1990) Pollination of the orchid Dactylorhiza fuchsii by longhorn beetles in Primeval Forests of Northeastern Poland. Biological Conservation, 51, 287-297. Hedrén, M. (2001) Conservation priorities in Dactylorhiza, a taxonomically complex genus. Lindleyana, 16, 17-25. Hedrén, M., Nordström, S., Persson Hovmalm, H. A., Pedersen, H. Æ. & Hansson, S. (2007) Patterns of polyploid evolution in Greek marsh orchids (Dactylorhiza; Orchidaceae) as revealed by allozymes, AFLPs, and plastid DNA data. American Journal of Botany, 94, 1205-18. Hedrén, M., Nordström, S. & Ståhlberg, D: (2008) Polyploid evolution and plastid DNA variation in the Dactylorhiza incarnata/maculata complex (Orchidaceae). Molecular Ecology, 17, 5075-91. Hörandl, E., Cosendai A.-C. & Temsch, E. M. (2008) Understanding the geographic distributions of apomictic plants: a case for a pluralistic approach. Plant Ecology & Diversity, 1, 309-320. Kropf, M. & Renner, S. S. (2005) Pollination success in monochromic yellow populations of the rewardless orchid Dactylorhiza sambucina. Plant Systematics and Evolution, 254, 185-97. Luyt, R. & Johnson, S. D. (2001) Hawkmoth pollination of the African epiphytic orchid Mystacidium venosum, with special reference to flower and pollen longevity. Plant Systematics and Evolution, 228, 49-62. Moore, J. J. (1977) Vegetation of the dune complex. In: D. W. Jeffrey, R. N. Goodwillie, B. Healy, C. H. Holland, J. S. Jackson & J. J. Moore (Eds). North Bull Island Dublin Bay –a modern coastal natural history (pp. 104-6). Dublin: The Royal Dublin Society. Neiland, R. M. R & Wilcock, C. C. (1995) Maximisation of reproductive success in European orchids under conditions of infrequent pollination. Protoplasma, 187, 39-48. Neiland, R. M. R. & Wilcock, C. C. (1999) The presence of heterospecific pollen on stigmas of nectariferous and nectarless orchids and its consequences for their reproductive success. Protoplasma, 208, 65-75. Neiland, R. M. R. & Wilcock, C. C. (2000) Effects of pollinator behaviour on pollination of nectarless orchids: Floral mimicry and interspecific hybridization. In: K. L. Wilson & D.A. Morrison (Eds) Systematics and evolution of monocots: proceedings of the 2nd international Conference on the Comparative Biology of the Monocots (pp. 318-26). Sydney: CSIRO, Melbourne.

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Nilsson, L. A. (1981). Pollination ecology and evolutionary processes in six species of orchids. Abstracts of Uppsala Dissertations from the Faculty of Science and Technology 593. Pacini, E. & Hesse, M. (2002) Types of pollen dispersal units in orchids, and their consequences for germination and fertilization. Annals of Botany, 89, 653-664. Pillon, Y., Fay, M. F., Shipunov, M. F. & Chase, M. W. (2006) Species diversity versus phylogenetic diversity: A practical study in the taxonomically difficult genus Dactylorhiza (Orchidaceae). Biological Conservation, 129, 4-13. Pline, W. A., Edmisten, K. L., Oliver, T., Wilcut, J. W., Wells, R. & Allen, N. S. (2002) Use of digital image analysis, viability stains, and germination assays to estimate conventional and glyphosate-resistant cotton pollen viability. Crop Science, 42, 2193200. Pridgeon, A. M., Cribb, P. J., Chase, M. W. & Rasmussen F. N. (Eds). (2001). Genera Orchidacearum. Volume 2. Orchidoideae (Part One). Oxford: Oxford University Press. Pritchard, H. W. (1985) Determination of orchid seed viability using fluorecein diacetate. Plant Cell and Environment, 8, 727-730. Proctor, H. C. (1998) Effect of pollen age on fruit set, fruit weight, and seed set in three orchid species. Canadian Journal of Botany, 76, 420-7. Randall, J. L. & Hilu, K. W. (1990) Interference through improper pollen transfer in mixed stands of Impatiens capensis and I. pallida (Balsaminaceae). American Journal of Botany, 77, 939-44. Rasmussen, H. N. & Whigham, D. F. (1993) Seed ecology of dust seeds in situ: a new study technique and its application in terrestrial orchids. American Journal of Botany, 80, 13741378. Rieseberg, L. H. (1997) Hybrid origins of plant species. Annual Review on Ecology and Systematics, 28, 359-389. Rieseberg, L. H. (2001) Chromosomal rearrangements and speciation. Trends in Ecology and Evolution, 16, 351-358. Roberts, D. L. & Dixon, K. W. (2008) Orchids. Current Biology, 18, R325-R329. Schmidt, J. M. & Antlfinger, A. E. (1992) The level of agamospermy in a Nebraska population of Spiranthes cernua (Orchidaceae). American Journal of Botany, 79, 501-7. Stebbins, G. L. Jr. (1959) The role of hybridization in evolution. Proceedings of the American Philosophical Society, 103, 231-51. Thompson, J. N., Cunningham, B. M., Segraves, K. A., Althoff, D. M, & Wagner, D. (1997) Plant polyploidy and insect/plant interactions. The American Naturalist, 150, 730-743. Vallius, E. (2000) Position-dependent reproduction success of flowers in Dactylorhiza maculata (Orchidaceae). Functional Ecology, 14, 573-9. Vallius, E. (2001) Factors affecting fruit and seed production in Dactylorhiza maculata (Orchidaceae). Botanical Journal of the Linnean Society, 135, 89-95. van der Pijl, L. & Dodson, C. H. (1966) Orchid Flowers – their pollination and evolution. Coral Gables. Florida : University of Miami Press. Zhang, S. Z. & O‘Neill S. (1993) Ovary and gametophyte development are coordinately regulated by auxin and ethylene following pollination. The Plant Cell, 5, 403-418.

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In: Pollen: Structure, Types and Effects Editor: Benjamin J. Kaiser, pp. 217-234

ISBN: 978-1-61668-669-7 ©2010 Nova Science Publishers, Inc.

Chapter 9

MICROSPORES AND THEIR APPLICATIONS IN BASIC AND APPLIED PLANT SCIENCES Mehran E. Shariatpanahi1,2, * and Alisher Touraev1 1

Max F. Perutz Laboratories, Vienna University, Vienna, Austria Department of Tissue Culture and Gene Transformation, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran

2

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ABSTRACT Nowadays, the use of isolated and in vitro cultured microspores is not limited for the production of doubled haploids which are important part of modern plant breeding programmes. Microspores became also a powerful tool to investigate important biological processes such as cell totipotency, differentiation, embryogenesis, cell cycle, plant reproduction and development. In the normal pathway microspores develop into mature pollen in vivo or when cultured in vitro in a rich medium with sugars, however they can be reprogrammed into the totipotent state and further induced to become embryogenic and produce embryos when subjected to various stresses, such as nutrient starvation, heat or cold shock. Microspore cultures are the most efficient method to produce doubled haploids, excellent system for in vitro mutagenesis and selection, attractive target for genetic transformation and for gene targeting in plants. This review paper describes the potential applications of plant microspores in plant breeding, genetics, cellular and molecular biology and biotechnology. Keywords: Microspore, embryogenesis, haploid, plant breeding, genetics.

