Genetic Manipulation in Plant Breeding: Proceedings International Symposium Organized by EUCARPIA, September 8–13, 1985, Berlin (West), Germany [Reprint 2019 ed.] 9783110871944, 9783110105964

Table of contents :
Preface
Acknowledgements
Contents
Part 1. Use Of Mutation In Breeding Research
Mutation breeding: a stepping stone between Gregor Mendel and genetic manipulation (a treatise for vegetatively propagated crops)
Induced structural rearrangements
Evaluation and selection of mutations on the basis of their conformity to plant ideotype
Chemically induced mutations for male sterility in Petunia x hybrida
Genome adjustment by breeding to balance yield defects in high-lysine mutants of barley
Selection of rust resistant wheat after mutagenesis
Use of maize mutants in breeding for improvement of protein quality
Promising rice mutants for developed production
Cytoplasmic male sterility in maize - nuclear and mitochondrial genome interdependence
Genetic studies and inheritance of seed storage protein of pea chromosome mutants
Part 2. Cytogenetics And Polyploidy
Cytogenetics In Breeding Programmes Dealing With Polyploidy, Interspecific Hybridization And Introgression
Tetraploid triticale - a tool in hexaploid triticale breeding
Instability in tomato lines with the breakage-fusion-bridge cycle
The present status of breeding autotetraploid cereals
Alien chromosome transfer from wheat into rye
Nucleolar competition in different (A/B)(A/B)RR and DDRR genomes of tetraploid triticales
Identification of interchanges in wild species of Pisum
Genetic regulation of meiotic recombination in Petunia hybrida
Attempts to transfer resistance to Phoma lingam from Brassica juncea and B. carinata to B. napus through interspecific hybridization followed by ovule culture
Analysis of ADH1 locus in tetraploid corn /Zea mays L./
C-banding of Avena species
A study of nucleic acids in the Symphytum officinale species complex and S. asperum: microdensitometry of nuclear DNA in leaf cells
C-banding and isozyme markers to analyze the segregation of rye chromosomes in the progenies of triticale x wheat hybrids
Breeding nematode-resistant sugar beets
Polyploidy in hop breeding, Humulus lupulus L.
Reconstitution of hexaploid wheat from Triticum dicoccoides (AABB, 2n=28) and T. tauschii (DD, 2n=14)
Preferential loss and gain of specific Hordeum vulgare chromosomes in hybrids with three alien species?
Genetic and cytological maps in Petunia
Biochemical characterization of cytoplasmic male sterility in Petunia hybrida
Cytogenetic studies of the polyploids of hop, Humulus lupulus L.
Cytogenetic studies of wheat lines with rye chromosome additions
Substitution of chromosome 5S of Aegilops longissima for its group-5 homoeologues of common wheat
Interspecific hybridization between V. faba and V. narbonensis: prospects and limitations
Alien gene transfer in groundnut by ploidy and genome manipulations (ICRISAT C.P. No. 277)
The potential for improving the primary distant hybrids of common wheat by anther culture
Part 3. Induction and Use Of Haploids
Criteria for the selection and use of doubled haploid systems in cereal breeding programmes
Haploid induction and production in crop plants
Effects of activated charcoal, cold treatment and elevated CO2-concentrations on embryogenesis in anther cultures
Utilisation of anther culture in breeding Brussels sprouts
The role of linkage and recombination in barley breeding with Hordeum bulbosum L.
Partial incompatibility between Scandinavian six-rowed barleys and Hordeum bulbosum L. and its genetical basis
Recent results on somatic embryogenesis in pea and bean
Sugar beet (Beta vulgaris L.) pollen quality assessment and effect of irradiation as measured by fluorochromatic reaction and in vitro germination
The detection of linkage using doubled haploids in barley
The Effect Of An Elevated CO2 Concentration In Combination With Cold Treatments In Maize (Zea Mays L.) Anther Culture
Obtention Of Embryos And Plants From In Vitro Culture Of Unfertilized Ovules Of Cucurbita Pepo
Agronomic Value Of Androgenetic Doubled Haploid Lines As Compared To Conventionally Selected Spring Barley
Pollen Plant Production In Triticum Turgidum Ssp.Durum
Production Of Haploid Sugarbeets (Beta Vulgaris L.) By Ovule Culture
Towards The Isolation Of Sperm Cells For Androgenic Purposes
Regeneration And Selection Of Isolated Microspores Of Hordeum Vulgare
Possibilities Of Obtainment And Utilization Of Doubled Haploids In Gerbera
Contribution To The Studies On The First,Generative Progeny Of Kragaria X Ananassa Polyhaploids
A Comparison Of Cross Prediction Methods In Spring Barley
Androgenesis In Oilseed Rape
Production Of Doubled Haploids In Oriental Lilies
Increasing The Efficiency Of Triticale Anther Culture
Genetic Gain For Some Agronomical Characters By Dihaploid Breeding In Barley
Effect Of A Gametocide On The Induction Of Haploids In Triticum Aestivum
The Induction Of Haploids Of Sugarbeet (Beta Vulgaris L.) Using Anther And Free Pollen Culture Or Ovule And Ovary Culture
Frost Tolerant Plants Obtained From Proline Accumulating Cell Lines
Somatic Cell Genetics Of Potato I. Use Of Monohaploids
Factors Affecting Callus And Plant Production In Anther Cultuses Of Tomato
Part 4. In-Vitro Propagation
Role Of Methodology In Facilitating Application Of Tissue Culture Techniques
In Vitro Propagation And Breeding Of Ornamental Plants: Advantages And Disadvantages Of Variability
Protoplast Culture And Use Of Regeneration Attributes To Select Somatic Hybrid Tomato Plants
Sexual Reproduction In Plants By Applying The Method Of Test Tube Fertilization Of Ovules
Cytogenetic Studies In Callus Cultures Of Asparagus Off
Systems For Regeneration Of Cucumis Sativus Plants In Vitro
Regeneration Of Temperate Fruit Trees In Vitro Via Organogenesis And Embryogenesis
Problems And Prospects For The Use Of Protoplasts In Beet Breeding
Shoot Redifferentiation Of Agrobacterium Transformed-Protoplasts And Plant Tissue- With Conventional Methods Not Achievable
Induction Of In Vitro-Regeneration Via Somatic Embryogenesis In Pea (Pisum Sativum) And Bean (Phaseolus Vulgaris)
Endogenous Cytokinins During Embryogenesis In A Carrot Cell Suspension
Tissue And Protoplast Culture In Cultivated Beets
Protoplast Formation In Cereals - An Assessment
Exploitation For Breeding Of In Vitro Culture Of Pea Explants
Some Aspects Of The In Vitro Culture Of The Beet. (Beta Vulgaris L.)
Interspecific Hybridization Of Red Clover (Trifolium Pratense L.) With Alsike Clover (Trifolium Hybndum L.) Using In Vitro Embryo Rescue
Pollen And Ovule Cultures Of Barley To Isolate, Manipulate And Transfer Sperm Cells In In Vitro Fertilization
Strategies In High Frequency Regeneration From Diploid And Haploid Cell And Tissue Cultures Of Barley
Somaclonal Variation In Plants Regenerated From Embryo Calluses In Rye (Secale Cerale L.)
Somatic Embryogenesis, Cell And Protoplast Culture Of Hordeum Vulgare L. (Barley)
Isolation And Culture Of Protoplasts From Callus And Suspensioncultured Cells Of Prunus Cerasus And Actinidia Chinensis
Somatic Embryogenesis In Protoplast-Derived Cells Of Cucumis Melo L.
A Comparative Study Of Callus Formation And Plant Regeneration From Different Explants Of Phaseolus Vulgaris And Ph. Coccineus
Somatic Embryogenesis, Cell And Protoplast Culture Of Triticale (X Triticosecale Wittmack)
Somatic Embryogenesis And Plant Regeneration From Meristematic Tissue Of Secale Cereale (Rye)
Somatic Embryogenesis Of Triticale
Part 5. Spontaneous And Induced Variation From In-Vitro Cultures
Variability In Tissue Culture Derived Plants - Possible Origins; Advantages And Drawbacks
Screening For Virus Resistance In Tissue Culture Adventitious Regenerants And Their Progeny
Chromosome Variation In Regenerated Plants
In Vitro Mutagenesis In Gerbera Jamesonii
In Vitro Mutagenesis In Maize
Somaclones Of Wheat Regenerated From Primordial Leaf Callus
Somaclonal Variation In Lotus Corniculatus L.
Potential System For The Specific Selection Of Plant Mutants Overproducing Methionine
Enhancement Of Asulam Resistance In Barley
Triazine-Resistant Nicotiana Mutants From Photomixotrophic Cell Cultures
Use Of In Vitro Culture For Inducing Variation In Rice And Fuchsia
Isolation Of Auxotrophic Mutants Based On Reconstruction Experiments With Nicotiana Plumbaginifolia Protoplasts
A Study On The Effect Of Different Initial Culture Media On The Chromosome Stability Of Solanum Tuberosum Cv. Maris Bard Protoplast Derived Regenerants
The Effect Of Explant Source, In Vitro Regeneration And Irradiation On Variation In Yield Induced In Chrysanthemum Morifolium
Transfer Of Genetic Material To Cultivated Barley From Alien Species Through Callus Culture (Preliminary Results)
Somatic Embryogenesis And Plant Regeneration From Leaf Tissues Of Secale Cereale L
Testing Of Salt /Nacl/ Tolerance And Regeneration In Callus Culture /n, 2n/ Of Rice
The Use Of Tissue Cultures For Obtaining Tobacco Male Sterile Forms
Somaclonal Variation In Triticale
Separation, Identification And Biological Effects Of A Toxin Produced By Phoma Lingam
Variability In Plants Of Sugar Beet (Beta Vulgaris L.) Regenerated From Callus, Cell-Suspension And Protoplasts
Chromosomal Variation In Regenerated Plants From Hybrid Callus From Crosses Between Hordeum Vulgare X H. Bulbosum
Part 6. Somatic Hybridization And Cybridization
Plant Cell Fusion As A Tool For Genetic Manipulation
Genetic Improvement Of Cytoplasmic Traits Through Cytoplasmic Hybridization In Cruciferae
Somatic Hybridization And Cybridization As Potential Methods For Widening Of The Gene-Pools Of Crops Within Brassicaceae And Solanaceae
Investigations Into The Transfer Of Genetic Information Between Solanaceous Species And Potato By Somatic Hybridization
Protoplast Culture And Fusion Of Red And Alsike Clover
Interclassical Protoplast Fusion Between Orchard-Grass And Petunia
Interspecific Chloroplast Recombination In A Nicotiana Somatic Hybrid
Loss Of Species-Specific Sequences In Somatic Hybrids, Obtained By Fusion Of Nicotiana Tabacum Cnx Protoplasts With Heavily X-Irradiated N. Paniculata Protoplasts
Somatic Cell Genetics Of Potato: Variant Cell Lines And Somatic Hybridization
Somatic Hybridization Between Two Nicotiana Plumbaginifolia Lines And Between Solanum Tuberosum And Phureja Using Electrofusion
Structure And Regulation Of Cytosol And Plastid Specific Isoenzymes In Higher Plants
Resynthesis Of Brassica Napus Via Somatic Hybridization: A Model For Production Of Interspecific Hybrids Within Brassiceae
Protoplast Culture And Plant Regeneration Of Solanum Pennellii And Lycopersicon Esculentum
Somatic Cell Genetics Of Potato III: Electrofusion Of Two Amino Acid Analogue-Resistant Cell Lines
Cp And Mt Genome Constitution Of Different Somatic Hybrids Between Brassica Napus And Brassica Hirta
Part 7. Isolation And Cloning Of Plant Genes
The Complexity Of Gene Regulation During The Lightdependent Development Of Chloroplasts In Barley (Hordeum Vulgare L.)
The Chloroplast Ribosomes Of Higher Plants And The Genes For Their Protein Components
Genetic Variation And Gene Expression; A Study Of The Ribosomal Rna Gene Loci Of Wheat
Cloning And Analysis Of Two Genes For Chalcone Synthase Of Petunia Hybrida
Cm-Proteins And Thionins In Cereals: Characterization And Cloning Of Cdna
Cin-Dna-Insertions Of Zea Mays
Part 8. Transformation
Recent Progress In Plant Genetic Engineering
Organ-Specific Gene Expression In Potato
Electric Field Mediated Transfer Of Nucleic Acids Into Carrot Protoplasts
The Pollen System Of Gene Transfer
Direct Gene Transfer To Plants
Genetic Manipulation In Potato
Culture And Transformation Studies With Maize Protoplasts
Transformation In The Forage Legumes
Part 9. Use Of Molecular Biology Methods For Plant Improvement
Integration, Expression And Stable Transmission Through Seeds Of Foreign Genes In Plants
Identification, Cloning And Transfer Of Chloroplast Genes Of Petunia Hibrida
The Need For A Multidisciplinary Approach To Genetic Manipulation In Plant Breeding
Appendix
Author Index
Subject Index

Citation preview

Genetic Manipulation in Plant Breeding

Genetic Manipulation in Plant Breeding Proceedings International Symposium Organized by EUCARPIA September 8-13,1985, Berlin (West), Germany Editors W Horn • C. J. Jensen W Odenbach • O. Schieder

W G DE

Walter de Gruyter • Berlin • New York 1986

Editors W o l f g a n g H o r n , Prof. Dr. agr. Lehrstuhl für Z i e r p f l a n z e n b a u Technische Universität M ü n c h e n D-8050 München Federal R e p u b l i c o f G e r m a n y C. J o h n J e n s e n , Dr. R i s o N a t i o n a l Laboratory Post B o x 4 9 DK-4000 Roskilde Denmark Werner O d e n b a c h , Prof. Dr. rer. nat. O t t o Schieder, Prof. Dr. rer. nat. Freie U n i v e r s i t ä t Berlin Institut für A n g e w a n d t e G e n e t i k Albrecht-Thaer-Weg 6 D - 1 0 0 0 Berlin 3 3 Germany

Library of Congress Cataloging in Publication

Data

Genetic manipulation in plant breeding. Bibliography: p Includes indexes. 1. Plant-breeding-Congresses. 2. Plant p r o p a g a t i o n - I n v i t r o Congresses. 3. Plant genetic engineering-Congresses. I. Horn, Wolfgang, 1925 - . II. Eucarpia. SB123.G396 1986 631.5'23 86-19721 ISBN 0-89925-100-5 (U.S.)

CIP-Kurztiteiaufnahme

der Deutschen

Bibliothek

Genetic manipulation in plant breeding : proceedings internat, symposium, Sept. 8 - 1 3 , 1 9 8 5 , Berlin (West), Germany / organized by E U C A R P I A . Ed. W Horn . . . - Berlin ; New York : de Gruyter, 1986. ISBN 3-11-010596-9 NE: Horn, Wolfgang [Hrsg.]; European Association for Research on Plant Breeding.

Copyright © 1986 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. N o part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike G m b H , Berlin. - Binding: D. Mikolai, Berlin. - Printed in Germany.

PREFACE

The international symposium, "Genetic Manipulation in Plant Breeding", was held at the Freie Universität Berlin, West Berlin, Germany, September 8-13, 1985. It was arranged as a joint symposium of the two sections of EUCARPIA (The European Association for Research on Plant Breeding), "Genetic Manipulation in Plant Breeding", and "Ornamentals". The symposium consisted of 9 sessions each of which concentrated on specific topics presented as invited papers, specialized papers, posters or workshop discussions. The purpose of the symposium was to provide a forum for discussion of scientific and technical achievements within the broad field of genetic manipulation of plants. The symposium promoted interaction between plant breeders and scientists interested in a wide scope of activities: from classical cytogenetic breeding aspects to current cell biological and genetic manipulation techniques. From the outset of the planning of the symposium the organizers thought that a written documentation in the form of proceedings would justify the effort of authors, editors and publishers especially as this would be a volume bridging the established genetic manipulation to the more futuristic activities in genetic manipulation. The proceedings deal with the use of mutation in breeding research, followed by cytogenetics and polyploidy to induction and use of haploidy. In-vitro propagation together with vegetative reproduction and regeneration and the occurrence of variation in in-vitro cultures is preceeded by somatic hybridization and cybridization. The isolation, cloning, transformation, and expression of foreign genetic material and actual transformation constitute the truly molecular part of the proceedings. The proceedings volume is aptly concluded by the combination of old and new techniques in the use of molecular biology methods in plant improvement. All papers bear with them the actuality of this en-

VI larging field of biotechnology. For this reason, this volume should be highly recommended for the busy breeder - to obtain a quick but thorough overview of basic plant biotechnology. In addition to the invited papers and the specialized papers the volume contains most of the poster material as brief papers. Although two to four pages cannot always do justice to the quality and information contained in a well presented poster, the organizers preferred this solution to the normal poster-abstract only policy of most current proceedings. It is therefore with great pleasure that the editors wish to thank the contributors to this proceedings volume for their own efforts. Special thanks are expressed to the secretary of the organizing committee, Heidi Jaiser-Gerlach, for her never-failing and energetic input to help to organize the presented material. It goes without saying that the organizers wish to express thanks for the generous assistance given to the symposium and to the cost of the proceedings. This help is gratefully acknowledged and detailed elsewhere here. Further, the proceedings would not have come to reality without the positive co-operation of the publishers. Lastly, the co-editors

(W.H., C.J.J, and O.S.) wish to express a

personal thank you to Professor W. Odenbach who carried most of the burden from the planning stage, throughout the symposium to the actual printing of the proceedings. W. Horn, C.J. Jensen, W. Odenbach and 0. Schieder

EDITORS ANNOUNCEMENT The manuscripts are printed as delivered and are not under the responsibility of the editors.

VII ACKNOWLEDGEMENTS The o r g a n i z a t i o n

of t h i s s y m p o s i u m

t h a n k s to g e n e r o u s

assistance

The S e n a t e Deutsche This

support

is o n c e

We f u r t h e r m o r e for

wish

travel

able to

has b e e n m a d e

by

of B e r l i n

(West)

Forschungsgemeinschaft again

possible

gratefully

to a c k n o w l e d g e

(DFG).

acknowledged.

special

c o s t s of p a r t i c i p a n t s

grants

otherwise

couldn't

be

attend

for p r i n t i n g from the following

the p r o c e e d i n g s

of t h e

Symposium

donors:

Daimler-Benz

AG,

Stuttgart-Untertürkheim

A. F i s c h e r ,

Hannover

Hoechst

Frankfurt

Imperial

AG,

Chemical

Industries

Ltd.,

Runcorn,

Cheshire

Krüss , Hamburg Pelargonien

Fischer,

Hillscheid

Saaten-Union , Hannover Schering

AG,

The N i c k e r s o n Walz,

Berlin. Seed Company

Stuttgart.

Ltd.,

Rothwell,

Lincoln

C O N T E N T S PART U S E OF

MUTATION

1

IN B R E E D I N G

RESEARCH

H a r t e n , A . M . v a n , a n d C. B r o e r t j e s Mutation breeding: a stepping stone between Gregor Mendel and g e n e t i c m a n i p u l a t i o n (a t r e a t i s e f o r v e g e t a t i v e l y propagated crops) H a g b e r g , A. Induced structural K r e f t , I. Evaluation conformity

3

17 rearrangements 37

a n d s e l e c t i o n of m u t a t i o n s to p l a n t i d e o t y p e

Harten, A.M. van, and E.C.J. Chemically induced mutations x hybrida

Bal for male

on t h e

basis of

their 43

sterility

M u n c k , l_. , K. B a n g - O l s e n a n d B. S t i l l i n g G e n o m e a d j u s t m e n t by b r e e d i n g to b a l a n c e h i g h - l y s i n e m u t a n t s of b a r l e y

in

Petunia 49

yield

defects

in

A b d e l - H a f e z , A.G., M.S. El-Keredy and A.A. B a s s i o n i S e l e c t i o n of r u s t r e s i s t a n t w h e a t a f t e r m u t a g e n e s i s

61

D e n i c , M . , S. R a t k o v i c , J . D u m a n o v i c , D. M i s e v i c U s e o f m a i z e m u t a n t s in b r e e d i n g for i m p r o v e m e n t protein quality

65 of

E l - K e r e d y , M . S . , a n d A. G. A b d e l - H a f e z P r o m i s i n g r i c e m u t a n t s for d e v e l o p e d p r o d u c t i o n K o n s t a n t i n o v , K . , M. D e n i c a n d V. S u k a l o v i c C y t o p l a s m i c m a l e s t e r i l i t y in m a i z e - n u c l e a r chondrial genome interdependence Rao, R., M.R. Mogno and M.S. Grillo G e n e t i c s t u d i e s and i n h e r i t a n c e of seed of p e a c h r o m o s o m e m u t a n t s

PART CYTOGENETICS

69 73

and

mito77

storage

protein

2

AND

POLYPLOIDY

C a u d e r o n , Y. C y t o g e n e t i c s in b r e e d i n g p r o g r a m m e s d e a l i n g w i t h interspecific hybridization and introgression Krolow, K.-D., and A.J. Lukaszewski T e t r a p l o i d t r i t i c a l e - a t o o l in h e x a p l o i d breeding

83 polyploidy,

105 triticale

X Ramanna, M.S. I n s t a b i l i t y in tomato lines with the cycle

breakage-fusion-bridge

Friedt, W. The p r e s e n t status of breeding a u t o t e t r a p l o i d Schlegel , R., R. Kynast and J.-C. S c h m i d t Alien c h r o m o s o m e t r a n s f e r from w h e a t into

119

123

cereals

129

rye

Cermeno, M.C., B. Friebe, F.J. Zeller and K.-D. Krolow N u c l e o l a r c o m p e t i t i o n in d i f f e r e n t (A/B)(A/B)RR and DDRR genomes of t e t r a p l o i d t r i t i c a l e s

137

Errico, A., and C. C o n i c e l l a I d e n t i f i c a t i o n of i n t e r c h a n g e s

141

in wild species of

Pisum

Farcy, E. , C. Mousset, D. M a i z o n n i e r and A. Cornu G e n e t i c r e g u l a t i o n of m e i o t i c r e c o m b i n a t i o n in Petunia hybrida

145

G e r d e m a n n , M., and M.D. S a c r i s t a n A t t e m p t s to t r a n s f e r r e s i s t a n c e to Phoma lingam from B r a s s i c a j u n c e a and B. carinata to B. napus t h r o u g h i n t e r s p e c i f i c h y b r i d i z a t i o n f o l l o w e d by o v u l e c u l t u r e

149

H a j o s - N o v a k , M., A. Balint, A.H. Nagy and G. Vida A n a l y s i s of ADH1 locus in t e t r a p l o i d corn /Zea mays

153

L./

H u t c h i n s o n , J., and J. Postoyko C - b a n d i n g of Avena s p e c i e s

157

Jaarsma, T.A. A study of n u c l e i c acids in the S y m p h y t u m o f f i c i n a l e species complex and S. a s p e r u m : m i c r o d e n s i t o m e t r y of n u c l e a r DNA in leaf cells

161

Jouve, N., A. B e r n a r d o , M. G a r c i a , P. G a r c i a and C . S o l e r C - b a n d i n g and i s o z y m e m a r k e r s to a n a l y z e the s e g r e g a t i o n of rye c h r o m o s o m e s in the p r o g e n i e s of t r i t i c a l e x w h e a t hybrids

163

J.ung, C., and H. L o p t i e n Breeding n e m a t o d e - r e s i s t a n t

167

sugar

beets

Kralj , D. , and M. Kump P o l y p l o i d y in hop b r e e d i n g , H u m u l u s l u p u l u s

L.

171

Lange , W. R e c o n s t i t u t i o n of h e x a p l o i d w h e a t from T r i t i c u m d i c o c coides (AABB, 2n=28) and T. t a u s c h i i (DD, 2n=14)

175

Lin d e - L a u r s e n , I., and R. von Bothmer P r e f e r e n t i a l loss and gain of s p e c i f i c H o r d e u m v u l g a r e c h r o m o s o m e s in hybrids with t h r e e alien s p e c i e s ?

179

M a i z o n n i e r , D., A. Cornu, E. Farcy and P. de G e n e t i c and c y t o l o g i c a l maps in Petunia

183

Vlaming

XI

Marrewij k , G.A.M. van, and L.C.J.M. Suurs B i o c h e m i c a l c h a r a c t e r i z a t i o n of c y t o p l a s m i c l i t y in P e t u n i a h y b r i d a M a s t n a k - C u l k , C., a n d F. S u s n i k C y t o g e n e t i c s t u d i e s of the p o l y p l o i d s l u p u l u s L. M i a z g a , D., Cytogenetic additions

C. T a r k o w s k i a n d s t u d i e s of w h e a t

187 male

steri191

of

hop,

M. C h r z a s t e k lines with rye

Humulus 195

chromosome

M i l l e t , E., Y. A v i v i a n d M. F e l d m a n S u b s t i t u t i o n of c h r o m o s o m e 5S of A e g i l o p s l o n g i s s i m a for i t s g r o u p - 5 h o m o e o l o g u e s of c o m m o n w h e a t

199

R o u p a k i a s , D.G. Interspecific hybridization between nensis: p r o s p e c t s and limitations

203

Singh, A.K. A l i e n g e n e t r a n s f e r in manipulations (ICRISAT

V.

faba

V.

PART AND

and

genome 211

distant

hybrids

3

U S E OF

HAPLOIDS

S n a p e , J . W . , E. S i m p s o n , B.B. P a r k e r , W. F r i e d t a n d B. F o r o u g h i - W e h r C r i t e r i a f o r t h e s e l e c t i o n a n d use of d o u b l e d h a p l o i d in c e r e a l b r e e d i n g p r o g r a m m e s Jensen, • C.J. Haploid induction

217 systems 231

and

production

in

crop

plants

Johansson,L.B. E f f e c t s of a c t i v a t e d c h a r c o a l , c o l d t r e a t m e n t C O 2 - c o n c e n t r a t i o n s o n e m b r y o g e n e s i s in a n t h e r Ockendon, D.J. U t i l i s a t i o n of

narbo207

g r o u n d n u t by p l o i d y C . P . N o . 277)

W a n g , X. The p o t e n t i a l for i m p r o v i n g the primary o f c o m m o n w h e a t by a n t h e r c u l t u r e

INDUCTION

and

257 and e l e v a t e d cultures 265

anther

culture

in b r e e d i n g

Bj 0 r n s t a d , A. The r o l e of l i n k a g e a n d r e c o m b i n a t i o n w i t h H o r d e u m b u l b o s u m L.

Brussels

sprouts 273

in b a r l e y

breeding

Bj 0 r n s t a d , A. Partial incompatibility between Scandinavian six-rowed b a r l e y s a n d H o r d e u m b u l b o s u m L. a n d its g e n e t i c a l b a s i s

275

B r e u e r , R . , W. Recent results

279

Kysely and on s o m a t i c

H.-J. Jacobsen e m b r y o g e n e s i s in pea

and

bean

XII

B u c h t e r - L a r s e n , A., a n d C . J . J e n s e n S u g a r b e e t (Beta v u l g a r i s L.) p o l l e n q u a l i t y a s s e s s m e n t a n d e f f e c t of i r r a d i a t i o n as m e a s u r e d by f l u o r o c h r o m a t i c r e a c t i o n a n d in v i t r o g e r m i n a t i o n

283

C a l i g a r i , P . D . S . , W. P o w e l l a n d J . L . J i n k s T h e d e t e c t i o n of l i n k a g e u s i n g d o u b l e d h a p l o i d s

287

D i e u , P., a n d M. B e c k e r t T h e e f f e c t of an e l e v a t e d C C ^ - c o n c e n t r a t i o n w i t h c o l d t r e a t m e n t s in m a i z e (Zea m a y s L.)

in

barley 291

in c o m b i n a t i o n anther culture

D u m a s d e V a u l x , R . , a n d D. C h a m b o n n e t O b t e n t i o n o f e m b r y o s a n d p l a n t s f r o m in v i t r o u n f e r t i l i z e d o v u l e s of C u c u r b i t a pepo

295 culture

F r i e d t , W . , a n d B. F o r o u g h i - W e h r A g r o n o m i c value of a n d r o g e n e t i c d o u b l e d h a p l o i d line c o m p a r e d to c o n v e n t i o n a l l y s e l e c t e d s p r i n g b a r l e y H a d w i g e r , M . A . , a n d E. H e b e r l e - B o r s P o l l e n p l a n t p r o d u c t i o n in T r i t i c u m D ' H a l l u i n , K., a n d B. P r o d u c t i o n of h a p l o i d ovule culture

Keimer sugarbeets

of 299 as 303

turgidum

(Beta

ssp.

vulgaris

durum

L.)

by

K e i j z e r , C.J., H.J. W i l m s , A.C. van Aelst and H.B. Leferink-ten Klooster T o w a r d s the i s o l a t i o n of sperm cells for a n d r o g e n i c purposes K ö h l e r , F., G. W e n z e l , I. A b e n t h u m a n d H . G l a s e r R e g e n e r a t i o n and s e l e c t i o n of i s o l a t e d m i c r o s p o r e s Hordeum vulgare M e y n e t , J. P o s s i b i l i t i e s of o b t a i n m e n t h a p l o i d s in G e r b e r a

307

311

of

315

319 and

utilization

of

doubled

N i e m i r o w i c z - S z c z y t t , K. C o n t r i b u t i o n to t h e s t u d i e s on t h e f i r s t , g e n e r a t i v e progeny of Fragaria x Ananassa p o l y h a p l o i d s

323

Powell, W., P.D.S. Caligari and J.L. Jinks A c o m p a r i s o n of c r o s s p r e d i c t i o n m e t h o d s in s p r i n g

327

P r a k a s h , J., Androgenesis

barley

and K.L. Giles in o i l s e e d r a p e

P r a k a s h , J., and K.L. P r o d u c t i o n of d o u b l e d

Giles haploids

331 335 in o r i e n t a l

lilies

R y ö p p y , P., J. H o n k a n e n and P . M . A . T i g e r s t e d t I n c r e a s i n g the e f f i c i e n c y of t r i t i c a l e anther

339 culture

XIII S a r r a f i , A., R. E c o c h a r d , C. P l a n c o n a n d M. A l i - S a d i q G e n e t i c g a i n for s o m e a g r o n o m i c a l c h a r a c t e r s by d i h a p l o i d b r e e d i n g in b a r l e y

343

S c h m i d , J., and E.R. K e l l e r E f f e c t of a g a m e t o c i d e on t h e i n d u c t i o n Triticum aestivum

347 of h a p l o i d s

in

S p e c k m a n , G . J . , J . P . C . van G e y t and M. J a c o b s The i n d u c t i o n of h a p l o i d s of s u g a r b e e t (Beta v u l g a r i s L.) u s i n g a n t h e r a n d f r e e p o l l e n c u l t u r e or o v u l e and ovary culture

351

S w a a i j , A . C . van, and E. J a c o b s e n Frost tolerant plants obtained from proline cell lines

355

U i j t e w a a l , B . A . , a n d W.M. H a t t h e i j S o m a t i c c e l l g e n e t i c s of p o t a t o . I. U s e of

accumulating 359 monohaploids

Z a g o r s k a , N . A . , M . D . A b a d j i e v a and H.K. O a n h F a c t o r s a f f e c t i n g c a l l u s and p l a n t p r o d u c t i o n c u l t u r e s of t o m a t o

in

anther

361

PART 4 IN-VITRO

PROPAGATION

B o r n m a n , C . H . , R. V a n k o v a a n d L.O. B j ö r n R o l e o f m e t h o d o l o g y in f a c i l i a t i n g a p p l i c a t i o n culture techniques P r e i l , W. In v i t r o p r o p a g a t i o n and b r e e d i n g of o r n a m e n t a l a d v a n t a g e a n d d i s a d v a n t a g e of v a r i a b i l i t y

of

tissue

plants:

S i n k , K . C . , L.W. H a n d l e y , R.P. N i e d z a n d P.P. M o o r e P r o t o p l a s t c u l t u r e and use of r e g e n e r a t i o n a t t r i b u t e s select somatic hybrid tomato plants Z e n k t e l e r , M., and A. SI u s a r k i e w i c z - J a r z i n a S e x u a l r e p r o d u c t i o n in p l a n t s by a p p l y i n g t h e m e t h o d t e s t t u b e f e r t i l i z a t i o n of o v u l e s B e c k e r , U., and G. R e u t h e r C y t o g e n e t i c s t u d i e s in c a l l u s

367

377

405 to

of

415

425 c u l t u r e s of A s p a r a g u s

M a l e p s z y , S., A. N a d o l s k a - O r c z y k and W. O r c z y k S y s t e m s for R e g e n e r a t i o n of C u c u m i s s a t i v u s p l a n t s

off. 429 in

vitro

J a m e s , D . J . , A.J. P a s s e y , K . A . D . M a c K e n z i e , O.P. J o n e s and E.C. M e n h i n i c k R e g e n e r a t i o n of t e m p e r a t e f r u i t t r e e s in v i t r o via o r g a n o g e n e s i s and e m b r y o g e n e s i s

433

XIV F o r d - L l o y d , B . V . , a n d S. B h a t Problems and prospects for the breeding

437 u s e of

protoplasts

in

beet

S t e f f e n , A., T. E r i k s s o n a n d 0. S c h i e d e r S h o o t r e d i f f e r e n t i a t i o n of A g r o b a c t e r i u m t r a n s f o r m e d p r o t o p l a s t s and plant tissue - with conventional methods not achievable

441

J a c o b s e n , H . - J . , a n d W. K y s e l y I n d u c t i o n of in v i t r o - r e g e n e r a t i o n v i a s o m a t i c e m b r y o g e n e s i s in pea ( P i s u m s a t i v u m ) a n d b e a n ( P h a s e o l u s v u l g a r i s )

445

A n d e r s e n , J . M . , F. O k k e l s , P. U l s k o v a n d J. M a r c u s s e n E n d o g e n o u s c y t o k i n i n s d u r i n g e m b r y o g e n e s i s in a c a r r o t cell suspension

449

B h a t , S., B . V . F o r d - L l o y d a n d J . A . C a l l o w T i s s u e a n d p r o t o p l a s t c u l t u r e in c u l t i v a t e d

453 beets

E r i k s e n , F . D . , C . J . J e n s e n a n d P. O l e s e n P r o t o p l a s t f o r m a t i o n in c e r e a l s - an a s s e s s m e n t F i l i p p o n e , E., a n d T. C a r d i E x p l o i t a t i o n for b r e e d i n g of e x pla nt s

457 461

in v i t r o

culture

of

pea

G e y t , J . P . C . v a n , K. C l a e s , A . H . S . S e n a n a y a k e and M.Jacobs S o m e a s p e c t s of t h e in v i t r o c u l t u r e of t h e b e e t (Beta v u l g a r i s L.)

465

H y r k a s , K . , M. K i v i n e n a n d P . M . A . T i g e r s t e d t I n t e r s p e c i f i c h y b r i d i z a t i o n of red clover (Trifolium p r a t e n s e L.) w i t h a l s i k e c l o v e r ( T r i f o l i u m h y b n d u m L.) u s i n g in v i t r o e m b r y o r e s c u e

469

J e n s e n , C . J . , A. B u c h t e r - L a r s e n , D. C a s s , E . C . T h o r n , K. E n g e l l a n d P. O l e s e n P o l l e n a n d o v u l e c u l t u r e s of b a r l e y to i s o l a t e , m a n i p u l a t e a n d t r a n s f e r s p e r m c e l l s in in v i t r o f e r t i l i z a t i o n

473

Jensen, C.J., and E.C. Thorn S t r a t e g i e s in h i g h f r e q u e n c y r e g e n e r a t i o n f r o m h a p l o i d c e l l a n d t i s s u e c u l t u r e s of b a r l e y

477 diploid

L i n a c e r o , R., and A.M. V a z q u e z S o m a c l o n a l v a r i a t i o n in p l a n t s r e g e n e r a t e d c a l l u s e s in r y e ( S e c a l e c e r e a l e L.)

from

L u h r s , R . , a n d H. L d r z Somatic e m b r y o g e n e s i s , cell and H o r d e u m v u l g a r e L. ( b a r l e y )

culture

and 479

embryo 483

protoplast

M a r i n o , G. I s o l a t i o n a n d c u l t u r e of p r o t o p l a s t s f r o m c a l l u s s u s p e n s i o n - c u I t u r e d c e l l s of P r u n u s c e r a s u s a n d Actinidia chinensis

of 487

and

XV M o r e n o , V., L. Z u b e l d i a , B. G a r c i a - S o g o , F. N u e z a n d L.A. Roig S o m a t i c e m b r y o g e n e s i s in p r o t o p l a s t - d e r i v e d c e l l s of C u c u m i s m e l o L.

491

R u i z , M . L . , M . I . P e l a e z , J. R u e d a , F . J . E s p i n o a n d A.M. Vazquez A c o m p a r i t i v e s t u d y of c a l l u s f o r m a t i o n a n d p l a n t r e g e n e r a t i o n f r o m d i f f e r e n t e x p l a n t s of P h a s e o l u s v u l g a r i s a n d Ph. c o c c i n e u s

495

S t o l a r z , A . , a n d H. Lorz Somatic e m b r y o g e n e s i s , cell and p r o t o p l a s t t r i t i c a l e (x T r i t i c o s e c a l e W i t t m a c k )

499 culture

Z i m n y , J. , a n d H. Lorz S o m a t i c e m b r y o g e n e s i s and p l a n t r e g e n e r a t i o n t e m a t i c t i s s u e of S e c a l e c e r e a l e (rye)

of 503

from

meris-

Zimny, J., and J.J. R y b c z y n s k i S o m a t i c e m b r y o g e n e s i s of t r i t i c a l e PART SPONTANEOUS

AND

INDUCED

5

VARIATION

L ö r z , H., a n d P . T . H . B r o w n V a r i a b i l i t y in t i s s u e c u l t u r e d e r i v e d origins; a d v a n t a g e s and d r a w b a c k s C a s s e l l s , A . C . , M. C o l e m a n , G. E . M . G o e t z a n d V. B o y t o n Screening for virus resistance r e g e n e r a n t s and their progeny K a r p , A. Chromosome

507

FROM

IN-VITRO

CULTURES 513

plants

Farrell,

R.

in t i s s u e

-

possible

Long,

culture

535 adventitious 547

variation

in r e g e n e r a t e d

W a i t h e r , F., a n d A. S a u e r In v i t r o m u t a g e n e s i s in G e r b e r a N o v a k , F . J . , T. H e r m e l i n and S. In v i t r o m u t a g e n e s i s in m a i z e A h l o o w a l i a , B.S. S o m a c l o n e s of w h e a t callus

plants 555

jamesonii Daskalov

563 577

regenerated

from

primordial

leaf

A r c i o n i , S . , D. M a r i o t t i , F. D a m i a n i , M. P e z z o t t i S o m a c l o n a l v a r i a t i o n in L o t u s c o r n i c u l a t u s L.

