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Biotechnology
 8187134909

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U, S tt cytt nttr etyetnet

Dr. U. Satyanardyana M .Sc..P h D .. F.l .C ..F.A ,CB . Professor of Biochemistry Siddhartha M edical Colle ge (NTR University of Health Sciences) Vijayawada, A.P., India

BOOf(S AND ALLIED (Pl Lto. No. 1-El2, "SHUBHenr Pr-eza" (ltt Ft-oon), 7OOO10 (INore) 83/I BauAGFIATA MarN Roao, I(olrera 3, 24I-84I1' Telefax : (+9 1 -33)241-857

e-mail : [email protected]

Biotechnologg FirstPublished: 2005 Beprinted: 2O06,2007,2008,2009 R e p ri n te :2 d 010

@ Copyright reserued by the Author. Publishing rights and Printing rights reserued by the Publisher. Maketing rights and Distributing rights reserved by the Publisher. Atl tights reserved. No pari of this publication may be reproduced or transmitted in any form or by any means, electronic, mechanical, photo-copying, recording ot any information storage and retrieval system, without the priot written permission of the Publisher. Exclusive rights reseNed by the Publisher for publishing, printing, sale, marketing, distribution, expoft and trunslation of this book for all editions and reprints thereof. About Cover Design Depicts the production of a recombinant DNAmotecule,and its utility in modern Biotechnologywith reference to microorganisms,plants and animals. (Note : This design is an oversimplification of the facts. For the sake of clarity,only a few helicesof DNAstructure are shown).

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UPPAIA AUTHOR_PUBLISHERINTERLINKS (A'P') D.No.48-16-10, NagarjunaNagar,MahanaduBoad,Vijayawada520008

is an exciting,rapidlydevelopingand revolutionary Biotechnology scientificdiscipline,with its roots in biologicaland technological sciences. Duringthe pastfew years,the majorityof scientificbreakthroughs particularlyinvolvinggeneticengineering.Bioin biologicalscienceshave come from biotechnology, technologyimpingeson everyone'slife, and is truly regardedas the scientifictechnologyof the twentyfirst century. THE NECESSITY FOR A COMPREHENSIVE EOOK ON BIOTECHNOTOGY Biotechnology is a subjectlearntby students with differentbackgrounds at the undergraduate and postgraduatelevels.lt is truethatthereare somebooksnow availableon biotechnology (by Indianand foreign authors).Thesebooks,however,deal with one or two specializedareasratherthan cdveringthe broad spectrumof biotechnology. Consequently, the studentshaveto dependon manybooksfor goodand latest informationon biotechnology. There is a long-feltneed for a comprehensive book on biotechnology providingupdatedinformationon the subject. THE BIRTHOF THE NEW BOOK It will not be out of placeto mentionhere how and when this book was born. The entirebook was writtenin the early hours(between2AM and 6AM, when the world aroundis fastasleep),duringwhich periodlcarry out my intellectualactivities.Aftera soundsleep,a freshmind ppckedwith creativeideas and innovativethoughts,has largelyhelpedme to write this book in a noveland uniqueway.Truly,each pageof this book was conceivedin darknessand born at day break! M A I O N HI GH T IG H T S OF T H E BOOK ' B I OTE C H N OIOGY ' The presentbook 'BIOTECHNOt-OCy',in colour,with severalnovel features,is comprehensively written with latestadvancesin the subjectto meet the needsof all categoriesof studentslearning biotechnology. 'The sectionon 'Basicsto learn Biotechnology'providesthe most essentialinformationneededto Thisis particularly understand biotechnology. usefulto students from diversqbackgrounds who areexposed to biotechnology all of a sudden.MolecularBiologyis.dealtwith iri good detail,as it laysthe foundations for geneticengineeringand modernbiotechnology. The other sectionsin this book include RecombinantDNA Technology,Medical/Pharmaceutical Biotechnology, Microbial/lndustrial Biotechnology, Animal Cell/AnimalBiotechnology, Plant/Agricultural Eachsectionis carefullycraftedto provideextensive, Biotechnology and Environmental Biotechnology. relevantand most recentinformation. headings,sub-headings, The book is written in a lucid stylewith colour illustrations, tablesand flou, charts.Coloursare usefulfor betterunderstanding and easy reproducibilityof the subjectmatter.An extensiveglossaryis addedto the book to makethe commonlyusedtermsand words in biotechnology very clear.

(tl

Besidesthe student community, this book will immensely help the personnelworking in biotechnology laboratoriesand industries.The languageand contentsof this book are made so simple and easy that even a lay m an wit h a m inim al k nowled g e o f b i o l o g i c a l s c i e n c e sw i l l b e a b l e t o u n d e r s t a n dt h e e s s e n ceo f biotechnology. As the subject of biotechnology is multidisciplinaryi books on the subject are written by authors with different backgrounds-botany, zoology, genetics,pharmacy, agriculture, engineering,etc, This resultsin an inevitable bias in the books. For instance, an author with a botany background tends to pay more attention to planVagriculturalbiotechnolgy.The presentbook is free from such bias. Written by an author with biochemistry background, covering all aspectsof life sciences, it is well balanced in the treatment of various branches of biotechnology. However, there is a perceptible bias in the book towards recent information on the applications of different branches of biotechnology to human healthcare,and for the ultimate benefit of mankind. This is justifiable from the fact that more than TOohof the budget earmarked for biotechnologl, vst.tt.^ in developing countries goes towards healthcare research.

AN INVITATIONTO READERS Thisbook is designed to caterto the needsof all categories of studentslearningbiotechnology in various disciplines-lifesciences,engineeringand technology,medicine,agricultufe,pharmacy,veterinaryetc. Thus,this book may be regardedas a buffettablewith somethingfor everyone(in everydiscipline).lt is ultimatelyfor the readerto do appropriateself-service and enjoy the menu. I invite the readersfor the wonderfuland excitingworld of biotechnology, and learnthe subjectto maximumpossiblewith easeand pleasure. The subjectof biotechnologyis very vast,thereforeit is not an easyjob to preparea concentrated capsuleof biotechnology. As this book is intendedto caterto the needsof studentslearningbiotechnologv f r omma n yd i ffe re ndt i s c i p l i n e sth, e r ei s a l ot of scopeto i mprovethe booki n future.Thi spri mari l ydepends on the feedbackfrom the facultyand studentswith specificrequestsand ideas. It is truethat I represent a selectedgroupof individualsauthoringbooks,havingsometime at disposal. besideshard work, determination and dedication.lconsider myselfas an eternallearnerand a regular studentof biotechnology. However,it is beyondmy capabilityto keeptrackof the evergrowing advances in biotechnology due to the exponential growthof the subject,arrdthis makesme nervous.I honestlyadmit that I have to dependon maturereadersfor subsequent editionsof this book. I welcomeconstructiveand helpfulsuggestions to improvethe book.

DR. U. SATYANARAYA\,{

(il1

I owe a deep debt of gratitudeto my parents,the late Sri U. VenkataSubbaiah,and Smt.Vajramma, for cultivatingin me the habit of early rising.The writing of this book would neverhave been possible without this healthyhabit.I am gratefulto Dr. B. S. NarasingaRao (formerDirector,NationalInstituteof life, and to my eldestbrother,Dr. U. Cudaru(former Nutrition,Hyderbad)for discipliningmy professional S angl i ),for di sci pl i ni ngmy personall i fe P r of es s oorf Po w e rs y s te msW , a l c h a n dC o l l egeof E ngi neeri ng, My youngerson, U. Amrutpani,has made a significantcontributionat everystagein the preparation M.B.B.S,M.S.,has etc. My elderson,Dr. U. Chakrapani of this book-writing, verification,proof-reading, with adequate emphasis ideas suggestions to orient the book with his and constructive helpedme creative of the book. I on human healthcare,besidesinvolving himself in the verificationand proof-reading acknowledgethe help of my friend, Dr. P. Ramanujam(Readerin English,Andhra Loyola College, Vijayawada), for his help at variousstagesof manuscriptpreparation. lexpress my gratitudeto Mr. ArunabhaSen (ExecutiveDirector,Books& Allied Pvt. Ltd., Kolkata) for his whole-heartedcooperationin bringing out this book to my satisfaction.I am gratefulto for the excellentpage-makingand graphics-workin the book. I thank Mr. Shyamal Bhattacharya I alsothank Mr. PrasenjitHalderfor the cover designof the book. Mr. DineshBhunyafor proof-reading. Last but not least, I thank my wife, KrishnaKumari,for her constantcooperation,supportand encouragement. for sponsoring and supportingme to Interlinks,Vijayawada, lam gratefulto UppalaAuthor-Publisher write this book.

DR. U. SATYANARAYANA

(llrI

ents in Brief

Introduction I

in 1l Manipulation of CeneExpression HostCells 137 12 HumanCenomeProiect 148

to Biotechnology

TheScope of Biotechnology3

Introduction Biolog]

X{e dic al/Pharrnaceu t i cal Iliotechnolog;' (Biotecluroloqy in llcalthcare)

to Nlolecular

13 GeneTherapy

157

2

1 1 14 DNA in Disease andStructure DNAandRNA-Composition Diagnosis andMedical

3

DNA-Replication, Recombination, andRepair 23 Transcription andTranslation 38 Regulation'of CeneExpression 59

4 5

Forensics 173 of DNA 15 Pharmaceutical Products Technology 189 16 Recombinant Vaccines 199 Antibcdies l7 Monoclonal (Hybridoma Technology) 213 Assisted Reproductive Technology 227 18

Genetic Engtureering/ Recornbinant. DNA Technolog.v 6 7 8

Ilicr 60' C) . Thes e ba c t e r i a can be used to extract copper and molybdenum re sp ective ly f r om c halc opy r it e ( CuFeSr ) a n d mo lyb de nite ( M oSz ) .

production)from iron or sulfurto oxygen.That is these organisms can obtain energy from the oxidationof Fe2"to Fe3+or from the oxidationof sul furand reducedsul furcompoundsto sul fareas A conrbination of two bacteria Leptospirillum illustratedbelow. ferrooxidans and Thiobacillus organoparpus can 4FeSO,+ 2HrSOo+ O, -----+2Fer(SOa)3 + 2HrO

effectively degrade pyrite (FeS2)and chalcopyrite tCu Fe Sr).The indiv idual or ganis m salone ar e o f n o u se in extrac t ing m et als . Pseudomonasaeruginosa can be employed in min ing lo w gr ade ur anium ( 0. 02% ) or e . T h i s org an ismh as been s hown t o ac c um ulat eabo u t 1 0 0 mg u ran ium per one lit er s olut ion in les s t ha n t e n seconds. Another organism, Rhizopus arrhizus is also effective for extractine uranium from waste water. Certain fungi have also found use in bioleaching. Thus, Aspergillus niger can extract copper and nickel while Aspergillus oryzae is used ior extracting gold. As already stated, among the various microorganisms, T. ferrooxidans and T. thiooxidans are th e mo s t widely us ed in bioleac hing . T h e u tiliza tion of m any of t he ot her oiganis m s is s t i l l a t the experimental stage. Mechanism

of bioleaching

The me ch anis mof bioleac hing is r at herc om p l e x n ot well under s t ood. The c he m i c a l a rd t'a nsforma tion of m et als by m ic r oor ganis m s m a y cccu r b y d irec t or indir ec t bioleac hing.