*

Corresponding author: Mehran E. Shariatpanahi, Department of Tissue Culture and Gene Transformation, Agricultural Biotechnology Research Institute of Iran, Mahdasht Road, P.O. Box 31535-1897 Karaj, Iran, Tel: 0098-261-2703536; Fax: 0098-261-2704539, E-mail: [email protected]

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INTRODUCTION Haploid is the general term for plants (sporophytes) that contain the gametic chromosome number (n). The haploid could also be called either monoploid (x) in a diploid sporophytic (2n) species in which only one set of chromosomes exists or polyhaploids in polyploidy species as they have more than one set of chromosomes. The haploid plant obtained from an autotetraploid (4x) with four sets of one genome was originally called a dihaploid (because 2n=2x). When the chromosome number of a haploid is doubled, it is called a doubled haploid (DH). It should be mentioned that a doubled haploid is different from dihaploid. The dihaploid is not homozygous since it represents two chromosome sets selected from four sets in the autotetraploid, whereas the doubled haploid from a monoploid or an allohaploid should be completely homozygous [Kasha and Maluszynski 2003]. The main advantage of doubled haploids in breeding is the reduction of the time required to develop new cultivars [Snape et al., 1986; Touraev et al., 2001; Thomas et al., 2003]. For annual self-pollinated crops it generally takes 10-15 years to produce a cultivar through a conventional plant breeding program such as the pedigree method, which includes selfing and subsequent selection. The time delay is costly and prevents breeders from responding rapidly to end users need. The production of haploids followed by chromosome doubling to produce homozygous lines, from which superior lines are selected, can reduce the time required for cultivar development by 3-4 years. For cross-pollinated heterozygous crops, doubled haploids are a rapid method to produce homozygous pure breeding lines, which can be used in the development of synthetic varieties or hybrids. The production of doubled haploids can improve selection efficiency as the phenotype of the plant is not masked by dominance effects. Traits encoded by recessive genes can be easily identified. A smaller population of doubled haploids is required when screening for desirable recombinants than would be the case for conventional diploid populations [Touraev et al., 2001].Over 200 varieties have been produced by deploying various DH methods [Thomas et al., 2003]. The vast majority of varieties derived from doubled haploidy are in barley (96), followed by rapeseed (47), wheat (20) and the rest [Thomas et al., 2003]. Application of DH systems for induction and selection of mutants has the benefits [reviewed by Szarejko 2003] such as (a) possibility to screen for both recessive and dominant mutants in the first generation after mutagenic treatment; (b) immediate fixation of mutated genotypes, which saves time in the production of pure mutant lines; (c) increased selection efficiency of desired mutants due to the gametic versus zygotic segregation ratios (1:1 vs 3:1, respectively) and the lack of chimerism; (d) possibility of applying in vitro selection methods at the haploid or doubled haploid level. Doubled haploid populations are ideal for genetic mapping [Forster and Thomas 2003]. DH populations are available for DNA extraction and mapping 1.5 years after the initial cross, i.e. almost as quick as an F2 or BC1 population and definitely much faster than a pedigree inbred or single seed descent population. DHs can be re-grown and distributed in seed form so that it is comparatively easy to screen it with a large number of markers. Map construction from a DH population derived from the F1 of a cross is relatively simple because the expected segregation is that of a backcross [Snape 1976]. DH populations are of benefit in the identification of Quantitative Trait Loci (QTL) as one can grow multi-site replicated trials,

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to derive the most meaningful data, three years after the initiation cross [Forster and Thomas 2003]. DH plants can be obtained by several strategies including wide hybridization followed by embryo rescue, parthenogenesis, gynogenesis or microspore embryogenesis (ME) in anther or isolated microspore cultures [Touraev et al., 2001]. Microspore embryogenesis is the most developed and documented approach to produce doubled haploids. ME can be induced either in excised and in vitro cultured anthers or in isolated microspore cultures. The induction of ME requires first to reprogram the development of the microspores from the gametophytic (pollen) pathway into the sporophytic (embryo) pathway via applying various stress pretreatments [Shariatpanahi et al., 2006a]. The phenomenon of microspore embryogenesis was first demonstrated by Guha and Maheshwari [1964, 1966], using the Solanaceous plant Datura innoxia. These investigators found that when anthers of D. innoxia were cultured on a mineral-salt medium containing casein hydrolysate, indole acetic acid, and kinetin, a substantial number of the enclosed pollen grains became embryogenic and within 6 to 7 weeks, developing embryos were observed emerging from the anther. Nowadays researchers have demonstrated that embryogenesis and plant regeneration could be achieved in isolated microspore cultures. The induction of microspore embryogenesis from the culture of isolated microspores was first achieved in D. innoxia by Nitsch and Norreel [1973]. In this procedure flower buds were pretreated for 48h at 3°C prior to microspore isolation. In comparison with other methods, some advantages of microspore embryogenesis are its applicability to all pollen-producing plant species and demonstrated feasibility in a large number of crop species, the good scientific basis of microspore embryogenesis, the high number of pollen grains in most species allowing a large number of doubled haploids to be produced by microbiological techniques, the possibility to double chromosome number during anther or microspore culture, etc. A disadvantage is the formation of albino plants (but only in cereals) and the somaclonal variation as a consequence of suboptimal tissue culture conditions [Touraev et al., 2001].

DEVELOPMENTAL PATHWAYS OF IN VITRO ISOLATED MICROSPORES Isolated and in vitro cultured microspores or immature pollen grains under certain conditions regenerate into embryos and whole plants. This phenomenon is called ―totipotency‖. Conversion of differentiated plant cells into totipotent cells depends on extraordinary conditions acting upon cells. A triggering factor in the form of stress is necessary to induce embryogenesis in cultured microspores [Nitsch and Norreel 1973, Heberle-Bors 1985, Touraev et al., 1997a]. Another important factor for successful conversion is the stage at which inflorescences, flower buds, anthers or microspores are excised, put into in vitro culture or isolated from the anther, respectively. An important issue, however, is to find and choose the right conditions at a certain stage in a given species [Heberle-Bors 1985]. In addition, physiological state and conditions of growth of the donor plants, isolation methods and the culture media are important in optimizing induction efficiency of microspore embryogenesis [Bajaj 1990; Ferrie et al., 1995].

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STRESS AS TRIGGER TO INDUCE MICROSPORE EMBRYOGENESIS

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Stresses widely used for the induction of microspore embryogenesis are cold, heat, carbon starvation and colchicine [reviewed by Shariatpanahi et al., 2006a]. Heat pre-treatment is usually carried out at 33°C to 37°C for a duration varying from several hours to several days, whereas cold treatment is carried out at 4°C to 10°C from some days to several weeks. Incubation of microspores in media containing non-metabolizable carbon sources, i.e. in mannitol-containing media, is also used with success [Touraev et al., 2001]. In addition, colchicine, a microtubule-depolymerizing agent, is being used as a stress pre-treatment [Zaki and Dickinson 1991, Zhao et al., 1996]. The neglected stresses such as abscisic acid [Imamura and Harada 1980a], feminizing agents [Heberle-Bors 1983], reduced atmospheric pressure [Imamura and Harada 1980b], ethanol [Pechan and Keller 1989], hypertonic shock [Wang et al., 1981], centrifugal treatment [Tanaka 1973] and gamma irradiation [Sangwan and Sangwan 1986] have been tested only in few species. Hardly any recent reports widening the application of these stresses in other species are available. Scarcity of reports may be due to the fact that, in general, widely used stresses, where applicable, are easier to handle and does not require extra labor. However, these stresses may prove effective to induce microspore embryogenesis in recalcitrant species in which the conventional stresses were not successful. Recently several novel stresses have been reported to induce microspore embryogenesis with success in some species. Incubation of microspores in a high medium pH [Barinova et al., 2004], or in the presence of inducer chemicals [Zheng et al., 2001, Liu et al., 2001], heavy metals [Zonia and Tupy 1995], carrageenan oligosaccharides [Lemonnier-Le Penhuizic et al., 2001] and 2,4-D [Shariatpanahi et al., 2010] are all novel stresses to be tested in detail on other species.

IDENTIFICATION OF EMBRYOGENIC MICROSPORES A number of early markers for the conversion of microspores towards embryogenesis have been described in the literature. Some of these markers, found in different species, i.e. Nicotiana tabacum [Dunwell and Sunderland 1974 a, b, Garrido et al., 1995, Touraev et al., 1996a], Brassica napus [Zaki and Dikinson 1990, Telmer et al., 1993], Datura [Sangwan and Camefort 1983, 1984] are: (a) fragmentation of the vacuole by formation of cytoplasmic strands from the peri-nuclear to the sub-cortical cytoplasm, (b) movement of the nucleus to the center of the microspore resulting in a central phragmosome, (c) increase in the size of the cell, (d) formation of a new cell wall below the exine, (e) size reduction of the nucleolus, (f) compaction of chromatin, (g) the appearance of a zone of multi-vesiculate bodies, resembling lysosomes, and the degeneration of plastids, (h) tannin-coated tonoplasts, (i) size reduction of the starch grains, (k) no marked structural changes of mitochondria, (l) symmetric division with a planar wall instead of the regular asymmetric cell division in gametophytic microspores [for details see Touraev et al., 2001, Aionesei et al., 2005]. But none of these markers were found to be universal in embryogenic microspores of all species. However, among them, the ―star-like‖ structure of a microspore after stress treatment, seen under the light microscope [Touraev et al., 1996a] and exhibiting a centralized nucleus surrounded by

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―star-like‖ cytoplasmic strands was shown to be sign of embryogenic microspores in a number of different species such as wheat [Touraev et al., 1996b], tobacco [Touraev et al., 1996a], apple [Höfer et al., 1999], and rice [Raina and Irfan 1998]. Furthermore, recent studies using tracking of the entire process of embryogenesis from single selected wheat microspores clarified that microspores with a star-like internal structure and a symmetrical cell division is essential for the initiation of the embryogenic development of isolated microspores [Indrianto et al., 2001]. In other species just the frequency of microspores with a symmetrical division is used as a marker [Telmer et al., 1993] although it has been shown that microspores with two cells of equal size can develop gametophytically [Touraev et al., 1995].