581

B r y , L. de, M. J a c o b s , R . M . W a l l s g r o v e a n d B . J . M i f l i n P o t e n t i a l s y s t e m f o r t h e s p e c i f i c s e l e c t i o n of p l a n t mutants overproducing methionine

585

Collin, H.A., P.D. P u t w a i n and S.C. G i f f a r d E n h a n c e m e n t o f a s u l a m r e s i s t a n c e in b a r l e y

589

XVI C s e p l o , A., P. M e d g y e s y and E. H i d e g Triazine-re sistant Nicotians mutants trophic cell cultures

593 from

photomixo-

D e k e y s e r , A., P. D a b i n a n d J. B o u h a r m o n t U s e of in v i t r o c u l t u r e f o r i n d u c i n g v a r i a t i o n and fuchsia

597 in

rice

D i r k s , R . , I. N e g r u t i u a n d M. J a c o b s I s o l a t i o n of a u x o t r o p h i c m u t a n t s b a s e d on r e c o n s t r u c t i o n experiments with Nicotiana plumbaginifolia protoplasts

599

F i s h , N . , a n d A. K a r p A s t u d y on t h e e f f e c t o f d i f f e r e n t i n i t i a l c u l t u r e m e d i a on the c h r o m o s o m e stability of Solanum t u b e r o s u m cv. M a r i s B a r d p r o t o p l a s t d e r i v e d r é g é n é r a n t s

601

J o n g , J. de, a n d J . B . M . C u s t e r s T h e e f f e c t of e x p i a n t s o u r c e , in v i t r o r e g e n e r a t i o n a n d i r r a d i a t i o n o n v a r i a t i o n in y i e l d i n d u c e d in C h r y s a n t h e m u m morifolium

607

Jtfrgensen , R . B . , a n d B. A n d e r s e n T r a n s f e r of g e n e t i c m a t e r i a l to c u l t i v a t e d b a r l e y alien species through callus culture (preliminary

611

Linacero, R., and A.M. Vazquez S o m a t i c e m b r y o g e n e s i s and plant t i s s u e s of Secale c e r e a l e L .

from results) 615

regeneration

Li Su N a m a n d L.E. H e s z k y T e s t i n g of salt (NaCl) t o l e r a n c e c a l l u s c u l t u r e (n, 2n) of r i c e

from

leaf 617

and r e g e n e r a t i o n

N i k o v a , V . M . , a n d N . A. Z a g o r s k a The use of t i s s u e c u l t u r e s for o b t a i n i n g sterile forms

in 621

tobacco

male

S c h i n k e l , C . , and T. L e l l e y S o m a c l o n a l v a r i a t i o n in t r i t i c a l e S j o d i n , C . , and K. G l i m e l i u s S e p a r a t i o n , i d e n t i f i c a t i o n and b i o l o g i c a l t o x i n p r o d u c e d by P h o m a l i n g a m

625 629 effects

of

a

S t e e n , P . , B. K e i m e r , K. D ' H a l l u i n , H . C . P e d e r s e n V a r i a b i l i t y in p l a n t s o f s u g a r b e e t (Beta v u l g a r i s L.) r e g e n e r a t e d from callus, ce11-sus pension and protoplasts

633

Thorn, E.C., and C.J. Jensen C h r o m o s o m a l v a r i a t i o n in r e g e n e r a t e d p l a n t s callus from crosses between Hordeum vulgare

637 from x H.

hybrid bulbosum

XVII

PART SOMATIC

HYBRIDIZATION

6 AND

CYBRIDIZATION

S c h i e d e r , 0 . , T. H e i n a n d H. K o h n P l a n t c e l l f u s i o n as a t o o l for g e n e t i c

641 manipulation

P e l l e t i e r , G . , C. P r i m a r d , F. V e d e l a n d P. C h e t r i t G e n e t i c i m p r o v e m e n t of c y t o p l a s m i c t r a i t s t h r o u g h c y t o p l a s m i c h y b r i d i z a t i o n in C r u c i f e r a e

653

G l i m e l i u s , K . , J. F a h l e s s o n , C. S j ô d i n , M. D j u p s j o b a c k a , H. F e l l n e r - F e l d e g g a n d Somatic hybridization and cybridization for w i d e n i n g of the g e n e - p o o l s of crops and S o l a n a c e a e

663

E. S u n d b e r g , H.T. Bonnett as p o t e n t i a l m e t h o d s within Brassicaceae

F o u l g e r , D . , N. D a r r e l l , N. F i s h , S . W . J . B r i g h t a n d M.G.K. Jones I n v e s t i g a t i o n s into t h e t r a n s f e r of g e n e t i c i n f o r m a t i o n bet w e e n s o l a n a c e o u s s p e c i e s a n d p o t a t o by s o m a t i c h y b r i d i z a t i o n

683

H o n k a n e n , J . , P. R y o p p i a n d P . M . A . T i g e r s t e d t P r o t o p l a s t c u l t u r e and f u s i o n of red and a l s i k e clover

685

J o s h i , C . P . , E. M ü l l e r - G e n s e r t , A. S t e f f e n , H . L ö r z a n d 0. S c h i e d e r Interclassica1 protoplast fusion between orchardgrass and Petunia

689

M e d g y e s y , P . , E. F e j e s a n d P. M a l i g a Interspecific chloroplast recombination somatic hybrid

693 in

a

Nicotiana

M u l l e r - G e n s e r t , E . , a n d 0. S c h i e d e r L o s s o f s p e c i e s - s p e c i f i c s e q u e n c e s in s o m a t i c h y b r i d s , o b t a i n e d by f u s i o n o f N i c o t i a n a t a b a c u m C N X p r o t o p l a s t s w i t h h e a v i l y X - i r r a d i a t e d N. p a n i c u l a t a p r o t o p l a s t s

697

Pennings, H.M.J. , L.J.W. S o m a t i c c e l l g e n e t i c s of somatic hybridization

701

G i l i s s e n a n d B. potato: variant

de G r o o t cell lines

and

P u i t e , K . J . , a n d S. R o e s t Somatic h y b r i d i z a t i o n between two N i c o t i a n a p l u m b a g i n i f o l i a l i n e s a n d b e t w e e n S o l a n u m t u b e r o s u m a n d S. p h u r e j a using electrofusion

703

K r u g e r , I . , M. H e r b e r t , R. W e n n i c k e , W. R e i t h , C. S c h n a r r e n b e r g e r , T. A m i r i a n d S t r u c t u r e and r e g u l a t i o n of c y t o s o l a n d i s o e n z y m e s in h i g h e r p l a n t s

705

W e i g e l t , B. P e l z e r J. S a l n i k o w plastid specific

S u n d b e r g , E . , a n d K. G l i m e l i u s R e s y n t h e s i s of B r a s s i c a n a p u s via s o m a t i c h y b r i d i z a t i o n : a m o d e l for p r o d u c t i o n of i n t e r s p e c i f i c h y b r i d s w i t h i n Brassiceae

709

XVIII

Tan, M.M.C., H.S. B o e r r i g t e r , A.J. Kool and J.G.T. Hermsen P r o t o p l a s t culture and plant r e g e n e r a t i o n of Solanum pennellii and Lycopersicon esculentum

713

Vries , S.E. de, E. Jacobsen, M. Tempelaar and M.G.K. Jones Somatic cell genetics of potato III: e l e c t r o f u s i o n of two amino acid a n a l o g u e - r e s i s t a n t cell lines

715

Primard, C., D. Martin, F. Vedel and G. Pelletier Cp and mt genome constitution of different somatic between Brassica napus and Brassica hirta

719 hybrids

PART 7 ISOLATION

AND CLONING

OF PLANT

GENES

Apel, a ~ E •O 3 O) c o — 3 •a -a c a) •H N

L-5 'I •— to 3 (-i O V "Ö Ö

to ^H

rH

cuttings were made and the resulting flowering plants were scored again for male sterility. Plants that still showed male sterility were indicated as 'stable' and entered into a crossing program. Control crosses were made with untreated BBF plants and with plants from

45 a non-segregating homozygous restorer line, indicated as R3-1. Fertility restoration is governed by dominant alleles. By combining the results from testcrosses and selfings of male fertile descendants the system of inheritance can be deducted for each MS plant, as is outlined in Fig. 1.

Results and discussion

In none of the treatments with EB, involving about 30,000 seeds, stable MS plants were produced. Rather unexpectedly, the EB treatments, which did not show MS plants, did not produce chlorophyll mutations either. This observation casts some doubt on the assumed specific activity of EB on extranuclear DNA. As the rate of survival of EB-treated seeds was much lower than in the controls, there is no evidence of inactivity of the chemical involved.

Fig. 1. Testing the nature of chemically induced male sterility in petunia.

TESTCROSSES 1

2

MS ^ x B B F '

MS x R3-1

SELFING F

PLANTS

3)

0% sterile: existing - 100% sterile:

CMS-type

CMS

y

100% sterile: new CMS-type

- 50% sterile: dominant GMS -

0% sterile:

- 25% sterile: recessive GMS -

0% sterile: physiological damage

1)

= male sterile;

2)

= Blue Bedder Fertile;

3)

= restorer line

46 From treatments with ENU, also with about 30,000 seeds, altogether 29 stable MS plants were obtained (Table 1). The highest concentration after which a reasonable number of seedlings survived, was about 25 mM ENU. In the first experiments the rate of emergence of treated seeds and the number of plants that has been transferred to the field was very low, i.e. not higher than 12%. Later (Table 1, Expt. 4), due to improved cultural conditions, up to 63% of the initially treated seeds resulted in plants in the field. From Table 1 it can be concluded that for ENU treatments the percentage of chlorophyll aberrations went up with an increasing concentration of ENU. In some of the MS plants only a single flowering branch showed male sterility. From such chimeric plants cuttings were made from different branches. These cuttings were kept separate. Since 1983 over 1000 crosses have been made. Crossing appeared to be rather difficult in some cases, requesting many pollinations for a single capsule with a few viable seeds. As Fig. 1 shows, CMS can only be expected when 100% of the progeny from crosses between MS and BBF plants are male sterile. Whether in such a case the observed CMS is of a new type or of a type that is already known, has to be further investigated in crosses with the restorer line.

Table 1. Results from Treatments of Petunia Seed with Different Dosages of Ethylnitroso Urea (ENU). Treatment Time 2 h in the Dark; Post-washing Time 1 h. Concentration (mM ENU) Expt. 3

treated

Chlorophyll--aberrations

'Stable' MS-plants

Number

% (3.6)

-

1

10

5,,000

12

15

7,,000

30

(7.5)

20

9.,000

90

(12.5)

1

25

8.,000

72

(13.8)

3

2,,000

-

-

Control Expt. 4

No. of seeds

18 24 Control

-

5.,000

192

(6.1)

8

5.,500

239

(9.2)

16

1 ,450

-

-

-

47 When in crosses of the type MS x R3-1 all descendants show restored fertility, evidence is provided that the newly found CMS type is apparently the same as the type already available. Only when no fertility restoration takes place a new type of CMS has been obtained. Up to now we have not detected such new types of CMS in our experiments, although it must be taken into account that sofar from only 50% of all crosses with MS numbers seeds have been obtained. Those plants from the cross MS x BBF that produce male fertile flowers, can be selfed in order to check whether monogenic recessive GMS has been induced,which is the case when the plants segregate in 75% male fertile and 25% male sterile. If, on the other hand selfed plants remain fertile, it must be concluded that the male sterility observed in the experimental plants in the field and the greenhouse must be due to physiological damage from the mutagenic treatment and such male sterility cannot be inherited. For several of our MS plant numbers it could not be decided yet as to which category they belong. When in the F^ from crosses between MS and BBF plants 50% male fertility and 50% male sterility is obtained, dominant genie male sterility may be acting. In that case a mutation from msms (male fertile) to Msms (male sterile) may have occurred in the treated material. Further crosses between the heterozygous male sterile plant and normal fertile plants are then expected to result in a 1:1 segregation ratio. This was observed in at least two of the induced MS plants. Dominant GMS seems to be a rather rare phenomenon. Some cases however have been reported, e.g. for petunia (10) and for snapdragon and pelargonium (9). Finally, analysis of crossing results of at least one of the MS plants obtained in our experiments suggests the involvement of more than one Ms-gene. In another part of the program the temperature-dependency of the MS plants obtained is checked in a phytotron. Cuttings have been made and the resulting plants have been investigated at 17, 21 and 25°C. Lack of space severely limited the number of cuttings that could be used at a time, but most experiments will be repeated several times. Sofar 20 out of 32 MS clones tested have shown stable MS expression at all temperatures. Recently we started to investigate the cause of the very low seed set observed in crosses between MS x BBF crosses. From a number of plants the penetration of pollen tubes in the style was investigated by the fluorescence method, using anilin-blue staining. In all cases the pollen tubes reached the ovule within 48 h after pollination, which indicates that other causes than incompatibility reactions must be responsible for the low seed set. In summary, it can be said that treatment of more than 60,000 seeds of Petunia x hybrida with the chemicals ENU or EB resulted in about 30 MS plants, all from the ENU treatments. Those plants were further investigated in a crossing program

48 with the fully fertile counterpart (BBF) and a homozygous restorer line (R3-1). No evidence of induction of (new or existing) sources of CMS was found, but in several cases dominant, monogenically inherited GMS seems

to have been induced.

Phytotron experiments revealed temperature-dependent as well as temperatureinsensitive reactions of some MS plants. There was no indication of the action of any incompatibility system in crossings between MS plants and male fertile control material.

Acknowledgement The authors wish to thank Dr. G.A.M. van Marrewijk and Dr. A.C. Zeven for critically reading the manuscript.

References 1. Marrewijk, G.A.M. van. 1979. In: Plant Breeding Perspectives (J. Sneep and A.J.T. Hendriksen, eds.). PUDOC, Wageningen, pp. 120-134. 2. Kinoshita, T.. 1980. Induction of cytoplasmic male sterility by gamma ray and chemical mutagens in sugar beets. In: Induction and utilization of malesterile mutation in plant breeding. Gamma field symposia 19, 27-48. 3. Burton, G.W. and W.W. Hanna. 1976. Ethidiumbromide induced cytoplasmic male sterility in pearl millet. Crop Science 16, 731-732. 4. Burton, G.W. and W.W. Hanna. 1972. Stable cytoplasmic male-sterile mutants induced in Tift 23DB-L pearl millet with mytomycin and streptomycin. Crop Science 22, 651-652. 5. Marrewijk, G.A.M. van and L.C.J.M. Suurs. 1985. In: Sexual reproduction in seed plants, ferns and mosses. Proc. 8th Int.Symp. (M.T.M. Willemse and J.L. van Went, eds.). PUDOC, Wageningen, pp. 39-43. 6. Bianchi, F. and P. Dommergues. 1979. Petunia genetics. Petunia as a model for plant research: genetics and mutagenesis. Ann.Amél.Plants 29, 607-610. 7. Kool, A.J., J.M. de Haas and G.A.M. van Marrewijk. 1982. In: Induced variability in plant breeding. Symp.Sect.Mutation and Polyploidy, Eucarpia, Wageningen, 1981. PUDOC, Wageningen, p. 127. 8. Pohlheim, F.. 1981. Genetischer Nachweis einer NMH-induzierten Plastommutation bei Saintpaulia ionantha J. Wendl. Biologische Rundschau 19, 47-50. 9. Hagemann, R.. 1976. In: Genetics and biogenesis of chloroplasts and mitochondria (Th. Bücher et al, eds.). Elsevier, Amsterdam, pp 331-337. 10. Singh, I.S.. 1976. Une mutation dominante pour la stérilité pollinique induite par le méthyle-sulfonate d'éthyle chez le Pétunia. C.R. Acad.Se.Paris 282 (série D), 859-862.

49 G E N O M E A D J U S T M E N T BY B R E E D I N G T O B A L A N C E Y I E L D D E F E C T S LYSINE MUTANTS OF BARLEY

L. M u n c k , K. B a n q - O l s e n ,

B.

IN

HIGH-

Stillinq

D e p a r t m e n t of B i o t e c h n o l o g y , Copenhagen, Denmark

Carlsberg

Research

Laboratory,

Introduction In the b e g i n n i n g of the what amino acid general

1960's

it w a s d e m o n s t r a t e d

i m b a l a n c e m e a n t to s t a r v i n g that something

feeling was generated

g e n t l y w i t h the a m i n o a c i d

balance

(1), and

this message also reached

in o r d e r

to m a k e

analyses than binding

large-scale

was proposed a c i d s and

technique The

screening

tion analysing

the w o r l d

a 20%

amino acids.

increase

the D a n i s h R e s e a r c h

for b a s i c

in l y s i n e and found

several mutants a lysine

Associa-

in s e v e r a l o t h e r

l e v e l of a full pig if c a l c u l a t e d

1508

feed

barley

including

on a protein basis.

the

1508

line It

essential

sorghum

since (8).

(4), a m o n g w h i c h the

i n c r e a s e of

45%.

barley

is l o w e r t h a n in the

(Table

The

mu-

nutritional

in f e e d i n g

trials.

(e.g.

However, when calculated in l y s i n e

full feed d i e t .

because The p i g

t h a t the g r o w t h r a t e of p i g s o n the 1508 d i e t w i t h o u t

the

sov) on a

the

protein

trial

shows

protein

a d d i t i v e s a p p r o a c h e s the g r o w t h r a t e of p i g s o n the f u l l

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

At

the

1) is s l i g h t l y a b o v e

protein additives

is l o w e r

(2))

amino

One Ethiopian

in m a i z e and

dye-

introduced

(3) w a s f o u n d .

the h i g h - l y s i n e m u t a n t s w a s c h e c k e d

l y s i n e c o n t e n t of

content

The

(review

I n s t i t u t e a t R i s o , D o l l e t al a l s o u s e d

found

tant M-1508 displayed

diet basis,

convenient

at the S w e d i s h S e e d

Hiproly

ur-

lysine

T h i s f i n d i n g w a s m e t w i t h a lot of o p t i m i s m

D B C - a n a l y s i s and

The

method

A

However,

needed .

orange-12

barley collection.

lysine called

also high-lysine mutants were

q u a l i t y of

were

s e n i o r a u t h o r of t h i s p a p e r

in 1965 w h e n w o r k i n g

displayed

to be d o n e

possible more

(DBC) w i t h a c i d

this technique

h i g h in p r o t e i n and

had

the p l a n t b r e e d e r s .

screening

as a convenient

lysine.

public

in c e r e a l s , e s p e c i a l l y

ion e x c h a n g e c h r o m a t o g r a p h y

capacity

to the

human populations.

feed

50 with

Pigs Restrictive

Without protein Normal feed b a r l e y P r o t e in Lysine 16g/N Lysine g/kg F e e d to o b t a i n 90 kg l i v e w e i g h t , kg Daily growth, g diet.

A drastic

with pigs

contrast

A drawback

with

the

tent

(5) r e s u l t i n g

feed

units, because

components acid

of

balance

209 635

206 703

fat, due

increase

had

seeds of H i p r o l y

improved. and

that

if the m u t a n t s w o u l d

of

started

to b r e e d

for

the h i g h - l y s i n e

nes from Hiproly

and

1508.

the H i p r o l y g e n e

lys 1 located

easily obtained increased, and

more

fiers a very

pointing

DBC-analysis was lys 3a, a l s o

several

with

It w a s ,

programme

too

located

the

that

lys

slow.

that less

trait

With regard

in the c h r o m o s o m e

be

value.

breeders

7, p l u m p

the

more modi-

that

for

were

yield

seemed

likely

ge-

with

seeds

several

selecting

For

to

work at Svalof

therefore,

1 trait.

had

However, when

it w o u l d

the

however,

there were

steadily

amino

trait using

the h i g h - l y s i n e out

(6),

considerably

barley plant

quality

in c h r o m o s o m e

lysine expression,

high yield

the

this difference

first crossings.

the major gene. of

fact was,

In the b r e e d i n g

the e x p r e s s i o n o f

large crossing

hest level bine

f r o m the

incomplete

for

1970's,

embryo

carbohydrate

have any economical

In the b e g i n n i n g

without

the hig

n o t be p o s s i b l e

to

such an o p e r a t i o n

to the

in

energy

trials while

1508 g a v e

con-

Expressed

p o i n t of v i e w , The

and

diets

starch

increased

in f e e d

two

a higher

in the n o n - s t a r c h

to be b e n e f i c i a l

the

energy.

to t h e

Thus from a quality

than normal varieties

these

addition.

barley was a much lower

the

eliminated

if o n e c o m p a r e s

barley without protein

starch content yield

feed

538 261

1508 m u t a n t d i s p l a y e d

to b e

Full

15.0 5.0 7.5

57% m o r e

seemed

shrivelled

barley

11.5 5.2 6.0

type

the

of a l a r g e (5).

1508

Danish

With protein add itive

additive

in l e s s e a s i l y a v a i l a b l e

however,

content because and

1508

to

10.5 3.7 3.8

is s e e n

fed w i t h n o r m a l

According

1508 m u t a n t

7, s e v e r a l

-

comthe

gene,

experiments

51 at R i s o w i t h e . g . c h r o m o s o m e - d o u b l e d

monoploid

1508

showed that the shrunken k e r n e l s a l w a y s followed trait

(27). B r e e d e r s found that o c c a s i o n a l l y

segregants

the

high-lysine

the b e s t

high-lysine

1508 s e g r e g a n t s in c r o s s i n g s could be as high y i e l d i n g a s n o r m a l barley in some trials at some y e a r s and l o c a t i o n s , but the yield stability over all t r i a l s w a s d i s a s t r o u s . 1970's, the c o n f i d e n c e

In the end of

in h i g h - l y s i n e barley b r e e d i n g w a s

and m o s t b r e e d e r s finished

their

W h e n joining the C a r l s b e r g R e s e a r c h L a b o r a t o r y felt r e s p o n s i b l e l e a s t to give

zero

efforts.

B r e e d i n g for H i g h - l y s i n e Barley at the C a r l s b e r g R e s e a r c h tory e m p l o y i n g the 1508 Gene (lys 3a).

a u t h o r g o t the o p p o r t u n i t y

the

Labora-

in 1973, the

senior

to c o n t i n u e his w o r k from S v a l o f .

We

to f u l f i l the p r o m i s e s from the 1 9 6 0 ' s or a t

it a hard try.

Because of the i n s t a b i l i t y of

e x p r e s s i o n of the lys 1 g e n e , we d e c i d e d lys 3a gene from m u t a n t 1508.

the

to c o n c e n t r a t e on the

As we were prepared

to d o a large

n u m b e r of c r o s s e s and to screen for m i l l i o n s of seeds, we could not use the DBC-method or any o t h e r c h e m i c a l method w h i c h w e r e too slow.

Fortunately,

v e r y large g e r m including even without dehusking.

scutellum

(Fig. 1) w h i c h can be

Our strategy w a s thus to s c r e e n

p l u m p seeds having the large g e r m c h a r a c t e r . one c r o p in D e n m a r k

available

the 1508 m u t a n t d i s p l a y s a

Being able to g r o w

in the summer and one in N e w Zealand

the w i n t e r , we crossed

during

first w i t h large seeded p l u m p v a r i e t i e s

such as N o r d a l , C i l i a , M i n e r v a , E l b o and G e r k r a , selected p l u m p r e c o m b i n a n t s and crossed variety.

seen for

back 2 times to the

In the o n s e t of the p r o g r a m m e we found

for the

high-yielding

that

reasonable

seed q u a l i t y could be obtained u n d e r D a n i s h c o n d i t i o n s in e . g . the c r o s s 1508 x N o r d a l but looking o n the same barley in New Z e a l a n d , they w e r e d e p r e s s i n g l y

grown

shrivelled.

In 1980 we w e r e near giving up b u t then something h a p p e n e d .

Our

2nd g e n e r a t i o n c r o s s e s involving b a r l e y s such a s T r i u m p h and

Nery

crossed w i t h the best 1508 x

Nordal^

type s u d d e n l y looked

accept-

able w i t h regard

to seed q u a l i t y w h e n they came back from m u l t i -

p l i c a t i o n in New

Zealand.

52 F i g . 1 . M o r p h o l o g y of B a r l e y Seeds Involving Crosses with l y s 3a ( d o r s a l v i e w )

12 In Fig.

3 4

1 is d e m o n s t r a t e d

morphologically Note

the

1) Borni 2) M - 1 5 0 8 f r o m Borni 3) C a 7 0 0 2 0 2 ( 1 5 0 8 x N o r d a l 2 ) x Triumph 4) C a 0 4 0 3 0 2 (Ca 7 2 2 7 0 1 x Mandolin).

best

1508

large g e r m and We

four years.

have

now

Table

2.

of c o m p a r i s o n

Summary of

the

1508

in y i e l d

and

Ca

720502

until

the

the

(Table

Trials with M-1508

Nery

in

not

standard

2).

Crosses

Yield % Zita

plump to

in N e r y a r e

to Z i t a ,

1982

the

Mandolin.

t y p e s and

segregants compared

compared

in D e n m a r k

Yield

1508

the c r o s s e s w i t h T r i u m p h and

in T r i u m p h a n d

significantly different variety

Mandolin

tested

Ca 7 0 0 2 0 2

of B o m i , M - 1 5 0 8

c r o s s e s w i t h T r i u m p h and

s c u t e l l u m of

g r a i n o f t h e T r i u m p h and M-1508.

the m o r p h o l o g y

1981-84

N o . of trials

P r e s e n t n a t i o n a l reference-*107 14 R 1508 ^ (lyg 3a) 88 18 2 Triumph 106 15 C a 7 0 0 2 0 2 2 (lys 3a in T r i u m p h ) 97 17 Nery3 101 13 C ? 7 2 0 5 0 2 3 ( l y s 3 a in N e r y ) 98 15_ T h e s e t r i a l s h a v e b e e n p e r f o r m e d in E n g l a n d ( 1 9 8 3 ) a n d a t 5 l o c a t i o n s in D e n m a r k ( 1 9 8 1 - 8 4 ) . 1) M i x t u r e o f V e g a , J e n n y , G i m p e l a n d T r i u m p h 2) 1 9 8 1 - 8 4 3) 1 9 8 2 - 8 4 . The Ca 700202 variety yield

to s e v e r a l

however, Table

judging

in y i e l d

significantly and

M-1508.

The

chemical

inferior

t h e l y s 3a g e n e .

absolute values given

The for

to produce

are

improved

level of same 1984

trials

fat

shown

for

is s e e n

in 1 9 8 3 .

trials

tendency

a high-lysine

in a l l

In

It

1983-84.

lines

is t h u s

barley with

lines

compared

is d e m o n s t r a t e d 4.

in

market,

the a b o v e - m e n t i o n e d

starch content

in T a b l e

is e q u a l

on the D a n i s h

to T r i u m p h e x c e p t

to Z i t a o f

composition

lines have

increased

it is p o s s i b l e

from four years'

the p r e s e n t v a r i e t i e s

3, the r e l a t i v e v a l u e s

Both high-lysine ning

of

to

contaiin

the

shown

stable

that

yield

53 Table 3.

Yield and Chemical Composition Relative to Zita = 100 in 1983-84. Levels of Significance: x) 0.05

0.01, xx) 0.01>P>0.001, xxx) P rH

0) (1)

•rH 4->

a>

O rH "

10 0 CO

> o

rH

IJ id CO

IW 0

c 0

•rH 4-1 •H

in

r-H

E 0

4-1

E

10 Q ÜH

T3 C

10

o

r-H

O o

co

^

rH

•

o

r-H r-H

0

ro

CN

T CN

a\

4J

4-1 (0

14H

-rH

•rH

C 0

4-1

+ • O VD

co •

CN in

CN •

u~) CO

rin

O

IO

•

m

O

u io

4-1

m

CO CN O •

L£>

rH

o

•

in

T

CN c£> • CO

— rH rH C^ • LD

CN CN CN •

^O

IN rH CD •

in

CQ

CD

0

c

•rH

•

CO

ra

-U

o

LTI

-IH

r-H 1

[SI

S

i

o o

^ CD Ci c a) u a»

-rH

X

ra

CTI

i-H

m

•rH

H

in

rH

ro

CO

E

I—I

rH

CO O rH

O rH rH

r—1

ja

rH

co

o ro

•c

CD

O

rH

c

0) CA

u

O O

rH

• CN

O

CO CD

•C 4J ü CO 1-1 ¡H CO T 3 E 4-1 > Q m x; 1 0 tfP c £> 0 in z (0 o

0

a

O

ro

•H - H

•rH

m

IO

r—H

•

o o

io o

J=

T3 \

CD T 3 rH

••

O

• CN

o o

CT'

•r-t r-H

c (0 a> s

00 • CN O rH

(0

T3 r-H

r-H

O O r-H

.c o \ o

W

(0 u 0

o

c 3 -rH h-t rH IS) CT

**

-P

O

cl) r-H UJ II 0 co

> u -y

J

CN

UH •H

T3

>

r^

CD

10 O

---rH

55

YIELD

Fiq. 2.

HKG/HA

Correlation between Yield and Starch Content (HyIdaqerqard, 1984, 4 repetitions).

(% DM)

In Table 5, the amino acid patterns of Triumph, Ca 700202 and M-1508 are shown.

The amino acid composition

chanqed due to the lys 3a qene increasinq

is drastically

lysine, threonine, gly-

cine and aspargine and reducinq qlutamic acid and proline. improved variety is equal or better in amino acid than the oriqinal mutant M-1508.

The

composition

In fact, the chanqed amino acid

pattern is very far from barley protein amino acid composition as commonly recognized, and the lys 3a qene produces the best pattern from a nutritional point of view amonq all cereal

hiqh-lysi-

ne mutants. Table 5.

Amino Acid Composition Asp Thr Ser

TRIUMPH Ca 700202 M-1508

(g/16 g N )

G l u Pro Gly Ala Val Met lie L e u Tyr P h e His Lys Arg

6.1 3.4 3.7 22.8 9.9 4.4 4.4 5.2 1.9 4.3 7.7 2.8 4.9 2.3 3.8 5.7 8.4 4.0 3.9 16.1 7.2 6.0 5.6 5.9 2.0 3.9 7.8 2.9 4.3 2.7 5.5 6.4 8.5 3.8 3.8 16.6 7.0 5.7 5.3 5.6 2.0 4.0 7.3 3 .0 4.3 2.8 5.2 6.7

% protein DM: Triumph 10.4, Ca 700202

11.6, M-1508

12.4.

Basic Research Triqqered by the Finding of Hiqh-lvsine Mutants and its Utilization in Plant Breedinq

Barley

It is natural to ask about the qenetic and biochemical basis of

56 the

improved

more

this, we could

high-yieldinq

perhaps obtain for

not yet

to d o a d e t a i l e d

time

1508

b u t we would

sing

such trials.

to be

like

high-lysine

here

In fact

theoretically

nutritional value

leading gene

Opaque

1508

2 gene

in m a i z e

the a p p a r e n t p r o t e i n protein

body

sium content genes are be

One

of

the

1508

major

the o p e r a t o r (see r e v i e w Hor

1, H o r

thesis.

b a c t e r i a and gene matic

Hordeins

ced

Ullrich sex

3c

the

biology

storage

so w e

in

with

the

characters

than

such as g e r m

free amino acids, these

manner

size,

pota-

recessive

and

can

easily

now

and

proteins

packed

5 control

smaller

Eslick and

large

a fairly

size

mutant,

containing compared

(12) e m p h a s i z e d proposed

that

the

the

in

idea of the

level

lys in

in their

3a the

endoplas-

bodies

(11).

the r o u g h

ER

a membrane

to called

bodies are

with quite

redu-

other

barleys.

shrunken

trait

seem

f r o m the

within

to n o r m a l

genes fact

the p r o t e i n material

maize

three

been cloned

good

through

structures contained

and

on

and

the p r o t e i n

secreted

syn-

years,

syn-

the c y t o l o g i c a l of

of

the h o r d e i n

Structurally

formation

1508

the

The g e n e s have have

15

focused

in b a r l e y

f o r m a t i o n of v e s i c l e s

In the

last

In b a r l e y

the

are

e f f e c t on the

the

the

barley

in the e n d o s p e r m

has extensively

(10)).

c h a n g e s on

characteristics

and

regulation

comparable

During

3 in c h r o m o s o m e

large

in n o r m a l

1508 p h e n o t y p e

seem

of g e n e

retarding

(8).

as their p r o d u c t s .

impairing

to m u c h

discus-

genes

of

synthesis

is t h e i r

& Miflin

sequenced

bodies.

staining

Hor

as well

tightly

protein

for

and

the o b j e c t

Mendelian

c l u s t e r s of g e n e s .

reticulum

have

beyond

Still,

h o r d e i n g e n e s are all q u i t e

introduces

endosperm

form

in m o l e c u l a r

2 and

We

700202

high-lysine

composition,

activity.

proteins

g e n e s of

to c o n s t i t u t e structure

2 genes

by S h e w r y

The

amino acid

effects on protein

storage

research

time-consuming

many other

in the e n d o s p e r m ,

specify

chromosomes.

and O p a q u e the

subject

affecting

in a r e g u l a r

less

in b a r l e y .

background

interesting

ribonuclease

o n the

the

t h e s i s of basic

and

inherited

located

and

some

3a s e e m s q u i t e

(8,9)

formation

trait

so-called

to the

lys

If w e c o u l d

c o m p a r i s o n w i t h Ca

to g i v e the

much more

plants.

The

types.

more elegant,

w a y s of b r e e d i n g had

the

1508

character

should

of

the

be d e s i g n e d

in a n a l o g y w i t h o t h e r m u t a n t s w i t h a s h r u n k e n

endosperm

as

57 showing

xenia.

This choice

of a p l e i o t r o p i c major gene.

complex

Choosing

illustrates

one

the

should

the p r o b l e m of w h a t

emphasize

shrunken endosperm

tant character

s e e m s to u s to p r e - c o n d i t i o n

titude

breeding

in a n gene

towards

interesting

(and

the

lection

work

see T a b l e

zed

interesting

through

non-plant enzymes lys

This gene

ters as compared ly s l i g h t l y caused

by a n

increase

We

have

Z which

further

24) w h i c h CI-2

is

have

shown

that

12 d a y s e a r l i e r timing

may

position

the C I - 2

lys

than

completely

in the r i p e

weight times

1 gene

the w i l d explain seeds of

700202,

trait

or

flexibility

of

quality

has been

and

and

mammal

inhibiting of

features

s p e c t r u m of

hordein

and

associated

hordeins proteases

is

is

(14, on-

mainly

medium-lysine CI-2,

the

charac-

level

level of H i p r o l y and

emphasi-

o n the n a t u r e

interesting

total

CI-1

pro-

6-amylase

to the

B-amylase.

protein

(17,18,19,20,21,22,23,

of 9 . 0 0 0

and

from potato,

u p to 50

the

inhibitor

is the p r o t e i n

inhibitors

increased

the

high-lysine

decrea-

inhibitors,

investigations

While

se-

a

proteins outside

enzyme

such

Our and

gene.

of a f e w h i g h - l y s i n e

has a molecular

its sequence

l y s 3a

in

for Ca

in s e e d

to a f f e c t a l e s s w i d e

the

studied

the

which

t o m a t o and

in H i p r o l y .

starts type

the

a

resulting

s t a r c h and

can admire

several

3a.

teins such as chymotrypsin and p r o t e i n

3a g e n e

the d r a w b a c k s

seeds

seeds.

the h i g h - l y s i n e

such as b a c t e r i a l

seems

affected,

lys

that

value

by c r o s s i n g

of p r o t e i n s w h i c h

Further

to l y s

high DBC

(especially

in e n d o s p e r m

1 displays

the p l u m p

the

l e v e l of

several different

insect amylases.

Hiproly gene

for

category

mainly

15,16).

Thus, we

the r e s e a r c h of

the

either

g e n e a s the

finding

restore

at-

Thus,

suggested

segregating

for

increased

to a d j u s t

such a major

the

carbohydrates

trait.

genome

is the and

of

barley.

(13)

it is p o s s i b l e

background

non-starch

large embryo

Another

some

that

4) w i t h o u t a f f e c t i n g

the b a r l e y of

of

a gene

could

to a

impor-

for a p e s s i m i s t i c

trial, Ahokas

1090

part

name

a s the m o s t

in h i g h - l y s i n e

without affecting

plump kernels,

level of

yield

C r y p t CI barley

has shown

to f i n d

in s t a b l e

the

1508

lysine value)

breeding

sed

preliminary

in the b a r l e y

crosses with

for yield

in g i v i n q

resembles

the c o m m o n

Further

studies

s y n t h e s i s of C I - 2

(25,26).

This difference

the d i f f e r e n c e s

in a m i n o

these

It

barleys.

in

leach.

acid

is t h u s

about in com-

possible

58 to s t u d y g e n e either

regulators

o n the p r o t e i n

w h i c h c a n be

by a n a l y s i n g

the b i o c h e m i c a l

level or o n the

transcribed

l e v e l of m e s s e n g e r

i n t o p r o t e i n Jji v i t r o and

racterized

by

To exploit

these options,

monospecific

Such antibodies

antibodies

needed.

important

p r o t e i n s w h i c h c a n be c o n v e n i e n t l y

1 and

also major

lys

3a.

genes

Genes

simply

for

the g r o w t h p a t t e r n of

with

same

the

major CI-2

genes

strategy.

We

to find o u t

protein 3a l y s

3a, g e n o t y p e ,

the

same d e f e c t i v e

(due

now available

called

BASI

supplied could been

arisen

to s u r v i v e

to p a s s

can

see

now

functions, involved

that

there

we could

also

For example,

and

are gene

nature

available

or are

they always occurring

fact, CI-1 content.

and

that Hiproly fraction

in q u i t e

CI-2

large

with presumed

and

which

and

could

is

repressor amounts

i n s e c t s and alive.

inhibitors

could

some

seed

be

plant protein

functions

nuclear isolate

the e x p r e s s i o n

important

proteins. repressor genes.

in t h e i r

& Chery band It

to

we

several

m o l e c u l e s of

to s e r v e

se-

also

When

in d i f f e r e n t

histones

and

have

the p l a n t

such as BASI with

whether

1,

on

configurations

helped

electrophoresis

to

some

1 lys

in j u s t a few c o p i e s p e r c e l l ?

s o m e c a s e s be p o s s i b l e of

lys

the

a-2-amylase

sequences

s t u d i e s by M c D a n i e l

has an unique

of

the w e a l t h of p r o t e i n

such

expression

proteins approach

Preliminary

inhibiting

bacteria

speculate

regulation

Inhibitor)

for b a r l e y

t r a c t of a n i m a l s

molecules

re-

is s y n t h e s i z e d

inhibitor

among

are

Thus

a double-headed

the e v o l u t i o n

are

recessives

hordeins.

sites

such

studied

be

a s the

Such

from moulds,

clue

complex,

recessive

template

Alternatively

the d i g e s t i v e

in g e n e

tissues.

found

just by chance

attacks

be a b l e

3a)

several

effects

in r e g u l a t i o n .

this protein

inhibitor

for d u r i n g

for

pro-

and e a r l i n e s s

make d o u b l e

in the d o u b l e

protease.

in the p l a n t .

selected

strength

a-Amylase/Subti1isin

separate

subtilisin

have

quences

(Barley

with

bacterial

cha-

to g e t a

pleiotropic

hierarchy

because

(28)

used

the p l a n t w h i c h c o u l d

to l y s

Mundy at our d e p a r t m e n t

for k e y

but biochemically

can also

their

is n o t e x p r e s s e d

lys

straw

inherited

gulating

a

are

the a c t i o n of m a j o r g e n e s d i s p l a y i n g

as lys

RNA

further

immunoprecipitation .

teins are of

phenotype

(29)

In

lysine point

out

in s e p a r a t i o n should

proteins

at

least

affecting

of in

59 We c a n hand

thus conclude

in h a n d .

breeders

first

that

suitable

lead

front and

the

b o t h a r e of p o t e n t i a l search objects the

for

step played

provements

and

plant breeding phase

practical

produce

theoretical methods,

genotypes

which

and

important

re-

scientists.

are

The r e s u l t s

back

to the p l a n t b r e e d e r s d e f i n i n g

work.

We a r e

with

scientists

they could now

the h i g h - l y s i n e

are

then

rationalize

looking

goes

plant

extreme

the m o l e c u l a r how

work

the

from

suggesting

in the w o r k

and

screening relevance

the m o l e c u l a r

investigations

second

the p r a c t i c a l

Provided

forward

barley

of in a

their

the

im-

further

to t h i s

second

mutants.

Acknowledgements T h a n k s are due Carlsberg since

to P r o f e s s o r

Plant Breeding,

1973.

We are

tensen. National and

to M r .

Denmark, land.

indebted

Institute

for h i s kind

England,

and

has also

b e e n of g r e a t

Sejersen and

Ms.

help

Mr. Birger

Kirsten

Science,

Landbrugets

Dr. J.

DSIRO,

for

and

for

yield

project

Mr. H.P.

the p i g

in

the

statistical

for c o m p l e t i n g

the

Zealand,

F i n a l l y we

analyses,

Jut-

Cambridge,

Christ Church, New acid

Mor-

trial

Sejet,

trials

Institute,

in t h i s p r o j e c t . the a m i n o

Larsen,

to t h i s

Kornforsd1ing,

in the e x t e n s i v e

Madsen

Kirkegaard

and

support

Plant Breeding

help for

keen

to D r . A r n e M a d s e n

Mr. Graeme Coles,

Corneliussen and

their

of A n i m a l

Kurt Hjortsholm,

Mr. P. H a n s e n , The

Ms. Bodil

D.v. Wettstein

for

Ms.

thank Lisbeth

calculations

manuscript.

References 1. 2. 3. 4. 5. 6.

O s b o r n e , T . B . , L.B. M e n d e l . 1914. J. B i o l . B i o c h e m . 17, 325-349. M u n c k , L. 1 9 8 3 . I n : F o o d P r o d u c t i o n , N u t r i t i o n , H e a l t h (S. R a j k i and Ä. B r u c e , e d s . ) A k a d é m i a i K i a d ó , B u d a p e s t , pp. 121-137. Munck, L., K.E. K a r l s s o n , A. H a g b e r g , B.O. E g g u m . 1970. Science 168, 985-987. D o l l , H . , B. K o i e , B . O . E g g u m . 1974 . R a d . B o t . 1_4 , 7 3 - 8 0 . D o l l , H . , B . K a i e . 1 9 7 8 . I n : S e e d P r o t e i n I m p r o v e m e n t by Nuclear Techniques. International Atomic Energy Agency, Vienna, pp. 107-114. M u n c k , L. 1 9 7 5 . I n : B a r l e y G e n e t i c s III (H. G a u l , e d . ) V e r l a g Karl T h i e m i g , M u n i c h , pp. 5 2 6 - 5 3 5 .

60 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

MacKey, J. 1981. In: C e r e a l s , A Renewable Resource, T h e o r y and P r a c t i c e (Y. P o m e r a n z and L . M u n c k , e d s . ) T h e A m e r i c a n A s s o c i a t i o n of C e r e a l C h e m i s t s , S t . P a u l , M i n n . , p p . 5 - 2 3 . N e l s o n , O . E . 1 9 7 9 . I n : A d v a n c e s in C e r e a l S c i e n c e and T e c h n o l o g y (Y. P o m e r a n z , e d . ) V o l . 3, 4 1 - 7 2 . M e r t z , E . T . 1 9 7 6 . I n : G e n e t i c I m p r o v e m e n t of S e e d P r o t e i n s . N a t i o n a l A c a d e m y of S c i e n c e s , W a s h i n g t o n , D . C . , p p . 5 7 - 7 0 . Shewry, P.R., B.J. Miflin. 1985. Adv. Cereal Sci. VII, 1-83. M u n c k , L. , D.v . W e t t s t e i n . 1 9 7 6 . I n : G e n e t i c I m p r o v e m e n t of Seed P r o t e i n s . N a t i o n a l A c a d e m y of S c i e n c e s , W a s h i n g t o n , D . C . , pp. 71-82. U l l r i c h , S.E., R.F. E s l i c k . 1977 . B a r l e y G e n e t . N e w s l . 66-73. A h o k a s , H . 1 9 7 9 . B a r l e y G e n e t . N e w s l . 9_, 3 - 7 . H e j g a a r d , J. 1981. Anal. B i o c h e m . 116, 444-449. H e j g a a r d , J., S. B j o r n , G. N i e l s e n . 1983. B a r l e y G e n e t . N e w s l . 12, 53-54. Boisen, S., C.T. Andersen, J. Hejgaard. 1981. Physiol. Plant. 56, 1 6 7 - 1 7 6 . J o n a s s e n , I. 1 9 8 0 . C a r l s b e r g R e s . C o m m u n . 47-58 . J o n a s s e n , I. 1 9 8 0 . C a r l s b e r g R e s . C o m m u n . £ 5 , 5 9 - 6 8 . S v e n d s e n , I . , I. J o n a s s e n , J . H e j g a a r d , S . B o i s e n . 1 9 8 0 . Carlsberg Res. Commun. £5, 389-395. S v e n d s e n , I . , B . M a r t i n , I. J o n a s s e n . C a r l s b e r g R e s . C o m m u n . 45, 7 9 - 8 5 . J o n a s s e n , I., J. I n g v e r s e n , A. B r a n d t . 1981. C a r l s b e r g Res. Commun. £6, 175-181. J o n a s s e n , I., L. M u n c k . 1 9 8 1 . I n : B a r l e y G e n e t i c s I V . F o u r t h International Barley G e n e t i c s Symposium, Edinburgh, pp. 330335 . J o n a s s e n , I . , I. S v e n d s e n . 1 9 8 2 . C a r l s b e r g R e s . C o m m u n . 4 7 , 199-203. J o n a s s e n , I. 1982 . C a r l s b e r g R e s . C o m m u n . £7_, 3 0 5 - 3 1 2 . M u n c k , L . 1 9 7 2 . I m p r o v e m e n t o f N u t r i t i o n a l V a l u e in C e r e a l s . H e r e d i t a s 7_2- 1 - 1 2 8 R a s m u s s e n , U. 1985 . Carlsberg Res. C o m m u n . 83-93 . Doll, H. 1981. In: B a r l e y G e n e t i c s IV, F o u r t h I n t e r n a t i o n a l Barley G e n e t i c s S y m p o s i u m , E d i n b u r g h , pp. 257-262. M u n d y , J . , I. S v e n d s e n , J . H e j g a a r d . 1 9 8 3 . C a r l s b e r g R e s . Commun. £8, 81-90. M c D a n i e l , R . G . , M. C h e r y . 1974. B a r l e y G e n e t . N e w s l . 50-51.

