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GENETIC TRANSFORMATION OF Cicer arietinum, L 

FOR INSECT RESISTANCE 

I&44Lhah!b 

SRI VENKATESWARA UNIVERSITY 

~~~ 

G . t ~ ~ ~ 4 ~ + 4 

DOCTOR OF PHILOSOPHY 

IN 

BOTANY 

GJ 

B. JAYANAND 

DEPARMTENT OF BOTANY 

SCHOOL OF BIOLOGICAL AND EARTH SCIENCES 

SRI VENKATESWARA UNIVERSITY 

TlRUPATl - 517 502 

INDIA 

JANUARY, 2003

CERTIFICATE 

Certified that the entire work embodied in this thesis entitled 

"Genetic Transformation of Cicer arietinum L. for Insect Resistance" 

has been carried out by B. Jayanand under my guidance in the Genetic 

Transformation Laboratory, International Crops Research Institute for the 

Semi Arid Tropics (ICRISAT), Patancheru, Hyderabad, India and that no 

part of it has been submitted elsewhere for any degree or diploma. 

I(\( ,L, ,% 

' 1 1 r 1 

Dr K. K SHARMA 

Research Supervisor 

Hyderabad, Inha 

January, 2003 

DR. KIK4RI '( St-14RM4 

Cen-'icT ' . .OlVtO v 

ICRIS"' . L 

Andhla FrdJ,sh 502323, INUIA.

Certificate 

Ger/$ed /La/ /Ae eldire morL emGoded ;I, 

/B>s /Aesis en/i//ed 

"Genetic Transformation of Cicer arietinum, L, for Insect 

Resistance" LUJ Gee21 curried ou/ 6j 8. Jayanand unr/er my 

yuidance ill /Le ~e,var/men/ of'~o/uny, 3'ri ve~~nh/erruuru Ulioesiiy, . 

1' 

Xrupu/i, 9ndLru %odes4 Yn;rJlu a11 J /La/ no par/ of i/ As 6ea1 

suln2i//edel;euLere/br a ~ deyree y or d+/otno. 

Tirupati 

January, 2003 

Dr, G. SUDARSANAM 

Sii,lleI'~~~O!' 

,>,,,* P - . ., / 

p, , ,, ' 

5 I 

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7, , -;.:,..;, ., ;, \ , ,, I,,,. .,

DECLARATION 

9 hereby aeclare that the bis~eratiorr entitlob "GENETIC 

TRANSFORMATION OF Cicer arietinum, L. FOR INSECT 

RESISTANCE" is an oriuinal and inbepenbent recorb o$ research work 

unbertaken by #re during the periob 06 my study nt S ri Uenkateswarn 

Mniuer~ity, qirupati, unbar the superuisidn 06 Dr. G. SUDARSANAM, 

Departuient 06 Sotany, 5.3.2.5., 5.3. Mniuersity, Ti+upafi, 

and that it ha$ nof prooiourly been subwritfed $09 the awarb o$ any 

other begree or biplorna 06 any Mniuersity. 

Tirupati - 517 502 

Date : Jan. 2003

I deem il as a great pleastire to exprcss my IicartSelr grat~tutie and profouiid 

respect to lily Researcli Supervisor, Dr. G. Sudarsanam, Associali: Professor, Dep:, or 

Botany, SBES, Sri Ve~ikateswara University, l'irupati. His colistant, ilispiril~g guidaiice. 

dedicntio~i, patieiicc, iinliri~ig, incessant e~icouragenient, eve~.last~~ig smile and unfl~gg~ny 

interest shown tliroughout my rcscntch is really i~nme~iior;ible for tile complclion oTrhis 

~esearcli ~\orl

"I'm convinced biotecllr~ology is going to help us. There's fear, 

but biotecllnology has been going on since the beginning of' time. Mother 

Nature was crossing plant genes loug before scierltific man and 

agricultural lrlall began doing it. If you like to eat spaghetti, you are 

eating a GlIO that Mother Nature made."

CONTENTS 

ABBREVIAI'IONS 

SUMMARY 

LIST OF TABLES 

LIST 01; FIGURES 

I. INTIIODUCI'ION 

2. lIE\'IEW OF LII'EIIAI'URE 

2.1 Gclietic trnnslorrnation 

2.1 1 Varioi~s liictl~ods olgclielic tralislbrinatio~i in 1pi:iliia 

2.1.1.1 Gcneric tr;~nslbnii;l~io~i by the biol~slic psoccss 

2.1 1.2 .AIlern;~tlve ~iietliutls olgullcl~c I~alislbl-iiial~o~l 11110 pI,:nl cells 12 

2 1 hl~laclc riiic~oorg,~~i~s~ii: ,,!gi.ohiir,/r,i.i~iii 17 

2 1 2 1 TI i~lasmitl ;111d IS C / ~ ~ I ~ ~ C ~ C I . ~ ~ ~ I C S 1 j 

2.1 2.2 ioleculiir ~~icclia~iisiii olT-DNA ~r:i~isicr ~nto pllillls 15 

2 1.2.3 Tools ofycnct~c ilansi'or~~il;il~o~~ 15

2.2.2.1 Organogenesis 

2.2 2.2 Sonlatic enibryogellcsis 

2 2.2.3 Ollier ~iietliods 

2.2 2.4 tic~~ullc 11,111albril1~1l1w 

2.3 Insect rcsistaiice ~~~a~iageiiicnt 

2 3.1 Pvls~liotls of co~iirol olhci. 1l1,iii b~olccllriolog) 

2.3 1 . I Ecological control 

2.3.1.2 Pliysicnl colltrol 

2.3.1.3 Chemical control 

1.3.1.4 B~ological conirol 

2.3.2 Biotechnology for insect resistance 

7_,3,?.1 BI: An amazing concept 

2.3.2.2 Genes employed [or 1nscc1 reslstnncc 01I1er tli~li DL 

3, hlATEKlALS AND METHODS 

3.1 Pli~li~ ~iiilteri;ll and culture co~id~l~o~ls 

3.2 Rcgelieration 

3.2. I SOII~YL~C e~iibryogeliesis 

3.2 I I Prepar,itio~~ of various csplants 

3.2.1.2 [~ltlilction of solilatic e~iibryos 

3.2. I .3 Maturation of sumaric eiilbryos 

3.2.2 Discct d11i1 Indi~ect org;inoyznssis 

3.2.2.1 Prcparntiorl orexplalits 

3.2.2.2 Inducr~ol~ of mi~lliple sllools

3.2.2.3 Shoot elongat~on 

3.2.2.4 Rooting oi'slioo~s 

3.2.2.5 Hardening ,ind trCi~isplaniatio~i orrootcd planti 

3.3 liisioloy~cal studies on 111~1it1plc SIIOOI 

i~~~iiaiioii !'ro~li tZM4 ehplant: 

3 4 Cic~ietic ~IXI~~~~IIIJIIOII 

3.4.1 'Irn~~sibr~iint~oi~ by b~oiistics ~iielliod 

3.4 I. I L,I~~ii-prcl):~r,~l~u~i 

~I'~II;I~IIIIcI DNA (AII

4.1.2.2 Eloiig;itio~~ ofllie siloor buds 

4.1.2.3 Rooti~ig of the elo~iyatcd sliools 

4.1.2.4 H:~rde~l~ng and \ra~isplanla~~o~i of rooted pla~its 

4.2 Histological slud~es of multiple shoo! development from AM4 expla~it 

4.3 Gc~ie~ic transfoniiat~on 

3.3.1 Biolistics ri~clliod of gc~ietic tra1lsfo1.1linr1o11 

3.3.2 T~arisforniatio~l by il,qi~oh~~c~~ei~iiiirr ~ilerliod 

5. DISCUSSION 

4 3.2.1 GUS liistocllem~cal assay 

4.3.2.2 Molecular analysis of To gelieratio~l pi~tative transgenic 

5 1 l'issi~c culti~rc suidics 

platlts ~sarisSorriied wilh pHS723.Bl and pHS737:SBI'I 

b~nary vectors conlaining B/Cr~i/lh and SBTI genes respect~vcly 

5 1.1 So~natic siubr)ogenes~s 

5. I .2 Ol.ga~iogencsis 

5 1.3 Histological stud~es of iiiuliipli. slioo~ dc~elop~netli 

from AM4 explanl 

j,2 Genetic t~.ansSor~ii~tioil 

5.3 Co~icliisio~is 

6. REFERENCES 

AI'IiENI)IN

ABBREVIATIONS 

2-il' 

2-isopci~~cnpl ;tdc~iillc' 

2,4-0 - 2,4-Dicl~loroncclic acid 

2,4,5-'I' ~,~.~-TI-I~I~I~I~~:IC~I~C 

acid 

AD ..lsc,oc~/~~~/cc I311gl11 

AUA 

BAP 

~\bsc~ssic ac~d 

Bcnzql Alllilio PLII-III~ 

BGM - UO/I:I /is Grey Molt1 

' 

Bt - ~trcrli~rs ililii-i~rgiel~srs 

Bti - L~~~ccli~r~ 

/~IIII~III~~~~IIAI.S-;A~~IL~~LJII.~~S 

CaMV 35s - C'alillo\ver Mos;iic V~ri~s 35s proniolcr 

CAT - Chlora~npl~iliical ,\cciyl Trallsferasc 

DNA - Ueosqr~bonucleic ticid 

FA0 -- Food anti A ~~IcLII~II~~ 

Orgnni~at~ol: 

GA - Gibbzl-cllic aciW 

IAA - Indolc-3-ncclic ac~il 

IBA - Illdole-3-bulyric iicid-. 

ICRISA'I' - Internaiio~l~ll Crop Rescnrcll I~~s~iti~tc. Tor Seini-Ar~d fropics 

ICAKDA - Illicl-ilatio~~:il C'cnlel- for Ay~.iculu~r,ll Kcse,lrch ill [lie Dry Arzai 

IPhl - Inrcprnled Pcsr Matlnscine~ll 

111111 - iiiill~iics ' 

MS medillm - Murnsllige and Skoog ~nediiini/ 

NAA - Naplitl~alene Acer~c Acid 

I - Nsonlyciil Pl~osplio Tsa~lsks~sc I1 

PCR - Polyllicrasc Clinill RsCic.tion 

Pls - Protz'isc Illhibilors 

QTL - Quo~itit~itive Tra~t Loci 

RNA - Iiibonucleic acid 

SB1'1 - Soybeall Trypsin lilliibilor 

SCRl 

Scottisll Crul) Rubi.;~rcl~ l~lblll~ilc 

sec - seconds 

1'-DNA Tr:ii:sSe~. DNA 

TDZ -- Thidinzuro~i ' 

'Ti plasnlid - l'i~rnol. i~itiuci~lg pl,~slllid 

iirtiA - (3-Gluciiro~ii~l~isc

SUMMARY 

Agriculture con~ributes major part of world's food especially i i ~ the developing 

world. Over 200 plant species are cultrvable ou~ of whicli rlce, wheat and maize make 

70% ofthc total output. Legunles have very important attributes such as protein arid lipid 

rich seeds and symbiotic nltrogen fixat~on, which qualifies them as best alternatii'e food 

crops. Legumes lire broadly divided Into food and forage legumes out of wli~ch forage 

legumes for111 major part. Chickpea is one of the most important leguminous, cool season. 

alternative food crop cultivated prevalently in Asin I'acific region. Even though it lias 

co~ivincing nutritional importance, its area ot'cult~vatio~l has been low anti i~iipro\ement 

virtually stagnant. Conve~itional breeding lias not been an effective crop improvement 

strateyp Ibr i.iiickpea and recclit advances in biotech~~ology s~rcli ;is plant t~ssue culture 

and gc~lctic irailsl'ormatiun, paved tlie way for aliernntive crop Iniprovcment 

metllodologies. 1'111h work \\as carr~ed out with principal object~ves of optimr~ation of 

tissue culturc iariables n11d tra~ihfo~~lldt~or~ of selected explo~its by ujlilg genes fro111 

Bacillirs 11iio.irigietisi~ Ui('ryL,lb ;itid soybeall trypsi11 inliib~tor (SUTI) geilrs io co11ti.r 

resistance to ~l~licovctprr cu.~~go.(i or tile legli~iie pod borer. The work can be broadly 

divided into tllree parts, I. So~liatic rnibryoge~lcsis, 2. 0rg;inogenrsis and 3. Genetic 

transforniation. 

S~iri(~iic e~~ibr):oge~~esis: Differo~it explnnts like mature embryo axis, parrs of 

nlatilre embryo axls, leaflets, stell1 seg~~ie~its anti root segrnci~ts \\ere derived bum 111 

vitro growl1 seedlings of different ages. Soinntic e~ubryos were induced by ilslng ?.1,5-T 

and 2,4-D as principal growtli rcguiarors ill co~~lbinntioli wit11 Iiorn~oncs like hllletiil, 

zeatin, TDZ and BAP. Efficient induction of sonl:itic embryos \\as observed and best

frequency was observed wit11 2,4,5-1' in combiriatio~i w~tli kinetin. However, efforts for 

maturation and rege~icration of embryos induced were unsuccessl'ul though good number 

of media coinbi~iatio~is involving ABA, zeatin, BAP and TDZ were testcd. Hence fu~.tlier 

work on the somatic embryogenic pathway of regeneration war not carried fonvard. 

Oqtriioget~csi~: Vnr~ous cxl~l;inh such as nlature e~itbryo axis, shoot tip, Icalleta. 

leaf' base, sten] seyn~enta, l~ypocotyl, epicotyl, root segmclits, root tip, ;~xillary bud. 

cotyledo~iary node and axillary nleriste~n explants (AMI, AM2, AM3 and AM4) werc 

prepared l?om 111 vitro grown sectllings and their multiple slioot regencrati~~g eflicicncy 

was tested via direct and indirect orgaliogcnlc pathways. Multiple shoot inductioli 

frequencies \\ere tested with slloot ~nduction mediilm (SIM) that consisted of4 p.LI TDZ. 

10 pM 2-iP and 2 pM klnetin. Explallts that do not co~itai~i ally traccs of pre-esishg 

meristerlis, si~cl~ ns liypocotyl, epicotyl, leaflets were also uscd so as to achieve 

rege~irratioii via callui phase. It was observed that i~tdirect orgauogenesis via callus pllasc 

cannot he acliievcd fro111 tlie abo\e-mentioiicd esplants with any of the tcsted ~nedia 

comb~nnt~oiis It \\as also observed that asyncliro~ious iiiultiple shoot regenerdtion could 

be achieved II-OIII 

pre-ex~sti~ig il~eriste~ns of expla~ita likc sliuot tip, axillary bud. Verq 

low frequency oisl~oot iliductio~i was obwrved wit11 l~ypocolyl and ep~cotyl and it was 

found th;i: k\v slioots tIi;it originateti werc fro111 traces of nieriste~~is associated n,~th sonle 

of the expl;~nls. Diised on ll~rsc resul,rs it \vas apparent that :~dve~ititious regeneration is 

very difficult wit11 o no st of the explants and inod~licario~i of the tested esplants. Four 

axilla~y meristem expla~its iianirly Akll, AM2, AM3 and AM4 were prepared so as to 

achieve adventit~ous rcgclielxtion 2nd tlirce of ~IICIII AM], AM2 and 1\hl4 were found to 

be better candidates. Alno~ig tliesr expla~~ts ALl4 \we ~iiorc respoiislve and prov~ded

adventitious slioot buds. Tliese seemed more applicable to gcnetic transformation 

experiments where negation of thc aprcal dominance of axillary bud and shoot t ~p was the 

salient feature of this explant. Removal of axillary bud followed by regeneration of 

multiple shoot buds ga\e bctter adventitlous regcneratlon and multiple wounding sites. 

Multiple shoot induction was found to bc betrur on liled~uin containirlg TDZ, when 

compared ro the medium contaiiiing BAP. However, it \+as observed that prolonged 

cul~c~rc uf llic cl~l;inls o~i 'l'D%-co~l~:~~~iiiig liicd~~~~ii ~ieg:~livcly i~it~rfcrcd wit11 I11rtI1cr 

elongation. Ilc~ice, [lie inclus~on of TDZ was restricted to gerriiination and first pliasc of 

induction. Sevcl.al other factors played an iniportant role 111 shoot bud induction. For 

example, acidic pH sliowed efficient ~~iductiuii of niultiple shoots. Age of the seedling 

was optimzed for all axillary meristeni explants wliere AM4 preparation required 5 to 6 

days for the rcn~oval of axillary bud and another week for removal of multiplc shoot 

buds. Inclus~on of cotyledo~i with AM4 explant was found to enhance multiple shoot 

regeneration frequency. Two step elo~igation us~ng shoot elongat1011 medium (SEMI; MS 

with 5 pM 2-iP and 2 1M klnct~n) in the first step followcd by 2 to 3 passages of sub- 

cultures on SEM2 (MS wit11 2 pbl GA,) resulted a better elongation frequency. A novel 

rooting sysrem was developed by employing filter paper br~dge tccli~iique aid tile liquid 

root inductloll mediuni (RIM) consisted of MS will1 5 pM IDA. This ~rietliod resulted in 

high rooring as ~vsll as transplantation frequelic~es. .All clficient liardeniiig nild 

transplaiiration method was standardized by optimizing variables such as potting 

medium, temperature, humidity, irrigation and phoroperiod. ~onviron~" growth chamber 

was found to be better Fac~lity for Iiardening arid i~i~tial phase of tnnspla~itation while pod 

maturation and harvesting was done in specially designed P2 facility. Best hardening of

in vitro grown plants w;is ocli~eved with srntic liydropo~iics systc~l~ contai~ii~ig Ar~~on's 

nutrie~it solutio~l. 

heric,iic irn~i~joj~nriuiio~i. Genetic tri~~isSor~~~;~tion ol' cli~ckpcu ivas achicvcd by 

using Ah14 eupla~its as ll~u startlllg plo~~t ii~atrrial and butli biol~st~c illid Agr~~h~icii~riii~)~- 

~iied~ated ~iietliods wcrc employed. A vector, ~RTO(I:GUS-III~ nla~i~tn~~led 111 E, cull was 

used 111 b~olisric process. 'fh~s vector li:rd irpill pc~ic ;IS ~lic aclcct;~blc ininrkcr and r~itli\ 

gem nb the rsporrcr. Prcpar;~t~on of thc cxpl,i~ils, niicrocarricr 

preparation and 

bombard~ncnr of tlie expl:i~~ts was ci~n-~cd out by al;~nd;lrd j~roccd~~rcs. Largc SIX 01' llic 

AM3 cxpla~~t was a li~liitatio~~ in 1111s process ns olily fi.w cxplu~ils could be grouped st tile 

center of the petri plate. l'ra~lsl'or~iiation by tliis ~lietliod d ~d ~iot result in creatio~~ of any 

transgenlcs as some of the selected slioots d~ed at tlie stri~~gerit seluction bkp. 

J~grobatie~.i~~~ri-~~lediated transfor~i~otion rcsuited in relatively higli IYeque~icy of 

transgenies as far as 111s TO results wcrc considered. Two binary veclors ~ia~iicly pl-IS 

723:Ut and pHS 737:SBTl based 111 stra~n C 58, wcrc used for co-cultivarions. Tl~c former 

has 81C'ryIAb as agrono~iiic;illy important gcne, ~lptll as thc seiectio~~ 111;lrker and iridA 

gclie as ~.cportcr \\l~ere i~s tllc later binary vcclor liad soybcc~~ t~-ypsin i~illib~to~ (.SljTi) as 

tlie gcne of agrono~n~c ~ntercst, ~ipill as tllc re1ect;iblc marker arid ilitlA gcnc as reporter. 

In both ~hc vectors ripill ;)lid iiidA genea ivc~c I't~hed ~nto ;I singlc unit tl~ougli their 

products exli~bit indepe~ident activities. AM4 ehpla~its ivc~c co-cult~vatcd with 

Agrobucicriirr~l stralli of lrltercst follow~rig tlie standard procedures. Ccfotaxiiiie was used 

to terminate tlie growl1 of Agrobucieriu~~i and kanamyein was used for select~un of the 

putative rransfornia~~ts. Starldtlrd~zed protocol of icgeneralio~l was used fu~ ~ege~ieration 

and recovely of the tralfsgenlc plants. Hardelled and transplanted rra~isgcnics wcre

initially maintained in ~onviron"" growth cllaniber 2nd li~ter in tlie P2 facility especially 

designed for growlng rra~isgi.nics. A total of I I plants w~lli UiC,?;lAb ,ind 9 pla~its w1t11 

SB77 genes were obta~iicd. Molec~~lar analysis of tlic>e pi~ti~t~ve tr,ins!'o~nia~its was done 

irlitinlly by GUS hisrocliemicnl assay followed by conlir~untion by PCR and Souther11 

blot analy~is. Soutlicrn blotting \\as done by 11o1i-rad~o~ict~vc ~iicthod by us~ng tile 

coniniercially available no~l-rnd~o;ict~ve AlklJlios clirect 1nbtl111g iir fro111 A~ncrsl~a~n 

(USA). PCll a~iiplificat~o~l of tlpill, uiiiA, B/Ci),lilD and SB1'1 gc~ics \r;;is carried out for 

preliminary screening of the putatlvc transformar~ts, About (10% of [lie Iputall\e 

transformants sho\ved positivc reaction for PCR for ~rpiil, 70% for ~iidi\ genes, 30% for 

BtCrylAb and 10% for SET1 gciic. Factors affecting restriction of geno~nic DNA such as 

enzyme conci'ntration, watcr and BSA were opt~~ilizcd. Southern blot analysis of 

B~CrylAb piarits sliowsd 70% ofpla~~ts ivill~ /lp/Il and BIC/yMb gene integrat~ons. 

In co~iclus~on tlic rcgencratio~i protocol developed during tlic course of this study, 

was tbi111d to be very eflic~cnt slncc culli~re coiid~r~ons for all tile stages of rcge~~cration 

and recovery of 111 vim grown plants tllat incl~ded seed geriiiirlatio~~, rnultiple shoot 

induction, elongatio~~, rooting hardening ;ind tra~isplantatio~i were optinlized, l'h~s 

protocol w:is effect~\,cly i~scd for succeasl'i~l geilctic trn~~ifor~~iatiu~~ of cllickpca w1t11 

insecticidal genes like BiCrjIAb and .SUTI genes. Follow~~ig tliis protocol, 40% of the 

sclccted putat~\c transgenic plants can be obtn111cd 111 per~ud of 90 to 100 days. In the 

present study, over 30tr;1nsgen1c plants carrqing U/C',),IAb wcrc obtained. Eight of these 

pl:iiits arc being 11in111taincd Sor cspcri~~ic~ir;~l iuorl< 011 illsect b~oassayi and field trials.

LIST OF TABLES 

Table 3.1 

Pr~~iier composlllons of all the Sour gellcs used Ibr penruc trdndirlwt~un. tlluir rr>pcr.t~vc 

annealing tempclatures. 2nd s~zc oSIhe rehpectlve liaglilellts umplliieti. 

Table 4.1 

Inductlu~l ol' aom;ltic clnblyuh oli MS ~UIII~IIIIII~ cu~lib~~i;lliulia ui 2.4.5- l (2.U aid 5 0 

IIM) with 'TDZ. Uhl', hlnelln or zearln, l lie results \\c~c recorded at tllc end 01 4 \vcch\ slid 

represent mcans of thee repllcalions. 

Table 4.2 

lnductlun of cmbryos on MS contnlnlng conlb~n:~t~ons oi' 2.4.5-'I' (10.0 o~id I5 0 phl) 

with 'I'DZ, BAP, klnetln or zealin. Tile results ac~c ~ecorded at l!ic elid or3 weks and rcproc~il 

means ofrhrev rcpl~cat~ons 

l~iduct~on of e1i1bryos 011 MS coninlnlng con~b~~lations of 2,4-11 (5.0 and 10.0 pM) wltll 

TDZ, BAP, klnetln or zearin. 'The result, were recorded at Ilir end of 4 wccks and rcprcaenl 

liicans oi'rluei. ~epl~cat~ona 

lnduct~uli oSenibryos on .CIS contalnlng cumb~narions 01 2,J-1) (15.0 and 20 0 phi) wllh 

TDZ, BAP, kllietln or zealin The resulls wc~c reco~dcd at thc c11d oi' 4 wceks and represent 

lneons ufthrec rc]~l~cal~uns.

I~iduct~o~; of e~nbryos from varlous ciplanis 'llic ehpla~its \\crc. culli~rcd 011 JEhl?!, 

~iied~um that conla~ned MS \b~tli 10 yM 2,J.S-'I' and 2 yM k11lru11. 'The reaulls are 111~. Ini.ali ul' 

thrcc rcililctlte5 

Table 4.6 

Table 4.7 

lnduct~on of ~liult~ple shoots from nialure embryo axla cxplatita on 111cd1a cuntalnllig HAP 

aa [he pr~ncipal giowlli rsgulalur. I\ total or36 exl)la~its pcr trealmclll acre cultured and lliere ivas 

100% response 111 terms of nu~iiber uCcxplants rcapond~np. I'hu rcsul~s wcrc rccordcd at tlie IIII;~ 

of 2"" and 4"' weeks. All llie results are the niean of t1;rc.c. repl~catca. 

'Sable 4.8 

Effect oi"I'DL, 2-11' a11d k111cl11i 011 slloot rCgCl;erdllo~i ho11i the c~~IJI;~ der~\cd Iio111 

ax111ary rner~steli>i uf cI11ckpe3. 'I he results here recorded at the end US 2"\11id 4"' \\,eeks and tl;c 

\.olues ore nirans of 1l11ee ~~eplicales. 

Effect oi pi1 oC [he culture ~i;ed~urii on r~iultiple sl~ool rcgclicrJllon li)r~ll llic ax~llary 

mer~stcm cxplants ot'cli~clilxa. Rcaulra were recurdud at tllc elid of 3 \becks and tlic values are 

liltan from three repl~catcs. 

'Table 4.10 

Effect of ~nclus~o~i of cotyledon t~ssucs along w~th tlic rcgeneratlng ax~llary mer~stc~n on 

shoot form~ng capaclty of the ax~llary ~iier~itenl explal~ls Resulta were recorded from 1 lo 5 

weeks to show the plomotlun oi'rege~ierat~ny abil~t!' and rate oC rnult~plc aliout ~~lducllun by the 

~ncluded cotyledon

Table 4.1 1 

Effect ul'age uf lhe expI0111 donor srl'dl~~igs oil regerlrrdilull ciip~city ot'dlfire~it ehplanls 

der~ved from axlllary nierlstenis. Ilegeneratrrig multrple rhuuts i\'ele counted In ~hc tlirrd wrek 

and the \aIuer are ~iiea~is ~ftlirr~ repl1~a1c.s 

Tablc 4.12 

Inducriun of ~ilt~li~pli. blluul buds liulii \JIIULI~ se~dlllig i.\pla~i~r de~~vcd ii.oni ;lx~ll:~sy 

inerrs~cm 1'11~. 11t1n1bi.1 01 ~ii~~lti))/c r1ioo1~ \\:I> coi~~ili.iI 111 tlic 11i11cl \\cch ~)~I,II to tlic~r 1r.11i~lcr to 

tlic sliool clung~l~un 11leLi1~11il 

Tablc 4.13 

El'tec~ of mtd~a cornposltlons on elo~ig~t~on ol Ihe rugericrated slluols. Results scrc 

recordcd li-urn Ili~ei. ~epl~c~lc c\pcrrliicnla ~niol\~rlg shoots III~UCU~ UI~ JCI(13 rncd~llrn 

ch~ckpea. 

Eficct ui' iiiedln co1islllucnla on loullng ol' 111 i'ltro furmcd arid elong~trd allools ol' 

Table 4.15 

L'tlial du,c Icjllng ,rnd cl'fect of'l LIZ oil Ictll~l cficl of ka~larnyc~~i, Sumhcr ol'ddyb of 

expluiil suri~val w.13 couri~cd u1111l Ilie cxplun~ bleached 01 sliu\bcd nu rlgns of gro\i.111.

LIST OF FIGURES 

Figure 3.1 

Diagr:~mn~attc rcprcsct~t;~[to~i of ll~c cxplanls dorivcd li-0111 111ati1re ct~lbryo i~xls. 

plumule, radicle, s~de asins and m~ddle portio~~. (An.ows shows sites of surgery) 

Figure 3.2 

Preparation of ax~llary ~neristem explants AM), AM2, Ah13 and Ahl-l. t:i11;11 

stage is the stage of regelleratio~i of l~~lllltple SIIOOIS nlier otie \beck of c11I1ure 011 slioot 

~nduclion medium. Large arrows sho\b progressloll of prepamlion of expiants. Mcdiu~~~ 

arrows show s~tes of surgcry and sniall ,isrows show sites of ~n~ultiple shoot rcgcncratior~. 

Figure 3.3 A - C 

Diagrammatic rcprescrllatioli Ibr preparallon oC nx~llary rnertsteni explant (AM2) 

showing the sites of surgery. A. 6-day old secdlitlg sl~owll~g pro~nincnt ;~xillary bud, B. 

Processing of the axilla1.y meristem explant (arrows shows tile sites of surgery), C. 

Axillary iilerls[cm sxpl,~nl (AM2) sl~owi~lg (lie ax~llary 111cris1e111 rsgioli w~th intacl 

cotyledoli. 

Figure 3.4 

Rcslrictiu~~ inlap of rhc plasmid pll'l'99:GUS-lnl used for biolistic-mediated ~CIIC 

transfer. 

Figure 3.5 

Figure 3.6 

Restriction map of the plas~u~d ptIS737:St31'1 used fo~ .4g1~obcic~eriiit11-1netI~od of 

transfomiatlon

Figure 4.1 A - D 

inducl~on of sonlatic enlbryos from mature embryo axis and leallei explants ;itler 6 

weeks of cullure on the 111edi1111l conla~ni~lg 2,4,j-T as pri~~c~pal growth regulator. A, B 

a ~ C. ~ d induction of enlbryos from Inlalure einbryo axis expla~il, D. Induction of soinatic 

embryos froin leaflet esplant (arrows iiidicotl: globular embryos). 

Figure 4.2 A, B 

lnductioil of sonlatic cmbryos SI-oin inlaiurc c111bryu asis CXPIOIII 

by uring 2.4-D as 

principal growth regi~lnlor. .A. Top \,icw of tlie einbryo axis sllowiiig inultiple globular 

embryos, B. Letefiil view of enibryo axis showing multiple globular embryos formed 

from plumiile region (arrows indicate globular embryos). 

Figure 4.3 A - E 

Effect of concentration of B.4P on ~nullrple shoot regeneration fro111 inalurc cinbryo 

axis explant after 15 d:iys of culture on slioot ~nduclion medi~lm. A. Multiplc slioot 

regeneration at 10 j~kl HAF concei~tralio~l, U. M~illipli: slloot rcgcncr~~ion will1 30 pM 

BAP, C. Miiltiple sliuot reguneration \$'it1140 pM BAP, D. Multiple sl~ool regeneralion 

with 50 pM BAP, E. Mult~plc slluot rcgenelntion w11Il I00 1tM BAP. 

Figure 4.4 A - F 

Multiple shoot regenel-ation fro111 dil'fcren~ cxplnnts al tile third week of culture 011 

sliooi iiiduclio~i ~iicdioni .A. 

Shoot lip sllowi~~g rn11111plc slioots cincrgitig fro111 

~neristeii~at~c region. B. Axillary bud showing nlultiple shools or~ginaling fro111 lip and 

basal regions, C. Mature enlbryo axis sliowing mulliple shoots cinerging rrom sllout tip 

as well as axillary bud regiu~is, D. AM1 explant showing ~liultiplc shoots from axillary 

region, E. AM2 explant showing swollen brisal region and multiple shoots emerging 

synchronously as cluster, F. AM4 explant show~ng synchroi~ous multiple shoots from 

different places oraxiIlai.y niel.istem region.

Figure 4.5 A - I; 

Stages of ~nultiple stloot regelleration from AM4 explant alter culture on the sl~oot 

induction medium A. Explant sl~owing the area of axillary bud re~noval (arrow sliows the 

sire of surgery), B. Muluple sl~oot buds oriyin;ltiiig fro111 the area loft by axillary bud 

removal (arrow sl~ows the s110ots buds e~ilcrging fru111 axillary ~nlcriste~l~ area). C. Esplii~~l 

showing swollen area after removal ofregcnc~at~~~g shoot buds (this stage of explant sas 

na~nsd 3s AM4 cxpla~it) akcr 7 days of culture OII shoot i~iduct~o~l rncdlulll, D. 

Regc~ierat~o~~ of ~il~iltiplc sl~oot biltls li.0111 tl~ cut ~po~tiul~s uftlic rcgclleratlng iiastiv after 

6 days ofci~lture on MS, E. Exubcra~lt nlultiplc sl~oot yrowtli Sron~ tile rcgcncrating tlssue 

after 10 to I? days of culture on MS, 1;. Mult~plc sl~oots originaiiny fro^^^ different p;~rts 

of the regellcrating area aCter 12 to 14 days of culture on MS. 


Various stages of tlongatio~i of regenerating sl~oot buds on shoot elo~lgation 111edium; 

A. Elongut~on of young shoot 011 shoot clo~igatio~~ medic1111 1 (SEMI) ancr 1 week uf 

culture, B. Elol~gatio~i of sliool on shoot elollgation 111cdiu111 2 (SEM2) nlicr 2 wccks of 

culture, C. Elongallon of sl~oots 011 SEM2 after 3 wecks. 

Figure 4.7 A , B 

Rooting of eloiipaled slioots on root induction ~niedill~~~ co~~tailli~lg 5 pM IBA, A. 

Rooting 011 filter paper bridye i~lllrlcrsed ill liquid root ind~iclio~~ nicdium, 8. Rooting OII 

solid root induction ~nlcdii~n~ co~~iaininy 5 pM IBA. 

Figure 4.8 

Stage 1 Ilardening of tissue ci~lture gro\an cli~ckpea after transferring to 8 cnl pot 

containi~lg 2-4 mm sand.


Alternative llletilods of hardening of rooted plantlets of chickpea prlor to 

transplantation; A. Hardening process by embedding !he root system ill the coarse sand 

wtth the cotton plug open. % Amon's solutlol~ \bas used for irrigation. B. Hardening 

process in static hydroponics system with root system immersed in tlie liquid mcdium. 

The Itquid med~i~ni co~llprlsed of % Arnotl's solution, C. A plant fron~ B showing prof~tsc 

groivth of root system 111 tile static hydropot~ics systel~~ after 15 days. Tllis plant is luady 

for transplantation illlo pols. 


Hardened and transplallted chickpea planls growing in the glasshouse; A. Plant 

growing without any post-hardening treatment, U. Plant with multiple branches aftcr 

post-hardening treatment of terminal bud ampiltution and juvenile flower bud removing, 

C. Group of plants being ~nlaintained in 20 cm pots after Ilardening. 

Figure 4.1 1 A - I 

A con~plete sclleme of regeneration and recovery of wliole plailts tllrougl~ tissue 

culture lnetllod using axillary t~leristel~~ explant (AM2) obtained froln it! vi11.o grohn 

seedlings olchickpea. A. Axillary mcristem explitllt (AM2) on the first day of culture on 

shoot inductton rnedlum, U. Sl~oot buds regenerating froln the region left by thc rcnloval 

of axillary bud after 7 days, C. Cluster of 11i~lltip1~ slloot buds or~gli~ating fro111 region lcli 

by the removal of axillary bud after 12 days, D. A closer view of the liiiiltiple shoots 

regenerating from the axillary nleristem explant after 15 days of culture 011 shoot 

induction medium, E. Elongation of the slloot buds after 7 days of culture on slioot 

elongatiol~ medium, 1;. 

Rooting of the elorigated slloot bud on the filter paper bridge 

immersed In liquld rootlng medium after 8 days of cullure, G. Picture showing rx vlrro 

static hydroponics systenl for hardening of the rooted shoot after 15 days, H. An 

alternative method for hardening of the planllel oblaincd through tissue cullure ill which 

the root system was immersed in sand wltli the cutton plug of tlie crilture tube kept open, 

I. Hardened and transplanted plant sl~on,ing normal nlotphology.

Figure 4.1 2 A, B 

Histological sectio~lsliowing status of axlllary meristem A. Longitudinal sectloti of 

the growing axillary bud just before its re11iova1 froni 6-day-old seedling sliow~ng 

actively dividing merislenialic cells atid uti~for~i~ oon-ineristematic tissue in rlie basal 

region. B. LS of explant aner removal of ahillary bud ill thc process orprcparing axillary 

nieristem explant. 

Figure 4.13 A - H 

Hlslological studles ofdcvelopme~it of ~il~lltlple ~iicrisremolds from ax~ll:~ry mcristcm 

region of AM4 explailt alier tile renioval of ax~llary bud. A. Longitudi~ial scctioii of rlic 

axillary nieristcm area oli day-l aner axillary bud removal. U. Developmenr ol' 

rneristenioids at tliz basal poriion of axillary bud on day-2 (tlie stndll b:~d appearing is a 

shoot but1 emerging li-0111 tlie basal portion of the removed axillary bud), C. Appeara~~cc 

of rneristemo~ds 111 the axill;iry meristelii area on day-3, D. l~icrcnsed !lumber of 

nieristemoids 111 the ax~llary meristem area on day-4, E. Increase ln the nnnibcr and 

yrowtli of ~ncr~steino~ds 011 day-5, F. E~po~ic~it~;tl ilicre;~se ill thc ~nun~bcr and growl11 of 

merlsteniolds on day-6, C. Loligiludlnal section of the rnult~plc shoot butls as seen on 

day-6, H. Meristemalic activity of d~viding cclls at various places in tlie ax~lldry 

meristenr ;Ilea 111 d~l'fcren~ d~rccriun on day-7. 

Figure 4.14 A - F 

Closcr observation of dcveloptnc~it and growth of rncristenioids in tlic axlllary 

nieristem area of AM3 explant A. Closer view oftlie mer~stemo~ds on day-4 after the 

removal of ax~llary bud, U. Growth and divis~on oi'meristeniatic cells in the meristernoid 

region on day-5, C. Growtli and division of mcristem;~tic cells around merlstemoid region 

on day-6, D. Growth and division oi'mer~stematic cells ofnicr~stemoid reglon on day-7, 

E. Closer view (40X) of meristemoid region and ccll division activity, F. Hectic 

meristematlc activity oftlie dividing and growing ~ells.


Restriction analysis of tlie plasniids used for tra~lsforniation. A. The plasmid 

pRTY9:GUS-lnt (6.9 kb) was tiscd In biolistic method ol'transfomiatio~~. Lane 1 sliows 

hDNA digested with UsrE I1 etlzynie as marker. Lanes 2 and 3 liave duplicate plasln~d 

un-restricted sample and lane 4 shows plas~iiid a!kr restr~ction with EcuRl showing Sour 

fragments. B. Two un-restr~cted plasmid constructs that were used in Agroboc.ierrii~,i- 

mzdtated transl'on~iation. Lane I shows hDNA d~gestcd w1i11 tiilid III ellzymc as 111arkcr. 

Lalies 2 and 3 llad pHS737:SBTI (14.3 kb) ~plasni~d wl~ile 1,111~s 4 and 5 llad pHS723:Bt 

(15 5 kb) plasm~d preparatluns. 


GUS histochenlical assay of the leaflets Srum pl~tdtivcly transfornlcd plants 01' 

chickpea. A. 4 closer view ol'tlle leaflet sllowing GUS activity in [lie vclns, B, C. GUS 

activity as seen 111 tlie pcliole and veins of lallets. 


PCK amplificat~on of 700 bp fragnlcnt of 1ipi11 gene fro111 \he gclio~~i~c DNA5 oSTu 

generation pla~its rratisSoni~ed w~th Brcr)>lAb and SBTl genes via Agrubricieriut~i- 

mediated transfonnalion. A. ~iplll-PCR of pli~nts lrallsforilled w~lli pMS723:Bt via 

Agroborieriu~~r-1n2dlatcd trallsfomiation. Lanes 1 to 10 lid transfomled samples and 

show the aniplification of ~tpfll gene. Lane I I negative co~ltrol and 12 lo 17 wcrc positive 

controls froni plasmid pHS723:Bt used for tralislbrniatio~~. Lane 18 IS - DNA and h 

DNA-BsiE I1 m:~rker was dddcd in the lane 19. Ll. ~rprll-PCR or plants lransfomicd \rill1 

pHS737:SBTI vector vln Agvobucieriu1?i-11ied1;itcd translorn~at~on. Lanes 1 to 9 had 

transformed samples CS5 to CS9. Lonc 10 L\,;IS negative coiitrol and l I 

to I5 were 

posltlve controls oSpHS737:SBTI vector tlial w;is iised In the transforni;ttion. ?, DNA- 

BsiE I1 marker was added in the lalie 16.


PCR arnplificatio~l of 1.2 kb fragment of tridA gene from genomic DNA samples of 

To generation putative trat~sgenic plants tra~isfonl~ed with pHS723:Bt and pHS737:SBTI 

vectors via Agrobocferiir~~t-inediatcd transfor~~~at~o~~. A. GUS-PCR analysts of plants 

transfom~ed wit11 pHS723:Bt vector. Lanes I to I0 \\,ere added \vitIl putntivc tratisfor~ll;i~~t 

samples, while lane I I \\as positive control and lane 12 Ineg;ltlve control. Satllple addcd 

In lane 13 was -DNA, h DNA-UbfE II m;~rket was added in tlie lane 14. U. GUS-PC'K 

analysis or plants tra~~slbrlncd wttl~ pHS737:SB'l'l. Lanes I 

to 9 wcre putarivu 

transformant samples, wliile lane 10 and I1 were positive co~ltrols of plasmid 

pHS737:SBTI and lane 12 was negative conlrul. San111le added in lane 13 was -Dh'A. h 

DNA-UsfE II ninrket- was added ill llle lane 14. 


PCR amplification of SBTI end BfCrj,I/IL gcncs fro111 ge110111ic DNhs of ptllative 

transforn~ants at To gene ratio^^ transformed vin Agrobacfn.ir~t~~-t~~cdi~~ted transforlnation. 

A. PCR analysis of plants rr:~nsfornied with pHS737:SBTI veclo~.. Ln~li: 1 tl~ruugh lane 9 

are putaive transfun~la~lt s;~tilples sliu~viiig an~pliiic;itio~~ ufJ97 bp fr:~gment. Ncgi~tive 

conrrol samples were added In llle lanies 10, 11 2nd 12 ptlS717,SBTI plasmid samples 

were positwe controls in the latles 13, 14, 15 atid 16. Lane 17 \+as -DNA and i, DNA- 

BslE I1 marker was added in lane 18. B. PCR allalys~s of pla~~ts trat~sformed wit11 

pHS723:Bt vector. Lane I 

rlirougli lane I I wcrc addcd with putative twnsforn~ants 

showing amplificat~on of 908 bp fragment. Lane 12, 13, 14 arid 15 had the negative 

control. Lanes 16, 17 and 18 were positlve controls of pHS723:Bt plasmid vector. Lane 

19 was -DNA and lane 20 was added wttll h DNA-U5tE I1 ~ilarker.


DNA profile of genomic DNA isolated from putative transfonnants of chickpea. A. 

Purified DNA profile of genomic DNA of BlCrylAb plants. DNA was prepared in 

duplicate samples. Lanes 1 to 10 shows first set and lanes 11 to 20 shows the second set. 

B. Effect of amount of BSA, water and enzyme in the reaction mixture to digest the 

genomic DNA sample of chickpea. Unless otlienvise stated, the Lola1 volume of reactton 

mixture was maintained at 25 p1 with 10 p1 of genotnic DNA, 2 pI (I0 units) of clizpe, 

2.5 pl of reaction buffer and the rest being water. In case of volume variations arising due 

to tile changing enzyme and water collcenlratioils, tlie reaction buf'fcr volu~ile \\,as 

maintained accordingly with its working concentralion always kept at IX. 

Lane 1: Unrestricted genoliilc DNA (5 pg, was added to tile well) 

Lalie 2: 1 p g genomic DNA digested with I0 ltnils of enzyme 

Lane 3: 5 pg, gelionitc DNA digested willt I0 units of enzyme 

Lane 4: 5 pg. getlomic DNA digested with 15 units of etizynie and dot~blc llic 

quanlily of haler 

Lane 5: 5 pg, ge~iolilic DNA digested \z;~lIt 20 units of eti/.ylne with double llle 

quanttly ofwaler 

Lanc 6. 5 ~tg ~CIIOI~IC DNA digested will1 10 utllts of elizytiie and 0.1% BSA 

Lane 7: 5 pg, genornic DNA digested w~tll IO units ofcn~yme, 0.1% of BSA and 

double tlie quantity ofwnler 

Lane 8: 5 pg. getiomlc DNA digested \villi IO ~~iitls ofcnzynle, 0 356 of BSA 

Lane 0: 5 pg, getioniic DNA digested wit11 I0 ullils of enLytne, 0.3% of USA and 

double tlie cll~antity of water 

Lane 10. 5 pg. gellolnlc DNA digesicd will1 IO i~rlits of cnzyme, 0.5% of BSA 

Lane 11: 5 pg. genomic DNA digesled will1 10 units of enzyme, 0.5% of BSA 

atid double the quantity of water 

Lane 12: 5 pg, genonitc DNA digested with I0 unlts of enzyme, 1.0% of BSA 

Lane 13: 5 pg, genonltc DNA digested with 20 itnits of enzyme


Southern blots of the To generation putatlvc lransfonna~~ts of chickpea transformed 

with i31CyIAb and SBTI genes via Agrobtrcleriior~-nlediated transfomiation. These blots 

show the signal accumulaled 011 tlie h~gher niolecular weiglit region due lo the li~liiled 