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MICROSPORE-DERIVED ALBINO PLANTS In cereals, the use of microspore-derived doubled haploids in breeding is limited by the occurrence of pigment-deficient (albino) regenerants. These albino plants lack chlorophyll and are unable to carry out photosynthesis. Albino regenerants are frequently found in cereal crop species such as wheat (Triticum aestivum, Triticum durum), rice (Oryza sativa), barley (Hordeum vulgare) and forage grasses like Lolium or Festuca, whereas it is less frequent in maize and not a problem at all in dicot plants [Touraev et al., 2001]. The expression of the albino phenotype in microspore-derived plants is dependent on a variety of factors, including genetic as well as environmental factors. Some of these factors are (a) the lack of genes required for chloroplast differentiation and chlorophyll synthesis [for ref. See Touraev et al., 2001]; (b) plastid DNA deletions as the result of a normal mechanism during pollen development ensuring maternal inheritance of plastids [Day and Ellis 1984]; (c) the absence of the plastid-encoded proteins [Hess et al., 1993; Hofinger et al., 2001]; (d) high temperature (32-34°C)-induced translation deficiency in plastids [Herrman and Feirerabend 1980; Hess et al., 1992].In the absence of evidence for deletions or transcription failure as primary mechanisms, a block in translation has therefore been postulated as the primary cause for albino formation [Hofinger et al., 2001]. It has been shown that manipulations of the microspore culture conditions may solve the problem. In one-step regeneration anther culture, which avoids callus formation, fewer albinos are produced [Liang et al., 1987]. In addition, direct embryogenesis system in isolated microspore culture of wheat in which embryogenic microspores are formed without any apparent stress treatment, enhanced significantly frequency of green plants [Shariapnahi et al., 2006b]. Recently, it was proved that stressful in vitro conditions in microspore cultures could make the plants fight their own plastids with antibiotic like compounds resulting in formation of albino [Torp and Andersen 2009].

GENE EXPRESSION DURING INDUCTION OF MICROSPORE EMBRYOGENESIS Tobacco and rapeseed in which microspore cultures have been developed to their highest efficiency in haploid production [Custers et al., 1994, Touraev and Heberle-Bors 1999] have

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been used to study the molecular mechanisms underlying the developmental switch in microspores from gametophytic to the sporophytic mode of development. Genes specifically expressed during embryogenic induction of microspores have been studied extensively [Touraev et al., 2001, Marashin et al., 2005, Malik and Krochko 2009]. The BABY BOOM (BBM) gene is one of many genes identified as being differentially expressed in embryogenic microspores. BBM was shown to encode a new member of the AP2/ERF transcription factor family and led to the formation of embryogenic structures on seedlings when over-expressed ectopically in Arabidopsis [Boutilier et al., 2002]. However, the role of this gene in microspore embryogenesis is not known. During heat-induced microspore embryogenesis in Brassica napus, both mRNAs and several specific proteins are synthesized de novo in response to the stress treatment and some of the genes activated during embryogenic induction have been identified as members of different families of heat-shock genes [Cordewener et al., 1994, 1995]. The question remains as to whether these stress-induced proteins are really involved in the process of microspore embryogenesis or are simply required for the microspores to survive the stress treatment. However, it was shown that much higher levels of a small heat-shock protein (18 kDa) are present in embryogenic tobacco pollen in which no heat shock was applied as compared with mid-bicellular pollen [Zarsky et al., 1995]. In maize, Magnard et al., [2000] showed using differential display that transcripts of two genes (zmae1 and zmae3), normally expressed in maize endosperm, interestingly also accumulated in microspore-derived multicellular pro-embryos after 5 days of culture. Three phosphoproteins, NtEPb1, -b2 and –b3, were also found to be abundant in embryogenic tobacco microspores [Kyo et al., 2002]. In vitro phosphorylation assays in extracts of mid-bicellular and embryogenic tobacco pollen showed quantitative and qualitative changes on protein kinase activities during the starvation treatment [Garrido et al., 1993]. Therefore, protein kinases are likely to be involved in the transduction of the hunger signal, mediating the effects of starvation on gene expression and cell-cycle regulation. Malik and Krochko [2009] used transcript profiling methods to identify differentiallyexpressed genes as well as shifts in metabolism during the early stages of microspore embryogenesis. Differentially- regulated gene clusters and 16 genes were identified that could be used as specific markers for microspore embryogenesis. It can be concluded that the switch of microspores towards embryogenesis is a complex of changes in gene regulation, transcription and metabolism. And, depending on the type of stress applied, different genes may trigger microspores towards sporophytic development.

MICROSPORE EMBRYOGENESIS AND DOULBED HAPLOIDS IN PLANT BREEDING Modern plant breeding programs depend on the availability of homogenous genetic resources with guaranteed levels of identity. Doubled haploids obtained by ME are the fastest route to homozygosity, in just one generation. They are absolutely pure lines with enormous advantages for line and hybrid breeding, as they speed up breeding programmes [Forster et al., 2007]. Most excitingly, in combination with genetic marker technology, they allow to define the best recombinant profiles early in a plant breeding programs and the most desirable

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lines can then be selected for further breeding. However, the technology is not available for all crop species and even genotypes of a given species, irrespective of the method used. For example, Brassica napus, var. Topas is highly responsive for ME whereas in many other varieties induction of ME and regeneration are still limiting factors [Swanson 1990]. The same is true for tobacco: Nicotiana tabacum varieties are, in general, highly responsive for ME and the large number of doubled haploids are produced routinely whereas Nicotiana alata is recalcitrant and ME is still not possible (unpublished data).

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MICROSPORE EMBRYOGENESIS AS A TOOL FOR MUTAGENESIS AND MUTANT SELECTION Microspore cultures in vitro can be used efficiently for mutagenesis and selection. Because the microspore is a single haploid cell, any genetic variation induced by the mutagen will be expressed in the entire regenerated plant and progeny, therefore eliminating chimeras [ Kott, et al., 1996]. All recessive and dominant traits are readily expressed and are easily selectable in culture. Such traits can be fixed by chromosome doubling to achieve homozygosity. This is unlike diploid cell selection where recessive traits can be masked by a dominant trait in the heterozygote. Since selection can be made at the haploid or doubled haploid state, plants carrying undesirable trait combinations can be readily discarded instead of being carried for generations as in heterozygotes. Apart from the highly regenerative potential of microspores, there are other advantages for their use in mutagenesis. Many uniform cells can be exposed to chemical or physical mutagens in a relatively small space. Appropriate selection pressure can be applied in the culture to select mutants. Mutagenesis was used to develop plants resistant to imidazolinone herbicides in Brassica napus cultures by mutagenising microspores with ethylnitrosourea (ENU) [ Swanson et al., 1989]. Microspores or microspore-derived haploids are also useful for selecting mutants that accumulate storage products. Mutants with high oleic acid were isolated from mutagentreated microspores of Brassica napus [ Kott et al., 1996].

MICROSPORE EMBRYOGENESIS AND DOUBLED HAPLOIDS ARE CORE FOR REVERSE BREEDING ME and DH plants also play an integral part in reverse breeding (RB), a recently developed technology [Dun et al., 2006]. This technology involves down regulation of meiotic recombination to develop a concept termed reverse breeding. DH are recovered from microspores of plants in which recombination has been suppressed through down regulation of genes like DMC1 and SPO11 via RNAi. Because of the lack of recombination only (1/2)5 normal (euploid) spores are expected to survive until plant regeneration. This means still about 3% of the spores will be normal. The population that will be generated from such RB events is highly unique because it will contain only combination of parental chromosomes without scrambling by recombination. More importantly, chromosome substitution lines are generated in only one step. By back-crossing a substitution line to its most related parent and subsequently inbreeding, Introgression Lines are obtained ―per chromosome‖.