61 SELECTION

A.G.

Abdel

OF

RUST

Hafez,

RESISTANT

M.S.

WHEAT

El-Keredy

A g r o n o m y D e p a r t m e n t , F a c u l t y of 33,000 Kafr El-Sheikh, Egypt

AFTER

and

A.A.

MUTAGENESIS

Bassioni

Agriculture

S e v e r a l m u t a n t s w e r e s e l e c t e d f o r r e s i s t a n c e to w h e a t r u s t s in field after Gamma irradiation and chemomutagenesis . Seeds from " G i z a 1 5 7 " w h e a t w e r e t r e a t e d w i t h G a m m a r a y s 0 , 5 , 1 0 , 15 a n d 2 0 Krad; s e p a r a t e l y or in c o m b j n a t i o ^ w i t h E^S 0.1, 0.2 or 0.3% for s i x h r , o r s o d i u m a z i d e 10 , 10 o r 10 M for three hr. Selection was c a r r i e d out after a r t i f i c i a l i n o c u l a t i o n in M^, M , and M . g e n e r a t i o n s . Several mutants e x h i b i t e d r e s i s t a n c e against leaf and stripe rusts during M and their r e s i s t a n c e was p r o v e d in M^ and M^ a l s o . O n e s i n g l e p l a n t f r e e of s t e m r u s t a n d s t i l l s h o w i n g r e s i s t a n c e to l e a f a n d s t r i p e r u s t s w a s f o u n d i n M^ . S o m e m u t a n t s w e r e c h a r a c t e r i z e d b y t o l e r a n c e to s t e m r u s t i n a d d i t i o n to r e s i s t a n c e a g a i n s t t h e o t h e r t w o r u s t s . T h e t r e a t m e n t 1 5 , 10 K r a d c o m bined with NaN ; 10" M a n d 20 K r a d , c o m b i n e d w i t h E M S ; 0 . 3 % o b t a i n e d the b e s t y i e l d of r e s i s t a n t m u t a n t s . F i n a l l y , t h e s t u d y p r o v e d t h a t s e l e c t i o n to c h a n g e h o s t / p a t h o g e n r e l a t i o n s h i p in h e x a p l o i d w h e a t c a n be r e a l i z e d in the M^ or in the f o l l o w i n g early generations.

Introduction

M u t a t i o n b r e e d i n g m a y b e of v a l u e to a c h i e v e h i g h l e v e l s of r e s i s t a n c e d e g r e e s , b e t t e r t y p e s of r e s i s t a n c e a n d / o r t o l e r a n c e . It c a n a l s o b r e a k u n d e s i r a b l e l i n k a g e s i n v o l v i n g g e n e s f o r d i s e a s e r e s i s t a n c e . R a d i a t i o n , c h e m i c a l m u t a g e n s or their c o m b i n e d t r e a t m e n t s w e r e a l s o u s e d to s e p e r a t e u s e f u l d i s e a s e r e s i s t a n c e g e n e s from associated genes that badly affect other traits by causing c h r o m o s o m e b r e a k a g e a n d t r a n s l o c a t i o n s ( 5 , 7, 9, 1 0 ) . I n s o m e c a s e s m u t a t i o n b r e e d i n g w a s s u c c e s s f u l l y u s e d to i n d u c e r u s t r e s i s t a n c e g e n e s ( 3 , 4, 6, 1 0 ) . T h e a i m o f t h i s s t u d y h a s b e e n t o s e l e c t rust r e s i s t a n c e m u t a n t s f r o m c o m m o n w h e a t c u l t i v a r G i z a 157 by G a m m a i r r a d i a t i o n a n d c h e m o m u t a g e n e s i s .

Materials

and

Methods

P u r e s e e d s of T r i t i c u m a e s t i v u m T h e l l . ; c u l t i v a r G i z a 1 5 7 w e r e t r e a t e d b y e a c h o f t h e G a m m a - r a v d o s e s 0 , 5, 1 0 , 15 o r 2 0 K ^ a d s e p a r a t e l y o r i n c o m b i n a t i o n w i t h s o d i u m a z i d e s o l u t i o n 10 1 0 ~ 3 or 1 0 ~ 4 for 3 hr at ph 3 or E M S c o n c e n t r a t i o n s 0 . 1 , 0.2 or

Genetic Manipulation in Plant Breeding ©1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

62 0.3% for 6 hr at ph 7. C h e m i c a l l y treated seeds were washed in running w a t e r for 30 minutes after sodium azide or 12 hr after EMS treatment. M^ plants w e r e raised , s e l f e d , individually h a r v e s t e d and their p r o g e n i e s were single plant w i s e s o w n . A m i x t u r e of five u n i v e r s a l s u s c e p t i b l e varieties acted as s p r e e d e r for p r e v a i l i n g leaf and stripe rust v i r u l e n c e s (2). At the 3-leaf s t a g e , plants were inoculated by d u s t i n g with leaf rust; P u c c i n i a recóndita and s t r i p e rust; P u c c i n i a s t r i i f o r m i s uredia . At jointing stage one M . plant from each M ^-progeny was inoculated by i n j e c t i o n and enougn epidemic of leaf and stripe rusts w a s i n i t i a t e d . M . plants w e r e assessed at F e e k e s 11 (8) and those exhibited r e s i s t a n c e to both rusts or one rust w e r e selected (Table 1 ) . In 1982/83 581 r e s i s t a n t m u t a t i o n a l lines were re-tested for r e s i s t a n c e in two sowing d a t e s with five w e e k s interval to avoid any escape s t a t m e n t . T h e reaction to d i s e a s e was assessed with respect to both type of infection and d i s e a s e s e v e r i t y . In 1983/84 s e a s o n , seeds from the 581 families b e l o n g i n g to M^ and M^ g e n e r a t i o n s were planted in o p p o s i t e rows, inoculated with stem rust; Puccinia g r a m i n i s tritici; at Feekes 7 (8). F i n a l l y , at F e e k e s stage 11 the d i s e a s e infection was a s s e s s e d .

Results

and

Discussion

D i f f e r e n t numbers of M ^ and M^ plants were originated from each treatment, both single and c o m b i n e d . A total of 9516 treated M^ plants and 606 u n t r e a t e d plants w e r e assessed for c o m p a r i s o n . Some treatments produced no v a r i a t i o n s in reaction to d i s e a s e , due to the low number of M ^ plants and or sterility caused by high m u t a g e n d o s e s or their combined t r e a t m e n t s (Table 1). High yield of mutants resistant to leaf and stripe rusts w^s achieved by G a m m a rays 15 and 20 Krad and sodium azide 10 M combined with 10 Krad t r e a t m e n t . Also, c o m b i n a t i o n of sodium azide was better than that of EMS with G a m m a rays. E v a l u a t i o n of r e s i s t a n c e of M^ across two sowing d a t e s enabled the v e r i f i c a t i o n of 27 m u t a n t s r e s i s t a n t to both r u s t s . Some m u t a n t s w e r e almost free of infection w h i l e others were slightly i n f e c t e d . In respect to s e l e c t i o n for r e s i s t a n c e against stem rust, selection was d o n e in M^ as m u t a n t s r e s i s t a n t to this d i s e a s e are more of interest, only if they show accepted r e s i s t a n c e to both other rusts . The artificial infection have been d e v e l o p e d to a high d e g r e e of severity and all the m u t a n t s were infected except for one single plant s e g r e g a t e d from a M^ family (Mutant 48) that was absolutely free of stem rust i n f e c t i o n . The progeny of this plant w a s also resistant in 1984/85 (M^) s e a s o n . M o r e o v e r , the mutants infected by stem rust produced s h r i v e l l i n g g r a i n s , but a few m u t a n t s produced normal not s h r i v e l l i n g g r a i n s showing t o l e r a n c e to d i s e a s e . Selection of m u t a n t s resistant to leaf and s t r i p e rusts in M2 and M ^ and stem rust resistant mutant in M^ g e n e r a t i o n s e n s u r e s the p o s s i b i l i t y to change the h o s t / p a t h o g e n r e l a t i o n s h i p in p o l y p l o i d s as hexaploid w h e a t , by s e l e c t i o n in early and f o l l o w i n g g e n e r a tions through m u t a g e n e s i s .

63 Table

1: M u t a g e n i c t r e a t m e n t s , M^ p l a n t s , M^ plants and selected in M^ for r e s i s t a n c e against leaf and rusts . No.of M plants

Total M2 plants

35

606

2

1

0

5 Krad 10 Krad 15 Krad 20 Krad

29 33 33 35

387 650 570 523

1 2 0 3

0 0 0 0

2 1 6 5

0 Krad + 0 . r/o EMS " 5 Krad + 0.1% " 10 Krad + 0.1% 15 Krad + 0.1% " 20 Krad + 0.1%

1 6 1 5 23 1 6 1 7

258 336 41 8 352 233

1 0 1 1 2

0 0 0 0 0

1 0 0 0 0

0 Krad + 0.2% EMS " 5 Krad + 0 .2% 10 Krad + 0 .2% " 15 Krad + 0 .2% " 20 Krad + 0.2%

1 1 1 1 1

2 5 9 5 8

75 250 41 1 295 337

0 0 1 0 0

0 1 0 0 0

0 1 0 0 0

0 Krad + 0 .3% EMS 5 Krad + 0 . 3% 10 Krad + 0 . 3% " 15 Krad + 0 . 3% " 20 Krad + 0.3%

1 1 1 1 1

8 5 5 4 5

323 253 241 223 1 37

1 0 0 0 0

0 0 0 0 1

0 0 0 0 2

0 Krad + i o - 2 N a N , 3 M " 5 Krad + " 10 Krad + 11 15 Krad + " 20 Krad +

1 1 1 1 1

7 7 8 8 6

31 2 242 263 330 260

0 3 0 2 1

0 1 0 0 0

1 2 5 2 0

0 Krad + 1 0 - 3 N aN M J " 5 Krad + 10 Krad + " 15 Krad + " " 20 Krad +

1 1 1 1 1

8 1 9 6 8

268 64 233 1 09 1 88

0 0 3 1 2

0 0 0 0 1

0 0 0 0 0

0 Krad + 1 0 - 4 5 Krad + " 10 Krad + " 15 Krad + " 20 Krad +

1 6 1 9 1 7 20 1 5

228 160 1 61 244 1 83

1 1 0 0 6

0 0 0 1 2

0 0 0 0 0

Treatment

"Giza

15 7'' untreated

NaN, M

••

J

No.of M^ plants resistant to leaf rust

No.of M^ plants resistant to yellow rust

mutants stripe No.of M 2 plants resistant to both rusts

64 References 1. A b d e l - H a k , T . , D . M . S t e w a r t , and A.H. Kamel . 1 972. The current stripe rust s i t u a t i o n in the Near East R e g i o n . R e g i o n a l w h e a t W o r k s h o p , B e i r u t , L e b a n o n , 1972. P r o c e e d . , the Ford F o u n d a t i o n , Vol . 1 - D i s e a s e s . 2. A b d e l - H a k , T., N. E l - S h e r i f , I. Shafik, A.A. B a s s i o n i , S. Keddis , and Y. E l - D a o u d i . 1 982. Studies on wheat stem rust v i r u l e n c e s and r e s i s t a n c e g e n e s in Egypt and n e i g h b o r i n g countries. 4th C o n g , of P h y t o p a t h o l o g y , A l e x a n d r i a , N o v . 30 - D e c . 3, 1982. E g y p t . 3. A b d e l - H a k , T., and A.H. K a m e l . 1977. M u t a t i o n b r e e d i n g for d i s e a s e r e s i s t a n c e in wheat and field b e a n s . In: Induced M u t a tions against Plant D i s e a s e s , P r o c . Int. Symp . I A E A / F A O , V i e n n a 1977, 305-314. 4. B o r o j e v i c , K a t a r i n a . 1977. S t u d i e s on r e s i s t a n c e to P u c c i n i a recondita tritici in w h e a t p o p u l a t i o n after m u t a g e n i c treatm e n t s . In: Induced M u t a t i o n s against Plant D i s e a s e s , P r o c . Int. S y m p . I A E A / F A O , V i e n n a , 1977, 393-401. 5. E l l i o t , F . C . 1957. X-ray induced t r a n s l o c a t i o n of A g r o p y r o n stem rust r e s i s t a n c e to common w h e a t . J . H e r e d . 48, 77-81 . 6. Konzak , C . F . 1 956. Stripe rust resistant mutants obtained i r r a d i a t i o n of Gabo w h e a t . P h y t o p a t h o l . 46_, 5 2 5 - 5 2 6 .

from

7. K n o t t , D . R . 1961. T h e i n h e r i t a n c e of rust r e s i s t a n c e . VI. T h e transfer of stem rust r e s i s t a n c e from A g r o p y r o n elongatum to common wheat. C a n . P l a n t Sci . 4J_, 1 09-1 23. 8. Large, E. 1954. G r o w t h stages in c e r e a l s . I l l u s t r a t i o n s Feekes scale. Plant Path. 3, 128-129.

of

the

9. Little, R. 1971. An attempt to induce r e s i s t a n c e to S e p t o r i a nodorum and Puccinia g r a m i n i s in wheat using gamma rays, n e u t r o n s and EMS as m u t a g e n i c agents. In: M u t a t i o n B r e e d i n g for D i s e a s e R e s i s t a n c e , Proc. P a n e l , V i e n n a , 1970, IAEA, 139-149. 10. S e a r s , E.R. 1956. T h e transfer Agilops u m b e l l u l a t a to w h e a t .

of leaf rust r e s i s t a n c e from B r o o k h a v e n Somp. Bio. 9_, 122..

65 USE OF MAIZE MUTANTS IN BREEDING FOR IMPROVEMENT OF PROTEIN QUALITY

M. Denic, S. Ratkovid, J. Dumanovic, D. Misevic Maize Research Institute, Belgrade-Zemun, Yugoslavia

Introduction Maize proteins contribute at large extent to the total plant protein production. However, due to the low lysine and tryptophan content maize proteins are considered as low quality proteins. Therefore in many breeding programs opaque-2 (02) mutant is used to improve protein quality. The incorporation of 02 gene is followed by reduction of yield and increase of grain moisture content. Studies of Glover et al (1) showed that some other genes, similar to the 02 gene, increase lysine and tryptophan content by incrreasing glutelin fraction and reducing zein fraction. The aim of this work is to study the influence of some mutant genes on the relationship between lysine content and moisture content and capacity of water imbibition in maize kernels.

Results In order to study the influence of some mutants on this relationship the amino acids were determined by ion-exchange chromatography (2,3) and capacity of water imbibition by simple gravimetric procedure as described earlier (4) . Data presented in Table 1 show that lysine content and lysine yield were higher in both experimental

(exp xv/18 02) and opaque-2 check

(ZPSC 72 02) than the commercial check with standard kernel type (ZPSC 704), which is one of the highest yielding commercial hybrids. The some data show that grain moisture content at harvest was higher in opaque-2 hybrids in comparison with the hybrid of standard kernel type.

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

66

Table 1. Performance of High Lysine Maize Hybrids

Hybrid

Grain yield (kg/ha)

L y s i n e content yield (% protein) (kg/ha)

Moisture content at harvest D.M.

°2

ZPSC 704 n

12.401

2.92

31.0

26.2

1.00

ZPSC 72 o 2

9.207

4.21

38.7

31.1

1.19

9.871

4.09

46.4

28.4

1.08

Exp xv/18 o 2

Table 2. Moisture Content and Capacity for Water Imbibition in Endosperm and Embryo of Different Maize Genotypes (% D.M.)

E n d o s p e r m Genotype

W 64A: N °2 du 02

E m b r y o

Initial Water Imbibimoisimbibi- tion ture tion mut:nor

Initial Water Imbimoisimbibi- bition ture tion mut:nor

11. 8

45 .2

38. 4

151 .3

14. 2

74 .4

1 .65

42. 4

176 .6

1.17

15. 1

108 .8

2 .41

49. 3

250 .6

1.66

-

-

fl 2 o 2

13 .4

82 .7

1 .83

53 .6

219 .9

1.45

h o2

17. 0

75 .9

1 .66

54. 4

177 .7

1.17

wx o 2

17. 2

87 .9

1 .93

76. 2

224 .2

1.48

Oh 43: du o 2

17. 7

74 .2

1 .64

48. 7

210 . 2

1. 38

fl 2 o 2

14 .3

78 .9

1 .75

41. 9

194 .4

1.28

h o2

14. 4

80 .1

1 .77

55. 2

272 .7

1.60

su 2 o 2

15. 0

104 .4

2 .31

46. 7

218 .3

1.44

13. 5

93 . 9

2 .08

40. 0

226 .0

1.49

16. 4

82 .4

1 .90

49. 7

211 .1

1.41

sh 2 o 2 Average

-1'All listed genes are homozygous

67 In order to see the influence of opaque-2 gene on grain moisture content and water imbibition special genotypes were constructed with the mutant genes affecting storage proteins and starch synthesis. Data presented in Table 2 show higher moisture content in embryo than in endosperm in all studied genotypes. Initial moisture contents of both endosperm and embryo were higher in mutants than in normal type of kernels. The same data show that water imbibition also was higher in embryo than in endosperm being higher in mutants than in normal genotype. In the case of endosperm higher water imbibition was found in double mutants as compared to the single opaque-2 mutant suggesting the additive effect of the other gene. This kind of influence is mainly due to the preferential gene action in chemical composition of endosperm in comparison to the embryo of the same genotype.

References 1. Glover, D.V., P.L. Crane, S.P. Misra, and E.T. Mertz. 1975. In: High Protein Quality Maize, Proc. Symp. El Batan, Mexico (Dowden, Hutchinson and Ross, eds.). Stroutsburg, p.p.228-240 2. Spackman, D.H., W.H. Stein, and S. Moore. 1958. Anal. Chem. 20, 1190. 3. Denic, M. 1968. Acta Chem. Scand. 22,

1809-1812.

4. Ratkovic, S., M. Denic, and G. Lahajnar, 1982. Period. Biol. 8±, 180-182.

69 PROMISING

M.S.

RICE

El-Keredy

MUTANTS

and

A.G.

FOR

DEVELOPED

PRODUCTION

Abdel-Hafez

Agronomy Department, Faculty Kafr El-Sheikh, Egypt

of

Agriculture,

Tanta

University,

R i c e g r a i n s o f " G i z a 1 7 2 " a n d IR 5 7 9 - 4 8 w e r e e x p o s e d t o G a m m a - r a y d o s e s : 0, 5, 7.5, 10, 12.5, 15 and 20 K r a d t o i n d u c e v a r i a t i o n s f o r r e s i s t a n c e to l o d g i n g a n d e a r l i n e s s . M progenies were planted indiv i d u a l l y i n s e p a r a t e r o w s 30 c m a p a r t a n d w i t h 10 c m b e t w e e n p l a n t s as w e l l a s u n t r e a t e d g r a i n s . T h r e e k i n d s of m u t a n t s w e r e selected; i.e. early ripening, erect wide leaved and vigorous mutants' in Giza 172. Early ripening and v i g o r o u s mutants were s e l e c t e d f r o m IR 5 7 9 - 4 8 t r e a t e d m a t e r i a l . T h e m u t a n t s w e r e r e p l a n t e d in M ^ a n d M 4 a n d p r o v e d to b e s t a b l e m u t a t i o n s . S t u d y of c u l m c h a r a c t e r i s t i c s of l o d g i n g r e s i s t a n t m u t a n t w a s c a r r i e d out by s p e c i a l e q u i p m e n t and i n d i c a t e d v a l u a b l e informations.

Introduction

L o n g d u r a t i o n in r i c e is u n d e s i r a b l e c h a r a c t e r , d u e to t h e n e e d f o r i n t e n s i v e p r o d u c t i o n s y s t e m w h i c h is a b l e to m a x i m i z e y i e l d f r o m t h e l i m i t e d a r e a of N i l e - D e l t a in E g y p t . S u i t a b l e c u l t i v a r s for m e c h a n i c a l h a r v e s t i n g are also h o p e d by E g y p t i o n farmers who a r e s u f f e r i n g f r o m l a b o r s h o r t a g e p r o b l e m . I n S o u t h e a s t A s i a (1) s e l e c t i o n of e a r l y a n d s h o r t c u l m m u t a n t s h a s b e e n d o n e by m e a n s of m u t a t i o n b r e e d i n g .

Materials

and

Methods

T h e r i c e c u l t i v a r s G i z a 172 a n d IR 5 7 9 - 4 8 a r e c o n s i d e r e d l o n g d u r a t i o n v a r i e t i e s ; n e e d a r o u n d 160 d a y s f r o m s e e d to s e e d u n d e r E g y p t i a n c l i m a t e . G r a i n s of t h e t w o c u l t i v a r s w e r e e x p o s e d to 0, 5 , 7 . 5 , 1 0 , 1 2 . 5 , 15 a n d 2 0 K r a d G a m m a r a y s . M^ p l a n t s w e r e p r o tected from outcrossing and harvested individually. The M1 prog e n i e s w e r e g r o w n in s e p a r a t e r o w s . P l a n t s h a v i n g e a r l i e r h e a d i n g or d e s i r e d p l a n t t y p e w e r e s e l e c t e d (2). T h e s e l e c t i o n s w e r e r e e v a l u a t e d in M ^ and M 4 g e n e r a t i o n s . F u r t h e r culm c h a r a c t e r i s t i c s of s o m e m u t a n t s w e r e m e a s u r e d by s p e c i a l e q u i p m e n t (3).

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

70 Results

and

Discussion

The mutants e x h i b i t e d induced variations in earliness, culm stiffness and thickness , wide leaved and lodging resistant , late heading a n d v i g o r (Table 1). G i z a 172 e a r l y r i p e n i n g m u t a n t s w e r e i n d u c e d b y 7 . 5 , 1 0 , 1 2 . 5 a n d 15 K r a d . E r e c t m u t a n t s w e r e o b t a i n e d

Table

Mutant

1 : M u t a n t s s e l e c t e d i n M_ G i z a 172 a n d IR 5 7 9 - 4 8 Original c u i tiv ar

D o s e of Gamma- r a y s Krad 7 .5

after the treatment with Gamma-rays.

Mutated

of

rice

characters

1a

Giza

1 72

2a

Giza

1 72

1 0

Erect, lodging resistant, stem, wide leaved.

strong

3a

Giza

1 72

1 0

Erect, lodging resistant, stem, wide leaved.

strong

3b

Giza

1 72

1 0

Erect, lodging resistant, culm, wide leaved.

thick

4a

Giza

1 72

1 0

Late

Early heading, resistant.

erect,

brown

spot

heading .

5a

Giza

1 72

1 0

Early

heading.

6a

Giza

1 72

1 2 .5

Early

heading.

7a

Giza

1 72

1 5

Early

heading.

7b

Giza

1 72

1 5

Early

heading.

7c

Giza

1 72

1 5

Early

heading.

8a

Giza

1 72

20

Late heading, lodging resistant, l o n g e r c u l m , p a r t i a l s t e r i l e as plant .

9a

IR

579-48

5

9b

IR

579-48

5

1 0a

IR

579-48

1 0

Taller

culm.

Erectly Early

strong

culm.

mutants.

1 Ob

IR

579-48

1 0

Early

mutants.

1 0c

IR

579-48

1 0

Early

mutants.

1 1 a

IR

579-48

1 5

Early

mutants.

11 b

IR

579-48

1 5

Early

mutants.

12a

IR

579-48

1 5

Early

mutants.

1 2b

IR

579-48

1 5

Early

mutants.

i n t h i s c u l t i v a r b y 10 K r a d . E a r l i e r m u t a n t s a n d t w o v i g o r o u s m u t a n t s w e r e o b t a i n e d i n c u l t i v a r IR 5 7 9 - 4 8 b y 5 K r a d . E a r l y ripening mutants were 5-10 days earlier than their correspondent parent c u l t i v a r . The mutants were g r o w n in M and M. g e n e r a t i o n s

71

and they proved to be true bred with d e s i r e d i m p r o v e m e n t s (Table 2) . Study of culms c h a r a c t e r i s t i c s of lodging r e s i s t a n t mutant 3b obtained v a l u a b l e i n f o r m a t i o n s (Table 3). The weight needed to cut T a b l e 2: Plant h e i g h t , panicle length, g r a i n weight per 1000-kernel w e i g h t and g r a i n yield per hill of mutants induced in Giza 172 by G a m m a - r a y s . Genotype

Plant height (cm)

Giza

1 08 .0 , 87..0 95 .4 . 89 .8 89 . .5 85 . .8 110 .0 123..0

1 72

Mutant Mutant Mutant Mutant Mutant Mutant Mutant

Table

1a 2a 2b 3a 3b 4a 8a

Length of panicle (cm)

Grain weight per panicle (g)

1000kernel weight (g)

Grain weight per hill (g)

1 9..70

3 .21

25 . .00

25 .60

24 26 25 26 25 22 23

3 .47 4 .08 3 .88 4 .08 3 .84 3 .51 1 .81

25 24 23 26 26 23 25

34 24 24 24 23 22 25

.30 . .23 .00 . .20 .83 .30 . .51

Characteristics

a) Qj -p 0 c 0) u Giza

. .24 .83 .60 . 72 .30 .30 .30

3: Culm c h a r a c t e r i s t i c s of lodging r e s i s t a n t rice from Giza 172 after G a m m a - r a y s t r e a t m e n t .

P •C CP •H 01 r — e p a c —

ra

p 0).— 0 P Ë C V -C -H— P CP -P 0) C 01 13 ai ra0 a rH C

E O x; -p CP

c

RRRR,

proved very useful. Again, the donor of the D- and E-genome chromosomes was the agrotricum amphiploid. It was crossed to the autoalloploid rye, and the F^ was crossed to hexaploid triticale (Table 9). Assuming full compensating ability of the D- and E-genome chromosomes, a staggering number of 279936 ((P ) chromosome combinations could theoretically be selected from such hybrids. The C-banding analyses of the materials are underway, and prove to be more difficult than originally thought. A. elongatum chromosomes do not show any clear and consistent banding patterns; thus they are difficult to identify. The results presented here are preliminary and limited to the D-genome chromosomes (Table 1o). In the materials analyzed the D-genome chromosomes show a tendency to be incorporated for the A-genome chromosomes (in homoeologous groups 3, 4, 6 and 7) rather than for the B-genome chromosomes

(in homoeologous groups 3 and 5).

It has to be pointed out, however, that the substitution pattern in this case depends primarily on the chromosome constitution of the wheat genome in the autoalloploid rye used in the original cross, as well as the compensating ability of the individual chromosomes. The D- and/or E-genome chromosomes can substitute only for those Aand B-genome chromosomes that are not present in the autoalloploid rye. The chromosome constitution of the autoalloploid rye was not known. The most frequent D-genome chromosomes in the analyzed sample were 3D and 6D.The frequent incorporation of 6D is of particular value as this chromosome increases spike lenght and fertility. To summarize the efforts to incorporate the D-genome chromosomes into tetraploid and hexaploid triticale it should be pointed out, that with the exception of chromosome 2D all homoeologous groups are involved in substitutions. Therefore, it is possible to enrich the gene pool of hexaploids and tetraploids with new and more D-genome chromosomes from different sources. Last year a new set of experiments was started, aimed at the incor-

110

poration of chromosomes from wild Triticeae species into triticale. From the wheat relatives, donors of the A, B and D genomes were chosen. The idea was to produce from T. monococcum, T. boeoticum, Ae . sharonensis, Ae. bicornis and Ae. squarrosa tetraploid amphidploids with rye. Unfortunately, most of these attempts have failed. Only the production of one Ae. bicornis x s. cereale (B^^B^^RR) and of several Ae. squarrosa x S. cereale ( D ^ D ^ R R ) amphiploids was successful. Since Sodkiewicz

(1984, 1985) has already obtained a T.

monococcum x S. cereale (A A RR) amphiploid, we have the three — mo mo tpossible wheat donor species in amphiploids with rye. The three amphiploids differ remarkably in fertility and the mode of propagation (Table 11). The T. monococcum and Ae. bicornis amphiploids appear self-incompatible and can only be propagated vegetatively. The Ae. bicornis amphiploid produced one seed which after a short period of development on culture medium ceased to grow and the seedling failed to develop into a normal plant. It appears that there is so much discord between the Ae. bicornis and

cereale genomes that the

sexual reproduction of this amphiploid is suppressed. Perhaps the same is true for the T. monococcum amphiploid. The crosses between several accessions of Ae. squarrosa and rye were quite successful, although embryos had to be rescued on culture media. At the present time we have four different amphiploids. They are self-fertile and show good seed

quality. Tetraploid Ae. squarrosa

triticales (or aegilocales) are therefore valuable for improvement of triticales at every ploidy level. Although the amphiploids of T. monococcum and Ae. bicornis with rye are self-sterile and cannot be propagated sexually, they can be crossed to hexaploid and octoploid triticales. This indicates that they produce viable egg cells, therefore can also be used in triticale improvement. The potential for new chromosome combinations at the hexaploid level are presented in Table 12. When the new amphiploids are crossed to octoploid triticale, hybrids result that have one A genome from T. monococcum and one A genome from hexaploid wheat, or one B genome from Ae. bicornis and one from hexaploid wheat, or one D genome from Ae. squarrosa and one from hexaploid wheat. The latter appears to be

111

the most interesting as it allows for testing the effects of the complete D genome combined with a mixture of A- and B-genome chromosomes in hexaploid triticale. Through hybridization of the new amphiploids with hexaploid triticale tetraploid triticales could be selected that contain chromosomes from T. monococcum, Ae. bicornis or Ae. squarrosa. In fact several tetraploids resulting from T. monococcum/rye amphiploid x tetraploid triticale hybrids have already been selected and karyotypically stabilized. The crossing program of Ae. squarrosa x tetraploid triticale is underway and looks very promising. Only the hybridization between Ae. bicornis/rye amphiploid x tetraploid triticale is difficult, as the resulting F^ hybrids are self-sterile as the amphiploid itself.

Summary Summarizing the results and perspectives presented in this paper one can conclude that indeed tetraploid triticales and their derivatives, the autoalloploia ryes, are a very useful tool in improvement of hexaploid triticale. They are of particular importance in the attemps to incorporate the D-genome chromosomes from hexaploid wheat and other alien chromosomes into the hexaploids. There are some indications that the D-genome chromosomes in particular can improve fertility, seed quality, baking properties and other agronomic characteristics of the hexaploids. The incorporation of Agropyron chromosomes is of interest in attemps to improve protein quality, drought and disease resistance. Incorporation

of Aegilops chromosomes may be useful in

the improvement of seed quality, protein content, baking properties and in the reduction of plant height. It is possible that sprouting resistance could also be improved. Although triticale acreage in the world is not great, it is slowly increasing. With the increase of production disease damages may become more dangerous. Tetraploid triticales with its extensive range of chromosome combinations and wide gene pool may become important for solving problems as they arise.

112

Tab. 1

C o n v e n t i o n a l M e t h o d s of P r o d u c t i o n of 8x-, 6x- and 4 x - T r i t i c a l e

AA BB DD (42) x RR (14) —

F 1 ABDR (28) —

Colch. —

AA BB DD RR (56)

8x-Trc.

AA BB (28)

F^ ABR

Colch. —

AA BB RR (42)

6x-Trc.

x RR (14) —

AA BB RR (42) x RR (14) —

(21) —

F 1 ABRR (28)

Selfing and Stabilization

AA RR (28)

Tab.

2

(AB) (AB) RR (28)

BB RR (28)

4x-Trc.

Genome Constitution of the Wheat Component in 4x-Triticale

Theoretical Number of Combinations

Berlin Triticales

- - -

( A 6 B l > < A 6 B l ) RR

7

- - -

( A 5 B 2 ) ( A 5 B 2 ) RR

21

( A 4 B 3 ) ( A 4 B 3 ) RR

. . .

1

1

1

3

4

35

6

Ol

1

Total

CD

AA RR

Polish Triticales

(A 3 B 4 )(A 3 B 4 ) RR

35

3

3

6

( A 2 B 5 ) ( A 2 B 5 ) RR

21

1

1

2

(AJBGJIA^G) RR

7

. . .

1

1

BB RR

1

—

128

11

18

29 (22,7%)

113

a c O "D •H O

p =1 P x: p P -H -H ÍH 3 p

T-i

:>

O IH CD CD

2.2.

C\J

CM CD r^

à? CD

Sí •y

CM C\J OJ >

•M

rH u.

rH O m

CM O CQ

M O m

"t a CD

~U 3 >-l O C -H

+

133 already from the third cycle. Despite this tendency there are some chromosome numbers which are more frequently present others. Among them the 35-, 21-, and 14-chromosome

than

individuals

are the most prominent genotypes. Considering the permanent

chro-

mosome number of n=7 of the pollen parent, it can be suggested that female gametes with 28, 14, and 7 chromosomes are transmitted preferentially. It can be speculated that those whole complements are not composed only by poor rye

chromosomes.

In the BC^ generation at least a total number of 54 monosomic rye-wheat additions with somatic chromosome number 2n=15 were observed, i. e. about 24 % of the offsprings. But 38 seedlings only were able to grow up to maturity. Many of them showed a vyeak growth habit combined with a low tillering capacity in e a r lier stages. Even under comparable environment several

morpholo-

gical characteristics may differ as can be taken from Fig. 1. The number of tillers, leaf morphology, leaf colour, maturity as well as less photoperiodic responce and different

susceptibility

to powdery mildew were modified depending on the extra chromosomes concerned. T o clarify the genome constitution the chromosomes of root tip squashes were checked first by their morphology in Feulgen stained slides which, however, did not reveal single wheat

chromoso-

mes. Although the wheat chromosomes appear somewhat smaller than the rye chromosomes they cannot be considered Therefore chromosome

unambiguously.

banding has been carried out to identify

the alien chromosomes by their patterns of heterochromatic

bands.

Beside C-banding most confident results were gained by the N - b a n ding procedure. The preliminary investigation showes that the monosomic additions of the wheat chromosomes 3A, 4A, 7A, 5B, 6B, and 2D are very likely. Meanwhile the added chromosomes 3A, 5B, and 6B have been confirmed also by isoenzyme markers. The

pat-

terns of GOT and EST gave evidence for the presence of chromosome 3A while NADP-AADH and GOT as well as AMP demonstrate chromosomes 5B and 6B, respectively

(see Fig. 2). Further

the cytolo-

gical and biochemical studies now are in progress to complete the series of the 21 rye-wheat

additions.

Compared to the previous study (8) there was a similar seed set in F^ hybrids of about 0.002 kernels per spiklet after

back-

crossing. This low fertility was slightly increased by subsequent

134

Figure 1

Plant morphology parents (varieties 'Petka' and 'Chinese Spring*) as well as a sample of eight of different 15-chromosome rye-wheat additions(

their from left

to right)

Isozymes

AMP-5 AMP-4 AMP-3 AMP-2 AMP-1

+ ) heterodimeric

i s o z y m e s , GOT-2c = h o m o - a n d h e t e r o d i m e r i c

Genes ( C h r o mosome a r m s )

Amp-B1 Amp-R1 Amp-D1 Amp-R? Amp-A1

(6BS) (6RS) (6DS) (6AS)

isozymes

a ) 1181-74. ( 2 n = 1 4 ) , b) 1 1 8 1 - 2 ( 2 n = 1 5 ) , c ) T. a e s t i v u m L . , cv. " C h i n e s e

Spring"

Figure 2 Zymograms of glutamate oxaloacetate transaminase (I) and aminopeptidase (II) of leafs in diploid rye (a), the rye-wheat addition( RVVA 6B) (b) , and hexaploid wheat (c)

135 backcross accompanied by a decrease of the number of the wheat chromosomes. Moreover, two of the monosomic rye-wheat

additions

(RWA 3A and RVVA 6B) were investigated more detailed as each of them has been propagated to establish three clonal plants. The first plant was used for a crossing with autoplasmic diploid rye (var. Petka), the second subjected to self-pollination, and the third plant was also self-pollinated, but before the anthers have been opened by hand to release the pollen grains. Since the induced rye-wheat additions led to an alloplasmic material by the crossing procedure mentioned above, they are almost caused by the cleistogamous flowering and unreleased

steril

pollen.

Open pollination within a population of 14-chromosome

plants,

therefore, showes a seed set of 0.04 kernels per spiklet

only.

Thus the backcrossing to diploid rye was proved as the most e f f i cient way in getting higher seed set, beside artificial

self-pol-

lination which demonstrates on some extent the viability of the male gametes in the additions. However, disomic additions have never been isolated after both natural and artificial nation. This is in striking agreement with previous

self-polli-

findings.

On the other hand, the female transmission of the extra chromosome, generally, correspondes to the observation of 8, whereby it ranged from about 3 ^ in RWA 3A to 13 % in R W A 6B. The

smaller

chr mosome 3A, surprisingly, is less frequent transmitted the larger chromosome 6B which can be attributed to the lar function of the nucleolus-organizer

than

particu-

chromosome.

Further experiments are initiated to maintain the whole set of rye-wheat additions as well as to stabilize alien wheat

chromo-

some transfer in the genomatic background of rye.

References 1. Anonymus. 1934. In: Statistisches Jahrbuch der DDR. S t a a t s verlag der DDR, Berlin 2. Blüthner, Vi.-D., D. M e t t i n . 1977. A r c h . Züchtungsforschg. 7, 15 3. Driscoll, C.3. 1985. Proc. 6th Int. Wheat Genet. Symp., S u p p l . 4. Gamborg, O.L., R . A . Miller, 0. Ojima. 1968. Exp. Cell R e s . 50, 151

136

5.

Mcintosh, R.A. 1983. Proc. 6th Int. Wheat Genet. Symp., 1197

6.

Miller, T.E. 1984. Can. 0. Genet. Cytol. 26, 578

7.

Schlegel, R. 1982. Biol. Zbl. 101, 641

8.

Schlegel, R., B.S. Gill. 1984. Can. 0. Genet. Cytol. 26, 765

9.

Schlegel, R., E. Weryszko. 1979. Biol. Zbl. 98, 399

10.

Schlegel, R., G. Melz, D. Mettin. 1985. Theor. Appl. Genet, in press

11.

Schmidt, O.-C., P. Seliger, R. Schlegel. 1984. Biochem. Physiol. Pflanzen 179, 197

12.

Zeller, F.3., S.L.K. Hsam. 1983. Proc. 6th Int. Wheat Genet. Symp., 161

137 Nucleolar genomes

competition

of t e t r a p l o i d

1

'cermeno,

2)

K.-D.

M. C . ;

(A/B)(A/B)RR

and

DDRR

triticales.

"''Friebe, B . ;

"''zeller,

F. J.

and

Krolow

"^Institut 2)

in d i f f e r e n t

für

Technische Institut

Federal

Pflanzenbau

Universität

für

Angewandte

Republic

of

und

Pflanzenzüchtung,

München Genetik,

Freie

Universität

Berlin

Germany

Introduction: Transcriptional regions

(NORs)

microscopy nique.

rDNA

in p l a n t

Recently triticales

Lacadena

et a l . ,

However,

since

complete

A, B and could

genomes

to a n a l y s e diploid Material

using

staining

of n u c l e o l a r

R genomes

triticales

in h e x a -

(Cermeno

in a m o r e

triticales

offer

analysed

of w h e a t w e r e

be s t u d i e d

et a l . ,

The

activity

present,

specific

aim

of

okto-

1984;

this

in d i f f e r e n t

and

D genomes

of w h e a t

nucleolar

with A/B

Different mixed

nucleolar

investigation combinations of

of

with

R

c

from

Secale

s cereale

and

R

1. D i f f e r e n t cereale

from Secale combinations

(2n = 2 8 ) ,

silvestre Tr i t i c u m

genomic

have

been

analysed:

t u rg id um, d u r u m - S e c a l e

constitution

(1B1B,

-, -, -, 5A5A,

6B6B,

-)RCR°

(1B1B,

-, - , 5B5B,

6B6B,

-)RCRC

(1B1B,

-, -, -, 5B5B,

6A6A,

-)R°RC

(1A1A,

-, -, -, 5A5A,

6A6A,

-)R°RC

2. Tr i t i c u m s q u a r r o s u m - S e c a lse s s i l v e s t r e genomic c o n s t i t u t i o n DDR R .

(2n =

is

wheat.

Methods: (A/B)

airways

way.

to a n a l y s e

(RR) w i t h A a n d B or D g e n o m e s

following

light

tech-

activity

and

up to now

(A/B)(A/B)RR

the o p p o r t u n i t y

detail.

nucleolar

and

organizer

of c o n v e n t i o n a l

a silver

suppression

obtained

D genomes

in m o r e

rye

nucleolar

1984).

not

of w h e a t

for

of

by m e a n s

A, D a n d

has been

of t e t r a p l o i d

competition

The

from

in t h e

activity lines

chromosomes

evidence

of S A T - c h r o m o s o m e s ploid

gene activity

has been analysed

28),

G e n e t i c M a n i p u l a t i o n in Plant B r e e d i n g © 1 9 8 6 W a l t e r d e G r u y t e r & Co., Berlin • N e w York - Printed in G e r m a n y

138 A comparative

a n a l y s i s of s o m a t i c m e t a p h a s e

out by m e a n s of p h a s e c o n t r a s t staining according

followed

G i r a l d e z et al.

cells was

by C - b a n d i n g

carried or

Ag-NOR-

(1979) and L a c a d e n a et al.