restrlctlon of genomlc DNA. Tll~s psoble~n was encountered wlie~i the factors affecting 

restriction of gelio~llic DNA, sucll as qu;~nl~ly of cnzyine, water and BSA were 1101 

standardized A. Southern blot will1 probe for BrCq~I/lb gene. Lanes I 

to 10 has 

puiat~~ely tl.i~~~stbr~lie~i s;~i~iples of CBI lo CBIO. U. So~~llier~i blot wit11 pl.obe for ~ip~ll 

gene. Lanes 1 to 10 were added with put,it~ve tl.a~isfosrn;il~ls transConucd wit11 biC~:i.iilh 

gene. Lanc I1 lias ~legativc co~ilrol, whilu Idlie 12 lias lllc pos~live control. Rcslricted 

genoltiic DNA samples from putative transgcnlc trs~lsfornled with SBTI gene wcre added 

in the lanes 13 to 2 1 

Figure 4.22 

Soutiicni analys~s of tlic To genelntio~i of BrC',ylAb p~~lalive transgclilc plants of 

cliickpea triinsibniied via Agruhtrcreri~~rrr-medlated lransfur~nat~on. Ge~iom~c DNA was 

restricted wit11 EcoRl enrynie. A~ialysis of copy nuntber w~lh respccl to the ~rplll gene. 

Lanes 1 to 11 were added ~v~tli transfonnant salliples CB I to CB I I. La~le 1: CBI, Lane 2: 

CB2, Lane 3: CB3, Lane4: CB4, Lanc 5: CB5, Lalie 6: CB6, Lane 7: CB7, Lane 8: CB8, 

Latie 9: CB9, L:tne 10: CBIO and Lalie I I. CBI I. Lane I2 11as the negotlve co~ltrol and 

lane 13 has the pos~tive con1101 (Rcslricled plasliiid pMS723,Bi that was used in gc~iel~c 

transformatio~i) 

Figure 4.23 

Southern a~ialysis of theTo generalion of br('ry1Ab pla~its. Genomic DNA was 

restricted with EcoRl eilzyllie that has two rcstricl~on sites within the RlCryiAh gciic. 

This double cut releases tile 3 lib fragnielit of UrCryIAb pc~lc. Analysis of the tra~lsgenics 

for the U~CrylAb gciic. Lanes I 

to I I were addcd w~lll Il-ansforman1 samples, Lane I: 

CBI, Lane 2: CB2, Lane 3: CB3, Lane 4: CB4, Lane 5: CB6, Lane 6: CB5, Lane 7: CB7, 

Lane 8: CB8, Lane 9: CB9, Lane 10: CBIO and Lane 11: CBI I. Lane 12 has the negative 

control and lane 13 has the posit~ve control Restricted plasmid pHS723:Bt with EcoRl 

enzyme).

1'1.0 INTRODUCTION 

L~fe is the ter~in uscd to explaiii the to-ordinate i~itegrity oi defitned nintter energy 

transacttons manifested in tlie living organisms. These tlansacho~ns hove been made 

possible with the ava~lnbility of fixed energy. Early life derived energy tiom anaerob~c 

environme~it and evolutio~i ofautotropliic orgoiiisms divcrsificd course of evolutio~i. Pla~its 

liavc bee11 thc I'irgcat source of lised energy, ,IS tiicy trd~isl'urm tile 1portio11 of solar ci~c~g! 

illto clietn~cal energy, ivliicli ~nakes theill cligiblc tbr tlic title of'thc Producers'. ' hs liked 

ericrgy is being used at vaiious levels of hod cliitin III v.iriuus Ibrrni. In platlts. lhis cncrgy 

has bccn uscd for various metiibolic oct~vit~cs 2nd stored in nialcrial fornis of tliocl-o 

molecules siicli as carboliydrates, protein itnd I~pids, collccrively struclured 111to orgalls like 

seeds, graltis, l'ri~~ts, vtgetobles ctc., to bc uscd at tlnc ti~iic of titcli, 'l'lic cot~ccpt ul 

agriculture was born \\lie11 11i:t11 rcltlizcd tlie ~il~tritio~iitl sig~nificii~itc 01' tlnesc plat11 parts. 

Agriculture brought about r~voli~tio~~iiry socii~l ~Iia~igcs by Ifiiiisfor~iiing no~iiad~c ir~bcb 

into inore adlanced static civiltzatiot~s, 2nd nictliods of:igricul~ur;il pr:~cticcs li,tvc been tliu 

principal inleasure to clsscss tlie ad\anceliiciit of'ctvilii;~t~o~is 

Abulid,nntly water supplyi~ig rcgioris and ailst,~ini~i'lc rainied areas l~ave becorrne tlic 

Inavens for settling and advancement of svutic civiliriittons. Agriculture gained sign~ticance 

priiicipally by ilourislii~lg llie Iiunian world dnd ll~ei~ dotncat~catcd aii~mals. Consistent 

discovery of plii~its with diversified ~outisliinp \:dues expanded tile span of :igriculture 

while one Faculty of agriculture concent~.ated oil d~scovery and cultivation of plants with 

added benefits siicll as niediciiial Iiiipottancc, titiiber, orn:imenlals etc., that have varted 

degrees of economic Ilnportance. Vi~ricty of' plutlt organs such as seeds, tiuits, leaves etc. 

were identitied io be nutr~ttonnlly very sign~:ica~it. A ~rritritious hod is identilied as a 

balanced diet illat prov~des optiii~al energy aiid :arious organic aiid itnorpzl~iic niolccules oi'

structurd and filnctiolial Impol.taiIcc. Carbol~ydr~tes and lipids are tllz prll~cipal sources oi' 

energy while llp~ds and some protelns are structurally s~gntficant. Another class of 

proteins, v~tamins, carbohydrates and various small organic and 1norg:lnic nlolecules arc 

function;~lly very important. Most of tllcsc org;lnlc n~acromoleculcs In conibinat~o~~ wttll 

several lnorganlc molecules must be supplied estcr~ially in the form of diet though tl~crc IS 

synthesizing machinery wlthin the body for sotlie of the molecules. Most of tlle countrlcs 

depend upon meat, poultry, eggs ctc, fo~ these d~ctary supplements. Ucvcloped world 

derives liiuch of its protein (46%) and cnrboliydrates (20%) from antnlal products llkc 

meat, poultry, eggs and fish and otlier seafoods, while developing coutitries der~ve thc 

protetli (64%) and energy source (65%) primarily iio~l~ agrl-based foods. Ilenceforth, 

economies of the later are directly deperide~>t on agriculture. 

Agricultural practices and crop selectiol~ dates back to ovcr ten tilousand ycura and 

ancient agriculture \+as mostly characterized by few crop v;lr~etics that nourislled relatively 

few peoplc and thcir domesticated anitnals. Tlic early moderate popul:~l~ona demanded 

equally tnodcrate agricultural outputs. Howcver, static civilizations stiniulnted wide array 

of social and cultural tratisfoiiiiat~ons tliat ste;ldlly increased the population as well, which 

in :urn nccc,s~tated better yield froni the selected crops. It was oi~ly stncc 19'~ century that 

dramatic advances took place in agriculture in terms a!' quality and quziotity i\,llicll was 

made posslble by well-concerted efforts of sc~cl~tists lion1 all ovcr tile world co~nmitted fbr 

a common czluse of food-for-all. 

Presently around 200 plant species with tliousnnds of cultivars address the issue of 

human diet. Alllong these only 15 species col~tribute 9OU/o of diet where rice, wheat and 

corn contribute up to 70%. Though these three crops arc kliown as staple food crops of thc 

world, some of the tllird world countries arc not in a position to cultivate and maintain

them. About 10% of the Earth's land surPacc ia arable that is f~st approaching saturatioil, 

with only 1% of water available for ci~lt~vat~o~i and wit11 inevitable population outburst 

there is an ii~creasiilg concern of how to feed people as cautioned by enliile~lt economist of 

yesteryears. Malthus. When domarid was n~orc than the supply, 1960a witnessed mqor 

cllailgss by revolutionary agricultural outpiils, nil put 1ogetll~'r under the iilsj)iring phrase 

'Greei~ Revolution' that was carefully crafcd by cini~le~~t brccdcr Nonnan Borlaug, who 

was riglltly awa~ded ll~e Noble Prize for pence. lilcepted \vilh wlleat and cxtendcd to 

almost all the crops, green rsvolution paved the way to light up smiles OII the faces of the 

hungry tl~ougll it shot down the world food prices. 'This led to a more sustainable 

agriculture, land use efficiency and opened tile doors to nlany hopeful avenues, wh~ch 

could address various other imminent problenls. These results came wllcn delllographic 

realities were seiiding ripplcs throiigh the scientific community about thc ways and means 

to manage {lie yioblcms of starvation and n~alnutr~tioii. With over 6 billion pcolllc on 

board, an est~inated increase of 1.7 bill~ons inore by 2020 and wit11 around 800 million 

people still statrving, tbcre are more questiuns t11ni1 answers. Nevenheless, all the efforrs 

put togetlier will record an est~mated increase in the oniu~al growl11 rate of 2.6% food grain 

productloti wllile popiilat~oii increase rate ii l:iUA. Uuto~.tunatcly evcii hit11 these results 

the iiunger atill rcmalns sii:ce the outputs are not reaching the nccdy because oi'tl~e low 

purchasing power and standard of living. Tllesc concerns led to cultivation of alternative 

food suppleincnts such as leguines other than the popular cereals. 

Pulses are dry seed legumes that h;i\e relatively lesser nutritional popularity. 

Nutritionally i~nportant pods bearing seeds and herbaceous naturc ci~aracterize these crops. 

More thaii 50% of the world pulse y~eld 1s being co~ltrlbuted by Asia-Pacific region. The 

proteii~ and oil rich seeds of these plants liave an ~nd~spcnsable coiisidcratiou in the human 

diet and aniil~al fezd as bod additives. Fcrmcntcd or processed seeds make preferred

dishes for people of the developing counlries as acccssoiy foods. Most i~~lportant feature of 

these plants is that they tix atmospl~eric nitrogen 111 symb~osis with Rhirobiunt, which 

qualifies them to be used in Inter cropping and rotation cropping practices in contblnation 

with the cereals to enhance the product~v~ty of tile later. However, despite their protein rich 

and n~troge~l fix~ng attr~butes, the production of pulses lids increased at nluch slower pace. 

This pace could have been the result of Pictors like rice-whcat rotation, illter croppirig w ~tl~ 

few other cereals and lesser ava~lub~iity of ~~u~rition;~lly dcpcildable Icgumcs in hot xa,oll. 

Most of the Important legumcs are cool season crops where the cereals relatively domi~~;itc 

the cool season. These hcts pushed the areas ot'lsgu~ne cnltivat~o~~ to ~nargitlal regions that 

are mostly ramfed. Sigiiiiicai~i crop losscs are observed due to varlous b~otic 2nd ab~ot~c 

stresses, as farnmrs of the developing countries are not inclined to use expensive remedial 

inputs such a5 fertilizers and pesticides. This lias resulted in a wide gap betwceil thc yiuld 

ratios of pulses to cereals tit 1:32 111 I990 (l'aroda, 1995). Howevcr, various scie~ltific. 

social and nutrition concerns 111dicate that the legu~lies must be given duc impo~lance in the 

regular agricultural practices which would be Illore beneficial to thc third world countries. 

In accordance with this pointer, various govcr~i~nental and 11o1l-goveriin1e11~;~l researcl~ 

institutes including ICRISAT, India; ICARDA, Syria; IATA, Nigeria etc., embarked onto 

understand~ng the metabolic and agrono~nic ~ntricacies of various food and pasture 

legumes and organize maneuvers for Improvelncnt and preiervatio~~ of the el~tc germplasn~ 

of peas, beans, lentils, grain legumes and various other pulses. 

During early i~griculture, selectio~~ of ci~ltivable varieties was principally based 

upon natur:~l selection. Iluuvever, ~t was in I:~ter par1 of 19"' century that dcti~al plant 

breeding programmes started. Varieties with elite phenotyp~c troits wele aelected and 

ciossed for co~i~bi~i:jt~on ~f neccssaly cIiar.icici,, in lhu otl'spr~ng. blendel'a famous pca 

experiments and observations provided a Inore logical basc for the brzeding trlals and

observations GkI Shull's experiments in 1907 gave an ilnpstus for plant breeding that 

resulted III an exponential Increase in brcedlng programs, which w~s in turn expanded to 

countless nlembers of cultivable variet~es. Socio-eco~lomic Importance of these progranla 

gave birth to thousands of varieties initially In cereals and later in otl~rr dol~~esticated 

crops. However, breeding is a highly t~~r~c consuming process and labor intensive as the 

crossing means polll~lat~ng every plant mailually. Crops wit11 lo~lg duration life cycles pose 

variety oT problcms in co~~ve~ltio~lal breeding. Another I~~liltat~o~l 01' the cunventio~i:~l 

breed~ng lies in the susu;~l ~ncompatib~lity of the cultivated varieties with their wild 

relatives, where the ancestral wild varieties were proven to tie the reservoirs of several 

agronomically important traits. Tools of modern biotechtlology have come to the rescue for 

some of the difficult constraints to crop improvelncnt by understand~ng their molecul;ir 

basis and providing ren~edles at the n~olec~~lar level itself. ldc~lt~ficalio~l of physical basis 

of life and niolzcul.ir cl~;iractcri~atlo~~ of i~ll~critn~~ce pattenls ~nads brecd~ng a more 

syste~llatic and mcani~~gfui science of crop improvement. Stat~stical appropriat~on ot 

polymorphisms ttlllcrlri'd with the help of techn~qucs l~lte Southern blott~ng and 

polymerase chn111 reaction (PCII) gavc birth to a new faculty of'rnode1i1 agriculture that is, 

marker assistcd selection (MAS). This was not only uscful ill identificat~on of quantitat~ve 

tra~t loc~ (QTLs) but also uscful III isolation of gencs for a specific trait. ldentificat~on and 

isolation of agronomically important gencs fro111 different prokejotes and eukaryotes 

ignited a desperate wisl~ ill sc~entists to ~ntroducr: Illem into plants and observe tiieir effect 

In the new env~ronnie~lt. Tile existing bacterial transformation systems (Mandel and Higa, 

1970) gave some logistic support to this idea ofplant transgenes~s by recombination events 

in the genetic material. Discovery of the ab~l~ly of a crown gall induc~ng soil bacterium, 

.4grobacfrriton f~rr~~ejiiciert~ to introduce the genetic material Into the plants (Drummond, 

1979), in late 70s revolutionized the genetic transfurn~ation research. Agrobacteriurn

tumefaciens, popularly kl~own as a 'natural genetic engineer' transfers genetic material 

with the help of the tumor inducing (Ti) plasmid (Chilton, 1983). The native genes of Ti 

plasmid perform the function of cancerous establishnient of the Agrobactrrium in the 

infected plant cells. Among various attributes of' this plasmid, the niost important one is 

that it transfers virtually any genetic sequence present between the left and right borders of 

the famous T-DNA. Tliougll this organihm's ilifectlvity 1s restricted to dicois, tcchnologlcal 

advancements made [his organism useful to tr,lnsfer genetic ~llatcrial to monocots (Slemcl~h 

and Schieder, 1996) atid even to animal and hun~an cell cultures as well according to rccclit 

reports (Kuriik et al., 2001). Various otlirr techniques evolved with the inspiration liol~l 

this organism, but tliey arc host and teclin~que spccilic monocots (Siemens and Scliiedrr, 

1996). Biolistics or part~cle bombardment teclinique is onc potential technique (Saliio~d, 

1993) that could solve tile problem ofliost incolnpnl~b~llty. 

\/Chickpea 

(211-16) is one of tile important food legumes prevalently cultivated in 

Asia-Pacific region \+liere large portiun oi' the yield is contr~butcd by the Indian sub- 

continent. Broadly [here are two vorletlcs of cl~lckpea, Desi arid Kabuli. It is a cool season 

pod bearing crop, seeds of wh~ch are excellent source of protclns rlch with nitrogenous 

amino acids espec~,llly lyaiiir and arglnlne 111;iklng the products of 1111s crop, very good 

food aildilivcs. Ue>ldrs, it 1s also ktlo\vn to Il111)rove soil fertility with the Iielp of'sy~i~biotic 

nitrogen fixation. It contributes 15% of the world's pulse Iialvest of about 58 rnill~on tons, 

anllually. Deap~te sign~iicant gains In world pulse production during the last two decades 

with an annual growtli rate of 1.9% cllickpeli production growtli has been slow. Ch~ckpca 

yields worldw~de have risen by 0.6% annually which :mounts to 800 kgha, and thc area of 

cult~vatioll has reniaiiled v~~iunlly stagn;lllt. Tll~slower p;lce has bee11 the result ol'vu~ lous 

refractory biotic aiid abiotic constrdlnts such as ,lrL~oc/iylu bl~gllt (AB), Liurrj'li~ Grey hloid 

(BGM), dry roo!jz, 

- - - 

coll,~r rot, Fi~~iri.iiirr~ wilt, pod borer, drouglit ;~nd low tempcratu!.c

(Nene and Haware, 1980). The legume pod borer has been the worst of ail accounting for 

over 20% of the total crop loss (Vyas et al, 1983). The enhancement of insect and disease 

resistance in chickpea could increase its yield potential by as much as three times. The 

available chickpea germplasm also lacks effective resistance sources for use in developing 

pest resistant genotypes. An attractive option is to introduce genes for insect resistance 

from other sources to chickpea by the iise of transgenic technology that has shown a great 

promise (Sharma and Ortiz, 2000). 

Attempts to create transgenic chickpea to combat tlie above constraints have been 

the short cut strategy adopted by various groups working for creation of el~te germplasm of 

chickpea. However, reliable regeneration and transfor~nntio~i protocols lmve been evaduig 

sucli efforts due to tlie perce~ved recalcitranl nature of chickpea towards tissue culture. 

Several regeneration protocols involving somatic ernb~yogenesis and orgallogenesis have 

been published during past one and half decade olily to show the difficulty in regelierati~ig 

chickpea in the in vltro cnvlroiiment. Micropropagation has inot been a serious problem and 

it can be ;ichieved using explant co~itai~iit~g prc-cxisting Illerlstema such as shoot tip and 

cotyledonary nodes (Rao and Chopra, 1987; Riazuddin et a]., 1988; Rao and Chopra, 

1989). Coiisiderable work has been done for regenerating wliole plants via sonlatic 

embryogenesis from mature leaflets (Rao and Chopra, 1989) and imn~ature leatlets (Barna 

and Wakiiulu, 19931, niaturc emrbryo axis (Sul~as~~ii ct ol., 1994) and initnature embryo 

axis (Sagare et a]., 1993) or cell suspension cultures (Prakasli et a]., 1994). Ilowever, the 

success rates on the niatiirat~on of indilced embryos into fully differentiated plants have 

been very low (

Wakhulu, 1994). However, regeneration of quiet a few shoots from traces of pre-existlng 

meristem with that of the explants such as hypocotyl and epicotyl were mistaken for 

indirect regeneration from callus phase. To date, effective chickpea regeneration has been 

possible only through the use of explants based on cotyledonary nodes or shoot apices 

derived from seedling explants. In most of the cases the shoot buds originated 

asyncbroiiously making these systems inefficient for genetic transformation studies. 

However, rooting and transplai~tation of (he in vitro recovered plants lias remained a major 

bottleneck in the meaningful application of this technology for serious crop improven~ent 

programmes. Such systems have been used to genetically transform chickpea and the 

transfostnation frequencies reportrd were very low (Fontana et a]., 1993; Kar et al., 1997; 

Krishnamurthy et al., 2000). 

J I ~ the ongoil~g efforts at ICRISAT to develop suitable tissue culture and 

trai~sfosn~;~tlo~~ protocols for cl~ickpea, the preseilr work was ain~cd at the improvenient of 

cxisting protocols for ;ill the atagcs or regenefi~tioii and transformation. The work was 

carried out with the ibllo\\ing object~ves. 

I. Development of efficient protocola for plant rcgcneration in tlssue cultures oi 

cliickpen. 

2. Optimization of varlous factors aficting legeneration via orgdnogenic 

pathway 

3. Optimization of factors i~ffecting liardenlog and transplantation of in vitro 

regenerated plantlets. 

4. Genetic transformation and successful recovery of chickpea transget~ic plants 

by using agronon~ically impoitant BlCryiAb and SLIT1 geiles. 

5. Molecular cl~aracterization of putative transgenic plants or ch~ckpea for future 

use.

jf.0 REVIEW OF LITERATURE 

Large part of the world's food is bring coi~tributrd by ogriculturc though nlost of 

the developed countries derive their food BOIII animal source. World food grain yruducl~o~l 

toucl~ed 2 billion ton mark at the turn of the last n~illet~niurn. The econoniic, con~niercial 

and social realities resulted in an uneven distribution of the food grains amply ava~lable 

that left 800 million people still undernourished (FAO, 2001). Tlie surpluses are used for 

exports and tl~e costs were dearer for the developiiig coul~tries that kept the number of 

undernourished people still alarming. Alter~iative food supple~i~e~~ts froin pulse crops 

gained all iiicreasii~g significance in tlic recent times that prompted developed cou~~try I~ke 

United States to include the lentils, broad beans atid cliickpeas in their 1:drnl bill 2002 so as 

to encourage the exports in this area (Food Outlook, FAO, 2002). Pulses are tile cd~ble dry 

seeds of legum~nous plants. They are of special liutr~tioi~al and economic i~nportance due 

to tlieir contribution to the diets of ~~~ill~ons ofpcople worldwide. Tlie nialn importnncc of 

pulses lies primar~ly 111 their liigli proteni content (two to three t~~iies higher than most 

cereals) as %ell as in being a valuable source ofenergy. In addlrion, pulses contai~i good 

anioulits of r~utritionally essential minerals sucli as calcium and iron. The use of pulses as 

-. 

hod is concetitrated in developing countries, accounting for about 90% of global l~uman 

pulsc consump~ioi~. In i~lost low-income countries, pulses contribute about 10% of the 

daily protein and about 5% of energy intakes 111 tllc diets of people (Paroda, 1995). Dur~ng 

the initial years of legume cult~vat~on brecd~ng was an important means of crop 

improvement. These efforts niet wit11 considcrablo succesa that resulted in iniproved yield 

of biomass in terms of wl~olc plant dry tnintter or thc secd. However, breeding Sor traits

agalnsr varlous biotic and ahiotic constraints met with low success rates where 

biotechnology may prove to be an effective altert~ative. 

Evolution is a process of natural creation of var~ations in the genetic ~~iake-up of 

organisms so that ~t firs into the variable conditions of the changing environnlent. This 

process takes ~iiillio~is of years :ind 11 goes 011. As the requ~rcmct~ts doubled and tripled. 

there is a ~necess~ty to induce val.iatiolls dellberatcly to lileet the 11ecd. This fart enierylnp 

licld oi' pl;i~ir sclc~~cc ~;IIIIS 1t5 s~pi~ilic;~ncc ;illd tidv;~~lt:igc o\cr tllc Ilnllt:~tio~~r oi' !tic 

genetlc recomb~nat~on by meatis of con\cntional breeditig. Thougll this tccliriiquc is bung 

applied to many urganisnis, ~ncluding ni~crobes atid animals, the scope of [his thcs~s 1s 

restricted to deal with plants only. Moreover, nlosl of tlie publislicd and on goilig \wrh IS 

on platits whcn compared to the animals. Microbe transfonnatio~l is rather a procedure Tor 

basic research and only a part of preparative tcchnique for the higher organibm 

tratlsforniation. 

/Genetic transformatio~i, 111 principle is ~ntegrntion of alien genes into the foreign 

organisn~. Stable ~ntcgratioll arid lriheritalicc of ubcl'ul gcocs ib the main object~ve of gene 

transfer enperlmentsy"&e 

concept of genetic transt'ornlat~oli started with Avery et al. 

(1944). In angiosperms, gene transfer, however, IS a regular process where paternal 

chromosomes iioin the explod~iig sperm arc tr:insfcrred to the egg cell of tlie fen~,~le 

gametopllyte (Frankel and Galun, 1977). So, the problem 1s to transfer tlie gencs 

deliberately. Advent of plant tissue culture espec~ally the protoplast sola at ion (Cocking, 

1960) and cybrid formation gave an encouragi~lg impetus to the concept of gene transfer. 

Protoplast isolat~on resulted in cell-wall-stripped cells and rcgeneratioo from the wholl:

plants from them enlhuscd scientists for adding new genetic elements and earliest reports 

were of Hess (1969; 1970) when no tools for transfer and characterization of transformed 

plants were available. Discovery of ability of the soil n~icroorgan~sm Agroboc!erhoii 

iu~iieJucie~ls to trensfer its T-DNA to the pinlit genollie revolutio~~ized tliis arca and crcated 

altogether a differcnt faculty of science. 

2.1.1 Various lnetl~ods of gel~etic tr:r~~sfo~.~~~atiol~ in plnnts 

Various neth hods of gene Iralisler ~nlo pl:ints have been designed w~tll 

Agrobacierii~nl-mediated transfer as the principal ~netliod. Its miraculous ability to transfer 

part of its ge~ietic material, T-DNA into the plan1 genonle for its own beliefit was one of 

the significa~lt discoveries of 20Ih century. V;ist literature accumulated in describing tl~e 

whole mcclianisnl and ~PP;II.:I~IIS used by ilic microbe Tor gene transfer. llence, this nietl~od 

will be dealt in detail in tile foilow~~~g sccrloll. Metliods oliier tl1a11 those relying 011 the 

Agrohiicie~~ii~~ii sucli ;IS b~olis~ics, n~icroi~ijcct~vii elc. ir~ll bc dsacribcd ill 1111s aection. 

"li~oiisuc" IS n short term for b~ological b;lIlistics; the proccss is one by whicli 

biological r~iolccule>, sucli as DNA and RNA, are accclcrated (usually on microcarriers, 

termed microprojcctiles) by gullpowder, con~prcsscd gas or other means. The biological 

molecules are driven nt liigh velocity inlo the torgct, i~i this contcxt, tile plant cells. A team 

of nanoiibr~car~on fac~lity of Col-11el1 Uii~vers~ty developed this tccl~nique, and Sanford 

(1988) gave early descriptio~~s. Tlils 1s b;~sically si~nplc device wllere the genetic lrlaterial 

is coated on to the tungstei~ or gold pal-ticlcs illid accelerated with 11igI1 pressures into the 

plant cells. Dur~ng subsciluent years, tliis device tcok serles of cha~~ges to fit cornmerc~al

equiren~ei~ts (Santbrd, 1993) The original dr~ving power - the real guripowder was 

replaced by safer cot~lpressed hel~uni system. A different acceleration system was also 

developed based on the spark di,cliarge cllaniber 111 wliicl~ a water droplet was placed 

between two electrodes and a h~gh voltage capacitor caused all instant vaporization of the 

water, creatlng a shock wave. This sl~ock wave ;~cceltrates DNA coated particles illto the 

target plant cells (Christou et al., 1990). Several labs tried their owti honlemade particle 

guns (Per1 el a]., 1992). Var~ous dev~ces illid ilitr~ci~cics of b1011stic I>I.OCCSSSS IVC~C 

reviewed by Potrykiis ;ind Spa~igenberg (1995). Tlic firat application uf tlic biolistic 

process was niadr: by its inventors uallig chlora~~~plienicol acetyl transferase (CAT), a 

riiarker gelie (Klein et al, 1987). Later ye;~rs saw [lie two i~~ipo~til~~t j)ublica~iolis ot' 

successful transforrriatio~~ of chloroplasts ill Cliln~tiydo~rio~~us and niitochondria in yeilsl 

(Jolinston et iil., 1988). 

Tills protocol becotiles significant w11eii tlic host cells are not complacent w~th t!lc 

methods llhe /Igrobuc~er~iorl-t~iediatcd and direct protoplast transfornialion. Especially 

Ayrobuc~o.i~i~i~-li~ediatcd transforniation is, to some cxtent restricted to dicots and it also 

requires wounding. Helice, several rzports appear~d using mctliod to obtain transforn~ed 

platits in rice (Dntta ct a]., 1990), soybcat~ (Cliristou el al., 1990), niai~c (Fronint el dl., 

1990, Kozicl et al., 1993) 2nd barley (Wan ;ind I.ciiiuux, 1994). 

2.1.1.2 Alternative nletl~ods ofgci~etic tra~~sl'orm:itio~~ into plant cells: 

Alternative nictl~ods can be div~ded inlo two types: direct physical i~~troduction of 

DNA and tr;insnilssion of gellettc tnaterlal by nlodlfied pl;lnt viruses. Viral gene transfer 

can also involve physical transmissioli to tlic plant (e.g., nib inoculation). Most iniportant 

method of direct introduction of DNA is thc protoplast transformation, ln~t~ally dircct

introduction into protoplasts using ply-L-omithine (Davey et al., 1980) and this compound 

was later replaced with calcium phosphateipolyethylene glycol (PEG) (Krens et al., 1982). 

However, the success of protoplast transformation lies in tlie successful regeneration of the 

whole plants from them. Most of the direct DNA transfoni~ation involves usage of E.coli 

plasniids such as pBR 322 and pUC derivativea. Both plant DNA 2nd RNA viruses offer 

possibilities as plant transi'ormntio~ vectors. 

~'2.1.~ Miracle microorgaois~~l: Agrobacferirr,rr 

Early ~n tweiit~eth century, S~ilitlillid Townsend (1907) studled these crow11 gall 

tumors of cultivated Paris daisy and for the first tinie astablishcd that this "plant tumor" is 

of bacterial origin. The ineffective bdcterid isolated fro111 these samples produced tumors 

011 tlie stems of other crops. This bacteriuni clia~iged its lialiie nialiy times froni Batferi~int 

riiniefucien~ through Pliyioiiroilo~ rtiiiirfiiciri~s, Uucillir~ ir~i~iejiicieris and finally settled at 

Agrubocrrrii~iri riiiiieJ~icie~is. lllkcr (1923) and 1'111ck;ird (1035) st~ldted llially i~itrlcacies of 

-.-. 

the plant-niicrobe ~l~teractiolis that resulted in varlous cell stiniulations in plants. The 

-. 

opines were dctccted 111 the tumors (Pctit, 1970) and tlic vttnl role of these opines in the 

establishine~~t of "genetic colonization" was revealed by Shell (1979). lliitially it was 

perce~ved that the genes for opme sy~itliesis is pltlnt borne n~id only after rigorous studies 

the source of opines was confirmed to be fro111 the ~nfecti~ig bacteriuni (Montoya et al., 

1977). Much earlier to that Kerr (1971) fouiid that the virulence could be transferred from 

Agrobncicriiriii to sapropliytic bacteria tlirougli DNA transforn~ation. Persistence of 

bacterial DNA in bacterial free tu~nor cells was observed by Johnson et al., (1974) and 

DrLica and Kado (1974) with the less efficient techniques of their ti~nes. A large plastnid 

with a size of around 200 kb, was fbund ro be iieccssdry for [lie virulence of the

Agrobactenu~ii (Zaenen et dl, 1974). Eventually, Chtltoli et al., (1977) found that the 

--- 

genes of TI plasrn~d were responsible for the synthes~s of op~nes which were necessary for 

the progression of turiior w~tliout Ilie additton of growth liorniones They also found that 

only 5% of the plarniid DNA war responsible for virulelicc (Chilton, 1978). Since then 

many groups embarked upon exploring the lntr~cacies of the Agrobocieriuni and its abil~ty 

to transforni the host cells. 

2.1.2.1 Ti plnsltlid a ~ its ~ cllarncterislics 

d 

Many scientific groups in early 70s found that tlie TI plasmid, precisely part of it's 

DNA is responsible for tlie tumorigenic nature of the infecting bacterium. Tile process of 

identification of causal sequence for the tumorigenic activity was many folds expedited by 

the d~scovery of Southern blot (Southern, 1975) and DNA sequencing metllods (Maxam 

and Gilbert, 1977). Hooykaas, Schilperoot anti their associates found additional evidence 

for the role of Ti plasmid III 

tu~iior induction (Hooykaas et al., 1977). A detailed. 

d~scription of eah-events In crown gall researcl! was provided by Shell et al., (1979). It 

a? becatlie evident fiom Cliilton's experimelits thnt part of Ti DNA (termed as T-DNA) 

- -. 

was transferred t ~ l k !$I. Transcriptio~i of tlie T-DNA was confirnied by northern 

. --- 

blot experiments (Drunimond ct a]., 1977). All tile T-DNAs were found to be similar and 

has a length of 23 kb flanked by alniost idelltical borders (Zambryski et al., 1980). 

Spontaneous deletions studies rcvenled the gclietic componelits of tlie T-DNA and their 

oncogcliic tiatuse was confirnied (Gelvin et al., 1981). Schilperoot atid colleagues revealed 

many aspects of the T-DNA by inducing mutations and tracking tl~em down in the host 

plant cells by transfortiling tile tobacco protoplasts (Hockcnia et al., 1984). By this time it 

was also evident that a set of vir genes was involveti ill the transfer of T-DNA. It became 

... 

14

evident by 1983 that the Agrobacteriuni IS a sure candidate for genetic trallsformation 

(Chilton, 1983; Herrera-Estrella et al., 1983). Where T-DNA of the Ti plasm~d call be 

--. . . 

transferred to the plant cells and that can servr. as an cxcelle~lt tool for the genetic 

transformation of plants by dlsarnming the T-DNA and introducing the genes of interest into 

that area. 

2.1.2.2 Molecl~lnr ~nccl~a~~is~~~ 

of T-DNA Ir:~~~sfcr illto pla~~ts 

Tlis dlrtails of tliesc ~nechanisms can be obta~ned from scveral ~revicws (Hooykans 

and Schilperoot, 1992; Zambryski, 1992; Grcene and Zambryski, 1993; Zupan and 

Zambtysk~, 1995). Wirlatls (1992) provided a critical review of chenlical signaling between 

Agrobacirriu~~i and plants cells. . 

2.1.2.3 Tools for ge~letic tl.ansformatio~~ / 

Understanding the plant gene structure and its essential components is n 

prerequisite for dcsig~~iny the tools for gellctic transformation. By the time the coilcept~~nl 

basis fbr the genctlc transfonnation was ready using Aprubaclei~iu~~i, the components of a 

complete plant gclie to be effcctivcly exl~resscd was also ready. The ainalgamation of 

various niolecular biology tech~iiques led to the dcs~gning of vectors for transforming the 

plant cells. 

i 

Tru~s/orrnuiio~~ vectors: Most widely used vectors are binary vectors (Bevan, 

1984) and cointegrate type vectors (Draper el al., 1988; Rogers ct al., 1987; Deblaere et al., 

1987). The co~ntegrate type vectors have bccoiile less popular since they are more difficult 

to engineer than binary type ones and are less efticiel~t.

~ - 

/'- 

Binury vectors: A binary vector should contain essentially at least one of the 

borders, it should have the ability to replicate in E.coli and it should contain a selectable 

marker (Armitage et al., 1988; Hood et al., 1993). In principle, a binary vector consists of 

two plasmid; a plasmid that is transferred and a helper plasln~d. In the initial years it was 

pBI101, and later Inany verslot?s were constructed by Beckcr et a!., (1992). Additional 

information on binary vectors was provided by several authors (Jones et al., 1992; Futterer, 

1995). Rcccnlly it lias been suggehted to use binary vectors that conla~~l two acparale '1'- 

DNAs (Crameri el al., 1996). The log~c of tlie authors is that the antibiotic resistance 

marker will be lost during subsequent generations. A helper plasmid contains the vir gene 

complements that are essential for transfer of T-DNA. 

Protfioiers.. Futterer (1995) reviewed the subject of promoters for genetic 

. . 

transformation of plants. In the early years of gcnetic transformation of plants, 

~nvestigalors were merely interested ill showing thal integration and expression of 

transgenes is a rcality 111 plants. So, initially promoters endogenous to thc T-DNA were 

used. Soon 11 was observed that the promoters ibr opine synthesis were weak. Cliua and his 

--.. 

collaborators (Odcll cl al., 1985) isola~ed tbc CaMV 35s promoter from turnip leaves 

~nfected w~rh the Caul~flower nlosaic virus (C;IMV). This promoter was found to be many 

folds stronger and resulted In constitutive expressior~ of tlie introduced genes. However. 

/ 

sub-domalns of this promoter were found to be exelling tissue specific expression (Benfey 

and Chua, 1989). Since then this proliloter became an attractive candidate for plant 

molecular biology research. Its fusion wit11 part of mannopine synthase (MAS) promoter 

- -- . 

increased the potency of this promoter (Kay et al., 1987). Valuable information can be 

found in the reviews by Benfey and Chua (1989), Wang and Cutler (1995).

The above promoters were found to be Inure efficient In dlcots a?d there was a 

.- - 

distinguished interest for finding out the promoters for monocots. In the early studies with 

/- 

rice (Shimanloto et al., 1989) the CaMV 35s promoter was used to activate tile selective 

7 

and repotter genes. However, it was found that this promoter was more efficient in dicots. 

Conlbination of this promoter with other pro:lioter segments and introns were even tried. 

This concept was followed by the wage of ccieul alcol~ul dehydrogenw I (Adlil) gene 

- +...- .- 

(Callis et dl., 1987; Kyozuka ct al., 1990). A silliilar approacll to integrate the first intron of 