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MICROSPORE EMBRYOGENESIS IN FRUIT TREES In perennial plants particularly, where conventional breeding is usually time-consuming due to their long reproductive cycle, high degree of heterozygosity and complex reproductive biology, the potential of microspore embryogenesis shows a great advantage in the development of haploids and doubled haploids from heterozygous fruit trees in a single step which is almost impossible through classical breeding methods [reviewed by Germana 2009]. In some fruit trees such as peach, F1 hybrids have been developed from microspore deriveddoubled haploids offering the production of uniform seedling scion cultivars which can be profitable since it is much cheaper to plant non-grafted seedlings than to use grafted plants. In future, haploid and doubled haploid technology in fruit trees will be optimized and widely used as a powerful fruit tree breeding tool [Germana 2009].

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OVERCOMING SELF-INCOMPATIBILITY USING IN VITO MATURATION AND GERMINATION OF MICROSPORES In cross-pollinated plants with homomorphic flowers, self-fertilization can be avoided on genetic/biochemical mechanisms. There are two quite different types of self-incompatibility including Sporophytic self-incompatibility (SSI) and Gametophytic self-incompatibility (GSI). Sporophytic self-incompatibility has been studied intensively in members of the mustard family (Brassica), including turnips, rape, cabbage, broccoli, and cauliflower. In this system, rejection of self pollen is controlled by the diploid genotype of the sporophyte generation. Because the plants cannot fertilize themselves, they tend to be heterozygous. In this system, Pollen will not germinate on the stigma (diploid) of a flower that contains either of two alleles in the sporophyte parent that produced the pollen. In such a cross-pollinated plant, Sporophytic self-incompatibility can be overcome using isolated microspore culture method. Microspores can be isolated in vitro and be matured and germinated. In vitro germinated pollen grains can be used for in vivo pollination of SSI plants. Thus, selffertilization can be restored [reviewed by Touraev et al., 2001].

MICROSPORE-BASED TRANSFORMATION The techniques, by which transformation are carried out on microspore or pollen [reviewed by Touraev et al., 2001], can be divided into three, i.e. (I) mature pollen-based transformation in which the DNA either is delivered into pollen before pollination or is applied to stigma before or after pollination (pollen tube pathway); (II) Male Germ Line Transformation (MAGELITR) in which the DNA is transferred biolistically into unicellular microspores in the G1 phase of their cell cycle and cultured in vitro to form mature pollen and then this pollen is used for pollination in vivo and the resulting seeds are selected for transformants; (III) microspore or immature pollen embryogenesis-based transformation in which the embryogenic microspores or immature pollen grains induced by stress are transformed and divided symmetrically giving rise to embryos and haploid plants under optimal conditions.

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Microspore is an attractive target for transformation because it is an easily available and accessible single haploid cell and ME is also available in many important crops [ Harwood et al., 1996]. In addition, the transgenes can be studied in both haploid and doubled haploid plants. Although the primary target is the uni-cellular microspore, cells or explants at all stages of ME and regeneration can be used as recipients for gene delivery. Applying haploid cells for gene transfer avoid hemizygosity [Touraev et al., 2001]. In the case when chromosome doubling occurs very early during microspore culture, either spontaneously or induced, homozygosity already exists in the regeneration process [Touraev et al., 2001]. Particle bombardment of barley embryogenic microspores has resulted in transgenic doubled haploids that were homozygous for the transgene [ Shim et al., 2009a, b], and similar results were reported in Brassica napus [ Abdollahi et al., 2007, Cegielska-Taras et al., 2008]. More efficient was the transformation of barley microspore-derived multi-cellular structures using Agrobacterium tumefaciens [ Kumlehn et al., 2006], demonstrating the feasibility of this technology for other cereals where ME is amenable. In tobacco, particle bombardment has been used to transform embryogenic and non-embryogenic microspores and immature pollen [ Aionesei ei al. 2006, Touraev et al., 1997b].

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DEVELOPMENT OF AN ENVIRONMENTAL-FRIENDLY F1 HYBRID BREEDING TECHNOLOGY Male sterility that facilitates F1 hybrid seed production and double haploid technology that help to speed up the breeding cycle are two most important technologies in modern breeding [Budar and Pelletier 2001, Ribarits et al., 2009]. A novel technology package has been developed that is able to combine these two valuable technologies and present an environment-friendly breeding system for both seed and non-seed crops [Ribarits et al., 2007, 2009]. Double point mutations in two critical positions were introduced into tobacco cytoplasmic glutamine synthetase (GS1), fused to the microspore-specific NTM19 promoter, and transformed to tobacco. Approximately 50% of pollen in T0 primary transgenic lines aborted close to the first pollen mitosis stage and homozygous 100% male-sterile T1 lines were produced via in vitro microspore embryogenesis. Male-sterile T1 progenies are able to set seeds after glutamine spraying and pollinated with wild-type pollen. Thus, microsporespecific inhibition of GS1 in developing pollen allowed the rapid generation of male-sterile inbred lines and the maintenance by fertility restoration. It widely avoids limitations of currently existing systems and, as a novelty, allows the generation of male-sterile doubled haploids via microspore embryogenesis. Such doubled haploid plants are 100% homozygous and nowadays used by many breeders to produce recombinant inbred lines. This technology allows maintaining the male-sterile lines by three different approaches (by in vitro maturation, glutamine sprays and microspore embryogenesis). The use of the microspore-specific NTM19-promoter renders F1 hybrids fertile due to pollination with segregating wild-type pollen. In any instance, the release of transgenic pollen is precluded in the production field where fertility restoration is not essential. The displayed technology avoids toxic substances or chemicals with potential side effects to induce male sterility or to restore fertility, and employs plant sequences. In addition, we believe that it is a technological advance, feasible in an agricultural setting, and provides a

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smart alternative to previously published systems. It is the first example of a molecularly designed breeding technology that combines reversible male sterility and doubled haploid production, and can potentially be applied to virtually any important staple crop. Currently we have established the same technology in important vegetable tomato [unpublished data].

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REPROGRAMMED AND EMBRYOGENIC MICROSPORES AS TARGET FOR TARGETED GENE REPLACEMENT Gene targeting (GT) by homologous recombination (HR) is a genetic tool allowing precise integration of genes at predetermined genome positions as well as the production of specific and predictable changes in the host genome [Reiss 2003]. Homologous recombination (HR) and gene targeting occurs efficiently in many lower eukaryotes such as bacteria and budding yeast and recently became available as a powerful tool also in some higher eukaryotes such as Drosophila, mouse, and human somatic cells [ Hanson, and Sedivy 1995]. In flowering plants GT technology is still inefficient and has limited reproducibility [Reiss 2003, Tzfira and White 2005, Puchta and Hohn 2005] . The successful gene targeting in rice [Terada et al., 2002] and moss [Schaefer and Zryd 1997] was attributed to the use of competent cells to HR and GT. In the moss (Physcomitrella) gene replacement occurs efficiently in the chloronemal cells which are haploids and arrested in the G2 phase of the cell cycle prior to gene transfer [Resch et al., 2009]. The plant male gametophyte as a target for gene targeting has been proposed earlier in the light of success of gene targeting in moss [ Puchta 1998]. Higher plant microspores and immature pollen similar to moss are haploid, gametophytic cells which experimentally can be arrested in the G2 phase of the cell cycle. They may, for these reasons, also offer high HR frequencies and, thus, be the best cell type for gene targeting experiments [ Resch et al., 2009]. Recently, immature pollen grains were used for GT to evaluate the potential of higher plant male gametophyte as a target for GT experiments [Resch et al., 2009]. The artificial B18/4 target locus inserted to tobacco genome was used to assess gene targeting in tobacco mid-bicellular pollen. In this system, a neomycin-phosphotransferase (npt II) gene which is expressed exclusively in seeds was converted into a constitutive npt II gene by insertion of the CaMV 35S promotor between the HMW seeds-specific promotor and a functional restored npt II gene at the target locus. The tobacco mid-bi-cellular pollen isolated from the B18/4 target locus plants were transformed biolistically with the repair construct. Southern analysis confirmed an ectopic GT event occurred in one transgenic line via modification of the repair construct by the target locus and subsequent integration elsewhere in the tobacco genome [Resch et al., 2009].