(1984). Results: The

r e s u l t s are p r e s e n t e d

Tab.1: Silver and

stained

nucleoli

in Tab.

nucleolar

visualized

organizer

Combination

1B + 6B

wheat-rye

regions

(Ag-NORs)

in s o m a t i c m e t a p h a s e s and

p h a s e c e l l s in d i f f e r e n t tetraploid

1.

(A/B)(A/B)R°RC

inter-

DDRSRS

and

combinations.

No. of M e t a p h a s e s A g - N O R s No. of n u c l e o l i

Total

plants

cells

1

24

14

2

54

3

19

4

2

3

1707 2 6 9 0

1 B + 6A

11

39

2

1254

1A + 6A

4

25

2

DDRSRS

3

15

2

4

963 1 0 0

5460

927

21 81

1057

845

1 902

576

407

983

Discussion: In

(A/B)(A/B)RCR°

chromosome satellite

chromoscme

those belonging maximum

number

corresponds Therefore,

combinations,

pair 1R of rye is s u p p r e s s e d

in t h e s e c o m b i n a t i o n s

1984). Similar

haviour

However

These

r e s u l t s are

(Cermeno

et al.,

chromosome

n u c l e o l i and s h o w i n g has been d e s c r i b e d 1B and 6B

in c o m b i n a t i o n s w i t h o u t

1B and

1B with

hybrids with Lacadena

1, et

combinations in

b a n d s . The same

ir, h y b r i d s c a r r y i n g

( C e r m e n o et al.,

which

pairs

pair 1B w a s a c t i v e

strong Ag-NOR

as the

in a g r e e m e n t

1984;

r e s u l t s w e r e o b t a i n e d for

of

detected.

only the c h r o m o s o m e

for d i f f e r e n t w h e a t - r y e

6B in w h i c h only

of c h r o m o s o m e

hand,

of p o s i t i v e A g - N O R b a n d s

2 or 3 d o s e s of rye g e n o m e

ducing

as w e l l

of n u c l e o l i o b s e r v e d at i n t e r p h a s e w a s 4,

to the n u m b e r

previous data reported al.,

nucleolar activity

to A g e n o m e of w h e a t . On the o t h e r

and 6B of w h e a t are a c t i v e .

missing

in w h i c h both 1B and 6B S A T -

p a i r s of w h e a t w e r e p r e s e n t ,

only

one

probe-

dosis

1984).

6B c h r o m o s o m e

pair

1R

139 was active, organizer maximum

exhibiting

secondary

constrictions

r e g i o n s and p r e s e n t i n g

number

nucleolar

of n u c l e o l i

activity

in

nucleolar

strong Ag-NOR bands. Since

observed

in these c a s e s w a s

of S A T - c h r o m o s o m e s

from A genome

that the n u c l e o l a r

organizer

the

2,

was

suppressed. T h e r e is e v i d e n c e , themselves

are

responsible

for n u c l e o l a r a c t i v i t y .

c a t i o n of o t h e r w h e a t c h r o m o s o m e s a given SAT-chromosome binations analysed,

constitution

in the

were found. However, by m a n y

the e x i s t e n c e

control.

of a g e n o t y p i c

Secale

silvestre,

chromosome the

S

DDR R ,

only

pair f r o m D g e n o m e

rye g e n o m e

remained

modifi-

a c t i v i t y , for c c (A/B)(A/B)R R com-

since nucleolar

g e n e s , we can not

in h y b r i d s b e t w e e n T r i t i c u m S

No

upon n u c l e o l a r

vity a p p e a r s to be r e g u l a t e d O n the o t h e r hand

chromosomes

squarrosum

nucleolar activity has been d e t e c t e d ,

for

acti-

exclude and

SAT-

while

that

of

suppressed.

Conclusions: 1. The r e s u l t s s h o w ,

that

in all c o m b i n a t i o n s

containing

6B as w e l l as in t h o s e c a s e s w e r e only one pair present,

the n u c l e o l a r a c t i v i t y

of s a t e l l i t e

f r o m R g e n o m e of rye and A g e n o m e of w h e a t , were

suppressed

pair 1R of rye w a s suppression

by the p r e s e n c e squarrosum

chromosomes respectively,

1B and 6B the s a t e l l i t e

chromosome

active. of c h r o m o s o m e

of s a t e l l i t e

has been

pair

1R of S e c a l e

chromosome

p a i r from

silvestre Triticum

observed.

References: C e r m e n o et al. 1 9 8 4 , C h r o m o s o m a Giraldez

and

completely.

2. In c o m b i n a t i o n s m i s s i n g 3. Total

1B

of 1B w a s

89, 3 7 0 - 376.

et al. 1979, Z. P f l a n z e n z u c h t g .

L a c a d e n a et al. 1 9 8 4 ,

Theor. Appl. Genet.

83, 4 0 - 48. 67, 207 - 213.

141

IDENTIFICATION

A.

OF INTERCHANGES

IN WILD S P E C I E S

OF PISUI1

Errico

I s t i t u t o di Agronomia G e n e r a l e di N a p o l i , P o r t i c i , I t a l y

e Coltivazioni

Erbacee,

Università

C. C o n i e e l 1 a Centro di S t u d i o Portici , Italy

per

il

Miglioramento

Genetico

degli

Ortaggi,

CNR,

Introduction

The w i l d gically

species (1,

2,

have a l w a y s

of

P i s u m have

3, 4,

5, 6 ) .

evidenced

the chromosomes to

involved

In

order

identify

of

two Pi sum s p e c i e s

med by u s i n g

the

been s t u d i e d

The g e n e t i c a l

presence

of

have never

a translocation

and c y t o l o g i c a l

reciprocal

involved

and f u l v u m )

tester

set,

in

cytolo-

analyses but

definitively.

the

translocations

a research

kindly

and

interchanges,

been a s c e r t a i n e d

the chronosomes (abyssinicum

genetically

given

was p e r f o r -

by d r .

Lamm

(Sweden).

Results

Crosses

have

slocation and p o l l e n

been p e r f o r m e d

testers

ble 1)

and t h e chromosome

abortion

The chromosome

between t h e w i l d

were a n a l y s e d

configuration

configurations

on F^

analysis

species

and t h e

tran-

at metaphase

I

hybrids.

allows

to c o n c l u d e

that

(ta-

1 ): no t r a n s l o c a t i o n

is

present

in

the a c c e s s i o n

1 of

Genetic Manipulation in Plant Breeding © 1 9 8 6 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

a b y s s i n i cum.

142 Table

2)

1

Chromosome

Configurations

one t r a n s l o c a t i o n

is

present

formation

formation

the c r o s s e s

the chromosomes 3)

1)

in

involved

two t r a n s l o c a t i o n s

are

are

in four with

3 and

present

in

association

1 and l i n e

7 and o f one q u a d r i v a l e n t

The f r e q u e n c y the c r o s s two

are T ( 3 - 5 ) (72.8%)

between

independent

i

2)

and

lines

F.

Hybrids.

2 o f _P. and one

abyssiniesavalent

2 and 5 i n d i c a t e

that

P. f u 1 v u m :

observed

in

in the

on t h e b a s i s

the c r o s s e s cross

with

of

with line

the

line 2 the

T(1-7).

of c e l l s

t h e normal

crosses

in

4.

octavalent

translocations

(fig.

I

in the a c c e s s i o n

cum. Two q u a d r i v a l e n t (fig.

at Metaphase

with

line

translocations

are

two q u a d r i v a l e n t s

8 and £ . present

(table

2)

fulvum demonstrates in

this

accession.

in that

One

143 Table

2

Frequencies

o f Chromosome

and P o l l e n A b o r t i o n

Configurations

Percentage

in

F,

at Metaphase

I

Hybrids.

CHROMOSOME CONFIGURATIONS AT METAPHASE I Cross

1 2 3 5 7 8

of

5II+1IV

411+1VI

(%)

(%)

75 57 51 61 58 59

16.5 87.8 7.8 11.5 22.4 27.2

59.3 8.7

x P. fulvum ii X ii X H X M X h X

the

two t r a n s l o c a t i o n s

videnced the

Cells Analysed (N°)

by t h e f a i l u r e

is

3II+2IV

311+1 VI 11

711

Pollen Abortion

(%)

(%)

(%)

{%) 3.9

59 67 74 65 62 62

20.3 3.5

92.2 88.5 46.5

27.5

3.4

72.8

characterized

o f one o f

by s h o r t

segments

t h e two q u a d r i v a l e n t s

as

e-

in 27.2%

of

cells.

References

1.

B e n - Z e e v , N. and D. Z o h a r y . 1 9 7 3 . S p e c i e s r e l a t i o n s h i p s Genus P i s u m L . I s r a e l J . o f B o t a n y 2j?, 7 3 - 9 1 .

2.

F o u z d a r , A. and S . L . T a n d o n . 1 9 7 6 . C y t o g e n e t i c a l t h e Genus P i s u m . C y t o l o g i a 4_1_, 9 1 - 1 0 4 .

3.

Lamp r e c h t , H. 1 9 6 4 . P a r t i e l l e S t e r i l i t ä t und t u r b e i P i s u m . A g r i . H o r t . Gen. 7 2 , 5 6 - 148 .

4.

R o s e n , G. Von. 1 9 4 4 . A r t k r e u z u n g i n d e r G a t t u n g P i s u m , i n s b e s o n d e r e z w i s c h e n P. s a t i v u m L . und P. a b y s s i n i c u n B r a u n . H e r e d i t a s 3_0 , 26 1 - 4 0 0 .

5.

S a c c a r d o , F. t e r 3, 38.

6.

S a n s o m e , E . R . 1 9 3 8 . A c y t o l o g i c a l s t u d y o f a F^ between P i s u m s a t i v u m and P. h u m i l e , and o f some t y p e s from t h e c r o s s . J . Genet. 36, 4 6 9 - 4 9 9 .

1971.

Crosses

among

in

evolution

the

in

chromosomenstruk-

Pisum s p e c i e s .

Pisum

Newslet-

145 GENETIC REGULATION OF MEIOTIC RECOMBINATION

IN PETUNIA

HIBRIDA

E. FARCY, C. MOUSSET, D. MAIZONNIER, A. CORNU INRA, Station d'Amélioration 21034 Dijon Cedex, France

des Plantes, B.V.

1540

Introduction When mapping genes

in Petunia it became obvious

enhanced recombination

in certain tightly

knowldege of Petunia genetic

line St43

quali-

to undertake a research on gene-

recombination.

(from the Genetic

(1). The

(2 -3) as well as its intrinsic

ties gave us favorable conditions tic regulation of meiotic

that some genotypes

linked groups

In this aim we used

Institute of the University

the

of

Amsterdam) which was able to increase recombination b e t w e e n certain genes comparatively with a "low recombination

frequency" line taken

as a control. Seven pairs of linked markers located on each of seven chromosomes

(table

1) were

the

tested.

Results a) Effect of St43 gametogenesis. combinations, seems

genotype

on meiotic

segments where is considerably

effect

to a particular chromosome

increased. It is decreased III

linked ones on chromosome

cases St43 genotype

tested

for

the

recombination during II and

VI clusters

male during

in the same conditions. In these two

has not modified

types which

rate

moderately

IV.

gametogenesis. Behaviour of chromosome male game togenesis was

for the

and it remains the same

b) Effect of St43 genotype on meiotic

recombinant

but

( table 1). For five of the chromosome

the markers are closely linked, recombination

linked markers of chromosome

However

female

St43 genotype effect, when used in heterozygous is not restricted

to have a general

weakly

recombination during

remained

the relative number of

comparable

this response cannot be generalized

to that of : very

Genetic Manipulation in Plant Breeding © 1 9 8 6 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

controls.

recent

146

Table

1 : Effect

Recombination of

Testcrosses

Chromosome

of

St43

Frequency on

F1

Genotype between

on

two

Macrosporogenesis Loci

Results

Plants.

P a i r s o f markers used

Linkage type

Recombination r a t e i n % Fl w i t h c o n t r o l

F1 w i t h St43

I

Hfl -

Phi

repulsion

1,.03

4..90

Lui -

Fl

coupling

0,.22

4 .24

*

III

H t l - Mf 1

coupling

6..78

2..89

*

IV

B_1

- An6

coupling

29..41

V

Po

- Mf 2

coupling

0..00

VI

An2 - Rt

repulsion

VII

An4 - Vs5

coupling

experiments effect male

of

meiosis.

owing

to

tially

recovery

of

significant

be symbolized

either being control

a

1:1

intermediate

might

0..15

5..63

*

2,. 15

11 .87

*

control

when

is

segregation, to to

depend range

factor(s)

and

is

used

of

or Fl

about

nuclear

slightly

of

higher

with

St43

one

major

factor.

suggests

that

there

on

and

(or)

than

(chromosome

genetic

and

chromosomes differenin

the

meiosis.

factor(s)• rates a

There

or

factor(s)

be

with a

male.

which we

can

Fl,

were the

markers). of

on

obtained

tested

control

background.

as

no

In e x p e r i m e n t s

plants

VI

is

obtained

female

Occurrence might

same

female

could

character

the

the

difference

modulator.

ability

half

as

progenies

affect it

to

male

rate

Recombination recombination

show

during

Thus

leading

recombination

St43

dependant

by Rm of

female

between

Fl's

rate

particularities.

competitivity

difference

markers

specifically

recombination

comparable

secondary

St43

modifying

comparable could

and V I I

of

transmissibi1ity nearly

III

from

the

*

16h-day). S i z e o f

recombination

recombinants

reciprocal

Therefore

on

structural

upon p o l l e n

Genetics

two

chromosome

genotype Therefore

there

act

N.S

2..00

different

with

St43

25.

*

CO

Significantly

T e s t c r o s s e s performed i n growth chamber (20°C, from 700 t o 2000 p l a n t s .

the

:

II

*

c)

Meiotic

on C h r o m o s o m e s

others

Thus

the

plants

interference

with

with

147 The

major

being

Rm

about

factor 2,5%,

is

lul

linked itself

to lul is on

locus,

the

linkage

long

relationships

arm of c h r o m o s o m e

II

(4) . Moreover

the

e f f e c t of

of chromosome

increasing

VI m a r k e r s

backcrosses

with

St43

level

30%.

It s e e m s

up

nation

to

rates

from

15%

the p l a n t

t e s t e d . As

0.3.

new

this

suited with

From

these

cular

linkage

the

tested

progenies

30%, o w i n g

compared level,

can p u t

to in

to

of Rm

forward

on c h r o m o s o m e

of

parts

of

of

which

is

about

is

better

loci

(4).

two

hypothesis on

recombi-

genotypes

Rm/Rm,

these

a working

VI and

recombination induces

different

t h a t of c o n t r o l

localization

recombination sucessive

exhibit

the p r e s e n c e

Rm on h e t e r o c h r o m a t i c

particularly

the

on

after

that h o m o z y g o s i t y

cytological

data we

action of

segments

line: . The

to

homozygosity,

An2-Rt, was

of a

parti-

pericentromeric

II.

Re fe r ence s

1.

C o r n u , A . , D. M a i z o n n i e r , H . W i e r i n g , P . de V l a m i n g . 1980 P e t u n i a G e n e t i c s . Ill The l i n k a g e g r o u p s of c h r o m o s o m e V. Ann. Amelior. Plantes . 443 .453 .

2.

C o r n u , A . a n d D. M a i z o n n i e r . 1983. The G e n e t i c s o f P e t u n i a . In: P l a n t B r e e d i n g R e v i e w s , v o l . 1 (J. J a n i c k , e d . ) A v i Publishing Company. W e s t p o r t Commecticut. pp. 11-58.

3.

De V l a m i n g , P . , A. C o r n u , E. F a r c y , A . G . M . G e r a t s , D. M a i z o n n i e r , H. W i e r i n g and H . J . W . W i j s m a n . 1 9 8 4 . P e t u n i a hybrida. A s h o r t d e s c r i p t i o n of the A c t i o n o f 91 g e n e s , t h e i r o r i g i n a n d their m a p l o c a t i o n . Plant Mol. Biol. Reporter. 2,21-42.

4.

M a i z o n n i e r , D., A . C o r n u , E. F a r c y , P. de V l a m i n g . 1985. G e n e t i c and c y t o l o g i c a l m a p s in P e t u n i a . P o s t e r p r e s e n t e d G e n e t i c M a n i p u l a t i o n in P l a n t B r e e d i n g . B e r l i n .

in :

149

ATTEMPTS TO TRANSFER RESISTANCE TO PHOMA LINGAM FROM BRASSICA JUNCEA AND B. CARINATA TO B. NAPUS THROUGH INTERSPECIFIC HYBRIDIZATION FOLLOWED BY OVULE CULTURE

M. Gerdemann, M.D. Sacristän Institut für Angewandte Genetik, Freie Universität, 1000 Berlin 33

The resistance to Phoma linqam (asexual form of Leptosphaeria maculans) found in a few varieties of rape (Brassica napus) is of "quantitative" type and not expressed at all developmental stages of the plant. This is true also for rape lines derived from selected cell cultures Q )

(^2). In contrast to rape, amphi-

diploid Brassica-species with B-genoma (B. juncea, B. carinata) possess a higher level of resistance, which is expressed, already at the seedling stage, as an hypersensitive reaction to the infection.

With the purpose of transfering this high degree of resistance to rape, interspecific crosses were made between B. napus (2n = 38, AACC) and the species B. juncea (2n = 36, AABB) and B. carinata (2n = 34, BBCC). A relatively high yield of hybrids could be achieved by culturing the ovules in vitro 12-24 days after the pollination O ) •

The results of these crosses are summarized in

Tables 1 and 2.

Tab. 1. Results of crosses between oil seed winter rape varieties and B. .juncea and subsequent ovule culture Cross*

No. poll. flowers 117

No. siliqua formed

Ovules cultured

Growing embryos

%

Hybrid plants

%

78

999

76

7..6

74

7..4

Lib

X

Bj

Gar

X

Bj

132

99

954

77

8..1

65

6..9

Lir

X

Bj

182

120

1723

178

10..3

157

9..1

* Lib = 'Librador 1 , Gar = 'Garant 1 , Lir = 'Liropa 1 , Bj = B. .juncea

Hybrid plants showed an intermediate morphology and a pronounced heterosis effect. Their hybrid character was also confirmed by chromosome counts: 37 chromosomes in .juncea-hybrids, 36 in carinata-hybrids • Both types of hybrids exhibit the same resistance to Phoma as the resistant parent.

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

150

Tab. 2. Results of crosses between oil seed winter rape varieties and B. carinata and subsequent ovule culture Cross*

No. poll. flowers

No. siliqua formed

Ovules cultured

Growing embry OS

/o

Hybrid plants

%

Lib x Be

877

436

1920

619

32.2

79

4.1

Gar x Be

927

402

2260

708

31.3

179

7.9

Lir x Be

909

529

2853

711

24.9

229

8.0

* Lib = 'Librador', Gar = 'Garant', Lir = 'Liropa', Be = B. carinata

The results of the first backcrosses are shown in Tables 3 and 4.

Tab. 3. First backcrosses: B. napus x (B. napus x B. Cross*

No. pollin. flowers

Ovules cultured

Lib x LibBj

535

1505

Gar x GarBj

634

Lir x LirBj

546

juncea)-hybrids

Growing embryos

R^-plants %

%

49

3.3

42

2.8

1225

30

2.5

21

1.7

1241

68

5.5

62

5.0

* Abbreviations see Tab. 1

Tab. 4. First backcrosses: B. napus x (B. napus x B. carinata)-hybrids Cross*

No. pollin. flowers

Ovules cultured

Growing embryos

R^-plants %

%

Lib x LibBc

213

572

22

3.8

11

1.9

Gar x GarBc

278

156

38

24.4

20

12.8

Lir x LirBc

332

345

77

22.3

62

17.9

* Abbreviations see Tab 2

As expected, R^-plants showed a great variability with respect to morphology and to the response to the infection with Phoma. Preliminary results of resistance tests in R^-plants seem to indicate a higher proportion of resistant plants among those from juncea-crosses than within plants of carinata-crosses. In the progeny of hybrids B. napus x B. juncea Roy (4_) (_5) found also resistant rape plants.

151 Acknowledgment

This research was supported by a grant of the Bundesministerium

für Forschung

und Technologie in collaboration with the Gemeinschaft zur Förderung der privaten deutschen landwirtschaftlichen

Pflanzenzüchtung.

References

1. Sacristan, M.D. 1982. Theor. Appl. Genet. 61, 193. 2. Sacristan, M.D. 1985. Hereditas Suppl. Vol. \

57.

3. Sacristan, M.D. , Gerdemann, M. 1985. Vortr. Pflanzenzüchtg. 9_, 91. 4. Roy, N.N. 1978. Euphytica 27, 145. 5. Roy, N.N. 1984. Euphytica 33, 295.

153 ANALYSIS OF ADH1 LOCUS IN TETRAPLOID CORN /Zea mays L./

M. Hajos-Noväk, A. Bälint Department of Plant Breeding, Agricultural University Gödöllo, Hungary H-2103 A. H. Nagy, G. Vida Department of Genetics, Eötvös Loränd University Muzeum krt, 4/a, Budapest, Hungary H-1088

Introduction

ADHl is the predominant ADH gene of corn. ADH is expressed in the mature scutellum and pollen grain. Alcohol dehydrogenase is a dimeric molecula. Different maize lines have two

electrophoretical-

ly distinguisable ADHl isoforms. ADH1-S and ADH1-F differ in their quantitative, organspecific expression and in the level of intragenic recombination. These can be explained by the gene competition hypothesis. Different ADHl alleles compete for a factor that limits ADHl expression. Using in situ staining of pollen grains for ADH activity various ADHl dysfunctional mutants have been obtained. Most of the interesting regulatory-type mutants at ADHl are in the ADH1-S allele.

Results Two allelic forms of ADHl were found in tetraploid I

corn fam-

ilies deriving from tetraploid synthetic population and in Wf9 tetraploid line. PAGE patterns of different dimeric ADHl are shown in Fig.l. Frequency of ADH1-S allele gradually decreased during inbreeding. F ^ n d F 2 hybrids of Wf9 /4x/ } x I 3 /4x/ 6 were homozygotes for ADHl-F form

/Table 1./.

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

154

Start

+ Fig.l. Electrophoretic Picture of ADH1 isoforms, Extracted from Tetraploid Corn Seeds /pore gradient PAGE 4-10%/. 1.5. homozygotes, 2-4 heterozygotes, a. SS c. FF homodimers, b. SF heterodimer. Table 1. Frequencies of ADH1 Alleles in Tetraploid Maize Line, Families and in their Hybrids

Plant material Wf9 /4x/ I o Z

1 J 2. I 3 F 1 F F

1

2 x Wf 9 /Bc-^ /

Allelic forms ADHl-S

ADH1-F

0..325 0..458

0.675 0.541

0..429 0..327

0.673

0..236 0..000 0..000

0. 764 1.000 l.OOO

0..125

0.875

0.571

155

The ratio of ADH1 heterozygotes was lower than expected in Bc^ generation. Lack of pollen-ADH was found among the Wf9 /4x/ plants, while the ratio of the ADH+ and ADH - pollen in the pollen grains of the Wf9 /4x/ $> x I 3 /4x/ 2-concentrations , and, the

effects

on

are

presented.

Material

and

Methods

used.

from Anemone Only

reached ge w a s

the

established

used. Where agar

(Difco)

ments,

used or

media rile The

the

double

contain this,

of

liquid

the

Results

and

double

free

were

the

sta-

and

pol-

(6),

was

bud

(Merck,

and

to

size

pH 5.8

p. a . ) w a s agent;

added.

either

In c e r t a i n

0.8%

experi-

polyvinylpyrrolidone

liquid

gelling

had

developmental

between

a gelling

L. grains

(BDH

culture medium.

agents

All

and AC, were

ste-

used

this,

0.5%

Anthers

the

in this

petri

activated are

study,

dishes

(O = 5

charcoal,

floated

on

the

and

surface

medium.

layer m e t h o d

has

to n u t r i e n t s

produced

by

the

medium.

possible,

If

embryoids are more

or

the

in

barriers,

as

that

the e x p e r i m e n t s In

with

liquid m e d i u m .

the a n t h e r s ,

means

of

(1).

solid medium,

4 ml

to d i f f u s i o n

When

in m o s t

Discussion

access

tances,

and

biopur)

layer m e t h o d

4 ml

above

due

domestica the p o l l e n

( K e l c o , K9AP1 for Both

Characters) in the G. Premise x A.Rcyal Cross. Obs: frcm SSD

Exp :frcm F3

Ht/IGW

2

Ht/foSW

3

7

Ht/Mat

2

0

Ht/AE

0

Ht/SPY

1

SPY/AE

0

SPY/Mat

1

:frcm DH 1

2 4 2

1 1 2

2 2 0

1

1

The simplest approach to prediction i s to observe the proportion of falling

into predefined categories in samples of F3 families.

lines

It i s then

possible to rank the crosses on this basis and use i t as a prediction (5). Rank correlations can subsequently be used to compare that predicted with

329

that observed in SSD lines, as shewn in Table 4. Although preliminary, the "progeny testing" of crosses would appear to hold premise as a means o£ ranking crosses for combinations of characters. •able 4.

Rank Correlation Coefficients Between the Predicted and Observed

Ranking Based on the Phenotypic Proportions in the F3 Samples. Ht/IGW Ht/MSW Ht/Mat Ht/AE Ht/GN Ht/SPY

>P1>P1 0.15 0.75

>P1P22.0 cm to 0 - 5 cm, obtained from the nurseries of Vletters Bros, and Den Haan, Oesterweg 204, Rijnsburg (Z-H), The Netherlands, were surface sterilized for 15 mins in 10% Domestos (Lever Bros., U.K.).

After rinsing two times in sterile distilled water, the

anthers with uninucleate microspores were dissected out and planted in 90 x 15 mm Petri dishes containing approximately 20 ml of solidified basal medium (1) with 0.1 mg/1 napthylacetic acid (NAA), 0.1 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D), and 8% sucrose. Cultured anthers were incubated at 25°C in continued darkness until the emergence of macroscopic globular embryo like structures or calli.

For ovule culture flower buds of 2.0 cm size were dissected

in a solution containing major salts of Murashige and Skoog (1) at half strength, 4% sucrose and 3g/l activated charcoal.

The

transverse section (2-3mm) of the unpollinated ovary was explanted on a solidified basal medium (2) with 400 mg/1 glutamine, 100 mg/1 serine, 50 mg/1 asparagine, 0.1 mg/1 NAA, 0.1 mg/1 2,4-D and 10% sucrose, and incubated in the dark at 21°C until the emergence of embryos.

When grown up to a size of 3-4 mm, the embryos were

Genetic Manipulation in Plant Breeding ©1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

336 isolated and grown in light on the hormone-free ovule culture medium with 2% sucrose. For histological studies the cultured anthers and ovules were fixed in Allen Bouin II, having the formaldehyde solution reduced to 5%, embedded in paraffin and serially sectioned at 15 to 18^4(3). The iron hematoxylin was found to be the most satisfactory stain with a very light counterstain of fast green.

Root tips of the

regenerants from cultured anthers/ovules were fixed in ethanol: acetic acid!chloroform (6:3:1), stained with acetoncarmine, and counts of chromosomes made using a light microscope.

Results and Discussion In the present studies anthers from unopened flower buds were excised and stages of the microsporogenesis were monitored in order to establish a relationship between the bud size and anther development stage.

In the buds of 2 to 2.5 cm size anthers were mostly

at uninucleate microspore stage and such anthers were explanted on different media recommended for anther culture, and experiments of temperature shock (37" , 35°, 30° and 25°C) for different durations, cold preconditioning of flower buds and excised anthers, and preconditioning and osmotic impregnation of 2,4-D into the anthers were performed.

The role of different amino acids, sucrose con-

centrations, and types and ratios of auxin/cytokinin was studied. However, in none of the experiments were microspore-derived embryos obtained.

Often, the swelling and enlargement of anthers was seen.

In liquid medium, with 35°C temperature shock treatment for one day followed by three days incubation at 30°C, anthers dehisced, but no further development of microspores into embryos or calli was seen.

Occasionally, globular structures and finger-like pro-

jections developed on the anthers which, after anatomical studies, were

found to be of anther wall origin.

In the entire period

of investigation, only ten plants were derived from the anthers. However, cytological analysis

revealed their diploid nature.

This indicated that these androgenic plants were derived through the callusing of filament, anther wall or tapetum.

The failure

in the haploid production via androgenesis in our case could be

337 attributed to the fact that no attention was given to the physiological stage of the donor plants, which in several cases has been demonstrated to play a vital role in pollen embryogenesis. In the studies with the induction of gynogenic haploids, within 25-28 days, one embryo emerged from the central part of each responsive ovule.

More than 70% of these embryos germinated into

normal plantlets within 3-4 weeks.

The sectioning of unpollinated

ovaries in a suspension of activated charcoal, the presence of high concentration of sucrose and the amino acid glutamine in the culture medium were found to be essential for the successful induction of gynogenesis in lilies.

In the present studies over

90 régénérants were produced through gynogenesis.

Anatomical study

of the cultured ovule confirmed the 'embryosac' origin of the régénérants .

Cytological studies of some of the régénérants showed

a mixture of haploid and diploid cells, revealing the spontaneous doubling of the régénérants.

To our knowledge, this is the first

report of the production of gynogenic doubled haploids from oriental lilies in the Western literature.

Acknowledgement The financial support and co-operation from Vletters Bros, and Den Haan (The Netherlands) during the course of present investigation is gratefully acknowledged.

References 1. Murashige, T., F. Skoog. 1962. Physiol. Plant _15, 473. 2. Keller, W.A., T. Rajhathy, J. Lacarpa. 1975. Can J. Genet. & Cytol. _17, 655. 3. Sass, J.E. 1958. Botanical Microtechnique, The Iowa State College Press, p.228.

339 INCREASING THE EFFICIENCY OF TRITI CALE ANTHER CULTURE

P. Ryöppy, J . Honkanen and P.M.A.

Tigerstedt

Department o f P l a n t B r e e d i n g , U n i v e r s i t y of H e l s i n k i , 00710 H e l s i n k i , F i n l a n d

Introduction E a r l y and hardy European and Canadian t r i t i c a l e m a t e r i a l s were grown as bulk p o p u l a t i o n s through ten generations i n F i n l a n d . The p o p u l a t i o n s have become w e l l adapted but they e x h i b i t wide v a r i a t i o n i n morphological and p h e n o l o g i c a l t r a i t s . To study t h i s v a r i a t i o n and to produce pure l i n e s f o r b r e e d i n g purposes an anther c u l t u r e technique was developed. The f i r s t

experiments

were promising (1) but there were l a r g e d i f f e r e n c e s between genotypes i n i n d u c t i o n of c a l l i

their

o r embryoids and a l s o v a r i a t i o n from y e a r to y e a r w i t h i n

the same genotype. Most o f the d i h a p l o i d l i n e s were a l s o too l a t e f o r the F i n n i s h c l i m a t e . To o b t a i n b e t t e r y i e l d s o f c a l l i , embryoids and p l a n t l e t s and t o enable the i n d u c t i o n o f e a r l i e r l i n e s pretreatment experiments were initiated.

M a t e r i a l and Methods For pretreatment experiments, three s t r a i n s were taken from the f i e l d m a t e r i a l : 0110 (bulk p o p u l a t i o n ) , 0130 and 0150 (two l i n e s s e l e c t e d from 0110 i n 1977). Stems o f the e a r l i e s t p l a n t s i n the f i e l d were cut about 20 cm below the ear when the p o l l e n was at the u n i n u c l e a t e s t a g e . The developmental stage o f the p o l l e n was checked by s t a i n i n g some anthers w i t h a c e t o - o r c e i n . Ears were i n s e r t e d i n the pretreatment medium and kept at +4°C f o r t h r e e , f i v e , seven o r ten days. Five d i f f e r e n t media were t e s t e d : El - d i s t i l l e d w a t e r ; E2 d i s t i l l e d water w i t h 2 mg/1 2,4-D and 0 . 5 mg/1 k i n e t i n ; E3 - E5 water was r e p l a c e d w i t h v a r i o u s c o n c e n t r a t i o n s

distilled

( 1 / l x , l / 1 0 x and l/100x)

of

Ng c u l t u r e medium w i t h o u t sugar but w i t h the same hormones as i n E2. As a c o n t r o l , anthers from 50 ears of each genotype were i n o c u l a t e d w i t h o u t any pretreatment. The c u l t u r e method f o l l o w s Chinese experiments w i t h Ng medium (2, 3). In the c a l l u s i n i t i a t i o n medium there are N, macro- and microminerals

Genetic Manipulation in Plant Breeding ©1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

340 and vitamins. Hormones are 2,4-D 2 mg/1 and k i n e t i n 0.5 mg/1. The sucrose concentration i s 5 - 8 %. In the d i f f e r e n t i a t i o n medium the hormone combination i s 0.2 mg/1 NAA and 1 mg/1 k i n e t i n and the sucrose concentration i s reduced to 2 - 3 %.

Results and Discussion In a l l experiments there was a p o s i t i v e c o r r e l a t i o n between c a l l u s production and the cold pretreatment time. For the s t r a i n s 0130 and 0150, the callus production i n i t i a l l y dropped but increased again after f i v e or seven days in the cold ( F i g . l . ) . Between media there were no s i g n i f i c a n t differences

(although

a low concentration of N g seemed to be a l i t t l e better than a high one) but differences were found between genotypes. Two of the s t r a i n s responded p o s i t i v e l y and one negatively to pretreatment media compared to the control (Fig.2.) D i f f e r e n t i a t i o n occurred only after short treatments, which may be due to pollen development in the cold. When taken from the f i e l d a l l pollen was at the uninucleate stage. Depending on the genotype, d i v i s i o n occurred after f i v e to

341

Fig. 2. The response of the genotypes to pretreatment media seven days and after ten days in the cold almost a l l the pollen was at the binucleate stage. One problem i s that a large proportion of the plantlets are albinos. By decreasing the sugar content from 3 to 2 % in the d i f f e r e n t i a t i o n medium and by lowering the growth temperature from 28°C to 24°C, one can obtain about 50 % green plants. Green plants adapt e a s i l y to s o i l and after colchicine treatment homozygous lines can be tested for agronomic characters.

References 1. Ryöppy-Ekbom, P. and T i g e r s t e d t , P.M.A. 1984 - In: Proceedings of the 10th Congress of the European Association for Research on Plant Breeding, EUCARPIA, Wageningen, the Netherlands, 19-24 June 1983. p. 345. 2. Chu Chih-ching, 1978. - In: Proceedings of Symposium on Plant Tissue Culture, May 25-30 1978, Peking, pp. 43 - 50. 3. Sun Jing-san, Zhu Z h i - q i n g , Wang J i n g - j u and Tigerstedt, P.M.A. 1980. Acta Botanica S i n i c a 22:27 - 31.

343 GENETIC GAIN FOR SOME AGRONOMICAL CHARACTERS BY DIHAPLOID BREEDING IN BARLEY

A. Sarrafi, R. Ecochard, C. Planchon, M. Ali-Sadiq Plant Breeding Department, Faculty of Agriculture, Toulouse University, Av. de Muret, 31076 Toulouse Cedex, France

145

Introduction

Several investigations have b e e n undertaken to produce dihaploids for barley breeding purposes. Among them, we can mentioned the most recent ones

: Callus

and suspension cell lines were derived from haploid barley embryos produced by the H. bulbosum method. The majority of regenerated plants were haploid (5). The percentage of embryos relates to cultivars of H. vulgare and H. bulbosum used in crossing programmes

(3). Comparison of the H. bulbosum method

(HBM)

and microspore culture (MC) w i t h the hap initiator technique (HIT) gave haploid production percentages as follows

: HBM 9.67 Z, MC 0.24 % and HIT 0.47 % (4).

Relationships between population size and chance of obtaining favorable genotypes in a barley breeding programme based on the use of dihaploids were established (1). Large differences were observed b e t w e e n dihaploid lines and some of them combined disease resistance and high productivity

(2).

The main purpose of this research is to study the genetic gain for some agronomical characters in dihaploid barley lines.

Material and Methods

Haploid plants were obtained by crossing selected F2 plants of "Robur x Platen" combination with H. bulbosum and Secale cereale. The percentage of haploid plants obtained by embryo culture in intergeneric crosses was

19.15 and two

times higher than in interspecific crosses. Dihaploids were produced by colchicine treatment. A total of 15 haploid lines were selected in field conditions. Dihaploids were compared with their parents in 1984 in a randomized blocks design with 4 replications, each 3 rows of 1.5 m. In 1985, twelve dihaploid lines selected through

15 were compared w i t h their parents in a replicated trail w i t h

3 replications, each 10 m2.

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

344 Results

Genetic gain estimated f o r three important characters

Table1Genetic Genotype

Platen DH - Hb

-

II

1

(cm)

1000 seeds weight

(g)

1984

1985

1984

90 53b

95 33 a

40 52 a

46.97 a

202 50 c

500 l b

102 25°

67 c

46b

52.30 b

147 0

a

4458a

40 66 a

49.41 a

152 95 a

4590a

39b

51.34 b

98 a

4700a

40 78a

49.58 a

172 15b

5078b

147 05 a

-

93 75b

116

101 17a

43

88

3

92 53b

It

4

99 o o c

II

5

75a

tl

6

93 oo b

103 17b

II

7

98 o o c

108 33b

50 3

50 a

89 c

52.04 b

46 16c

54.28 C

86

25 a

1

83

II

2

95 75b

II

3

94

It

4

87 oo a

It

5

100 53C

It

6

99

25C

II

7

95 oo b

It

8

75a

L S D 5 %

(g)

1985

2

-

yield

1984

II

DH - Sc

1.

Gain f o r some Agronomic Characters in Dihaploid Barley Lines

Plant height

Robur

is resumed in Table

84

97 0

a

102 83b

41 06 a 99

101

50 a

101 50 a

43

193 53°

4943b

43 72b

54.69°

195 20C

4683a

41 2 l a

59.09 b

181

33b

4745 a

213

58 d

5028b

226 88 d

54 82

44

105 oo b 115

67C

98 17a 104

33b

7.13

95b

-

40b

43 l l b

51.5

154

4b

44

03b

5.88

44

1985

-

170

38b

-

-

154 98 a

-

41 72a

50.55 b

192 53 c

4706a

46b

51.74 b

195

90 c

4676a

40 89 a

50.92 b

195 63C

4967b

42 89b

51.72 b

10b

4488a

1.83

3.01

43

169

19.90

298.65

Means with the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t DH- d i h a p l o i d l i n e , Hb- H. bulbosum, Se- Secale c e r e a l e

Results show that : 1. Some d i h a p l o i d l i n e s l i k e "DH-Hb-2" and "DH-Sc-1" are as short as "Robur" v a riety 2. The "DH-Hb-6" and "DH-Sc-2" l i n e s have b i g g e r seeds than those of

"Platen"

variety 3. Genetic gain f o r y i e l d i s observed in "DH-Sc-1" and "DH-Sc-2". These two l i n e s are more productive than "Robur" v a r i e t y .

345 The whole study shows that dihaploid line "DH-Sc-2" presents genetic gain for all characters and should be a promis sing line.

References

1. England, F.J.W. 1981. In : Barley Genetics (Proceedings of the Fourth International Barley Genetics Symposium). Edinburgh Univ. Press UK. pp 176-178. 2. Friedt, W. and B. Foroughi-Wehr. 1983. Field performance of androgenetic doubled haploid spring barley from F1 hybrid. Zeitschrift fur Pflanziichtung. 90. 177-184. 3. Pickering, R.A. 1984. Crossability relationships between certain species in the Hordeae. Barley Genetics Newsletter. 14-17. 4. Powell, W. and W. Wood. 1984. A n assessment of the hap initiator gene for haploid production in Hordeum vulgare. J. Agric. Sci. 103, 253-255. 5. Seguin-Swartz, G., L. Kott and K.J. Kasha. 1984. Development of haploid cell lines from immature barley embryos. Plant cell Rep. 3. 95-97.

347 EFFECT OF A GAMETOCIDE ON THE INDUCTION OF HAPLOIDS IN TRITICUM AESTIVUM

J. Schmid and E.R. Keller Department of Crop Science, Swiss Federal Institute of Technology (ETH), 8092 Zürich, Switzerland

Introduction

Genotype and environment have an important influence on the induction of androgenesis in wheat. We have at our disposal several wheat lines with a good androgenetic response. The main objective is to induce the ability to produce haploids by anther culture, independent of the genetic background of the breeding material. The genotype/environment interactions are numerous; we want to develop a method which utilizes all those factors which will have a positive influence on the induction of androgenesis. Out of a series of anther culture in wheat with an average of 6.7 embryos and 0.1 plant per 100 cultured anthers, one male sterile genotype was striking with 69 embryos and 5 plants per 100 anthers. Working with male sterile tobacco lines (1), the number of haploid plants was higher than with normal tobacco plants. One reason for this is the increase in certain types of microspores which seem to be more able to induce androgenesis. We therefore started to work with gametocides which could have the advantage of inducing male sterility and androgenesis on all of the interesting genotypes.

Results and Discussion

The following results form a part of preliminary studies which have not yet been completed. a) Variation of gametocide (CGA) concentrations in the potato-2-medium. Details of anther culture methods are described in (2). Best results were obtained with combinations of 2,4-D and CGA but without kinetin (Tab. 1). The induction of androgenesis, shown by the number of embryos formed, was high in the combination 1.5 mg/1 2,4-D and 1.0 mg/1 CGA. Treatments with 3.0 mg/1 2,4-D combined with several CGA concentrations resulted in an essentially lower percentage of embryos (18.1 %) as compared with 1.5 mg/1 2,4-D (52.7 %). The number of anthers

G e n e t i c Manipulation in Plant Breeding © 1986 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

348 cultured was low; in spite of this fact we observed a great variation among the petri dishes within the same treatments, indicating that many unknown interactions with the gametocide influence the success of anther culture.

Table 1.