the 3hru1iki.11 I gene of maize was also followed in cereal tratlsformat~on (Mass et al., 

1991), but I: becatue less popular in the subsequent years. Rice actin gene pro~l~oter (Acll) 

- . 

was found to be even nlo1.e potent than the above two (Zliang ct al., 1991). This pronioter 

showed more or less similar potency as that of Ettiu pron~otcr tllat is a recombii~allt 

promoter cotltaining a ti-ul~cated Adlil~n?_oter with other elements (Last et a]., 1991). The 

-- 

curretlt most effective promoter is tile Ubiyuiiiii I (Ubii) of n~aize (Clrrislcnseit et al., 

1992). Tii~a proilioter was used successfully to tlnnsfornl !\,\.heat (Weeds el al., 1993) 

barley (Wan and Lemaux, 1994) and rice (Toki et nl., 1992). Another pronloter of tile rice 

Aldviure P (Aidl') gene was ibund to be one of tile bctter alternat~ve (Kagaya et al., 1995). 

Tcnrii~zrrlars: Knowledge of the ele~lleiits for geilc expression 1s as important as the 

promoters. It is considered that fundamentally n1RN.A is stable unless destabilizing motifs 

are involved. Specific examplcs of the studles that handled the polyadenylation signals in 

plants are investigations of Mogen ct al. (1990). 1:otiinie et al. (1994) studied the essence 

of the terminator regions and impact of 3'-end ~.cgions on the level of gene expression of 

octopine syntliasc gene and otllcr gene constructs was studied by lngelbrecht et al. (1989). 

Hence, the usage of tern~inator region at [lie 3' end of tile transgene was found to be

essential. In practice, temllnator of ~iopali~ie synthase gelle or of the CaMV was fused into 

the respective chimeric gene. 

Selectablr O I I ~ rryorriilg nrarkers: These are essential comporlents of the total 

cassette that is to be tr:rrlsferrcd lo llle pla~ii cells, These genes select ihe transfor~lled cells 

from that of the untransibrnled. Sorlle of the popular selectable markers arc ant~b~ot~c 

markers such as kanamycin, hygromycin, strepto~nycin etc. Other groups of' selcctablc 

nlarkers are the ones that co~lfer resistu~lce to herbicides, such as phopbinohtrici~~. 

biolophos, glyphosate. dalapllon etc. As noted above this group of selectable nrarkers can 

serve a dual pull,ose: to select transfom~i~nts and to render crops resistant lo respective 

herbicides (D'Halluin et al., 1992). The third group is ti~verse, including gencs that cause 

resistance to high nitrate, 111gIl a~lli~~o ac~d levels (lys~ne or tl~rconine) or amino ac~d 

analogues (Schroit, 1995). Most commonly used selectable genc IS the kanamyc~n 

rcsista~~ce gcnc ineo~~lyci~l pl~ospl~otra~~sfcrase (~rpfll). This gene producl dctuxilics 

an~inoglycoside ;~~~ub~otics such as ka~~alnyci~~, neonlycin, gencticin and paromomyci~i 

(Vurdi et al., 1990). The gene Iipt was isoloied f'rom licoli. It codes for hygrornyc~n 

phosphotransfrrasc that deroxilies antibiotic hygronlyci~~. 

Kcporter ge~~cs are coding sequences that, upon expressloll in the transgenic plant, 

provide a clear 111dicat1011 tlliit gc~letic transfor~llat~o~l did take place. They are useful also 

for transient expressio~~ experimenis, in wlirch tl~c iransgene is not necessarily integrated 

into the lhost gcnolne. Scllrott (1995) rcvicwcd a review of genes used and tllcir assay 

methods. blost co~ll~no~lly used genes used, as ~cporters arc the ones that code for CAT, 

GUS, Luciferase and Glee11 1:luorescent I'rotein (GFP). Thc assay for the riidA that codcs 

for GUS was developed by Jefferson and his associates (Jefferson, 1987). This gene has

gained an illstant popi~larity owi~ig to 11s cfticic~icy and localiza~ion of the exprcssio~l 

without extrnctiiig llie tissue. The luoiferase reporter gelie bas developed by de Wet a1111 

associates (De Wet el al., 1985) and was reviewed by Luelirsrn el al. (1992). The 

gene 

that codes for green fluorescelit prote~~i (GFP) *as the recellt one isolated from jcllytish by 

Chalfie et al. (1994). Many reports apprarcd In support snd against tlie usage of GFP 

(Ilaseloffai~d Amos, 1995). 

f 

2.2 Legume tissue culture and tra~~sfor~iiatio~~ 

Leguminosae is a very i~i~portant falllily of angiospcrnls comprising of ~nany 

species in relation to Iiuman nutr~tion, pasture and tbdder needs. Important protein rich 

seed bear~ng plants, n~ostly lierbaceous, such as pros, lentils, beans collectively known ss 

pulses are members of this filmily, They rank next to cereals in tenns of human nutrition. 

In qua~ititative significance they are far bchind the cereals, however, gaining some due 

inlportancc as food additives ill tlie recent yc,irs. Domi~~ot~ol~ of tlic cereals in tlie food 

sector allowed oi~ly miirpinal ~~icreascs in tile overall yield of pulses. I

2.2.1 Food legumes 

There are several apecles and subspec~es classified as food legumes. But, only few 

(15 to 20) genera are vely important, Hundreds of cultivnrs witl~i~i these genera ore 

included in the agricultural practices, each having sorue selected attributes. Most ilnportaut 

of tliese species are Glycitie irrar, Amchis /i)y~ugoeo, CI~CL'~ 

(~ri~iiir~iiri. L~IIS (1111i11~rris. 

Pisurri ~crfivu~ri, Lor/r)v.irs saiivirs. C(ijnrrres ccijair, Yigiiri ruiiiirio, Vig~in rriu~rgo, Yi,qiitr 

ocoirirfo/icr, Viyrrci rtr~ibcllntc~, Vigiia rirrgrricril~i/ir, Plroseulir~ vrrlgriris, Mrrrro~~~lorrrri 

nuj7onrr11 etc. These species col~stitute over 80% of total food legume output. 1111t1;1lly 

many of these specles were thougl~t to be recalcitlant ill 

tissue culture and latcr 

advancements ol' biotcchnological tecliiiiqlles gr;rdunlly e;ised the technical difficult~cs. 

Micropropagation was relatively easier wl~cli colnpared to adve~~titious shoot ~~rgenerauon, 

where the shoots originate fro111 pre-existing nieristems. Advelitilious regeneration is a pre- 

requisite for a successful ge~iet~c transforniation. Each species responded differently In 

tissue culture and some of tlie protocols were successf't~lly used for gcnctic transforniation. 

Organogenesis is a widely used tissue culture strategy for rcge~icration of whole 

plants via direct and indirect illduction of varlous plant organs such as sl~oots atid roots. 

Generally shoots are induced ~~iitially fiom selcc~ed expla~its followed by roots. Mult~ple 

shoots are il~duced e~ther directly or through callus phase where the techniques are termed 

as direct and ind~rect ol.gal~ogenesis respectively. 

Direcr organogeriesis: Following are some selected repons where organogenesis 

without any intermediary callus phase lias been reported in various ecol~omically in~portal~t

legume crop species. Cotyledon explants for the indirect regeneration shoots was 

developed for soybean (Glycine niar) using 2,4,5-T for embryogenic callus induction. 

Somatic embryogenesis was best on SE (soyabeen embryo) medium supplemented with 

BAP and best regeneration of shoots was found on hormone free medium or on medium 

/ 

with IBA (Cho et al., 1992). Multiple shoot regeneration was obtained from leaf and 

hypocotyls explants of Glycine wigtii. 3-4 day old seedlings cultured on NAA and IBA 

containing medium gave rise to multiple shoot buds (Pandey and Bansal, 1992). A wild 

relative of soybean, Glycine clandeslina, was induced with brown, compact and nodular 

callus and plants were regenerated from it (Sharma and Kothari, 1993). Culturing of 

zygotic embryos and nlultiple shoot regeneration was studied envisaging their use for 

micro projectile bombardment in Arachis l~ypogaea (Schnall and Weissinger, 1993). 

Effects of auxins (IAA, NAA, IBA and 2,4-D) and cytokinins (kinetin and BAP) were 

studied for multiple shoot regeneration from cotyledons and cotyledonary node explants of 

Arackis liypogaea (Venkatachalam and Jayabalan, 1997). Various concentrations of BAP, 

2-iP, chloropyridylphenylurea (4-PU), TDZ and zcatin in co~nb~nation with NAA were 

used to i~~lprovise the regeneration from cultured leaf segments in Aruchis hypugueu 

(Akasaka et al., 2000). Explants such aa petioMes, ep~cotyl sections and other seedling 

explants were used for regeneration of AI.uL.~~J liypoguea (Cheng et al., 1992). 

Regeneration via caulogenes~s (shoot organogenesis) was achieved in Arachis /~ypaguru, 

from plumular explants. Tile shoot buds rcgeilerated on medium containing brass~n, BAP 

and P-naphthoxy acetic acid (Ponsamuel et al., 1998). In vitro regeneration of Arachis 

hypogaea was achieved via organogenesis by employing BAP as the principal multiple 

shoot inducer. Optimal temperature for culturing of leaf explants was standardized, and

effect of silver nitrate was studied (Pestana et al., 1999). Wild species are generally less 

responsive in tissue culture. However, differentiation in tissue culture using mature leaf 

expiants was first reported i~i Aracltis vil/os~r/ic.orpu (Johnson atid Pittman, 1986). A 

protocol for tlssue culture based regeneration of Artichis pitrrui, a wtld perennial peanut. 

was developed. Day length and media effects were directly correlated with variations In 

regeneration (Ngo and Quesenberry, 2000). Effect of aluminium on the tissue cultures of 

Piiuseoliis t'iilgirvis was studlcd (Esp~tio et ol., 1994). Direct plant regeneration and 

multiplicat~on was obtatiicd ftom the embryo and cotyledons of cominon bean Pkusc.olus 

vlugaris that were inibrbed for 3 days atid cultured on a soil rnediun~ for 7 days (Mohamed. 

1990). A small portion of split embryonic axis sliowed a genotype dependent multiple 

shoot regeneratioti in Pliuseolirs vulgaris and P, cocci~~rus (Santalla ct al., 1998). Various 

factors sucli as l~ght intensity and duralion plant growtli regulators etc, were studied whilc 

regeneratitig multiple al~oots from mature en~bryonal axes of Cujanus Cajar~ (Franklin et 

al., 2000). Epicotyl, hypocotyl, leaf, and cotylcdo~iary nodal explants were shown to 

regenerate a h~gli frequency of multiple shoots with high BAP and kinetin of pigeon pea 

(Cajoirus caja~~) (Gretha et al., 1998). Multiple shoots %ere regenerated from distal ends of 

cotyiedo~~ary segments of. Caja~rirs cajan. This was achieved using conibinations of BAP. 

kinetin with adenine sulfate (Molian and Krislinarnurthy, 1998). Cotyledonary node culture 

using BAP and IAA was reported in pigeonpea, Cajirtlus cujut~. A mass of~nultiple shoot- 

initials formed at the axillary bud region of. tlrc cotyledonary node of the seedlings within 

two weeks. The cotyledotia~y node along witli the mass of shoot-init~als excised from the 

seedling, continued to form new shoot-initials on MS medium containing 6- 

benzylaminopurine and supplemented topically with indole-3-acetic ac~d. (Prakash et al.,

1994). Clonal propagation of FI inte~spccific hybrids of Vig~rci radiaia atid K ~rtungc, was 

done. Mult~ple sliuots were induced fro111 the cotyledoliary node explants of FI hybrids 

(Aven~do et al., 1991). Effect of culti~re ~iledi~i~n o~i plat11 rcge~~eratioii fron~ cotyledons of 

Yigrrcr radiaia was studled. Genotypc size, oriclitation 2nd age of thc explant showed very 

sign~ficant effects on plant regeneration (Gulati and Jaiwal, 1990). Diftkrsnccs 111 siioot 

regeneratio11 liolii coryledo~iaiy llodc expla~its 111 Asiatic Vigrtci apccies were used for 

gelioliiic group~~ig wtth~n subgei~us ('rro/oiro/~/rb(Avcnido and Ilattor~, 1099). Shoot lip 

cultures were establislied for plant regeneration of n~u~igboa~i, Vigtzucirliolci Complete 

plants njere regenerated d~rectly without an intervening callus phase from shoot tips on 

basal nlediuril (MS salts -. I35 vitamins). Regeneration frequeilcy var~ed with cultivara, 

explant slze and growth regulator conib~natioii; 111 [lie ~ncdiu~n. Additio~l of cytok~~lins 

induced a var~able atnount of callus at the base of tlie slioot tip, followed by multiple shoot 

formation. BAP, kiilrtili and zeatiii each ~nduccd i~~ulliplc slioors ill 100% of the cxplants 

but thc liigliest ~luiuber of regenerntits per explants (9) was produced with BAP. (Gulati 

and Jaiwal, 1992). Regeneration was achieved using cotylcdo~laiy nodes giving rise to 

axillary shoots of I'ignu 

i~tuttgo (black gram). Regz~ierntion lias been acli~eved through 

organogcnesis usiiig explilnts from axillary slioota or~ginatiilg froni the nodes of seedlirigs 

ger~n~~iated in cytoki~li~i co~i~ai~lillg rnedluni. Seeds gerulinated in tllidiazuron (TDZ) 

supplemeilted MS produced 11 axillary shoots/cotyledonary node. Stern and petiole 

explants derived froin these axillary-shoots produced callus along with shoot-buds after 2 

weeks of culture on half stl-engtll MS supple~nentcd with NAA. Shoot-buds were also 

produced from varlous sites of injury caused by ~ncisions on the stem explants. Full 

strength MS salts tnh~b~ted bud for~natio~i (Das et al., 1998). Cotyledo~l explant5 der~ved

from germinated seeds of a multipurpose legulnino~is tree. Srsboi~in graildifolia. showcd u 

high percentage (96%) of expli~nts prod~icilig .n least 30 slioot bud per expinlit (Iletre~ c~ 

al., ~YY~J. 

Irii111~~c.l o~~flriogcvw~i~: Regcncri~tioli ~III(I ~IIJI~SIS ur c;~llus li.olii Ilu\v-sorted 

heterokaryolls of soybeall (Gli,c,~ric 1rici.1) mid 6' c.ciir~,sc,c.irs was donc (Haliiluatt ct a1 . 

I9S8). I'lcces 

of 1e:rvcs li-o~n sectll~ligs ot' ,liii~,/ii\ jirii/oi ivclc IC~L'IICI;IIC~ \I;I 

orgaliogelieals a~ld so~li;~tlc e~iibryogcncsis path\\.;lys. Plant rcgelleratioli was obtaillrd 

I;I 

two developmentnl path\vays o,-ganogenesls and soniatic eniblyogenesis. Orgaliogcnic 

callus cultures were i~illiatcd Sroni pleces of leaf oli MS niediunl suppleliirlitcd wltli NAA 

or 2,1-D ill culiiblliatiuli \villi BAP, kinetill or 2-IP. Tlie niust sui~able co~~ibi~iat~u~i for 

plant rege~ieratioii tliruugl~ orgn~iuge~icsis was a11 iliitial lliediuli~ cornposed ol' NAA and 

BAP fbliowed by transfer ot'the callus to a slioot inductioli medium (MS+ BAP) Routing 

of regenerated shoots was read~ly acli~cved by culture on MS w~tli NAA. Embryogenic 

callus cultures were inltialcd froni plcces of leaf on MS n~ediurn supplenlented with 

picloram ~n coliib~lietio~l w~ih kitletin, Aeatlil, BAP or 2-iP, alid the iiioat suitable 

comblnat~ons wcre picloram, BAP or 2-il'. 

(Iky ct al., 2000j. A comparative study of 

callus t"ormation and pl;~nt regelicratlon was dolie using d~fttrciit explants of Phuseolus 

vulgaris and P. coccitieus (Ruiz et al., 1986). A different sylithctic auxin 2,3,5- 

trilodobenzoic acid was sllowii to ~nduce callus atid roots on stem cuttings of niungbeali, 

Phaseul~is aureus (Ali and Jarvis, 1988). A novel liietliotl of culturing lcaf disc explnnts of 

pigeon pea (Cojrirruv ccijtrri) on ~~iulril)lc slioot ~liduction niediuni with IAA and BAP where 

the shoots originated via callus phase (Itathore ct al., 2000). Plants were regenerated fro111 

leaflet-derlved callus of Aescl~)~noir~oie setisiliva, A. urrie~icunu and A, villosu. Explants

were induced to for111 callus when asept~cally cultured on Murashige and Skoog medium 

solidified w1tl1 0.8% ag;ir ,lnd colitallnlig NAA 311d b~~i~ylilde~i~ne. Slloot regeneration was 

readily ach~eved and roots were induced when shoots were transferred to medium devoid 

of growth regulators or w~lh NAA. Callus from A. jnlcciicl failed to regenerate shoots. 

Explants froni leaflets of A. jluri~i~~ei~sis d~d not produce callus w11e11 cultured ill vltro. (Rey 

and Mroginskl, 1996). Pre-soaked sccds of Viglici rtrtliiiici showed variable callus growth 

wile11 exposed to varioua liite~lsit~es of light whcrc tho slloot regenciatlon was also vnr~ablc 

under dlferellt intens~tics of light. Hormonal supple~nents of tlii: culture rnediun~ liad 

some proniotive effect on regeneration under various light intensities (Narciso et al., 19L)7). 

Analysis of tissue culture borne genetic variations (somaclonal variations) was done in 

Pislo11 sotiviirii (Griga et al., 1995). One hundred and forty six son~aclo~ies were generated 

that were resistant to the puritied toxin of Cc.rcuipuru cciilesctvii (Kaush;~l et al., 1997). 

Somatic cnibryugcnesis is anuther el'ticient strategy where regenerating tissue 

Initially attalns some dctined globul'ir, ell~pt~ct~l etc, shapes and .tilose units gradually 

regenerate illto whole planls. Usually tile globular proenlbryos split into torpedo shaped 

ones and shoot primordium regenerate from tile axillary portion. Relatively regeneratloll 

via somatlc embryogeilesis pathway consumes more ti~ne than organogenic pathway. A 

fast and efficient regeneratloti system vla somatlc enibryogei~esis was developed uslng 

BAP and NAA on cotyledo~l explailts of Glycirie rliux. Plants were ready w~thin six weeks 

from explant stage (Fu et al., 1995). Ail efiic~cnt regeneratton system was developed for 

Glycirre io~r~ertrc.ito via sonlatic embryogenesis pathway. Effect of plant growth regulators 

and pi3 was studied ;ind condit~ons were standardi~ed (Lee, 1992). Proinotive role of

thidiazuron (TDZ) was studied to induce direct somatic embryogencsis and regeneration 

from seedl~ng explants of Arochis l~ypogaeii (Gill and Saxena, 1992). Direct somatic 

embryogenesls from zygot~c embryos derived from 40-day-old immature pods of Arachi~ 

i~ypoguea using 2,4-D were studied where various factors like growth regulators, sucrose, 

genotypes and length of elnbryo~iic axis influenced frequency (Reddy and Reddy, 1993). 

2,4-D-induced somatic elilbryoge~iesis was obtained from embryogenic calluses derived 

from hypocotyls explants of Arucilis iiypoguea. High concentration of 2,4-D decreased the 

frequency of somatic embryogencsis (Venkataclialam et al., 1997). Synthetic seeds oS 

Arucl~is iijpogueu were obtained by encapsulatilig 5 to 30-day-old somitlc embryos and 

geminated by on medium with various concentrations of sucrose, maltose, BAP and NAA. 

25% of these embryos were filially converted into plantlets (Padmaja et al., 1995). A 

refinement of embryo rescue technique to improve plant recovery from early heart shaped 

embryos of interspecific hybrids of Pi~ascolur poiyutlrtlus and P. viilgaris was reported 

(Geerts et al., 1999). Rates of ethyle~ic production were determined in highly embryogenic 

and virtually non-embryogenic tissue cultures of Aledicago sarivu ssp. faicata during a 10- 

day induction period on medium containing 2,4-D and kinetin, and during the first 10 d of 

somatic embryo ibrmation on growth regulator-frcc medium. It was concluded from thesc 

experiments that the high rates of ethylene product~on during embryo iilduction are not 

essential for subsequent enibryo differentiation (Meijer, 1989). Efiicient plant regeneration 

via somatic enibryogenesis has been developed in pigeonpea (Cajanvs cajan). Cotyledon 

and leaf explants from 10-day-old seedlings produced embryogenic callus and somatic 

embryos when cultured on MS supplenlented with 10 pM thidiazuron (TDZ). Subsequent 

withdrawal of TDZ from the induction medium resulted in the maturation and growth of

' 

the ernblyos into plantlets on MS basal rnediun~. (Sreenivas et al , 1998). Distal ends of 

cotyledons were used to induce somatic embryogenesis in Cajanus cujan by applying 

BAP, kinetin and adeilitie sulfate atid whole plants were regenerated (Patel et al., 1994). 

f 

Direct somatic embryogerlesls was induced from excised seedling leaf segments of 

/ 

vegetable legume, Psophocnrp~is reirngo~iolobiis by using NAA atid BAP and the 

coliversioll frequency of cotyledonary embryos was 53.3% upon culture on MS medium 

cor;taiili~lg ADA for 7 days followed by transfer to MS medlum supplemented with 1BA 

and BAP. (Dutta Gupta et al., 1997). 

2.2.1.3 Other methods 

Besides orgal~ogellesis and sotllatic enlbryogenesis the techlliqucs such as 

regeneration of platits from isolated protoplasts, microspore, anther and ovule cultures etc. 

are less frequetltly applied in the tissue culture studies. Excellent ylelds and quality were 

achieved for soyabean (Giyci~ie ~llax) protoplasts and l~latits regenerated from agitation- 

derived protoplast preparations had a higher cllance of being derived from intact cells. 

(Zaghnlout et al., 1990). I1rotopl;~st isolation in Araciiis iiypogu~a is relatively rare 

phenomenon and there is one repor1 for isolatioll and regeneration of plantlets through this 

method and tiis method was effectively used Ibr elcctroporatioll mediated tralisforniatioll 

(Li et al., 1995). A different method us111g 1hi11 cell layer tech~i~que and transverse thin cell 

layer (iTCL), where the ffCLs were cultured on TDZ for Piiaseolus vulguris was 

employed. Shoot multiplication was enhanced uslng BAP with silver nitrate (Cawallio et 

al., 2000). A reproducible protocol for plant regeneration from seedling hypocotyls 

protoplasts using varlous growth regulators such as zeatin riboside, GA, and 1Bh was 

reported in Vigr~a rubloba~a (Bhadra et al., 1994). Callus regeneration was achieved fro111

protoplasts isolated from mesophyll tissue of sweet pea Lofi~yrus odorolus (Razdan et al., 

1980). Protoplasts were isolated from leaf tissue of Lens culinaris by using cellulase, 

macerozyme dissolved in 0.5 M mannitol witit pental salts. However, the callus cultures 

could not be regenerated into plantlets (Stiff et al., 1991). Genetically variable plants were 

obtained from anther derived callus cultures obtained from microspore cultures of Cuja~ius 

cajun (Kaur and Bhalla, 1998). Cryopreservation is an excellent method for germplasrn 

conservation provided an efficient method is abailable for regeneration. Methods for pollen 

embryo cryopreservation and conservation of germplasm of Arachis, Rrossica and 

Triticunl sp. were explicitly reviewed (Bajaj, 1983). 

2.2.1A Genetic transforniatio~i 

h/ 

Aerobacierii~m-~nediated frat~sJ?~r~t~urio~i: Direct crown galls were induced by 

infecting stem explants of Lentil (Le11s a~lbiuris) with four strains of Agrobacieriuni 

lurne/iucie~is. Opines were detected in the crown gall and Southern analysis showed that T- 

DNA was transfersed (Warkentin and MctIughen, 1991), inclusion of potato suspension 

culture in the culturc niediuni cnilanced the trailsformation frequency of the callus obtained 

from In vitro grown seedlings ofthe Glyci~~e 1lia.r (Chang and Chan, 1991). An efficient 

protocol for Agrobacreriuiii-n~ed~ated transfornlatio~l of cotyledon explants from in vitro 

grown seedlings of Amchis 1typogoc.u that resulled in a very h~gh frequency of 

transforniation (55%) was reported. The expla~lt, were transformed with binary vectors 

pBI12I and pROK1I:IPCVcp that consisted oC1ipt11 as seleclable marker gene and lndlan 

Peanur Clump Vlrus coat protein gene as the agrono~llically important gene (Sharma and 

Anjiah, 2000). Co-cultivation of cotyledonary node explants of Ari~cir~s Iiypuguea was 

done with A. IUIII~~UC~L'IIS strain Ilarbor~ng binary vector containing uidA gene as reporter

and nprII for selection. PCR and Soutllern analyses confinned the integration of transgene 

(Venkatachalam et al., 1998). Transformation of A. hjyogaea was also done using the 

somatic embryogenesis pathway. The cotyledons were co-cultivated with A. rumefaciens 

strain LBA4404 containing uidA and 11pill genes. Somatic en~bryos were ~nduced with 

NAA and BAP and later regenerated into whole plants with a transformation frequency of 

47% (Vcnkatachalam et al., 2000). Embryo axis explants were uaed for thc 

Agro6ucie1~iiii1i-ri1ed1ated transform;~tion in /lriiciri;, I~ypogcieu (McKenlly et al., 1995). In 

vitro grown seedlings of Crijoirrrs crijait were inoculated with three types of wild strains of 

Agrobncirriu~ir A281, A6 and T37 and cultivar-Agrobacleriiiir~ strarn specilic induct~on 

tumors was found (Rathore and Chaod, 1997). Lcaf disks of pigeon pea cv. ICPI5164 were 

transfor~iied by il iur~iejiciens strain LBA4404 plasniid pBAL2 carrying kanumycin 

resistance and GUS reporter genes under tile control of tile 35s promoter. The optimul~i 

period of coc~ilt~v;i[ion was 4 days, giving 47.8% tronsfor~ned calluscs (Arundhati, 1999). 

Tr;~nslbrn~alion of pigconpea wila acli~evcd LISII~~ 

A. r~i~rlefu!/a(.if~t~ strain GV2260, 

containing the construct of isolated cowpea proteinase inhib~tor gene, pCPI. The gene was 

dr~ven by CaMV 35s promoter containing kanamycin resistance as plant selection marker. 

hlolecular analysis of tile putative transibrniatits was done by Northern blotting technique 

(Lawrence and Kuundal, 2001). /Igrobocleriiii11-n1ed1ated transformation of Vigi~u 

sesquipeiiuiis was achieved using cotyledonary node explant, where 2% of the shoots 

showed integration of riprII, phosphinothr~cin acetyl transferase bur) and uidA genes. 

Integration was confirmed using GUS histo-chemical assay and Southern blot analys~s 

(Ignasimuthu, 2000). Hypocotyl and primary Icaves excised from 2-day-old in vltro grown 

mungbean (Vigrla rudiata) were used for tlle transfomiation studies. This particular gram

legume was considered to be liighly recalcitrant. A convincing transforniation frequency 

was obtained and tlie frequency was confir~ned using GUS li!stochelliical assay and 

Southem blot analysis (Jaiwal et al., 2001). Culture and co-cultivation of priinary leaves 

of Vig~a mungo resulted In transfor~~ied calluaes tllat dld not regenerate into whole plants 

althougli the selected calluses exhibited positivr NPTll assay (Karthikeyan et al., 1996). A 

genomtc fragment e~lcodiny Plraseolrrs vulgaris arcelin-5a protein that confers resistance to 

an insect pest Zalrrores sirbjisciatus, along with 11pil1 and ~ridA genes were constructed 

into binary vector which was used to tmnsfon~i Pliasrolur ucrtri/o!ius 

where bud explants 

were used froiii genotypes (Dillen et al., 1997a). Callus of Phasrolus acutifolius var. 

acut~alius, the Lepary bean, was co-cultivated with Agrobacterium tumefaciens strain 

C58ClRif. Due to the high regeneration co~llpetence of P, ~curijolius, transfonned plants 

could be raised and transformed seed was obtn~ned. It is suggested that by interspecific 

hybridization of rransfbrnied I-', acurijoliirs witli tlie regeneratioil recalcitrant P, vulgclris, 

inrrogress~oii of desirable genes into P. virlycrris could be achieved. The relevance of thia 

approach with reference to alternative techniques aimed at reducing or omitting the ~ieed 

for in vitro regeneration (e.g. pollen transforniation, mcristem transfoniiation) is assesscd 

(Dillen et al., 3997b). Epicotyli and iiodal explants of' Pisii~~r surivlrm were transformed 

using binary and co-integrate vectors. The transformntion frequency was found to be the 

funct~on of explant source, A, tu~~~rfaciens straio, pea gellotype and duration of co- 

cultivatiori (Katlien et al. 1990). Sonx co~nniercial breeding lines of Medicago sutiva (alfa 

alfa) were transfornied by Agrohacteriuni method. Stable transgenic material was screened 

with nptII speclfic PCIl amplification and Soulliern hybridization (Desgagnes et al., 1995). 

Shoot and leaf expla~its of non-regenerable Mrdicago sp, were infected w~th

Agrobacleriuiii and ill otlier experiment transgenic platits were obtained by eloctroporating 

protoplasts (Kuchuk et a]., 1990). A. lui?teJ'ucieils med~ated transformation was performed 

with some members of a population of Medicogo sii/ivu into wli~ch a trait of somatic 

elnbtyogenes~s was ~ncotporated vla breeding and the transge~iic pla~its were analyzed by 

PCR (Du el al., 1994). A. i~oiie/i:cieilr mrdia~ed tra~isformetian of Liipiiitrs riiuiubilis was 

done using shoot apical explants. A first report in tliis liarticular platit, tratisforman~s werc 

contirlned with 11oi1-'idionctive Soutlicri~ iiybi.~dtfiition (U;iboogln et al., 2000) AII clllc 

accession, ClAT 184, of an important paslure legume, Sly/osutilh~'~ guiuiie~is, was 

transformed by Agrubac./eriiiiii-111ediated oictl~od with binary vector harboring itpill and 

11idA genes (Sarria et al., 1994). A rapid and reproducible protocol for l'orfoliirr~i 

subterrar~eiiin, a subterranean clover, was standardized uslng itpill, uidA and an alplia- 

amylase i~iliibitor gene. Tlie protocol shows that glucose and acctosyringo~~e was required 

in thc co-cult~vation medium. Four cointiiercial cultivars were successfully traiisfomed 

(Khan et al., 1994). Genetic tral~sfortnation of tllc broad bean, Vicici jhbu, was done using 

.4, tu~?iejircie~is and A, rhizogencs. Ttiree cultivars and mutants wcre used for the 

transforniai~on stud~es (Jelenic et a]., 2000). 

,,&lisiics: 

Biolistic trnnsibrniation of Gl),ciiie iiiclx was done using a bovine milk 

protein, p-casein cloned under seed specitic lectin promoter (hlaughan et al., 1999). The 

method of electroporatlon was used to transform the protoplasts of Glycbie urgvreu. 

Protoplast colonies developed into callus and 78% develop into transformed shoots that 

showed nplII activity (Jones and Davey, 1991). Elnbtyogenic callus tissue from various 

cultivars of Aracliis liypogaeu was used for bioiistic-mediated transformation. Callus from 

mature seeds, escape free selection on liygron~ycln, brief osmotic desiccation followed by

sequentlal subculture on cytokinin medium are the salle~lt features of this protocol 

(Livingstone and Birch, 1999). A novel method for transfor~natiol~ of Cajo~lus cajatt via 

biolistic bombardment was developed uslng a vector contailling heterologous oat arginine 

decarboxylase cDNA that is an Important gene ill polyami~ie metabolisni. An increase of 

putrescine levels was found in the transgeliic lines (Sivarnant et a]., 2001). Genetic 

transforn~ation of Pi~aseoius corcineris and P. v~ilgiiris was done using novel nylon micro 

projectiles. Ethanol co-prec~piration niethod for DNNpartlcle preparatio~l was superlor to 

that of ~a'*/s~ermidine (Genga ct al., 1992). A ~iovel method of electric discharge partlcle 

acceleration was used to transform seed l~ieristenl explants of Phusrolus vlgaris. 0.03% of 

confirmed transformed plants were recovered (Russell et al., 1993). 

Orher ntetho[ls. Electroporation, microinjection etc. are sonic methods of genetic 

transforn~ation of plants that were used less oftcn. Mesophyll protoplests of Cajutius cuju~i 

were elctroporated wit11 pias111id construct conta~ning 11p1ll gene as selectable ~narker and 

the transgenic callus was produced. Tlie transforn~ation freql~e~~cy was observed to be 30% 

(S;ira~lg~ ct :I\ , 1991), 

2.2.2 Forage atid pasture legumes 

Legunlinoceae is famlly of over 18,000 species, which has a distinct economic 

importance by virtue of processing protein rich seeds and ability to fix atmospheric 

nitrogen through symbiotic nitrogen fixation. Over 70% of the legumes are forage and 

pasture legumes (NAS, 1979). Many domesticated and wild animals feed on these forage 

legumes deriving most of their nitrogen require~nents from this class of pasture legumes. 

Owing to the magn~tude of their importa~lce several groups carried out studies on

ioteclinologicsl i~iiprovemel~t of the same so as to prov~de better food for the 

donlesticated anltnals. 

2.2.2.1 Orga~~ogencsis 

Llirecr orgai~age~iesis: A complete in vitro plant regeneration syste111 was 

developed for nzuki bean, Vigils migiiiuris wllerc advent~tious regeneration was observed 

from hypocotyls segments of cotyledonary node explants by using BAP (Avenido and 

Hattori, 2000). Elhylciie inl~ibnurs such :IS sliver nitate. 2,5-i1orbun1ad1ene 2nd cobitlt 

chloride were shown to enllailce the orgallogenic frcqucncy from the cotyledon explants of 

cowpea, Vigila ur~yuicuiulo (Brar et al, 1999). Regeneration wus achieved uslng 

hypocotyls and co:ylcdonary explants exc~ssd from green ~nln~ature pods of cowpea 

(Vigi~rc uiiytiicuiuru). A primary polyani~ne putrescine was also used and some son~aclonal 

variation was also observed (Pellegrineschi, 1997). Morphogencc potentials of shoot 

regeneration from root explants of Loius coriricuiurus were exploited to regenerate whole 

plants. Explants were ~soli~ted fro111 3 different parts of the root from 4, 8 and 16-day-old 

seedlings and buds were ibr~ned on the proxinlal end of explants, and roots on the distal 

end. Explaiits located proximally regenerated mow shoots than those orig~nat~r~g from the 

distal end. In the presence of BAP the ~nu~llbcr of rcgcneratcd plants was higher because 

numerous meristi'matic zones formed in the secondary cortcx. In contrast to the explant 

response oil horoione-fret: medium, disturbance of explant polarity wcre observed in the 

presence of BAP. (Rybczynsk~ et al., 1995). The pie-lrealrne~lt of immature inflorescence 

with phytohornlones, especially BAP, and culturing on horrnor~e free medium, resulted in 

shoot bud induction and shoot dlfferentiat~on, of an inlportant pasture legume Medicago 

lupulina (Li and Demarly, 1995). Preconditioned multiple shoots were obtained by

germinating the seeds of pasture legumes, Lutlt,~~us ticrra, L, oclr~~ris and L, sativus. Best 

nlultiple shoot frequency was obtained on ~ilrdiuili contain~ng 50 pM BAll (Malik et al., 

1992). Mulliple shoots were induced born slioot tip explan~s of iii vitro grown seedlings of 

iWacror)~io~~~ii ii~iijluriri~i. Effects of ;tdclllne sulfatc. BAP and IBA were studied 

(Varisaimoliamcd ct al., 1999). High frequency regeneratio11 of adventitious shoot buds 

was observed in Pi~~ilit suti~~ir~ii. Silver nitrate did not sllow :my promotive effect on 

nlultiple slioots but resulted in slloots \vttli ucll-dcvelopsd tetldrtls and large stipulcs 

(Ozcan et al., 1992). Young i~lflorzscenccs of Cerutu~iic~ silic/uu were cultured 011 MS 

mediuni supplenlerited with BAP with various concentrations of casein liydrolysate fbr 

obtaining multiple shoots (Bl~alerao and Chinchanikar, 1992). Luthyrlis sj~lvestris (flatpea) 

is an important forage legume especially in acid~c soils. Hypocotyl derived multiple shoot 

systeni was developed for the clonal propagation of this species (Foster et al., 1991). All 

efficient micropropagation metllod was developed for Scsbo~licr rostru~u by opt~~li~z~ng 

parameters 11ke variations in the basal mcdiulli and pilotoperiod changes (Pellegrtneschi 

and Tepfer, 1993). Mult~ple shoot rcgenvrurion was observed fro111 Immature seedl~ng 

explants of Lupiriu~ ~riu~ubilis by using TDZ with modified Schenk and Hildebrandt 

lnediulrl (Rah~m et a]., 1999). Complete plants of Lupi~rus iutetrs were regenerated from 

hypocotyls segments and were eficielitly ~nodulated by Brudyrliizobiu~n sp. (Dam and 

Chamber, 1993). A~~tityilis cytisoides, a legulne shrub used for aforestation and reclamatiori 

was successfully regenerated fro111 both juvenlle (cotyledonary nodes and apical buds) and 

mature (axillary buds) explants (Gavidia et al., 1997). 

Indirect orgunoge~~esis: Plant regeneration was achieved via callus phase using 

hypocotyls explants of an irnporta~it forage legume Astruguius udsurzens. The

combinat~ons and concentrdtions of different growth regulators such as 2,4-D, BAP and 

NAA were sl~own to br crit~cal factors for both the frequency and \he type of callus 

format~on as well as for the potential of callus differentiati011 (Luo and Jia, 1998). Petiole, 

stem, lzaf and cotylcdonaty explants of Medicoyo saliva werc used to induce callus by 

ustng 2,4-D and whole plants were regenerated (Moursy et al.. 1995). 

NAA in 

combi~iation w~th BAP was used to regenerate plants via callus phase by using stem, rach~s 