MICROSPORE EMBRYOGENESIS AS A SYSTEM TO STUDY PLANT CELL REPROGRAMMING, TOTIPOTENCY AND EMBRYOGENESIS One of the most intriguing questions in developmental biology is the reprogramming of a somatic cell with restricted developmental options into a cell, which is able to give rise to an

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embryo and a reproductively competent organism [ Heberle-Bors and De Vries 1997]. This phenomenon, called ―totipotency,‖ has first been proposed by the Austrian botanist Haberlandt [Haberlandt 1922] and experimentally induced in higher plants [Reinert 1959]. Despite a large body of experimental data, obtained in almost 50 years, little is known about the mechanism of cell reprogramming and formation of totipotent cells [ Halperin 1995]. Several experimental systems have been developed and used with various levels of success to study this process. Reprogramming and totipotency have been demonstrated also in male reproductive cells [Touraev et al 1997a, 2001, Guha and Maheshwari 1964]. Isolated and in vitro cultured microspores or immature pollen of flowering plants can be induced to undergo a developmental switch by certain physical and chemical stress treatments, such as cold, heat or starvation [ Touraev et al 1997a, Shariatpanahi et al., 2006a]. More precisely, microspores convert from their intrinsic gametophytic development through a stage of totipotency towards a sporophytic pathway, resulting in the formation of haploid embryos and plants [ Touraev et al., 1997a]. Microspore embryogenesis has several unique properties which make this system ideal to study the molecular and cellular biology of plant cell reprogramming, totipotency and embryogenesis: direct embryogenesis from a single, isolated cell, free of surrounding tissues, large amount of synchronised embryogenic cells, and the possibility to study the formation of the embryo founder cell.

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CONCLUSION Plant microspores, when isolated, stressed and cultured in vitro, can be diverted from their normal gametophytic pathway towards embryogenesis, with the formation of haploid embryos and, ultimately, doubled haploid plants. This process, called microspore embryogenesis (ME), is a very attractive system to study the mechanism of plant cell reprogramming, totipotency and embryogenesis. In addition, ME is widely used a) to generate haploids and homozygous doubled haploids, which are core technology for many genetic studies and plant breeding, b) in plant transformation, mutagenesis, gene targeting etc. Doubled haploids obtained by microspore embryogenesis are the fastest route to homozygosity, in just one generation [Touraev et al., 2001; Thomas et al., 2003]. Isolated microspores are unique plant haploid cells as they can develop in vitro sprophytically forming embryos via stress treatment or gametophytically producing mature pollen grains. This ability enables microspores to be used in both fundamental and applied science [Touraev et al., 1997a , 2001]. Recently, the NtDCN1 gene (the Nicotiana tabacum DCN1 ortholog) has been identified in our group to play a significant role during the transition of microspores into pollen grains in vivo, into embryogenic microspores to initiate sporophyte formation in vitro, and from the globular stage of both microspore-derived and zygotic embryos to the heart-shaped stage (Hosp et al., submitted). It has been demonstrated that loss of function of NtDCN1 caused by RNAi blocked these transitions. Over-expression of NtDCN1 accelerated the formation of embryogenic microspores by reducing the duration of the stress treatment required to reprogram microspores into sporophytic development. These results clearly indicate that

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NtDCN1 is involved in phase changes during gametophyte development and embryogenesis in plants. Biochemical experiments defined that NtDCN1 binds both ubiquitin and RUB1/NEDD8 and associates with cullin, suggesting that indeed cullin neddylation followed by targeted protein degradation may be required for the above mentioned transitions. Development of a novel and reversible male sterility system using targeted inactivation of glutamine synthetase presents an environment-friendly breeding system for both seed and non-seed crops [Ribarits et al., 2007, 2009] which is very useful for breeders. We think that in future F1 hybrids via this technology will be developed in more species. In the end, it can be concluded that microspore is a remarkable multi-functional haploid cell which can be used for various purposes.

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Zheng, MY; Liu, W; Weng, Y; Polle, E; Konzak, CF. Culture of freshly isolated wheat (Triticum aestivum L.) microspores treated with inducer chemicals. Plant Cell Rep, 2001, 20, 685-690. Zonia, LE; Tupy, J. Lithium treatment of Nicotiana tabacum microspores blocks polar nuclear migration, disrupts the partitioning of membrane-associated Ca+2, and induces symmetrical mitosis. Sex Plant Reprod, 1995, 8, 152-160.

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Chapter 10

THE ROLE OF ANION CHANNELS IN POLLEN GERMINATION AND TUBE GROWTH Maria Breygina, Anna Smirnova, Natalie Matveeva and Igor Yermakov Department of Plant Physiology, Biological Faculty, Lomonosov Moscow State University, Moscow, Russia

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ABSTRACT The basic feature of polar growing cells is uneven distribution of organelles, forming distinct cytoplasmic zones. It is closely related to the cytosolic free ion gradients and transmembrane ion fluxes, which may cause uneven membrane potential distribution along the cell surface. Participation of Ca2+, H+ and K+ in pollen tube growth has been proved in numerous studies. Data on inorganic anions contribution to the growth process are scarce and controversial. In somatic plant cells anion channels have vital functions, including membrane voltage and turgor pressure regulation. In this chapter we give an overview of recent findings in ionic regulation of pollen germination and give evidence for the important role of anion channels in this process. The use of inhibitory analysis combined with fluorescent methods has allowed us to observe both temporal and spatial changes of membrane potential and reveal the involvement of anion channels in the regulation of this value. Our data on the key role of anion channels in structural and functional compartmentalization of the polarized pollen tube cytoplasm are considered. A contribution of mitochondrial anion channels to the pollen tube growth regulation is also discussed. Keywords: pollen germination, pollen tube, anion channels, membrane potential.

INTRODUCTION Germinating pollen grain provides the formation and delivery of the sperm cells to the ovule, and therefore plays a central role in sexual plant reproduction. Moreover, it is

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relatively simply organized, and became a widely used model in studies of plant physiology, including those of morphogenesis regulation and polar growth. An important feature of male gametophyte is a capability for polar growth based on structural and functional cytoplasm compartmentalization. Numerous studies demonstrated common mechanisms controlling the apical growth of pollen tubes, root hairs, fern rhizoids, fungal hyphae and axons of neurons (Geitmann, Emons, 2000; Palanivelu, Preuss, 2000; Bushart, Roux, 2007). The pivotal role in polar growth organization is played by inorganic ions (Hepler et al., 2006). Heterogeneous distribution of ion fluxes, intracellular ion gradients and their connection with morphogenesis has been shown for different cells, including growing fungal hyphae, frog and fish oocytes, drosophila and brown algae eggs (Nuccitelli, 1988). As early as in 1975 it was shown using nonselective microelectrodes that lily pollen grain activation leads to the appearance of transmembrane ion currents (Weisenseel et al., 1975). The inward current reached maximal values in the place of the would-be germination, while the outward current – on the opposite pole. After germination the inward currents were distributed along the tube, while the outward currents were shifted towards the pollen grain. According to these authors (Weisenseel, Wenisch, 1980), membrane potential values of the vegetative cell of lily pollen grain varied from -90 to -130 mV. In the later studies similar results (from -110 to -150 mV) were obtained for lily pollen using improved microelectrode methods (Obermeyer, Blatt, 1995). For pollen grains of other plant species, more positive membrane potential values have been reported: -30 mV in Petunia hybrida and -37 mV in Narcissus (Feijo et al., 1995), while potential of isolated pollen protoplasts of Brassica chinensis was -79 mV (Fan et al., 2003). Pollen tube membrane potential also varied in different plant species: e.g., in Agapanthus umbellatus it amounted to -55 mV (Malho et al., 1995), whereas in Arabidopsis – around 100 mV (Mouline et al., 2001). Recently the data on heterogeneous distribution of ion currents in the pollen tube has been approved using ion selective microelectrodes. It has been shown that potassium, calcium and protons enter the pollen tube in its apical part (Michard et al., 2009); outward proton current is located in more distal parts of the tube, and its intensity changes along the tube length (Michard et al., 2008, 2009). The question about possible changes of membrane potential during pollen germination has been brought up in early studies of Weisenseel and Wenisch (1980), Obermeyer and Blatt (1995). These authors found out that ungerminated pollen grains divided into two populations with more or less negative membrane potential. The potential of ―more negative‖ group became less negative when pollen was treated with cyanide or incubated in cold. The potential of ―less negative‖ group didn't respond to these treatments. However, the question about temporal or spatial membrane voltage alterations has not been solved in microelectrode studies. Essential progress could be reached by means of fluorescent methods that had earlier been successfully applied in studies of animal and fungal cells. Among different ion-transport systems responsible for pollen germination anion channels are the less studied. Attempts to find anion channels in pollen grain and tube using microelectrodes didn`t succeed (Dutta, Robinson, 2004), though transcriptomic analysis showed high expression of two pollen-specific chloride channels (Moreno et al., 2007). On the other hand, according to Zonia et al. (2002), the tube growth is accompanied by intense oscillating Cl¯ efflux. Messerli et al. (2004) used the same ion-selective electrodes to demonstrate that Cl¯ efflux can be mistaken for H+ influx. According to Hepler et al. (2006),