2So + 30, + 2HrO ------+2HrSOo 2FeS,+ 70, + 2HrO ->

2FeSOo+ 2HrSOo

As is evident from the third reaction given above,iron is extractedin the solubleform the rron ore pyrite (FeSr). lndirect bioleaching: In this indirect method, the bacteria produce strong oxidizing agents such as ferric iron and sulfur.icacid on oxidation of sol ubl ei ron or sol ubl esul fur respecti vel y. Ferri c i ron or sul furi c aci d, bei ng pow erful oxi di zi ng agents reacf with metals and extract them. For i ndi rect bi ol eachi ng, aci di c envi ronment i s absol utelessenti y al i n orderto keepferri ci ron and other metal s i n sol uti on. l t i s oossi bl e ro conti nuousl ymai ntai naci di c envi ronmentby the oxi dati on of i ron, sul fur, metal sLtl fi desor by dissolutionof carbonateions. COMMERCIAL PROCESS OF B IOLE A C H IN G

The natural l yoccurri ngmi nerall eachi ngi s very sl ow . The mi crobi albi ol eachi ngprocesscan be opti mi zed by creati ng i deal condi ti onstemperature,pH, and nutrient,O, and CO, supply Direct bioleaching : ln this process, there is a of general direct enzymatic attack on the minerals (which are etc. A diagrammaticrepresentation' ;-s,:eotible to oxidation) by the microorganisms. bioleachingprocessis depicted in Fig. 32.1. The :'tr -:';':a denTedmtcroorganrms wrtri nulrtenl4 aad efq. are cedatn baclerta /e.9., Z,/erraas'tVarzd iliiiillr-rii,r0.5 mmolil-) anci micronutrienfs (concentration \

lsLeaf II

+

The explants for good organogenesisshould be mitotically act iv e im m at ur e t is s ues . I n gener a l ,t h e bigger the explant the better the chances for o bta inin g via ble c allus / bells us pens ionc ult ur e s l t is advantageousto select meristematictissues(shoot tip, leaf, petiole)for efficient indirect organogenesis. This is because their growth rate and survival rate are much better. For indirect organogenesis,the cultures may be grown in liquid medium or solid medium. Many culture media (MS, 85 White's etc.) can be used in 'of organogenesis. The ' concentration growth regulators in the medium is critical for organogenesis. By varying the concentrations of auxins and cytokinins, in vitro organogenesiscan be ma nio ula ted . . Low auxin and low cytokinin concentration will in du ce callu s f or m at ion. . L ow au xin and high c y t ok inin c onc ent r at ionw i l l promote shoot organogenesisfrom callus . Hi8h a uxin a nd low c y t ok inin c onc ent r at ionw i l l ind uce roo t f or m at ion. SOMATIG

E M BRYO G ENESI S

The process oI regeneration of embryos from somatic cells, tissues or organs is regarded as somatic (or asexual) embryogenesis. Somatic embryogenesismay result in non-zygotic embryos or somatic embryos (directly formed from somatic

Plant Fig. 47.5 : Micropropagation of plants by organogenesis (A) Direct organogenesis (B) lndirect organogenesis through callus (C) lndirect organogenesis through suspension cell culture.

B IOTE CHNO LO CY

558 organs), parthogenetic embryos (formed from r-rnfertilizedegg) and androgenic embryos (formed from male gametophyte) In a general usage,when t he t er m s om at ic em br y o is us e d i t i m p l i e s t h a t i t i s formed from somatic tissues u nder in vitro conditions. Somatic embryos are structurally similar to z.ygotie (sexually formed) embryos, and t hey c an be ex c is ed f r om t h e p a r e n t t i s s u e s a n d induc ed t o ger m inat e in t is s u e c u l t u r e m e d i a Development of somatic embryos can be done in plant cultures using somatic cells, particularly epider m is , par enc hy m at ous c e l l s o f p e t i o l e s o r s ec ondar y r oot phloem . So m a t i c e m b r y o s a r i s e f r onr s ingle c ells loc at ed wi t h i n t h e c l u s t e r s o f m er is t em at icc ells in t he c allu s o r c e l / s u s p e r r s i o n . Fir s t a pr oem br y o is f or m ed w h i c h t h e n d e v e l o p s int o an em br y o, and f inally a p l a n t . Two routes of somatic embryogenesis are known - direct and indirect (Fig a7.6). Direct

somatic

ennbryogenesis

When the somatic embryos develop directly on the excised plant (explant) without undergoing callus formation, it is referred to as direct somatic embryoglenesis(Fig 47.64). This is possible due to the presence of pre-embryonic determined cells (PEDQ found i,n certain tissues of plants. The characteristic features of direct somatic em br y ogenes is is av oiding t h e p o s s i b i l i t y o f int r oduc ing s om ac lonal v a r i a t i o n s i n the propagated plants. Indirect

somatic

embryogenesis

I n indir ec t em br y ogenes is,th e c e l l s f r o m e x p l a n t (excised plant tissues)are made to proliferate and form callus, from which cell suspension cultures can be raised. Certain cells referredto as induced embryogenic determined cells (IEDO from the c ell s us pens ion c an f or m s o m a t i c e m b r y o s . Em br y ogenes isis m ade pos s ib l eb y t h e p r e s e n c eo f growth regulators (in appropriate concentration) and under s uit able env ir onm e n t a l c o n d i t i o n s . Som at ic em br y ogenes is( di r e c t o r i n d i r e c t ) c a n be c ar r ied on a wide r ange o f m e d i a ( e . g . M S , W hit e' s ) . The addit ion o f t h e a m i n o a c i d L-glutamine promotes embryogenesis The presence of aux in s uc h as 2, 4- dic hlor o - p h e n o x ya c e t i c a c i d is es s ent ialf or em br y o init iat io n .O n a l o w a u x i n o r no aux in m edium , t he em br y o g e n i cc l u m p s d e v e l o p into mature embryos.

Plantletwith shootsand roots

Plantletwith shootsand roots

\,,\// \

Plant

of plantsby Fig. 47.6 : Micropropagation embryogenesis(A) Direct embryogenesis(B) lndirect emDryogenes6

(MIC R OP R OP A C A TION ) 47 : C L O N ALPR O P AC AT ION ChA P I CT

s59

Ind irect som at ic em br y ogenes isis c om m er c i a l l y Production of seeds in some crops rery attractive since a large number of embryos Micropropagation, through axillary bud ca n b e ge ne r at ed in a s m all v olum e of c u l t u r e proliferationmethod, is suitable for seed production nredium. The somatic embryos so formed in some plants This is required in certain plants are synchronous and with good regeneratron where the limitation for seed production is high cao ab ilitv. degree of genetic conservation e.g. cauliflower, on ton. embryos Artificial seeds from somatic Artificial seeds can be made by encapsulation of The embryos, coated with somatic embryos. sod ium a lgin at e and nut r ient s olut ion, ar e dip p e d in calcium chior ide s olut ion. The c alc ium i o n s ind uce ra pid c r os s - link ing of s odium alginat e t o p rod uce sma ll gel beads , eac h c ont aining a n Thes e anif ic ial se e d s e ncap su late d em br y o. (e ncap su late dem br y os ) c an be m aint ained i n a viab le sta te till t hey ar e plant ed.

Micro pro pa gat ion has bec om e a s uita b l e alternative to conventional methods of vegetative propagation of plants. There are several advantages of micropropagation High

rate

of plant

propagation

Through micropropagation, a large number of plants can be grown from a piece of plant tissue within a short period. Another advantage is that micropropagation can be carried out throughout the year, irrespective of the seasonal variations. Further,for many plants that are highly resistantto conventional propagation, micropropagation is the su itab le a ltern at iv e. Th e small s iz ed pr opagules obt ained i n micropropagation can be easily stored for many years (germplasm storage),and transported across international boundaries. Production

of disease-free

Micropropagation requires minimum growing space. Thus, millions of plant species can be maintained inside culture vials in a small room In a nursery. T h e p r o d u c t i o n c o s t i s r e l a t i v e l yl o w p a r t i c u l a r i y in developing countries (like India) where the manpower and labour charges are low Automated

APPL ICATION S OF MICROPROPA G ATI O N

plants

It is possible to produce disease-free plants through micropropagation. Meristem tip cultures are generally employed to develop pathogen-free plants (details given later in this chapter). ln fact, micropropagation is successfully used for the production of virus-free plants of sweet potato (lpomea batatus), cassava \Manihot esculenta) and vam (Discorea rotundata).

process

Gost-effective

micropropagation

It has now become oossible to automate microprcpagation at various stages. In fact, bioreactors have been set up for large scale multiplication of shoots and bulbs. Some workers e m p l o y r o b o t s ( i n p l a c e o f l a b o u r e r s )f o r m i c r o propagation, and this further reduces production cost of plants. DISADVANTAGES OF MICROPROPAGATION Gontamination

of cultures

During the course of micropropagation,several slow-growing microorganisms (e.9. Eswinia sp, Bacillus sp) contaminate and grow in cultures. The microbial infection, can be controlled by addition of antibiotics or fungicides. However, this will adversely influence propagation of plants. Brewing

of medium

Micropropagation of certain plants (e.g. woody oerennials) is often associatedwith accumulation o f g r o w t h i n h i b i t o r y s u b s t a n c e si n t h e m e d i u m . Chemically, these substances are phenolic c o m p o u n d s ,w h i c h c a n t u r n t h e m e d i u m i n t o d a r k colour. Phenolic compounds are toxic and can inhibit the growth of fissues. Brewing of the medium can be preventedby the addition of ascorbicacid or citric acid or polyvinyl pyrrolidone to the medrum.

560

B IOTECHNO LO CY

Genetic

variability

When micropropagation is carried out through s hoot t ip c ult ur es , genet ic v a r i a b i l i t y i s v e r y l o w . However, use of adventitious shoots is often associatedwith pronounced genetic variability. Vit r if ic at ion During the course of repeated in vitro shooL m ult iplic at ion, t he c ult ur esexh i b i t w a t e r s o a k e d o r almost translucentleaves.Such shoofs cannot grow and even may die. This phenomenon is referredto as vitrification. Vitrification may be prevented by increasing the agar concentration (from 0.6 t o1% ) in t he m edium . How e v e r , i n c r e a s e d a g a r concentration reduces the erowth rate of tissues. Gost

factor

. Virus replication is inhibited by a high concentration of endogenous auxin in shoot a p i c e s . T i s s u e c u l t u r e t e c h n i q u e s e m p l o yi n g meristem-tips are successfully used for the production of disease-free plants, caused by several pathogens- viruses, bacteria, fungi, mvcoptasmas. METHODS TO ELIMINATE VIRUSES IN PLANTS In general,plants are infectedwith many viruses; the nature of some of them may be unknown. The u s a g ev i r u s - f r e ep l a n t i m p l i e s t h a t t h e g i ve n p l a n t i s f r e e f r o m a l l t h e v i r u s e s ,a l t h o u g h t h i s m a y n o t b e a l w a y s t r u e . T h e c o m m o n l y u s e d m e t h od sfo r vi r u s elimination in plants are listed below, and briefly described next.