and leaf explants of beach pea, Lurliyr~rs jcipuriicrrs (Debnath et al., 2001). Callus, 

organogenesis and plantlet fornution was observed from seeds of Clltoriu renlutru in the 

presence of high kinetin and IAA levels (Lakshmanan and Dllanalakshmi, 1990). 

Canavanine and canaline were detected in the callus cultures induced with BAP and IAA 

in Cariavuliu lirieum (Hwang et al., 1996). Shoot buds were regenerated either directly or 

through callus phase from leaf explants of fodder legume Vignu ocorti~i$olin and various 

factors affecting regeneration were also studied (Bhargava and Chandra, 1989). Four 

species of Srsburiiu, S brspirrosa, S, caniiubiria, S. l;'oniiosu and S, se~buii were 

regenerated in vitru by using root, hypocotyls and cotyledon explants. Callus was induced 

with 2,4-D and shoots regenerated with BAP (Zhao et al., 1993). Callus was obtained from 

mature leaves, stems, petioles and roots of young seedl~ngs of Psoruleu corylifolia and 

regenerated to whole plants on BAP contai~ling n~edium (Saxena et al., 1997). Hairy rools 

that were induced by infection wltll A.rl~izoger~es were used for the regeneration of 

rnulr~plc slloots In Crotolureu juilcea followed by colrlplete plant regeneration and 

confirmation of their transgenic nature through Soutllcrn blot analysis (Ohara et al., 2000). 

Seventeen accessions of Medicagopolyr~iorplicl were screened for their capacity to produce 

callus. Hypocotyls proved to be the best for the regeneration of whole plants via callus

phase (Scarpa et a]., 1992). Direct and indirect nlultlple shoot regeneration was reported in 

the winged bean, Psophocorprrs tetrugo~~olohris. Shoot tip, epicotyl, hypocotyl and 

internode explants were cultured on Ms rnedia supplelnented wit11 different concentrations 

and combinations of BAP, NAA and IAA. Plant regeneratton was acllleved from internode 

and hypocotyl explants via direct orga~iogenesis and fronl internode and ep~cotyl explants 

via indirect orgallogenesis drpend~ng on tllc gsuwti~ regulators (A~~julll;iilara et sl., IYc)X). 

Callus derived shoot regeneration was achieved from root explall~s of Lutilyr~a sor~vus by 

using various concentrations of kinetin and rootillg was done on IBA (Roy et al., 1992). 

Macrufiiiurn uirupurpureuni is a ~uodei plant wit11 broad synlbiont range fbr ~lodulatlon. 

First report of in vitro plant regeneration 111 th~species by using hypocotyls expiants was 

reported by Ezura et al. (2000). A novel rnethod of ind~rect organogenic regeneration was 

developed for Pisuni soiivirn~ using thin cell layer segments of nodal explants I'roni which 

leaflets and axillary buds were removed (h'aue1.b~ et al., 1991). Indirect regeneration of 

shoots via callus phase was obtained In paature legu~lles Lolo~~urlls Darrie~ii (Bovo et al., 

1986), Cer~iruse~rla brusilionurr~ (Angclo~u et al., 1YY2), Desr~totliur~i uJJine and D. 

unciirofu~r~ (Iley and Mroginski, 1997). Various explants like hypocotyls, root and leaves 

of pasture legume Slyloso~rfhrs g~lyane~~~is were used to regenerate shoots via callus pha~e 

(Meijer and Broughton, 198 1). 

2.2.2.2 So~natic enlbryogr~~esis 

Five species of Metiicagu, M. ciliuris, M. ~r~erc.~, M. orbicuiaris, M. polyniorpha 

and M, truncuiulo were assessed for somatic en~bryo induction with 2,4-D. Incorporation 

of PEG resulted in better maturation of the embryos (lantcheva et al., 2001). Callus was 

induced from stloot-tip cult~ires of eight species of Tri/diurn and whole plants were

egenerated via somatic eliibryogenesis of T. rubens (Parrot and Collins, 1983). Direct 

somatic embryogenesis and plant regeneration was obtained from protoplasts of red clover, 

Trifoliitm pruretrse (Radionenko et al., 1994). Plants were regenerated by the direct sonlatic 

embryogenesis from the cultured embryos of genus Trijoliu~i~ (Repkova, 1991). High 

kequency somalic enibryogenes~s and plant regeneration was achieved from the callus 

cultures of Asrrugulus udsurge~ls (Luo el al.. 1999). A protocol for regeneration vla 

somatic erlibryogenesis pathway in the pasture legume Cliloria iertiuieri was developed. 

Manipulation of kinerill in combi11at~o11 with IAA was found to be usethl (Dhanalaskhnl~ 

and Lakslimanan, 1992). Methyl jasmonate and Abscisic acid were used generally for the 

maturation of induced soniatic embryos. However, their application in the soniatic embryo 

induction medium was tested and they were found to be inllibitoly to tlie embryo 

induction. Somat~c enibryo induction in Metliccigo sarrvu was also fol111d to be Inhibited by 

ami~ioetlioxyvi~iylglycine, amino-oxyacetic 

acid, 2,4-dintrophenoi and salicylic acid 

(Meijer and Brown, 1988). With a view of obtaining plailts free lion1 neuroloxin, a qulck 

and efricielit system was developed for r.egeticrating plants by using explents from wide 

range of tissues of Lailyrur sciiiviis. An embryo rcscue method was also described to 

fiicil;tate inter-spec~tic hybridizations (Misra et al., 1994). Immature cotyledons of Vignu 

sinensir gave rise to somatic embryos on 2,4-D and BAP-containing medium, which 

eventually developed into whole plants (Li et al., 1995). Treatment with 2,4-D followed by 

BAP treat~llclil reger~ct;itcd whule plants vla ;III exuberant sonlatic embryogenesis from 

leaf sections of h.letliccigo ~ujjrrtricosn (LI and Denlarly, 1996).

~$2.3 Other metltods 

Protoplasts were isolated from immature co~yledons of G/yciile soja. These 

protoplasts started to divide after 3 days of culture and the division freqtle~lcy of 

protoplasi-der~ved cells counted at 12 days was 36.8%. Slioot buds were regencrated on tl~e 

surface of the nodular celluses with a frequency of25% \\hen tlic cnlluses werc placed OII 

MSB medium with IAA and BAP. Wliole plants were regencrated uiio~i transfcr of 3-4 cm 

shoots to 50% MS mediu~ni witli IDA (\\lei n11d Xu, 1990). A brcnhtl~rougli In the pl,lnt 

regencratlon from tile protoplasts isolated from Vicia jrrba and V. tror6oire1r~i.s. I'rotupl;i,i~ 

of 10 cult~vars were isolated l'rom etiolated slloot lips and tested for their regcnc~~lt~o~~ 

capacity. After purification, protoplasts were embedded in sodiun~ algitiatc and cultured 111 

the medium contait~ing 2,4-D, NAA and BAP. Division frequencies of up to 40% werc 

obtained. Six weeks afier cmbcdding, protoplast-dcrivcd calluscs were transfcrrcd to 

Gelrite-aoiidified mcdin wit11 different colnbinationa of groivtli rcgulalors (Tegeder et al.. 

1995). Low voltage treatment and nurse cells from Medicngo ~crriva were used to 

regenerate callus from protoplasts isolated from seedling aiid suspcnsio~n cultures of 

7i.i/olirii11 slrb~errri~~eii~ir (Li et al., 1990). A protocol Tor the isolatiorl of root proloplasts 

from Vigi~ri rridiara and leaf nlesopl~yll protoplasts of ~niotlibean, V cico~ritijolia gave a 

plating efficiency of 1.3% and 2.81% respectively. Shoot mcristemoida devcloped o~lly 

from mothbean, into shoots and later lrlto ivholc piants (Avenidu et al., 1993). Genotype 

dependent protoplast ~regencration into wlloli: plants was observed in red clover (Trfiliioli 

pruiense). 

Protoplasts were derived from leaf and suspelision cultures of the culi~var 

(Myers et al., 1989). Cull suspension cultures were grown and plantlets were regenerated In 

Indigofera 

eni~eapl~ylla (Bharal and Rashid, 1984). In vitro conditions for plant

egenerat~on from protoplasts and callus cultures of Hedysarirnr coro~~ariur~i were 

optimized (.4rcioni et a., 1985). Protoplasts were ~soiated tkoni ~~icsopi~ylls of Mcdicago 

sari\,a and various phytohormoncs tested for a better frequency of regeneratloll (KIIII and 

Cho~, 1989). Frotoplasts of Lolus coniiculurus were ~solated us~t~g pre-plasn~olysaiio~i of 

green cotyledo~i, in CI'W salts containing 13% iliann~tol. Plantleta were also regeiicrated 

w~tll two lines being somaclo~ial varlanta (Vessabulr atld Grant, 1905). Callus and 

protoplast CUI~LI~SS were used to Iregenerote ~)ln~lilcts ol"Wedinigu and leaves of hleilicagu 

Iirroralb, an anlllial legunic reslstat~t to li~llgl~s l's~'i~i/op~'~i~n ~tii'di~~giiris. I'lalltlets wcrc 

regeiicrated 011 niedium co~ltai~ii~ig 2-IP cornbi~icd wit11 IAA, andlor DAP w~th NAA (ZaSar 

et al., 1995). Pla~lt regoleration fium cotyledo~l p~oloplasls was achlcvcd in Meiiicagu 

saiiva cv. Krnsnovodopadskaya 8 by culturing cell aggregates on agar tiiedium aftcr tlic 

immobilizat~on of protoplasts in agarose. Regeneration was also achieved from tissue 

cultures of tile wild species M. prosrrara, hi, urbiculuris, M. rruurverleri, M. borealis, M. 

cueriilea, M. rigiduila and M fulcnrn (Svanbaev, 199 1) Cotyledon protoplasts served as 

useful tools for regeneration of Sesbatiia bispi~losa. 111 a liquid-over-agar culture systetn 

with MS ~nediutii supplemented with 2,4-D, BAI', glutanline and mannitol, 84% of these 

protoplasts divided and formed callus. Callus fosn~ed from the protoplasts differet~tiated 

shoots on MS medium supplemented wit11 IBA arid BAP. These slioots developed into 

complete platitlets when excised and cultured on MS containing IBA (Zhao et al., 1995). 

Protoplasts der~vcd froin 3 species of Siylosarrrl~es were cultured in K81' medium at 

densities of 5 X 1041t1iI and I X 1051tiil I'rotoplast-derived colonies tratisferred to MS 

tiiediu~n suppleme~ited wit11 cot~ibitiatiot~s of NAA and BAP formed compact, green 

microcalluses. Shoot regeneration, which occurred after 28-56 days of culture, was by

organogenesis rather than somatic embryogenesis, with leaves and stems developing 

directly on the callus surface (Vie~ra et al., 1990). Protoplasts were isolated from epicotyls 

and shoot tips of Vicin ~tiirboriersis etiolated seedl~ngs. They developed into plantlets vla 

somatic e~nbryogenic pathway when cultured on less auxin medium, arid via organogenic 

pathway when cultured on TDZ contai1111ig medium (Tegeder et al., 1996). 

Cotylcdotlary explants of Sesba~~~o grci~itlifilicr were irrad~ated with ganlnia rays for 

callus growth and regeneralion. Cytoge~iet~c studies showed dist~ilct cliro~~iosomal 

aberrations (Sinlia and Mall~ck, 1993). Enhanced shoot regeneration was observed us~ng 

homogenized callus oELoius cort~iculaius (Orsbinsky et al., 1983). 

2.2.2.4 Genetic translormatioo 

4' 

Agrobocieriir~n-mrdiated traiistbrniation: A rapid and reproduc~ble protocol for 

transformat~on of Lorirs cornicirlaius by using cinilamyl-alcolid dehydroge~iase (CAD) 

was developed for the purpose of Iignin reduct~on. The transgenics were confirnled by llie 

polymerase chain reaction (PCR) and CAD activity. The gene was derived fro111 Amliu 

cordura (Akashi et al., 1998). Tllrec cultivars of Medicc~go surivu and one cultivar of 

Onobtycl~is viciijoliu were evaluated for their response to inoculation witli A, rliizoget~es 

strain A4T (containing pRiA4b). A cultivar-dependent response was observed in M. suiivu 

with 94%, 25%, and 4% of infected stem explants producirlg transfornled roots in the 

cultivars Vertus, Rcgen-S, and Rangelander, respect~vely. In 0, vrciqoliu cv. Hampshire 

Giant, an cxplant-dependent respolise was observed with 78% and 50% of seedling 

cotyledon and hypocotyl explants responding, respectively (Golds et al., 1991). An 

accession-dependent genetic transfornlation was observed when Glycine cunescens and 

Glycine clarrdes~it~u were transformed with A, rllizogenes (Rech et al., 1988). 

The

transformants were identified by sylitllesis of opines in the shoots. An important fodder 

legume, Astrogalus sinicus was transformed by A rhizogrr~es atid the uidA gene activity 

was confimied (Cho et al., 1998). The regenerants exhibited Ri-plasln~d syndron~e (sn~all 

thin lcaves and short internodes) arid 55% of tlle seedlit~gs diowed GUS activity and 

positlve gene integrations. Genetic transforniation of tile pasture legurne and the model 

plant for study~ng Rllizobilrn~ legume symbiosis, Medicugo truncorulu was reliably 

transforti~ed wtth b~nary vector harboring ~iptll and rriclA and NOS atid CaMV 35s 

pronioters respectively. The TZ geileration of the platits showed Mendelian inheritance of 

the integrated transgenes (Wang et al., 1996). A rapid regeneration system from 

cotyledotiary node explants was used for stabbing and injection co-cultivation of 

Agroboclcri~r~rr, In Pisu~ir sutivunr (Jordan and Hobbs, 1993). Genetic transibnnatron via 

somatic embryogenesis pathway with immature leaflets and l~odal explants of pasture 

legume Lutlryr~rs sarivus was reported by Barlia and Mehta (1995). Mature de- 

enibryonated cotyledons with intact proximal end of Yigrto rorgrritulutu were used for 

Agrobacreriu~?~ transfortnation. Over 15% of the shoots were selected on selecttoti 

medium. 

Iritegration of Itpt gene was detected by usi~ig Southerti blot analysis 

(Muthukumar el a]., 1996). Ri plasmid atid dlszir~iied 1 i plasmids were used to transfonn 

Mediccigo trnnccrttrlo. Genes of Ri plas~iild negatively ~nterf'ercd with 

soinatlc 

embryogenesis. Only Ri plasmid with an inactivated rol A gene regenerated transgenic 

plants (Thomas et a]., 1992). Loitis jupoilicris is a ~iiodel plant for Rhirobium host 

interactions. A. rhizogenes mediated transformation was perfomied and hairy root 

formation was observed. Most virulenl strains A, rliizogenes for this species were also 

found (Stiller et al., 1997). Lorrrs cor~riculatus and L. lenuis were transforn~ed wtth A.

l~izogeites for the integration of 11pt gene (Daniiani et ai., 1993). An efficient protocol for 

A. rhizogerres transformation was developed for Lorus attguslissimus. This was receded by 

an efficient regeneration systerli of indlrect organogenesls using hypocotyl, leal; stem, 

cotyledon and root explants (Nenz et al., 1996). Bean yellow rnosalc virus rcsistenr 

transgenics were obtalned by tr;~~lsforiiia~~oi~ of 7i.ijoiiiiiri subterru~rerirrt with vorlous 

segmellts of virus coar protein (Chu et al., 1999). Rapid and efficient trdnsfomlation of 

.I4etl1~.cigo ii.ri~rctririh~ i~nd ,M. ~ci/i~,n w;i> tlo~lc \\,ill1 twu ciiily iiodulin and latc nodul~n 

genes where pl;ilit rcgrncratlon occ~irred tlirougl1 somatic e~nbryogelies~s (Trinli et al.. 

1998). Two different regeneratioll systems were used to obtain transibr~iled plants of 

~Medicagu fulcaia. Tlie A. turnefacierrs ilioculated plants were regellcrated via direct and 

indirect somatic embryogeliesis pathways (Sllao et al., 2000). Mrdicago trtiricatulu was 

tralisfoniled with A, iiirr~cjhciens using put selection marker. Trailsforn~ants could be 

obtained In just 2.5 months (Trieu atid Harrisoii, 1996). 

Biolisfics. Plasmids containing 11ptI1 gclie under CaivlV35S promoter were used for 

particle bombardment experiments into Lucerne calluses derivcd from petiole and stem 

sectiolis of Med~ccigu srrlivn. Analysis of Irailsge~~ic plallts showed ~ntegrntion while 

progeny showed I: I Mendelian segregatioli ratio (Pereira and Ericksoli, 1995) 

LM111sect resistance nlallagement 

World-wide crop losses without the use of pesticides and otl~er non-chemical 

control strategies is estlniated to be about 70% of crop pruduction, amountillg to US S 400 

billion. The world-wide pre-harvest losses due to insect pests, despite, the use of 

insecticides is 15% of total production representing over US $ 100 b~llion (Krattiger, 

1997). The annual cost of insect control itself amounts to US $ 8 billion, thus warranting

urgent econoniical control measures. Many of tlie crop varieties developed in the past 30 

years were high yielders, but had poor storage characteristics (Kerin, 1994), Insect pests 

are capable of evolving to biotypes that can adapt to new situations; for instance, they 

overcome the effect of toxic materials or bypass iiatural or artific~al plant reslstance, w111ch 

fu~ther confounds the problem (Rousli and McKennc. 1987). An iiltegrated pest 

nianagenient (IPM) prograni, con~pris~ng a combination of practices including tlie 

judicious use of pcstic~des, crop rot~t~on, lield sanitation :ind abovc all ex]>loitation oi' 

~nherently resistaiit plant varletles would prov~de the best option (Meiners arid Eldcn, 

1978). The last option includes the use of transgenic crops, expressing foreign insecticidal 

genes, which could ~iiake a significant contribi~tion to sustailinble agriculture and tllus 

could be an impo~tant coniponent of IPM. 

Insect reslstance nlanagelilent is iiot a concept evolved from a single method of 

operation, but a11 integrated approach, as opined by niany others. Classification of 

methodologies has taken a different turn with advcnt of bioteclinology. Biotechnological 

control conceptually is a part of biological control nietliod, however, separated itself from 

other methods owing to vast literature and logical base it has acquired. Hence this sectlon 

describes all the tliethods other than biotechnology, which have been an integral part of 

Integrated Pest Manage~nent 

2.3.lJEcological 

control: 

This is a strategy in which various methods are employed. It is somewhat similar to 

the "biointensive" control described by Frisbie and Smith (1991) that rely mostly on

natural biological controls with prescriptive chemical input as last reson. The strategy is 

primarily based on ultderstandilig the interaction of pest with i(s environment, in the 

present context, agro-ecosystem, defined as the effective environment at the crop level 

(Altieri, 1994), or at thc level of local lalidscape (Duelli, 1997; Collins and Qualset, 1999). 

It is defined that an insect become5 a pest when general equlllbrium populatioll exceeds 

econonlic lnjury level (EIL) (Higley and Pedigo, 1996). Exploitat~on of species divers~ty to 

decrease Ihe herbivore population was done et'li.ctively (Altier~, 1994). Biodiiersily 

increase in agroaosystems results ill di.\~cloplirent of tilore inter~ial links with111 the food 

webs resulting in fewer pest oulbreaks (Altieri and Niclrolls, 1999). These understandings 

led to some practical approaches such as poly harvesting (Pral~cis, 1990) and interplanting 

(Alticri and Whitcoinb, 1980) are potent~al eco-control mcnsurss. 

2.3.1.2 Pliysicnl control: 

J 

Tile methods in lliis strategy are relatively ancicnt, but stood for ayes, as they are 

farmer and eco-friendly while bcing easy-to-use. They requ~re leaser scientific kriowledge 

and are based on physical destructioli of inscct populations. These havc attracted 

considerable attention In the recent past because of the development of effective food and 

visual attractants (Lindgren and Fraser, 1994). Understanding the insect behavior is a 

crucial prerequisite for making traps (Alm et al, 1994). Usage of baits such as pheromones 

and novel trap designs made insect trapping devices attractive tools in the industrial point 

of view (Dowdy and Mullen, 1998). Usage of some sort of banding material especially 

arourid the trunks of the trees had some degree of antagonist effect on insects (Raupp et al, 

1992). Live stock insect traps (Tozer and Sutherst, 1996), colored traps (Vernon and 

Broatch, 1996), arid fermentation traps (Norris, 1933) are some of the effecttve

applications of traps. Technically more advanced lllethods such as light traps (Pickens and 

Thimijan, 1986), electrocuting traps (Gilben, 1984) micro ii~adiatiolls (Biron ct al., 1996), 

gamma irradiation (Metcalf atid Metcalf, 1993) and otlier irradiations, temperature, 

coiltrolled atmospheres with COi ~n combination w~th other gasses and varlous othcr 

physical methods designed at the coliverilelice of the user and (ype of insect pest are being 

widely used In various industr~ol and domestic envll.onmcnts. 

2.3.1.3 Cl~e~~~icill colitrol: 

., ' 

Use of chemicals to fight Insects, dates back to 1200 BC. This is thc most popular 

but cco-unfr~tndly strategy. Global market for insect~cides was estilnated to be over 20 

billion in 1989 (Environnlent Protection Agency, 1989). Wide varietics of i~isecticidcs 

were discovered due to the~r instant resul~s despite their adverse effects on thc ~ntegrity of 

the ellviron~iient. To name a few, orga~ioclilorilles (DDT, BHC), orgaliopliosphatcs 

(Carbophenothion), Carbarnates (aldicarb, carburyl), pyretliroids (nllethrin, cyalotllrill), 

averniectins are some of the widely used insecticldes. Many plant based insecticides have 

been used for ages, such as pyrethrin, rotenone, azadirachtin etc. Keeping as~de their 

popularity, ~nsect~c~des usage is highly alarnling as they negat~vely affect many of the 

living things living in their respective ecosystems. These concerns have bccn published by 

many authors, emphasizing the adverse eficts of the insecticide use (Rand, 1995; Edwards 

et al., 1996). Their effect on soil microorganisms (Domsch, 1983) microorganisms of the 

aquatic environments (Parr, 1974; Curlley and Robinson, 

1989), soil inhabiting 

invertebrates (Edwards acid Thompson, 1973), liematodes (Yardim and Edwards, 1998), 

alukatic invertebrates (Brown, 1978), fish, amphibians, birds (Hardy, 1990) and many 

other loving components has been a point of serious concern. Despite many precautions

ased on these reports, an estimate of over 1 rllill~on people worldwide is poisoned, and 

over 15,000 eventually die. 

2.3.1.4 Biological co~:trol: 

Various pest control strategies llavc bceu developed where each one provldlng 

partial answer to the singular or a sct of pest problems. 1540s witnessed d~scovery and 

application of wide array of chemical pesticides and people thought a "panacea" lias 

evolved for all pest problems. Th~seemed true till it was ioglcally d~sproved firstly by 

Cars011 (1562) and was repeatedly e~npllasized by various authors In the follow~~~g years. 

For example, an estimated 7% of crops were destroyed by inscct pests pr~or to 1940s, and 

by late 1980s crop destructio~i has risen to 13% despite a 12-fold increase in the pesticide 

use (Environmental Protection Agcncy, 1985). These alarming statistics divertcd tile peat 

management strategies to opt Ibr age-old control practices such as phys~cal and biolog~cal 

meiliods. 

Definit~ons of biolog~cal control var~ed through ages, and one of the latest itlcluded 

the use of predators, parasitoids, pathogens, pheromones, and natural plant producta. 

According to this defi~litiorl biotechnological approacli becot~les an integral part of 

biological con~rol. Oldest known method was colonizaiio~~ of ants in china and Yemcn 

(Coul$on et al., 1982). There are three bn51c types of biological control, na~nely, 

conservation, i~ltroduction and augmentation (Waage and Mills, 1992). introduction of 

natural enemies beco~lles a necessity when tllere 1s a sudden outburst of foreign insect 

pests, which are tnot native to ;I particular region. In such cases their natural enemies are 

imported, multiplied and introduced (Waage, 1996). Augmentation is increasing the 

natural enemy numbers from the existilig populat~ons, by producing and multiplying them

in the laboratory and releasing tl~en~ (HoiTniann et al., 1998). Pathogells used for biological 

control include bacteria, fungi, viruses, protozoaus and nematodes. Mass production ant1 

marketing of these agents tire relatively easy co~npared to tlie otller two (Cook et al., 1996). 

Over 700 species of fungi were bel~eved to infect Illsects (Jaronski, 1997); for exan~ple. 

Beauveria bussianu is a potentla1 biological cotitrol ~igcnt of many arthropod insects 

(Maddos. 1987; Georg~s, 1992). The mctliod of lis~~~g bacteria for b~ological co~itrol I> 

relat~vely donlin;~ted by Uncillii~ //iir~.i~igie~isis speclcs and tlie S-endotoxins twlenacd by 

them, molecular genetlcs of the resistance ~n~eclla~iis~n lias bee11 the order of the day 111 tile 

recent bioteclinoiogicai approaches. 

2.3.2 Biotechnology for illsect resistance 

1 

One ofthe methods of b~otechnology, m;~rker assisted selectio~l could not cater to 

the inirnincnt needs of the IPM specialists, as resistance phenotypes and their QTLs could 

not be conclusively ide~ltitied. Hence there was a need of short cut methods to combat the 

worst biotic constraint, lnsecl pests. Discovery of 6-endotoxins produced by the famous 

Bucillus liiuringiettsis gained considerable sig~nficance in the recent past. Along with U 

thuringiensis tnany other genes conferring resistoncc partially have bccn discovered out of 

which protease inhibitors are of significant importance. 

>:3.2.1 Ut: All A~nazi~ig Concept: Pcrhaps n~ost widcly used entomopathogen is the 

bacterium BuciU~~s thiirinyiensi~ Berliner. Many sub-species of th~s bacter~u~n release w~de 

array of larvicidal S-endotoxins that confer resistance to various insects. Other species of 

Bacillus, l~ke B, spliaericus (Porter et al., 1993) and some anaerobic bacter~a such as

Closrridi~r~n (Barloy et al., 1998) were identified to pocess potential mosquito larvicidal 

properties. 

Bacillrrs ihuringiel~sis subsp. isrocliet~sis (Bti) was the first subspec~es of Bt that was 

found to be toxic to Dcptcra larvae (Margalith, 1990). It was also foulid to be effcctive 

against many species of mosquitocs and black fly larvae. Extensive srud~es wcre crimed 

out to demonstrate the toxicity of these orga~iisnis towards nianinials and it was not foulid 

to be highly host specific thus ensuritig its s;~fcty (Murtliy, 1997). 1r was also Iprovcli to 

have an expanded host range sucii as snails, sonie liunia~l ;~nd avian parasites and few othcr 

insect pests (Saraswati and Ranganathan, 1996). 

Tlie toxin pruteins rind ilreir ge~~e.s: Thc fanlily of irlsccticldal crysral proteins (ICPs) is 

iiomally associated with larger plasnllds of various sizes rangllig Ilct\ceeil 5 to 210 

kilobase pairs that liave been broadly class~fied as Cry atid Cyt 6-c~idotox~ns(Lereclus et 

al., 1993). Though these toxins are liot related structurally, tliey arc fi~nctlonally related in 

their membrane permeating activities. All 

insccticidol 6-cndotoxiiis are initially 

syntliesized as a larger protoxin that is eventually cleaved at specific s~tcs to form the 

actual toxin. In total there are seven classes of Bt insi.:ticidal proteins, but four proteins 

and few accessory proteins and their molecular genetics associatcd hitb Bti are well 

characterized. Tlic major four proteins arc locallzcd in pnrasporal crystlillinc cndotox~iis 

sy~itlies~zed during sporul;itio~i and tliey arc sylltllesized by rcspcctivc genes: B~Ct~l,lo, 

BfC~jllA, BiCry4B ant1 HtCry4A (Fcder~ci et al., 1990). Much of the ~nosquitocidal 

properties is attributed to the synergistic i~iteract~oils of these four proteins, but the wh0k 

crystal was found to be niucl~ niore 10x1~ (Crickmore et al., 1995). Vi'ealtil of Information 

was accumulated regarding the molecular biology, biocliemistry and ~truclural biology

have been exhaustively reviewd (Dai and Gill, 1993; Douek ct al, 1992; Wirth et al, 1997; 

Guerchicoff et al, 1997; Purcell and Ellar, 1997). Two accessory proteins ~iarncly PI9 and 

P20 that arc requ~red ill tile usse~nbly ol'a~i i~~clus~o~i body linvc also been cliaracter~zsd 

(Wu and Federici, 1993; Tlliery et nl, 1997; M;~~i;isiierob et al. 1997). 

Mode oJuctio~~: Early studlcs showed that the prunary target of Bti toxicity is tlie 

niidgut epithelium, where e~izynialic systems tra~isfornis the protoxin tiito an active toxin 

under alkaline coi~ditions [Al-yahyaee 2nd ElI;ir, 1995) Tliese toxins act coordinately and 

synergistically to distupt tlie epithelial cells of tlie larval gut where niidgut cells vacuolisc 

and lyse (Lahkim-Tsror et al., 1983). Tliese sym)~tonis are more or less snnie for toxins of' 

all Bt strallis other than Bti. Though Cry and Cyt toxi~is are structurally dissi~n~lar, tiley 

have the similal. ti~embrane-pem~eatirlg ab~lity, where Cry toxi~is bind to nicmbranal 

protcilis and B~Cvtiilu binds to ulisatt~ralcd pliospholip~ds acting as "bind~ng sites" 

(Feldriiann et ai., 1995; Gill ct al., 1992). 

Mode of action takes place in two steps; binding to a cell reccplor and subseque~it 

pore formation (Knowlcs and Ellar, 1987). Soon after conversio~i of proto-toxin into active 

toxin by gul proteases, the toxin is distiliguished illto two doiliains (Donlain I and Donio~n 

11) wherc Doiiia~~i I1 bt~tds to a brusli border membrane receptor, acts as an anchor, while 

Domain I inserts ~tsclf illto the ~iiembra~ie for~ning ;I pore (Deal1 et al., 1996; Flores et al., 

1997). Binding of the toxin beco~iies irreversible alier llie i~isertion of Donlain 1 illto 

mernbra~ie (Che~i et al., 1995). ~4 and =5 liel~ces 1nscl.t into the membrane and a7 serves 

as sensor to initiate tlie structural rearrangelllent of the inserting dornaln (Gut and Shai, 

1998). Many otlier fi~~ictio~is of seglnents of the toxin have been elucidated. It was found 

tliat substitutio~i of glutamine at 149 by prohne in the center of helix 4 results in co~ilplcte

loss of toxin activity (Uaw~thya et al., 1998). W~tll reference to cytosolic (Cyt) toxins Cyt 

1Aa was studied ill detail. It was found tllnt plasma membrane liposonles contain~ng 

phospholipids arc the target of this loxi11 (Tl~onlas ilnd Ellnr, 1983). Tox~n binding leads to 

a deterge~~t-l~ke rsarraligenieilr of the bound I~pids, re,ult~ng in hypcrtroplly disruptio~t of 

the membrane ~ntegrity and eventually cytolys~s. I'ore ibrmatio~l was observed prior to 

cytolys~s (Gill st al., 1992). The 24 kDA active toxi11 \+as lbund to be three times actlve 

tlla~l the protoxin (Butko ct al., 1996). Anotllcr ~nlcrest~ilg aspect of the ~ilode of:~c[~o~~ 1s 

that thc tux111 monolllrrs display a synel-gist~c aclio11 towards spec~fic i~isecls. D~ffcrc~~t 

synerg~st~cally exert~ng some host specific action (Crickmore et al.. 1995) and tllis ,was 

dcmonatl.;lted by cloning a conlbinatlon of cry 4A and cry I IA of Bti into E.coli (Ben-Uov 

CI :I\., 1995). 

2.3.2.2 Genes employed l'or illsect resislancc otller t11a11 Bt: 

Protease Inllibitors: 

The p~.oduction of transgenic crops has seen rapid c~dvnnces during the last decade w11h the 

commerc~al ~ntroductio~i of Br tra~isgenics However, the n~ajor concern with tllese crops 

has been the developmellt of resistance by pest and public acceptabil~ty, Hence, there has 

been a need to discover new effective plant genes, which would offer resistance and 

protectio~l against these pests. Protease i~ihibitora (Pls) are one of the pritne candidates 

with highly proven inllibitory acrivity against insect pests. 

Pla111 proteitse bshibilors: Tlle possible role of protease inh~bitors (Pls) In plaill 

protection was investigated as early as 1947 when, Mlckel and Stand~sll observed that the

larvae of certaln Insects were unable to develop l~orli~ally on soybean products. 

Subsequently the trypslti inhibitors present 111 soybean uere shown to be toxic to the larv;ie 

of tlour beetle, Tri6oiilr111 co~rfirslr~~~ (Llpke et al.. 1954). Following tliese early studies. 

there I~JYC been riiany exa~i~ples of protcase ~~~l~ibrrors active against certalli inscct species. 

both in ~n vltro assays agalnst insect gut proteases (Panneticr et al. 1907: Koiwa et al. 

1998) and in in vivo artificial diet bioassays (Urwi~i ct al. 1997; Vain ct al. 1998). The tcrrn 

"protcase" includes both "endopeptidases" and "exopcptidases" wlierc:is, thc tcrlii 

"prote~nabe" 1s used to describc only "endopeptidases" (Ryan, 1900). Several lion- 

lloniologoi~s farnilres of proteinase 

inhibitors arc recognized anlong tlie animal, 

microorgaliisriis and plant kingdom. Maijority of protei11;ise rnhibitors studicd in plarlt 

kingdom originates fro111 three main fiimllies liarnely Ieguminosae, solanoceae and 

graniineae (Ricliardsoli, 199 I). 

A Iiirgr 11u111ber of protease rnliib~tor gc11i.s wit11 distilict niodes ofactioli liavc beeti 

isolated from a wide range of crop species. Drvelop~ne~it of tra~isgenic crops lia\,e coriic ;I 

long way from tlie tirst transgeliic developed by H~ldcr et al. (1987). Considering tlic I11gl1 

cornplex~ty of proteaselinhibitor interactions ill host pest systems and the d~versity of 

proteolytic elizyliies used by pests and palllogens ro liydrolyre dietary protelns or to cleave 

peptide bonds in more specific processes (Graham et al. 1997), the choice of an appropilate 

proreinane inhtb~tor (1'1) or set of PIS represents a primary determ~rl;int In the succcsa or 

failure of any pest control strategy relying 011 protease inliibition. Firstly, tlle choice of 

su~table PIS should be based oil a detailed understanding of the biological system assessed. 

Resistant biotypes of insects may evolve aAer prolonged exposure to selection pressure 

that is medlated by an insect~cidal proteln or plant resistance gene (Sparber, 1985). Seco~ld

point to consider would be the targeted expesslon of Pls ill response to pest attack. Tills 

will be controlled by using inducible plolnoiers, sucl~ as !liosu of PI-1185 al~d TobRB7, tllat 

are act~vated at tlle site of lnvaslon by pests, pa!lioge~l atid nematodes, respectively 

(Oppemian et a1.1904). An ideal promoter should be highly responsive to ~nv;~sion of tlic 

lios! plallt by ;I pest, or rcglilated by i11ducel.s just prlor to pest attack. Tlic promoter should 

be sufficiently active to mediate 8 sub~tal~t~al defenbc, spscinlly loculiiecl to tile site of],cat 

usefulness of reconlbinont Pls in plant protection still re~l~ains to be dcmonstriltcd 

111sect resista~~ce genes other tllnn Cry cl:tss of genes transferred to crop species 

Bean 

I ----- 4 

Snowdrop lect~n-GNA 

I 

Cereal 

I 

Pea leclin 

I 

1 1 

I 

I 

Protra~e ii~/ribi/ors 

Wllrat germ ;lgglutinin-WGA 

Soybean (serine protease) Jacalill 1 

I 

I 

F 

Barley (tryps~n) 

- --. - -- 

lllcc Iectin 

1 _ 

1 

. . ' 

I 

Cowpca (trypsin) Bcan cliit~r~nse ! 

I 

I i .-I 

I Mustard (ser~nc protease) 

Tobacco peroxitlase 

~ 

I 

i Rice (cystelnc proteuse) 

Tomato chitinase 

I 

I 

Potato (protease inl~ibitoss 1 2nd ,I) Tlyptophan decarboxylase i 

I 

Soybean (Kunitz trypsin inhibitor) 

Atibi~ul gcties 

'I I 

Torneto (protease ~nllibltors I and 11) 

Various enzyme inh~b~tors

3.1 Pl:~nt 111nteri:il sllrl culture col~ditiul~a: 

hfatorc hccdh ut'cliiclq~c~~ (('I~~,I,~IIC,/IIIIIIII 1.) CLI~II\,I~ C.235, ,I !\~cIcly gro\\~i ~LIIIIV;IS 

111 Il~di;~, ncrc s~~rl';~cc s~cril~~cd NIIII ~(I'?U (1 '\) c111,11iol (or 1 1i1i11 , U loo I~~CI~II~IC ~l~lor~(lc for 

10 11111i . ii~i~l r111seci 5 l1111es 111 h~ct.11~ d~>tillcd \v,~ti.r ~IILIS 10 ~U,II\III~ u~erl11g111. The dc-~ii~ted 

5ecda \rere Lcpt iir~ gcniii~l,~~~oii 011 hli~~;lzll~gi' ,111d SI\OU;'> (IOO?) I~IC~ILIIII (\IS: we 

;~ppe~i(i~\ I), cir 011 ;I II~~L~ILII~: q)ec~licci tiir tllc cyil,~~il \>I~~>\IKIIIOII ('LIIILIIC ~i~ccl~;~ nc~c ohcd ;I, 

liqul(I or ;IS 

scln~-,olld Iho\111g 0.8'io (\v \) I)ilku-lj;~cto :I:I 

:I rcq~~~r~d 

dilcl 1pII \\;I\ ;iiljusted 

to 5.8 i~lilcss o~licr\\~sc ~iie~itio~ictl. All llic ~~hsui. cultuscb \\cl.c I~I;IIII~~III~C~ ;II 20ilYi' u~idsr 

contlnuou:, cool irl111c l~glit ~proi~dcd by lluo~~c~cci~t I;iinp\ (30 11Lln 'S I)

!Chrirlre e~!ib~~,o rr.ri~: Tl11s call be cunsidercd :IS 

\verc soaked uicril~glit ;iiid tile sscd co;it \\,is ic~ilo\cd IC 

tile O-do)-oltl accdlilig. Thc seeds 

i'illo\\ III~ I~IU~I~III~. Tlic eo~yledo~~~ 

\+ere bplit ope11 JII~ IC CIII~I:)~ ;I\I~\!i~\ 

ici~~o\c~l c:~~cliill~ ,111cl CLIIILII:~~ 011 11ic ciiibryo 