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controversial results obtained by these two groups can be explained by distinctive technical features of experiments. One can conclude that microelectrode method can not answer the question about Cl¯ fluxes, and alternative technical approaches are needed to study this problem. Among the most useful methods there are quantitative fluorescent microscopy and fluorometry. However, data obtained using inhibitory analysis demonstrate an important role of transmembrane anion transport in germination. Matveyeva et al. (2003) studied the contribution of Cl¯ transport to the germination process using ionselective electrodes. They found out that anion channel inhibitors (NPPB - 5-nitro-2-(3-phenylpropylamino) benzoic acid, DIDS - 4,4′-diisocyanato-stilbene-2,2′-disulfonic acid and niflumic acid) totally suppress germination (Matveyeva et al., 2003). NPPB was the most effective inhibitor, and it also blocked Сl¯ efflux from pollen grains, while DIDS had no effect on it. Zonia et al. (2002) showed that NPPB, nuflimic acid and DIDS suppressed the growth of pollen tubes and caused their swelling. These authors assumed that the main function of anion channels in pollen tubes is osmotic balance regulation. In somatic plant and animal cells anion channels are an important part of growth and development regulation. Anion channel activity is closely related to cytosolic calcium dynamics. This connection is most studied in stomata guard cells (De Angeli et al., 2007) and in plant cells attacked by pathogen or treated with elicitors (Ebel et al., 1995; Wendehenne et al., 2002). There is also an interrelation between anion and potassium transmembrane transport, which has been clearly demonstrated in situations of osmotic stress (Shabala et al., 2000), cell growth (Heslop-Harrison, Reger, 1985) and stomata movements (Barbier-Brygoo et al., 2000). One can assume that these conceptions cover the process of pollen germination. However, to understand clearly the role of anion channels in germination and pollen tube growth one must study their contribution to the membrane potential regulation, osmotic balance and organelle compartmentalization. Another matter of interest is functioning of intracellular anion channels in male gametophyte, and, first of all, channels of mitochondrial membranes. In plant somatic cells anion channels have been found in both internal and external mitochondrial membranes. These channels provide for ion and metabolite transport between cytosol and matrix and, together with potassium channels, hold membrane potential (O‘Rourke, 2007; Laus et al., 2008). They also serve for ROS transport to the cytosol (Godbole et al., 2003; Han et al., 2003). One can suppose an important role of anion channel activity in mitochondria functioning in pollen grain and tube. These questions haven't been previously studied.

MATERIALS AND METHODS Plant Material and Sample Preparation Objects were cut plants of lily Lilum longiflorum Thunb., variety White Europe and plants of tobacco Nicotiana tabacum L., variety Petit Havana SR1, grown from seeds in a climatic chamber (25°С, 16-h light day). Anthers were removed from flowers on the eve of their opening and placed into a thermostat (25°С) for 3 days. Pollen from the opened anthers

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was collected in test tubes and stored at -20°С. After thawing-out the pollen grains were washed out from tryphine with hexane and air-dried. Dry pollen samples were incubated in a moist chamber at 25°С (1-2 h) before they were used to obtain the pollen grain cultures or protoplasts. Pollen was incubated in the standard medium in 2.5-cm Petri dishes or in the 35-μl plastic cultural chambers (CoverWell, Schleicher and Schuell, Germany) covered inside with 0.01% poly-L-lysine. In the latter case, the pollen tubes attached to the upper chamber wall were analyzed. For protoplast isolation, lily pollen was incubated in the medium with enzymes (see below) for 2 h at 30°C, then washed with the same medium without enzymes and used immediately for staining or fixation. Protoplasts isolation was controlled by the commonly used method (Tanaka et al., 1987) by staining samples with Calcofluor White M2R fluorescent dye (Fluorescent Brightener 28, Sigma) that reveals the presence of the cell wall.

Mitochondria Isolation

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Mitochondria were isolated from pollen after 2.5 hour incubation, according to Hajek et al. (2004). Pollen was collected on nylon 20 μm filter, then rinsed with washing buffer and transferred into isolation buffer. Pollen was broken with Diax 900 homogenizer (Heidolph, Germany) at 17000 rpm for 45 s. Mitochondria purification procedure included two step centrifugation (5 min 1500 g, then 10 min 6000 g), sedimentation at 12000 g (10 min), transfer into new buffer portion and fractioning in Percoll gradient (40 % and 23 %) at 12000 g (30 min). Mitochondrial fraction formed a band in the upper Percoll layer. It was washed on membrane 0.22 μm filter with washing buffer, stored and stained at 4 °С. Mitochondrial protein concentration in tested samples was approximately 114 μg /ml (Lowry test).

Reagents DIDS - 4,4′-diisocyanato-stilbene-2,2′-disulfonic acid, NAO - 10-N-nonyl acridine orange, DiОC5(3) - 3,3'-dipentyloxacarbocyanine iodide, DiВAC4(3) – Bis(1,3dibutylbarbituric acid(5)) trimethine oxonol and Di-4-ANEPPS – 3-(4-(2-(6-(dibutylamino)2-naphthyl)-trans-ethenyl)pyridinium)propane sulfonate (Molecular Probes, the Netherlands), fusicoccin (Serva, Germany), cellulose, pectinase and sodium orthovanadate (ICN, USA), NPPB – (5-nitro-2-(3-phenylpropylamino)benzoic acid, DCFH-DA - 2‘,7‘dichlorodihydrofluorescein diacetate, СССР - carbonyl cyanide m-chlorophenylhydrazone, PMSF – phenylmethanesulphonyl fluoride, Fluorescent Brightener, Protease Inhibitor Cocktail and poly-L-lysine (Sigma, USA).

Composition of Growth Media and Added Reagents The standard incubation medium included 0.3 M sucrose, 1.6 mM H3BO3, 3 mM Ca(NO3)2, 0.8 mM MgSO4 and 1 mM KNO3 in 25 mM MES-Tris buffer, pH 5.9. Mitochondria isolation buffer (Hajek et al., 2004) included 0.3 М mannitol, 2 mM EGTA, 2

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mM MgCl2, 4 mM cystein, 0.4 mM PMSF, 1 μl/ml proteinase inhibitor cocktail in 25 mM HEPES buffer, рН 7.5. Mitochondria washing buffer was the same composition without cystein and inhibitors, рН 7.2. Fusicoccin, sodium orthovanadate, and NPPB were added to the pollen grain suspensions after the 75-min incubation in the standard medium. The final fusicoccin concentration amounted to 1 μM, of orthovanadate – 1 mM, of NPPB – 40 μM, of DIDS – from 10 to 80 μM. Time of action of each of these reagents on the pollen tubes was 10 min. Changes of the membrane potential value were revealed using two dyes: DiВAC4(3) and Di-4-ANEPPS. DiВAC4(3) belongs to the group of slow dyes; charged molecules of these dyes are distributed between the cell cytoplasm and surrounding medium in correspondence with the Nernst‘s equation (Plašek, Sigler, 1996). The potential was calculated as described elsewhere (Emri et al., 1998) from the equation

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E = (RT/F)·ln(I/I0), where I and I0 are the fluorescence intensity of the tested cell and cells in a fixed sample (completely depolarized cells), respectively. The fluorescence values for pollen grains were corrected for the dye nonspecific binding to the pollen cell wall. For this purpose, fragments of cell wall after pollen grain crushing were stained together with the studied cells. The cells were stained with 5 μM DiВAC4(3) solution for 10 min. Di-4-ANEPPS belongs to the group of fast dyes that are inserted into the membrane. With the change of membrane potential the charge in the dye molecule migrates, which leads to a shift of the excitation and emission spectra (Loew, 1996). Usually an intensity of fluorescence excited in two spectral regions (the blue and green ones) is measured. Their ratio (Fb/Fg) is a measure of membrane potential at a certain membrane area. Thus, the use of this dye allows detecting local changes of membrane potential within a single cell. The pollen tubes were stained in a drop on a microscope slide by mixing suspension of germinated pollen grains with the Di-4-ANEPPS solution (10 μM) in the 1:1 ratio and were microscoped immediately. Cl¯ transport through the plasma membrane was blocked by two methods: (1) using anion channel inhibitor NPPB in final concentration 40 μM or DIDS in different concentrations and (2) by adding Cl¯ to the medium to equalize its electrochemical potential on both sides of the plasma membrane. Extracellular concentration of Cl¯ (Cout) was calculated from the following equation: ∆=in - out = RT ln Cin/Cout zFЕ, if ∆= 0, then Cout= Cin e zFE/RT, where in, out are the electrochemical potential values of the ion on either side of the membrane, Cin, Cout are the concentrations of the ion on either side of the membrane (M), E is