For m ic r opr opa g a t i o n t e c h n i q u e s , s om e ex pens iv e equipm ent , s ophis t i c a t e df a c i l i t i e s a n d t r ained m anoower ar e needed . T h i s l i m i t s i t s u s e .

r Heat treatment of plant

PRO DUCTI O N O F DI SEASE. FREE PLANTS

o Other in vitro methoos

plant species are infected with Many pat hogens - v ir us es , bac t er ia , f u n g i , m y c o p l a s m a and nem at odes t hat c aus e s y s t e m i c d i s e a s e s . Although these diseasesdo not always result in the death of plants,they reduce the quality and yield of plants. The plants infected with bacteria and fungi frequently respond to chemical treatment by bactericides and fungicides. However, it is very difficult to cure the virus-infected plants. Further, v ir al dis eas e ar e eas ily t r a n s f e r r e d i n s e e d propagatedas well as vegetativelypropagatedplant species. Plant breeders are always interested to dev elop dis eas e- f r ee plant s , p a r t i c u l a r l y v i r a l disease-freeplants. This have become a reality through tissue cultures.

l.leat treatment of plants

wit h l o w Apic al m er is t em s of v ir us es c onc ent r at ion In general,the apical meristemsof the pathogen inf ec t ed and dis eas e har bou r i n g p l a n t s a r e e i t h e r free or carry a low concentration of viruses,for the f ollowing r eas ons .

. Meristemtio culture . C h e m i c a l t r e a t m e n to f m e d i a

(therrnotherapyf

In the early days, before the advent of meristem cultures, in vivo eradication of viruses from plants was achieved by heat treatment of whole plants. T h e u n d e r l y i n g p r i n c i p l e i s t h a t m a n y vi r u se s tn p l a n t t i s s u e s a r e e i t h e r p a r t i a l l y o r co m p l e te l y i n a c t i v a t e d a t h i g h e r t e m p e r a t u r e sw i t h m i n i m a l injury to the host plant. Thermotherapy (at temperatures 35-40oC) was carried out by using hot water or hot air for elimination viruses from growing shoots and buds. There are wvo limitations of viral elimination by heat treatment 1 . M o s t o f t h e v i r u s e s a r e n o t s e n s iti veto h e a t treatment. 2. Many plant species do not survive after thermotherapy. With the above disadvantages,heat treatment h a s n o t b e c o m e p o p u l a r f o r v i r u s e l i m i n a ti o n .

. Abs enc e of v as c ular t is s u e i n t h e m e r i s t e m s culture t hr ough whic h v ir us es r ead i l y m o v e i n t h e p l a n t Meristem-tip body. A general description of the methodology . Rapidly div iding m er is t em a t i c c e l l s w i t h h i g h adopted for meristem and shoot tip cultures has metabolic activity do not allow virusesto multiply. been described (see Fig 47.2).

(MICROPROPACATION) Chapter47 : CLONALPROPACATTON

561

Terla 47.1 A selected list of the plants wlth vlrus elimitation by neristem cultures Virus eliminated

Plant species

- A, X, Y, S Leafroll,potato viruses Tobacco mosaic virus - Mosaic virus virus Mosaic Mosaic virus virusturnipmosaic virus Cauliflower/mosaic Fealthery mottlevirus Veinbanding virus Hoplatentvirus Turnip mosaic virus mosaic virus Cucumber Hycinth mosaic virus Dahlia mosaic virus VirusB Tobacco mosaic virus lrismosaicvirus mosaicvirus Cymbidium Pallidosis virus,yellow viruscomplex Freesia mosaic virus

(potato) Solanum tuberosum (tobacco) Nicotiana tabacum (sugar cane) officinarum Saccharun Allium sativu m (garlic) (pineapple) Anenas sativus (caulillower) Brassica oleracea (sweet potato) lponoea batata grassularia Ribes Humulus lupulus Armoracia rusticena Musa sp(Banana) Hycinthus sp Dahlia sp Chrysanthenun sp Petunia sp lrissp sp Cymbidium Fragaria sp Freesia sp

Fo r vira l e lim inat ion, t he s iz e of t he m e r i s t e m u se d in cu ltur es is v er y c r it ic al. This is due to t h e fact that most of the viruses exist by establishinga gradient in plant tissues. In general, the regeneration of virus-free plants through cultures is inversely proportional to the size of the meristem used . The m er is t em - t ip ex plant us ed f or v i r a l e limina tion cult ur es ar e t oo s m all. A s t er eo s c o p i c microsco pe i s us ually em ploy ed f or t his pur p o s e . Me riste m - t ip c ult ur es ar e inf luenc ed b y t h e following factors. . Physicilog ic alc ondit ion of t he ex plant growing buds are more effective.

ac t i v e l y

Ghemical treatment

of media

Some workers have attempted to eradicate virusesfrom infectedplantsby chemicaltreatment of the ti ssuecul turemedi a. The commonl yused (e.g.cytokinins) chemicalsare growth substances (e.gthi ouraci lacetyl and anti metabol i tes , sal i cyl i c acid). There are however,conflictingreportson the el i mi nati onvi rusesby chemi caltreatmentof the medi a. For i nstance,addi ti on of cytoki ni n suppressed the mul ti pl i cati onof certai n vi ruses w hi l e for someothervi ruses,i t actual l ysti mul ated. Other in vitro methods

. Cu lture m edium - M S m edium wit h l o w con ce ntra t ionsof aux ins and c y t ok inins is i d e a l .

Besidesmeristem-tipculture, other in vitro methodsare also usedfor raisingvirus-freeplants. ln this regardcallus cultures have been successful to someextent.The callusderivedfrom the infected tissuedoesnot carrythe pathogens throughoutthe cel l s. In fact, the unevendi stri buti onof tobacco mosaicvirus in tobacco leaveswas exploitedto developvirus-freeplantsof tobacco.

A selected list of the plants from which viruses h ave b ee n el im inat ed by m er is t emc ult ur es is g i v e n in Table 47.1 .

Somatic cell hybridization, gene transformation and somaclonal variations also useful to raise disease-free olants.

o Thermotherapy prior to meristem-tip culturefo r certa in plant s ( pos s es s ingv ir us es i n t h e meristematicregions),heat treatment is first given and then the meristem-tios are isolated and cu ltured.

Biotechnology [36]

B IOTECHNO LO CY

562

. Wh e n t h e e m b r y o s r e m a i n d o r m a n t fo r l o n g r:eriods. . Low survival of embryos in vivo. Bes idest he elim inat ion of v i r u s e s , m e r i s t e m - t i p . T o a v o i d i n h i b i t i o n i n t h e s e e d f o r g e r m i n a ti o n . c ult ur es and c allus c ult ur es a r e a l s o u s e f u l f o r . F o r c o n v e r t i n g s t e r i l e s e e d st o v i a b l e se e d l i n g s. er adic at ion bac t er ia, f ungi a n d m y c o p l a s m a s . Som e ex am ples ar e giv en S e e d d o r m a n c y i n p l a n t s p e c i e s i s a co m m o n ELI M I NATI O N O THER THAN

O F PATHOG E N S VI RUSES

. The fungus Fusarium roseum has been s uc c es s f ullyelim inat edt hr o u g h m e r i s t e mc u l t u r e s f r om c ar nat ion plant s . o Certain bacteria (Pseudomonas carophylli, Pectobacterium parthenii) are eradicated from c ar nat ion plant s by us ing m e r i s t e m c u l t u r e s . M ERI TS AND DEM ERI TS O F DI SEASE, FREE PLANT P R O D U C T I O N

o c c u r r e n c e .T h i s m a y t r e c j u et o c h e m i ca l i n h i b i to r s or mechanical resistanceexerted by the structures c o v e r i n g t h e e m b r y o S e e d d o r r n a ncy ca n b e s u c c e s s f u l l yb y p a s s e db y c u l t u r i n g t h e e m b r yo s r n vitrc E m b r y o c u l t u r e i s r e l a t i v e l ye a s y a s th e y ca n b e B r o w n o n a s i n r p l e i n o r g a n i c m e d i u m su p p l e m e n t e d w i t h e n e r g y s o u r c e ( u s u a l l y s ucr o se ) .Th i s is possible since the mature embr'.vosexcised from t h e d e v e l o p i n g s e e d s a r e a u t o t r o p h i c i n n a tu r e ,

Anrong the culture techrriques, meristem-tip culture is the most reliable method for virus and EMBRYO RESCUE ot her pat hogenelim inat ion T h i s , h o w e v e r , r e q u i r e s Embryo rescue involves the crrlfure of immature good knowledge of plant pathology and tissr,re embryos to rescue them from unripe or hybrid c u lt ur e. seeds which fail to germinafe. This approach rs Vir us - f r ee plant s ex hibit i n c r e a s e d g r o w t h a n d very useful to avold embryo abortion arrd produce v igour of plant s ,higher y ield ( e . 9 .p o t a t o ) ,i n c r e a s e d a v i a b l e p l a n t . flower size (e.9. Chrysanthemum),improved rooting Wi l d h y b r i d i z a t i o n i n v o l v i n g c r o s si n g o f tw o of siem cuttings (e.g. Pelarg,onium) different species of plants from the same genus or Virus-free plants are more susceptible to the d i f f e r e n t g e n e r a o f t e n r e s u l t s i n f a i l u r e . Th i s i s same virus when exposed again. This is the major mainly becausethe normal development of zygote limitation. Re,infectionof disease-freeplants can a n d s e e d i s h i n d e r e d d u e t o g e n e ti c b a r r i e r s be m inim iz ed wit h good k no w l e d g e o f g r e e n h o u s e C o n s e q u e n t l y ,h y b r i d e n d o s p e r m f a i l s to d e ve l o p m aint enanc e. l e a d i n g t h e a b o r t i o n o f h y b r i d e m b r yo . Th e e n d o s p e i m m a y a l s o p r o d u c e t o x i n s t h at u l ti m a te r y kill the embryo.

Embryo culture deals with the sterile isolation and in vitro growth of a mature or an immature embryo with an ultimate objective of obtaining a v iable plant . Conv ent ional l y , t h e t e r m e m b r y o culture refers to the sexually produced zygolic embryo culture. This is different from the somatrc em br y ogenes is alr , eady des c r i b e d i n t h i s c h a p t e r There are two types of embryo culture - mature em br y o c ult ur e and im m a t u r e e m b r y o c u l t u i " e (embryo rescue). M ATURE

EM BRYO

CULT U R E

Mature embryos are isolatedfrom ripe seedsand cultured in vitro. Mature embryo cultures are c ar r ied out in t he f ollowine c o n d i t i o n s .