~~~cilictiu~i 1iicd1i1111 (Elhli 111:11 cunsiatcd of I0 11hl 2.4.T-1 :ind 2 1111 killc1111 

Synthetic ourci~is. 2.4.5-T and 2,4-0 iirc lllc ba\ic co~iipoi~cii~~ ui' il~e enlbryoge~lic 

indiiur~uii media. All tile 111cdi:i were re,ted 

un ~lintilrc cniblyo akis eul>lalil li~r opli~i~izatio~~. 

The ~~iducrio~i ilicdi;~ were tiiu type!, 01ic 2.4.5-l' based cilitl llie olhcr 2.4-U based. Tllc 

lornicr was iiariied ;is JEJI scrics niid llic lalcs \\a> ilamcd na JIIM serlcs. 111 JLIhl series tlic 

2,4.5-T \+\.as :idded ;it 2. 5, IO ;ind 15 phl :~nd in cach tscntniclil ~ar~able COIICCII~~B~IOI~S or 

TDZ at 0.5 and 1.0 phi. BAP ;it 0.5 ;!lid 1.0 j1J1, Lcnrlii ;II 0.5. 2 O ,ind 5.0 phl nlld kinetin at

0.5. 2.0 and 5.0 pM were added. 111 JDM series. 2.4.1) was added ;it 5. 10. 15 and 20 pM and 

III C~CII trcatmcnt \:irl.lble collceliil.nrloii> ol'TDL ;it 0.5 ;III~ l .O libl. BAl' ;n 0.5 a11d 1.0 pM. 

zcat11i ;it 0.5, 2,0 ;iiid 5,O pM iii~d LIIICIIII 

;it 0 5, 2,0 ;IIILI 5.0 pbl ucsc ;iddcd, 

Bsst iiied111r11 tior III~LIC~IOII of I~I;IYIII~LIII~ 11ii11lbcr 01' C\PI;IIIIS \V:IS JtS \\irIi I0 hihl 

2.4.5-'r :iilil ? lib1 hiticti11 (J1:Jl 20: T~blc 42) !\I1 tllc c\~)~,II~Iz ~ C Z C ~ I ~ ;IIJO\C 

C I ~ \$CI.C 

CII~ILI~C~ 011 JllXl 20 II~~~~ILIII~ I~I tcd 111cir el'lici~~y lus II~~LICIIOII ~I'e~~ibr\o\ '~IIc IIIC~I~II~I \\;IS 

~~rc~).~ri'ii eill~c~ 111 ,I jolid 01 II~LIICI ii)r111 SUII(I IIIC~IOI~I \\:I\ ~~OLII.C(\ III tlie ~~DIII pl;llc\. ;III~ tile 

induction w~th liquid n~ctl~i~ni \r.:~s dolie 111 cullilrc ~iibc\ I:or 

tlie Iincr ;I \lcr~lc lilter p;lpcr 

bridgc was 1111l11cr\cd III tile Iiq~lid illcdi~~il~ :iiid 1\11' CYI;I 

\\ere pI:~ced 011 1111' brlilgc. 

Sitice moat of~l~r cl'fons lor inaturatloli and coil\crsloii of tile iirduccd cl~ib~yus were 

iinsucccssful soille dddl~iun;~l ~nccIion~c;il ~iictliods \\ere ;ilbo rcstcd. 

55

Cold shock: Tlir explants benr~ng the eli~bryos \\.ere pl;iceci on MS containing 5 pM 

ABA and 10 1iM GA: and the pl:itcs were pl,icctl nt 4 OC tbr 1. 3. 5. 7 2nd 9 hours on frcsh 

~iicdiu~ii \\ it11 5d11ie coiilpo~~c~i~s. 

Heo! .\/rock. The expl;~l~tbcar111g c11lbr)oz \vcrc pl;iccd o~i 1111. II~~~I~III~ 

;12 I~ICIII~OIIC~ 

iibove ;111d \\SI.C IIICLI~;IIC~ :I\ scl);~s:~te O,IICI~CI ,II 40 'C' :ICI 5U 'C' li)r 30. (10. 9U ;111d 120 

IIIIIILI~CI 

fiill~\\i'~i by 111~1s II.;III~~;.I 10 fi.esI1 II~C~ILIIII iil~d IIICII~~IIIOI~ 111 IC 

CLI~III~~ 1.00111. 

Type ut' cu[)l:1111 i, cl.i~cl;rl Il~clor lcir tile :icl~ic\c~lic~lt ol au~t,~blc ~rcgcllcl.;ilio~i ibr 

gc11e11e ir~~~isl~r~~i,~t~u~i 

~Y]>c~II~~~III> 

and proccsslllg cond~t~ons were P~CI);ISC~ 

\',I~I~LI~ 

~XIII;II~I~ IYo~ii >cc(ll~~ig~ 

01 il111k1e111 ,igc, c~~lturt 

;iltriiir.c o~rbl.i o ir.uii Thc scctls \vc~c ~oahed ui.cr~ugl~[ ‘inti llic sccil cui~t iraa rclnovcd 

rile follo\+,~lig nior11111p 'Tile cutylcdo~ir \vcsc j)I11 opeli :111d the cll~bryo :IAI\ W;IS silrgically 

exc~sed ;ind ci~l~urcd OII 111~1li1pli: IUOI 

II~~LIC~IUII lilcd~~~~ii tlii11 Ilrid hlS iv~lli BAP wltlliti il 

mnge of 5 lu IOU pM III co111b11!;111ufl \vllh ? ul 5 jlL1 klllcl~ii. 

Sirool tip Tile dc-coaletl seed> \rere pcsl~i~~i;~lcd un L1S fur 2 dtiys, [he slloot tip (2 

mm) \\as tiie~i cxclscd susgicolly 2nd culti~rcd on 111c shoot III~U~IIOII 

~ni~diulii (SIM) that 

co~lslsted of hlS UIIII 4 pV TDZ, IU KM 2-IP and ? ubl kinellli 

56

Lec!/i?is: Young anci juve~iile leallers, semi-iiiaturi. leatlets ti.oni thc ni~ddle portion of 

the seedli~ig and ~ii;~tiire leallcts ti.otn tlie bilsal p;itls of7-day-old scedli~ig were sep;lr;~ted and 

cultured oil SIM. 

Lrq Dii~e. 

Tlie dr-co:itcd scuds wcrc pcr~ii~~i,~tcd u~i hlS lbr 7 d.~yb 11r1or to obt;111111ig 

tile leal'cxplti~it. Lcal'b;~sc (pct~olc base) II~;I ~iic~~stircd ;~but~t 3 111i1i W;I~ C\CI~C~ ;111il c~~lttircd 

on tiit SIM. 

//ip~~~o(~/: The orcr~i~giit so;iked ;il~ dc-co;rlcd jccds \vcrc eriii~~i;i!cd oil MS and 4 

to 5-day-old seedlings were bclcclcd for dcr~v~iig tlic ~~I~;IIII 'Tlic stelii 11;11t that was just 

abo\e tile cotylcdon,iry liotlc jt~~iclio~i iv~~h csc~vtl a~~tl 111s sliout 111) rugion was rcniovcd. Tlic 

explant tha ~iieasurcd :iboui 4 iiim \v\.;ls cululred on SIM. 

Epicu~l: Tile ove~.nigIit soohctl and dc-co;~tcd scccis \+ere pcr~iii~i:ilctI on MS and 4 to 

5-day-old sccds \\ere stlccted ib~. obl;ri~iil~g tlic c\pl;~~it Tlic ~~ly)er niost Iparr ol'lhc tap root, 

just below the cotyledoii:rry iiodc jt~~ictiu~i \+;I\ exc14 and tlie cxpl;i~it ~iicasuring 4 mm 

devoid ol'roor 1111 rcglon was cult~~rcd on the ~~iductioti ~ncdiu~ii. 

Roo, A?~I?I~,II/.Y "1111 I.UIJI /I/). Root \egliielilb ;111tl root lip MCSC ehcibcd fro111 7-day-old 

sccdlingr and cultiired ~epa~.ately on tlie i~itlt~ctio~i iiicd~ii~ii. 

A.~rIlory bi11i. T\io types axillary bud uxpla~ila wcrc prcparcd. Tlie ~iia~jor v:iriatioii for 

preparat~on of these cxpla~its wt~s their gsl-~iii~lation paltcrli. Tlic first onu, dcsignatud aa ABI, 

was prepared by excisi~ig tlie avillary bud from a 7-day-old secdli~ip hat was gerniiliated on 

hlS medium. Tlils nx~llary bud ~iieoaured around I to 1.5 mm In leiigtll. Tile second one. 

57

des~gnated as AB2, !\.as prepared by excising the ax~lla~y bud ti0111 a 6 to 'i-J;~y-old seedlings 

tliat were ger~~linated on MS conr;~ining 5 pM tliid~i~/~~ron (TDZ), This nxllla~y bud nieasured 

approx~niatcly ? to 3 nil11 

('r~iilc~~li~~ilr~~i~ 

liori, Tlle oicr111g11t ~ooLed ,~nd tic-co,itcd 5ci.d. 

\\ere gcr~i~~~i;~tcd on 

11s. 3-dnyold scedl~~ig was t:1ke11 ;\lid tlie slioul t~p, root tip u~ltl cotylsdo~~s ucrc re~uovcd. 

Tlie cotylcdonasy node ju~ic~iu~i th,it ine;~surcd 3 lo 4 lii~ii \\';IS CIIIIII~C(I 

011 Slhl 

A.v~//oI~I~ 1111~1~1~1~~111 L'Y/J/~III/\' ~\~>~;llll ~~~CpLIRlll~ll ;llld tl~l~lll~lllOll 011 111~ ~Lll~~lr~ 

mediuni Is cr11cl:l~ tbr o~~lIl11lllll U1'g,lIIoQeI11~ I.L!S])OllhC. O\\lll@ I0 I~IC d~lksc~~~cs ill t l l ~ 

gesmlllatloli lp:~ttcrli, age ol'tlic hcetll~ng ;inti proccasllip. four typch of lllc ;~xlll:~ry l~~cr~slclii 

cuplal~ts were prcparcd anti they \+ere n;~mctl ;IS 11M I. Ahl?, AM3 ;111d Ah14 (see 171g 3.2 for 

diagr;i~i~lii;~~~c 1relxe%nt:1t1011 oi'e~l)l;~~n ~)scl~:~~tiuli). 'rliclr I~~L!~;II';IIIUH ii ;I\ li~llow\. 

AM1 explant: Seed coiit ol' the sust~ce stcsili~cti a~itl oicr~iiglit su;lhed seeds W;IS 

re~iioved nlid tlic dc-coatcil aced\ \+ere gcrni111;ltctl 0111pl;iiii MS. 2-d;iy-old scedli~ig~ were 

selected slid roo! portloli \baa 1c111ovcd by 1cav111g suliie oi'tlie Ih~~)ucu~~l rcyiol~ 'l'l~cn two 

cuts here 111:ide tl~roi~gh tlic ax~ll~sy ~ncr~stc~i~, 'I'his rcaultcd 111 1l1ri.e cxl,1;111ts 111;1I 111cludcd 

two axlllilry nier~steni, one hiloot tip witli cpicotyl. 'lliu iix~lli~ry ~~icr~hle~ii cxpli~~ils ~vllli 

cotylcdo~intact was na~lied as AM1 i~nd itas cilltllred on thc lnduct~oli ~iiediuni. 

AM2 explant: Tlic dc-coated secds were gsriiu~~;~ted 011 SIM al~tl ;ifIcr pruniine~~l 

appe;il.ancc ul'axlllary bi~d, in ,iboul 4 ro 5 tl,~ys. tlley were c;~reli~lly runloved and two cuts 

were given through rhe ;~x~ll;isy Iiieslstem, dlhcasd~ng rlic root ponlon, rlic rcsulta~it axillary 

mcristem explants w~th co~yledons i~itacl (A.112) wcse cult~~rcd 011 tile above-mcnt~oncd SIM 

again.

AM3 explant: This was a by-product during the preparation of AM2 explant as 

indicated above. Removal of AM2 explants resulted in shoot tip with eplcotyl region. Further 

removal of shoot tip provided [lie AM3 explont that contalned au~llnry nieristeni on either side 

of the eplcotyl and was cultured on SIM. 

AM4 explant: The de-coated seeds were gerlti~n;itcd OII SIM at ;I dclisity of 10 to 15 

per plate. They were grown for about a week ~tlit~l :inlllaiy bud was prollilnent. Then the 

axiilary bud \\as rcltioved up to tlic base two cuts \\,ere give11 as 111 the casc oTprt.pol.;~tioll of 

AM2 explont. The resultant axillary nieristeiti explant, were sub cillturcd on tlic same 

niediuni for another 6-7 days. Tile b,lsc ofaxillary bud enlarged and sotiic ~ii~~ltiplc shoot buds 

cnierged. Tlie emerglng shoot buds were reniovcd apolil by canli~lly acr,lpping the shoot buds 

irith ;I sharp blade Tlir res~ilt;inr cxplnlit corit:i~li~tig cotyledoli uitli ;I bulge on \he 

cotyiedu~iaty ~iode rcg~oli \\CIS cultttrcd 011 IIU~IIIUII~ lice MS. 

The ~iirdia tbr itiductiott ul'~iiult~ple ~Iiou!) liutil tlie givc~i cx~luttt~ ciili be d~v~ded in 

rive m;~jur cl;isscs. I. TDZ-ba~cd ;tiiil 2. DAl'-bt~scd. Thc\c conslats oScltllcl- 'TDZ or UAI' as 

pril~cipnl niultlplc slioot iliti~~ctilig cytuk~n~ii ;III~ a111c acc~s\ory C ~ ~ O ~ I I ;ilid ~ I I ~UXIIIS ~ S werc 

i~icludcd ~n tile tnedi;] ;is per tlis rcquircn~ciit. Tl~c orhcr cyt~kilillls illid d~~xillj ilddcd were 2- 

iP (5 and I0 ~IM ill TDZ-b;ised mctli;~), k~llelili (2 ;iild 5 liZ1 I 'rU%-b:~scd:ti?d 2 2nd 4 pM in 

BAP-bnscd illsd~;~ conib~n.itio~is). Optinliutlon of'tllc i~~tiuct~oli nicdi~~m was dolie initially 

w~tlt tnaturt. elnbtyu 3x1, and I;iter w~tli ,~xillnr) meristem explant, Ah12. Unless otherwise 

mentioncd. the explants were cultiiled 011 tlic 111duc1iori iiiedium Sor abuut IX to 20 days alicr 

wliicli they werc tri~nsverred to the clongat~on ~nediulti. An average of 6 to X expl;l~lts werc 

c~llt~lrcd per plate and the re,puliies werc atudlcd.

EJJkcr 

ofpH on 1~111l11ple slloor rtlditcrio~r: The shoot induction nlediuni (SIM) was 

prepared with pH mriations of 4.0, 4.5, 5.5, 6.0, 6.5, 7.0, 7.5 ailti 8.0 prior to autoclavi~lg, 

Solidificatio~~ of the ~iiedii~~~i \\as corufully ~ilon~torrd and the prepared AM2 cxpla~lts were 

culturud on [lie SIM with abo~e-nlcntio~ied pi4 v;iriatio~is. 

Role of 

co(vletlui~ni~' iis~~~r: Co~iiplete or parti;il inclusioil of co~yledon w~tll the 

regenerating expla~lt. 011 mult~ple slioot i~ldiictiun ficcjilcncy wiis slud~ed. Tlie AM2 explallts 

were prepared accord~~ig tu tlie proci.durc nisntiuncd abovc and illc cotyledon portion was 

surgically exc~sed ci~lier co~iiplstcly or pa~.ti;~lly (I~~ilt) and one set ol'c~pI,iilts were cultured 

witli inclusion of co~nple~u cotyledo~l. 'The rcsulti~ig cxpl;inta ivcrc ci~ltured on SIM witli a 

tlclis~ty of6 to 8 sxpl;~~its per 1)1;1tc. 

CUIII~)OI.II~I~~L, 

IIII~/~I/I/~, ,/iooi iiiilirti~o~~ liviii i/~/]e~.r~~ii ci.i~/coio M:il;irc c111Oryo axis. 

sliool lip, i~x~llary butl. i\hl I, AM?. AM3 011~1 Ah14 CAI)~;III~> \\ere P:c~~~I:c~ d~c(~rcliilg to tllc 

procedul-es rncntlo~icd III tllc 5ccti{1113.2 2.1 ii~ld cu1111:cd UII Slhl. Nu111bi.r uf rcslio~iding 

exl~la~lts and inunlbcr oi' I~ILIIUIIIC 

before their trunslir to tlic ~lioul clu~lp:~iiun mctliii~ii 

$110uts ]per C.X~~;IIII bere rcc~r~lud 111 lllc third ivcck just 

Age o/ [lie \cctl/iiiy: I3as11ig UII tllc ~[>li111t I>~UI):I~;I~IUI~ sIr:~Iegy, d~lli'rc~il ehplillits 

\sere derived boll1 hecdllngs of iarluu ;I~A. Ahll. Ahl!, 

AM3 a~id Ah14 cnplanls were 

prepared accord~~lg to tl~eirc,pectivc proccdi~rca ~ncn~ioncd abow konr 2.1. 6. X, 10, 12, 14 

and I6 day-old seedlings. Tlic rcsuli~iig cxpl;ints werc culiiired o11 SlM. 

3.2.2.3 Shoot elongatioll: 

The hiloot elo~igatio~i 11iedi11m (SEM) bnslcully coiis~stcd ol'lo\rcrcd co~~cc~~tration of 

plant growth regulators, %lie11 compared to tlie r~tdi~ction rncd~~~ln Various plant growth 

regulators ~ ~sed for this pnrposc werc 2-il' (2 and 5 pM), BAP (2 i111d 5 pM), kiiiet~n (2 and 5 

60

PM) 2nd GAJ (2 

5 PM), eltller singly or ill conibilinlion with c;sh otlier. Tlie induced 

nlultiple shoot buds Here caretiilly excised ti.0111 llic e\pl:in~a ;IS 

;I bu11211, rhoill ill ;11ly extn 

gro\rtlis of c;lllus or elobulnr structl~rcs ;IIILI CLIIILIIC~ 011 I~IC C/OII~:IIIOII 

111ciliii111111tl:illy ti)r 

about 10 days. They \\ere sub-culttired Ibr 2 lo 3 p;lss;iges of IU ti;iys c.icli 011 li.csh ~ncdiu~ii. 