Induction of Androgenesis by a Gametocide (CGA) Combined with 2,4-D in Addition to the Potato-2-medium with Two Spring Wheat Genotypes (Dadora and 83Z118.32). CGA concentration (mg/1) 0.5 0.75 1.0 1.5 2.0 3.0 control 1

Dadora

CN t-H oo

6

83Z118.32

m

1 :: No. anthers 2 •: No. embryos 3 :: 2/1 %

604 55 9.1

1 ;: No. anthers 2 :: No. embryos 3 :: 2/1 %

409 120 29.3

821 54 6.6 -

1462 643 10 0 0.7 0 220 266 120.9 -

-

206 63 30.6

258 0 0

1670 4 0.2

113 51 45.1

686 18 2.6

For example, the best petri dish in the treatment with 1.5 mg 2,4-D and 1.0 mg CGA formed 212 embryos from 46 cultured anthers; the poorest petri formed only 19 embryos from 62 cultured anthers. b) The gametocide was applied to the anther donor plants (run-off treatment, spike length: 2cm) in order to synchronize and optimize all factors leading to the induction of androgenesis and to obtain more information about the reaction of the gametocide. Because of the limited material in these preliminary studies, the small differences between the treatments are difficult to explain. Best results were obtained by applying the gametocide when the spike had reached 2 cm in length. The concentration 0.1 mg CGA/ plant resulted in a good embryo as well as in good plant production (Tab. 2). The gametocide has a positive effect on the precondition of the donor plant and thus on the induction of androgenesis.

Table 2. Induction of Androgenesis by the Application of a Gametocide (CGA) to the Anther Donor Plant of the Spring Wheat Genotype 83Z118.32. CGA (mg/plant)

No. anthers

No. embryos

No. embryos ^ No. anthers 0

No. plants

No. plants y No. anthers

0.1 1.0 2.0 5.0

637 325 458 238

193 56 143 70

30.3 17.2 31.2 24.7

44 12 13 18

6.9 3.7 2.8 6.4

Control

686

18

2.6

3

0.4

c) A cytogenetical analysis was made to determine the effect of the gametocide on the possible change in the type of microspores. The aim of this study was to

349

find out if a correlation exists between the p-pollen (with high androgenetical potential) and the induction of androgenesis..The addition of CGA to the medium did not lead to a higher number of p-pollen, but only to the expected increase in t-pollen (dead pollen). In the trial with the application of CGA to the donor plants, we did not observe a positive correlation between p-pollen and the number of embryos formed. Further experiments are needed to determine whether the gametocide has an effect on the type of microspore which could cause an increase in the induction of androgenesis. d) The gametocide induces male sterility and may have a positive influence on the induction of androgenesis. In addition, it has an effect on female development which could be utilized to induce gynogenesis. We looked for a gametocide treatment which would result in the induction of haploids through androgenesis and gynogenesis simultaneously; preliminary studies are under way. Haploids from male and female parts of the same flower are of interest to wheat breeders.

Conclusions

A gametocide added to the potato-2-medium can lead to a higher number of embryos as compared with the control (2,4-D, kinetin, without CGA). CGA combined with low concentrations of 2,4-D (1.5 mg/1) showed the best effect. Direct application of the gametocide to the anther donor plants resulted in good haploid induction, possibly due to a preconditioning effect.

Acknowledgements CIBA GEIGY Limited, Basle (Switzerland) made the gametocide available for our experiments. We wish to thank Dr. E. Fankhauser for his advice concerning the application of the gametocide.

References

1. Heberle-Bors, E. 1982. Planta 156, 396 2. Schmid, J., H. Winzeler, P.M. Fried and G. Kleijer. 1985. Mitteilungen fUr die Schweiz. Landw. 8^, 187.

351

THE INDUCTION OF HAPLOIDS OF SUGARBEET (BETA VULGARIS L.) USING ANTHER AND FREE POLLEN CULTURE OR OVULE AND OVARY CULTURE.

G.J. Speckmann jr. Nickerson-Zwaanesse bv, Stompwijk, The Netherlands J.P.C. Van Geyt, M. Jacobs Instituut voor Moleculaire Biologie, Vrije Universiteit Brussel, B1640 St-Genesius Rode, Belgium

Introduction Haploid

and

diploid

plants

and

cultures

can

have

important

applications in sugarbeet breeding. Haploids are useful for the selection of induced mutations. Dihaploids can be used for the homogenisation understanding

of

the

breeding

material

and

of the genetical background

for

the

better

of single as well as

polygenic traits. Results Androgenesis. Nuclear and cell divisions could be induced in anthers with microspores

in

late

meiosis

until

early

uninucleate

state.

No

correlation could be found with regard to floral characteristics and the stage of development of the microspores. The reaction was highly

dependant

propagation response.

and High

on

the

genotype

vernalisation sucrose

did

used. not

of

the

cells

of

the

division

in

the

microspores.

anther After

vitro

vegetative

the

androgenic

affect

concentrations

reaction

In

(15 wall

two

to

%)

inhibited

the

and

favoured

cell

three

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

weeks,

the

352

microcalli had to be isolated out and subcultered on media with lower sucrose content

(5 %) and charcoal

(0.5 %). After spraying

the inflorescence with 2-chloroethyl phosphonic acid, proembryoids were

detected

in the

anther

after

approximately

three weeks of

culture. No plantlets could be regenerated. In

order

induction

to

induce

period

of

divisions

at

least

in

four

free

days

pollen

was

culture,

necessary.

an

Nuclear

divisions could also be induced in culture media supplemented with an amino acid mixture derived from an analysis of anthers in the right stage for androgenesis (1). Gynogenesis. Haploid culture

plants

could

be

regenerated

from

ovule

(3). The reaction was highly dependant on the

and

ovary

genotype.

The number of ovules or ovaries regenerating at least one plantlet ranged

between

0

and

2.2

%.

The

plantlets

originated

from

embryogenic structures. The development of callus originating from the mothertissue hampered the development of the plantlets. This callusformation could be inhibited by adding 0.5 % charcoal to the medium

(D'Halluin,

pers.

comm.).

This

treatment

increased

the

plantlet induction to a maximum of 6.1 %. The plantlets seldomly formed a good root system. Therefore

the plants were propagated

vegetatively as described before (2). The ploidy various

and origin of the regenerants was checked by

methods.

The

origin

was

determined

by

comparing

heterozygous isozyme patterns with the patterns of the regenerated plants

(4).

All

plantlets

proved

to

be

homozygous.

Chromosome

counts in root tips and chloroplast counts in the guard cells of the

stomata

of

the

leaf

epidermis

showed

a haploid

chromosome

number. The corresponding root tips however were mostly diploid or

353 tetraploid. Chimaeric shoot or root tips were seldomly found. In some

cases

different indication

different

chromosome that

regenerants numbers

in

polyploidisation

of

the

their occurred

same

clone

roottips. after

This

showed is

an

the process

of

gynogenesis.

Acknowledgements We want to thank mr. Dua for excellent technical assistance and Miss. D'Halluin

for helpful

discussions.

This

research

was

supported by the IWONL, Belgium.

References 1.

Van Geyt, J.P.C., D'Halluin, Pfanzenzucht. (in press).

2. Van Geyt, J.P.C. 4:66-69. 3.

K.

and Jacobs, M.

and

Jacobs,

M.

1985.

Z.

(1985). Plant Cell Reports

Van Geyt, J.P.C., Speckmann, G.J. jr., D'Halluin, K. Jacobs, M. (1985). Theor. Appl. Genet, (in preparation).

and

4. Van Geyt, J.P.C., and Smed, M. 1984. Z. Pfanzenzucht.: 92, 295308.

355 FROST TOLERANT PLANTS OBTAINED FROM PROLINE ACCUMULATING CELL LINES

A.C. van Swaaij, E. Jacobsen Department of Genetics, University of Groningen, Kerklaan 30, 9 751 NN Haren, The Netherlands

Introduction Within the genus Solanum large differences exist in frost tolerance between species (1). Efforts to transfer frost tolerance of wild species via sexual crosses to the frost sensitive cultivars of S. tuberosum have resulted in hybrids with increased frost tolerance, but the further selection of frost tolerant cultivars has been rare (2) .

Proline has often been suggested to be protective in plant cells during environmental stress (3). In a number of potato clones frost tolerance could be increased by inducing a high proline content of the leaves (4). Here we describe a method to obtain frost tolerant S. tuberosum plants by the regeneration from cell lines which were selected for proline accumulation. Additionally, methods are described to measure the frost tolerance in plants as well as in cell cultures.

Methods And Results Proline accumulating cell lines were selected from a cell suspen2 sion of a dihaploid potato clone H 578, after plating on media with hydroxyproline (Hyp, 5-10 mM) (van Swaaij, in prep.). From 67 selected colonies 6 0 showed a proline content varying between 2 and 25 times that of the wild type when grown away from Hyp for 1 month. One of these proline accumulating lines was further characterized. Tab.l shows that Hyp-resistance and proline content in H4a decrease in time when callus is grown away from Hyp. However, callus growing for 7 months without Hyp and also, callus derived from regenerated plants of H4a (like LC1H4a/lO) still show higher Hyp-resistance and

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin New York - Printed in Germany

356 T a b l e 1: Some C h a r a c t e r i s t i c s o f C e l l L i n e H4a, C a l l u s D e r i v e d from a R e g e n e r a t e d P l a n t (LC H4a/10) and o f W i l d t y p e C a l l u s . 1, 3, 7 = Months o f Growth on N o n - s e l e c t i v e Medium. H y p - r e s i s t a n c e = R e l a t i v e Growth on 5 mM Hyp Compared t o Growth on H y p - f r e e Medium. - = n o t determined. H4a 1 Proline

(umol/g. FW)

Amino a c i d s

(%)

probably higher p r o l i n e lected

trait

3

2.2

(umol/g. FW)

Hyp-resistance

LC^a/lO

Wild

type

7 0.9

6.0

7.1

74

20

content

0.3

0 . 16

0 . 25

2.0 27

0

than t h e w i l d t y p e .

appears t o be p a r t l y

stable

Thus the

se-

and i s p a s s e d through

re-

generation . Apart

from p r o l i n e

acids,

there

with a r e l a t i v e

t i v e decrease in glu, Frost tolerance of of

is

an o v e r a l l

larger asp and

cell

increase of

increase

in pro,

l i n e H4a was d e t e r m i n e d .

regenerated

L e a v e s were f r o z e n i n t e s t

of

the e l e c t r o l y t e

s u s p e n s i o n s were f i r s t c o l l e c t e d t r e a t e d i n t h e same way as the

leaves.

After

resulting

the e l e c t r o l y t e s

i n an e l e c t r o l y t e

of

the l e a v e s

filters

(4).

in both

Cell

and than

t h e s o l u t i o n o b t a i n e d by

50% o f

The

temperature

t h a t o b t a i n e d by

f r e e z i n g i n l i q u i d n i t r o g e n was used as measure f o r f r o s t (FKT)

as

thawing damage was d e -

in d i s t i l l e d water.

leakage of

to

a t 0°C dam-

l e a k a g e from the c e l l s on g l a s s f i b e r

t e r m i n e d by measuring t h e c o n d u c t i v i t y exosmosis of

of

plants

tubes

and a f t e r thawing o v e r n i g h t

age was d e t e r m i n e d by measuring the c o n d u c t i v i t y an i n d i c a t i o n

amino

tyr.

c u l t u r e s as w e l l as o f

predetermined temperatures

free

and a l a and a r e l a -

tolerance

cases.

T a b l e 2: F r o s t T o l e r a n c e and P r o l i n e Content i n C e l l Suspensions and L e a v e s o f R e g e n e r a t e d P l a n t s , t = 0 and t = 3 R e f e r t o the Months o f Growth on N o n - s e l e c t i v e Medium. Proline cell

suspension

Regenerated plants

FKT

(umol/g.FW)

(°C)

H4a

(t=0)

1.6

-5.6

H4a

(t=3)

0.6

-4.7

Wild type

0.2

-2.5

H4a

0.3 to

15.5

Wild type

0.2 t o

0.8

-3.2 to

-4.7

-2.5

-3.3

to

357 Frost tolerance in the regenerated plants of H4a varied considerably but was in most somaclones substantially higher than in the wild type (Tab.2). This increase was accompanied by a high leaf proline content, however, proline content and frost tolerance were not always correlated, suggesting that other factors, possibly the total free amino acid content, could play an important role too. Frost tolerance of cell suspensions of line H4a varied with the time of growth on non-selective medium, but was always higher than that of the wild type cell suspension. Thus frost tolerance of H4a seems to be determined by cellular factors.

References 1. Chen, H.H. and P.H. Li. 1980. Plant Physiol. 6J5, 1146-1148. 2. Estrada, R.N. 1982. In: Plant Cold Hardiness and Freezing Stress (P.H. Li and A. Sakai, eds.). Ac.Press, New York. 3. Aspinall, D. and L.G. Paleg. 1981. In: The Physiology and Biochemistry of Drought Tolerance in Plants. (L.G. Paleg and D. Aspinall, eds.). Ac.Press, Sydney. 4. Swaaij, A.C. van, E. Jacobsen and W.J. Feenstra. 19 85. Physiol. Plant. 64, 230-232.

359 SOMATIC CELL GENETICS OF POTATO I. USE OF MONOHAPLOIDS B.A. Uijtewaal, W.M. Mattheij Dept. of Plant Breeding, Agricultural University Lawickse Allee 166, NL-6709 DB Wageningen, The Netherlands

In a joined project of the Department of Plant Breeding tural University, Wageningen), the Foundation ITAL

(Agricul-

(Wageningen)

and the Department of Genetics of the University of Groningen, financed by the Foundation for Technical Sciences

(STW), research

is going on to construct a gene map of potato to pave the way for genetic engineering of that crop. Via prickle-pollination

(pseudogamy) and anther culture, more than

400 monohaploid lines of different Solanum species and interspecific hybrids have been produced. These single genome lines (12 chromosomes) are screened for stability of ploidy level after several cycles of shoot multiplication and growth in vitro. From the first 200 monohaploids ten different lines from four different genetic sources could be selected. These lines were then screened for the ability to produce callus and to regenerate plants after protoplast isolation and fusion. At this stage of research we are able to produce calli out of protoplasts from most of the genotypes 2+

and regenerate plants from some of them. By way of PEG (Ca

, high

pH) method, fusion frequencies of more than 5 % have been realized. Fusion products are being selected for hybrid vigour. Another aim is to produce from each monohaploid a series of homozygous clones with the ploidy levels x, 2x, 3x and 4x in order to study gene dosage effects. At this moment x-2x-4x series of six monohaploids have been obtained. They are being investigated for vigour and fertility.

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

361 FACTORS AFFECTING CALLUS AND PLANT PRODUCTION IN ANTHER

CULTUSES

OF TOMATO

N.A. Zagorska, M.D. Abadjieva, H.K. Oanh Institute of Genetics, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria

Introduction

The interests of a large number of research workers nowadays are directed towards the establishing of methods for obtaining h a p loid plants and increasing the quantity of haploids by using different treatments. The aim of this study w a s t.o identify some factors w h i c h influenced the induction of callus and plantlet formation in anther cultures of tomato. Twelve different variants of pretreatment /temperature and radiation/were tested on flower buds and anthers of 2 genotypes. The anthers were cultivated on Murashige and Skoog /MS/ nutrient medium /1/+2ip or zeatin after a method previously described /2. 3/

Results

The results obtained showed that, the influence of gamma r a d i a t i o n and temperature pretreatment on callus induction was unimportant while both of them affected shoot formation and plant r e g e n e r a tion. The best results for organogenesis and plant regeneration

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

362 for the male

sterile variety Roma, homozygous for the recessive

gene ms 1 0 ^ / R o m a msms/were observed with a pretreatment of 4°C for 48 hours/22,6% of the anthers produced plantlets/, and with 35°C for 4 days - 21,„4% /Table 1./.

Table 1 The Influence of Temperature on Callus and Organogenesis Induction in Anther Cultures of Tomato Pretreatment

Nutrient Medium MS +

Anthers w/th Callus - % Roma Msms msms

Anthers w/th Shoots - % Roma Msms msms

Control

2ip zeatin

78,0 70,5

1.6,5 14,2

1,9 2,%

4°C 48 hours

2ip zeatin

88,2 68,1

3,0 1,5 2,8 2,0

25,5 26,2

22,,6 10,9 8,6 1'7,4 16,6 7,2

0,9 0

47,7 39,5 51,,2 51 „0 21,0 17„2

4°C

6 days

2ip zeatin

7°C

7 days

2ip zeatin

36,7 55,9 68,0 51,6

10°C 9 days

2ip zeatin

72,.3 56,4

69,1 39,8

14,3 8,0

4,5 2,0

35°C 4 days

2ip zeatin

82,9 66 „4

42,2 44,8

21,4 4,4

0,1 1,7

In Table 1 the data of

three years' investigations /1982-1984/

were presented. The anthers were collected from the donor plants grown in the field and greenhouse conditions. It was necessary to notice that the anthers collected from the field conditions had given higher percentage of organogenesis, than those cultivated in greenhouse. 32,2% of the anthers from the field pretreated with 35°C for 4 days and 29,3% of anthers pretreated with 4°C for 48 hours had given shoots,, while those of greenhouse conditions with the same pretreatment have respectively 11,4% and 5,6% shoots. The role of the genotype for the organogenesis was also of great importance.In the same growing conditions the percentage of

the anthers from the fertile variety Roma, heterozygous for •7 IT

the recessive gene ms 10

/Roma Msms/ that gave callus was the

highest - 69,1% at 10°C for 9 days and 4,5% organogenesis, at 4°C for 48 hours the percentage was respectively 51,1% and 3,0%.

363 Por the variety Roma msms with the same pretreatment the results obtained were higher - 88,2% and 22,:6%.

The phytohormonal compo-

sition of the nutrient medium influenced organogenesis and regeration. In all variants of pretreatment the medium with 1 mg/1 2ip gave better results, than the medium with zeatin /Table 1/. In respect of the influence of gamma radiation on callus and shoot formation the results showed that, the best, pretreatment was when using 400 rads only or in combination with low temperature. In our

investigation 400 plantlets were regenerated. They

showed great variability of morphological properties /height of the stem, size and shape of leaves and flowers/ and continuation of the vegetation. The cytological analyses of the regenerants' root meristem demonstrated great variability of chromosome number. Most of

the regenerants were mixoploid, but there were hap-

loids, diploids, triploids and tetraploids also. The results obtained showed that the genotype, the growing conditions of donor plants, the phytohormonal composition of nutrient medium and the pretreatment with low temperature and gamma radiation affected the frequency of organogenesis and regeneration in anther cultures of tomato.

References

1. Murashige,T., F. Skoog. 1962. Physiol. Plant.

473.

2. Zagorska, N.A., M.D. Abadjieva, H.A. Georgiev. 1982. Compt. Rend.Acad.Bulg.Sci. 3£, 1, 97. 3. Zagorska, N.A., M.D. Abadjieva, H.A. Georgiev, R.A. Georgieva. 1982. Proc. 5th Int.Cong.Plant Tissue and Celi Culture.Tokyo. 539.

365 PART 4 IN-VITRO

PROPAGATION

Bornman, C.H., R. Vankova and L.O. Björn Role of m e t h o d o l o g y in f a c i l i a t i n g a p p l i c a t i o n of tissue culture t e c h n i q u e s

367

Preil, W. In vitro p r o p a g a t i o n and breeding of o r n a m e n t a l a d v a n t a g e and d i s a d v a n t a g e of v a r i a b i l i t y

377

plants:

Sink, K.C., L.W. Handley, R.P. Niedz and P.P. Moore P r o t o p l a s t culture and use of r e g e n e r a t i o n a t t r i b u t e s select s o m a t i c hybrid tomato plants Z e n k t e l e r , M., and A. SI u s a r k i e w i c z - J a r z i n a Sexual r e p r o d u c t i o n in plants by applying the m e t h o d test tube f e r t i l i z a t i o n of ovules Becker, U., and G. Reuther C y t o g e n e t i c studies in callus

cultures of A s p a r a g u s

to

of

off.

405

415

425

M a l e p s z y , S., A. N a d o l s k a - O r c z y k and W. Orczyk Systems for R e g e n e r a t i o n of Cucumis sativus plants in vitro

429

James, D.J., A.J. Passey, K.A.D. M a c K e n z i e , O.P. Jones and E.C. M e n h i n i c k R e g e n e r a t i o n of t e m p e r a t e fruit t r e e s in vitro via o r g a n o genesis and e m b r y o g e n e s i s

433

F o r d - L l o y d , B.V., and S. Bhat P r o b l e m s and p r o s p e c t s for the use of p r o t o p l a s t s breeding

437

in beet

Steffen, A., T. Eriksson and 0. Schieder Shoot r e d i f f e r e n t i a t i o n of A g r o b a c t e r i u m t r a n s f o r m e d protopl.asts and plant tissue - with c o n v e n t i o n a l m e t h o d s not a c h i e v a b l e

441

J a c o b s e n , H.-J., and W. Kysely I n d u c t i o n of in v i t r o - r e g e n e r a t i o n via s o m a t i c e m b r y o g e n e s i s in pea (Pisum sativum) and bean (Phaseolus vulgaris)

445

A n d e r s e n , J.M., F. Okkels, P. Ulskov and J. M a r c u s s e n E n d o g e n o u s c y t o k i n i n s during e m b r y o g e n e s i s in a carrot cell s u s p e n s i o n

449

Bhat, S., B.V. F o r d - L l o y d and J.A. Callow Tissue and p r o t o p l a s t c u l t u r e in c u l t i v a t e d

453

beets

Eriksen, F.D., C.J. Jensen and P. O l e s e n P r o t o p l a s t f o r m a t i o n in c e r e a l s - an a s s e s s m e n t

457

F i l i p p o n e , E., and T. Cardi E x p l o i t a t i o n for breeding of in vitro c u l t u r e of pea explants

461

366 G e y t , J . P . C . v a n , K. C l a e s , A . H . S . S e n a n a y a k e and M.Jacobs S o m e a s p e c t s of t h e in v i t r o c u l t u r e of t h e b e e t ( B e t a v u l g a r i s L.)

465

H y r k a s , K . , M. K i v i n e n a n d P . M . A . T i g e r s t e d t I n t e r s p e c i f i c h y b r i d i z a t i o n of r e d c l o v e r ( T r i f o l i u m p r a t e n s e L.) w i t h a l s i k e c l o v e r ( T r i f o l i u m h y b r i d u m L.) u s i n g in v i t r o e m b r y o r e s c u e

469

J e n s e n , C . J . , A. B u c h t e r - L a r s e n , D. C a s s , E . C . T h o r n , K. E n g e l l a n d P. O l e s e n P o l l e n a n d o v u l e c u l t u r e s of b a r l e y to i s o l a t e , m a n i p u l a t e a n d t r a n s f e r s p e r m c e l l s in in v i t r o f e r t i l i z a t i o n

473

J e n s e n , C.J., and E.C. Thorn S t r a t e g i e s in h i g h f r e q u e n c y r e g e n e r a t i o n f r o m h a p l o i d c e l l a n d t i s s u e c u l t u r e s of b a r l e y

477 diploid

Linacero, R., and A.M. Vazquez S o m a c l o n a l v a r i a t i o n in p l a n t s r e g e n e r a t e d c a l l u s e s in r y e ( S e c a l e c e r e a l e L.)

from

L u h r s , R . , a n d H. L o r z Somatic e m b r y o g e n e s i s , cell and p r o t o p l a s t H o r d e u m v u l g a r e L. ( b a r l e y )

culture

and 479

embryo 483

M a r i n o , G. I s o l a t i o n a n d c u l t u r e of p r o t o p l a s t s f r o m c a l l u s sus p e n s i o n - c u I t u r e d cells of Prunus c e r a s u s and Actinidia chinensis

of 487

and

M o r e n o , V . , L. Z u b e l d i a , B. G a r c i a - S o g o , F. N u e z a n d L . A. R o i g S o m a t i c e m b r y o g e n e s i s in p r o t o p l a s t - d e r i v e d c e l l s o f C u c u m i s m e l o L.

491

R u i z , M . L . , M . I . P e l a e z , J. R u e d a , F . J . E s p i n o a n d A.M. Vazquez A c o m p a r i t i v e s t u d y of c a l l u s f o r m a t i o n a n d p l a n t r e g e n e r a t i o n from d i f f e r e n t e x p l a n t s of P h a s e o l u s v u l g a r i s and Ph. c o c c i n e u s

495

S t o l a r z , A . , a n d H. L o r z Somatic e m b r y o g e n e s i s , cell and p r o t o p l a s t t r i t i c a l e (x T r i t i c o s e c a l e W i t t m a c k )

499 culture

Z i m n y , J., a n d H. L o r z S o m a t i c e m b r y o g e n e s i s and plant r e g e n e r a t i o n t e m a t i c t i s s u e of S e c a l e c e r e a l e (rye) Zimny- J., and J.J. R y b c z y n s k i S o m a t i c e m b r y o g e n e s i s of t r i t i c a l e

of 503

from

meris507

367 ROLE OF METHODOLOGY

IN FACILITATING APPLICATION OF TISSUE CULTURE TECHNIQUES

12 • 3 2 Chris H. Bornman ' , Radomira Vankova ' and Lars 01 of Björn •'•Cell and Tissue Culture, Hilleshög Research AB, Box 302, S-261 23 Landskrona, Sweden ^Department of Plant Physiology, University of Lund, Box 7007, S-220 07 Lund, Sweden 3 Institute of Experimental Botany, Czechoslovak Academy of Science, Ke dvoru 15, 166 30 Prague 6, Czechoslovakia

Abstract The use of phytological methods for the selective elimination of chloroplasts as well as their application to the removal of the radish (Raphanus sativus) chloroplast genome from male sterile oilseed rape (Brassica napus) is described. This example of controlled cybridization is used to support the argument that in vitro culture when used in conjunction with basic scientific methods, is expected to increase the opportunities for genetic manipulation in plant breeding. Introduction Plant cell and tissue culture is assuming ever-increasing attention as a potential biological tool in contemporary plant breeding.

However, with few

exceptions such as haploidization via anther and ovule culture, major difficulties are often encountered in the adaptation of in vitro culture techniques to practical plant breeding. Examples include the failure hitherto of somatic hybridization, the inability to fully utilize somatic embryogenesis because of embryoid incompetence, and the unavailability of useful engineered genes in order to take advantage of transformation systems such as electroporation.

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y

368 It seems appropriate to emphasize that plant cell and tissue culture is a tool, not a science.

Its rational application is dependent upon our understanding

of the complexity of cell, tissue and whole plant biology.

Therefore, the

imaginative combination of biochemical, physiological, biophysical, immunocytochemical and other methods with in vitro culture is expected to extend opportunities for genetic manipulation in plant breeding in future. This presentation deals generally with cybridization and specifically with the elimination and substitution of the chloroplasts by either photobleaching or sensitization, with or without high intensity irradiation.

The

problem chosen was the chlorophyll defect displayed by male sterile Brassica napus plants when grown at low temperature.

The aim was to determine whether

available physiological methodology could be used in conjunction with existing in vitro culture technology so as to reduce the complexity of the subsequent selection procedures. Construction of cms-cybrid Brassica napus Pelletier et al. (1983) demonstrated that the loss of physiological vigour resulting from the transfer to Brassica napus (oilseed rape) of the male sterility conferred by the cytoplasm of Raphanus sativus (radish) (Bannerot 1974, 1975) could be corrected by fusion of protoplasts of normal (male fertile, mf) and cytoplasmically male sterile (cms) oilseed rape, followed by extensive selection.

While observing post-fusion site specific recombination of mitochon-

drial genomes Pelletier et al. (1985) did not find combination of the chloroplast genomes; the elimination of either the one or the other was attained during the first few mitoses.

We (Carin Jarl, unpublished results) have found over

60% of the plants regenerated from the one-to-one fusion products of male-fertile and male-sterile oilseed rape protoplasts to be amphitetraploid, necessitating a complex programme of backcrossing and selection to recover amphidiploid,

369 male-sterile plants that display normal photosynthetic behaviour under conditions of low temperature. The selection of hybrids or cybrids from a heterogeneous population of cells or microcalli following somatic fusion is usually based on mechanical or complementation selection.

Complementation selection frequently involves

chlorophyll deficient or non-allelic mutants and has largely been confined to Datura spp., Nicotiana spp. and Petunia spp.

Ideally, the fusion of a single,

selected evacuolated protoplast or nucleoplast with a single, selected enucleated protplast (cytoplast) would eliminate the problem of post-fusion selection altogether.

Therefore, the techniques of Koop et al. (1983, 1985), and Spangenberg

et al. (1986) who worked with Brassica napus, could come to play an important role in test tube plant breeding. The two essential components in the synthesis of a cms oilseed rape cybrid are (1) a Brassica napus cytoplast containing Raphanus sativus cytoplasm from which the radish chloroplasts have been eliminated and (2) a normal Brassica napus protoplast. Elimination of chloroplasts From a practical point of view there are two ways by which chloroplasts can be eliminated: (1) inhibition of plastid replication by ultraviolet radiation, high temperature, application of antibiotics such as streptomycin and nalidixic acid (Schiff et al., 1961, Ebringer 1972), and (2) sensitization and destruction (Marschner 1964, 1965).

Plastids can be destroyed directly by very intense

red light, by making them more susceptible to light by pretreatments which cause a pathologic accumulation of protochlorophyllide (for example, 5-aminolevulinic acid), or by inhibiting carotenoid systhesis. Enrichment of cytoplast and nucleoplast fractions Density gradient centrifugation (Lorz et al. 1981, Griesbach and Sink 1983) can be used to provide enriched fractions of cytoplasts (enucleated

370 protoplasts of varying diameter) and nucleoplasts (evacuolated protoplasts ca 10-20 pm in diameter).

Removal or inactivation of the nucleus using cyto-

chalasin B or X-irradiation, respectively, are not recommended. Fusion Fusion is carried out by using either polyethylene glycol-induced or electric field-induced treatment.

However, in order to take advantage of

Koop's (Koop et al. 1983) method namely that of fusion of two single selected protoplasts, only electrical field fusion can be employed.

A recent study

Hahn-Hagerdal et al. 1986) in which the effects on membrane properties of polyethylene glycol- and electric field treated-oilseed rape protoplasts were compared, showed that whereas both treatments caused the same apparent changes in surface hydrophobicity and destabilization of membrane components, the electric field-treated protoplasts resulted in considerably less leakage of intracellular components, probably explaining the increased viability of such protoplasts as compared with chemical fusogens. Analysis of cybrid plants In order to confirm the nature of the putative cybrid plants a comparative analysis of the regenerated cybrid plants, male-sterile and male-fertile oilseed rape parental plants and radish cms-donor plants should be carried out.

Cytological (chromosome number, karyotype), morphological (flower and

gross plant morphology), biochemical (restriction analysis of chloroplast and mitochondrial DNA, isozymes, fraction I protein) and physiological

(variable

chlorophyll fluorescence) methods can be used. Procedure Two procedures, one actual (A) and one potential (B), for the establishment of cms-cybrid Brassica napus are summarized below and a few alternative combinations (C) are also indicated.

371 A.

Fusion of chloroplast-free cytoplasts of a cms Brassica napus line with nucleoplasts of a normal regenerative Brassica line. 1.

Germinate surface-sterilized seeds of the cms line on an agarified medium containing macro- and micronutrient elements of Murashige and Skoog (1962), 3% sucrose and 100 pg ml" 1 SAN 9789 (norfluorazon, filter-sterilized).

Grow for 2-3 weeks in constant light, photon

fluence rate ca 150 (jmol m " 2 s " 1 , wavelength 670-680 nm, 22 + 2°C. Grow seedlings of the normal line under similar conditions, omitting SAN 9789 and reducing sucrose to 1%. SAN 9789 results in accumulation of phytoene, leading to the inhibition of carotenoid synthesis, to the excitation of chlorophyll to the triplet state as well as to the oxygenation of chlorophyll to chlorophyll peroxide. 2.

Isolate protoplasts from the hypocotyls of photobleached SAN-treated seedlings.

Enrich the cytoplast fraction by subjecting the protoplasts

(5 x 10"* per 5 ml tube)to an iso-osmotic three-step (Lorz et al. 1987, but using 50, 30 and 20% Percoll) discontinuous density gradient centrifugation (35000 g, 40 min).

Withdraw the cytoplast band with

a hypodermic syringe by inserting the needle into the tube just below the band; wash twice to remove Percoll. Isolate protoplasts from either petioles or mesophyll of normal, mf-seedlings and enrich the nucleoplast fraction as above, or by subjecting the protoplasts (10® per 5 ml tube) to continuous density gradient centrifugation (150000 g, 1 h) according to Griesbach and Sink (1983).

Withdraw the nucleoplasts with a hypodermic syringe;

wash twice to remove Percoll. 3.

Carry out bulk one-to-one fusion of chloroplast-free, cms-Brassica napus hypocotyl cytoplasts with chloroplast-containing, mf-Brassica

372 napus petiole or mesophyll nucleoplasts using either polyethylene glycol or electric field treatments (Hahn-Hagerdal et al. 1986). Transfer all the material subjected to fusion treatment to 0.5 M sucrose and layer over 0.9 M sucrose.

Centrifuge at 100 g, 5 min.

Fused cytoplasts and nucleoplasts tend to collect at the 0.9 M interface, non-fused and fused cytoplasts float, and non-fused and fused nucleoplasts sediment.

Culture fused cells in liquid medium

and plate in nutrient agarose after ca two weeks. 4.

Vernalize regenerated plantlets, 45-60 days, 4-6°C; check for absence of chlorosis, but also use other parameters to verify nature of cybridization.

B.

Fusion of two single selected protoplasts following laser microbeam elimination of radish chloroplasts in a cms Brassica napus cytoplast.

(See Koop

et al. 1983 for detailed description of protoplast selection and fusion procedures.) 1.

Prepare cytoplasts and nucleoplasts as described under Al, 2 omitting SAN treatment and using petiole protoplasts of the cms Brassica napus line and mesophyll protoplasts of the normal line.

2.

Using a micromanipulator, introduce a single cms petiole cytoplast into a 0.1 pi droplet of fusion medium.

Focus the beam of a helium-

neon laser (4-6 mW) mounted on an inverted microscope to a 5-8 pm spot.

Because light of ca 634 nm is maximally absorbed by proto-

chlorophyllide, the He-Ne laser which emits at 632.8 nm is ideal. We have found that addition of 5 mM 5-aminolevulinic acid to the enzyme incubation medium (overnight incubation) results in a large accumulation of protochlorophyl1ide.

Both pigments and plastid

components are then readily destroyed by the laser's very intense red light.

373 3.

Following irradiation of the cytoplast, introduce a single mesophyll protoplast or nucleoplast into the same microdrop.

Bring the two

protoplasts into focus and lower two electrodes mounted on the condenser of the microscope into the droplet.

Align the protoplasts

via dielectrophoresis by applying an AC-field. DC-pulse to induce fusion.

Apply a single

Transfer the fused protoplasts to micro-

drops consisting of freshly prepared culture medium.

(Thus far we

have concentrated on the technique, which requires patience and skill, and have not attempted to regenerate callus from the fused protoplasts.

However, the method is sound in principle and may

possibly also be used to selectively alter mitochondria by making use of a blue-green argon laser.). C.

Alternative approaches in cybrid formation As was noted earlier, fusion of cms Brassica napus protoplasts with mf Brassica napus protoplasts tends to result in synkaryony.

The methods

indicated under A and B above are based on the fusion of a cms Brassica napus cytoplast with a mf Brassica napus nucleoplast.

However, since the

napus nucleus is common to both, cms Brassica napus protoplasts can also be fused with mf cytoplasts. As regards the elimination of chloroplasts following the pathologic accumulation of protochlorophyl1ide, the most practical sequence is: tissue irradiation

protoplast isolation

fusion ->• regeneration.

However, reversing protoplast isolation and irradiation in this sequence has also been found to work, although the regeneration frequency is greatly reduced presumably as a result of damage to the protoplasts as a result of direct irradiation.

When irradiation follows after fusion, the

frequency of regeneration is further reduced.

374

References

Bannerot, H., Boulidar, L., Cauderon, Y., and Tempe, J.

1974.

Transfer of

cytoplasmic male sterility from Raphanus sativus to Brassica oleracea. Proc. Eucarpia Meeting Cruciferae, 52-54.

Bannerot, H., Boulidard, L., and Chupeau, Y. met with the radish cytoplasm.

Ebringer, L.

1972.

1977.

Unexpected difficulties

Eucarpia Cruciferae Newsletter, 2-16.

Are plastids derived from prokaryotic micro-organisms?

Action of antibiotics on chloroplasts of Euglena gracilis.

J. Gen. Microbiol.

71:35-52.

Griesbach, R. J., and Sink, K. C.

1983.

Evacuolation of mesophyll protoplasts.

Plant Sci. Lett. 30:297-301.

Hahn-Hagerdal, B., Hosono, K., Zachrisson, A. and Bornman, C. H. 1986. Polyethylene glycol and electric field treatment of plant protoplasts: characterization of some membrane properties.

Physiol. Plant, (in press).

Koop, H.-U., Dirk, J., Wolff, D., and Schweiger, H.-G. hybridization of two selected single cells.

1983.

Somatic

Cell Biol. Int. Repts

7:1123-1128.

Koop, H.-U. and Schweiger, H.-G.

1985.

Regeneration of plants from

individually cultivated protoplasts using an improved microculture system. J. Plant Physiol. 121:245-247.

375 Lörz, H., Paszkowski, J., Dirks-Ventling, C., and Potrykus, I.

1981.

Isolation and characterization of cytoplasts and miniprotoplasts derived from protoplasts of cultured cells.

Marschner, H.

1964.

Chlorophyllbildung und Blattschäden bei Gerste unter dem

Einfluss von Cäsiumionen.

Marschner, H.

Physiol. Plant. 53:385-391.

1965.

Flora 154:30-51.

Anreicherung von Porphyrinen und Protochlorophyllid in

Gerstensprossen unter dem Einfluss von Cäsium.

Murashige, T. and Skoog, F.

1962.

Flora 155:558-572.

A revised medium for the rapid growth and

bioassays with tobacco tissue cultures.

Physiol. Plant 15:473-497.

Pelletier, G., Primard, C., Vedel, F., Chetrit, P., Remy, R., Rouselle, P., and Renard, M.

1983.

protoplast fusion.

Intergeneric cytoplasmic hybridization in Cruciferae by

Mol. Gen. Genet. 191:244-250.

Pelletier, G., Vedel, F., and Belliard, G. breeding.

1985.

Cybrids in genetics and

In Proc. of the 1st Nordic Cell and Tissue Culture Symposium on

Research, Breeding and Production of Crop Plants, Frostavallen, Sweden, March 5-9, 1984 (Eds Bornman, C. H., W. K. Heneen, C. J. Jensen, and A. Lundqvist). Hereditas Suppl. Vol. 3:49-56.

Schiff, J. A., Lyman, H., and Epstein, H. T.

1961.

Studies of chloroplast

development in Euglena II. Protoreversal of the U. V. inhibition of green colony formation.

Biochim. Biophys. Acta 50:310-318.

Spangenberg, G., Koop, H.-U., Lichter, R. and Schweiger, H.-G. culture of single protoplasts of Brassica napus.

1986.

Micro-

Physiol. Plant. 66:1-8.

377 IN VITRO PROPAGATION AND BREEDING OF ORNAMENTAL PLANTS: ADVANTAGES AND DISADVANTAGES OF VARIABILITY

W. Preil Federal Research Centre for Horticultural Plant Breeding Bornkampsweg 31, D-2070 Ahrensburg, F.R.G.

Introduction In vitro propagation methods have become more and more important during recent years, especially with many ornamental plants like Gerbera, Nephrolepis, Saintpaulia, Lilium, Cordyline, etc. (1). Variations have been rarely observed among plants that have been produced in large numbers, e.g. orchids (2). In other cases high variability could be detected in progenies derived from tissue culture

(3). Two groups of horticulturists use these facts for

different practical purposes: The plant propagators, on the one hand, apply those multiplication methods which result in the lowest possible variability, e.g. by initiation of axillary shoot development and by suppression of callus or adventitious bud formation. The breeders, on the other hand, take advantage of those techniques which increase variability for selection of any kind of improved cultivars. Some years ago the new term 'somaclonal variation 1 was created and recognized as a novel source of plant improvement. From the literature of the last 20 years it was concluded that plant cell culture itself generates variability

(4). It is very unlikely that tissue

culture could induce biological processes that follow pathways different from those in the entire plant. There is, however, no doubt, that tissue culture may uncover preexisting variability in somatic cells of the donor plants and may also enhance processes responsible for different kinds of genetical or physiological changes. In horticultural history clonal variation has proved to be a common phenomenon, well known since the early days of Dutch tulip breeding in the 17th century. Variations have been frequently observed

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

378 when plants were propagated vegetatively over a period of several years. In all important clonally multiplied ornamentals

'sports',

i.e. spontaneous somatic mutants or chimeral rearrangements, were found in sometimes surprisingly high frequencies. For example, among some 1300 varieties of tulips mentioned in the 1951 - 52 registration, 'Murillo' bred in 1860, produced about 60 sports expressing an amazing range of colours (5). Out of 274 azalea cultivars listed by Heursel (6) 144 (= 52.5 %) were sports. Evaluating the 1984-catalogues of 22 companies from Europe and the USA offering Chrysanthemum cuttings for the German market 585 (= 48.1 %) out of 1217 cultivars were sports or induced mutants (7). Some other examples of clonal variation in ornamentals have been reviewed by Wasscher (8) and Horn (9). The reason for such large numbers of introduced varieties may be seen in the fact that new types are much more appreciated in ornamentals than in any other crops which are less subjected to trends of fashion. The selection and cultivation of 'sport-families' in horticultural practice has, furthermore, the advantage that these near-isogenic lines can be grown under the same environmental conditions. Therefore, in general

breeders and growers of ornamentals

have turned their attention more intensively to all kinds of somatic mutants than agriculturists do.

So we have to put the question

whether the recently emphasis on'somaclonal variation' represents a 'novel' source for plant improvement or is only a new view of an old situation made more accessible by improved techniques. This paper will review some reasons for variability observed after in vitro propagation of ornamental species and will discuss the use of in vitro methods for screening and selection of spontaneous or induced mutants.

Chimerism Chimerism is the result of intra-apical mutations or of incorporation of mutated somatic cells into de novo formed shoot apices. For definition of different types of chimeras see Brabec Tilney-Basset

(11), and Bergann

(10),

(12). In species vegetatively propa-

gated over several years chimerism seems to tie the rule rather than the exception. Most of the chimeras remain undetected and, therefore.