I n t h e n o r m a l c i r c u m s t a n c e s ,e n d osp e r m ftr st develops and supports embryo development nutritionally. Thus, majority of embryo abortions a r e d u e t o f a i l u r e i n e n d o s p e r m d eve l o p m e n t. Embryo abortion can be avoided by isolating and c u l t u r i n g t h e h y b r i d e m b r y o s p r i o r t o a b o r ti o r r . The most important application of embryo rescue is the production of interspecific and intergeneric hybrids from wild plant species Gulture

technique

for embryo

rescue

The isolation of immature embryos often poses some difficulty. The aseptically isolated embryos c a n b e g r o w n i n a s u i t a b l e m e d i u m u n d e r o p ti m a l c o n d i t i o n s . I n g e n e r a l , a c o m p l e x n u t r i en t m e d i u m i s r e q u i r e d f o r c u l t u r e m e i h o d s i n v o l vi n g e m b r yo rescue.

(MICROPROPAGATION) Chapter47 : CLONALPROPACATION

Endosperm with failed development 'Hybrid embryo

Ovulewithhybridembryo j

563

Normal enoosperm Normal emoryo Ovule with normalembrvo

I

J

t

Hybridembryo Normalendosoerm

Fig. 47.7 : Embryo-endosperm transplant technique used in embryo rescue (or immature embryo culture).

1 . Heterotrophic phase : This is an early phase F or ade q u a ten u tri ti o n a sl u p p o rto f i m mature and the embryo is mostly dependent on the em br y osemb , ry o -e n d o s p e tra rm n s p l a nits used. endosperm and maternal tissuesfor nutrient supply.

Embryo-endosperm transplant: The endosperm t r ans plantt e c h n i q u eu s e d fo r c u l tu ri n gi mmature 2. Autotrophic phase : This phase is embryosis givenin Fig.47.7,and brieflydescribed characterized by the metabolic capability of the below. embryo to synthesize substances required for its T he hy b ri d e mb r:y ofro m th e o v u l e i n w hi ch endospermdevelopmenthasfailed is takenout by excision.Another normallvdevelopedovule with endos per me n c l o s i n ga n e m b ry o i s th o s e n. Thi s and the normalembryois pressed ovule is dissected w i th a n exi t out . T his le a v e sa n o rm a le n d o s p e rm hole. Now, the hybridembryocan be insertedinto t he nor m a l e n d o s p e rmth ro u g h e x i t h o l e . Thi s r es ult sin em b ry o -e n d o s p e tra rm n s p l a nwt h ich can be c ult ur edi n a s u i ta b l em e d i u m. transplant,many By using embryo-endosperm plants have been intergeneric interspecificand r ais ede. g. ,h y b ri dp l a n tso f l e g u m e s . NUT RI T I O N A L R EOU IR E M EN T S OF E M B RY O C U L T U R E S

growth which slowly makes it independent. The critical stage is the intervening phase between the heterotrophic and autotrophic phases. The nutrient supply is highly variable at this phase which mostly depends on the plant species. In general,the composition of the medium for culturing immature embryos is more complex than that required by mature embryos which can grow o n a s i m p l e i n o r g a n i c m e d i u m F u r t h e r ,t h e t r a n s f e r of embryos from one medium to another is frequently needed in order to achieve full develooment of embrvos. Composition of the medium : Some salient features of medium and culture conditions are listed below

Therearetwo phasesin the embryodevelopment . Inorganic constituents of MS, B5 or White's media are adequate. l q u i re me nvta ri e sa c c ordi ngl y and t he nut ri ti o n are

BIOTECHNOLOCY

56,4

Tlow 4:7.2A selectedlist of dlstant plant speciessossed and the reslstancctralts developed throughembryorescuetechnique Resistancetrait(s)

Distant plant species crossed

sativax O.minuta Oryza x S. etuberosum Solanum tuberosum x S. khasianum melanogena Solanun x B. oleracea Brassica napus x L. peruvianum Lycopersicon esculentum x H. vulgare Hordeun sativum x Thinopyrun Triticum scipeum aestivum . Sucrose is most commohly used energy source . Ammonium nitrate is the preferred source of n itrogen. . Cas ein hy dr oly s at e,r ic h in v a r i o u s a m i n o a c i d s i s frequerrtlyused. o Certain natural plant extracts with embryo factor pr onlot e em br y o c ult ur es e . g I i q u i d e n d o s p e r m of coconut milk. The embryo factor is believed to s upply c er t ain am ino ac i d s , s u g a r s , g r o w t h regulatorsetc.

blight andblast Bacterial Potato leafrollvirus Brinjal shoot andlruitborer vduudvs ^^hL^^^

^^hiillu oPl

fungiandnematodes Virus, mildewandspot blotch Powdery Salttolerance

2. Overcomingseeddormancy: Seeddormancy inhibitors, is causedby severalfactors-endogenous embryo immaturity,specificlight and temperature etc. Further, dry storagerequirements requirements, in someplantsthe naturalperiodof seeddormancy itself is too long. Embryoculture is successfully andto produce appliedto overcomeseeddormancy, i n thesepl antspeci es. vi abl eseedl i ngs

o An incubation temoerature of 24-26"C is idear.

3. Shorteningof breedingcycle : Sonreof the pl ants i n thei r naturalstate have l on g br eeding cycl es. Thi s i s mostl y due to seed dor m ancy attributedto seed coat and/or endosperm.The enrbryoscan be excisedand cultured in vitro ro devel op i nto pl ants w i thi n a short p er iod. For plantcan decorati on H ol l i es,a C hri stmas i nstance, l>e grown in 2-3 weeks time through embryo cultures in contrast to 3 years period required throughseedgermination.

. Bettergrowth of embryos is observed in darkness which are then transferred to lieht for ger m inat ion.

4. Productionof haploids: Embryoculturehas been successfullyused for the production of haploid(or monoploid)plantse.g. barley.

. ln general, growth regulatorsare not required, as t hey induc e c allus f or m at io n . . Embryos grow 5- 7 . 5.

well

in

the

pH

range of

5. Overcoming seed sterility : Certain plant speciesproducesterileseedsthat do not Serminate e.g.earlyripeningvarietiesof cherry,apricot,plum' with incomplete Seedsterilityis mostlyassociated 1 leads to the death of which development embryo APPLI CATI O NS O F EM BR Y O C U L T U R E embryo cultures,it Using germinating embryo. the .l . Prevention of embryo abortion : Incompatiis possibleto raiseseedlingsfrom sterileseedsof bilit y bar r ier s in int er s pec i f i c a n d i n t e r g e n e r i c earl y ri peni ngfrui tse.g. apri cot,pl um. During the culture conditions, the embryos are grown into plantlets,and then transferredto sterile soil for full-pledged growth to maturity.

hy br idiz at ion pr ogr am m es l e a d i n g t o e m b r y o abortion can be successfullyovercome by embryo rescue. In fact, many distant hybrids have been obtained through embryo rescue techniques. A selected list of distant plant speciescrossedand the resistancetraits developed by employing embryo rescue technique is given in Table 47.2.

6. Clonal propagation: Embryosare ideally This is due to suitedfor in vitroclonalpropagation. the fact that embryosare juvenile in naturewith potential.Further,it is possibleto high regenerative and somaticembryogenesis organogenesis induce from embryonictissues.

ermplasnr broadly refers to the hereditary I material (total content ot genes)transmittecltr-r the offspr ing t hr ough ger m c ells Ce r m p l . r s n r pro vid esth e r aw m at er ialf or t he br eec lert o d e v e l o p va riou s cr ops . 1- hus , c ons er v at ion of ge r m p l a s r l r assu mess ignif ic anc ein all br eeding pr ogr a m m e s . As th e pr im it iv e m an lear nt about t he u t i l i t y o f pla nts for f ood and s helt er ,he c ult iv at ed t h e h a b i t of saving selected seeds or vegetative propagules fro m on e seas ont o t he nex t one. I n ot he r w o r d s , this may be regardedas primitivebut conventional germplasm preservation and management, which is hig hly v aluable in br eedr ng pr ogr am m e s The very objective of germplasm conservation (or storage) is to preserve the genetic diversity of a particular plant or genetic stock for its use at any time in future In recent years, many new plant sp ecies wi t l. r des r r ed and im pr ov ed c har a c t e r i s t r c s h ave star t ed r eplac ing t he pr im it ive a n d co nven tionally us ed agr ic ult ur al plant s . l t r s imp orta nt t o c ons er v e t he endanger ed p l a n t s o r else so me of t he v aluable genet ic t r ait s pr e s e n t I n the prtmttive plants may be lost. A global body namely lnternational Board of Plant Genetic Resources (IBPGR) has been e sta blishe df or ger m plas m c ons er v at ion. It s n r a i n o bie ctive is t o pr ov ide nec es s ar y s upp o r t f o r colle ctio n, c ons er v at ion and ut iliz at ion o f p l a n t genetic resourcesthroughout the world There are two approaches f or germpl.rsm co nservati onof plant genet ic m at er iais

I

/n-situ conservation

'2 Fx silrr conservation

The conservation oi germplasm in their natural environment by establishingbiosphere reserves(or national parks/gene sanctuaries) is regarded as irr-.slfuconservation This approach is particularly u s e f L rflo r p r e s e r v a t i o n o f l a n d p l a n t si n a n e a r n a t u r a l h a b i t a ta l o n g w i t h s e v e r a lw i l d r e l a t i v e sw i t h g e n e t i c diversity fhe in-situ conservationis consideredas a h i g h p r i o r i t y g e r m p l a s mp r e s e r v a t i o np r o g r a m m e The major limitations of in-situ conservationare listed below . The risk of losing e n v i r o n m e n t a ]h a z a r d s

germplasrr

due

to

. T h e c o s t o f m a r n t e n a n c eo f i r l a r g e n u m b e r o f B e n o t y p e si s v e r y h i g h . r:i jl

; ij :ri-..r ili :+Pi i il fl *l.f.I! Tilr,

I I

Ex-situconservation is the chief method for the preservationof germplasntobtainecJfrom r:ultivated and wild plant materials.The genetic materials in the form of seeds or from in vitro cultures (plant cells, tissuesor organs) can be preserved as gene banks for long term storage under suitable c o n d i t i o n s . F o r s u c c e s s f u l e s t a b l i s h m e n to f g e n e b a n k s , a d e q u a t e k n o w l e d g e o f g e n e t i c s t r u c t u r eo f plant populations,and the techniques involved tn sampling,regenerationm , a i n t e n a n c eo f g e n e p o o l s etc .lre essentral. 565

t I

B IO TECHNO LO CY

566 Germplasm in the form

There are mainly three approaches for the ln vitro conservation of germPlasm

conservation of seeds

Usualfy, seeds are the most common and convenient materials to conserve plant germplasm. This is becausemany plants are propagatedthrough seeds, and seeds occupy relatively small space' Further, seeds can be easily transported to various places. There are however, certain limitations conserVationof seeds

1. Cryopreservation(freeze-preservation) 2. Cold storage 3. Low-pressureand low-oxygen storage

in the

Cryopreservation (Creek, krayos-frost) literally means preservation in the frozen state. The . Viability of seeds is reduced or lost with passage principle involved in cryopreservation is to bring the plant cell and tissue cultures to a zero of time. metabolism or non-dividing state by reducing the . Seeds are susceptible to insect or pathogen temperature in the presence of cryoprotectants. attack, often leading to their destruction. Cryopreservationbroadly means the storage of o This approach is exclusively confined to seed germplasm at veri low temperatures. propagating plants, and therefore it is of no use o Over solid carbon dioxide (at -79"C) for vegetatively propagated plants e.g. potato, . Low temperature deep freezers (at -80"C) Ipomoea, Dioscorea. . lt is dif f ic ult t o m aint ai n c l o n e s t h r o u g h s e e d conservation.