During each passazc k\v alioo~a clo~~g,rtctI ;)lid ~lic u~~-clu~~g;itctI butis \\cw sub culu~rcd OII 

tlic lroh 1ncdit1111. Tlie bu~lch ol'~ili~l~iplc \11uo1 but15 gl.c\\ rl1i11 \\ 1111 ~cspccl 111 1111. I~IIIII~C~ OS 

\Iiuut btlds thruugll c:lcii p,iss:lgc I1rckr,1iil). tilt sli~llg;llillg sliutllr ,111il a/lt)ol l~~idr li'~lll1 lllc 

,ecuiid pass;igs \\ere cul~urcd 011 b1S \\i\li tiA; (2 1111) I.lo~~g;~lctl rliuols tu :ihuul 5 c111 In 

Tile I~~C~~IIIIII 

used \\;I CIIIIS~ III sei~i~-r(~l~il lb1111 01. li(l~~i(l li~rl~i, l'lis clo~igilcil s11oots 

\lerc ~~sctl to optinli~c lie rouliny 1lcdi;i \;I~I;IIIVII~ 

\verc IIIC~III tlic litll~id ~i~ed~tilli iib lie 

selliisolid iiicd~u~ii rcsulied 111 ;I \try low li.cquc~icy ol'root~l~g. IC I i111d 1)/1;1sc 2. DUI tllc j~lliiscs M~CI.C ~ii;li~i~,ii~~ed 111 the culture 

roo111 ~~iidi'r ;I~CI)IIC CIII I~OIIIIICI~I. I lic sht)ut\ tliill dill 1101 IOUI III bo111 pl~,~\e\ WC~C ci~lried to 

ilic phase 3 Dark grceil 

lic:~ltlly \llooir ol'oroilnd 5 ciii lcilylli \\c~.s 1dc;il lor ruotlnp, TIC 

rootable slioots wcrc ci~ltiircd ii~ cult~irc ti~bcs (!Sx!OO 

IIIII?) ~~I~~~IIIIIII~ 

lil~cr 1);1j)er bndges 

immerscd I liquid roo( ~nductlon mcditlm (Itlhl) lliat consi\tcd of MS MI 

~nod~licd lcvcls 

of KNOl (9.4 ~IM: 11,lll'of the CUIIC~I~II.~IIIOII 

111 \IS) Il3A \v;I~ lillcr ~~c~III/c~ a11d added at 5 

pM. Wliilc 60 to 805; ~1'111s cloi~garcd >tloo15 roo~ctl In thc pli;iac I tllc slioc~i, (>8 clii) devoid 

of roots were canied on to phase 2. ljiicli shoots were briclly dippsd ill filter stcrili~cd 

solution of 100 pM IBA and placeti 011 lilter paper bridge in cuhure lubes contain~ng 

hormone-free liquid MS. Effect of half MS and MS devold of ;~ny growth regulalors and 

61

effect of varlous co~iccntriirio~is of NAA (5 31iJ 10 ~IM) a11d IBA (5 and 10 yM). Effect of 

sucrose on rootiilg \vns stlldled by add~ng s~icrosc at 0.0. 1.0. 1 5.2.0. 2.5 and 3.0% v,Ilile all 

llic other co~lstit~~c~its ol'RIM 

groivlli rrguliitor 111 lllehe ~IL'~I;I \\;IS 

~ii;ii~~tii~~~cd S:III~L' :IS II~CIIIIOII~~ ;~bo\,c. Tlic root lliducing 

IBA illitled ;it 5 li\l cullcellrrntlon. 

tllis sl~p 

IS :I 

Rootirig 111 ilii, lii~(liu/~oiiii.\ .\I \/ciii Tll~s aptciil 15 Icl~llcti ;I> pliiihc 3 oi' ruut111g ; i~~d 

O~IIUII;II o~lc .\boil1 10 lu 2O"o ui'tlic rooi;~blc al~oota. \\l~icl~ tlid ~iot root cveli 

ii1ii.r ! lo 3 aub-ci~ltliri.\ un IKILI ncrs C,I~~ICL~ to 111~ II~~IO~)UIIIC \)\ICIII tll,it \\;IS ge11e1.11lIy 

~ised fir 11ardv1111ig i111ri11g tile I~,III~~II,IIII~III~I~ lproce\a. 75 strc~~gtli I~~IIOII's \u11111oii \V;IS 

it1 8 CI~I Miigeiit,~ 1,it 

lillcci 

;III(I tlic 511001 \\;is s~is~)c~icIcd \v1111 ji~pliort h11cli !list I el11 or 1I1e sl~out 

basc \$ah imnlcrscd III tlic bulutiu~i 111;1t coiltn~~lctl 3 ~IM ILL\, Tlic IIIC(~IIIII~ 

\&;I> c11;111gcd every 

H:~lilci~iiig ;ind ~I~I~\~~,IIII;I~IUII proccja \+,is blu;~dli dib~dcd illlo tlllcc atagcs: akge I: 

i~iltlnl tr,inskr into X c~ii (~I~I) )pol\ \\it11 coicr (i lo IU d,~).,): \t;~gc?: ~ICCIIII~.I~I~I~IOII phase in 

wliicl~ the plniils ucrc gi;iduoIly cul)oacd to tlic a~~lb~cncc by ~plticli~ng l~ulcs and rcniovel of 

the corncrs oi'the cover (I5 to 20 days): stage 3: tl.ill~\ti.r oI'l11c ~pliilita 10 tllc 20 CI 

(d~a) puts 

and mainteliance in glnsahousc lbr fi~lllier growlli. 

I-lnrdeliinp a1:111cd u 1111 

tlic rcmurnl ofconu~~ 11lug.i ol'tllc culturc lubo for I lo 2 days. 

Tlie plants wcre careliilly t;iken ULIT ofthe liibe and rhe roo15 ivcrc tlioroiiglily washed, dipped 

ill diluted tliiram"' 

(fi~nglclde) solutiun nild tr:in\ferred to 8 cni (dia) cunr;ilnlng co;irse rand 

(2 to 4 nlm dia) as the potting 1iicdi11111. Tllcy wcre completely covcrcd will1 1r;insparent 

polypropylene bags and allo\ced to grow fur 7 to IU days. Coi~densarioi~ on tlie inner surface 

62

of the plastic bag was removed rwlce di111y. 'The plallts were cxposcd to ~lic anibiclit 

conditions gnduall) by pi~i~liilig lhules aid cu~ti~~g the cor~icss of tlic polyprol~ylelie bags. 

F~lially after 10 &)a tlic cater \\ns upelled on lop slid nllo\\ed to SI;I~ lilr ;ihout a \beck 

Ibllo\\i~ig \\llicli Ilii. plallt \\as c:irci'~~lI) lr;~~ral;.rrctlo 20 CII~ (dlii) 1put II,I\III~ tlic 1p11111119 ~riix. 

'Tile potl~rig 1111x ~UI~I~SISC~ 01'il IIIIS~LI~C 01' a~iioiitli :III~ c

3.3 Histological studies oa multiple sltoot ii~itiatio~~ I'I.OIII Ah14 e\pli~~tt: 

Preparation of Ahl4 cuplant 1s described 111 tile srctio~~ 3.2.2.1. The liistulogical 

studlea \\ere carried out ti.oni llic z~;~gc ~~llcil the ax~ll,ll.y bud \\;15 

~ciiloved. Tlic clay of 

a\illai) bud rctiiu\;il \\:\a 

co~is~dercd ;IS d~y- I The dilielopii~e~i~ ~1'hl\ll4 c\l)I;ltit \.,IS 

st~idied 

111)to Y days ;ilier the rc~~lo\.~l oS;ixill;~s) b~lcl i.c . 111) IU d,i!-X 

Pre/~iii'~~iou. linrriotr iiiid ileln~~irociu~~ ul ii,\\i~r, .\,i~rrp/c.s'Tile c\pIa~llr \vcrc ~prcp.lrud 

,111d buli)re !i\3tio11 CUI~IC~OII 

\\JS re~lioied D:I\J/ IIS~IIC of IC r~gc~icl.;~ti~ig arc.1 \\,la ,iIau 

rc~nuicd ni'ikl~ig 1l1c 5;11nple Into ;I bluck of1 to 5 II~III bide. TIIC 'i~ilpli' \.,IS 

~~i~t~~cd~;itely 

plilccd In 1ix;hiie so1~1tlo11 (see ilppc~iiii~ Ibi. co~ilpus~t~ul~) i111d ~~osccl OVCIIII~I~I ill 4 'C', l'he 

IisaIiic u;I:, d~sc;lrded ,111d IC s;~lllpIcb \\e~.e del~ydr'ilcd \$llli IO"',i, 30U'~i. 4006, 50%. ;1111170% 

alcohol scq~lentl:~lly for % Iiui~r c;icli and 1iii;illy 111~) wcle storeil 11170% ;~lcul~ul. 

Tlir S~CCIIII~II 

\r;l m;lrkctl \villi ;III ~clc~nil)i~ig nllilibcr 1'111s ~iuiiibcs W~IS kept wit11 

1Iie t~ssire block tlirouglioul procv\lng. Tlic li,we bloclc i\ilb co~ivcycd IIIIOLI~II 

a acrio of 

Ibllo\r ing aolients :I, pcs tlic sclicdulc Ibs ileliytl~,~~iu~i, clu,lrlng ;III~I p;~s;ll'li~~ ~i~liltr;~tioii.

- . -- 

laopropyl alcoliol ; Absolulc. I hour 

During ttic process 01' 

e~i~bctltl~~ig, tt~e II~SLIC block~ here or~c~ilctl so tli;~l sectiolia 

\+ere cut in the dcsiscd pl;i~ie ul' llic II,~IIC. 

'Tiru L-~liapcd nletal nluultis wcrc laid on nlctol 

plate so as lo cnclohc 3 rectangular or squ;lre yyacu. l'li~s IS 

paraftfill (58 lo 60 'C ~iieltrd p,~~.iiSti~i \\;I) 

tlicl~ parlly lilled with ~iiellcd 

LIJCL~) iind tlie tls>lle w;is plt~ccti i~i the deslrcd 

posltio~i. Tlir colitalncr xas rlicri tilled w~th nieltud p;iraffin and allowed to cool untll 

rcaso~iably tinil so that tlie set block of paraffin wilh 111e tissue car1 be rcli~ovcd f'rotn tlie 

moulds. The block was tlic tr~nirned to a ,u~tabte size atid fixed on a ~liclal ubjrct liolder. Thc 

65

lock was fiirtlier trimmed so that paraftill o~erlyrng the piece ol'tissue was excluded and an 

adequete area of the tlssile thc111g the knlfe was exposcd. Tl~e block \\:la the11 kept I'or coo1111g 

at 0 "C. 

"C'. Tllc bectiolls \\ere cut u\ilip Lclc,~ 11h1 !155'", 

111illi1 I~I~C~OIUIIIC (ICVICC. 'I.11~ ~UCIIUIIS 

ii.om the water \\err niou~ircd 011 clc,111 513s~ AIIIIC~, \\IlicI~ II;I\C 

bcc11 ari~e~~rcd \~IIII a droll 01' 

Strii~ii~~g u/ tlrt, sii111ple~ 011 r/iili\. S~~ii~iilig ol'tl~e slide5 \+:19 

~OIIC ~15illg tic~~idtoxyliri- 

Losin btairi (see Al)li~'l~iii~ lbr COII~IIOSII~OI~) 'I.11~ sl~dc COII~;IIIIII~~ IC ~CCIIOII \$;I\ s~.r~aIIy 

processed as ibllo~rs. 

x}lul I 3 111111 

Xylol I1 3 111111 

Ruliriing \$;lter 3 111111 

tlclll~llo~yllll \l~llll I? 111111. 

Wash 111 I~UI~I~III~ t;lp u:itcr I? 111111. 

Los111 working solut~un 

I niili. 

95% alcoliol 2 to 3 dip5 

9SU/" ;ilcol~ol - 2 cI1~111ges 

Acetone - 2 clia~iges 

Xylol - 2 changes 

3 m111 each 

I to 2 min cecli 

3 rnln each

The sl~des were Illell muiaited and b'icued ulldcr ~ni~croacclpc. The nuclei stained 

bluish violet and cytoplasm in ranous sh;ides of piilk. 

Ge~ietlc transfiir~~i;ii~o~i of rllc AM4 c\p1;1111> \v,i:, 

dtil~c \in b~ul~a~ic :I> \i'cll :IS 

.~~~O~~IC/~I~~~IJII-III~~I;II~~ 

111et11otls. BI~II~IIC IKII~\~U~III;ILIOII \\;I> LIOIIC liy LI\III~ 111c ~>l~~s~ii~d 

pllTL)Y:(;US-ll~t (6.7 ib) (i:lp 3 3) (\'cclor \\,IS Lllltlly jpiu\idcd b) Dr I 111!tll gellc .is aclccr:ible ~~~orker, IIII/I\ gc11c \\ 1111 ;it1 ~lllcr~~lcrsild 1111ro11 (GUS-llll) 

;is the reporter allel ;IIII~)~CI~~II~ IC~I~~CIIICC pe11e I~II. OIi~tcr~~~I >CICCIIOII. 11~11111/1/11 i111e1 iiidA 

genes \\ere under lllc rcgiilat~o~~ ul'C;1I\1V-355 proiliotcr. I\ \;illel) (11' 

\111glc ;lntI n~t~lt~plc 

cluiiil~g altes wcrc prcaclit ;it diili.relit luc;~~ioiis 011 tlic pl,i>lnid 'Tlic ; ~,~Iu~~I~I~~~IIIII 

nletllod ir,ls ~>crhri~ied by 11ai11g t\io iiii~,ir) \~CIOII, 

ii;i~i~cIj, pI1S 723:131 (I,ig 3.4) and pIIS 

737:SUTl (Flp 3.5) (bull1 the \cc~ora\vcl.c kl~itily ~)rov~clcil hy 111. 

(; Scli;lr;~j, I'I;IIII 

131otecl111ology li~~l~~utc. 

Siiaka~uon. C'ii~~id,iJ \~'IIICII II;II~OIC~ U/('~:~l:ll) ;tiid SU77 ;la 

;~grono~~~~c;illy inlpurtal~t genes real~e~tivcly. Uutl~ rlic vscton 1i;lic 111iiA r ~ 11/1/11 ~ ~ gc11es d 

ll~sed Intu ailigle UIIII 

tlrlveli by C'aMV 35s prolilotcr. Ui0.1.l:lh gcnc \v;li ilr~vei~ by doublc 

35s proniotcr where ;la SUl7 gciic ir;ia dr~vcii by \~iiglc 35s Iproilll~c;~lio~~ t11;il C~II 

operate both III E. (oii as i\cill aa ilgrubot/ei.~ii~i~ ;III~ otl1e1. at111b~1tcs aucI1 ;IS ~~i~~ltiple C~OII~II~ 

SllCS. 

In borl~ biolistic ;is \$ell as dyrohot /eri~i~ii-~?lciIinlcd procesw procedi~res tl~e putolive 

transforn~iints iverz obt;i~~~cil b) ~elccling llle t~-;iiirliir~i~n~n, using 111~111 as tile sclcctlng gcnc 

and kananiycin as tlie ant~b~otic I'or iclcctioli. Control uxplanb wurc used lo test the lethal

dose (LD-50). This cxperimel~t \bas done by ciiltilri~~g AM? expl:~nts on MS wit11 kanaolycin 

(5, 10, 15, 20,25 and 30 111glL) iiiid with viiryillg COIIC~IIIMI~OIIY ofTDZ (0. 2. 4. 10 pM) 6 to 

8 explants xerc cultured per plat 

3.4.1 Tra~~sfornintio~~ by biolistics nlvtl~otl: 

C:I~I~FIC 

111:1s111id ~sol;it~t>~~ i111d I~LI~I~~C~III~II 13 IIIC Ii13t '11~1) LI I>IO~I~II~'I 

111vt11od of 

tt.ansforil1a1io11 iblloi\etl b) CU;IIIII~ oi' IC pl:~s~ii~d IJI~IU tllc I~I~C~UC;II.~ICI.'; ;III~ ~III~I~,I~~IIICIII 

uf tile c.xpIo111s wit11 tl~c n~icruc~~s~icrs. DOIII~;I~~II~CIII C\CI~I\ 

\\c~c I)CI~~IIIIIC~ 

by I~IuI(:I~ 

1000/llc PIIS systcln (IjioI~~s~)il~~dcd III IUO 111. (; 1'1: 

EITrA) bi~l'lkr ;111d\\ah I,cpl VII lee l'or 5 111i1i 

(Cjlucow-Tris- 

3 200 ILL lyals b~11'ii.r \i.,ls iitldctl: ii~bc \v;~s illvcrtcd .;c\cr,~l IIIIIC> to IIIIX IC CUI~ICII~S 

lcli for 5 nlin on Ice. 

3. 150 ~ IL 5 31 polu,s1~1111 ~cciolc u;~i otl~lcd, \crrlc\ccI ant1 lcli UII I ~ lilr C 5 111111. 

5. The seaelion m ~xt~~re cenrrifilped Ibr ilhuut 5 111111 at 14000 rpnl ~III~ the supernata~ll 

wo, traiisli.~red to n ft.csli tube (care \$as 1;1kc11 nut to c;lsry okcr tlic prccipilate or lloatl~lg 

ll~,llcrlal).

6. The bupcniatant \\,I,, t,ikcli illid [IIC IINA \\;IS prcc~pit~~~cd \\1t110.8 iol. (400 IIL) of 

isopropanol. The I ~!I~~LII~ \$:I$ l~ll~\~tld to slatid ;it roo111 tcnip. Ibr ? Inln and ce~itrili~ged :~t 

12.000 1q>t11 for 10 illill :I! routii te~t~j>cs,~t~~rc 

7. The pellet \\:11 u.i,lied \ill11 icc-cold 70"'. ctll:~nol. LtIi,~~iul \\;IS 

rc111o\cc1 (by cIc~;~~lt~ltio~i 

or ~S~IKI~IOII) ;111cl 111~ pcllct \\,I, 

:III-~~I~L~, 

8. Tllu pcllel N;I~ dls~olicd 111 3U-50 ~IL \\,IIc~ ur Sl: S h~1111.1 (11 \Iniulil ~111110111 

approsi~li,ttely I 11g;10 ~tl-), fliis I)Nr\ C~II b~ t~\cil lur rcstrict~u~l ;III~I~!\I~ or Ii)r l

4, ~IICI~L)I)LII~~IC~C'~ 

\\ere J~I'IIL'IICLI by SI)IIIIIII~S 1;1r 5 \C~OII~\ III ,i ~lilcr~li~ge 

5, The IIC]LIIC~ \\:I> rc1110icd ~11111 ~Ih\c;i~dc(l. 

6. The l'ulloi\~l~g slcph \\~rc 

re~)c,~led 3 111111's: 

;\, i ml ofs~crllc iv,llcr \\,IS 

b. Vor~cscil Ibr 1 111111. 

.~ddctl 

c. The p;lrllclch i\cvi' :lll~liictI 111 hclllc hr I lllll1lllC 

ti. 

Mic~opnrrlclcs iicri. pcllcltcd by spln~li~~g lor 2 ,ccu~id~ III ;I 1111cr~)111gc 

e. Llijiiid waz rcmovc~l and dl\i.:inicd 

7. 1 1111 sterile 50"iu ~I~CCIOI ivi~s ,iddcd lo bs111g llle 1111ero IS:ICIC CUICL' I.,IOI 10 00 111gl1. 

8 Tllc ~n~sropa~l~clcs v,el.c aiorcd ,II roclnl tcl!lpcr'ul~lre li)r 1111 lo ? wccRs. 

3.4.3.2 C'o:~ti~lg I)S.\ u~lttr ~i~icror;~rricl"is 

Cuat111g uf UNII onto Inlcrocarrlcrh \\a\ dune by u~111g tl~c pt.o~ucoi ticicloped by 

5:111ford ( 1993). 

I The ~lllcrucarlieliic~c \u~lc\ctl li~ 

d~\rupr ;~gylo~iicr,~~ctl P~IIIICIC~. 

5 IIIII~LIIL'~ (111 'I I)/;IIIUII~~ 

IUIICYC~ 

2 50 p1. (3 nlg) ul IIIIC~II~LI~~ICI? iicrc 1;1j\e11 11110 ;I I 5 1111 IIIIC~O~~I~L: lube 

3 lVl11lc vol~ex~ng \~goruusly, lkc IbIloi:i~~g \+c~c ;iddcd III oidc~ 

I. 5 1iL DNA (I LI~}II.) 

11. 50 111. C;ICI 2 (2.5 Mj 

I~I. 20 111. apunliidlne (0.1 Mj 

4, Vo~tcx~~~g 

wa> cunl~nuctl for 2-3 ~ilirlulsz 

111 rcs~~\[)elld il~ld

5. Microcal~lers were allowed to settle for 1 111111. 

6. Microcarriers \\ere pellcttcd by sp~iili~iig hr 2 sccuiids ill ii IIIIC~UI'U~I' 111be. 

7. Liquid was relllo\ed and dihc;lrded. 

S I-IU 111. 01 70'; ctI~;r~~ol \ins ;iddcd \\II~ULII dl\t~irht~lg tll~ pellcl 

9. Llquid \\;ij renloicil ;III~ di\cilrticd 

lU. l4U 1iL of IOU"; 

etIl,lilol \\;is ,idilcd \r~tliuut d15turb111g 11111 ~pcllci 

1 I, L~ilu~d \+;15 re111oi cci ;111d tl~s~;~rclc~l. 

12, 48 } ll~ 01 IOU0" c1l1.111c1l \\$IS ~lllll~~ll. 

13. Tile pcllcl \v.i, gellily r11~~1~j)cticIcd b) i;ij)/)i~~g tlie .\liIe 11I'tlic iubc \c\,cr;11 li~llcs, illid tllell 

by vorlcxlilg ;it low apecd lbr!-3 

\CCOIIL~~. 

Si\ allquuta 01.6 pi. c,lcli ul' ~n~croc;irricrs \\ere collcclcd a~rd triln\li.rrcd 

to tile center ol'a 

~ii;icroc:~rrlcr. Equ,il ;III~O~II~I~(500 big) ol' ~nicriic;irr~crs c;icli IIIII~ ;iiiiI 10 rpl.cad cicllly over 

tlic cc1itr~11 I el11 ctr~ 11li11c :I\ ;I g10i11) \\ IIIIIII ;I di;i~i~c~cr 01' I ,5 111~11 Thc la11iiii;ir 

hood consislillg ol'll~e biollsl~cs gene g(111 ibil\ 1l1i110~1gIlly CIC~IIIC~I \~ilIr IOUUh ethnnol. The 

petri plate colltaii~ing tile cxpla111\ w;is placed ;II ;In ciplxupri;lte d~at:~nce li.oln tl~e device 

consistllig of rbe macrocarricr, 

The explat)ls iwrc bo~ilbardcd with the lnicrocarriers by 

applyllig appropriate pressure ill tlie r,ingc of000 lo I 100 psi. Sprcadilig oftlie iniicrocarriers 

was ensured and tlic eupl;l~its \\ere sepa~ited atid \\ere culrurcd 011 shoot induction nlediuin 

(SIM) containing h.1S with 4 pM TIIZ, 10 pM 2-IP and 2 pM kinctili. They were incub;lted

for about 3 to J Jaya alid \\ere sub-ci~lturcd un the SIM consisti~~p 01'25 nl&'L kanaiiiyc~n for 

about two weeks. The rcgcncratlllg ahuot buda \$ere clilti~ird on tIu slioot clo~igst~on iiiedium 

(StMI j. I~t;~tetl cells \bere s~~apc~~dccl 111 25 1111. 01'~1crilc 12 MS. 1'111s CIIIIUI.~ was 

dcbcribed ill tiit prcviuii~ \ectlun (ace 3.2 1.1) Tllc cxl)la~ita 14c1.c bl~elly dipped llito tlic 

dprobiic~rc~riioli culture poured 111 rlie petri pl;~lc Ibr 1 to 2 xc. 'fhcy wcrc cultured in MS 

niedlunl co~it~llii~lg 4 /IM TDZ. I0 phl 2-11' ,ind 2 pM kitletin (SIM). The crpl;~lits wcre co- 

cultl\ated iiitli llie bacrcrl;~ lor 18 IU~I, 

;III~I Mcle CLII~LI~C~ VI MS ~ncd~uni cunt;~~ni~ig 250 

mg'L ceibtaxlme 

Tlic ontib~ouu celbti~n~~ilc bras uacd to tcrlnlniitc Ihe growth of tlie 

agrobscterial cells, ll~clusio~~ of cctbtax~nic in thc culturc nicdla co~it~nued fbr I lo 2 passages 

72

till the growtli of bacterial cells \Val complet~ly tcrn~in~ted. Tlic cultl~red explaits were 

maintamed in the inon-select~on induction nlrdli~nl tllal d~d 1101 co~lsibt of kanil~nycin, for 

clbout 3 to 4 diiys. TIIC l'\jild~~ts \\ere tlle~l trG~nsl;.rrcd lo tile MS IIIC~IIIII~ 

co~lti~i~ling 25 III~IL 

k;~noniyc~n i111d III~II~~~IIIIL'~~ tbr OIIC \\eck. TIICII the c\l>l;i~~t> \\ere CLIIIII~C~ 1111hlS III~~~~IIII 

COIII;IIIIIII~ 

50 1113 L /\~II~~III~!C~II ;III~\\ere I~I;IIIII;IIIIC~ liir ;10o11t 7 10 lU d,iy\. 'rile slloot buds 

~III~VIII~ CIS bi111c11ca \\ere ~;~rc(i~ll! ~C/I;II.:IIC~ liulil tl~e CUI~IC~OII~;III \\ 1111 >OIC bi15;ll ci~lliia 

IIII~IC~ i~~lJ IIIc! !\CIS ~ri~~~>li'rrciI 10 111c 51111111 ~I~II~~III(III IIIC~~I~IIII C~IIII~IIIIIII~ 5 11hI 2-11) :III~ 2 

pb1 ~III~IIII (SEhll) i111ti 75 1n1g'L Lc~~~,~~li! 