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the electrochemical potential on the membrane (V), T is the absolute temperature (K), F is the Faraday constant, and z is the ion charge. The membrane potential (E) values determined in preliminary experiments equaled -37 and -77 mV for unactivated pollen grain and pollen tube, respectively. The intracellular concentration of Cl¯ in a hydrated pollen grain of tobacco (Cin) is about 50 mM (Andreyuk et al., 2001); no experimental data are available for the pollen tube, so the value Cin = 5 mM was chosen based on the analysis of different plant cells (Taiz, Zeiger, 2006). According to our calculations, the addition of 200 and 100 mM Cl¯ should suffice to inhibit Cl¯ release from the pollen grain and pollen tube, respectively. These concentrations were used in the experiments. Medium with high Cl¯ concentration was prepared by the addition of 4 M HCl and adjusting pH to 5.9 with 0.5 M bis-tris propane. To stabilize osmotic conditions, sucrose level was accordingly decreased in media with high Cl¯ concentration. Mannitol was used instead of Cl¯ in control samples. In the experiments on pollen grain germination, NPPB or 200 mM Cl¯ was present in the medium from the beginning of incubation. In the experiments on pollen tube growth, NPPB or 100 mM Cl¯ was added to the medium after the tubes reached the length exceeding the pollen grain diameter in standard medium. Efficiency of pollen germination (proportion of pollen grains germinated after 60-min cultivation) was determined in fixed samples. In each sample, 500 cells were counted. Oxygen consumption by pollen grain suspension was determined with Clarke electrode.

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Cell Fixation After culture, 2% paraformaldehyde in 50 mM sodium phosphate buffer (pH 7.4) was added to cell suspension (1 : 1, v/v). After fixation at 6°C for 15 h, the samples were washed from fixative twice in the same buffer.

Identification of Transport Vesicles and Mitochondria Transport vesicles were identified by the method of Parton et al. (2001) by staining the pollen tubes with the lipophilic dye N-(3-triethylammoniumpropyl)-4-(6-(4(diethylamino)phenyl)hexatrienyl) pyridinium dibromide (FM4-64; Molecular Probes, the Netherlands), which enters living cells only by endocytosis and stains different populations of vesicles (Samaj, 2005). The final concentration of the dye was 8 μM. Mitochondria were identified by staining the pollen tubes with the fluorescent dye 10-N nonyl-acridine orange (NAO; Molecular Probes, the Netherlands) (Mileykovskaya et al., 2001). The final concentration of the dye was 5 μM. The distribution of the transport vesicles and mitochondria in the tube was studied by germinating pollen grains in the presence of FM4-64 and NAO, after which the incubation continued in dye-free medium containing NPPB or 100 mM Cl¯ (or no inhibitors in control). Anion release from pollen grains was studied using the fluorescent dye 6-methoxy-Nethylquinolinium iodide (MEQ; Molecular Probes, the Netherlands). Weighed samples of pollen grains were incubated in medium containing 5 μM MEQ for 2 or 10 min. Pollen was

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removed by centrifugation (3000 g, 60 s) and the dye fluorescence was measured on an RF5301PC spectrofluorometer (Shimadzu, Japan). Fluorescence was excited at 344 nm and recorded at 445 nm. The autofluorescence of incubation medium was negligible.

Staining of Isolated Mitochondria Isolated mitochondria were stained with 1 μM DiОC5(3) or NAO for 10 min at 4 °С or with 10 μM DCFH-DA (Halliwell, Whiteman, 2006) for 60 min. Respiratory chain uncoupler СССР (20 μM) and anion channel inhibitors NPPB (40 μM) or DIDS (80 μM) were added to the suspension 5 min before staining. ROS excretion from mitochondria was detected by DCFH oxidation (Cathcart et al., 1983; Smirnova et al., 2009).

Flow Cytometry Isolated mitochondria were analyzed by flow cytometry using FACSCalibur cytometer (Becton Dickinson, USA) equipped with argon laser (488 nm). In each preparation 10000 events in R1 region were counted. NAO, DiОC5(3) and DCFH-DA fluorescence was counted in F-1 channel (530 ± 15 nm). Data was analyzed with FlowJo software (Treestar Inc, USA).

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Microscopy and Computer Image Analysis An Axioplan 2 imaging MOT microscope supplied with the corresponding filter sets, a mercury lamp, and an AxioCam HRc digital camera (Carl Zeiss, Germany) was used in the study. Fluorescence was excited in the range of 475-495 nm and recorded at 515-565 nm (DiВAC4(3), NAO) or excited in the blue (475-495 nm) spectral range and recorded at wavelengths higher than 590 nm (FM4-64); excited in blue or green (540-552 nm) spectral range, while recorded at wavelengths higher than 590 nm (Di-4-ANEPPS). Objects were photographed using a high-rate automatic shutter that allowed illumination of the preparation only at the moment of shooting and allowed making series of photographs with certain exposure. The images were obtained and analyzed with AxioVision 4.7 software (Carl Zeiss, Germany).

Statistics All experiments were performed in no less than in five biological repeats. Statistical significance of differences was determined by Student‘s criterion at the level of 0.05 or 0.01. Figure and table present the mean values and their standard errors.

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RESULTS The Role of Transmembrane Anion Transport in the Regulation of Pollen Germination and Tube Growth NPPB was shown to be the most effective inhibitor of pollen germination (Matveyeva et al., 2003) and tube growth (Zonia et al., 2002). It has been assumed that an important role in these processes is played by transmembrane chloride transport through NPPB-sensitive anion channels. To verify this assumption we compared pollen tube growth under blockage of anion channels activity by NPPB or selective blockage of Cl¯ efflux. In both situations pollen germination and tube growth were suppressed (Table 1), which shows participation of NPPBsensitive anion channels in the regulation of germination.

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Table 1. Pollen germination and tube growth in the presence of anion channel inhibitor 40 μМNPPB or Cl¯ (200 mM Cl¯ in germination medium, 100 мМCl¯ - in pollen tube growth medium)

Index

Control

Cl¯

NPPB

Germination efficiency, %

47 ± 2

3±1

0±1

Tube growth rate, μm/h

83 ± 11

4±4

1 ±2

To find to what extension NPPB and Cl¯ inhibit anion efflux from pollen grains, we used fluorescent dye MEQ, which is widely applied in studies of animal cells (Wöll et al., 1996). It hadn`t been used for studies of pollen. It is known that MEQ fluorescence is sensitive to Cl¯ concentration in medium (Verkman et al., 1989), but we found out that it is also sensitive, to less extension, to organic anions citrate and malate. So this dye could be thought of as anionsensitive dye with higher sensitivity to chloride. MEQ fluorescence intensity in control samples was significantly quenched after 2 minutes incubation of pollen grains (Fig. 1), which demonstrates quick anion efflux during the very first minutes of hydration. NPPB suppressed this effect almost totally. One can conclude that NPPB-sensitive anion channels are the main path for anion efflux from pollen grains on the first stage of germination. In the presence of 200 mM Cl¯ fluorescence quenching was nearly the same as in control sample. High chloride concentration inhibits efflux of this anion, so one can assume that quenching in this case is caused by some other anions coming out of pollen grains. Data on anion efflux and its inhibition by NPPB agree well with the results that had earlier been obtained by using Cl¯-selective electrodes (Matveyeva et al., 2003). It is very likely that in the situation of selective chloride efflux blockage organic anions, which are necessary for germination, come out of pollen grains instead of Cl¯. This leads to suppression of pollen germination and tube growth.

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Figure 1. Anion release from pollen grain revealed by MEQ fluorescence quenching: white bars fluorescence intensity of the solution without pollen, hatched – 2 minutes incubation of pollen, grey – 10 minutes.

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Taken together, obtained results demonstrate an important role of NPPB-sensitive anion channels, through which Cl¯ comes out of pollen grains, in male gametophyte germination. It has been proposed that the main function of plasmalemma anion channels in the pollen tube is osmotic balance regulation (Zonia et al., 2002). Comparing the effects of two anion channel inhibitors, NPPB and DIDS, we found out that DIDS, unlike NPPB, caused pollen tube swelling (Fig. 2). With rising DIDS concentration the number of burst tubes increased (Table 2). So in experiments with pollen tubes we used 20 μM DIDS, which caused osmotic effect but didn't suppress tube growth.