. In vapour phase nitrogen (at -1 50'C) i I n l i q u i d n i t r o g e n ( a t - 1 9 6 'C )

Among these, the most commonlY used cryopreservation is by employing liquid nitrogen. At the temperatureof liquid nitrogen (-1 96'C), the c e l l s s t a y i n a c o m p l e t e l y i n a c t i v e s ta te a n d th u s for germplasm In vitro methods can be conserved for long periods. In fact, conservation cryopreservationhas been successfullyapplied for In vitro methods employing shoots, meristems germplasm conservation of a wide range of plant and embryos are ideally suited for the conservation s p e c i e s e . g . r i c e , w h e a t , p e a n u t, ca ssa va , of germplasm of vegetatively propagated plants. sugarcane,straberry,coconut. Severalplants can be The plants with recalcitrant seeds and genetically regenerated from cells, meristems and embryos engineered materialscan also be preservedby this stored in cryoPreservation in vitro approach. of cryoPreservation Mechanism There are several advantagesassociatedwith in The technique of freeze preservationis based on v itro germplasm conservation the transfer of water present in the cells from a . Large quantities of materialscan be preservedin liquid to a solid state. Due to the presenceof salts a n d o r g a n i c m o l e c u l e s i n t h e c e l l s , th e ce l l w a te r s m all s pac e requires much more lower temperature to freeze . The ger m plas m pr es er v e dc a n b e m a i n t a i n e d t n (even up to -68'C) compared to the freezing point an environment, free from pathogens of pure water (around 0"C). When stored at low metabolic processes and r lt can be protected against the nature's hazards temperature, the b i o l o g i c a l d e t e r i o r a t i o n si n t h e c e l l s/ti ssu e sa l m o st r From the germplasm stock, large number of come to a standstill. plants can be obtained whenever needed. for successful Precautions/limitations . Obstaclesfor their transportthrough national and cryopleservation int er nat ional bor der s a r e m i n i m a l ( s i n c e t h e Cood technical and theoretical knowledge of ger m plas m is m aint a i n e d u n d e r a s p e c t i c I i v i n g p l a n t c e l l s a n d a s w e l l a s c r yo p r e se r va ti o n c ondit ions ) . Certain seeds are heterogeneousand therefore, are not suitable for true genotype maintenance.

567

AND CRYOPRESERVATION CONSERVATION Chaoter48 : CERMPLASM

t2

------l

tr

Shoottips Pregrownon medium with DMSO

in culturemedium Ampoulethawing

Ampoulestored in liquidnitrogen Shoottips in ampoule frozenin liquidnitrogen Fig. 48.1 : An outline of the protocol for cryopreservation of shoot tip (DMSO-Dimethyl sulfoxide).

technique are essential. Other precautions (the cryopreservationof plant cell culture followed by limita tion stha t s hould be ov er c om e)f or s uc c es s f u l the regeneration of plants broadly involves the following stages cryopreservationare listed below .l . Development of sterile tissue cultures . Forma tion ice c r y s t als ins ide t he c ells s hould b e prevented as they cause injury to the organelles 2. Addition of cryoprotectantsand pretreatment a nd th e cell. 3. Freezing . Hig h in tracel lular c onc ent r at ion of s olut es m a y 4. Storage a lso da mag e c ells . 5. Thawing o Sometimes,certain solutesfrom the cell may leak 6. Reculture o ut d urin g fre ez ing7 . M e a s u r e m e n to f s u r v i v a l / v i a b i l i t y . Cryoprotectants also affect the viability of B. Plant regeneration. cells. . The physiological status of the plant material is also imoortant. TECHNIOUE

O F CRYO PRESERVATI O N

The salient features of the above stages are briefly described. Development

of sterile

tissue

culture

The selection of plant species and the tissues An outline of the protocol for cryopreservation particular referenceto the morphological and The with 48.1 . in Fig. of shoot tip is depicted

B IOTECHNO LO CY

568

phy s iologic alc har ac t er sla r g e l y i n f l u e n c et h e a b i l i t y freezing technique is used for the cryopreservation of shooi tips and somatic embrYos. of the explant to survive in cryopreservalion.Any tissue from a plant can be used for cryopreservation 3. Stepwise freezing method I This is a e. g. m er is t em sem , br y os ,en d o s p e r n t so, v u l e s ,s e e d s , combination of slow and rapid freezing procedures c ult ur ed plant c ells , pr oto p l a s t s ,c a l l u s e s . A m o n g (rvith the advantagesof both), and is carried out tn these, meristematic cells and suspension cell a s t e p w i s em a n n e r .T h e p l a n t m a t e r i al i s fi r st co o l e d cultures,in the late lag phaseor log phaseare most t o a n i n t e r m e d i a t e t e m p e r a t u r e a nd m a i n ta i n e d suitable. t h e r e f o r a b o u t 3 0 m i n u t e s a n d t h e n r a p i d l y co o l e d b y p l u n g i n g i t i n t o l i q u i d n i t r og e n . Ste p w tse and ; \ odlt ion of c r y opr ot ect a n t s freezing rnethod has been successfully used for pretrelatment c r y o p r e s e r v a t i o n o f s u s p e n s i o n c u l tu r e s, sh o o t Crvoprotectants are the compounds that can prevent the damage caused to cells by freezing or thawing. The freezing point and supercooling point of water are reduced bv the presence ot cryoprotectantsAs a result,the ice crystalformation is retarded during the process of cryopreservation Ther e ar e s ev er al c r y opr o t e c t a n t sw h i c h i n c l u d e dim et hy l s ulf ox ide ( DM S O ) , g l y c e r o l . e t h y l e n e , pr opy lene,s uc r os e,m ann o s e ,g l u c o s e , p r o l i n e a n d acetanride. Among these, DMSO, sucrose and glycerol are most widely used. Cenerally, a mixture of cryoprotectantsinstead of a single one, is used for more effectivecryopreservationwithout damage to cells/tissues.

aoices and buds

1 . Slow-freezing method : The tissue or the r equis it e plant m at er ial is s l o w l y f r o z e n a t a s l o w cooling rates of 0.5-5oC/min from 0'C to -100"C, and t hen t r ans f er r ed t o l i q u i d n i t r o g e n . T h e advantage of slow-freezing method is that some amount of water flows from the cells to the outside. This pr om ot es ex t r ac ellu l a r i c e f r o m a t i o n r a t h e r t han int r ac ellularf r eez in g . A s a r e s u l t o f t h i s , t h e plant c ells ar e par t ially d e h y d r a t e d a n d s u r v i v e better. The slow-freezing procedure is successfully used for the cryopreservation of suspension cultures.

The ultimate obiective of storage is to stop all t h e c e l l u l a r m e t a b o l i c a c t i v i t i e sa n d m a i n ta i n th e i r r,iability For long term storge, temperature at -196"C in liquid nitrogen is ideal. A regular and c o n s t a n t s u p p l y o f l i q u i d n i t r o g e n to th e l i q u i d nitrogen refrigeratoris essential.lt is necessaryto c h e c k t h e v i a b i l i t y o f t h e g e r m p l a s mp e r i o d i ca l l y i n some samples.

4. Dry freezing method : Some workers have reported that the non-germinated dry seeds can survive freezing at very low temperature in contrast t o w a t e r - i m b i b i n g s e e d s w h i c h a r e su sce p ti b l eto c r y o g e n i c i n j u r i e s . I n a s i m i l a r f a s h i o n , d e h yd r a te d cells are found to have a better survival rate after cryopreservatton. Storage

M a i n t e n a n c eo f t h e f r o z e n c u l t u r e sa t th e sp e ci fi c temperatureis as important as freezing. ln general, the frozen cells/tissues are kept for storage at temperatures in the range of -70 to -196'C. However, with temperatures above -1 30'C, ice Fl a+ ; illr g c r y s t a l g r o w t h m a y o c c u r i n s i d e t he ce l l s w h i ch The serrsitivityof the cells to low temperature is r e d u c e sv i a b i l i t y o f c e l l s S t o r a g ei s i d e a l l y d o n e i n v ar iable and lar gely depe n d s o n t h e p l a n t s p e c i e s . l i q u i d n i t r o g e nr e f r i g e r a t o r - a t 1 5 0 'C i n th e va p o u r Four different types of freezing methods are used. p h a s e ,o r a t - 1 9 6 'C i n t h e l i q u i d p ha se .

2. Rapid freezing method : This technique is quit e s im ple and inv olv e s p l u n g i n g o f t h e v i a l c ont aining plant m at er i a l i n t o l i q u i d n i t r o g e n . During rapid freezing, a decrease in temperature -300" to -1000"C/min occurs. The freezing process is c ar r ied out s o quic k ly th a t s m a l l i c e c r y s t a l sa r e f or m ed wit hin t he c ells . F u r t h e l t h e g r o w t h o f int r ac ellular ic e c r y s t als i s a l s o m i n i m a l . R a p i d

Proper documentation of the germplasm storage has to be done. The documented information must b e c o m p r e h e n s i v ew i t h t h e f o l l o w i ng p a r ti cu l a r s. . T a x o n o m i c c l a s s i f i c a t i o no { t h e ma te r i a l o History of culture . Morphogenic potential o C e n e t i c m a n i p u l a t i o n sd o n e . Somaclonalvariations . Culture medium o Growth kinetics

ChAPICT 48 : CERMPLASM CONSERVATION AND CRYOPRESERVATION

569

Thawing Tlrrr 48.1 A selected list of plants in varlous forms that are successfullycryopreserved

Thawingis usuallycarriedout by plungingthe frozen samples in ampoules into a warm water (temperature 37-45"C)bathwith vigorousswirling. By this approach,rapidthawing(atthe rateof 500750" C m in- l) o c c u rs ,a n d th i s p ro te c tsth e c el l s from the damagingeffectsice crystalformation.

PIant material

Cellsuspensions

As the thawingoccurs(icecompletelymelts)the ampoulesare quicklytransferred to a waterbath at temperature2O-25"C. This transferis necessary sincethe cellsget damagedif left for long in warm (37-45'C\ water bath.

Callus

For the cryopreservedmaterial (cells/tissues) where the water contenthas been redur-edto an optimal level before freezing, the process of t howingbec om e sl e s sc ri ti c a l .