CII~ Ali~~r LILI{I IU ~I,I)\ 111~ICIII~;IIC~ :IS \$,ell 21s 111c 

~~~i-clo~l@,~tcd sllootb iicre tr;~~~sli.rretI to tile slioot clongatioll iniedii~n~ 111.it ~olltaincd MS \vilh 

2 pM GA, (SFhl2) ,~nd IUU m$L L;~I~;~III~~III. 'She ~~n-lr;~~~slurn~cd >11oo1~ICLICIIC~ i11 every 

sli~gc oi'tllc III~IIC~I[III 

,III~ ~ICIII~~II~UII Tile ~CICCIC~ \IIUUI) 

III~II \\CIC g111\1111g II~;II~IIII~ \vcrc 

carci'i~lly separntcd liom lllc elotipotii~g slioot builcll :lilti irere cuhl~rcd ill the root inducrlon 

II~~~~IIIIII \\IIIc~~\\;I, 

~~re~i,~red \VII~IOLII 

;I~~III~ tl~c \CICCIIUII ;IIII~~~~IIC ~~III~IIII~CII~ i~\ 

kil~~ili~iycin 

\ 11u

sectior~ :uid c\l~bl~blird pln111s \\crc t~.i\libfcrreJ IU 1111' !U C I (d~a) POIS fur lilslll~r growtli 2nd 

malntenalice, l'heac pl;llits ,Ire lr.rii!ed ;IS 

p~~t,~ti\cly tr,i~~sibr~~~ed pl;i~irs. 

200 ILL (or autliclcllt to d~p 111s iia>iic) of assay Iiilxtlirc w;ih acldcd to tlbcuc aaniple 

(sections or dlscs 01. strips) and vaciluni ~nlilttated for 3-5 ~iii~iurcs. Tlic {anilile was iricubated 

74

;I! 37 'C for 3-24 Iiours 111 d;irL. Tlie iias,iy 1111~t111.c \\.;I\ 

rc~ii~\ed ;i~id tlie ~ I~SUC \\;IS 

cleared 

otT chloropli)Il by accji~c~lt~:il cli;~~~ge~ III 70-Ili(i"~ ctl1;111ol LII~II~ llsallz Iiod 110 cliloropliyll. 

Allcnioli\eIy, for ditticillt tu clear tissuc ;~tid 75"" I;ictic i~cld \vnr iltidcd .ind ,111tocl;i\cd tbr 15 

mlllutcs. This gl\c> ;I \cry good 111idge Cur ~)IiuIugr~~~Iiy Tlie tia5~1~ \\'I> IIIULIII~C~ III gIyce1.01 

illid ob\er\ed ~lnJer a illlcroacope 

~rail.dcrreti Ilno 30 1111 ILI~S 

2 I5 1111 ot'e\~rac!~o~i t~i~l'lbr :~iicI I ,0 1111 oS20!o SUS \vcrz ;i~l~Ic~l, hli,~kc~i n~cll for IU scc 

;i11d lilt lubes werc i~icub~tcil a1 05 degreca Ibr I0 111111 

3 5.0 1111 5hl I'OI~I\~ILII~~ ~~CCIJI~ \L;I~ ;~ddcd. ~ ur~c~etl :III~ IIICLI~~I~C~ :I( U degrees Ibr 2U 

llllll 

1. hlo\t Iprutclllh il~i~l lpolyiic~Ili~ride> ~CIIIU\C~ iiltli llie IIIIOI~IIJIC li-tluilecyl ,ultiitc ppl. 

5 Tlic snnlplc \\:15 ce~lt~~~i'~~gctl ;it 25000g ibr 20 111111 ;III~ lilte~cd lllru Miril cIol11 illlo 30 

nll tube contalllilig 10 ~ nl ~supropn~iol, ~ll~xcd aiid IIICII~~IICLI ill -20Y(' for 30 111111. 

0. U\A \\;la precip~t;~led '11 2OUUiJy Sur I5 111111 illid ~IIVCI.III~@ tlie lubes 011 piipcr towels 

tbr 10 111111 dried pellct. 

7 Tllr pcllct was rcauspended ill 5UU ~IL 'I'L 8 111id IU ILL of' IKU;lse A solution was 

added (5 mdnil). 

8. The samplc w;ls i~icubated n\ 37'C for 3U 111111. I vu1 (500 jiL) PI~uIIoI: ~hlorohr~n 

nllxture \\as added, lii~xcd \~goruuhl) ;III~ cc~ilr~S~~gcd ;it 12,000 I~II~I /br 2 mill. Tliu 

7s

SII~)~I~XI~VI~~ \\,la t;~hen (;I~~UCOIIS plldst) alij I 101 (COO 111.) ~I'cIIIo~~I~~)sII~ \\as added, mixed 

and cc~itnf~~gcd ;11 I!.OUU Il1m lor! nili1. 0.1 yo1 (50 111.) of3hl soclil~iii :lccl;itc (pll 5.2) \,;IS 

nddsd to tlic rllpeni;ltelir ;111d lllc DhA \\,I> prcc~p~tnrcci \r111i U.S ~ o(400 l 111.) ~soplops~iol. 

Tlie s;~~ilple \\:IS 

nllo\\d lo slalid ;II roo111 te1111) 1;)s ! IIIIII 

;111d cc~i~r~li~gcd $11 12.000 ~11111 hr 

10.15 111111. Tlie supcr~~.~lc~ir \\;IS rc~lio\tti ,111cl IC 

I~CIICI \\,ir \\;~\l~cd b11cl1) \\ill> ice-cold 

XlUu CIII,II~UI. 'I Ilc lpcllc~ \\,I\ :~~s-elr~cci 11rio1 to cli\~ol\ ills IIIC l)h 1 III ?i!l!-.~OO 111, 1'1;. 

Gc~ion~lc [IN/\ of piir,ilivc ~r,~~islbrni;in~\ ol'cllicLpc:~ \

PCN for 111cl.4 ge!iiJ: I'C'I< tbr ir111.A 3c11e \ins dolie \rith Iractloll colld~tions of initial 

denaturation of 94 'C for 3 111111 (one cycle) ;111d c:icIl cycle o1';111iplili~;1t1011 \villi steps of 

dsli,ltl~l.;~t~on (04 'C for 1 liiilil. :IIIIIC;I~II~~ (57 'C1 (01. I 111111). ,111d C\ICII>IOI~ (72 'C li)~. 1.5 

~~IIII), i111d CA~~IISIUI~ ('2 'C lilr I , IIIII~) lur 30 C!CIC~ ;III~I .I 1i11.1l C\IL'II'~IUII ;II 72 'C li)r 5 111111 

ions c!i'lc). 

/'('I? lo!. ijf( !.I,/.I/I gi2iii' I'CII lor Ui('r.\!.l/i CIIC 

\\ah tlul~c \\1l11 rc.~ctioi~ cu~iil~t~o~i\ ol'111111;1l 

ilci~.ilur.~~~o~i oI'04 -i' lur 3 111111 ((IIIL' 

C)CISI $111il i':~~li cycle or ~~~i~lililic~t~o~l 

\v~tIi >ISI)S 01' 

~~II,I~LI~.;I~IUII (94 'C' 

lii~ I III~II), ;IIIII~~I~II~~ ((13.1 'C' I'CX I 111ilij. ~IIICI C\~CI~\IUII (72 "C' livr 1.5 

IIIIII), 

;III~ C\ICIISIOI~ 

(72'i' lbr I 5 111111) hr 30 C)LIC~ ;III~:I li~i:~l L'X~~II~IUII :I[ 72 'C' k)r 5 111111 

(Olli. c)clC). 

P('N /vr SUT! gc~rc. I'CI< Ibr .S'/~iI gule ":I\ d011s \\IIII 

rc;iitlon ianiI~lioli\ ol' in1ti:ll 

dc~l.~tur;ltlon oS04 "C' lur 3 111111 (one C~CIC) ;III~ C:ICI~ L')CIC 01' l~~~ll~l~li~;~t~u~i 

\LIIII steps OS 

de~i,~tu~.,~t~oii (04 T C'UI I 111ill). I ~ ~ ~ ~ (5Y.U ~ e C i ~ li~r l 1 ~ IIIIII), ~ ~ 1 g ~ ~ ~ ( 1 

C\~~II\~UII 

(72 'C' lur 1.5 

III~I~). ;III~ CX~CIISIOI~ (72'C 101. I 5 111111) li)r 30 CYCIC~ i111d ;I li~li~l CXICIISIOII 

;II 72 "C' Ibr 5 mln 

(ulie c)clv). 

3.4.4.4 Soutller~l blot 11ybritlir;ltion: 

Thc ertractcd ;~nd pu~~licil I)NA ~111ckl)ea I~III;III\C 

II-:III\SU~II~~III~S used ior Suuthcrn 

blot lhybr~diz;ltiol~s \\IIII 

a specilic p~obc lulloir~rip tllc 111c11lod glieli by Soutllcrn ct al., 

(15175). 

Th~s tcclln~que w;i, i~scd to ldcntitj rlic ililcgratlon of ~iprll dnd UtC'rviAb gencs 

from plants translbr~~isd \v111i agronon?icaIly Important UiCi;ililh 81ld YU7'1 ~CIICS. 

11piII

gclle was probed uirh rlie PCll ti.ng~iic~lt 01'700 bp ,111ii Bi('r~,l:lb gclii' \\,;is probed with 

908 bp fiagmenr I'CK '1fnii.111. Tlic prube \\;IS lnbclcd w~rh tlie co~~i~ncrci;~lIy available 

:\I~PII~~s' i11rcc 1'1bel111g AII pro\ ~ddd b! :\~i~c~\li~i~ii (L!Sj\i 

Hc,\ii.ir~iioi~ o/ go~uitlil /),\',I 

ciiiil c~ii~c~o~ripIir~i~!~\i\ 

I 

Tlie ge1iu1111c DNA \\;I> ~ II~c~Ic~ 

\\IIII LIII,IL)IC 

I~!,I~~CII~II c11/y1iic 

;I, rollo\\b: 

!U 111. cliru~iiosu~ii.~l L)NA (15-20 11g1 

5 IL IOX rcalncnon bul'l'cr 

5 111. rcblr1c11011 cll/~lllc (50 \lllll>i 

1'11~ lirl~il vo!~i~ilt 111;1dc ilp to 50 ~IL \vi\l> htcr~le \\~ttr ;IIKI\\;I> i~ic~~bt~tcd l'or 

211 nr 37 C. 

2. 2 ILL oi' gel loud~ng but'li.r \.,IS ddcd lo eocii rotriciet! DNA belbre 

clccrropliorc~~s Tlic rc~tricictl DNA \\;I> ~i/c-li;~ct~u~i.ircci ill O Xu" ;~g.~rusc gcl prcparcd III 

IX TDE buffer nlollg \\111i 

5 111, pl.~s~iiitl DNA rc>rr~crcd ~1111 I:colll ;IS \I;i~ld;ird 11i:irkcr 

;III~ IS clcc~~opliu~c~ed o\e~-n~gIi~ ;I( 30 volt\ III IN TDI: bufir 

3. Tlie gels wcrc i1;1111cd tiur 20 iil~ll UIIII 

~lli~iiii~~~i br~ili~ilc ;it 1 11g;riiI bull;.r 

and tlie 

rcbtr~crcd DNA ii.ngmcnt b:inds wcre v~suali/cd on ;I UV I~o~is~ll~~i~ii~i~lur 

:illd plio~oglaphcd. 

Prui r,\.siiig titi!/ i rii~~~iltrri hloiiiiiq o/ ihc g1.i 

1 Tlic sue fi.ecrioriatcd gcrio~il~c DNA In !lie gel bvi15 covered LII~~I 25U n~bl 11CI and 

nglrated ~lii~il lie b~~oiiiupl~c~i~~l blue lurlib lo ycllo~v color (5-15 n~inutcs) tu depurintilc 

DNA. 

2. The gel was asslicd three ~inici w~rli dcnll~iefi~lized ukircr. 

3. Tlic gel was ~ncubated w~th dcnzirurarion solur~o~i (l0X gel volumc. I .5M NaCl + 0.5 M 

NaOH) for 15 miiiiltes twlcc, c;ich at room ~ciiipcraturc on a sliaker

4. Tliu pel \\as \r;~alicd 3 ti~iirs \VIIII 

dc1ii11icrali7cd \\:lter ;111d co\urud \vltli ~ieut~-dlizi~i,g 

aolutio~l for ? tinlcs 30 Iniilutes c;1?11 ( I 5 L1 NoCI + 0 5 hl l'r~s-HCI. pli 7 5 ,i

2. The nie~iib~;~~ir n;ir c.~~eli~ily ~nscrtctl 111 tlic Ii~hridi/;~lio~i tube ;III~ llic pre-llyb bufkr 

carefully ;~ddsd Tlic ~ ~~~-I~!~~I~IL~I~IUII 

\\,15 tIo11c ~LII ,111t1111 I IICILI~ t~t 0UL'C, 

3. Whlli. rlic prc-hybr~iil/;~t~w is 111 prugrcss. ~lic ~irobs \\*I\ builcd ~ br tibout IO ~iii~i. :It 

100°C [or dc~~;irurnt~oii It \\as Lcp~ 111 lee ~~i~~ncd~;ircly. I'ollo\\ III~ re.~ctio~i C~II~S~IILIL'II~S 

\\ere ;idded to t11c probe. 

rcp:~rctl I~cslily L~IIII 2 111 c,ruhh.li~ikcr \VIIII 8 111 

i\atcr suppl~sd b) tiis colilp;~~~! 1. 

4 Tlic ~C:ICIIOII II~I\III~C i\:lr IIICIII?.I~~~I ,it 3jllC iur 3U 111111. ,111d 11 M:I~ ;l(I(led 10 ~IIC 

liyl)r~di/;~t~o~i Ix~ttlc, 

5 Tile liybrldl/;~t~(i~~ \\;I> dolic o~cr~iiglit 

6 The ~nc~nbl.;ilic \\;I> lal,e11 o~it o~i tlic Ibllosring niornllig ,111tl\rab \v:~slicd w~tli ~)rilil;~i.y 

iv;~sli b11t'ii.r (1;~ biii'f'cr colilpo>itio~i. rcc Appcndlu) twlcc krr I0 111i1l. C;ICI 

;III~ \villi 

sccu~itl,lry ua4i bull21 (fur bullbr cunipo,iliu~i, see ;'\~>~~IIL~IAJ luicc li~r 5 nii~i. c:lcll. 

7 Tllc blol \v;ih trcatsd \LIIII CII)IJ-SI;I~I'I for ;ibo~t llircc I~~IIIII~C\ ;i~id\\;I\ [~rlcketl ill llie 

d,:\ e1o1111ic1it C;I>SCII'. 

8. AII X-ray fill11 wni kepi oil tile blot :III~ CXIIUSII~C ivi~, ~UIIC ;IS I~CI. ~IIC SI~II~I~ recorded. 

9. The X-ra) film \vas \v;~sIistl ill tile folluw~~ig o~tler. 

;I. Developer for ? 1ri111 

b. V4;lti.r ior 30 scc. 

c. Fixer for 2 tii~n. 

d. Wiltcr for 2- 3 mln. 

10. The developed X-r;ly filni \\'as dricd and v~cwed on the sl~de-viewer

4.0 RESULTS 

$loaf of tllc c\l~cr~~llc~lls UII ~UIII;I~IC snlbryogc~ic>~s ncre cio~~c II~III~ llic c111bryo 

;I\)> cupl;l~it S~o~~ti;irtI~/;~riul~ oI' .11111ro1)11:1ti. IIIC~I~III~ 1i.r IIIC~IICIIUII 01' CIII~I.YOS \c:I~ ~UIIC 

LI~III~ 2,4,5-T ;III~ 2.4-1) ;I\ lpr111ci1>;1I 1pl,111l gru\\ll~ IC~III;II~I~. I:I$ 4.1 ~IIU\VS Ille i~id~~ctii~~i 

oI'c11111r)us I'ru111 III~IIIII~ c111h1)u ,1ii5 c~p1~11il rill 11511ig 2.4.5-T '15 I~IIIII;II~ $~o\\lli regulator 

and 1:1g 3.2 clio\vr llic ~ndi~ct~tr~i ol' enibr)u* 11s11ig 2.-1-1). Add~l~il~i~~l ~~OLCIII reg~~li~tors 

s~lch ;is TDZ. DAI'. /t.,~titi.:III~ ki1icI111 \I.CI.C 01>0 119cd 111 co~iibi~i;it~i~~i UII~ llie 2,4.5-T illid 

2,4-D. ?,4,5-T co~~la~~i~ig ~iiudi;~ (JEM ssrics) T:~ble 4.1 sliowa Ihc cfict of 2 and 5 pM 

?,4,5-'1' in co~iibrii,~t~on wltli TDZ, MI', ~III~IIII ur /c,~ti~l uli su~ii;it~c~iibryu ~nductioii. 

Comb~~l;n~una colitainllig TDL ,111d UAI' sllowc~l \cr) luw liciji~c~icy ul'c~iibryoye~icais 111 

terms of the nu~iibcr oI'rxpl;inrs respo~ldl~ig ;111d average ~ii~nlbcr oi'c~iibryoa per explant. 

- -\C__- 

The expltinta on tliese ~iied~a 

-- 

con~binat~olis prodi~ccd awdl yrcc~i niass of callus on which 

two to four embryo$ ;ippt,lrcd Tlic cnibryos ;~ppc:ircd ;IS I;lrge ylubular entiricb and lliey 

-. 

did not look l~ke well-dcli~lcd c~~~bryu, III;II ;~pl~e;lrcd ill ci~li~lre ~iicd~a co~l~a~~ii~ig 2,4.5-T

w~tli kltlctin. More iii~niber ol'capl,i~lts responded \\lie11/cat111\\:IS ;iddctI \vitIi 2.4.5-T, but 

the number oienibryos per espl.~nt \\as verb lo\\. Eliibryo axis gre\\ 11i1o a sa.ollell Inass 

on wli~cli embryos appc;ircd I~nlc I;lrgsr \\1tI1\\sll-delilied globul;~r ali.~lie. Irilcdio with 

kl~ietiii 111 coiiibili;ltio~l iinh 2.4.5-'I' i\ci.c I;)IIII~ to he bvst ,IIIICIII~S~ ;ill ~IIC CUII~~II~~IIIOIIS 

used. ,411 ;lveragc of 7 6 eliibryoa \\'crs i~lditccd \per c\l>l~~lt flo~ll tile bcat rcal)utidl~lg 

COI~~~IIIJIICII~ 

I e., 2 lib1 2.4.5-1' ;1101ig\VIIII 2 pkl h111cri11, 'l';~blc 4.2 ~lio\vs i~icrs,ise 111 tllc 

concelitrJtioti ot'2.4.5-'I' to IO ;~nd 15 [~hl \\IIIIP :IPI)J~III~ 

tlic otlicr Ii~r~iii~i~ss ;I\ 111 tile c;ise 

ui'luble 3.1 \\,liere tilers \\,la gr;~cili:il ~IICIC;I~C 111 tlic I~CL~IICIIC~ ol'c~iibiy~ i~iducli~~i~ \+it11 

incre;lae In ?.4,5-'l'. ;~nd 10 11R1 2.4.5-I' was I'oui~d to be bc\t. I';II~S~II ui' 111iluct1011 i~tld 

tilorpliolo~y ofcali11~ ;i~id cti~hryos \\;IS 

li1111li1 to be s ~~il~li~r ill ill1 111s ~LII. XIS 

~~'I~CLIII~~CIIIS 

I.c., lo\% Ireq11c11~) III~UCIIOI~ 

;111d grcc~i callus \vitIi 'I'D/. ~III~ lj/1IJ c~~iit;~iiii~ig 

co~~ib~nat~un~, al~glitly iiicrcaaed t'rcilr~ency ,ind irlliie c;illu~ \v~tIi rc;~rii ;III~ besl 111ductio11 

iicqucnc! iritl~ broivii c;illu in tllc c;~hc oi' h~~ist~ii CUIII;IIIIII~~ 111c(/ii1, Aiiiu~~gst ill1 tlie 

conib~~i;tt~ui~~ LI~CII. CUIII~III;IIIUII 

01' IU lib1 2,J.S-'1 \villi 2 lihl ~IIIC~III iilduced all ;Ivcrage 

ut' 19.0 cillbryol per cspl;int. Tllcre W;I\ 

cnl;lrgc~licnt of tlic e~nbryu ;]xi\ ~n 2.4.5-T and 

kinct111 c~~nib~~i;iiro~is \\llcrc (lie cliibryos or~g~~iiitcd di~cctly li.0111 tlic ,II)IC;II 

;itid ax~llary 

reglolls Tli~ set of embryos :lppcarcd crcaliiy ~Iiltc w~th well-delinuti globular littad and a 

stt~lk. All the otlici. co~iib~ii;~tions co~it;ii~iing 2.4,5-T \villi 'I'D%. UAI' ;ind zcatlli showed 

embryos wltli no st;ilks aiid looked litrls Imrgcr tiinn rile fornlcr. I'rolongcd c~ilturr of~he 

etiibryogcnlc explants fol. 6 to X wceka slio%ed indtiction of sccoiidary embryos. Tlie 

secondary en~bryos or~ginnted from the surlilce of tlie globular licad ol'priliiary eiiibryoa. 

Tablcs 4 3 ;~nd 4.4 sho\\s tlle inductio~i li~eq~iencics on tllc liledia cuntaitiing 2,4-D 

as principal grourh regt~lator ;~ppl~cd at 10. 15, 20 and 25 pM concc~i~ratio~is (JDM series).

These combi~iatiori sl~owed liiorc cnllua cu~iipored to tlir ?.4.S-l'-co1itai1ii1iy media where 

the embryos originated liul~i tl~ callus III~IS~CII~. TIic iii~~iibcr ~I'expla~~ts respo~idi~~p was 

s~m~lar but tllc aicrage I~LIIII~C~ ut'e~~ibryu~ per exl>Ialit \\.;IS 

cu~iil);~~.;~~i\eI! Io\ver tl1i111 the 

abo\e-nient1u11cd JELl aeiies ol'~~icd~;~. Tlic c~iib~qus :~l>l>c;~rcd I~~rgcr tliii~~ tlic UII~S III JEM 

scrle, 

;III~ tiley l;~cbctl ;illy atalhs. Fig. 4.2 sllu\rs 111~luction ol' e~~~bryos fro111 111;lturc 

e111bri.o ;ial:, cul)l;~~ir. Ucs~ rcsl)o~idlng COII~~III~I~IUII \\,IS 

?O 11hl 2,411 w~tli 2 1lM kkieti~i 

tli;ir ilidi~ced 9.3 c~iibryos per c\pl;iiit No sccontl;~ry enlbryos ~ c ubscrvetl ~ e e\en VII 

prolonged CLIIILISC ul' tlic c\pl;i~it\ 111 11ic b~~~lie 1iiec11111ii \\li~Ic tl~c 111d11ccd c~iibryos 

gfiidunlly entered c;~lli~s pli;isc 

V;il.~oua otllcr ~ hpl~~iti s~lcli :la 1);1r1:, 

o1'111:1lurc e~iibryu ~1x15 (~>lu~iii~l~, radicle. side 

;irlns ;lntl ~ii~tltllc purrion). Icaflcl>, stelii aeglnclits ;uid root acg~ilcl~t\ wcrc ci~ll~~rctl on bcst 

CII~~S)O~~IIIC COIII~II~~IIIUII ui'JLhl20 rIi;~t CUIIL;I~I~C~ I0 piVl ?.1,5-T ;III~ 2 11M ~IIIC~III, I'hc 

results ;ire allo\\li 111 rlic 1;lblc 3.5. I'lu~nc~le iiolil 1ii;iture c~iibryo ;IXIS 

;111tlcaflel:, g;~vc bcst 

c~iibryi~ ~iiJui.t~o~i li.cij~lcllc) ol'70 ;IIILI JO'% I~II~SCII\CI~. All IC UI~ICI CSPI;II~~~ CKCCP~ 

r.id~clc ti.uni c~iibryu ,isls sliu\rccl luglicr iiccltlc~icy of II~~LICII~II \+liere ;I t~~iic tlepc~idc~~t 

~nducuo~i \ids obhcric~l. 111 ge~icl.;il, clilbryu ~~~~luctioli \V,I~ observed u~tliin 4 to 5 weeks 

and s~icli phcnornc~lui~ \\;I, obser\cd ii~tli iiinlllre enibryu ;ix~s dc~.~vcd cxpla~its and 

le:111c1h IIo\+cYcI.. at~111 ii~icl 1.001 \egIIiCliti sllu\\i'd III~IICIIUI~ 011 ;I 1)ruIu11gcd CUI~LI~C on tlic 

:,;i~iic 1nedi~1111 Iur ;~buut X lo 9 \\ccks. Tlic cnibryo\ ;~ppcared a~ni~l:ir tu tllc ones on cliibryo 

nsls and leaflet explnl~ls. Tile epldernial I;iyer ol' SI~III 

and root scgmcnts broke opcn into 

crevasses and embryos apl)c:~rcd orig~~ii~ti~ig fro111 tlic ililicr tissue.

'I'lle c.;pesllilclith 

011 slluol osg;llloycnc>lb wusc Il~u;~tlly d~\~dcd illlo two types 

namely dlrvct ;111ti ~nillrcct ~nle~liods B;ISIII~ on lliis co~iccpt tllc expl;ll~ts used were 

6 

c;lt;lgo~i~etillto two groups. I, lixl)l;i~i~s tli;~~ g:l\e tl~rec~ slloot org;~nogenes~s und 2. 

Euplants that gave liltllrect slioo~ org;inopenesls. E.\pla~ll\ 1i;iving prc-cx~stllig ~nicsistenis or 

any traces ol'mes~s~cms come u~lticr lis,~ gloul) i111i1 llic olhcri like Ic;~ilc~~, Ic;ll'basc, stel11 

segments, ep~cotyl, liypocutyl, root segnients n11d root tip tllel were 11o1 ;lssociated w~tll any 

mrl.istemat~c tissue conles ulidcr tllc seco~id clabi. Tile ubscrvnt~o~is slioued that ~ndirect

egeneratloll \!;IS 

dli'licull ;~nti could 1101 be ocl~icvctl n.itl~ 111s tcsted 111cdiu. M;lture elnbryo 

axli, shout tip. ;lxlIl;iry bud, cot)li'llo~i.~ry node .ind d\~li;lr) III~~I\~~III (AM) explnllts 

sl~u~vcd sl~uut rcge~~cr;~l~u~~ 

;IIIIUII~~I \\ IIICII ;1\111;1ry II~~~I\I~III c\p1;111ts ,AM2 :III(I 

AM4 were 

~CICCICJ ,IS [lie best UIICL OL'C F I J ~ 4). 

Sla~~d:~rdi~:~tio~~ 

ol' il~~ll~cliu~~ ni~di11111: SI,III~~:I~~I/;IIIUII oi';111 ;I~I)~UPII,IIC I~I~L~I~IIII 

ii~r III(~LIC~I~~II LII'II~LII~I~)~~ hl~out\lio\\h II~IIIIII)/C hliuut II~~UCIIUII I~UIII III~IIIIIC 

CIII~~)O iixih by 

11511ig DAl> ;IS 111c 1pri11~1p;iI grot\ tli regt~l:i~ur ivlt~lt~~~lc slioul~ urigi~i:itcd o11c al'tcr tllc otl~cr 

~~iy~~cliru~ioti~l~ 

;it IIIC \IIUUI 111) ;IIICI iixlll;ir) bud rcg1011~ 01' I~I,IIIIIC eillbryo :IXCS. Thc 

~spli~~it gre~i I~CC 

IU ~ULII t1111es III sl/e ;illil Iiicri~tcIii;iLIc IC~IUIIS br,~~~cli~d IU 111uIt1ple 

\tiout\ 'rllc otllul cullib~~~;~l~u~l~ 

~ulll;llllitly 2-11' ;III~~\I\;'+ 

\4llll Di\l' 111 ~CIICI.;II sI10i~ed 

ciicuuraylilg results, h1;11iy ~I:IIII gru~,th rcgl~l;i~or\ lilw LC~IIII~, ~ICIU~~IIII, lAA iilld IDA 

ucse 

citlicl- i~liIividu,illy or 111 combtnolion \LIIII UAI' sliowcd soinc ~rcdundiint rcsults 

of lo\+ li.eqiic~~cy 111ul:iplc illout ~~iduct~oil and llrcy were IUI 

tzibulalcd due to ltlclr 

~iiiig~iiticant rcsponic I'ur ilioot i~lduct~o~i 

Separate crperime~us for ~ hc sla~~d;~rdi~,itiut~ oi'sliout illduct~o~~ mcdlum were done 

uslng TDZ as pr~~icipal niillt~plc slioot ~nduclng grow111 regulator 111 cun?b~nnt~o~i w~th 2-if'

and klnet111 by using AM! ,mil A>,l4 c\l1l.i11tr Hoth 111c c\;l,l;~~~t\ hliu\\cd lllorc or less 

s11i11lar ~iuiiibcr ot' ~nilliiplc rIiou~\ per e\l)l.ii~r (l',~blc 4 X i rI)L \\;IS 11act1 ;II various 

conccntr,itioii, ralig111g liutu 2 lo IOU phi. Tile 111rdln C~III~III:UI~IIS \\ere 11;1111cd 11s JCR 

acrles \\liicli tilso cu~is~atcd ot'?'i)Z \\nil 2-11' ,111d LIII~IIII. SI;III(I;~ILI~/:~~I~II ot' he I~~C~IIIIII 

\\;la done hccpll~g tile I,IIU st,i~cs ui ~IUII~:IIIOII ;IIIJ suo1111g 111to ~,o~is~tIcl.;i~~ol~ :is 

cu~~ci'~itl.;~l~ui~ ,IIIL~ IIIIIC vl cllIl!lrc 01' c\lll;1111\ 011 I'~)/-COIII:IIIIIII$ III~.~I;I r110\v \1g11ilic;1111 

ctf'cc~ UII 

a!~bsci]t~c~~t diuul cIo~ig:it~o~~. IIIC~\ISIUI~ 01' !.il' i111tI L~IICIII~ \\;I< :IISO t'o~111ci 

easc1111~11 ;IS 111c) \\ere ;IISO IIIcI~I~I~~I 111 ~IIC SIOUI cIoi~g,itio~~ 111c~li~11i1 [SEM) ;I( 

Iu~~cr 

~UII~~III~~~IIUIIS. 11 Iun le\cl IJI'I'DZ (41141, J['l

decreased a[ cllkalinc pH \+li~le 11ic shoot buds iliduccd oli nlcdia \\it11 pll of 5.0 to 5.5 

clollg:~ted \+ell 011 ~lic ~II~IUI ~I~I~~;IIIUII 1i1c111111ii 

Effect of cot!Icdollary 

liswe 011 rlluot ilnluctiul~: l'hc et'tet,t uf ~liclusion of the 

Intact co~yledun or .I pur11011 of it :~lolig\villi 111c rcgclicr.nllig II~~IIC IS alio\\~i 111 tlio Toble 

1 10. Iliclusio~i ol' c~I)Icdo~i Ibis :I co~lr~~l~r:~liIc ;I~\;II~I,I~~ l'or ~~~CIIC'IIIOII 

of ~ii~~l~iplc 

alluulr ti.oiii :l\~lI~r! 11ltc111 ~ C ~ I ~IIICC U I ~ COIPICIS C\C~II\IOI~ of IIIC co~).Icdc)~i ?~Iio\ved 

dcl;~ycd rel)u~isc ;III~ ,I \~gli~lic;i~it ~Iccrc.~~~ 111 IC 11l111iher 01' SI~UUIZ 1)cr rcsl>o~id~~ig 

explant. E\cIi~sio~l of col~lctlo~i 1111>:11.1\ 

I c . CII~III~III~ oI';I'LIII;I~~ IIICI.I\ICIII IISSLIC \VIIII h;lIl' 

arid LC~O cu~yledo~i aIio\+ctI 111iic ~ICI)CI~L~L'III cI1;111yca 111 Ill? 111111lbcr of\Iiut~tb per ehpl;lnt. 

cu~nl~;~~cil ru tllc e\~il;llir a11l7 11.11l' cu~ylctlo~~ Ibllv\~ecl by /cru i.otylctio11. Iluwever, 

\\lrIi rcjpect to tile nll~iihcl. of siioo~s per expl;lnt in c.cl~l:lli!\ WIIII 

fill1 ;III(I lin1fcutyIcdo11. 

Sub-culturiiig oftlic rcgciicratllig hoot butis In clu\lera, Iio\rcver, rho\\'cd a11 cxpolientlal 

II~C~C,I~C 111111111ibcr 

01'11111111~11~ \11001\ 1pcs UY~I;IIII\ Tl11\ ~ I~CIC:I~ W~IS li11111d 10 be two to 

tlirer 111i>o5 al~licr~ur 111 !lie cspl:1111i \+IIII III!;ICI 

cot~lcdu~i. 

Effect ol'expl;~~it dul~ur scctlling .nge: i\gc oftlie accdllllg lj.0111 ivl11cIi the CXPIBII~S 

ucre derived, playa 'III 11iil)ortn111 role 111 rcgclicl-;itiun oI'1ii~1l11plc slioota (Table 4.1 1). All 

i~x~llnry Illerlatelii rliItint\ slluuetl ;lgc tlcpel~tlc~it rcspoiirc>. A.11 cxp1:11it difircd ti.oln 

other tllrre b,is~c;~ll) tlur tu 115 ~II~I~,IIIUII lp.lr~cln. 2-

Sl~uot regeaeruliu~~ ill dil'fercl~t espla~lls: 1111vari;~t~uns ot'tllc csplalits colisisted 

of rnerirlei~~;ltlc /olics 11irougl1 \rliicl~ dircct rcgencr;ltioil \\,IS 

;~cliicvcd. Fig 4.4 shows 

r~g~'11cr~111011 pillter11 i~'ulll dltli'rclll c\pl;llili l~llil 1';lble 4 I! aIiu\ir, llic rc$eIIer;tlltig obililies 

of \;iriuils cspI;i~its, .All tile c\pI;inis e\pccl ,4113 al1uni.d IOO",, li.c~luc11i.y of IC\~,UI~~III~ 

eupl;ints, 

\\it11 vdried 1i~111lbcr 01' SII~UIS 1pcr c\plil~~t, E111bry0 ;IXIS 

c\~)~;II~I ~II;II \\:IS 

liciliicllily iiscd 111 rcge~~er.~liu~i CXI)C~IIII~'I~IS \l~uiii'cl :I\)IIC/I~~~IIOII\ II~LIIII~~IC s11u1)ts 

origiii,it~i~g ~ZCILICIIII~III) li.u~il ~ UUI 111) ;iliil l~\~/I,~r) bud I~UIIILIII~ 'foi;il 11111nhcr of zlioo~s 

origin.~llng li.u~il 5hou1 111) ,111d t\$o ;~\ilI:ir) bilil I)U~~I~UII~ NCIC 

C~ILIIII~~ ,111d ~~OIIIIC(/ 

luge[lier 111 recordil~g !he ~caults A ~icor \)~icliruny ot'~cgc~ici:~~~~~p 

II~~IIII~)/C I~OOIS ti.0111 

sliuot t~p \us obscrvcd. I'rolongctl cului~c 01' 111c cujil,~~~~.; 1.11 SIM ici~~l~cil 111 de~rciiscd 

I'rcil~lc~~c) oi'tllc \llool clu11&1t1u11, i\x~ll:iry bull \\,IS 

I,llic11 a\ a bl-limdi~ct ol' AM1 311d 

,'tL12 r\p1;111ts Yc~r 

~~IIC~IIUII~I~I\ 111~111iplc \l~uuti \vcrc l)rucl~~~ccl by IIII~ e\/~Iii~~t. N~i~ilhcr 

u1' 11i11ltil1lc \/IUUI~ jpcr ~A~~;IIII \i.'ia 

1)cltcr tl1:111 cllibryo :Ihla ;III~ \Iioot III) IIolvcvct~. llle 

;~x~ll;lry bud cxpl,~~it ubi,~incil ;I:, ;I byp~utl~lct ul' AZll c\pl;1111 hilowed IIIIIC~I 

Ivivrr 

li-cilucllc) uf' III~IIIII)IC ~IIuI~I~. 1 IC 11i1111ber o!' 1li11111l)lc >IIou~~ \lit19 Iu\\cI. tIl;~li cll~bryo :IXI~ 

allti aliuoi 111). Abuw OOYI ol'ilic ;i\~lli~r! Ih\;ds aIiu\vcd III~IIII~IC \11uuta OSI~II~~~III~ Ii-0111 llie 

bar :if the ;lslllar) lilld. 'i'li~i type US i'cgc~~c~;lt~oii wai aiail~iictl to bc u.icfi~l for guiict~c 

tr:~~isl'ur~i~;~tiu~i, Tllc axill;~~,y bud 1purtiu11 c~~l;irgccl ;IINI tlic ii1111111)lc sI1ou15 origi11;itcd fi.o~~i 

siiollcn portion oi'AL1 I cuplatli. AM2 cxl11,iili tI1,1i g:~i,c riic lo ~nii~ltiplc ,Iioots lio~ii olie 

oi boll1 sldcs ot'illr ;ire;! ol tllc ;IIII~)LI~;II~~ ;~rlll;iry IILI~. 111 ~uo\t ol'tlic caho ~iii~ll~plc sllools 

\+ere produced Crolli ollc sldc oi' illc ;ixlllar) bud ;irc;i wlicrc syiichronoua mtrltlplc slic~ots 

were observed from ll~is espl;liit. 111 AM3 expl;lni llie rcgcncr;llioli I'retjuc~~cy \+as vcry low 

when compared to oilier tested explnnis. A vcry low nulilbcr of nlul~iple sliools, ci1111c from

either side of'bws;tl portiol~ oillle espl;~~~t bur d ~d nur elongste well. AM4 cnplant that can 

bc cons~dcred as itic:ll tbr gene IV,III~(~.I CK~~~IIIICIIIS yve risc to lllultiplc s11oots 3s 

nllllt~ple cl~iatcra \rl~cre tllc hiloot.; ;II.C 

lbr~~~sd 1rci111 TIII 

1p0r11011~ 011 tl~c si+ollc~~ 1p;ltt of' 

,~k~llilr! 11icr1>lt111. I:IC 1.5 ~IIu\\~ regi'~icr,~l~~~g IIIIIIII~>IC ~IIUI)I\ litllli 4\11 ~\~)I,IIII \+l~cre 

III:III~ 

~Iloutb U~I~IIIJIC~ li.0111 tllc ~;Is;II I~U~~IUII:, ui 1I1c ~CIIII)\C~ 

SI~~I~II h~ld\ I'r~lu~~gcd 

CLIIILIIC ul' ill1 1llc ;~bu~c-~~~c~~tiu~~ccl 

L'\~~:IIII> 011 sl~oul ~t~d~~eliti!> III~~IIIIN 1~h111tetl ill 

L'\~~ 11er C\JI~~IIII \\~lli C:ICI 

sl~uut but1 ll~\ 1118 tlic ab1111) tu tlc\clul) 111tu CUIII~~~CIS ~)I:IIII 

4.1.2.2 Elol~g;~tiul~ ul'tlle slloot buds: 

I'lilllt gro\\tll r~g~ll~ltorh \bere elll~lloyccl :It IU\V cullcclltr,ltloll:, 10 lllcrc~lhe tI1c 

~nunlbcr ut'sllooti elo~~giling ~ C cul,l;1111 I ('l:~blc 4 13) Ijhl'. 2-11], ~IIICIIII 

;III~ GAi kcre 

IC 

key COIII~)~~I~CII~> 1;ir b11iiot C~OII~;IIICIII, 

Tllc rlluul cl~~lg~ltio~l 111eil1,1 H~I.C I; IC~ iis C'I:L 

scrio koln \\111cli Cl:l.? iuln;lllrlllg 5 11hl 2-11' ,111d 2 lib1 ktiieull ;111tl CI:L? collrallllng 2 

11hl GAI sl~u\ved beat I.~LLI~I~ by C~OII~:I~III~ 7 3 :III~ X 7 SIIOUI I.CI;I)~CIIVCI~ (1.1~ 4.6 C). 

EIollg;~tion 011 ~~IIC~III \+,IS 

iu1111d IU be I~~IIIIIII;I~, E~UII~,I~IUII W:I\ 

ill50 tcated 011 basal MS, 

bur ll~c rcsillts were 11ot s~~coui.ng~~~g irlic~i cu~lil);~rcd tu tlic ullicr II.U;I~IIICIIIS. IIowcv~~, 

prolo~lgcd cul~ure UII MS ioultcd III ille ruuul~g 01.5 lu 10% ul'tlic clo~igallilg shoola. All 

lilt tested conib111,111o11 sliu\rcd elollg;~t~un uS0.1 to 3 sl~uuls per uxpl,lnl 111 the illirlul step 

(elong;ll~on I), k111cli al~u\vcd ;I lei~gtl~ ul' 5 tu X c111 111 anutller I\+O wceks. I(ca1 of the 

explat11 bas placed 011 fl-csli clo~ig;itiun II~C~ILIII~. Ucat results IVC~C ubralncd ~vl1e11 tile 

elollgatlon \+us done \\~rli CkL? 111 the clulig,it~o~~ I ;III~ CLL7 111 tlic I;ltcr stilgca. The 

sllool buds gro\~ing 111 cluster'; \+ere cultrlrcd 111 CEL7 fur two to tlirec pas\:~gcs ;11 IO to 12

day inlervals. CAI naa fourid lo be ploy~l~g cruc~al role ill tile clur~p;~t~on 1 phi~sc where its 

application showed lncre;~sed internodal Ic~~ptli illid bctlc~ Icat' ~~~orliliulupy. Tile slioots 

cluriil;ited In cocI1 p;ias,l$e \vcrc tr~~~ist>rrctl lu tile louti~~p III~~IILIIII. 

I cupu\ud to tllc ~uutlng incd~;~ dr;~b~lcally di'crc;~actl the rootilig 

Srequency. Late roots rnent~oncd abobe, sliowcd \low growth illid they ~uritil~r~ed several 

root hairs. Sliools, wll~ch did 11o1 lout, ~ crc;~rrlcd lo the P!I;I~C 2 III HIIICII they were pulsc 

trc;~tetl \v~lli 100 lihl IUA li~r 2 to 3 scc. I

IBA sl~owed bro\\~~i~~g i~lld de:~ll~. A~u\c IC 

de~d llllll. roots \\we t'urll~ed 111 buncl~cs. 

Shoot growth slo\\ed do\\il u1111l ll~c 11c!\ r~.ou~s r~;~rlcII,I>~ I ~lc~cril~c~l III ll~c II~,I~L,II,I~> ,111cl III~IIIO~I\ 

S~CIIUII), Sucrose 

I 5':" ~III~ ?",, ~110i\ctl Oc\l rc.;t1115 III ICIIII\ 01 Iicq~~c~icy ,IC~ r ~ t 

~~~orpliolog) L~trlc or ~io rc\po~~\c !\;I\ obscr!c~l III cull~lrc?, ilc\uid crl'\ucru\c 

Effect of ~lletliul~~ on rooting: Ll'lkcl ut'v;~riuu\ cu~~cc~~lr;~t~u~rs ol' IDA ,111tl NAA. 

\IS 1111\ ;IIY >IIU\VII III 111c I',~t?lc 4 18 lI3tl ;I\ 5 pM 

\\;I> 1ii~111cl ICI 112 L)e\l IUI IUOLIII~ !\ l)r~el' C\I>U.;IIIC 01 1I1c \II

co~llbinatio~i \villi rncli otl1c1- ncre tested ill llle sr;lge I ufil~s II~I~~CIIIII~ II~OCCSS (tig 3.8). 

Coniblnationa of procesbed org;llilc m:iltcr SICI 

Cell l

humidity uf 50% 111 the dayrinie a11d uier 80Uh 111 the 111gh1 'rllc pl:1111s ivrre inltlally 

exposed to humidity as lilgli as over 90% b) ~I~ILIC 01. cuver~~ig 1lic111 w~tll PI'ISIIC 

b;igs. 

Tliry were sloicly i~cclltilnti~cd to tllc i~~iib~clil IIIIII~I~II~ b) grililu;illy c~l~cliilig [lie cover. 

Prolonged coierlng. 1.c.. oicr c\liohi~rv 111 li~gl~ I~III~II~II) c:ii~~cd \\IIIIII~ ;111d dccrc;~sed 

exposure caused preinillllrc dl.).lllg, b011i ~.~il~ll~iig III dciilli of Illc ~llii~il. IICIIC~ llic iili~11~ 

hu~nidlty accliniat~?at~ol~ procesh rcqi~~retl ;1bou1:I ~iiunlli ;III~ i11111c1\1 C;IK 

bc1i)rc c~itcril~g 

illto the stage 3. 

lhc pl:ilil. Iicl;ce, tllc e1illl.e II;I~~CI;III~ I)IOCC~\ \v;I\ 

duli~ ;II ,I II~III IIIICII>II~ OS 10.000 lo 

12,000 lux ill ll~e C~II\ II.~II'" gron Ill cli;~~iibcr. I)lrccr CS/)U~LI~U 10 llic SIIIIII~III 111 llie grcc11 

house caused dry111g 01' 111 ~I:IIII. lY1111 ~C~~ICCI IU IIIC I~IIOIO~)CI IU~. \I;I~U 

I W:I> lii;~~~itillll~d 

at I0 Iioilr l~glit ;IIILI4 Ihot~r dark, \t~igc 2 ,II 12 liu~ir l~gl;~ ,ilc cultllrc growl; cl;~

addition to tile tubes collt:lllllllg llcli~itl 11ltdii1111. 111 th~s 111st11od IC 

1011 1101.11011 01'11111 plilllt 

was exposed to tllc ULII side COI~~I~IUI~~ 

,111d hlo\\ly c ~ e e l ~ \\IIIIS ~ l gro\\illg ~ ~ ~ ~ I:igure ~ ~ 4.10 ~ d 

H, C sliows onulilcl mc~hud ol'sl;~l~c l~)drol~o~!~i.a s)sIcl!i ill i\lilcl~ 1I1c rouiillg ayslclil \+as 

completely 111111i~'racd 

[lie 4 a11e11g111 A~'IIOII'\ 

~O~~IIIOII. 111 1111, 111crI10~\. IC 

I~~,IIII \\I18 

direclly cxpoaed 10 1I1e ;III!~ICIII ~UII~IIIUII$ IYVIII ~IIC lir>r cI,I\ ll~c\er, (1ry111g\\;I> 

pl.evented bccauac ol' Ihc Il(l111d 11;11i1rc 01' 111c IIULISII~IIII~ IIIC~IIIIII. 'Illla ~i~e~li~d 

CZIII 

supple~ne~ll rile atagc 1 ;111d 2 ~Isacr~be~l ,~bi)\c;111d 

ll!~ plil~il\ CJII 

dlrc CIIIPII))C~I ~~I~CCIIIII~ \\ I~CII IIICIC \\:la 110 g~u!\lli ol'lli~ 10ti1 \)\~cI~I. \\IIICII 

dirsctlj i11111blti11g lllc g10\\111 of I11e ~I;IIII. ' ~III~ IIICI~IU~ \\;la 

IIII~~UI~,III\ III ~II~IJIICIII~ tllc 

survi\;il ticq~~u~lcy by irouble>llout~ng In 1Iic IIJI~CIIII!~ I)IUCCI\ IIICII~IOIIC~ 111 llie III;I~C~~~I~S 

:~nd methods section. C;ro\vlh of llic i'uor jysli.~!!, wliicl! 1s .I Ley I:lctor Ibr Ills II;I~~CIIIII~ 

appeared III nbou~ 15 ti;lys alicr IC I~.III~~)~:IIII,I~I~~II i1!10 ?O CIII 1po1.;. lllc Ilu\~ei':, kcre 

renloved aa sooll tiley ;ippcorcil ior ;lboi~t 3 \vi.cL\ >u ;I$ 

10 II~CI~.I\~ 

llic icgelanvc grow111 

and lliiis ilici'ease the ~ii~mbci' ol' accda per ~I,IIII I~CIIIU\,II 

ul' Il~i\\'cr\ :II\u rcs~111cd III IIIU 

111creabe of br:111cl1111g ;III~ IC 

~I;IIII~ ;i5$11111e~l

4.2 Histological studies ~II nlultlple slloot de\tloplllel~t I'I.~III Ahl.1 espl~l~~t: 

As described 111 secliull 4.1.2,l. Ah14 elplants alio\\.cd ;~dvcn~~t~uus niul~iple shoot 

induction ir~lll alloul bud> OII~II~;I~III~ (i.0111 dilji'reiit 11;1r1\ uI' IC ~C~CIICI.,IIIII~ ;I~c;I. 