Figure 2. Pollen tube swelling in the presence of 20 μM DIDS and absence of the effect in the presence of 40 μM NPPB.

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Maria Breygina, Anna Smirnova, Natalie Matveeva et al. Table 2. Bursting of pollen tubes in the presence of anion channel inhibitor DIDS DIDS concentration, µM 0 10 20 40 80

Proportion of burst tubes, % 2,8±0,2 5,2±0,9 20,6±0,9 27,4±1,7 45,1±1,1

We can conclude that the involvement of anion channels in pollen germination is more complicated than it had been earlier assumed: DIDS-sensitive channels contribute to the osmotic balance regulation, NPPB-sensitive – to the growth processes. In this regard we brought up a question whether NPPB-sensitive anion channels are also involved in the maintenance of compartmentalization of the pollen tube cytoplasm and vesicle traffic that underlie the polar growth process. One could suppose that inhibition of pollen tube growth in the presence of NPPB or Cl¯ was caused by disarrangement of organelles.

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Disturbance of Functional Compartmentalization of Pollen Tube Cytoplasm in Case of Anion Transport Blockage Growing pollen tubes were analyzed by fluorescent microscopy with double staining of mitochondria and vesicles. Mitochondria were stained by specific dye NAO, which binds to cardiolipin of mitochondrial membranes (Mileykovskaya et al., 2001), transport vesicles were stained with FM4-64, which binds to plasma membrane and enters the living cell only by endocytosis (Samaj, 2005). Experiments with double staining showed severe disturbance of polar distribution of both vesicles and mitochondria. In control samples, in agreement with well-known conception (Cheung, Wu, 2008), vesicles were concentrated in the apical part of the tube, forming a cone; mitochondria were located more distally (Fig. 3a).

Figure 3. Effect of NPPB and Cl¯ on organelle distribution in pollen tubes: vesicles stained with FM464 (red), mitochondria – with NAO (green). Pollen germinated in medium with two dyes, grown tubes were incubated in dye-free medium with NPPB or Cl¯. a: control, b: 40 μM NPPB, c: 100 mM Cl¯.

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NPPB significantly altered the organelle distribution: the vesicle cone disappeared while mitochondria entered the apical zone (Fig. 3b). Thus, NPPB had an effect on polar organelle distribution and movement, causing disorganization of the apical compartment. In the presence of 100 mM Сl¯ pollen tube growth was inhibited, though polar cytoplasm organization was preserved (Fig. 3c). We can conclude that cytoplasm zonation is compulsory, but not sufficient condition for growth maintenance. One can suppose that, in situation of selective blockage of Сl¯ efflux, anion fluxes essential for polar organelle distribution are maintained at the expense of important anions` loss. These results reveal anion channels` involvement in the maintenance of polar organelle distribution in the pollen tube and their movement underlying the apical growth process. Earlier the role of ion mechanisms in the regulation of these processes had been discussed mostly in connection with calcium channels (Cheung, Wu, 2008).

The Contribution of Anion Channels to the Regulation of Plasmalemma Membrane Potential during Pollen Germination and Tube Growth One of the most important functions of anion channels in plant somatic cells is the regulation of membrane voltage, which, in turn, controls ion and metabolite transport and interactions between cell and environment. If we assumed that pollen grain activation and pollen tube growth were connected with membrane voltage alterations or its uneven distribution on the cell surface, we could expect the participation of anion channels in these processes.

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Membrane Potential Dynamics during Pollen Germination The plasmalemma potential value was measured by fluorescent microscopy and computed image analysis with voltage-sensitive ―slow‖ oxonol dye DiВAC4(3). Charged molecules of ―slow‖ dyes are distributed between the cell cytoplasm and surrounding medium in accordance with the Nernst equation (Plašek, Sigler, 1996). After measuring fluorescence intensity of living and fixed cells one can calculate membrane potential values in millivolts (Emri et al., 1998). In a preliminary study we optimized the staining procedure and calculated the correction for nonspecific dye binding with lipophilic exine. During pollen grain activation, which is reflected in the increase of oxygen consumption, membrane voltage became more negative (Fig. 4). Membrane potential of the pollen tube plasmalemma was even more negative and different in the two zones of the tube (Table 3): the apical part was less negative then the distal part. To verify this fact we carried out membrane voltage mapping using the ―fast‖ dye Di-4-ANEPPS (see below). Obtained values of membrane potential (Table 3) are in the same range as previously measured with microelectrodes (-55 mV for Agapanthus, pollen tubes, -100 mV for Arabidopsis) (Malhó et al., 1995; Mouline et al., 2001). For pollen grains membrane potential had been shown to vary from -30 mV to -150 mV (Weisenseel, Wenisch, 1980; Feijó et al., 1995). Great difference in these values can be explained by differences in the germination processes in certain species and also by different experiment duration. Indeed, comparative analysis of tobacco and lily pollen grains and lily pollen protoplasts revealed species

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differences and showed that during protoplast isolation 2-hour incubation in liquid medium caused significant hyperpolarization of plasma membrane (Table 3). So, it was important to use noninvasive fluorescent method, which allowed us to study membrane potential on the early stage of germination.

Figure 4. Plasma membrane hyperpolarization during pollen activation in vitro and the influence of NPPB on this process. Е – plasma membrane potential value. White bars – control, hatched – in the presence of 40 μM NPPB. Grey bars – oxygen consumption by pollen grain suspension.

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Table 3. Plasma membrane potential of the pollen grains and tube, determined with aid of potential-dependent dye DiВAC4(3)

Object

Membrane potential value, mV tobacco

-37 ± 1,5

lily

-23 ± 1,0

Hydrated pollen grain Lily pollen protoplast Tobacco pollen tube

-108 ± 3,0 apical part

-61 ± 3,0

25 μm from the tip

-80 ± 4,0

Revealed membrane potential changes during pollen germination (Fig. 4) can be caused by the proton pump activation. The idea of H+-АТPase as the most important electrogenic mechanism on the surface of plant cell is widely accepted (Sze et al., 1999). Indeed, the data on pH changes during germination (Rodriguez-Rosales et al., 1989; Matveyeva et al., 2002) confirms this assumption. It has been also shown that drugs stimulating and inhibiting the H+-

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АТPase activity respectively change the germination efficiency (Rodriguez-Rosales et al., 1989). In the presence of NPPB evident depolarization appeared in pollen grains, while hyperpolarization found in control samples was suppressed. Comparing these results with the fluorometery data on anion efflux (Fig. 1), one can assume that in the presence of NPPB, when anion efflux is completely blocked, the activity of different transport proteins closely connected to the anion channels can be altered. This, in turn, can cause plasma membrane depolarization and block membrane voltage alterations preceding the pollen tube appearance.

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The Mapping of Membrane Potential on the Surface of Pollen Tube For analysis of membrane potential value in different points on the cell surface the ―fast‖ styryl dye Di-4-ANEPPS was used. ANEPP molecules are known to incorporate into plasmalemma, changing spectral characteristics depending on membrane voltage (Loew, 1996). We recorded fluorescence intensity excited in blue (Fb) or green (Fg) spectral ranges and calculated Fb/Fg ratio, which reflects membrane potential value (Montana et al., 1989). This dye hadn't been used before for staining plant cells, so we needed to define its limitations in a preliminary study. We found non-specific binding to hydrophobic exine, but not to the pollen tube wall. So, we couldn`t use Di-4-ANEPPS for staining of ungerminated pollen grains. When we calculated Fb/Fg ratio along the tube plasmalemma, we found that this ratio substantially decreased from 3 to 20 μm from the tip, demonstrating a gradual hyperpolarization of plasma membrane. In the zone more distal from the tip (21 – 35 μm), Fb/Fg flattened to plateau (Fig. 5). This results agree well with the data obtained in experiments with DiВAC4(3) (Table 3), revealing the membrane potential gradient along the tube (Fig. 5). Which ion transporters could play a crucial role in the maintenance of this gradient? We assumed an important role of anion channels, which had been shown to participate in membrane potential changes, and proton pump, which is the main electrogenic force on the plant cell plasmalemma. To reveal the participation of Н+-АТPase in the gradient maintenance we studied the effect of fusicoccin, which stimulates the proton pump activity, and its inhibitor orthovanadate on the distribution of membrane potential along the tube. Both influences altered the shape of the curve (Fig. 5). Fusicoccin caused hyperpolarization in the region close to the apex (р