Meristems

Beculture In general,thawedgermplasmis washedseveral times to removecrvoorotectants. This materialis t hen r ec ult ure di n a fre s h me d i u m fo l l o w i ng standardorocedures. Some workers prefer to directly culture the thawed materialwithout washing.This is because certain vital substances, releasedfrom the cells during {reezing,are beiievedto promote in vitro cultures. Measurement

Protoplast

of survival/viability

Zygotic embryos

Somatic embryos

Pollen embryos

Plant species 4^,-^ vr yzd

^^t:.,^ JdIIva

Glycine nax Zeanays Nicotiana tabacun Capsicum annun Oryzasativa Capsicun annum Saccharum sp Zeamays Nicotiana tabacum Solanun tuberasum Cicerarielinun Zeamays Hordeum vulgare Manihot esculenta Citrussinensis Daucus carota Coffeaarabica Nicotiana tabacum Citrussp Atropa belladona

The viability/survival of the frozencells can be measuredat any stageof cryopreservation or after appropriate plant growth and regeneration, the thawingor r ec u l tu re . The techniquesemployedto determineviability of cryopreserved cellsarethe sameas usedfor cell cultures (Refer Chapter 42). Staining techniques us ingt r iphenylte tra z o l i u mc h l o ri d e(T T C ),E v an' s blue and fluorescein diacetate(FDA)are commonly used. T he bes t ind i c a to rto m e a s u reth e v i a b i l i tyof c r y opr es er v ed c e l l s i s th e i r e n try i n to c e l l d i v i s i on and regrowthin culture.This can be evaluatedby t he f ollowingex p re s s i o n ^ No. of c e l l s /o rg a nBsro w i n B * ,O O No. of cells/organs thawed

cryopreserved cells/tissues have to be carefully nursed, and grown. Addition of certain growth promoting substances, besides maintenance of appropriate environmental conditions is often necessaryfor successfulplant regeneration. A selected list of plants (in various forms) that have been successfullyused for cryopreservationis siven in Table 48.1 .

Cold storage basically involves germplasm conservation at a low and non-freezing Plant regeneration temperatures(l-9"C). The growth of the plant of materialis sloweddown in cold storagein contrast The ultimate purpose of cryopreservation germplasmis to regenerate the desiredplant. For to completestoppagein cryopreservation. Hence,

t.-

B IOTECHNO LO CY

570 Other gases (2%)

z E c) o

6 seo o o0

Normalatmospheric storage

LOW-pressure slorage

LOW-oxygen storage

Fig. 48.2 : A graphic representation of tissue culture storage under normal atmospheric pressure, low-Pressure,and low-oxYgen'

cold storage is regarded as a slow growth The major germplasm conservation method advantageof this approach is that the plant material (cells/tissues) is not subjected to cryogenic injuries.

c o n s e r v a t i o n . A g r a p h i c r e p r e s e n t a t i o no f ti ssu e culture storageunder normal atmospheric pressure, Iow-pressure and low-oxygen is depicted in Fig. 48.2.

Long-term cold storage is simple, cost-effective and y ields ger m plas m wit h g o o d s u r v i v a l r a t e . Many in vltro developed shoots/plantsof fruit tree s pec ies haVe been s uc c es s f u l l y s t o r e d b y t h i s approach e.g. grape plants, strawberryplants.Virusfree strawberry plants could be preserved at 10'C for about 6 years, with the addition of a few drops of m edium per iodic ally ( o n c e i n 2 - 3 m o n t h s ) . Several grape plants have been stored for over 1 5 years bv cold storage(at around 9'C) by transferring t hem v ear lv t o a f r es h m edi u m .

Low.pressure storage (LPSI

As alternatives to cryopreservation and cold storage,Iow-pressurestorage(LPS)and low-oxygen storage(LOS) have been d'evelopedfor germplasm

B e s i d e s g e r m p l a s m p r e s e r v a t i o n , LPS r e d u ce s t h e a c t i v i t y o f p a t h o g e n i c o r g a n i s m s a n d i n h i b i ts s p o r e g e r m i n a t i o n i n t h e p l a n t c u l t u r e syste m s.

pressure theatmospheric storage, In low-pressure s u r r o u n d i n g t h e p l a n t m a t e r i a l i s r e d u ce d . Th i s resultsin a partial decreaseof the pressureexerted by the gases around the germplasm. fhe lowered partial pressure reduces the in vitro growth of plants (of organized or unorganizedtissues). Low-pressure storage systems are useful for short-termand longterm storageof plant materials. T h e s h o r t - t e r m s t o t a g e i s p a r t i c u l a rl y u se fu l to i n c r e a s et h e s h e i f l i f e o f m a n y p l a n t m a te r i a l se .g . f r u i t s , v e g e t a b l e s ,c u t f l o w e r s , p l a n t c u tti n g s. Th e g e r m p l a s mg r o w n i n c u l t u r e sc a n b e s t or e dfo r l o n g term under low pressure.

571

AND CRYOPRESERVATION CONSERVATION Chaoter48 : CERMPLASM Low'oxygen

storage

(LOSI

In the Iow-oxygen storage, the oxygen con ce ntra tion is r educ ed, but t he at m os phe r i c pre ssure(2 50 mm Hg) is m aint ainedby t he addit i o n o f in ert g ases(p ar t ic ular lynit r ogen) The partial pressureof oxygen below 50 mm Hg re du ce s p lan t t is s ue gr owt h ( or ganiz ed o r u no rga nizedtiss ue) .This is due t o t he f ac t t hat wi t h r e du ce d availa bilit y of O r , t he pr oduc t ion of CO, is low. As a consequence, the photosynthetic activity is reduced, thereby inhibiting the plant tissue growth and dimension. Limitations of LOS : The long-term conservation of plant materialsby low-oxygen storageis likely to in hib it the pla nt gr owt h af t er c er t ain dim ens ion s .

2. Cryopreservationis an ideal method for long term conservation of cell cultures which produce secondary metabolites (e.9. medicines). 3 Disease (pathogen)-freeplant materials can be frozen, and propagated whenever required. 4. Recalcitrant seeds can be maintained for ron8. 5. Conservation of somaclonal and toclonal variations in cultures.

game-

6. Plant materials from endangered species can be conserved. 7. Conservation of longevity.

pollen

for

enhancing

B Rare germplasmsdeveloped through somatic h y b r i d i z a t i o n a n d o t h e r g e n e t i c m a n i p u l a t i o n sc a n be stored

The germplasm storage has become a boon to plant breeders and biotechnologists.Some of the ap plicatio ns a re br ief ly des c r ibed. 1 . Maintenance of stock cultures : Plant mate rials(ce ll/tis s uec ult ur es )of s ev er als pec iesc a n be crvooreservedand maintained for severalyears, an d used as a nd when needed. This is in c ont r a s t to an in vitro cell line maintenancewhich has to be subcultured and transferredperiodically to extend via bility. Th us, ger m plas m s t or age is an id e a l meth od to a vo id s ubc ult ur ing, and m aint ain c e l l s / tissu esin a viab le s t at e f or m any y ear s .

9. Cryopreservation is a good method for the s e l e c t i o n o f c o l d r e s i s t a n tm u t a n t c e l l l i n e s w h i c h could develop into frost resistantplants. 10, Establishment of germplasm banks for exchange of information at the international level. LIMITATIONS STORAGE

OF GERMPLASM

The maior limitations of germplasm storage are and the trained the expensive equipment personnel. lt may, however, be possible in the near future to develop low cost technology for c r y o p r e s e r v a t i o no f p l a n t m a t e r i a l s .

' y the conventional plant breeding techniques, ." . ." ' 6..' -c, s ignif ic ant ac hiev em ent s h a v e b e e n m a d e i n I t i s o n l y t h r o u g h t h e r e c o m b i n a n t a p p r o a ch the improvement of several t,ood crops. These age( g e n e t i c e n g i n e e r i n g )o f b i o t e c h n o l o g y,n e w g e n e s old c las s ic al m et hods , inv o l v i n g g e n e t r a n s f e r with desired characters (that may or may not be through sexual and vegetative propagation, fake p r e s e n t i n o t l . r e rp l a n t s )c a n b e i n t r o d u ce d i n to th e very long time. For instance, about 6-8 years may p l a n t s F u r t h e r , i t i s p o s s i b l e t o m a n i p u l a te th e be required to develop a new rice or a wheat by e x i s t i n g g e n e s t o m a k e t h e p r o t e i n s w i th su i ta b l e s ex ual pr opagat ion. Rapid a d v a n c e s i n g e n e a l t e r a t i o n s e . g . i n c r e a s e i n t h e c o n te n t o f a n s t r uc t ur e and f unc t ion, c oup l e d w i t h t h e r e c e n t e s s e n t i a la m i n o a c i d . T h e n r o s t i m p o r ta n t r e a so n s dc v elopm ent s m ade in lhe g e n e t i c e n g i n e e r i n g for developing transgenic planfs are listed t ec hniques hav e dr am at ic all y i m p r o v e d t h e p l a n t br eeding m et hods t o y ield t h e d e s i r e d r e s u l t s i n a o T o i m p r o v e a g r i c u l t u r a l , h o r t i cu l tu r a l o r s hor t oer iod. ornamentalvalue of plants.

,

Plant genet ic t r ans f o r m a t i o n t e c h n o l o g v . To develop plant bioreactors for inexpensive basically deals with the transfer of desirable m a n u f a c t u r eo f c o m m e r c i a l l y i m p o r t an t p r o d u cts gene(s) from one plant species to another (or p r o t e i n s , m e d i c i n e s , p h a r m a ce u ti ca l e.g. insertion of totally new genes) with subsequent compounos. int egr at ionand ex pr es s ionof t h e f o r e i g n g e n e ( s )i n o T o s t u d y t h e a c t i o n o f g e n e s i n p l a n ts d u r i n g the host Benome. The term transgene is useci to d e v e l o p m e n t a n d v a r i o u s b i o l o g i c a l p r o ce sse s. represent the transferued gene, and the genetic transformation in plants is broadly referred to as Genetic traits ir;trorlueed intei plant transgenesis fhe genetically transformed transgemi* n*aile'.; new plants are regarded as transgenic plants. S i n c e p l a n t c e l l s a r e t o t i p o t e n t ( i .e . a si n g l e The dev elopm ent of t r an s g e n i c p l a n t s i s t h e p l a n t c e l l c a n r e g e n e r a t ei n t o a w h o l e p l a n t) , th e out c om e of an int egr a t e d a p p l i c a t i o n o t g e n e t i c a l l y e n g i n e e r e d c e l l s w i t h n e w g e n e ( s)ca n r ec om binant DNA ( r DNA ) t e c h n o l o g y , g e n e p r o d u c e a t r a n s g e n i cp l a n t T h i s p l a n t ca r r yi n g th e t r ans f er - m et hods and t is s ue c u l t u r e t e c h n i q u e s . d e s i r e d t r a i t w i l l g i v e r ai s e t o su cce ssi ve (Note : The reader must refer Chapters 6 and 7 for generations. Many genetic traits have been bas ic inf or m at ion on genet ic e n g i n e e r i n g ,a n d f o r introduced into plants through genetic engineering c om m on t ec hniques em p l o y e d in rDNA . R e s i s t a n c et o h e r b i c i d e s t ec hnology )