379 are called

1

crypto 1 -chimeras. Only a few groups can be recognized by

their phenotype, e.g. the chlorophyll chimeras caused by gene and/ or plastid mutations. In flower colour chimeras some can be easily identified, e.g. in azaleas

(Rhododendron simsii) anthocyanin de-

fect mutations in the L^ result in flowers with white edges and lighter coloured central regions as compared with the original cultivar. Of all azalea sports registered in Europe (6) 37.5 % are anthocyanin defect L^-periclina1 chimeras. Proof that a plant is a chimera may be often obtained from adventitious buds arising from internodes of the stem or sometimes from roots, as already mentioned seventy years ago by Bateson (13). A reason for the accumulation of chimeras in vegetatively propagated plants is the induction of immense numbers of axillary and adventitious shoots incorporating mutated somatic cells into the developing apices. According to the official statistics, e.g. 20 mil]. azaleas were produced in the FRG in 1984 (14). Each of these azaleas was pinched five times during an 18 months cultivation period, i.e. several hundreds of million of shoots were decapitated every year. Therefore, it is not surprising that spontaneous somatic mutations have a good chance to participate in the development of axillary or adventitious buds expressing different kinds of chimerical structures. The mutation rates vary, of course, depending on the genetic

background of the cultivars. In some cases azalea

seedlings, flowering the fi rst time after the fifth pinching, already had branches with periclinal anthocyanin defect L^. The first L^-chimeral sport of cv. 'Hellmut Vogel 1 was introduced after 6 years, and the first sport of cv. 'Ambrosiana' only occurred

after 27 years. In 'Violacea1,

a

variety more than

100 years old, no pigment deficient L^-chimera has been found to date. A similar situation can be expected in all vegetatively multiplied crops. Many mutations influence physiological characters which may cause plant improvement. Cultivars, probably periclinal chimeras, could have been improved in the past by selection in clonal progenies. Clonal selection, therefore, is an important tool for breeding purposes

(12, 15). Because the economic

value of many cultivars

depends on their characteristic chimeral composition, unique traits may be lost or altered after in vitro or in vivo vegetative propagation when adventitious shoots develop which are not chimeral or

380

consist of a rearranged apical constitution. In case two different mutations happen in two of the three histogenic layers, a periclinal trichimera may arise. Rearrangement of the three genetically different layers would theoretically result in 27

different

apical

meras, and 3 homohistonts

constitutions: 6 trichimeras, 18 dichi(12). This high number of possible re-

arrangements in histogenic cell layers after two or more mutations explains the appearance of sport-families after a relative short period of time in many vegetatively multiplied ornamentals. For the true to type multiplication process of chimeras the maintenance of the original apical constitution is of extraordinary importance. For this purposes the in vitro cultures have to be started from intact shoot tips or axillary buds. For breeding and selection of improved variants, however, the segregation of the histogenic cell layers has to be induced, e.g. by callus proliferation, cell suspension cultures and development of adventitious buds.

Mosaicism In the literature the term 'mosaicism' is used in various meanings. According to the definition of an IAEA recommendation

(16) mosai-

cism is the 'instability in the progeny of a cell due to chromosome, genie or cytoplasmatic cause'. This includes the usually observed instability of cell- and callus cultures (17, 18, 19, 20, 21). Genetic instability of cells in intact plants resulting in mosaicism is well known since the investigations on transposable genetic elements in maize (22). In the past 20 years similar elements have been identified in every kind of organism been examined

that has

(23). Mosaicism caused by stable extra-apical mu-

tations in organs or callus systems can convert to chimerism after incorporation of mutant cells into adventitious buds of multicellular origin. In periclinal chimeras mutations affecting the colour of plastids influence different kinds of leaf variegation of mosaic like character (11,24, 25, 26, 27, 28).

381 Chimerism or mosaicism related to in vitro culture of some ornamentals: Poinsettia, Chrysanthemum, Saintpaulia, Begonia, Pelargonium Poinsettia

(Euphorbia pulcherrima)

The detailed studies on chimeras in poinsettia demonstrated that sport families can be obtained by rearrangement of the histogenic cell layers (29, 30, 31, 32). Investigating the inheritance of bract colour of poinsettia, anthocyanin synthesis was found to be determined by a single dominant gene WH (33). Plants homozygous for the recessive allele whwh develop anthocyanin free (white) bracts. The origin of pink bracts was either determined by a single recessive gene pk, whose effect was to reduce the amount of pigment formed

(34) or by the appearance of periclinal chimeras

lacking anthocyanins in the L^ (30). It is a common observation that the L^ in heterozygous poinsettia cultivars

(WHwh) mutates

after a certain period of in vivo clonal propagation into whwh resulting in a periclinal chimera with pink bracts. Thus the old cv. 'Imperator' sported into 'Ecke's Pink1 consisting of histogenic constitution

'white-red-red'. After chimeral rearrangement of

'Ecke's Pink' the two cultivars 'Trebistii alba' and 'Ecke's White'

(white-white-red)

(white-white-white) originated in vivo (30, 32).

Today these processes of sport development can be considerably shortened by in vitro techniques: From cross sections of petiols of the monoekto-chimera

'Annemie-Pink'

(white-red-red) cultivated

on MS-agar medium, three histogenic cell layer rearrangements beside the original 'Annemie-Pink'-plants can be achieved variants obtained were 'Annemie' (white-white-red), and

1

(red-red-red),

Annemie-White 1

(35). The

'Annemie-Marble'

(white-white-white). These

experiments demonstrate the multicellular origin of adventitious buds ir. "-h^ case of the regenerated chimeral 'Annemie-Pink' and 'Annemie-Marble'-plants. Other experiments using cell suspension cultures of 'Annemie-Pink', however, resulted in regeneration of plants with red or white bracts indicating that the de novo formation of meristems was initiated by single cells only

(36).

According to the statement of Broertjes and Keen (37), 'the apex of the adventitious shoot is formed by only one (epidermal) cell of the meristem or the callus. Another possibility is that the apex formation is not a random process and that the first dividing

382

cell, forming the future meristem or callus, soon occupies the major part of this meristem, on top of which a few cells

(most like-

ly genetically identical vegetative daughter cells - mutated or unmutated) form the apex of the adventitious shootlet'. From in vitro cultures of the chimera

'Annemie-Pink', we can conclude

that the origin of adventitious shoots may develop de novo either from single cells or from groups of cells depending on in vitro cultural conditions. The identification of crypto-chimeras in any species is of interest for both, propagator and breeder. In case of chimerism care has to be taken to prevent all cell layer rearrangements during in vitro propagation. In breeding experiments one has to bear in mind that L^ mutations are not transmitted sexually, and, furthermore, that the phenotype of many important chimeral cultivars is the result of the combination and interaction of genetically different histogenic cell layers. After plant regeneration segregated

from

layers, variants of diminished value must be taken into

account when important characters are the result of a unique chimeral constitution. This

can

be demonstrated

'Annette Hegg 1

nating from cell suspension cultures of cv. sport

'Brillant'

(syn. Diamond)

'Annette Hegg' cell

(A) plants with short internodes, oak-like

and bracts similar with

and its

(36). Two different groups could

b« 3ist in-juishcd among the 37 3 plants from suspensions.

for plants origi-

'Annette Hegg',

leaves,

(B) plants with elongated

internodes, narrow bracts and 10 days delayed flowering. The leaves were of the same type as in group A. Axillary branching, a typical and horticulturally important character of

'Annette Hegg'

and its sports, did not appear in any group. The suspension culture of cv.

'Brillant' yielded

139 uniform vari-

ant plants lacking axillary branching, but with elongated

inter-

nodes and oak-like leaves. The appearance of only one type is possibly a result of diplontic selection influenced by in vitro culture conditions. Many morphological characters of these plants appeared similar to those in cv.

'Paul Mikkelsen 1 . This

supports

the opinion maintained by a group of poinsettia experts that 'Annette Hegg' may be a chimeral sport of

'Paul Mikkelsen'.

In order to determine the genetic constitution of histogenic layers of seven pink cultivars, plants were regenerated

cell

in our lab

383 from cell

suspensions

(38). T h e

the seven cultivars were found 1

in t h e i r p r o g e n i e s .

Ziegers-Pink',

were

achieved.

and

Six white

The

1

Annemie-Pink1

Hegg-Pink1

a n d 221

which was

that

five

no pink b r a c t s

were

of

only

red plants d e r i v e d

a n d 51 w h i t e

already

'white'

The two c u l t i v a r s 116'

can be c o n s i d e r e d

ture methods procedures

and

13' a n d

culture

the chimeral

for

those

low

propaga-

conditions. Seedling

due to their

that these

bracts

'Christi-

chimera.

'W. S ü p t i t z - P i n k

is n o d o u b t

an a d v a n t a g e

to b e a

indicate

suspension

red

from

355 r e d f r o m

identified

to be h o m o h i s t o n t s

There

represent

since

under

'Gutbiers V

form pink progenies.

easily

cells

of

'Preduza-Pink', plants with

small n u m b e r of p l a n t s w i t h w h i t e b r a c t s

t i o n rates of No

because

From suspensions

'Annette

ane Zieger-Pink'-suspensions, of

results demonstrated

chimeras,

uni-

suspension

further practical

n a t u r e of a p h e n o t y p e

cul-

breeding

now can

be

ascertained.

Chrysanthemum Although

in C h r y s a n t h e m u m

in v i t r o

(39, 4 0 , 4 1 ) ,

tice because of

a rooted

high multiplication

c o s t o f a n in v i t r o

the

appearance

is a n o t h e r

handicap of

servations

of Stewart and Dermen

tions, chromosomal

to result

a suppressor

Variants

originating

tions

chimeras

21

napolis'

types

e.g.

suggested

instability

The most loss

frequent

in m i t o s i s

that

to

supposed

to be

are known proved

less

type

in

sporting

chromosome

chromoplasts.

frequent

layers than

of

those

of

muta-

(43).

s e r i e s of c h i m e r a l composition.

(49). T w e l v e o f to b e p e r i c l i n a l

explanations

t y p e of

Ob-

muta-

involved

on the nature

is w e l l d o c u m e n t e d

of a p i c a l

are

of t h e a p i c a l

literature

and van Harten

that gene

of a

f o r m a t i o n of y e l l o w

For more

cultivars

cultivars

cases different cultivar,

to t h e

in C h r y s a n t h e m u m

48). From m a n y

(42)

chimeral

in vivo.

are

d u c e d d u e to c h a n g e s family

prac-

exceeds

of p l a n t s not true

from rearrangement

loss.

see Broertjes

Chimerism

plant

achieved

in

in v i t r o p r o p a g a t i o n of C h r y s a n t h e m u m .

from the

carrying

from chromosome

of h i g h n u m b e r s

loss,and

the somatic variation

periclinal

produced

can be

a role

cutting.

Occasional

is a s s u m e d

rates

this method does not play

(42, 4 4 ,

45, 46,

s p o r t s c o u l d be From

the

intro-

'Indianapolis'

16 i n v e s t i g a t e d chimeras

47,

'India-

(42). I n

some

e x i s t for t h e a p i c a l c o n s t i t u t i o n o f one

'Indianapolis-Pink'

was

identified

as a

trichi-

384 mera, whose L2

apex consists

and a yellow

changes

L^

in flower

(44). T h i s w a s b a s e d o n

layers:

cream,

and bronze

yellow

lateral

from the o r i g i n a l

'No 3 I n d i a n a p o l i s - P i n k '

Stewart They

original

homohistonts.

cultivar.

by any m u t a t i o n .

It s e e m s ,

only

in the upper

florets. cipating

o f I^

the genetic

for s y n t h e s i s

To b r i n g m o r e

into

light

regenerated

plants

remaining

67 p l a n t s

for

156 h a d

93 p l a n t s

showed

4 plants

It s e e m s tations

8 weeks

that the investigated ly o f

the a p i c a l

scribed

plants may

anthocyanins carotenoids

of p r o d u c e d

anthocyanins,

suspensions

lab

genetiwere

(50). O u t o f

into

pink

four

249

colour.

groups:

flowers.

culture,

types were

consist

was

a trichimera, ( P W Y ) , as

156 n o r m a l p i n k

PPP,

because

exist

some

layers which

resulting

PWY,

PWW.

interactions influence

deare

all of them

PWP,

probab-

flowering

cells of the petals and PPW, PPY,

mu-

conclude

of u p to s i x t y p e s , w h i c h

epidermal

cell

caused by

and, therefore, we

'pink-white-yellow'

that there may

different

gene-

flowers

in the m e s o p h y l l :

thermore, we assume

parti-

as t h e

9 developed more dark pink

in t h e i r p h e n o t y p e ,

in the upper

the genetically

in o u r

(44). T h e r e g e n e r a t e d

not distinguishable

cell

'Indianapolis-Pink'

theoretically

(not

as w e l l

flowers

constitution

by W e a v e r

anthocyanin

discussion on the

that all variant

suspension

ligulate

13 h a d c r e a m - w h i t e

yellow

to b e u n l i k e l y

not

gene-

expressed

and L^

flowers of the o r i g i n a l flowers,

that

carotenoids.

c o u l d be c l a s s i f i e d

light pink

exhibited

during

unknown

the controverse

w i t h t r a c e s of a n t h o c y a n i n s , and

for

the

periclinal

is

of C h r y s a n t h e m u m

'Indianapolis-Pink',

initiated and cultivated The

of

unlikely

pink

synthesis

potential

remains

'Indianapo-

ideotype was

identical,

epidermis

t i c b a s i s o f t h e L^

cal c o n s t i t u t i o n of

tho and

it w a s

that several

(origin of the p e t a l - m e s o p h y 1 1 )

in p e t a l d e v e l o p m e n t )

that

however, very

the original

anthocyanin

(L^-derived)

In s u c h c h i m e r a s

synthesis

In c o n t r a s t , f r o m

It is m o r e p r o b a b l e

because

white,

'Indianapolis-Pink'

furthermore,

tically different, but phenotypically chimeras were derived

type

to b e a c h i m e r a b e c a u s e

after decades of clonal propagation affected

composition

flowering

a n d D e r m e n (42) c o n c l u d e d that b o t h

argued,

not be e x p e c t e d

seedling

altered

pink

buds of

unpigmented

radiation-induced

sports were derived.

and adventitious

types were

lis-Pink' would

pink L^, an

colour due to a s u p p o s e d l y

of histogenic s t u d y of

of a g e n e t i c a l l y

the

have lack Fur-

between amount

in l i g h t e r o r d a r k e r p i n k

pig-

385

mentation or only in traces of anthocyanins in the petals. Such interactions between cell layers influencing pigmentation were described by Bergann (31, 32). Supposed interactions may also explain the results of Bush et al. (46), who reported on in vitro cultures of petal segments of 'No 2 Indianapolis Bronze'. They observed among regenerated plants individuals which had besides carotenoids in the petal mesophyll 'normal', 'some', 'traces' and 'no' anthocyanin production. This corresponds to the four possible genetical constitutions of the petals for anthocyanin synthesis in L^ and L 2 tissue: PP, PW, WP, WW. The combination PW and WP may possibly be responsible for 'some' and 'traces' of anthocyanin. The genetical situation of 'Indianapolis-Pink' seems to be much more complicated than expected so far. Our own results allow the assumption that homohistonts and heterohistonts had been regenerated from cell suspension cultures, i.e. apex formation started from single cells as well as from groups of cells, in contrast to the poinsettia experiments. From in vivo experiments it is known that adventitious shoots may arise either from single cells or from more than one cell (42, 51). Results obtained from in vitro culture of petals indicate that adventitious bud formation may be of multicellular origin

(46, 52). From histological examination

it was infered that cell divisions in petals started in the L 2 tissue (52). However, corresponding observations by Broertjes et al. (51), suggested, that adventitious shoots develop from single epidermal cells (L^) resulting in almost exclusively solid

(non-

chimeral) individuals after X-irradiation. Abnormalities in plants regenerated from leafy callus of cv. 'Indianapolis white Giant No 4' after 9 years of in vitro culture were described by Sutter and Langhans (53). These plants were less vigorous than their controls. About 15 % were characterized by lack of apical dominance. Delayed flowering and morphologically aberrant flowers of reduced diameter were observed. Genetic instability, chimeral rearrangement as well as residual hormone effects are offered as possible explanations for the abnormalities. Of 87 flowering plant three had anthocyanin pigmentation in 50 % of the florets. A few had yellow stripes down to the centre of the florets. This flower colour variation seems to be unexpectedly low, because 'Indianapolis White' is assumed to be a periclinal chimera, which L 1 and

are unpigmented, but the L, is genetically yellow

386

(able to synthesize carotenoids) according to Weaver (44) or genetically pink (able to synthesize anthocyanins) according to Stewart and Dermen (42). In both cases more flower colour variation has to be expected due to rearrangement and separation of the cell layers. Investigations on chromosome numbers in Chrysanthemum showed distinct variations in callus cells and in vitro derived plants from different explant sources

(54, 55). In the cells of the callus

chromosome numbers varied largely from 49 to about 230 after two weeks of culture. The frequency of the cells identical with the original plant was only 32 %. Cells with more than 100 chromosomes reached 2 0 %. In plants, however, regenerated from capitula and from almost all shoot tips after 5 1/2 months of culture the chromosome numbers were not different from the parental tissue. In contrast, 36 % of the plants from various stem segments showed variations in chromosome number. Changes in the phenotype such as flower colour

inflorescence size, floret shape, and plant height

were observed. Frequency of the variation in flower colour depended on the explant sources. Shoot tip culture produced 7 % of plants with different flower colour, capitula 22 % and stem segments 37 %, respectively. Saintpaulia The importance of in vitro propagation of Saintpaulia increases continuously in horticultural practice. The number of plants produced in the Netherlands was about 0.6 mill, in 1980 and 4.4 mill, in 1984 (1). Spontaneous mutations are rare in some clones, but more frequent in others

(43). Off types derived from in vitro cul-

ture do not exceed the percentage usually found in conventional propagated clones (56). In general chimerism seems to be rare, because adventitious buds predominately arise from single cells (57). Experiments with a white flowering unstable clone, showed that about 10 - 30 % pink off types regularly developed

(58). Solid

white and pink clones as well as segregating progenies could be selected after two steps of in vitro culture of leaf sections. In cv. 'Inge', a blue flowering clone, plants not true to type with pink and lightviolet spots can be usually observed in frequencies of about 0.01 % among conventional propagated clones. The chimeral nature of such off types had been demonstrated after in vitro mul-

387 tiplication. Blue, pink and lightviolet flowering individuals with a large range of variations in morphology and flowering time could be obtained. Uniform progenies were established from selected single plants after in vitro culture

(58). Investigations on varie-

gated cultivars demonstrated that parental leaf pattern could be maintained after in vitro regeneration. This led to the assumption that in vitro adventitious buds may arise from multicellular units (59). From leaf variegated cv. 'Marge Winters' 90 % of the in vitro regenerated plants were similar to the parent variegation. Progenies from cv. 'Bold dance 1 were 100 % identical to the parent (60). It is surprisingly that from a chimera uniform

(chimeral) proge-

nies

buds,

could

be

obtained

via

adventitious

all

of

multicellular origin. Leaf variegation pattern in Saintpaulia, however, can also be explained by plastom mutations followed by segregation of intact and mutated plastids during cell divisions (61, 62). This assumption would correspond to the well documented observation that adventitious buds originate

from only one cell

in Saintpaulia. Begonia Begonias demonstrate high regeneration capacity in in vivo and in vitro propagation systems. In the horticulturally most important species Begonia X hiemalis in vitro methods are used for both elimination of bacterial pathogenes from stockplants for commercial propagation

sulted in numerous sports of commercial interest authors reported uniformity

(63, 64, 65) and

(66). Spontaneous variation in vivo re(67). While some

in vitro progenies or did not find in-

creased variation in tissue culture derived plants as compared with propagation by cuttings (64, 66, 68, 69), others found varying numbers of plants not true to type (70, 71, 72, 73). Recently Westerhof et al. (74) compared the variation after one, two and three cycles of in vitro propagation. Whereas the population of cv. 'Aphrodite-Pink' obtained after one cycle was uniform, 13.1 and 10.7 % variants were found after two and three cycles. Among cv. 'Schwabenland-Red' 0.7, 2 3.7 and 45.2 % deviations occurred after one, two or three in vitro propagation cycles. When subculturing three deviating types of 'Aphrodite-Pink' with 'round leaves', 'extreme branching of shoots' or 'early flowering', uniform offspring were obtained indicating stability of variation.

388

From three subcultured off types of

1

Schwabenland-Red' , namely

'extremely tapering leaves', 'thick, brittle leaves, short internodes' or 'late flowering', only one plant of the type 'late flowering' yielded a stable offspring resembling the deviating parent. The other variants returned to the normal type and, therefore, can be classified as epigenetic changes due to effects of the in vitro culture environment. These findings should have consequences for further strategies of commercial in vitro propagation. No detailed information basing on experimental data are available on the cellular origin of stable in vitro derived variant plants. A comparable situation, however, can be assumed to exist in Saintpaulia, because adventitious buds predominantly arise from single epidermal cells also in Begonia (75), and, thus, solid mutant plant may develop from chimeral or mosaic tissue. Pelargonium In vitro multiplication rates of Pelargonium are usually not high enough for mass propagation. Nevertheless, meristem culture is an important technique for freeing clones from bacterial and viral diseases resulting in healthy stock plants for in vivo propagation by cuttings

(76). Many of the cultivars are supposed to be peri-

clinal chimeras with inherent possibility of subsequent cell layer rearrangements. Cv. 'Madame Salieron' should be mentioned as a classical example for variation in Pelargonium zonale due to chimerism. This cultivar, known since 1877, is one of the best investigated periclinal trichimera, with green L^, white

(chloro-

phyll deficient) L^ and green L^, which is responsible for reduced length of internodes, a typical character of 'Madame Salieron' (77, 78). Twelve different rearrangements of the original apical cell layer constitutions ABC (L^ ,

, L^) have been described,

probably

those

many

more

exist.

From

combinations

contain-

ing the green ideotype C in the apex develop plants with short internodes. After removing C from histogenic layers plants develop with elongated internodes. This demonstrates impressively that a mutation in only one cell layer may influence distinct changes in the morphology of the whole plant. Other cultivars of Pelargonium have been recognized to be chimeras identified by the use of conventional techniques

(11, 79) or in vitro methods

Experiments of Skirvin and Janick

(80, 81).

(82) demonstrated a high varia-

389 bility in callus culture derived plants of scented Pelargonium varieties. The investigated cultivars are between about 20 and more than 200 years old

(e.g. 'Rober's Lemon Rose' introduced about

1950, 'Old Fashioned Rose' in 1774 (!) and 'Altar of Rose' in 1690 (!), and, therefore, represent in all probability periclinal chimeras. In callus cultures of such heterohistonts variation have to be expected due to segregation of the original apical constitution. Low calliclone variability was found, however, in some cultivars, e.g. in the 'Old Fashioned Rose'. One should keep in mind, when interpreting such results that the regeneration capacity of genetically different cells in callus culture may be restricted to only one ideotype simulating homogeneity if examination is based on the developed plantlets only.

Spontaneous and induced mutants The use of in vitro culture methods for the selection of variant types in ornamentals has been documented for many traits especially for flower colour, plant morphology, and some physiological characters. From available information

it appears that in vitro

methods may shorten breeding cycles and, therefore, can reduce the costs

of the development of a new variety.

Induced variability does not seem to be different from that known to occur spontaneously. Mutagen treatment, however, can increase mutant frequency drastically. Although some variants, e.g. changes in flower colour, may emerge from spontaneous mutations at relatively high rates, mutation frequency of many useful traits is very low. Mutagen treatments, therefore, are of outstanding importance for practical breeding purposes. The ontogenesis of a plant can be extended by vegetative propagation over a long period of time, e.g. several years, decades or even longer. As a consequence, in tissues of every clonally multiplied 'old' cultivar, many somatic mutations become cryptically accumulated resulting in the well known clonal variations

found in

in vivo propagation in the horticultural industry. From in vitro cultures of such old cultivars representing genetic mosaics (result of extra-apical mutations) or chimeras

(result of intra-

apical mutations), (Fig. 1), variant plant types can be expected

390 after short term tissue or callus culture followed by the regeneration of adventitious shoots. This pre-existing genetic variation in the donor tissue was often misinterpreted and classified as 'in vitro culture induced'. Many observations indicate that the frequency of variant types increases with duration of in vitro culture. This suggests that processes responsible for mutations are promoted or de novo induced under in vitro conditions, especially in cell and callus cultures. The potentially responsible mechanisms for the in vivo and in vitro origin of chromosome structural changes and gene mutations have been recently discussed by D'Amato

(83).

spontaneous mutations

spontaneous and in vitro culture induced mutations

mutations induced by mutagenic treatments

age (number of subcultures)

increasing mutagenic doses

In Vivo C u l t u r e

In Vitro Culture

In Vitro Mutagenesis

e x t r a - a p i c a l mutations (mosaicism)

somaclonal variation

homohistonts (solid mutants)

ontogenesis

intra - apical mutations (chimerism)

Fig. 1: Increase

heterohistonts (chimeras)

in the number of mutations during ontogenesis,

the in vitro culture and after in vitro mutagenesis Mutant-characters like flower colour or different morphological traits have to be selected at the whole plant level in the greenhouse or in the field. Selection at the cellular level is applicable only when the genetic changes are expressed in the cultured cells or tissues and are correlated with the behaviour of the whole plant. This may be the case only for some physiological traits like

391 low-temperature tolerance, salt tolerance or resistance to herbicides and toxins released by pathogens. As known from practical breeding work most of spontaneous or induced mutants are without

importance for horticultural purpo-

ses. Selection procedures,therefore, can be successfully applied only to those cultivars which are easy

to multiply in vitro, re-

sulting in sufficiently large populations for screening. For numerous ornamental species mass propagation methods have been developed - the most important prerequisite to combine in vitro culture techniques and mutant selection in practice. Selection for changes in flower colour or plant morphology According to literature (43) many ornamental species are very suitable for mutation breeding, since flower colour and other mutations can be produced without altering any other character of the original idiotype. In roses, for example, numerous in vivo mutation induction experiments have been described, but only three commercial mutants were published. The reason for this restriction may be that it sometimes takes a few years before mutants can be isolated and propagated by conventional methods. Recently Walther and Sauer (84) could demonstrate that in vitro mutagenesis can be applied very efficiently to roses since micro propagation methods are now available. A period of only 9 months was required from X-irradiation of in vitro plantlets producing axillary shoots to mutant selection in the greenhouse. The mutation spectrum comprised 73 % flower mutants (colour, size, number of petals), 13 % mutants with modified leaves and 14 % with a changed growth type. The mutant rate increased with increasing irradiation doses between 25 and 6 0 Gy. The selection of mutants from in vitro cultures has been reported by several authors in the last years. In Gerbera X-ray doses between 10 and 25 Gy applied in vitro to axillary shoots resulted in the appearance of mutants with changes in length of the stalks or with varied size of the capitulums and leaves. From this it was concluded that 'cultivar families' can be developed consisting of members differing only in the length of their stalks to be used as pot plants (85). High numbers of flower colour changes and variations in plant morphology were found in Kalanchoe after in vitro X-irradiation as well as in untreated

392 cultures

(86, 87). Leaf explants of clone M produced 10 % (control),

33 % (15 Gy) and 39 % (30 Gy) of plants with changed flower colour. In progenies from callus cultures 31 % (control) and 32 % (30 Gy) of plants with altered flower colour were observed. In populations from suspension cultures the percentage of flower colour variations increased from 34 % (control) up to 38 % (30 Gy) (86). This indicates surprisingly high spontaneous mutation rates. The number of plants with abnormal growth habit regenerated from untreated suspension cultures of Kalanchoe cv. 'Montezuma' was 30 % (87). A polyploid variant originated from calli clones of Pelargonium 'Rober's Lemon Rose' and was released because of its general attractiveness and vigour (88). After irradiation of Begonia rex mutated leaf sectors were isolated and used for regeneration of mutant plants

(89, 90). From irradiated detached leaves of Be-

gonia x hiemalis plants developed of which 30 % were mutated in colour, size and form of the leaves and flowers. Most of the mutants (98.5 %) proved to be homohistonts

(91). In Kohleria mutants

were found in EMS (Ethyl methane sulfonate) and NMU

(N-nitroso-N-

methy1-urethane) treated cultures. Some of them started flowering earlier, produced significantly more and larger flowers, or showed altered leaf characters

(92). As demonstrated by Broertjes et al.

(51) high mutant frequency can be induced by X-irradiation of in vitro cultured explants of Chrysanthemum. To increase the genetic variability for yield in cv. 'White Spider' pedicel segments were irradiated and cultivated in vitro. All treatments yielded variability in flower morphology. Clones were selected that had retained the morphology of 'White Spider', but outperformed the controls in flowering time and flower number (93). Mutated petal tissue from sectorial chimeras can be easily isolated and cultivated in vitro. Thus, from Chrysanthemum cv. 'Golden Princess Anne' pink flowering mutants could be obtained after plant regeneration (47). Out of 15.000 Saintpaulia plants from NMU-treated in vitro cultures 40 % showed leaf pigment alterations from green, to light green, yellow, redish or blueish. Only few flower colour mutants could be selected

(94). Comparing in vivo and in vitro mutagenesis

in carnation, treated with gamma rays, better results could not be obtained from in vitro methods, which proved in this species to be more complicated than in vivo manipulations

(95).

393 Selection for physiological mutants A general review on the progress of in vitro isolation of mutants was given by King (96). In ornamental plant breeding recent research on the in vitro selection of various stress tolerant plant types is focussed mainly on low-temperature tolerance, one of the most important trait of greenhouse grown plant species. As demonstrated by Jung-Heiliger and Horn (97) and Broertjes et al. (98) low-temperature tolerant variants could be obtained after gamma or X-irradiation

of in vivo cuttings of Chrysanthemum. Several of

these variants lost their low-temperature tolerance after clonal propagation during the following years , rrost probably because they were periclinal chimeras and reverted unnoticed to the original apical cell layer constitution tures Preil et al. (99)

(98). Using X-irradiated suspension culselected

cold-tolerant Chrysanthemum

after in vitro application of long term temperature stress. These plants proved to be mutants confirmed by subsequent clonal propagation and cultivation under greenhouse conditions for

more than

three years. All mutants flowered earlier in winter than the original cultivar under a 10° C temperature regime. In summer at high temperatures the photoperiodical reaction changed: only some flowered earlier, others at the same time compared to the control, or even later (100). Recently Broertjes and Lock (101) and Huitema et al. (102) reported on the selection of low-temperature tolerant Chrysanthemum after irradiation of pedicel segments or suspension cultures. According to these results in Chrysanthemum in vitro mutagenesis can probably be used successfully for induction of low-temperature tolerance in other ornamentals. Up to now is not possible to decide which in vitro technique will yield the highest efficiency for practical breeding purposes. The induction of adventitious shoot formation after mutagenic treatment of pedicel explants, callus or suspension cultures enables ]arge populations to be produced for screening under low-temperature pressure in vitro or in greenhouses. Application of low temperatures in vitro for screening may have the advantage that mitosis of non mutated cells will probably be more inhibited by the stress temperatures than that of the mutants. Thus, the number of plants will be reduced for confirmation of mutant character under greenhouse conditions. However, an unexpected high number of callus colonies from X-ray treated suspen-

394 sion cultures of Chrysanthemum had survived after the exposure of plated cells to 8° C stress temperature over 170 days (99). Among 3.300 regenerated plants only 24 proved to be low-temperature tolerant mutants when grown at 10° C in the greenhouse. From this it can be concluded that during temperature stress numerous epigenetic variants appeared which could not be distinguished from mutants under in vitro conditions. For definition of the term 'epigenetic variant' see Binns (103), Chaleff

(104), and Meins (105).

The adaptability of non mutated cells to unfavourable conditions is a serious problem in the application of any in vitro screening system for practical purposes. Therefore, in vitro conditions have to be developed which restrict the physiological adaptation of non mutated cells resulting in a clearer separation from cold-tolerant mutant cells. First attempts of selection for low-temperature tolerant mutants in Saintpaulia were published by several authors. Schlegel (106) and Amberger et al. (107) tested up to 30 cultivars to get more information

on in vitro regeneration ability at low temperatures.

They investigated, furthermore, the suitability of different methods for selection. Mutagenic treatments with EMS or NMU were carried out by Geier

(92) and Warburton et al. (108). However, no

low-temperature tolerant mutants have been described up till now. Plants could be regenerated from X-irradiated cell suspensions of poinsettia

after exposure of plated cells to 12° C stress tempe-

ratures over 170 days. Only three out of 997 regenerated plants proved to be slightly better adapted to low temperatures showing reduced leaf fall under cold stress compared with the original cultivar. The improvement was statistically significant but small, and, therefore, without practical value (99). The appearance of epigenetic variants indicates similar adaptation processes to low temperature in vitro as already observed in Chrysanthemum. From the above mentioned experiments we conclude that in Chrysanthemum morifolium, a species from temperate climate, the genetic variability for low-temperature tolerance seems to be much higher than in Saintpaulia or poinsettia, which represent genera from the tropics and subtropics. Mutations influencing

low-temperature

tolerance must be considered as rare events in both genera. In order to enhance the efficiency of in vitro mutagenesis,

395 methods have to be developed which induce a maximal mutability. Using a marker gene WH for anthocyanin synthesis in poinsettia, it has been doses

demonstrated in model experiments that increasing X-ray from

10 to 60 Gy caused an increase

of

mutation rates in

cell suspension cultures. The effect of the mutagenic treatment was further improved by application of fractionated X-ray doses resulting in higher survival rates of embryogenic cells as compared with acute dose irradiation. It was shown

that the mu-

tation of the anthocyanin locus can be used as a marker for over-all induceable mutability including low-temperature tolerance in poinsettia (109). No experimental data is presently available on the effects of different in vitro screening conditions on mutant selection

(e.g. duration of stress period or the most sui-

table stress temperature). In those cases in which a very low induceable mutation rate has to be expected a 'post propagation screening' was proposed

(110). This method gives the mutated cells

the chance to propagate under optimal temperature conditions before stress temperature

are

applied. Thus organogenesis of mu-

tant cell clusters probably will be encouraged. Tentative experiments on the selection of cold tolerant Fuchsia after in vitro treatment with chemical mutagens were recently described by Bouharmont and Dabin (111). Mutagenesis using callus and plantlets was employed to select for NaCl tolerant Chrysanthemum by Dasou and Short (112). Isolated salt tolerant clones were more succulent than control plants but exhibited comparable growth characteristics.

Conclusion For commercial in vitro propagation all variations from the original type are unacceptible

Suppression of callus formation, limi-

tation of the number of in vitro propagation cycles and axillary shoot initiation in chimeral cultivars generally result in uniform clonal progenies comparable with those from conventional propagation. Unstable cultivars have to be excluded from the mass propagation process. For breeding purposes spontaneous or induced variability, however, represent a valuable source for selection of improved types, and, therefore, the interest in this special field has tremendously increased in recent years.

396 References 1. Pierik, R.L.M.. 1985. Vermeerdering in kweekbuizen stijgt tot 30 miljonen. Vakblad. Bloem. _40 (26), 44-45. 2. Holdgate, D.P.. 1977. Propagation of ornamentals by tissue culture. In: Plant Cell and Organ Culture (J. Reinert and Y.P.S. Bajaj, eds.). Springer Berlin, Heidelberg, New York, 18-43. 3. Beauchesne, G.. 1983. Appearance of plants not true to type during in vitro propagation. In: Variability in Plants Regenerated from Tissue Culture (D. Earle and Y. Demarly, eds.). Praeger Scientific, 268-272. 4. Larkin, P.J., W.R. Scowcroft. 1981. Somaclonal variation - a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. £0, 197-214. 5. Doorenbos, J.. 1954. Notes on the history of bulb breeding in the Netherlands. Euphytica 3, 1-11. 6. Heursel, J.. 1977. Beschrijvende Lijst van Rhododendron simsii (Azalea indica) cultivars. Rijkscentrum voor Landbowkundig Onderzoek, Gent. 7. Preil, W.: unpublished 8. Wasscher, J.. 1956. The importance of sports in some florists flowers, Euphytica fj, 1 63-170. 9. Horn, W.. 1968. Genetische Ursachen der Variation bei Zierpflanzen. Gartenbauwiss. 3_3, 317-333. 10. Brabec, F.. 1965. Pfropfung und Chimären. Handb. Pflanzenphysiol. 15/2. Springer Heidelberg, New York, 388-498. 11. Tilney-Basset, R.A.E.. 1963. The structure of periclinal chimeras. Heredity ]_S, 265-285. 12. Bergann, F.. 1967. The relative instability of chimerical clones - the basis for further breeding. In: Induced Mutations and their Utilization (H. Stubbe, ed.). Abh. Dtsch. Akad. Wiss. Berlin, Kl. Medizin 1967, 2, 288-300. 13. Bateson, M.A.. 1916. Root-cuttings, chimeras and 'sports'. Journ. of Gen. 6, 75-80. 14. Statistisches Bundesamt. 1984. Land- und Forstwirtschaft, Fischerei, Fachserie 3, Reihe 3.1.6. Bodennutzung - Anbau von Zierpflanzen 1984, 1-15. 15. Bergann, F.. 1955. Einige Konsequenzen der Chimärenforschung für die Pflanzenzüchtung. Z. f. Pflanzenz. 3_4» 113-124. 16. Anonymous. 1983. Conclusions and Recommendations of FAO/IAEA Research Co-ordination Meeting on 'In-Vitro Technology for Mutation Breeding', Vienna, 1-13.

397 17. D'Amato, F.. 1977. Cytogenetics of differentation in tissue and cell cultures. In: Plant Cell, Tissue and Organ Culture (J. Reinert and Y.P.S. Bajaj, eds.). Springer Berlin, Heidelberg, New York, 343-357. 18. D'Amato, F.. 1978. Chromosome number variation in cultured cells and regenerated plants. In: Frontiers of Plant Tissue Culture (T.A. Thorpe, ed.), 287-295. 19. D'Amato, F.. 1984. Nuclear cytology of tissue culture. In: Plant Tissue and Cell Culture Application to Crop Improvement (F. Novak, L. Havel and J. Dolezel, eds.). Proc. of Int. Symp., Olomouc, 295-303. 20. Bayliss, M.W.. 1980. Chromosomal variation in plant tissues in culture. In: Perspectives in Plant Cell and Tissue Culture (I.K. Vasil, ed.). International Review of Cytology, Suppl. 11A, Academic Press, 113-144. 21. Constantin, M.J.. 1981. Chromosome instability in cell and tissue cultures and regenerated plants. Envir. Exp. Bot. 21, 359-368. 22. McClintock, B.. 1948. Mutable loci in maize. Carnegie Inst, of Wash. Yearbook 47, 153-169. 23. Fedoroff, N. . 1984. Transposable genetic elements in maize. Scientific American, June 1984, 65-74. 24. Stewart, R.N., H. Dermen. 1975. Flexibility in ontogeny as shown by the contribution of the shoot apical layers to leaves of periclinal chimeras. Amer. J. Bot-, 935-947. 25. Bergann, F., L. Bergann. 1982. Zur Entwicklungsgeschichte des Angiospermenblattes. 1. Über Periklinalchimären bei Peperomia und ihre experimentelle Entmischung und Umlagerung. Biol. Zbl. 101, 485-502. 26. Bergann, F., L. Bergann. 1983. Zur Entwicklungsgeschichte des Angiospermenblattes. 2. Über die Blattmusterbildung bei mesound diektochimärischen Formen von Peperomia-Arten, insbesondere über die Beteiligung des "Dermatogens" an der Mesophyllbildung. Biol. Zbl. 102, 403-429. 27. Bergann, F., L. Bergann. 1983. Zur Entwicklungsgeschichte des Angiospermenblattes. 3. Über unmaskierte Binnenfelder in den Blattspreiten periklinalchimärischer Buntheiten von Eleagnus pungens, Coprosma baueri, Ilex aquifolium, Hoya carnosa und Nerium oleander. Biol. Zbl. 102, 657-673. 28. Pohlheim, F.. 1985. Nachweis einer nichtchimärischen Scheckung bei Peperomia obtusifolia A. DIETR. Wiss. Z. Päd. Hochsch. Potsdam, 29, 12-18. 29. Bergann, F., L. Bergann.. 1960. Über die sogenannte Kräuselkrankheit der Poinsettie und die Beteiligung des "Dermatogens" bei der Mesophyllbildung. Flora 149, 331-344.

398 30. Bergann, F.. 1961. Eine weitere Trichimäre bei Euphorbia pulcherrima WILLD. Biol. Zbl. 80, 403-412. 31. Bergann, F.. 1961. Über zwischenzellige Genwirkungen (Partnerinduktionen) bei der Pigmentbildung in den Brakteen der Periklinalchimäre Euphorbia pulcherrima WILLD. "Eckes Rosa". Ber. Dtsch. Bot. Ges. 73, 40-41. 32. Bergann, F.. 1962. Über den Nachweis zwischenzelliger Genwirkungen (Partnerinduktionen) bei der Pigmentbildung in den Brakteen der Periklinalchimäre Euphorbia pulcherrima WILLD. "Eckes Rosa". Biol. Zbl. 8J_, 469-503. 33. Stewart, R.N.. 1960. Inheritance of bract colour in poinsettia. J. Heredity 5J_, 175-177. 34. Stewart, R.N., T. Arisumi. 1966. Genetic and histogenic determination of pink bract colour in poinsettia. J. Heredity 57, 217-220. 35. Sauer, A.: unpublished 36. Preil, W., M. Engelhardt. 1982. In vitro Entmischung von Chimärenstrukturen durch Suspensionskulturen bei Euphorbia pulcherrima WILLD. Gartenbauwiss. 4J7 , 241 -244 . 37. Broertjes, C., A. Keen. 1980. Adventitious shoots: do they develop from one cell? Euphytica 2_9 , 73-87. 38. Preil, W., M. Engelhardt, A. Lorenz. 1983. Analyse der genetischen Konstitution des Sproßscheitels bei verschiedenen rosafarbigen Poinsettiensorten durch Suspensionskulturen. Bundesmin. Ernährung, Landwirtschaft und Forsten. Forsch. Jahresber. 1983, L 22. 39. Ben-Jaacov, J., R.W. Langhans. 1 972. Rapid multiplication of Chrysanthemum plants by stem-tip proliferation. HortScience 1_, 289-290. 40. Earle, E.D., R.W. Langhans. 1974. Propagation of chrysanthemum in vitro: II. Production, growth, and flowering of plantlets from tissue cultures. J. Amer. Soc. Hort. Sei. 9_9 , 352-358. 41. Roest, S., G.S. Bokelman, 1975. Vegetative propagation of Chrysanthemum morifolium Ramat. in vitro. Sciencia Hort. 3, 317-330. 42. Stewart, R.N., H. Dermen. 1970. Somatic analysis of the apical layers of chimeral sports in chrysanthemum by experimental production of adventitious shoots. Amer. J. Bot. 5J7, 1061-1071. 43. Broertjes, L., A.M. van Harten. 1978. Application of mutation breeding methods in the improvement of vegetatively propagated crops. Elsevier Sei. Publ. Comp., Amsterdam, Oxford, New York. 44. Weaver, G.M.. 1963. The effect of Caesium 137 gamma radiation on plant growth and flower colour of greenhouse chrysanthemum cultivars. Can. J. Gen. Cytol. 5, 73-82.