572

CF PLA N TS -ME TH OD OLOC Y Chapt er49 : C EN ET ICEN C IN EE R INO

573

Tmu 49.1 Gene transfer {DIiA delivery) nethods ln plants Salienl features

h4ethod

l. Vector-mediatedgene transfer genetransfer (Tiplasmid)-mediated Agrobacterium

groupof plants Veryefficient, butlimitedto a selected Ine{fective method, hencenotwidelyused

Plantviralvectors ll. Direct or vectorlessDNA transfer (A) Physical methods Electroporation (particle Microprojectile bombardment) Microinjection Liposome fusion

Mostlyconfined to protoplasts thatcanbe regenerated to viableplants.Manycerealcropsdeveloped. Verysuccessful method usedfor a widerangeof plantsr Riskof genereanangement tissues. high. Limited usesinceonlyonecellcanbe microinjected at a personnel time.Technical should be highly skilled. Confined to protoplasts intoviable thatcanbe regenerated wholeplants. Requires regenerable Thefibres,however, cellsuspensions. require careful handling.

fibres Siliconcarbide (B) Chemical methods glycol(PEG)-mediated Polyethylene (DEAE) Diethylaminoethyl dextran-mediated

to protoplasts. Confined Regeneration of fertileplantsis problematical. frequently Doesnotresultin stabletransformants.

r Protection against viral infections o Insecticidal activity The gene transfer techniques in plant genetic transformation are broadly grouped into two categories

. lmp roved nu t r it ional qualit y o Altered flower pigmentation . Tolerance to environmental stresses

Vector-mediatedgene transfer

ll. Direct or vectorlessDNA transfer

. Se lf in co mpat ibilit y Griteria for commercial genetically transformed

l.

use of plants

For large-scale commercial application of genetically engineered plants, the following reouirementshave to be satisfied o ln trod uction of des ir able gene( s ) t o all p l a n t cells.

The salient featuresof the commonly used gene (DNA) transfer methods are siven in Table 49.1.

Vector-mediated gene transfer is carried out transformation either by Agrobacterium-mediated or by use of plant virusesas vectors.

Expression of cloned genes in the appropriate AG R O B ACT E N I U M.MEDI AT ED c ellsat t he ri g h tti me . GE N E TR A N S FE B of new gene(s)inserted. Stablemaintenance Agrobacteriumtumefaciensis a soil-borne, Transmissionof new senetic information to Cram-negativebacterium.lt is rod shaped and generations. motile, and belongs to the bacterial family of subsequent

l.;

t'

B IOTECHNO LO CY

574

Wound

f i.o Erf ruenl

Excessand waste sludge Fig. 57.4 : Flow diagram of activated-sludge process.

Factors affecting performance : There are several factors that influence the efficiency of activated sludge process,the most important being the type of the reactor, aeration, food microorganism (F/M) ratio, nutrients, sludge recirculation rate, besides pH and temperature. Advantages : The activated sludge process is a very compact, low-cost and an efficient biological treatment system for sewage treatment. lt is worked o ut th at un de r ideal c ondit ions , up t o 95% o f BOD', 98% of bacteria (particularly coliform) and 95% of suspendedsolids can removed by activated sludge process.The excessand waste sludge has a higher fertilizer value compared to other treatment processes. Disadvantages : There is production of large r olumes of sludge which sometimes becomes ctifficultto ha nd le. Power c ons um pt ion is r elat iv e l y h igh for o pe ratio n.Super v is ionby s k illed per s on n e l s necessary. Conventional

activated

sludge

process

ln the normal treatment of sewage,the activated sludge is preceededby primary sedimentationtank. The conventional activated sludge system consists of a separationtank, settling or sedimentationtank and sludge removal line (Fig. 57.4\. fhe sewage after the primary treatment is introduced at the h ea d of the tan k . lt is des ir able t o s upply O, u nifo rmly thro ug hout t he t ank . Modified

activated

sludge

processes

For increasingthe performance of the activated 'several modifications have been sludge system, years. Most of them are directed in the recent done to bring out efficient aeration Aeration can be

done by step aeration, tapered aeration, high rate aeralion by complete mixing and extended aeration. AERATED

LAGOONS

Aerated lagoons, also called as aerated ponds, are the faculative stabilization pcnds wherein surface aeratorsare installed to overcome the bad adours (due to overload of organic materials).The microbiological treatment of aerated ponds is comparable to the activated sludge process. The major difference is the large surfacearea in aerated p o n d s a n d t h i s i s m o r e s u s c e p t i b l ef o r t e m p e r a t u r e effects. It is possibleto carry out continuous nitrification in aerated lagoons. This however, depends on the design and operating conditions of the pond (particularly the temperature). SEQUENCING

BATCH

REACTOR

S e q u e n c i n gb a t c h r e a c t o r( S B R )i s a m o d i f i c a t i o n of activated sludge treatment system.The processes namely aeration and sedimentationare carried out in both the systems.The major difference is that while in the conventional activated sludge system, a e r a t i o na n d s e d i m e n t a t i o no c c u r s i m u l t a n e o u s l yi n separatetanks, these two processesare carried out sequentially in the same tank in SBR. Thus, the sequencing batch reactor may be regarded as filland-draw activated sludge process. The operating sequence of a typical SBR is depicted in Fig. 57.5. The process is carried out in aeration a sequence of five sreps-- filling, (reacting) sedimentation (settling), decanting and idle. Severalmodifications and impi'ovementshave been made in the SBR for more efficient or;eration.

B IOTECHNO LO CY

692

Aeration status

digesters.More details on aerobic digestion are eiven elsewhere(Referp . 7 1 1 ) .

Air onlolt

Air on

Aerobic attached-growthtreatment processesare commonly used to remove the organic matter found in the sewage. These processes are also useful for the nitrification (conversion of ammonia to nitrate). The commonly used attached-growth processesare listed. . Trickling filters . R o u g h i n gf i l t e r s

Reacting(aeration)

. R o t a t i n gb i o l o g i c a l c o n t i a c t o r s Air off

. Packed bed reactors. A m o n g t h e s e ,t r i c l a

Genotype- aa Phenotype- Normalfemale (B) Autosomal recessive PARENTS Mate(d) Genotype- Bb Phenotype - Carriermale Femate (Q)

,/

d'

Genotype-Bb Phenotype - Carrierfemale

)Y

/'

CHILDREN Genotyperatio-1:2: 1 BB/Bb/Bblcb Phenotype-25% affected -25"k normal -507ocarriers

t*o

(C) X-chromosorne(sex chromosome)-linked inheritance PARENTS

CHILDREN Genotyperatio-1 : 1 : 1 : 1 WXY/X'XA"Y of malesaffected Phenotype-50%

Mate (d) Genotype- XY Phenotype-Normal male Female (Q) Genotype-X"X Phenotype- Carrierfemale

y'x d'

\"

Fig. 69.1 : Patterns of inheritance-autosomaldominant, autosomal recessive and X-linked (Note : Genotype refers to the description of genetic composition, while phenotype represents the observable character displayed by an organism).

2. Ar r t os om al r ec es s iv e in h e r i t a n c e : I n t h i s c as e,t he nor m al allele is des ig n a t e da s I w h i l e t h e disease-causingone is a (Fig. 69.1B). The gametes of c ar r ier m ale and c ar r ier f e m a l e ( b o t h w i t h genotype Bb) get mixed. For these heterozygous carrier parents,there is one fourth chance of having an af f ec t edc hild. Cy s t ic f ibr os i s ,s i c k l e - c e l la n e m t a and phenylketonuria are some good examples of autosomal recessivedisorders. 3. Sex (X)-linked inheritance : In the Fig. 69.1C, s ex - link ed pat t er n of inher it a n c e i s d e p i c t e d . A nor m al m ale ( XY) and a c ar r i e r f e m a l e ( X C Y )w i l l pr oduc e c hildr en wher ein, half o f t h e m a l e c h i l d r e n are affected while no female children is affectet-r. This is due t o t he f ac t t hat t h e m a l e c h i l d r e n

possessonly one X chromosome, and there is no dominant allele to mark its effects (as is the case w i t h f e m a l e s ) .C o l o u r b l i n d n e s sa n d h e m op h i l i a a r e good examplesof X-linked diseases. A selected list of genetic disorders (monogenrc traits) due to autosomal and sex-linked inheritance i n h u m a n s i s g i v e n i n T a h l e 6 9 . 1. GENETIC

DISEASES

IN HUMANS

The pattern of inheritanceand monogenic traits along with some of the associateddisorders are described above (Table 69.1). Besides gene mutations, chromosomal abnormalities (aberrations) a l s o r e s u l t i n e e n e t i cd i s e a s e s .

Chapter69 : CENETICS

425

Tlrrr 69.1 Selected eranples of genetlc dlsorders (monogenlctraltsf In humans I n he r it ed patter n/cli sease

Estitnated incidence

Salient features

Autosomaldominant 1 500 1 5000 'I 12000

Highriskfor heartdiseases Nervous dementia disorders, Tumcrsof retlna

1 800

Highriskforbreast andovarian cancers

1 : 2500(inpeople of Meditenanean descent)

A blooddisorder; thebloodappears to be blueinstead of red

Sickle-cell anemia

1 : 100(inAfricans)

Cysticfibrosis

1 : 2500(inCaucasians)

lifethreatening Severe anemia; confers resislance to malaria Defective iontransport; severelung infections andearlydeath(before they reach30 years)

Phenylketonuria cr,-Antitrypsin deficiency

1 : 2000 1 : 5000 1 : 3000 (inAshkenazi Jews) Rare(only100cases reported worldwide)

immune Highly defective system; early death

1 : 50 males males 1 : 10,000 1 : 7000males

Unable to distinguish colours Defective bloodclotting Muscle wastage

Not known

mayleadto Damage to opticnerves, blindness

Familial hypercholesterolemia Huntington disease Familial retinoblastoma Breastcancergenes (BRAC1 and2) B-Thalassemia Autosomalrecessive

Tay-Sachs disease immunodeficiency Severecombined (SCID) disease

Mental retardation dueto braindamage Damage to lungsandliver Nervous and disorder; blindness pararysrs

Sex-linked Colour blindness (li/B) Hemophilia dystrophy Duchenne muscular Mitochondrial Leberhereditary opticneuropathy

Aneuploidy : The presence of abnormal number of chromosomes within the cells is referred to as :ne up loid y. Th e m os t c om m on aneuploid c ondit i o n , trisomy in which three copies of a particular :r'omo so me are pr es ent in a c ell ir r s t eadof th e - rr.nal two e.g. trisomy-21 causing Down's stndrome; trisomy-1B that results in Edward's *ndrome. These are the examples of autosomal .-e up loid y. in case of s ex - link ed aneuploidy , t he s e x --'Dmo so mes oc c ur as t hr ee c opies . e. g. phen o -.::ca lly male c aus ing Klinef elt er ' ss y ndr om e h a s t r\: triso my-X i s phenot ic ally a f em ale wit h XX X .

Selected examoles of chromosomal disorders along the with the syndromes and their characteristicfeatures are siven in Table 69.2. EUGENICS Eugenics is a sclence of improving human race based on genetics. lmproving the traits of plants and animals through breeding programmes has been in practice for centuries. E u g e n i c si s a h i g h l y c o n t r o v e r s i a ls u b j e c t d u e t o social, ethical, and political reasons. The proponents of eugenics argue that people with

826

B IOTE C H NO LO CY

Tlru 69.2 Scte€fedexanples of chromosonal dlsorders in humans Condition

Chromosome

Syndrome

Salient features

Normal Females

2n (46,XX)

Males

2n (46,XY)

Autosomalaneuploidy

2n + specific chromosome

Trisomy-21

4 7 ,X Y+, 21

Down'ssyndrome

lQo t u u o n s , . / b 2

BIOTECHNOLOCY

870

S o m a clo n a l va r ia n ts, 5 4 7 S o m a clo n a l va r ia tio n s, 5 4 6 S o ma clo n a l va r ia tio n sap p lica tio n s,5 4 9

Streptomyces-Benetic 342 manipulations,

S yntheti c vacci ne,202 1B 3 C X ,-S ynucl ei n,

Streptomycin, 337 Stroma,753

S o ma clo n a l va r ia tio n sl im ita tio n s,5 5 0

760 Structuralisornerism,

S o ma clo n e s,5 4 7

528 Sub-protoplasts,

S o m a tic ce ll g e n e th e r a p y, 1 5 7

768 Subcellular organelles,

S o m a tic e m b r yo g e n e sis,5 5 3 , 5 5 7

Subculture,445 monolayercultures,445 suspension cultures,446

T-DNA,575

Substratespecificitv, 792

Tandemgenearrays,138

Substrate straintheory,794

TATAbox, 42

S u b t i l i s i n1,3 5

Tautomericforms,12

l ita tio n s, S o m a tic h yb r id iza tio n - im 537

Subunitvaccines,201

30 1'elomerase,

Subzonalinsertion,235

Telomere,30

Sorghum beei', 370

Svedbergunit,768

codons,46, 53 Termination

Sour-doughbread, 365 S o u th e r n b lo Vb lo ttin g , 6 9 , 9 8

water Sugarindustry-waste treatment,705

S p a wn , 3 8 1

Suicidegenetherapy,168

624 of transcription, Terminators Tertiarystructureof protein, 783

Spectrophotom eter, 7 67

S u i c i d ei n h i b i t i o n 7 , 91

S p er m se xin g , 2 2 8

S u l f o n y l u r e a6s1, 0

S p he r o id s,4 T l

610 resistance, Sulfonylureas

S p i n o ce r e b r a la ta xia , 1 8 0

S u l f u ra m i n o a c i d s ,8 1 1

Spirulina SCP, 379

Superbug, 725

S o m a tic e m b r yo s, 5 5 8 S o m a tic h vb r id p la n ts, 5 3 2 S o m a tic h yb r id iza tio n , 5 2 8 S o m a tic h yb r id iza tio n ap p lica tio n s, 5 3 6

S p l i ce o so m e ,4 4 Sporophytes, 755 S p r ay to we r s, 6 7 3 , 6 7 7 S p r i n klin g filte r s, 6 9 2 S t a b ility o f p r o te in s, 1 3 9 Starch, 622, 774 S t e m ce ll cu ltu r e s, 4 4 7 S t e m ce ils, 4 4 7 Stem cells-charactetization, 448 S t e re o iso m e r ism ,7 6 0 Stereospecificity, 791 S t e r iliza tio no f b io r e a cto r , 2 4 5

StufferDNA, 84

Supercoils,26 Supermouse,481 Supercritical fluid extraction,276 Superovulation,22S 18'l dismutase, Superoxide Surfacecondense,671 Surfaceimmobilizedplant cell t e c h n i q u e5, 1 1 Surfacetension,765 Suspensioncloning,467 Suspension cultures (of plants),502

T

T o l y s o z y m e1, 3 3 , 1 3 5 281 Takadiastase,

Tertiarytreatmentof sewage,698 Testtube babies,233 Tetrac),clines,33B 45 B-Thalassemia, Thaumatin,367 oxygendemand,683. Theoretical 300 Thermalbiosensors, Thermal incinerator,677 297 Thermistors, 300 biosensors, Thermometric ELISA,301 Thermometric of plants,560 Thermotherapy Thiobacillus ferroxidans, 402 T h i o n i n s6 , 06 Threedimensionalcultures,47-1 Threonine,352 .l Thrombosis,95

S t i cky e n d s, 7 8

T h y l a k o i d sT, 5 3 Suspension cultures,446 , 0, 574 icrobialproduction,U., T i p l a s m i d 7 Sweeteners-m peripheralnerve Tissueengineered i m p l a n t s4, 7 6 Symbaprocess,378 skin,475 Tissueengineered Symbaproduct,378

Stirred tank reactors, 240

Symbioticnitrogenfixers,646

urothelium,476 Tissueengineered

S t i r r e r cu ltu r e , 4 5 3

Symbioticnitrogenfixing,640

472 Tissueengineering,

S t i r r e r fla sk, 4 5 3 Stratagene, 'l 23

Symmetrichybrids,534

Tissuemodelling,477

Synchron ization (of plant cultures),504

activator,135, Tissueplasminogen 194

S t e r iliza tio n o f cu ltu r e m e d ia ( o f fe r m e n ta tio n ) ,2 5 0 >terotost / / /

S t r e p to kin a se ;19 6

471 geneexpression, 65 Tissue-specific TK suic idegene,169 To mat or ipening,613

method, 482 for human di seases,487 mi croi nj ecti on method, 481 retroviral vector method, 481

u

Topatoes,533

Transgeni cmosqui toes,493

Ubiquitinpromoter,592

Totalorganiccarbon,683

Transgeni cpi gs, 488

768 Uftracentrifugation,

Totipotency,490, 497

Transgeni cpl anl s, 572, 585, 622

Uniformresourcelocator,828

Totipotent,572

Transgeni cpl ants as bi oreactors,

Urea biosensor, 300 Urea cycle,809

Traditionalvaccines,199

622 plants-therapeutic Transgenic proteins,638 'lransgenic sheep,488

Transamination, 809

Transgenic snails,493

Transcriptional genesilencing,593

Transgenic tsetseflies,493

Transcriptionfactors,43

Transgenic veggievaccines,637

Toxoids,200 Tradesecrets,745 Traditionalbiotechnology,3

Urokinase,196

v

Transcription inhibitors,45

Translation, 46

Transcription, 39 in eukaryotes, 42 in prokaryotes, 39

Transposable elements,32

Vaccines,l98

Transposition, 32

Vaccinesin plants,636

Transposition of genes,67

V a c c i n i av i r u s , 2 1 1

Transcriptome, 38

I'ansposons, 32

Vacuoles,753

Tra ns c r ipt om ic s , 3S

Transposontagging, TO

Transduction,88

fray towers, 677

Variablenumber tandem repeats, 186

TransferredDNA, 70

Trehalose,624

Vectorrecombinant vaccines,211

Transfer RNA, 21, 45

l r l ac y l gl y c er O l S/ / /t)

Vectorvaccine,211

Transformation, 87

Tricklingfilters,692

Transformation assays,462

(plant)gene Vector-mediated rransret5/J

Transformation efficiency,87

Triosephosphate isomerase, 133 Triple repeatdiseases,-180

VectorlessDNA transfer,583

Transformation of cells, 463

Triple-stranded DNA, 17

Vectors, 82

Transformedcells, 430

Trophophase, 254

Vegetablevaccines,636

Transformed cells-characteristics, 463

Trypsinization,438

Veggievaccines,636

Tryptophan, 353, 811

Vermicomposting, 714

Transgene,480, 572 Transgenesis, 480 Transgenesis in largeanimals,487

Tryptophanoperon, 62

Viable celfs-separalion, 44'l

Tryptophanrepressor,62

Vinegar,325

Tuberculosis-diagnosis, 176

v r r a to r s e a s e s , , / 5 / Virapap,178

Transgene stability,593

Tubercu losis-recombi nant vaccine, 203

Transgenic animals,480

Tubularbowl centrifuge, 272

Virus resistanceof plants,602

Transgenic bollworms,493

Tumornecrosisfactor,168

gene transfer,58'l Virus-mediated

Transgenic Btplants,743

Viruses-characteristics, 757

Transgeniccattle, 488

Tumor-infiltrating lymphocytes,168 gene, .l69 Tumor-suppressor

Transgenic ihickens,489

Tumorigenicity, 465

Virusesin gene therapy,159

Transgenic crop plants,620

TUNELassay,436

Viscosity,764

Transgenicfish, 49O

Turbidbstatbioreactors,262

Vital force theory, 758

Transgenicgoats,486

Two-geneexpressionvector,144

V i t a m i nB r 2 ,3 5 5

Transgerric medflies,493

Two-vectorexpressionsystem,144

VitaminC, 327

Transgenicmice, 2"17,481 applications, 485 embryonicstemcell

Tyrosine,€09

Vitamins-microbial production.355

TyrosylI-RNA synthetase, 136

Volatileorganiccompounds.618

Transgenesis in plants,572

Virus coat proteins/602

Viruses-importance, 757

BIOTECHNOLOCY

6?2

w

Water recycling,705

36 Xerodermapigmentosum,

Westernblotting, 100

X y l a n a s e1, 3 3 , 6 3 3

Whey,249 439 Warm trypsinization, Waste water, 682, 686 Wastewater treatment,6B6 Wastewater treatmentfor dairies,703 Wastewater treatmentfor distillery,T04 Wastewater treatmentfor sugar indus t r y , 705 Wastewater treatmentfor tannery, 7O4 Water blooms, 665 Water borne disases,680 Waterdeficitstress,610 Water deficit tolerance,6'11 Water hyacinth,396, 4O4,667 Water fettuce,4O4, 667 Water pollution,679 683 Water pollution-measurement, quality DNA testingby Water .l analysis, 84

Whiskers,587 White buttonmushroom,381 302 Whole cell biosensors,

Y

Windro system,7l2 Wines,370 48 Wobble hypothesis, Wood-richplants,399

Yeastartificial chromosome,86, 1 4 0 ,4 8 4 Yeastexpressionsystem,141 Yeastthree-hybridsystem,72 Yeasttwo-hybrid system,72 Yoghurt,365

x Xanthan,383 Xanthangum, 383

z

Xenobiotics, 718 Xenogeneiccells,473

Zinc finger motif, 66

Xenograft, 821

Zoo-blotting,99

493 Xenotransplantation,

Zygote intrafallopiantransfer,234