399 45. Dowrick, G.J., A. El-Bayoumi. 1966. The origin of new forms of the garden chrysanthemum. Euphytica J_5, 32-3 8. 46. Bush, S.R., E.D. Earle, R.W. Langhans. 1976. Plantlets from petal segments, petal epidermis and shoot tips of the periclinal chimera, Chrysanthemum morifolium 'Indianapolis'. Amer. J. Bot. 63, 729-737. 47. Shigematsu, K., S. Hashimoto. 1978. The isolation of mutants by petal tissue culture from chimera flowers in chrysanthemum. IV.Int. Cong. Plant Tissue and Cell Culture. Univ. Calgary. Book of Abstr., 135. 48. Langton, F.A.. 1980. Chimerical structure and carotenoid inheritance in Chrysanthemum morifolium (RAMAT.). Euphytica 2 9, 807-812. 49. Yoder Bros., Inc. 1967. Evolution of the Indianapolis family. Grow. Circ. Newsl. No. 51. Yoder Bros. Inc., Barberton, Ohio, 1-4. 50. Preil, W. , M. Engelhardt. 1982. Bestimmung der chimärischen Konstitution des Sproßscheitels der Chrysanthemensorte "Indianapolis-Pink" durch Suspensionskultur. Forsch. Bundesmin. Ernährung, Landwirtschaft und Forsten. Jahresber. 1982, L 11-12. 51. Broertjes, C., S. Roest, G.S. Bokelmann. 1976. Mutation breeding of Chrysanthemum morifolium RAM. using in vivo and in vitro adventitious bud techniques. Euphytica 2_5, 11-19. 52. Cassells, A.C., D. Kelleher. 1984. Chimeral instability, breakdown and resynthesis as sources of somaclonal variation in Chrysanthemum. In: Plant Tissue Culture and its Agricultural Applications (P.G. Alderson, L.A. Withers, eds.). Proc. of 41st Conf. Easter School Ser. in Agric. Sei, Univ. Nottingham, Book of Abstr., p. 87. 53. Sutter, E., R.W. Langhans. 1981. Abnormalities in Chrysanthemum regenerated from long term cultures. Ann. Bot. 4_8 , 559-568. 54. Miyazaki, S., Y. Tashiro. 1977. Tissue culture of Chrysanthemum morifolium RAMAT. II. Variation in chromosome number of plants regenerated from stem segments in vitro. Agric. Bull. Saga Univ. 42, 27-42. 55. Miyazaki, S., Y. Tashiro. 1978. Tissue culture of Chrysanthemum morifolium RAMAT. III. Variation in chromosome number and flower colour of plants regenerated from different parts of shoots in vitro. Agric. Bull. Saga Univ. 4_4, 13-31. 56. Grunewaldt, J.: personal communication 57. Broertjes, C., B. Haccius, S. Weidlich. 1968. Adventitious bud formation on isolated leaves and its significance for mutation breeding. Euphytica V7, 321-344. 58. Grunewaldt, J.. 1980. Spontane und induzierte Farbschecken von Saintpaulia ionantha H. WENDL. Gartenbauwiss. 45, 124-128

40( 59

Norris, R.E., R.H. Smith. 1981. Regeneration of variegated African violet (Saintpaulia ionantha WENDL.) leaf chimeras in culture. Plant Physiol. 67_, 117.

60

Smith, R.H.,R.E. Norris. 1983. In vitro propagation of African violet chimeras. Hort Science 436-437.

61

Pohlheim, F.. 1974. Nachweis von Mischzellen in variegaten Adventivsprossen von Saintpaulia, entstanden nach Behandlung isolierter Blätter mit N-Nitroso-N-Methylharnstoff. Biol. Zbl. 93, 141-148.

62

Pohlheim, F.. 1981. Genetischer Nachweis einer NMH-induzierten Piastommutation bei Saintpaulia ionantha H. WENDL. Biol. Rdsch. V9, 47-50.

63

Reuther, G.. 1979. Die Gewebekultur als phytosanitäre Maßnahme bei der vegetativen Vermehrung von Elatior-Begonien. Gärtnerbörse und Gartenwelt 7ji, 840-842 .

64

Reuther, G.. 1980. Elatiorbegonien. 1. Weitere Untersuchungen zur Gewinnung von befallsfreien Elitepflanzen durch Gewebekultur. Gärtnerbörse und Gartenwelt ^0, 876-881.

65

Hakkaart, F.A., J.M.A. Versluijs. 1983. Control of leaf curl and Xanthomonas begoniae in Begonia 'Elatior' by meristem culture and an isolation test. Acta Hort. 131, 299-301.

66

Takayama, S., M. Misawa.. 1982. Factors affecting differentiation and growth in vitro, and a mass-propagation scheme for Begonia x hiemalis. Scientia Hort. 65-75.

67

Lindemann, A.. 1968. Mutationen bei Elatiorbegonien Rasse Rieger. Gartenwelt 68, 266-267.

68

Hilding, A., T. Welander.. 1976. Effects of some factors on propagation of Begonia x hiemalis in vitro. Swedish J. Agric. Res. 6, 191-199.

69

Theiler, R.. 1981. Einsatz der Gewebekultur zur Anzucht pathogenfreier Pflanzen: Möglichkeiten und Probleme ihrer Anwendung. Erwerbsobstbau 2_3, 18-20.

70

Mikkelsen, E.P., K.C. Sink. 1978. In vitro propagation of Rieger Elatior Begonias. HortScience J_3, 242-244.

71

Roest, S., M.A.E. van Berkel, G.S. Bokelmann, C. Broertjes. 1981. The use of an in vitro adventitious bud technique for mutation breeding of Begonia x hiemalis. Euphytica J30, 381-388.

72

Bigot, C.. 1981. Multiclication végétative in vitro de Begonia x hiemalis ('Rieger1 et 'Schwabenland'). 2. Conformité de plantes élevées en serre. Agronomie 441-447.

73

Bigot, C.. 1982. In vitro propagation of Begonia x hiemalis Fotsch: Plant conformity. In: Plant Tissue culture 1982 (A. Fujiwara, ed.). Proc. 5th Int. Cong. Plant Tissue and Cell Culture, 695-696.

401

74. Westerhof, J., F.A. Hakkaart, J.M.A. Versluijs. 1984. Variation in two Begonia x hiemalis clones after in vitro propagation. Scientia Hort. 2_4 , 67-74. 75. Reuther, G., N.N. Bhandari. 1981. Organogenesis and histogenesis of adventitious organs induced on leaf blade segments of Begonia elatior hybrids (Begonia x hiemalis) in tissue culture. Gartenbauwiss. j46 , 241-249. 76. Reuther, G.. 1983. Propagation of disease-free Pelargonium cultivars by tissue culture. Acta Hort. 131, 311-319. 77. Bergann, F., L. Bergann. 1959. Über experimentell ausgelöste vegetative Spaltungen und Umlagerungen an chimärischen Klonen, zugleich als Beispiele erfolgreicher Staudenauslese. I. Pelargonium zonale Ait. "Madame Salleron". Der Züchter 2_9, 361-374. 78. Bergann, F., L. Bergann. 1962. über Umschichtungen (Translokationen) an den Sproßscheiteln periklinaler Chimären. Der Züchter 32, 110-119. 79. Pohlheim, F.. 1983. Vergleichende Untersuchunaen zur Änderung der Richtuna von Zellteilungen in Blattepidermen. Biol. Zbl. 102, 323-336. 80. Cassells, A.C., G. Minas. 1983. Beneficially-infected and chimeral Pelargonium: implication for micropropagation by meristem and explant culture. Acta Hort. 131, 287-297. 81. Cassells, A.C.. 1985. Genetic, epigenetic and non-genetic variation in tissue culture derived plants. In: In Vitro Techniques, Propagation and Long Term Storage (A. Schäfer-Menuhr, ed.). Martinus Nijhoff/Dr. W. Junk Publ., 111-120. 82. Skirvin, R.M., J. Janick. 1976. Tissue culture induced variation in scented Pelargonium spp.. J. Amer. Soc. Hort. Sei., 101, 281-290. 83. D'Amato, F.. 1985. Spontaneous mutations and somaclonal variation. Proc. Int. Symp. on Nuclear Techn. and in Vitro Culture for Plant Improvement, Wien, IAEA-SM-282.(in press). 84. Walther, F., A. Sauer. 1985. In vitro mutagenesis in roses. Proc. 1st Int. Symp. on Research and Cultivation of Roses. Rehovot, Israel.(in press). 85. Walther, F., A. Sauer. In vitro mutagenesis in Gerbera jamesonii.(in this volume). 86. Horn, W.. 1984. Treating in-vitro cultures of floriculture crops with mutagens. Mutation Breed. Newsletters, 2_4, 13-15. 87. Schneider-Moldrickx, R., W. Horn. 1985. Vermehrung von Kalanchoe-Blossfeldiana-Hybriden in vitro. II. Kallus- und Suspensionskulturen. Gartenbauwissenschaft 5_0, 9-13. 88. Skirvin, R.M., J. Janick. 1976. 'Velvet Rose' Pelargonium, a scented Geranium. HortScience 11, 61-62.

402

89. Shigematsu, K., H. Matsubara. 1972. The isolation and propagation of the mutant plant from sectorial chimera induced by irradiation in Begonia rex. J. Japan. Soc. Hort. Sei. 41, 196-200. 90. Matsubara, H., K. Shigematsu, H. Suda. 1974. The isolation and fixation of a wholly mutant plant from a sectorial chimera induced by gamma irradiation in Begonia rex.. J. Japan. Soc. Hort. Sei. 4J3, 63-68 . 91. Roest, S., M.A.E. van Berkel, G.S. Bokelmann, C. Broertjes. 1981. The use of an in vitro adventitious bud technique for mutation breeding of Begonia x hiemalis. Euphytica 3_0, 381388. 92. Geier, T..1983. Induction and selection of mutants in tissue cultures of Gesneriaceae. Acta Hort. 131, 329-337. 93. De Jong, J., J.B.M. Custers. 1985. Induced changes in growth and flowering of Chrysanthemum after irradiation and in vitro culture of pedicels and petal epidermis. Euphytica (in press). 94. Grunewaldt, J.. 1983. In vitro mutagenesis of Saintpaulia and Pelargonium cultivars. Acta Hort. 131, 339-343. 95. Silvy, A., Y. Mitteau. 1985. Diversification des Varietes d'oeillet (Dianthus caryophyllus) par traitement mutagene. Proc. Int. Symp. on Nuclear Techn. and in Vitro Culture for Plant Improvement, Wien, IAEA-SM-282.(in press). 96. King, P.J.. 1984. Selection for plant variation using cultured cells. In: Efficiency in Plant Breeding (W. Lange, A.C. Zeven and N.G. Hogenboom, eds.).Proc. of 10th Congr. of Eucarpia.Pudoc Wageningen, 199-214. 97. Jung-Heiliger, H., W. Horn. 1980. Variation nach mutagener Behandlung von Stecklingen und in vitro-Kulturen bei Chrysanthemum. Z. Pflanzenzüchtg. 85, 185-199. 98. Broertjes, C., P. Koene, Th. Pronk. 1983. Radiation-induced low-temperature tolerant cultivars of Chrysanthemum morifolium RAM.. Euphytica 32/ 97-101. 99. Preil, W., F. Walther, M. Engelhardt. 1983. Breeding of lowtemperature tolerant poinsettia (Euphorbia pulcherrima) and Chrysanthemum by means of mutation induction in in vitro culture. Acta Hort. 131, 345-351. 100. Preil, W., M. Engelhardt. Flowering of in vitro selected lowtemperature tolerant mutants of Chrysanthemum cultivated under summer and winter conditions.(in preparation). 101. Broertjes, C., C.A.M. Lock. 1985. Radiation-induced low-temperature tolerant solid mutants of Chrysanthemum morifolium RAM.. Euphytica ¿4, 97-103.

403 102. Huitema, J.B.M., G. Gussenhoven, J.J.M. Dons, C. Broertjes. 1985. Induction and selection of low-temperature tolerant mutants of Chrysanthemum morifolium RAMAT. Proc. Int. Symp. on Nuclear Techn. and in Vitro Culture for Plant Improvement, Wien, IAEA—SM-282. (in press). 103. Binns, N.A.. 1981. Developmental variation in plant tissue culture. Env. Exp. Bot. 2A_, 325-332. 104. Chaleff, R.S.. 1981. Genetics of Higher Plants. Applications of Cell Culture. Cambridge Univ. Press. 105. Meins, F.. 1983. Heritable variation in plant cell culture. Ann. Rev. Plant Physiol. 34, 327-346. 106. Schlegel, G.. 1982. Influence of low temperature on regeneration of Saintpaulia ionantha H. WENDL.. Book of Abstr. 21st Int. Hortic. Congr. Hamburg 1982, Vol II, p. 1712. 107. Amberger, S., G. Schlegel, W. Horn. 1984. Selection for chilling tolerance in Saintpaulia ionantha. Proc. 12th Meetg. Eucarpia Sect. Ornamentals, Arslev, DK. 108. Warburton, J., B.W.W. Grout, K.C. Short. 1984. In vitro selection for cold-tolerant cell lines of Saintpaulia ionantha (WENDL). In. Plant Tissue and Cell Culture Application to Crop Improvement (F. Novak, L. Havel and J. Dolezel, eds.). Proc. of Int. Symp., Olomouc, 355-356. 109. Kieffel, B., F. Walther, W. Preil. 1985. X-ray induced mutability in embryogenic suspension cultures of Euphorbia pulcherrima. Proc. Int. Symp. on Nuclear Techn. and in Vitro Culture for Plant Improvement, Wien, IAEA-SM-282. (in press). 110. Walther, F., W. Preil. 1981. Mutants tolerant to low-temperature conditions induced in suspension cultures as a source for improvement of Euphorbia pulcherrima Willd. ex Klotzsch. In: Induced Mutations - A Tool in Plant Breeding, Wien, 399-405. 111. Bouharmont, J., P. Dabin. 1985. Application des cultures in vitro a l'amélioration du Fuchsia par mutation. Proc. Int. Symp. on Nuclear Techn. and in Vitro Culture for Plant Improvement, Wien, IAEA-SM-282. (in press). 112. Dalsou, V., K.C. Short. 1986. Selection for sodium chloride tolerance in chrysanthemums. Acta Hort. (in press).

405

PROTOPLAST CULTURE AND USE OF REGENERATION ATTRIBUTES TO SELECT SOMATIC HYBRID TOMATO PLANTS

K.C. Sink, L.W. Handley, R.P. Niedz and P.P. Moore Department of Horticulture, Michigan State University, East Lansing, Michigan 48824

Introduction The cultivated tomato is an ideal plant species for somatic cell genetics.

It is an important vegetable crop throughout the world,

has a diploid genome, and an extensive genetic map.

Cell manip-

ulations based on protoplast systems, however, have been hindered by the lack of efficient protoplast culture and shoot regeneration methods for this species.

Herein, we report new protocols de-

vised for Lycopersicon esculentum cultivars taxonomic species, Solanum lycopersicoides of their somatic hybrid plants

(1), a related (2) and the selection

(3) based on differential regen-

eration attributes of the parental and hybrid protoplasts and resulting calli.

Results Lycopersicon esculentum Protoplasts were isolated from the basal, fully expanded leaves of 3- to 4-week old seedlings of eight tomato cultivars grown in a controlled environment chamber

(CEC).

(Table 1)

The CEC

conditions were 200 pEm _ 2 s -l from cool white fluorescent bulbs on a 16 h photoperiod and a 27°C/22°C day-night temperature regime.

Slicing the leaves transversley, using preplasmolysis

and the appropriate enzyme combination resulted in consistent yields of 1 x 10 6 protoplasts per gram of leaf tissue.

Older

seedlings, 5-6 weeks, had a 70-90% decrease in protoplast yield. The eight cultivars varied in plating efficiency

(PE) as deter-

mined by the number of viable protoplasts 24 h after plating that

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

406 formed colonies after two weeks in liquid modified 8E (M8E) medium (1).

In this study, the PE ranged from 27-87% with

Lukullus being the lowest (28%) and LA 1268 (87%) the highest. As indicated in Table 1, PE, degree of browning and ability to regenerate shoots are responses observed in vitro that are apparently independent.

For example, P.I. 126914 had a high PE

(80%), and minimal browning, yet failed to regenerate shoots. Table 1.

Morphogenetic Responses of 8 Genotypes of L. esculentum.

Line

Plating efficiency 3

Browning13

LA 1268 Lukullus Red Cherry P.I. 126914 Ventura Walter Sub-Arctic Maxi LA 1622

87 ± 28 ± 65 ± 83 ± 58 ± 35 i 75 ± 0.1

+ ++ + + ++ ++ ++ +++

a

4 5 6 4 4 5 14

Calluses (No.)

75 81 57 0 45 34 40 0

Calluses regenerated (no.)

Shoot regenerntion m

2 4 13 0 4 1 5 0

2., 7 4 .. 9 22.. 8 0 8., 0 2.9 12.. 5 0

Data reported as mean ± S..E.

^Browning:

+, minimal ; ++,, moderate; +++, severe.

This new protocol alleviated the previous constraints exhibited by tomato protoplasts in culture.

These constraints included

low PE, browning and deterioration of protoplast-derived calli, and lack of consistent shoot regeneration.

In essence, the use

of protoplasts with high viability and changes in the protocol that led to maintaining vigorous cell division to the macrocallus stage were beneficial.

In specific, following a 4-5 d period in

the dark, the cells will form 35-50 cell colonies at 21 d.

To

promote sustained growth, the liquid cultures are divided in half; thereby, decreasing the medium volume to about 2 ml per 60 x 15 mm petri dish.

During this initial culture stage, the

frequent addition of fresh medium and the removal of used medium also stimulates growth.

Finally, the transfer of 8-10 week-old

macrocalli onto filter paper overlaid on agar resulted in calli ready to transfer to regeneration medium in 10-12 weeks.

As

indicated in Table 1, the differences in degree of browning suggest genetic factors also play a role in this trait.

However,

the protocol used permitted the production of ample macrocalli of the moderate browning category genotypes to test shoot regeneration.

407 Shoot regeneration was likewise promoted by in vitro cultural procedures designed to maintain active cell division and the subsequent early dissection of young shoot primordia.

The shoot

regeneration medium was Murashige and Skoog (4) salts and vitamins + zeatin (2 mg/1) + sucrose (30 g/1).

When calli growing on the

filter-paper on agar attained a 0.5 - 1.0 cm size, they were placed on MS2Z medium for shoot regeneration.

Shoot primorida

became visible for some genotypes within 6-10 weeks on MS2Z. Among the six genotypes that exhibited shoot morphogenesis, the range was from 1 to 22% of the calli among the lines studies. Shoot regeneration and elongation was promoted by excising shoot primorida from the parental callus and culturing them individually on MS2Z medium.

Primorida left intact would remain static without

exhibiting elongation.

Gibberellic acid at 1 mg/1 stimulated

shoot elongation, but was not required for excised primorida. Excised shoots (0.5 - 1.0 cm) rooted in MS medium containing 10 g/1 sucrose.

Solanum lycopersicoides The protocol established for this species was aided by previous cell and tissue culture studies conducted in our laboratory

(5).

Since seeds of the accession LA 1990, obtained from C.M. Rick, University of California, Davis, were not in abundant supply, a callus cell source had to be devised to provide consistent yields of protoplasts for the experiments.

This was done by initiating

cell suspension cultures (CSC) with callus taken from stem internode cross-sections placed on MS + 15 mg/1 NAA (5). Protoplast

yields of 9-12 x 10^ per 10 mis of packed cell volume

were routinely obtained from CSC 2-5 d after subculture.

Standard

procedures were used for enzymatic isolation and washing of these protoplasts prior to plating in the two media used (2).

After

initial culture in the dark for 3 d at 28°C, both media yielded abundant cell colonies in 6-8 weeks.

No benefit was observed by

deleting NH .NO, from the culture medium (medium B, Table 2).

408 This is in contrast to other Solanaceous species where deletion of NH^ has been found beneficial (6, 7).

Likewise, no benefit

was found by using the liquid on agarose culture method employing agarose type VII (Sigma) at 0.7%.

In both media A and B, the use

of agarose reduced PE almost 40% (Table 2) and in some cases produced only a few large colonies. Table 2.

Plating Efficiency and Percent Shoot Regeneration of

Suspension Culture-Derived Protoplasts of Solanum lycopersicoides in Media A and B. P.E. (%) (± S.D.)

No. calli placed on MS3ZG

Shoot regeneration 2nd month (No. calli)

Percent shoot regeneration

A w/o agarose

33 + 3

470

338

72

A with agarose

20 ± 4

270

184

68

B w/o agarose

28 + 3

165

112

68

B with agarose

16 ± 5

135

67

50

Medium

When the microcalli were 0.5 - 1.0 mm in size (3-5 weeks old), they were transferred to the surface on filter paper overlaid on solidified MS medium + 3 mg/1 zeatin + 0.1 mg/1 gibberellic acid (MS3ZG) for shoot regeneration.

After 6 weeks on the filter

paper-agar setup, the calli were transferred to MS3ZG medium without filter paper. approximately a month.

On this medium shoot initials emerged in Such regenerating calli were subcultured

to new MS3ZG medium to promote shoot elongation. consistently yielded 1 to 5 shoots.

Most calli

Calli originating from

medium A had high shoot regeneration frequency

(70%) irrespective

of whether they were from liquid or liquid on agarose culture (Table 2).

However, calli from medium E showed a marked differ-

ence in shoot regeneration relative to culture method.

Those

calli taken from the liquid on agarose dishes had about 25% lower shoot regeneration than those grown in liquid medium.

409 The total length of time from protoplast isolation until harvest of regenerating shoots was from 14-18 weeks.

The shoots were

rooted in 1 to 2 months by insertion of the basal end into solidified MS + 0.1 mg/1 GA medium.

Plants were subsequently

acclimated in soilless planting medium to low humidity conditions and then transferred to a greenhouse. Somatic hybrid plants between tomato and Solanum lycopersicoides During the protoplast culture and regeneration studies involved with tomato and Solanum lycopersicoides it was apparent that a potential selection system was available that may permit recovery of hybrid calli (Figure 1).

This scheme was based on the fact

that protoplasts of the 20-22nd subcultures of S. lycopersicoides would not divide in M8E medium.

Tomato protoplasts divided in

M8E and would only exhibit low frequency shoot regeneration. This model was further enhanced as will be discussed later. Initial fusions, however, were hindered by the tendency for lycopersicoides protoplasts to float in the medium used to initiate fusion.

This problem was alleviated by following the

isolation protocols previously outlined, but resuspending the protoplasts in W5 salt solution (8). Figure 1.

Selection System for Hybrid Cells in Medium M8E.

Lycopersicon esculentum

Solanum lycopersicoides

(mesophyll)

(suspension)

I

protoplasts

colonies callus

I

Fusion

protoplasts

PEG - DMSO

no growth

I

colonies hybrid callus somatic hybrid plants

410 Thus, a mixture of 2 x 10® leaf protoplasts of the tomato cultivar Sub Arctic Maxi and 9 x 10® suspension culture derived protoplasts of S. lycopersicoides in 12 mis of W5 was prepared.

A modifica-

tion of the procedure of Menczel and Wolf (8) using 12% PEG (8,000 mol. wt.) and 10% DMSO was employed.

One ml of this

fusogen was placed in a 16 x 100 mm glass test tube, overlaid with 0.5 ml of the protoplast mix, and allowed to stand for 3 min.

The mixture was gently agitated and allowed to stand for

an additional 5 min.

This suspension was then diluted by gently

adding 8.5 mis of W5 solution containing 50 mM MES buffer at pH 5.6.

Protoplasts were incubated in this solution for 30 min.,

centrifuged

(60 x g; 5 min.), and the supernatant discarded.

The protoplast pellet in each tube was resuspended in 4 mis of M8E and 2 mis were placed in each 60 x 15 mm petri dish. Control dishes of tomato and S. lycopersicoides protoplasts did not survive the PEG/DMSO fusion.

No viable protoplasts were

observed after a week in culture and no colonies subsequently appeared.

Thus, in addition to the model selection system as

depicted in Figure 1, division of tomato protoplasts was inhibited by the PEG/DMSO fusion procedure.

In contrast, p-calli appeared

in fusion dishes following a feeding regime with M8E without 2,4-D and gradually decreasing the level of mannitol

(2).

The

culture sequence was essentially the same as used for S. lycopersicoides .

It was obvious that some macrocalli arising on the

filter paper overlaid on MS3ZG medium had a different phenotype than calli from the parents as observed in previous studies. Tomato calli from M8E were characteristically light to dark brown in color with a watery appearance; whereas, those of £>. lycopersicoides were yellow to light green.

Such parental calli did

appear on the filter paper on agar stage, but the majority were very dense and hard; had a lime-green to whitish color. calli grew much more rapidly than either parent.

These

Such hybrid

vigor of fusion products has previously been used as a selection system in Datura (9) and Solanum (10).

These calli were individ-

ually selected and placed on MS3ZG medium without filter paper.

411

Shoot regeneration of these calli began after only one month on MS3ZG.

One hundred separate calli out of 480 which regenerated

shoots were randomly selected and retained.

Eight of the first

calli to regenerate shoots have been examined by biochemical and cytological methods to verify their hybrid origin (Table 3).

One

of these, number 67, did not root; thus, has not been completely examined.

Differences between tomato and S. lycopersicoides were

found for 5 enzyme systems.

These five enzymes represent seven

loci which are mapped to five chromosomes in tomato.

They in-

clude Skdh-1 on chromosome 1, Pgm-2 on chromosome 4, Got-2 and Got-3 on chromosome 7, Got-4 on chromosome 8, and Pgi-1 and Pgdh-2 on chromosome 12 (3).

For example, Skdh-1 is a monomer

expressed in roots and mapped to chromosome 1 (11) and both parental bands were detected in 7 somatic hybrids (Figure 2). Table 3.

Cytology and Seed Set of L. esculentum + S. lycopersicoides Somatic Hybrids Chromosome no.

% Pollen fertility

Self fruit set

S. lycopersicoides

24

91

+

L. esculentum Sub Arctic Maxi

24

96

+

L. esculentum x S. lycopersicoides

24

2

23

68

30

36

64

8

-

47

60

39

-

57

53, 54

49

5

67

NR

-

-

69

48 , 53, 54, 55

43

1

165

48

36

1

204

48

2

Line

NR - No rooting

-

2

-

412

Figure 2. Skdh-1 isozyme patterns (left to right): S. lycopersicoides, sexual hybrid, tomato, typical somatic hybrid.

No differences in the large subunit of fraction 1 protein were observed.

However, for the small subunit (SS), two double bands

occurred for S. lycopersicoides and three for tomato.

The seven

somatic hybrids analyzed had the SS bands of both parents. The number of chromosomes for the seven somatic hybrids regenerated to plants varied from the expected 2n=4x=48 to 2n=68 (Table 3).

Two plants, 57 and 69, had mixoploid roots.

In

general, the somatic hybrids have a vegetative and floristic phenotype primarily like S. lycopersicoides, which was previously observed for the unilateral sexual hybrid

(12).

References 1.

Niedz, R.P., S.M. Rutter, L.W. Handley and K.C. Sink. 1985. Plant regeneration from leaf protoplasts of six tomato cultivars. Pit. Sci. 39, 199-204.

2.

Handley, L.W. and K.C. Sink. 1985. Plant regeneration of protoplasts isolated from suspension cultures of Solanum lycopersicoides. Pit. Sci. (In Press).

413 3.

Handley, L.W., R.L. Nickels, M.W. Cameron, P.P. Moore and K.C. Sink. 1985. Somatic hybrid plants between Lycopersicon esculentum and Solanum lycopersicoides. Theo. Appl. Genet. (In Press).

4.

Murashige T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497.

5.

Handley, L.W. and K.C. Sink. 1985. Plant regeneration of Solanum lycopersicoides Dun. from stem explants, callus and suspension cultures. Plant Cell, Tissue and Organ Cult. (In Press).

6.

Bokelmann, G.S. and S. Roest. 1983. Plant regeneration from protoplasts of potato (Solanum tuberosum cv. Bintje). Z. Pflanzenphysiol. 109, 259-265.

7.

Boyes, C.J. and K.C. Sink. 1981. Regeneration of plants from callus-derived protoplasts of Salpiglossis. J. Amer. Soc. Hort. Sei. 106, 42-46.

8.

Menczel, L. and K. Wolfe. 1984. High frequency of fusion induced in freely suspended protoplast mixtures of polyethylene glycol and dimethylsulfoxide at high pH. Pit. Cell Rpts. 3, 196-198.

9.

Schieder, 0. 1980. Somatic hybrids between a herbaceous and two tree Datura species. Z. Pflanzenphysiol. 9j3, 119127.

10.

Austin, S., M.A. Baer and J.P. Helgeson. 1985. Transfer of resistance to potato leaf roll virus from Solanum brevidens into Solanum tuberosum by somatic fusion. Pit. Sei. 39, 75-82.

11.

Tanksley, S.D. and C.M. Rick. 1980. Isozymic gene linkage map of the tomato: Application in genetics and breeding. Theo. Appl. Genet. 57, 161-170.

12.

Rick, C.M. 1951. Hybrids between Lycopersicon esculentum Mill, and Solanum lycopersicoides Dun. P.N.A.S. 37_, 741744.

415

SEXUAL REPRODUCTION IN PLANTS BY APPLYING THE METHOD OP TEST TUBE FERTILIZATION OP OVULES

M.. Zenkteler Faculty of Biology, Adam Mickiewicz University, Poznari, Poland f

A.. Slusarkiewicz—Jarzina Institute of Plant Genetic®, Polish Academy of Sciences, ul. Strzeszyriska 30/36, Poznari, Poland

Introduction The method of in vitro pollination of ovules makes it possible: 1/ to study in detail the processes of fertilization and embryogenesis in controlled conditions; 2/ to obtain viable seeds capable of germinating in situ; 3/ to raise interspecific and intergeneric hybrid embryos and plants in those cases when normal in vivo hybridization fails; 4/ to induce parthenogenesis and the development of haploid embryos and plants; 5/ to interfere in the development of the female gametophyte following pollination of immature ovules. In vitro self-pollination of placentas with ovules has been performed successfully with about 35 species and usually with those whose ovaries contained a large number of ovules / 1.2.3/. The process of sexual in vitro hybridization has been successfully applied to obtaining hybrid embryos and plants among several species of Caryophyllaceae and Solanaceae. In the case of monocotyledons it was possible to obtain well developed embryos after direct pollination of ovules of Zea mays with Z. mexicana / 7/. The present report contains the preliminary results of experiments carried out on self- and cross-pollination of ovules of certain species of Solanaceae, Scrophulariaceae.Primulaceae. Tritlcum aestivum and self-pollination of immature ovules of Melandrium album.

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

416

Material and Methods The following species growing in the Botanical Garden in Poznari and in the greenhouse of the Institute of Plant Genetics, Polish Academy of Sciences in Poznari, were used f o r the experiments: Scopolia carniolica. Physochlaina praealta. Nicotiana tabacum cv. Samsun, N. alata. N. l o n g i f l o r a , N. debneyl. N. knightiana. N. g y l v e s t r i s . N. sanderae. Hyoscyamus albus. Petunia hybrida, P. parodli. Lyciurn halimlfolium, Primula pubescens. P. auricula. D i g i t a l i s lutea. D. purpurea. Torenia f o u r n i e r l . Melandrlum album and Triticum aestlvum cv. Dlamand. Flower buds of those species which ovules were to be obtained f o r culture work were emasculated and bagged 2-4 days before pol l i n a t i o n . P i s t i l s were b r i e f l y s u r f a c e - s t e r i l i z e d with 70 % ethanol, then with 0.1 % of mercuric chloride or chlorine water f o r several minutes. Following s u r f a c e - s t e r i l i z a t i o n the p i s t i l s were rinsed 3-5 times in s t e r i l e water. Isolated placentas with ovules were cultivated on media prescribed by Nitsch / NJ £ / r Murashige and Skoog / MSJ / and Rangaswamy / 6 /. The same day, anthers were excised from the s t i l l - c l o s e d flower buds and kept during several hours in the s t e r i l e chamber, l a t e r the pollen grains were scooped out and spread on the surface of the cultured ovules. A l l the experimental material was cultured in the dark at a temperature of 22 - 26°C. Isolated placentas, with ovules of Melandrium album at various stages of megasporogenesis and megagametogenesis were immediately pollinated following inoculation. During the next 4 days of culture ovules were f i x e d and l a t e r on sectioned on the microtome. Ovules containing hybrid embryos at d i f f e r e n t stages of development were cultured in the above mentioned media with various concentrations of kinetin, zeatin, 6-benzylaminopurine / BAP /, 2,4-dichlorophenoxyacetic acid / 2,4-D /, indoleacetic acid / IAA /, naphtaleneacetic acid/ NAA /.

Results Table 1 shows the process of sexual reproduction in v i t r o in self-pollinated species. In a l l the species of Nicotiana and

417

Petunia as well as in Sconolla carniolica pollen grains germinated several hours after inoculation. Fertilized ovules started to increase on the 3-rd day. Table 1 Self-Pollination Of Placentas With Attached Ovules Species Nicotiana alata Nicotiana tabacum cv, Samsun Nicotiana longiflora Nicotiana debneyi Nicotiana knightiana Petunia parodii Petunia hybrida Scopolia carniolica Primula pubescens Primula auricula Digitalis purpurea Digitalis- lutea Torenia fournieri

Embryos

Plants

+

+

+

+

+

+

+

-

+

+

+

+

+

+

+

+

+

+

+

-

+

+

+

+

+

+

Dissect ion of enlarged ovules / Fig. 1 / at various stages of their growth revealed the presence of embryos and endosperm» Depending on the species used in our experiments fully developed embryos were observed after 3-6 weeks after pollination. After isolation of the cotyledonary embryos they started to grow and developed into plants. The ovules^ of both species of Primula were usually pollinated 24-48 hours after inoculation. Fertilized ovules increased and after 5 weeks of culture mature embryos were present. In dissectioned enlarged ovules it was found that some of them contained only remnants of degenerating embryos which probably collapsed shortly after fertilization. Plants were developed from isolated embryos of Primula pubescens. The pollination of ovules of both Digitalis species and of Torenia fournieri took place the same day as inoculation. In Digitalis most of the ovules attached to the placentas increased in size during the next

418

Fig. 1. Developing ovules of Scopolia carniolica 21 days after self-pollination /20X/J Pig. 2. Embryo with cotyledons of Torenla fournieri 4 weeks after self-pollinationj Pig. 3.. Germinating pollen grains / arrow / of Primula pubescens oil the ovules of P. auricula /5X/. several days, however, only in some ovules embryos and endosperm were developing. Seedlings were obtained from cotyledonary embryos of Digitalis purpurea isolated from ovules after 5 weeks of culture.. Ifeture embryos were also obtained by self-pollination of ovules of Torenia fournieri / Pig. 2 /. A few days after pollination certain ovules enlarged and among those some contained only endosperm, many ovules were empty and only sporadically embryos at various stages of embryogenesis were found. Cotyledonary embryos isolated from ovules 6 weeks after pollination germinated on media and produced fully formed plants. The technique used in cross-pollination of ovules was similar to that applied in self-pollination. Hybrid globular embryos were obtained in the following crosses: Nicotiana longiflora x N. deb— neyi, N. longiflora x N. knightiana, N. sanderae x N. debneyi, N. sylvestris x N. knightiana, Primula pubescens x P. auricula / Figs. 3,4 /- Proembryos developed usually till the 15-th day after pollination in vitro and later on they collapsed. The development of proembryos concomitantly with the endosperm,-of endosperm only, or of proembryos only occurred in various combinations. The efforts to culture ovules with hybrid proembryos on various media were unsuccessful.

419

Pig. 4. Globular hybrid embryo of P. pubescens x P. auricula 15 days after cross-pollination. Pig. 5 . Hybrid plant of Nlcotlana tabacum x N. knightlana in test tube /0,5X/. Pig. 6. Developing ovules situated on the placenta of N. tabacum after pollinating in vitro with pollen of Petunia parodli. 4 days after cross-pollination /5X/.. Pig. 7. Hybrid embryo of N. tabacum x P. parodli 14 days after cross-pollination.. Hybrid embryos and plants were obtained only from cross N. tabacum x N. knight lana.. After 3 days of culture in some of the enlarged ovules developed proembryos and endosperm. Enlarged ovules, 8 days after pollination, were transferred to MS medium supplemented with 2 mg/1 of 2,4-D. Three weeks later certain ovules burst and a mass of white calluses appeared. Small fragmenta of calluses were again transferred on MS medium supplemented with 4 mg/1 of kinetin and 3 mg/1 of NAA. During the next several weeks of culture calluses produced shoots which, after transferring on MS medium containing 2 mg/1 of NAA, developed into fully formed plants / Pig. 5 /. Prom among 14 plants growing in pots, in 8 plants the chromosome numbers were 2n=36, remaining 6 plants were aneuploids. Promising results were obtained in the cross Nicotiana tabacum cv.. Samsun x Petunia parodli. where after 2-3 weeks of culture hybrid embryos reached the stage of torpedo / Pigs. 6,7 /. Those embryos or whole ovules were excised and transferred on MS medium with 2 mg/l of kinetin and 1 mg/1 of IAA„ During 3 weeks of culture the embryos produced calluses. Unfortunately, this medium did

420 not support the organ formation. In the crosses Scopolia carnioIlea x Physochlaina praealta and N. tabacum x Hyoscyamus albus. in certain ovules the endosperm was very poor but the hybrid embryos reached the preheart stage. Some of the ovules, or whole globular embryos, were transferred and cultured on MS medium with various concentration of kinetin and NAA. In the case of Scopolia x Physochlaina. these media did not support a good growth of embryos and after a few days of culture the embryos collapsed. About 30 % of the hybrid embryos of N. tabacum x H. albus produced calluses on MS medium with 2 mg/l of kinetin and 1 mg/1 of IAA. Unfortunately, calluses did not differentiate and no organ were obtained. A much more complicated technique was the isolation and culture of single ovules of Trlticum aestivum. During opening the ovaries and isolating the ovules a lot of them became injured and degenerated already before transplanting on medium. The intact ovules were carefully transferred on medium and immediately pollinated. Pollen germinated sporadically and short tubes burst soon. However, pollen grains of Melandrium album when put directly on ovules started to germinate after several hours and later on pollen tubes grew in various, directions. Microscopic analysis of pollinated ovules cultured for 24 hours revealed the presence of pollen tubes inside the embryo sacs. It was not possible to find the process of fertilization, however, in certain embryo sacs additional -nuclei of unknown origin - probably hybrid endosperm — were distinguished. There are no data on pollen germination and pollen tube growth on the immature ovules. We have presumed that a pollen tube when present on the surface of an ovule may enter the ovule in spite of the lack of a mature embryo sac. On the basis of our preliminary investigations we found that in cultured immature ovules of Melandrium album the process of megagametogenesis was not distorted and that mature embryo sacs were formed several days after inoculation. Knowing this we cultured and pollinated ovules at the stages of 1-, 2- and 4-nucleate embryo sacs. Pollen grains germinated abundantly and their tubes spread all around the ovules / Pigs. 8,9 /. In the dissectioned ovules remnants of pollen tubes were sporadically observed inside the micropylar end of the embryo sacs. At present we are not able to explain what happened with the

421

Pig. 8. Germinating pollen grain / arrow / of Melandrium album on the immature ovule of M» album / ovule at the stage of 2-nucleate embryo sac /» Pig. 9. Pollen tube of M» album entering the micropyle / arrow / of the immature ovule of M» album» two: male gametes after entering the pollen tube inside the micro— pylar region. In certain immature ovules which were cultured for 72 hours the number of nuclei inside the immature embryo sacs increased. Their size and location were different from those of the control material»

Discussion There are certain species which can be reproduced sexually by applying the method of test-tube pollination of ovules» Unfortunatelly, the number of hybrids obtained in test tubes is very limited. One of the main obstacles which hinders the development of hybrids among species belonging to remote taxonomical groups is the lack of suitable methods for culturing the globular hybrid embryos» A s was shown in our report as well as in other papers / 8,2,1° /» present we are not able to induce proembryos to develop further on media. There is a n urgent need to carry out investigations in this direction in order to find the most efficient methods for the culture of proembryos. Only then can the value of method of wide crosses in test tubes be appreciated. There are no data on ovule cultures at the stages of megasporogenesis and megagametogenesis. It can be presumed that in vitro both processes may proceed differently than in vivo and consequently

422 new or aberrant female gemetophytes might develop. It is quiet probable that pollination of immature ovules can also evoke certain deviation from normal development of female gametophytes. Our preliminary experiments carried out on only one species show that male gametophytes germinate on immature ovules and that pollen tubes enter immature female gametophytes. It would be of great value to induce the nuclei of immature female gametophyte or male gametes to divide and develop into embryos. The method of test-tube pollination of immature ovules may also provide answers to the following question: do pollen tubes enter the ovule at the stage of megasporogenesis if so, then can the male gametes fuse with megaspores? Whatever the value of this question its confirmation or refutation requires thorough further research.

423

References 1 • Z e n k t e l e r , M. 1980.. I n t . R e v . C y t o l . . S u p p l . 11B. 137-156» 2 . S t e w a r t , J-McD- 1981. E n v i r o n . E x p . B o t - 21_, 3 0 1 - 3 1 5 . 3.. S l a d k y , Z . , M. G r i g a , J.. J u r o c h . 1982. S c r i p t a Univ.-Purk.Brun. 1_2, 371-376. 4 . N i t s c h , J . P . 1951. Am-J-Bot. 2 8 ,

Fac.Sci.Nat.

566-577.

5.. Murashige, T . , F . Skoog. 1962. P h y s i o l . P l a n t . 1jj_, 4 7 3 - 4 9 7 . 6 . Rangaswamy, N..S. 1961. Phytomorphology 11_, 109-127. 43-46. 7 . D h a l i w a l , S . , P . J . King. 1 9 7 8 . T h e o r . A p p l . G e n e t . » 8„ S l u s a r k i e w i c z — J a r z i n a , A», M.. Z e n k t e l e r . 1983. E x p e r i e n t i a 39.1399-1400. 9 . Z e n k t e l e r , M.., G„ M e l c h e r s . 1978. T h e o r . A p p l . G e n e t .

581-90.

1 0 . Z e n k t e l e r , K.,. W„ N i t z s c h e . 1984.. T h e o r . A p p l . G e n e t . 68, 311-315.

425

CYTOGENETIC

U. Becker,

STUDIES

G.

IN C A L L U S C U L T U R E S

OF ASPARAGUS

OFF.

Reuther

I n s t i t u t für Botanik, Germany .

Forschungsanstalt

6222 G e i s e n h e i m ,

West-

Introduction Asparagus plants

is a d i o e c i o u s

are heterogametic

(xx). The

homogametic

ones

(xx)

and supermale

(yy)

In A s p a r a g u s

of e q u a l

sized spears.

called all-male of t h e

Therefore

induced.

After

from all male

testing

for

a higher

are

Subcultures of

0.5 m g / 1

lets were

1 mg/1

seed

of t h e

of t h o s e

supermale

plants

explants placed on modified

1 mg/1 NAA

and 1 mg/1

for o r g a n o g e n e s i s w e r e I A A + 0.1 m g / 1

soil.

In t h i s m a n n e r

52) of s o m e

and totally

Asparagus,the

dedifferentiated

of t h e r e g e n e r a t e s

is of g r e a t

subculture

DNA-content

steps

(jj)

of n u c l e i w e r e

com-

male

steps,

and female highly

(3)

(up

to

cultivars

organogenic uniformity

(4). T h e m a j o r p r o b l e m

in c a l l u s c e l l s , m a i n l y

(6). T h e r e f o r e

trans-

attained.

of F-^-hybrids t h e

importance

IAA

the c a l l u s b o r n p l a n t -

lines c o u l d be

for b r e e d i n g

is t h e a l t e r a t i o n of t h e p l o i d y

was

set up w i t h hormone

long-term callus cultures

callus

(2J.

Linsmaier/

IAA a n d subsequently

a few subculture

callus born plants

and the

Kin callus growth

For rooting

selected supermale,

established. After

so

BA for m a l e p l a n t s a n d 1 m g / 1

k e p t on m o d . L/S + 0.5 m g / 1

subculture

pro-

productivity

p r o p a g a t i o n m e t h o d for

2 - i P for f e m a l e p l a n t s .

ferred into

can

female

combining

for F ^ - h y b r i d

reveal

to

plants

The results

the cultivars consist

depression

from meristematic

binations

rous

selfing.

male

(xy).

vegetative

Skoog basal medium with

Using

vary

of p a r e n t p l a n t s b y c a l l u s c u l t u r e h a s b e e n t e s t e d

Starting

were

or b y

the male plants

inbreeding

are

so c a l l e d a n d r o m o n o e c i o u s

individuals.

F-^-hybrids

l a c k of a c o n v e n t i a l cloning

female plants

t h e y c a n b e u s e d as p a r e n t p l a n t s

duction.

Because

the

(1). T h o s e

fertilized with each other

ability

+

1:1. The

(xy),

f l o w e r s of g e n e t i c a l l y m a l e p l a n t s

hermaphroditic be

s p e c i e s w i t h a s e x r a t i o of

comparative

after

c a r r i e d out by c y t o p h o t o m e t r i c

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

nume-

studies of

the

measure-

426 ments, on Feulgen stained slices

(]_) • For each staining series con-

trol measuring was done at root sections of diploid and tetraploid Asparagus plants. In order to eliminate variations in staining density, they were used as standards for the classification of the DNAvalues found in the callus

sections.

Results The nuclear DNA-content of the roots of diploid and tetraploid Asparagus plants is shown in Fig. 1 and Fig. 2. Very clear

is the

main peak at the 2C level in the diploid root, while the tetraploid tissue has the main peak at the 4C level, as it is expected.

20

Fig.

2C

4C

8C

2C

«

8C

DNA

The DNA-values of an apical meristem of an in vivo grown spear, as it is used as expiant for starting culture, are expressed in Fig. 3. It is the normal DNA-distribution of a diploid tissue in mitotic activity, with a distinct peak at 2C. Fig. 4 is the histogram of an in vitro grown shoot tip after the first subculture step on mod. L/S + 1 mg/1 NAA + 1 mg/1 Kin. It shows a very similar DNA-profile as it is demonstrated in Fig. 3.

427 Comparing the two different callus types, the organogenic one shows greater ploidy stability than the dedifferentiated one. In Asparagus callus of high organogenic competence the 2C and 4C phases are dominant in the meristematic areas, ture steps

(Fig. 5) even after 52 subcul-

(Fig. 6).

30Fig. 5

Fig. 6

li.

2(r

2C

4C

8C

2C

i,C

8C

DNA

The measurements of the dedifferentiated callus types contrast sharply to the results of the organogenic ones. The pure callus areas show a shift from diploidy to polyploidy. Also the number of abnormal nuclei and high polyploid cells even up to 14C or more is increasing

(Fig.

7+8).

JL Fig. 8

Fig. 7

20'

2C

4C

2C

UC

5, 309-320. 18. Karp, A . , R.S. Nelson, E. Thomas, S.W.J. Bright. 1982. Chromosome v a r i a t i o n in protoplast derived potato plants. Theor. Appl. Genet. 63, 265-272. 19. Creissen, G.P. , A. Karp. 1985. Karyotypic changes in potato plants regenerated from protoplasts. Plant Cell Tissue Organ Culture 4, 171182. 2a

F i s h , N., A. Karp. 1986. Improvements in regeneration from protoplasts of potato and studies on chromosome s t a b i l i t y . 1. The effect of i n i t i a l culture media. Theor. Appl. Genet, (submitted)

21. Nelson, R . S . , A. Karp, S.W.J. B r i g h t . 1986. Ploidy v a r i a t i o n in Solanum brevidens plants regenerated from protoplasts using an improved culture system. J. Exp. Bot. (in p r e s s ) .

554 ¿L. Karp, A., R. R i s i o t t , M.G.K. Jones, S.WJ. Bright. 1985. Chromosome doubling in monohaploid and dihaploid potatoes by regeneration from c u l t u r e d leaf expiants. Plant Cell Tissue and Organ Culture, 3, 363373. 23. Sree Ramulu, K. , P. D i j k h u i s , Ch. H. Hanisch Ten Cate, B. de Groot. 1985. Patterns of DNA and chromosome v a r i a t i o n during in v i t r o growth in various genotypes of potato. Plant Sci. 41, 69-71T. 24. Kasperbauer, M.J. , G.B. C o l l i n s . 1972. Reconstitution of d i p l o i d s from l e a f t i s s u e of anther^ derived haploids of tobacco. Crop S c i . 12, 98101. 2 5. Ooms, G., A. Karp, M.M. B u r r e l l , 0. Twell, J. Roberts. 1985. Genetic modification of potato development using Ri-TDNA. Theor. Appl. Genet. 70, 440-446. 26. Ooms, G. , A. Karp, J. Itoberts. 1983. From tumour to tuber; tumour cell c h a r a c t e r i s t i c s and chromsome numbers of crown-gall-derived t e t r a p l o i d potato plants (Solanum tuberosun cv. ' M a r i s B a r d ' ) . Theor. Appl. Genet. 66, 169-177:

555 IN VITRO MUTAGENESIS IN GERBERA JAMESONII

F. Walther Institute of Radioagronomy, Nuclear Research Establishment Juelich, D-5170 Juelich A. Sauer Federal Research Centre for Horticultural Plant Breeding D-2070 Ahrensburg

Introduction The demand for Gerbera jamesonii plants will be covered more and more by in vitro propagation. The laboratories in the Netherlands produced in 1984 about 10 million Gerbera plants by this method and a further increase is expected for the near future (1). Such a well known technique provides the opportunity to include mutation experiments in commercial breeding programs for Transvaal Daisy. This may have a similar significance as for other species, namely the development of a 'family of cultivars' from an economically important one as practised for many years in several plant species such as Chrysanthemum, Dahlia, Achimenes, Streptocarpus, Saintpaulia etc. (2). With the intention of applying mutation induction procedures for further improvement of Gerbera Walther & Sauer (3) very recently completed investigations with the aim of finding a parameter to determine the radiosensitivity of different idiotypes. Such experiments have to be considered an important prerequisite for any mutation induction experiment. It was the aim of these investigations to learn something about the X-ray induceable mutability in Gerbera.

Material and methods The pot-grown advanced clonal line 'A 26' with dark red coloured flowers was in vitro propagated on solid MS-medium Skoog-medium)

(Murashige-

(4) containing 0.1 ppm NAA (naphthaleneacetic acid),

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

556 2 ppm BAP (6-benzylamino purine), 3 % sucrose and 0.6 % Oxoid-agar. Regeneration of axillary shoots was sufficiently high under 16 h photoperiod

(1.000 lx warm tone Philips TL 65W/34) and a tempera-

ture regime of 25° C. In vitro derived microshoots with leaflets 5 - 7

mm long were placed in petri-dishes on the above mentioned

solid MS-medium and X-irradiated with doses of 10, 15, 20 or 25 Gy (1 Gy = 100 R). Ten in vitro shoots represented one replication/ dose. The experiment consisted of three replications resulting in an X-ray treatment of 30 explants in each variant. The technical data of the X-ray machine were: 12 mA, 150 kV, 1.7 mm Al-filter resulting in a dose rate of 0.9 Gy min ^. Regenerated shoots were cut off four weeks after X-irradiation and on three subsequent dates with intervals of 4 weeks each. After the cut off the shoots were placed for a further two weeks on the same MS-medium as before for shoot elongation. They were then rooted on 1/3 strength MS-medium

(macro- and micro-components) containing

2 ppm IAA (3-indoleacetic acid) and cultivated under the same environmental conditions as for shoot growth. About 21 days later the plantlets could be transferred from growth chamber to the greenhouse. Pricking them into a special Gerbera peat-soil mixture and keeping the young plants for about 1.0 days under humid conditions resulted in a survival rate of 91 % in the mean. Later the plants were potted into another kind of peat-soil mixture and cultivated up to flowering. The developing plants were often inspected and those carrying changed character(s) as compared with the control plants were marked with a sticker and thoroughly described as mutants. During several months of cultivation each mutant developed numerous flowers and the selected plants were checked two to three times for control of their mutant type. In case of differences to the first description as mutant they were classified as control plants and rejected as such.

Results Type of X-ray Induced Mutants The total number of 622 M^-plants regenerated from X-ray treated explants arose from 30 explants/dose and from the cut off on four subsequent dates.

557 Classifying the different phenotypes in groups of mutants with a similar main character facilitates the description of the X-ray induced population. Most of the mutative changes referred to decreased length of the stalk: 42 % of all mutants. The next frequent mutant type belonged to that with changed flower size and colour and here the most striking mutative event concerned the alteration of the length or width of the petals (30 %). The colour of petals was expected to be altered in many mutants, but this was not the case (19 %). Only some types had small changes of the red tone to tint or to shade. Some of the mutants showed dark red petals with yellow stripes differing in their number and width from flower to flower in one and the same plant. Later a flower with a pure yellow sector was found. As already mentioned above, the size of the flower differed in some mutants as a result of a mutation determining the shape or size of the petals but there were also some types with an increased number of circles of petals resulting in an almost filled flower.Another notable mutation affected the size and shape of the leaves (6 %). To get an impression on this mutated character some examples are presented in Fig. 1.

Fig. 1: X-ray induced alteration of leaf size and shape

558 A few mutants flowered about one week earlier than the control plants. - Among the mutants a wide range of variability was present. The above described mutated traits appeared as solely altered ones in about 41 % of all mutants. Two mutations in one plant were detected in 40 % and more than two in the remaining types. X-ray Dose Dependent Mutability In every mutation induction experiment the mutation rate will be influenced by the applied dose, by the number of treated plants, shoots etc., by the radiosensitivity of the cultivar as well as by the number of plants inspected for mutants. It should be valid also for in vitro mutation induction experiments to receive the highest 'output' by applying a minimum expenditure of 'input1. In this context the regeneration rate of X-irradiated explants on subsequent dates of cut off may become important. In Fig. 2 the influence of X-ray doses on shoot regeneration is demonstrated.

control

10 Gy

15Gy

20Gy

25Gy

Fig. 2: Influence of X-ray doses on regeneration rate; total number of regenerated microshoots = 100 % The control explants regenerated the most shoots on the date of the second cut off. In the variants 10, 15 and 20 Gy the maximum portion was found on the third date whereas in the 25 Gy variant the highest number was produced on the fourth date. This dose de-

559 pendent shifting of the maximum regeneration rate must be considered a typical aspect of in vitro induction experiments

(5). It should

be pointed out that this delay results in every case also in a decrease of the total number of produced shoots over a limited period (3) . In general, the X-ray dose dependent mutability is expressed as mutation or mutant rate. Only the last mentioned mutant rate can be calculated here. Among 662 M^-plants cultivated up to flowering, 90 mutants were detected and confirmed, i.e. the overall mutant rate was 14 %. As mentioned before numerous plants carried more than one mutation and this resulted in a higher number of mutations as compared with that for mutants. In total 149 mutations were registered and the percentage of mutations/dose was calculated as follows: 10 Gy = 21 % ; 15 Gy = 30 %; 20 Gy = 38 % and 25 Gy = 11 %. In Fig. 3 the mutant rate per dose is demonstrated.

307.

20-

10-

10 Gy

15Gy

20Gy

25Gy

Fig. 3: Percentage of mutants/X-ray dose; number of M 1 -plants/dose = 100 % The portion of mutants among the different induced populations increased up to a maximum after 20 Gy and was decreased in the 25 Gy variant.

560 The cutting off of M^-microshoots took place on four subsequent dates and it may be worthwhile to investigate which date is the most effective one with regard to mutant selection.

7.

10 Gy

15Gy

20 Gy

25Gy

Fig. 4: Relationship between date of cut off shoots and mutant rate; total number of shoots/date of cut off = 100 % The first and second date of cut off yielded the highest portion of mutants with the exception of the 25 Gy variant; the last may be influenced by the extremely low number of shoots regenerated on the first date. Considering the total mutability induced by a certain X-ray dose and calculating the percentage of mutants induced on the four subsequent dates it can be recognized that after relatively low doses (10 & 15 Gy) the maximum portion of mutants arose on the second date of cut off. This maximum effect will be shifted to a later date in the case of increasing doses: 20 Gy = 3rd and 25 Gy = 4th date.

Discussion and conclusions The mutation induction experiments conducted as model investigations resulted in the development of plants with mutated traits, i.e. in the expected increase of variability. Mutants could be selected

561

carrying changes of almost every character being important for further improvement of .Gerbera cultivars. With regard to the present efforts made towards the development of pot-grown Transvaal Daisy, the high portion of mutations for stalk length and for varying flower size, as well as for decreased leafsize being induced independently from each other, should be of certain interest for Gerbera breeders. Now, it may be possible to develop lines - by means of in vitro mutation induction - differing only in stalk length, but by retaining all the other important traits of the economically significant original cultivar.

Fig. 5: Mutants with different length of the stalk For a cultivar already propagated by in vitro culture the procedure to provide a mutant collection needs a space of time as long as for the development of flowering plants from seeds (about 5 months). The number of mutants with altered colour of the flower was an unexpectedly small one. This may depend on the idiotype used

(nonchimera (?)) or on the strength of the X-ray dose applied.

Possibly such mutants can be induced only by very high X-ray doses also in Gerbera jamesonii as is known from carnation (6, 7) and roses (8). Taking the results described above as a basis, the following recommendations can be made for practical mutation induction experiments in Gerbera: - Gerbera jamesonii is well suited for mutation induction using X- or gamma-irradiation of in vitro derived microshoots. - The X-ray dose to be applied on a certain cultivar should be determined in test-experiments; the doses may be in the range of about 20 Gy of hard X-rays or gamma-rays resp.

562

- Axillary shoots regenerated by explants treated with ionizing radiation should be cut off on at least two or even more subsequent dates. - Plants carrying distinctly changed characters as compared with the original ones have to be checked for their altered trait(s) several times during their growth time for confirmation. Summary Applying X-ray doses between 10 and 25 Gy on in vitro derived axillary shoots of Gerbera jamesonii resulted in the induction of mutants with changes of the length of the stalks and the petals and in such with different size and shape of the flowers and in numerous types with a varied size and shape of leaves as well as in others with a different physiological behaviour. The percentage of mutants was highest in the 20 Gy population. Most impressive was the great portion (42 %) of mutants with a decreased length of the stalk compared with the original plants. This allows the breeder to develop e.g. a 'cultivar family1 consisting of members differing only in the length of their stalks.

References 1. Anonymous. 1985. Deutscher Gartenbau

1448.

2. Broertjes, C., A.M. van Harten. 1978. Application of Mutation Breeding Methods in the Improvement of Vegetatively Propagated Crops. Elsevier Scientific Publ.Comp. Amsterdam/Oxford/New York. 3. Walther, F., A. Sauer. 1985. Proc.Int.Symp. on Nuclear Techn. and in Vitro Culture for Plant Improvement, Wien, IAEA-SM-282 (in press) 4. Murashige, T. , F. Skoog. 1 962 . Physiol. Plant. _15v 473. 5. Walther, F., A. Sauer. 1985. Acta Hort, (in press) 6. Sparnaay, L.D. 1974. In: Meeting of the Mutation Breeding Contact Group, Wageningen, Oct. 1974. (Broertjes, C. Ed.) External Rep. No. 23, Euratom-ITAL, Wageningen, p. 28. 7. Sparnaay, L.D., J.F. Demmink, F. Garretsen. 1974. In: Eucarpia Meeting on Ornamentals Frejus Inst.Hort.Plant Breed., Wageningen p. 39. 8. Walther, F., A. Sauer. 1985. Proc.Int.Symp. on the Research and Cultivation of Roses, Tel-Aviv, Israel (in press)

563

IN VITRO MUTAGENESIS IN MAIZE

F.J. Novak, T. Hermelin, S. Daskalov FAO/IAEA Agricultural Biotechnology Laboratory A-2444 Seibersdorf, Austria M. Nesticky Maize Research Institute Trnava, Czechoslovakia

Abstract The objective of our work is to assess somaclonal and radiation induced variability in totipotent (somatic embryogenesis) in vitro culture of maize. Plants of the inbred line CHI-31 were selfpollinated and immature embryos (1-1.2 mm long) were irradiated with gamma rays at 0, 5 and 10 Gy, respectively. They were immediately excised and cultured in vitro. The somatic embryogenesis and plant regeneration were induced on N-6 medium supplemented with 2.5 yM 2,4-D. The variability was evaluated in the R^ and M^R^ plants and their progenies. Irradiation did not change the spectrum of morphological and chlorophyll variants in R^ and generations, respectively. Progenies of R^ plants were tested in the field and increased variability was found in five characteristics, i.e. height of plants, ear position, length of ear, number of rows on the cob, number of kernels in a row, when compared with the standard line. Tests were made of the progenies of regenerants for combination ability by means of top-cross method. The differences between somaclonal and radiation induced variability are discussed and the in vitro system for maize mutation breeding is considered.

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

564 Introduction In the past few years there has been an increased interest in a new phenomenon of tissue culture induced genetic variation. This variability - referred to as somaclonal variation (1) occurs in plant tissue culture systems without mutagen application. However, physical and/or chemical mutagens may significantly influence the frequency and spectrum of mutants among the progenies of regenerated plants from tissue culture. Evaluation of mutagen induced variability must be considered in relation to the somaclonal variation when induced mutagenesis is applied in tissue and cell cultures. The objective of our work is to assess somaclonal and radiation induced variability in in vitro culture system of maize.

Materials and Methods Plants of the inbred line CHI-31 were self-pollinated and immature embryos (1 to 1.2 mm long) were used in the following experimental procedures (Fig. 1): (1) ES - Plants from zygotic embryos in situ: The immature cobs were irradiated with gamma rays at 0, 5 and 10 Gy, respectively, and caryopses were maturated on the plants. The plants from irradiated embryos in situ (M^ generations) were selfpollinated to obtain seeds of M^ generation. (2) ET - Plants from somatic embryos in vitro: The zygotic embryos were exposed as stated above, but immediately after gamma irradiation they were excised and cultured in vitro. Embryos were cultured individually in test-tubes with the scutellum facing upwards and the plumule and radicle sides in contact with agar. The basal nutrient medium was N-6 (2) containing 2.5 yM 2,4-D (2,4-dichlorophenoxyacetic acid) and 120 g/1 sucrose. After 120 days in the culture the embryogenic calli were transferred onto the hormone-free N-6 medium with 60 g/1 sucrose. Some plantlets were cultured on MS medium (3) supplemented with 2 yM NAA (anaphtha-leneacetic acid) to promote rooting. The fully developed plantlets were aseptically transplanted into perlite saturated with half-strength MS mineral solution and after 14-day-culture in

565 Fig. 1.

Schematic representation of experimental procedures to assess induced and somaclonal variations in maize

SELF POLLINATION / A

iK

SELF

/\

POLLINATION zygotic embryo ( Imm) in situ irradiation

7 \

o * o »••'Ï

r

SEED MATURATION

n I

HARVEST © Q

SH 1 3 C 3 a 0 W 3 O

K Cn

fi

g

£ IN

1

*—

^

•a in ai -P .C fi o m (d h a; CM u

•

m

a) T I 0

>

•rH

cu T i (U VH 3 -P rH 3 O T I c

«

rH 0

^

4-> a 0 o

•

•H K

ra 0 > i

fi

0 -H -P Cd

^

Q)

fi

01

en 0) in

-M c rd

IH a i 0 c a) 3 Ö1 01 (-1 h

i N

£

«

fi •rH

>1 Ü C

— ,•

Q 1 T

m ai SH

3 •P rH 3 O

— •

m

-P c m rH a X ai

—

n i n

CN

m CO CN

n

—

—

.

rH en

kO

m

en

CN

o a i oo

CN r-» o rH

CO O i n

•

T-S" CN

•

u) (U ra a)

si

•P c

1 M X) g U

> i -p •rl m Ü " 3 C T 01 •H t d < * > 01 C DJ — ^ UH a) (d 0 PH 0 Cn

m m

n

CN T

•

«

n i n

c •H

Ln

100

R

H

0

(1983-84) plant

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

year

after

regeneration

582

Analysis of progenies obtained from régénérants

Regenerated plants

X

Original population

Plants obtained regenerants

were

-15 contrasting R - •propagationby scions 72 R

»half-sib pollination

0

»15 plants-

from self compared

•self pollination

• self pollination

and half-sib pollination plus scions of under

field

conditions

according

to

a

randomized block design with three replicates during 1985.

Results

Evaluation of callus-derived population

Table

1.

Significance

between seed-derived

levels

(sampling

and callus-derived

within seed-derived population, population,

1983-84) of:

a) variance

populations,

b) variance

c) variance within

callus-derived

d) ratio between variances of seed and callus derived

populations.

Plant height

83—^—84 ** *

83-^-84 ** **

83-^-84 ** ns

83——84 ns **

DMY

**

**

**

**

**

**

ns

**

Stem number

*

**

**

**

ns

ns

**

**

Flowering time

*

*

**

**

**

**

ns

**

Stem diameter

**

ns

**

**

**

**

ns

ns

Leaflet length

**

ns

**

**

ns

ns

*

**

Leaflet width

**

ns

**

**

**

ns

ns

**

Number of pods

ns

**

**

**

**

**

ns

ns

Number of seeds/pod **

**

**

**

**

**

ns

ns

Seed yield

**

**

*

**

*

ns

ns

ns

ns=not significant; *=P$0.05; *»=P*0.01

583 1)

Within

callus-derived

significant

phenotypic

regeneration. plant

2)

"in

variants

completely

vitro and

several

variance

Minus

regeneration

depressive

population

effect"

seed

characters

three

years

observed

derived

after

during

disappeared; decreases

2nd

it

with

year

after

indicates

time.

3)

that

Variance

callus

years.

4) F o r all t r a i t s e v a l u a t e d , v a r i a n t s s u p e r i o r to the p l a n t

superior

to

the

best

induction were

plants

of

is

plant

within

i n i t i a l l y u s e d for c a l l u s

populations

displaied

similar

observed.

original

5)

in

both

Régénérants

population

were

not

present. A n a l y s i s of p r o g e n i e s o b t a i n e d f r o m

T a b l e 2.

Significance

of c a l l u s - d e r i v e d

levels of:

plants;

b)

régénérants

a) v a r i a n c e among self

ratio b e t w e e n

variances

of s e e d

c a l l u s d e r i v e d self p r o g e n i e s ; c) v a r i a n c e among h a l f - s i b of c a l l u s - d e r i v e d p l a n t s ;

d,e)

progenies and

progenies

regression between régénérants

and

t h e i r respective h a l f - s i b and self p r o g e n i e s . b

c

*

*

ns

*

*

*

ns

*

Leaflet width

*

*

ns

*

*

Petiole

ns

*

*

*

*

ns

ns

*

*

a N u m b e r of Leaflet

Stem

seeds/pod

length

length

diameter

1) A m o n g

the

self

p r o g e n i e s of

d

e *

ns

ns

ns

ns

ns

ns

ns

*

ns

*

*

callus-derived plants

the

presence

of g e n e t i c v a r i a n c e w a s o b s e r v e d for some traits, this v a r i a n c e not

significantly

progenies

of

different

seed-derived

respect

plants.

to No

that

observed

significant

f o u n d b e t w e e n r é g é n é r a n t s and the m e a n v a l u e s of self 2)

Among

significant

the

half-sib

genetic

progenies

variance

was

of

the

observed

among

all

self

regression

was

progenies.

callus-derived for

was

the

plants

evaluated

traits, m o r e o v e r for some of them a s i g n i f i c a n t r e g r e s s i o n w i t h the régénérants was

observed

584 References

1.

Larkin, Novel

P.J. Source

a n d W . R . S c o w c r o f t . 1981. S o m a c l o n a l of

Variability

Improvement. Theor.Appl.Genet.

2.

from

Cell

Cultures

Variation-a for

60,197-214.

M a r i o t t i D., M . P e z z o t t i , E . F a l i s t o c c o a n d S . A r c i o n i . 1 9 8 4 . Regeneration

Plant

Plant

from L e a f - d e r i v e d C a l l u s of L o t u s c o r n i c u l a t u s L.

cv.Franco. Genet.Agr.

38,219-223.

585 POTENTIAL SYSTEM FOR THE SPECIFIC SELECTION OF PLANT MUTANTS OVERPRODUCING METHIONINE De Bry L. '•2, Jacobs M. 1 . Wallsgrove R. M . 2 & Mlflln B. J. 2 , (1) Plantengenetica, Vrije Universiteit Brussel, Paardenstraat 65, B - 1 6 4 0 Sint-Genesius Rode, Belgium. ( 2 ) Biochemistry, Rothamsted Experimental Station, Harpenden, Herts, AL5 2JQ, U.K. An increased level of free methionine is a desirable agricultural trait in plants, and is the ultimate goal of this work. Methionine biosynthesis is traditionally presented as one end-product of the aspartate pathway (I). This pathway shows the close relationships between methionine and several of the other essential amino acids: lysine, threonine and isoleucine. We prefer to present is as a "cross" of some biochemical pathways: aspartate pathway, sulphate assimilation pathway and one carbon unit pathway. (flg.l).

Fig. 1: Methionine pathway. This presentation emphasizes the three origins of methionine biosynthesis, and its three ways of utilisation. It also shows the possible main sites of action In vivo of:Propargylglycine on cystathionine - ) f - synthase. I

1 —'

Ethlonlneon methionine transaminase, end Selenomethionine on methionine adenosyltransferase end methlorryl - tRNA synthetase

586 The regulation of amino acid biosynthesis Is widely Investigated by the use of amino acid analogues to select mutants which are altered in their ability to regulate the synthesis of the natural amino Kids (2). Resistance to amino acid analogues can arise by a number of mechanisms: (a) inhibition of analogue transport, (b) decreased affinity for analogue of competitively inhibited or end-product sensitive enzyme, (c) overproduction of analogue-sensistive enzyme, (d) aminoacyl-tRNA synthetase with decreased affinity for analogue, (e) activation of DNA repair against a mutagenic analogue, (f) increased catabolism of the analogue, and (g) overproduction of natural amino acids. A positive procedure, In which wild-type and undesirable resistant mutant cells would be killed, would be rapid and simple. Methionine reverses the combined inhibition of propargylglycine, selenomethionine and ethionine (fig. 1 & 2) (3). The isolation of mutants resistant to this combined inhibition (fig.3) may result in the specific selection of plants overproducing methionine.

587 Leeues of Nicotiniana Plumbaginifolia Uiuiani

i Treatment luith cellulolytic enzymes

i

Protoplasts Mutagenic treatment

I

Colonies Screening in a selection medium containing propargylglycine, selenomethionine and ethionine

i

Identification and propagation or the resistant mutants

F i g . 3 : S e l e c t i o n scheme for the potential s p e c i f i c i s o l a t i o n of mutants o v e r p r o d u c i n g methionine

References: (1) Chalef R. S. (1981). Genetics of Higher Plants. Cambridge University Press, pp 184. (2) Negrutiu L.Cattoir-ReynaertsA., Verbruggen I. and Jacobs M. (1984). Theor.Appl. 6enet. 68: 11-20.. (3) De Br/ L., Walsgrove R. M., Bright S. W. J., Miflin B. J. & Jacobs M. (1985) (In preparation).

589 E N H A N C E M E N T OF A S U L A M RESISTANCE IN B A R L E Y

H . A . Collin, P.D. Putwain, S.C. Giffard Botany Department, University of Liverpool, P.O. Box 147, Liverpool L69 3BX U.K.

Introduction

The development of new herbicides within the current registration regulations is a long and costly process.

An alternative approach would be to make use of the variability

shown by crop plants to herbicides, then by selection to obtain crop varieties resistant to the herbicides (1).

This approach has been succesful for perennial rye grass and paraquat

resistance (2, 3) and oil seed rape and atrazine and simazine resistance ( 10»

0 x R

22

11

65

(0 x R) selfed

32

18

¿16

77 x M

0

0

77 x R

12

6

(77 x R) selfed

25

in

> 104 39 37 > 1011

118 x M

0

0

118 x R

17

5

27

(118 x R) selfed

65

26

55

0

0

27 x M

> 10«

27 x R

till

0

56

(27 x R) selfed

80

0

19

0

0

> 10«

58 x M 58 x R

13

0

51

(58 x R) selfed

81

0

20

85 x M

0

0

85 x R

33

0

27

(85 x R) selfed

65

0

21

> 10«

Fig. 2: Differential segregation into fertile, 1/2 fertile and sterile plants among the progeny of sterile plants bearing an Ogura radish cytoplasm (0) or cybrid cytoplasms (77. 118, 27. 58, 85) crossed by either a maintainer line (M) or a heterozygous restorer line (R) and among the progeny of self-pollinated restored plants.

658 - The original c.m.s "0" radish cytoplasm as well as cybrid cytoplasms (77, 118, 27. 58, 85) are restored by the R. line, confirming that cybrids actually retained the male sterility character present in the c.m.s parent of fusion and ruling out two other possibilities :

in vitro

induced variation leading to c.m.s.

and a new c.m.s. character created by recombination between parental Mt DNA. - In the case of 0, 77 and 118 cytoplasms the progeny segregate into three different phenotypes: fertile, half fertile and sterile. So at least two restorer genes are evidenced. In the case of cybrids 27, 58, 85 the progeny segregate into only two different phenotypes, and these ratios are in agreement with the need of only one restorer gene. In order to interpret these results, taking into account Mt DNA recombination as shown by molecular analysis, we must assume that 0 mitochondrial DNA bears more than one c.m.s factor and that 27, 58 and 85 recombinants possess fewer c.m.s factors. The simplest hypothesis is to consider that 0 mitochondria lead to c.m.s for two independant reasons : the "Ogura" male sterility already expressed in radish (13) and the alloplasmic male sterility expressed when radish mitochondria are in the presence of a Brassica nucleus (Id). Mt DNA recombination makes it possible to genetically separate these two factors and to give a simpler system of restoration in some cases (cybrids 27, 58, 85). In fact this male fertility restoration system raised an unexpected problem rendering it up to now of no use for hybrid seed production. Among segregating progeny of self-pollinated restored plants, a dramatic decrease of female fertility is observed (15) as illustrated in fig. 3. Male fertile plants are always less female fertile than male sterile plants in the same progeny and we were unable to observe a genetic segregation between male fertility restoration and female sterility traits. Female sterility is due to the abortion of embryo sacs, Just after female meiosis curiously at the same time male gametogenesis is blocked in c.m.s 0. If the restorer genes are actually the genes responsible of this female sterility, this observation would be of great interest in the understanding of the physiology of sterility.

659

Male sterile Seeds per silica

+

+

18.6 -

Restored male fertile (type II)

Restored male fertile (type I)

1.3

¡1,28 -

+

0,6

0,58 -

0,25

Fig. 3: Numbers of seeds per silica in male sterile and restored male fertile plants showing the decrease of female fertility. Strategy for localizing the c.m.s factors on Mt. DNA The comparison between physical maps of Mt DNA of cybrids could makes it possible to localize the c.m.s factors. To reach this goal, we have to obtain a sterile plant and a fertile plant differing only by these genes. The strategy used consists in back-fusion experients in which c.m.s cybrids are fused again with fertile plants, in order to get an almost normal ( B. nanus ) Mt genome with only the c.m.s genes from radish, and

reciprocally,

fertile cybrids are fused with plants bearing 0 cytoplasm to get a radish Mt genome with only the fertility genes from

B. napus .

Cybrids in these experiments are screened, according to Fig. U, by taking advantage of the following two cytoplasmic

traits:

c.m.s/fertility and atrazine resistance sensitivity. The Mt DNA analysis of the first generation of back fusion cybrids is now in progress. Conclusion In summary, it is possible in Brassica napus to create new cytoplasmic associations which are of great interest for plant breeding. The material we have obtained is now used by breeders of rape and cabbage. This material also offers the opportunity to better understand the mechanisms of Mt DNA recombination which appears to be site specific, and in the future, to know which genes are implicated in the c.m.s.

phenomenon.

660 m.fertile

c. m. s Atr

s

Atr R

(+)

Screening for c.m. s Atr R

m. fertile (+)

Atr s

Screening for c.m. s Atr s Fig. 1: Back-fusion cybrids are obtained by screening "recombined" combinations between c.m.s/fertility and Atr R /Atr^ traits present in reverse forms in the parents. In this figure, almost normal ( B. napus ) mt genomes with only c.m.s genes are sought.

References

1. Bannerot, H., L. Boulidard., Y. Chupeau. 1977. Unexpected difficulties met with the radish cytoplasm. Eucarpia Cruciferae Newsletter 2 , 26. 2. Hirschberg, J., L. Mc Intosch. 1983. Molecular basis of herbicide resistance in Amaranthus hvbridus . Science. 222 . 1346-13*19. 3. Belliard, G., F. Vedel, G. Pelletier. 1979- Mitochondrial recombination in cytoplasmic hybrids of Nlcotlana tabacum protoplast fusion. Nature. 2&1 . 101-103-

by

1. Levings, C.S.III., B.D. Kim, D.R. Pring, M.F. Mans, J.R. Laughnan, S. Gabay-Laughnan. 1980. Cytoplasmic reversion of c.m.s-S in maize: association with a transpositional event. Science 2£2 , 1021-10235- Bannerot, H., L. Boulidard, Y. Cauderon, J. Temp6. 1971. Transfer of cytoplasmic male sterility from Raphanus satlvus to Brassica oleracea . Proc. Eucarpia Meeting Cruciferae, 52 . 6. Pelletier, G., C. Primard, F. Vedel, P. Chetrit, R. R6my, P. Rousselle, M. Renard. 1983. Intergeneric cytoplasmic hybridization in cruciferae by protoplast fusion. Mol. Gen. Genet. , 211-250.

661

7. Vedel, F.. C. Mathieu, P. Lebacq, F. Ambard-Bretteville, R. Remy. 1982. Comparative macromolecular analysis of the cytoplasms of normal and cytoplasmic male sterile Brassica nanus . Theor. Appl. Genet. £2 , 255-262. 8. Maltais, B., C.J. Bouchard. 1978. Une moutarde des oiseaux ( B. EâEâ . L.) résistante à l'atrazine. Phytoprotection. , 117-119. 9. Beversdorf, W.D., J. Weiss-Lerman, L.R. Erickson, Souza-Machado. 1980. Transfert of cytoplasmically triazine resistance from birds rape to cultivated ( B. campestris and B. nanus ). Can. J. Genet. 167-172.

V. inherited oil seed rape Cytol. 22. .

10. Chetrit, P., C. Mathieu, F. Vedel, G. Pelletier, C. Primard. 1985. Mitochondrial DNA polymorphison induced by protoplast fusion in cruciferae. Theor. Appl. Genet. , 361-366. 11. Palmer, J.D., C.R. Shields. 1984. Tripartite structure of the Brassica campestris mitochondrial genome. Nature. 307 , 437-440. 12. Chetrit, P., C. Mathieu, J.P. Muller, F. Vedel. 1984. Physical and gene mapping of cauliflower ( Brassica oleracea ) mitochondrial DNA. Curr. Genet. £ , ¿113-421. 13. Ogura, H. 1968. Studies of the new male sterility in radish with special reference to the utilization of this sterility towards the practical raising of hybrid seeds. Mem. Fac. Agric. Kagoshina University, £ , 39-78. 14. Mc. Collum, G.D. 1981. Induction of an alloplasmic male sterile Brassica oleracea by substituting cytoplasm from "early scarlet globe" radish ( Raph'anus satlvus ). Euphytica , 855-859. 15. Sahli, M. 1984. Etudes sur la restauration de la fertilité du colza mâle stérile porteur du cytoplasme ogura du radis. Dipl. Etud. Approf. Université Rennes 1.

663 SOMATIC H Y B R I D I Z A T I O N AND CYBRIDI Z A T I O N AS FOR AND

WIDENING

OF

POTENTIAL

THE GENE-POOLS O F CROPS WITHIN

METHODS

BRASSICACEAE

SOLANACEAE.

K. G l i m e l i u s ,

3. F a h l e s s o n , C . S j ö d i n , E. S u n d b e r g ,

M.

Djup-

s j öbacka. Department tural

of P l a n t B r e e d i n g ,

Sciences. Uppsala,

Swedish University

of

Agricul-

Sweden.

H . Fe 11 n e r - F e 1 d e g g . Department

of P a t h o l o g y ,

Sciences. Uppsala, H.T.

Swedish

of

Agricultural

Sweden.

Bonnett.

Department 97403

of B i o l o g y , U n i v e r s i t y

techniques

genes

of

one plant

terest

to p l a n t

incompatible bination genes

have made cell w i t h

breeders,

species

can

can be p r o d u c e d .

combined

Eugene,

respects. The genetic

extreme

likely

Oregon

to

species

any

comthat

is u n l i k e l y

will

function

and

speciation

following

in-

sexually

In p r i n c i p l e it

the

is of

between

in a d e g r e e of

development

combine

together which

diversity fusions

which

between

combinations.

Isolated

plant

ridizations combined

protoplasts

where

both

g e n o m e of

only

hybrids within

will have

species

for

be d i s c u s s e d been

for

genome

interspecific and

species. The other

the c y t o p l a s m

the o t h e r

aspects

can be u s e d

the n u c l e a r

f r o m two p a r e n t a l

combine

cybrids

the b a r r i e r s

isolation

results

prevent

possible

of a n o t h e r . T h i s

in p r a c t i c e

from unrelated

during evolution

two

Oregon,

be b y p a s s e d . But

in all

will m o s t

it that

since

occurs

where

of

U.S.A.

Protoplast

to

University

are

possibility

is

f r o m one s p e c i e s w i t h production and

produced

of

within

the

cybrids.

illustrated

hyb-

the p l a s m o n

by

Genetic Manipulation in Plant Breeding © 1986 Walter de Gruyter & Co., Berlin • New York - Printed In Germany

These

experiments

§£.a§.!i£aceae

SoUnaceae.

total

and

664 A.

SOMATIC

In

order

used

to

investigate

a

complement

as

various

HYBRIDIZATION

techniques

brassicaceous tolerance

to

seed

rape

(1,

sexual

e.g.

the

2)

cabbage

pulated

¿n_\u_tj-o

In

of

some

genetic These most

of

traits

producing oil

too

been in

Attempts

have

limited

there

are

Therefore,

been

sexual

success

tions.

several

with

broaden

Culture

and

differentiation

culture

high

plating

Young tyl

conditions

within

efficiences

hypocotyls

protoplasts

start

to

divide

nies

with

then

plated

division high

10-20 on

and

are come

within

media

the

growth

of

the

culture

of

results

in

to

most

or

oil-

mani-

pool

(1,

an

calli,

most

a

2 days better

in

after

these

spite

of

species,

the

combina-

interest

as

a

Brassiceae.

from

various

resulting

frequencies

protoplasts.

Hypoco-

tissue

culture.

Small

are of

l