Histological slut11i.a ol' ~iitlltiplc slloo~ III~IICIIUII 1j.oi11 ~llebe c\lll;ii~ts \\ere co~iductctl 10 

colitirm tile for~ii;lt~oli oT~iit~ltiple ~~dvc~~ti~iou~ 

~ilet~iate~ili~~ila ,ilicr the i.s~iiuv:~l ol',~\~llo~~y 

bud. 'I'hc dey \+Iicii ;ixill,~ry but1 \+';is rc~iiuvcd \\;IS 

eu~isiilercd as d.ly-I ;\lid tile slud~cs 

were coiiduclcd 1111 7 diiyb. Fig 4. I3 A tu H ~ll~\\'b 11c\cIu~i111~1it ol' I~~C~I~ICIIIUI~~ ;II 

dlff~~~lll ~ 1 ~ ~~;l.\l~~;l~) 

~ 1 ~ ~ 5 

lll~~l\l~lll 

;lrC;l \\ 11~1C 15 10 ?(I lll~Vl\lClll~llt~~ ~~L'\c~u~JL'~~ 

~1~1\~~!~11 

4 to 6 days oi'culrurc on tlic slioot IIIC~UCIIOI? I~IC(/IIII~~ 1.1s 4.1) I5 sIio\v\ ;I \11iiilI ~p~rlio~i 01 

axillary bud rc~iiaii~iiig cvcci alicr ~lic cvcislu~l of tile gruw~lig ,~\~llary but1 and 

dcvelopmeii~ of rnerlstelllo~d\ ;I[ the base of 11. .fliis rcsiilt alluiv\ l~islolt~g~c;~l c\ldcncc lor 

llie cnicrgc!ise of I~ILIIIIIIIC ;1tl\c1it111011\ 4 1 1 ~ 1 bt~d~ I'ro111 llic b;is;~l 11;1itll' IIIC ,~\~ll:iry bud 

:is sho\+~i 111 lllc 1'1g 4 -I U MCI.I\ICIII;I~IC :1et1\11y 01 IIIC ;ICII\CI! IIIVI~IIII~ ;III~ grt)iviiip cell:, 

could be ubscr\,cil 1i.ol11 tllc e\pl;~~~ts obt,~iiicti I~.OIII d;i)-5 tl~ii\,ii.db (1.1~ 4.13 I), 1'. F lllld 

I{). 

Tliese tIi\isio~ib rcullcd 111 [lie Ibr~ii,~r~o~i ol' II~C~I~ICII~~IIII. /OIICI 

IIl;it ~C\LIIIC~ ill llic 

f'urniatlon of 11lul1il)ie ili(10t bud> oh >liu\vll 111 ilic l:ig 4 13 (;, lb~g 4 1.1 A to I sliuws 

nicrlstcniatic ;icti\wl! ol'd~i i~l~~ig ecI1 iIi;~t C\CIIIII;III~ ~c~cIuI~c~I ill10 ~ ~iiilt~lll~ sliouts. 

Ccnetic tl~;insfuriii;it~on iraa carr~cd uiir by LI,III~ b ~ul~\ti~ ;IS IVCI 3s Afirohoi!i,~.ili~~~- 

lncdiilted lllelhoda In boll) llie proccd\ire> llic p~~l,~l~\c rr:iii~l'orniaiira \vcrc obtalllcd by 

selccti~lg the trallhl'urlllallt, 

liilllg il/illI ii\ lll~ 

s~~c~l~lble lll~.l.k~~. gCI1S iilld k:~naillycill 2s llle 

antjblouc br selcc[lon Conlrul cxplanls \r.crc ubcd lu tesl thc Ictl1,il tluse (LD 50) by 

culturing AM4 explants oil MS \rltI~ ~~III~IIII~CII~ (5, 10, 15, 20. 25 mid 30 11iuL) with

varying concenlr;~lions ol'TD% (0. 2. 4. 10 jlhl). 6 lo 8 cspl.~~it\ \rcrc ci~lluled per pl;~tc 

(Table 4.15). TDZ \\;is eniployed so .IS 

lo Itst tile clli.c~ ol' TI)% ti11 c~lli;~~~ci~ig survibal of 

thecultured explants 011 k;~~ianiyc~ii COI~I:IIIIIII~ 

III~LIIIIII~ hi~r\l\:~I \\.I, IC IIIC;I\II~L' tlf ;III~ 

collcel~~lble gro\\lll 01' tI1c ~\l~I~llll ~1lcI~lLllllg llllllll~llc \lltlcrhl>lellcc of 

chlorophyll plgliicnt In llic eul~l,1111. Tl~oi~yl~ llic c\l~lLl~~r p~o\\~l~ \\;I.; 111li11111cd c\cn ;it 20 

11i9'1 ka~ia~n)ci~~ COI~C~IIII~II~~II. cliloro~~l~yll 1pig111c11t 11crh1\1etI (or re\\ ti~~irc cI;iy, 

,~ttcr 

wli~cli expl;l~~ta blcuclictl. F.\~>I~IIII sro~111 ;111d aI1tlo1 b11tI II~~~~ICI~OII \\;I\ COII~PICICI~ 

lnliibitcd :11it1 CX~I~IIII ~OI~II~ICICI) blisc~cl~cil 111c C~JI~~CIIII.;IIIIJII 

LII' 30 111s I. I\;III;IIII)ICIII .A 

gradu;il dccrc;~sc In si~rvi\;~l I.;I~C US tlie ~YIII;IIII\ \\,I\ li1i111\1 \\II/I illcre;~hc ill L;III,IIIIYCIII 

co~ice~~tr;~t~o~i. Grti\vtl~ lctli,~l tloac \\$la fi\c

sliowetl no liirtlie~ yrowlli Tllc ~clccicd slluut~ yru\il~ig I~c.iltl~~ly ~cic tlal~ilkrrcd to 

rooting medium 1Iii11 dltl II~I 1i;iic ;my I\:III;II~I~L,III >IIICC II ivi15 IOLIII~ to d ~cr~i~sc IC ro0l111g 

Ii~cil~~c~icy, l'bc ruute~l I~III,III\C II,III~~UI~III~IIII ucrc I~ii~clc~~~d ;illel t~:~~~,pl;iiilcd tl~,~l sI~u\~'cd 

11onnal gro\\,tli ;lnd ~iior~iliull;~gy cu~lll>,ircJ tu tile coinrul\ they \bcsc I~I;III~I;IIIIC~ 111 tlic 

spsc~nlly dcs~giictl 1'2 iiic~l~t) for trclnagslili.i. I lie ~)I;III~~ lloirc~cd III ;I~OLI~ 15 l;li~ys illlor 

the tra11splentatioli illto tlic 20 el11 put, 

The 1)1d111r MCS~ pr~1)~18;11cl;I ~cgctilt~vcly Sor ill1 

extended limo b) alill,ut.itllig 

llle IC~I~~III,II bud> ol' ,oiiie 

bl-alichc ;III~ rc~liuvi~ig llle 

enlerglng flo\vera. Thii nrc~liutl could plu\ itlc rLiSlic~cnl IcaS tlrs~ic ~\II. ~ln)lccul;~r ,~iialys~s. 

Emergence ;~nd matur.~tioii ul'liotl\ \r ;I\ Iuuild tu be nurillal, liu\\,cvcr, tllc ~lun~bcs illid b i ~ ~

oftlie seeds was tiound 10 be lo\~t.r coi~~p,il.cci lo cu!ilrula The 111;iturcd IILILI\ \\,ere dr~ed and 

s101-ed ;it 4 'C Svr I'LII.I~IC~ use '1.11~ ~III;III\~ IKIIIS~~I~III,II~I~ III~II iicru I~;IIIS~LIVI~~C~ \\it11 

BICI:~.,I/J 

9c11e \\c~e i1;1111eel ;IS CB (CB I, ClQ, .,, 111 series ,111~1111c O I I ~ IV,II~\VO~IIICL~ ~ 

\vitl~ 

T I I c I:III S ' I 2 . . 11) \er~c\ .I 1u1;1101 1 I ('13 llh1111\ :IIIL~ 0 C(S 

~)lants \\.ere obt;i~~ictI ;111,I llicy \\CI.C L ~LI~~C~~IICI~I~! ;111;1ly/i,t1,II IC 

III~IICZLII;II le\cl 

IIIIII,I/ >el 01' II~IIIS~UIIII;IIIUII CX~ICIIIIICIII~ 

1\41 i1011e \\1111 111iS723 131 2nd pIIS 

737,SBTI bln;ll-y vcclurs CUII~;I~IIIII~ Ull'i~./,lh ;III~I .S'li/7 ~CIICS ~csl)ecl~iely. I'u~;~livcly 

rranslbrmed ~planlr w1t11 Ui(',~l,.lh 

\rere ,i~~al!/ctI by 11it11g IICI< ;III~ Suulller~i blulli~ig 

tccll~l~que\ and p~~t:iti~eiy ~~a~isiur~~~cil pl,1111i c;lrrylIlg .5'1$77 gcnc wurc it~~i~ly~cd with PCIL 

Molscul;ir ;in;~lya~s irnh dune 11l1t1~11ly by do111g 1'('1< 

lollowcd by Soulhcr~~ blorl~ngs.

Conlirmntioll of plllati!e 

t~.~ll~afor~~~a~lts II~~II~ ~)ulyll~crasr rl~ai~~ re;~ctioa 

(PCK): PCR wi15 fbu~~d to be 011s ol'111c II~\ISI 

e1'1;.ct1\c ;III~ ~ULIIIIICI~ IISCJ tecll111q~1~1 hr 

Initial co~~lirfiii~l~u~i ui' rrI,IIIII \\'~tIl rcqlcc~ ICI L'lj (/11('r1,/..1/1) ~I;IIIIS. 

amphtic;iliu~l ol'lhc c\pi.ctcd 7011 bp I'I~III~I~I apcc~lic to i~/)ill gcllc \\;I> ub>c~\cti 1115 uul 

oi' 10 ICSI~CI pl;ll~la. (~CIIOII~I~. 

I)\ \ \,1111lili.\ 111 ('111. C'I12, C131. C111 i~~ld ('117 >II(I\vc~ 

proliilncllt t ~ ~ ~ ~ ~ ~ I .2 l LO ~ :1111pI11ictl 

l i c ~ ~ l ~ K ~ o g spcc~l.~~, 

~ ~ b ~ 1i)s c ~ iii,/,\ ~ ~ gc11e \\,I> ob~crvctl 

111 X out ol' I0 L~~l~lllcb \VIIII ~i~rllccl lo C'S ~)l.~n!\ \\ 1111 \Hi/ gcllc. 5 our ot") pl;1111> (C'SI. 

CS2, CS4, < S5 :111d('50) ~IICI\$~CI ,IIIII)III~~~IIIL~II vl'c\~~cc~c~l 700 bl1 I'I,I~IIICIII ul II/JIII gc11e 

;IC~ 

5 li't1111 0 ]I~.II~!$L h2, C'S?, C S5, C Sf1 ~11tl L'S')) jI~t)\\cd 1c\pcc11\e I 2 Mi I~;I~III~III 01 

iiii/A pclic i~~iipl~licd ,\ 1111111 b,tlliI ,llhu :~l)lic:~secI III ('SX II~~ uI'I3r ,111il 5131'1 I)~~IIII\ ;IIC 

I~~I\\II 111 IIIC 1.1014. I4 ,111d 

re>~~ltb vl't11c l'Cl< ;III~]IIII~IC,III~II~ vl 1.2 kb ~I~I~I~ICIII \pcc~lic tu III~/I~ ge11c ;~rc SIIU~VII III 111c 

1,'1g 4 15. \YI~II sty~ccl 10 /~I('I.I/,~~/I gc11e ,II~I~III~~.,IIIUII~ livv \ct\ ~I~~II~~IIILIcI~~I~cI~ 

1pri111crs 

\b3iiI~(hi. tiil~cr he1 ol' I)I.IIIIC~~ I~c~IIII~ ul' IIIC I'CI< :III~~~III~C;I~I~III\ 01' I~,I~I~IUIII rl)cclIic to 

I~I('I:I/~~!J gc11e CI~C 5Iiu\\11 111 ~IIC 1'1g 4 I6 (L3) 4.10 (I$ \II{I\\\ ~C\LIII\ oI',S/1/Y gc11c 

~IIII~I~I~C~IIIUII :I 497 b11 I~;I~IIICIII \\;I> IULIII~III L S2 lh.111 0 til'tl~c Ic~lccl >c~~~~plcj 

F(rr/ur,\ trjjei,/iitg rrt~ric~ioit ~~Jgi~riun~ic 1)V:l: UIII li~rll~ i111d CI'I~CICIII re~tr~ct~u~~ 

01' 

genomlc DNA 15 ;I prcrcqu~\~tc lus CI 

cl'lkcuv~ Suurllcrn II~~I.I~I/~IIIUII :III~ IIIC COIIS~~~UCII~S 

ui' 111c ~C~I~ICIIUII 

~C'ICIIUI~ II~II~LII~ 111,iy CI ~11rcct role III 111c lprucc~~. llo~cvcr, dur111g tile 

cuur\c of rl115 atlid). II \$

~~IIOII~IC 

DYA ror SOLIIII~SII L~~~:~ly,~> ~CIIOI~IIC 1)'4,\\ uf IIIC p111;111\cly II;III~U~III~~ 

ch~ckpea pl;1111s \\ere dlgeali.~l enl~cs \\ 1111 I.'< oIIIu\\~~ III;II USA IS ri.q~~~rc(I III IC 

I~;~CIIOII 

1111xture. ~\tId~riol~.il ~IICIIIIII~ or l\lllllr \ill11 I.tbll~CCII~C 111obc 01' 1i011-rildiuil~l1~~

5.0 DISCUSSION 

During tile la31 dccadc, btu~cci;~;ulug~ I1i1, 111~1(lc i~ill)~.e\hivc ;I~V;II~CCIIIL.II~S. 

Genetic ~ranstbrrn~r~o~; oi'crop l)la~il\ I\ 11, \:ilil,~blc ,I~J)cc~. Ie;1d111g tu~~irdb \lie h~ildt~ig 

of an organized and hcallliy ,ig~~culti~re \).>IL.III 

ficc li.ulii 111c u>c ol'pullr~l~ng i~~acc~icidca 

and funglcidea. Explor~tig 111~' poa\~btl~rlc\ (11 I~anilcrr~ng pch rci~cla~~cc pcllcs In pea

(Shade et al. 1991: blorru~i ct ol. 20011) II;I~ opcncii 11cr\ \15t;is lor tra~~sli.rrl~lg 

agronoln~cally 1lnport:lnt tr;l~ts lilto othcr crup pl.iilr\ tu dcvclup cllte c~ilt~\ars (Hirch. 

19971. Thc trcliliology c~llpll.~t~c~illy aupport\ lllc ir,l~~sl;.l, III~~.~~.III~II ;III~ c\l)rcsaloll ol' 

foreign gsries 111 l~etcrologou> orga~iisiiis. I~~IUIIUIIIIC;I~~~ IIII~)O~I,IIII ~CIIC:, 10 1111l1ru\c tll~ 

crop ylrld qualirativcly ;IS 

\\,ell as qu:~i~tiiat~\~cIy II:I\~ beell ~;ul.~tcd ;III~I TIOIIVLI 1'1.0111 

vartous sourccs. TI~csc 111clu~lc gclics Ibr li~l~giil r~h~st;i~lcc- C~II~III:I~C~ :ICI 

~IUC~III;I~CS: 

blral I.es1st:llii.e- cu;it prutc~l~. rcplic.~>c ;lnd r~bu\~i~lc III~II[)IIIII~ I)~U~CIII\: lpc:,i ~CII~I~III~C- 

a~iiylasc ~~il~ib~torr, ~irutci~~.i\e i~ili~b~tvir ;IIIJ 111 ~O\III gcilca ,111c1 ~CIIL,~ hr IIII!II~ILIII;I~ 

improvenicnl- ?S iilbi1111111 ~CIIC 

B~ulceI~~iuIog) c~~~o~iip,~~~lllg 

gc11e11c 

CII~IIICC~III~ 011;'r:, 

ave~it~es to o ~crco~~~c tlle b;lrr~cr> 111 ~c111e\ III~ I~IJ\IIIILIIII 

cl~~chl~c;~ ~~I~ILILICIIUII 

5.1 'l'issuc r~lllure atlldirs 

p~.otocol.; aplx,iscd 111 il~ickl)c;i rutlr,lllc cnlbr)o~c~lcs~\ 111:it (lid IIO~ \;I~I>I) Ihc 

~ ~ l ~ L l l ~ C 1 ll ~ b l l ~CIICIIL ~ S ll,lll~~~~llll~lll~~ll k,,ll~) lCllUll\ ~11' l(

leatlets (Dilieshkuniar er al.. 1094). %earill ;ind I&\ \\;re 

ii>cii IU iildiice soiii;nic ciilbryos 

from inimature uotyletlo~~s \'cry liigli ti.cql~elicy ofpI;i~itlc~~ \\ere regcner;~lcd tio111 tllcse 

embryos (Hila el al. 1997) Hunc\e~. .ill l / l ~ 6 I)~I~IOL,U~\ ~ re~t~r,~l~~d 

111' II~~I~CIIIIY 01 

rnaturat~on and regcliclniioli ol' nliole p1~11tb ii.1 \olii,nic c~i~br~o~ei~c~~~ 

~p;iili\\i~~, rliis 

was rc-co11firmt.d 111 our c\perlliitIi1\ A tcr! good ~~.CLIIICIIC! ul' ~II~~~ICIIOII \\;IS obhcr\ed 

tion1 m;iturc eiiibryo ;I\IS :iiit! Ic;itl~.i c\l)l;iii!s I:~iibryopi'l~li II~\IIC 

\\.,IS ~hsci\ed to 

iiiducc bcticr e~iibryugcnc.;ia 1Llc~c1l~l1)ll liarlle ol')utliig lc.~Ile~\ \\.;I\ 

;II\ 1'11~ p~ihli~llcd prolocolh 

were repeatedly ~ricd \rill1 Iirilc ur 110 ~LICCC\A. I'ICIC 

r~i1111> s~~ggesI 111~11 ~C~CII~~:IIIO 

protocol vla soiiinlic ei~ibryugciics~h calilior be ;I rcll.ibls t11ic lirr gci~cl~c ~~~iislbrin;lt~oli 

exper~iiicnts 

5.1.2 Ori~g~~~~ogc~~csis: 

~!IC~O~)~U~);I~~IIIOII ,iiitI ~~~~I~V'IIIUII \),I tlirtc~ or t11~11rcc1 

organogclie>ls 1i;ive beell e\le~~\~\cli ~itlti~cd :III~\CVCI:II 01 

ICI~UIIZ \\ere ~p~lbl~~~l~rd. 

liiclus~o~i of I~~L'~I~ICI~I:IIIC IUIICI 

ii~lli IC c\I)I,II~I\ \L,I\ liltl~id ti OC cruci;iI for 

regcncr;ltiuil of slluoi butla .ind i\liulc 1)1;111t\ 111 CIIIC~~)C:I. I.;II.I) rcj)ort\ ~III cli~ckpca 

sho~ed callu> III~II;II~UI~ ,111tl ~IU\\III \\JIII 11u regciici,~t~

selection arid prepordtloii ul' c\llliilita. 'Tlie I~C~IIII~ILI~ I)IU\~S LI~C~LII \\IISII 

I;irgc-sc,~le 

multipl~catio~i of a speclea is leqilircd 111 oilier \\orti> IIIIS c\pI;ili~:, IS USC~III tbr 

ni1croprupng;itloli :1101ic ;ilid i)gutic cliibr)o b;lsctl c11Ilu1i.a \\ere IIOI coliaitlcrcd :IS 1dc;ll 

for l~~~isfur~ii~~t~o~i 

stiid~eb, Ullicr e\pl,1111h II~S 

IIIII~I;IIIII~ co~~lclluiis (Sli11,111~1 L)

used with an add111ou;ll care :u~d obscrvat~on. Shoot buds i~~ii~~ccd UI I~AI)-CUII~:I~~I~~I~ 

medlum sho\ved n better elongi~tio~~ cu~~~p:~red to 111c ones nn\li 011 '1'1)L 

Ilu\\c\er. 

~iu~iiber ofsl~oors per e\p1,11l1 UII TIIZ II~C~I~IIII 

\\.I\ I i 10 ? IIIII~\ bct~cr III:III 

l3:ll' IIIC~I;I. 

As IS III~IIIIOI~CL~ 

,~bo\c.:I~~IIIOII;II C;ISC 

~IIOLII~ be c\crc~,ct but 

efftctivc TLIZ concenrratiotl ,~nd kept on TD% ~~ic(li~~~ii 1'~r lllc 1cdhl ~U~:~II

appropriate conce~itralio~i of TDZ 111 gerllill1,irlun and III~LICIIUII 111edi.i 15 SI~III~~C~IIII 

nlodifical~on over euls~lng protocols 

cotylcdo~inry node ere. ilu\ri.\cl., sclcct~u~i ,11111 ~irocc\\ll~g ul' ~p~ol)cl s\l~l:lnl I:, 

other. Rcnioval ol' a\ill,iry 

b~~il. CLII 

111ro~1gl1 tl~c ;i\iIl,~~j IIICII\ICIII ,iiid ~IIC~U\IIIII 111' 

-- - 

cotyledon .- -~ were tile s ~y~i~lic:~~~l v:~ry!ms~:l!~~j:~ 111 llic prcsc111 brudy. '\ I~OIIIP~C~ICII\~\.C 

---- 

~irolocol for rcyci1cr;iiluii ontl ~sccuvcry ol' \\llulc ~IJIII\ oI'cli~cl\l~c;i Ii) II\III~ :i\~ll:iry 

tlinr tlir All? ;III~ A\l4 c\pl:~l~l\ bliu\v\ :~IIII~I\I \111111:tr \11oot I.C~CIICI;IIIOII CI'I~CICIICIC~ ;III~ 

\%,Iiicli the A~~O/IOL/L,I~IIIIII C,III IC,ICI~ IIIC ~~~CIICI',I~III~ II\\LIV l

tissue to regenerate illto lesser i i u ~ i ~ ~ ~ ~ 111 utl~e 

oC;IS~ t 01' s ;l\ill;lry . 

-- .-- 

meristen~, removal of axillas) bud liiilllliea tile .~pic;il ~OIIIIII~IIIC~ 

111 :idtlilit111 to tllis. IC 

medlun~. The tissue a~lj;~cci~~ tu llie ahlll:~ry bud ;IIL';I t11.1r II,I> III~II\ICIII;IIIC IIISLIC I.CTCI\C~ 

shoots. Et'rcct of age ur tllc >CCLIIIII~ I~UIII \\IIICII llic ckl11~1111\ \\ere dcri\cd sIIu\\\ 111,it 

expl:~nts \\.ill be derived. Uoci;ll .II~L~;i\lll.iiy mcrlhicilla II.I\~ bee11 I~~LIIIII\II~~ II~\IIC~ lili 

regeneraling eapacily \+IIII cul~\i~~ci~~gly ;IC~~~.IIIIIIULI> 11:ilurc ul'lhc rcgci~ctill~~ig IIILIIIII~IC 

rrgellerarloil protocol, ol' v.il.lou> 111,1111\. I\\I/~;II) IIICII>IL'III 

\\.I\ ~LIIIIIIC~I IIII~C~ \:IIIOLI\ 

IISSU~ has a direct correlation \\IIII 

il~uu~ ui ruul ~II~'~~~-CIIII.III~II I~(I~CIIII;II in N~,(i>\i(.ii

cotyledoli incl~ided. Aalllary ~iii.~~atc~li e\pl;in~s ilc\oid oi cot)ledoli ,llo\vcd lesser 

- - 

it afkctr upt:ikc of~:~r~uuh I~LI~WICI~I 11111>. 1'111.1Lc 111' 111li~i1~ 

;III~,1111111~111~1111 

%ire I~~IIII:III~~ 

affected by the mediiilii 111-1 (Ilclirc~itl ;illti \l:!ti.l~\. lCi:5. l(,i\sn. ICjSi)i 11 \\;is ubscl~\cd 

that nltr;lte 11pr.ike la 1:1\uurrd :!I lo\\ 1111 :1nd ~1111111011111111 .II III~IICI 1111 .\\lll,~r! 011d 

111ult1pli~a11011 III shoot CII~~~IIC:, UI' C'i~il~~llc:~ \\:I\ II~UII 

\i~~~\l';~~~~r~ 

\\IIcI~ 111~' 1111 (11' hlS 

\\,as red~~ccti to 4 (Cllsvrc el :I]., IOh'3). lC~\C~ \\IIII cIiich11c:1 tll~t \I~.C~C~I\C ill 111~ 

C~IIICCIII~,IIIOII ~i IIIIKIIC\ 

in the ibrm ol'KY01 \\':IS t;~~or.~blc Ibr luul~iig Ths III~~~I~C 

LIIII;II\C 

I\ I;I\II~CCI :I( IL)IYCI pII 

and iininio~iiulii at IllgIle~ plI. it c.111 bc li)putl~c\i/ud LII:II l ~ ~ loll\ ~ IIII~III ~ I~c ~ ~ 

lil\orlng routing at li~glicr pli ;111d III~~,I~~S 111v~~r111g SIOI 

III(~UCIIOII 

S~;i~~d;irtiiiat~u~i ul' IIII~UCIIUII IIICL~IIIIII \V;I~ LI~JIIC L.(III\I~CIIII~ iill \I,I~c\ 01' 111.111t 

rcg~1ier;ilIoli \i~cIi ;IS 

clo1ig:111011 'III~ ~OOIII!~ 1 IS\II~, lo\\s\l 11111 sli2ct1\ c COIIL~II~~;I~IOII ul' 

TL)z \KIS i0Lll1li IU bt !3~1\~~~11 4 I0 11) ~ \ 1 Ill \I?\\ 11~'~~lL' i':l~l ld1;lI ~ 1 1 ~~J11CC11~~1~1011 

~ ~ 1 ~ ~ 

ol'TDZ negati\cI) ~llrcrli.~c\ \r 1111 tl~c IILIIII~CI 01 >IIUUI bull\ ,111~1 IIICI~ ~IOII~;I~IUI!, 4 ILM 

was conaidered 45 u1111111:11 iiorh~llg COIICCIII~~IIIOII 

\LII~I~CI ui'\I~uut hi~ds ;~lro(ICC~C;I\C~ 

~vitli illcrease 111 TL)% CUIICCII~I,III~II 

L'cI~ 111gIi CUII~CIIII~III~JII 

III'IIIC TI)% M:I\ ~bher~cd lo 

gibe stunted slio~~t biiil, \\IIII l~ttlc or no ~IUII~~IIIUI~ In atldlt~on to tills exlcndcti I111ic of 

culturc on TUL ,ilsu 11;1a ,umc ncg;ltl\r cllka on lllc I;llcr at;lgo 'l'llougl! 4 pM 'I'D% was 

optlmnl work~ng cunceilrratloli. CLII~LI~III~ ills C\PI~IIL~ UI 

Lllc s~ille IIIC~IUIII Sur 5 tu 6 

necks dccrcabed tlic clu~ig~t~uti l'ic~~ucrlc~ !\~II~IL,IIILJII 

uI';i~iy gru~tli rvg~11;11urs :iI'tcr lllc

prololigcd culture on TUL sho\\~d 110 pru~llot~\c cii~ct UI 

~IUII~;III~II. Iicr~cc, illc 

lnductloii tlnir \\as re:,trlcted tu ! to 3 uccLa \\'nil ~~SI>L.CI to :\hl4 ~\~)I,IIII, tlie \VIIUIC 

preparation \\as dollr OII 

TDZ collt;1lliing III~~I~I ~CCIC,I\CLI I'rv111 :\A14 c\li1:1111 ~LIIIII~CLI oli J1'l ~ ~ g . 01' ~ IILIIIIOC~ \ c ;III~ IIIICIIIU~I,II ICII~III 

----.. 

i'otllalrlitig 5 lib1 2-11' ;iliil! 11Cl LII~C~III IIIC~~IIIIII 

('I:[.! 

CIUII~~IIC~I lilil I),IIC/I 111 ~11oot~ 111 iibut~i ? 

shoo15 \\ere CIUII~~III'II. 

I Io!\c\cI. ~LI~-CLI~ILI~III~ l11c il~oota UII C L1.7 CLIIII.IIIIIII~ 

2 IIM (i,41 

alier tliey neru cullt~rvd UII CI.I.2 C~I~IIIII~II~~ 5 ~IM 2-11' ;III~: 2 lit1 ~IIIC~III tin :II)OLII 2 

\veeks cunsldcrably ~iicrc:~icd ~IOII~;IIIUII 

~'~CCIIICIIC~. (;,Ii ~~ICICII~ III ~IIC 1'1.1.7 IIIC~IUIII 

is 

5l1o&1i to pi;]) :III~ 1111purla111 rule 111 CII~I~IIICIII~ IIIC ~IOII~;IIIUII ~ IC~ILICIIC~ I or UII~IIU\VII 

reasons, frequency of >houla clu~lgattd 011 CIA, cullt.llnllly II~C~ILIII~ rrglll lro111 llic lirst 

pliaac was 1101 ;IS goud tllc li.cilui.~icy U~I,IIIICL! III \lcp\v~>c m;iliiier 1\11 ~hc Ialcl 5ub- 

cultures oil CEL7 rc5ul1cd III nur.~blc IIICSC,I\C 111 ilic 11uinber of alloola clongalcd. Sub- 

culture of CEL2 elu~lgatsd >llool> uli CLL7 ag,illi rc~illcd In a bcller ~~~orphulugy 111

terms of leaflel ~norpholugy atid ilitcr~lud~il Ie11gll1 I'rt~lo~~ged CIIIILI~I' ~t'sliuu~s 011 CEL2 

medlu~n drled earl~er ilia11 tile o11cs culli~red UII ('EL7 

Hs~icc, clonyst~uii \\;is dolie 

~n~tially tbr about 2 \veeka o ~i C'EL? fullu\red b) CELi 111 ,111 I.lrc~. p;ih\;lcca. 

p, 

(13t 

. 

1ccort1 

. 

or rc10111ig ol' clu11g~11ctl \I~~UI, 

cIl~cLpe:~ \\ 1111 ;II,~(11' 

llic ~>CI~>III;I~ 

11ied1ci for root111g dicl 11ot slio\\ i.u~~siJcr,~hlc li.cqi~cilc) I'~I~I\~II! L,I ;il . ( lL)0o) rcl,urtc~l 

\el-) lllgh freq~iu~lcy ul'rootii~g. ;I 111c1l1od \\ l11cIi \\;I\ 11o1 rc1~ru~l1ic1blc. !\ IIII\C~ ~~~IIIIIL~IIC 

of ruotlllg on filter p;~pcr b~~tlgus III;II 

\rc~c 1111111craud III l~tli~iil ruuflnp nictli~~~n \v.I~ 

developed. Root~ng n:lr .~lau tlo~~c UII \cll~~-wlltl I~IC~~IIIII~ i111t1 ;I e111111).11;111\~ b~utly \\:is 

dolie. Rooling li~cq~~c~~c~ III IILIIII~ ii1cd1i1111 \\%I\ 5 10 S III~IC~ ~CIICS 111~111111e ru0t111g 

frequency 111 se~ii~-sulid 11icd1t11ii. I~~C~~LICIIC~ 

\\;IS \cry li~gh III I~q~iiil IIIC~ILIII~ ;I[ 70 IU 

90% cu~npared lo 5 to Ill?',, 011 ac~l~~-aul~tl 1iiet11111il IIIC ~ic\\Iy CIC~CIUI)C~ ~~~clliud 1101 

only clisurcd better li.cql~cncy but ;ilsu sIio\vcd bc~ter iiiur~>I~olc~g ;111tl IIICI.C;I\V~ 

I;LI~\I\'~I~ 

tili~c ol' tlie pl;1111s. 111 u11ie1 \+t~rcl\. e:irI) ~I~\I~~;IIICIII uf ~IIUC)I\ ei111111cd (111 ~~I~II.\~II~ 

med~uiii \+as prc\.cmcd In IIC~III~ I~IC~I~II~I 11~11,111~ lllcili :IIIICII,I~~C tot \LI~-CII~ILI~III~. 

Hu\+e\,cr, tlicrc \yere t\\o I~I:III~ tl1\:1tl\:1111;1gca uI'111c 11c~ 1) [levcl~~~~ctl 111clllt~~l l;~rilly~ llie 

shout, acqu~rcd liypcrliydric~ty u11 \crl;ll ~LI~-CII/III~III~ ,ICI 

\CCOIICII~, IIIC 1111ots blio~cd 

;In ~ncrc;~scd grow~li ralc '1'11~ Iiy~~crli~tlr~c~~y 1problc111 \\.I, i.eairlc~~~I 10 111c aI1001\ OIII~ 111 

the sub-culruring slags, .III~ 1per51a1111g /)robIc111 CULII~ be III~III'I~C~ by pl.1c111g 1l1c I.LIUIIII~ 

CUIILI~~ tubes 111 the srcr~le clivilonlliclir (I~III~II~I llu\v) hill1 CUIIOII plugs O~CII Ibr bout 

kw hours. The second problcln cuuld bc 111:111dgcd by usltig lu~~gcr culrurc tiibcs (25x200 

mm) and reltluvlng llic C~CC>>I\C Iuv,cr lll~lt US tll~ \lci11 diir~tlg thc >II~-CLII~LI~CS. 

Addltlona[ care was exercised to ;i~htc\c the high li.cq~lclicy of routing. I:irstly, dark 

greeii, healrhy ,hoots ir.1111 \r.cll-de\clupcd Ici~ilcta \bere iclcclcd ILr routlllp 1I1;it tiid no1

exceed 5 cni Icngtl~. Loiigcr shoots posed tlic sbo\c-ii~r~it~o~ied I>~UOICIII I I I C ~ ~ ~ I lcilgtl~ ~ C ~ 

in the culture tubes. Shoots III;II 

d~d IIUI 

rout aliould 1101 st.i) ior lollger riil~c 111111s aanie 

culture rnediiiill and be tKlnsli.rrcd tu 111c licrl~ IIIC~IIII~~ ,ilicr II~C tirht 0;11cl1 II;I~ IULIC~. 

Care should be riikrii iiut lo ;illon tlic aliouta 10 II~L~~III~ 

IIYIIL'I./I!~~II~II! 

bi'c;iii~c SLICII 

shoots do no1 rout \\ell a~lci Ii.i\c tu be c,iriicd IU IC ~)II~IZS ! ul'r~ot~~~g. 

tli~rdcn~iig iiiiil trC~~is~>li~~it~it~o~i 

(11' cliick~~c:~ li:i\ I)cc11 ,I ,L~~IL!LIS IJILIIIICIII 1 ~C IVI 

10 ~~~cl'licic~it 

111 \ 11ro $ri~(iiiig 

by utilizing tlic roo1 sysle111 I'rolii tile prc-gcr~i~in;~~e~l sccd1111gs (Ki~~liii;i~~~~~rll~y 

CI ill. 

~iiiiiiber ~t'Iic~~~~le~ic~l 

I>~;IIII\. 'l'lic co~ii~ircIie~~\~\ c I)IOIO~OIi)i l~;i~.clc~i~~ig ,IIII,IIIIS ivctc l;ick or 

gro\+~h of roo ti^^^ ~~,ICIII 

t~r~cl 1i~1111icli1y ;I~~~III~,III/;III~~II (~IIIE 10 111c cllic~c~~cy ~~f\I;itic 

hydlopo~n~cs s)ste~ii III,II CII~LI~C\ IC ~IO~III 111' roo^ \y\Ic111 C;III lhc coi~\~dvrv~I ;IS llic best 

nicthod for hardcn~np. I'lii. ollicr 111c1Iiud ui II;I~L~SIIIII~ ,111d II;III\~)~~I~~I;I~IOII III~OLI~II tlircc 

different stages \baa al,u 

cific~~~it ~ n rc11;lblc. d Ihu\rci'cr, \ ii~~pl~c~lj, ~i~ti. ;III~ III 1ot;iI tllc 

efficiency of Ilydropoilic\ a)slenii\ si~pcr~(~t In ;I~~I~IUII. Ilic 3-bI;igc n~ctliod cil>plu~s 

hunlldily eblabll\]inlcll[ bj uu\crlily ,ICI III~I~ gr;~dii:~l O/>CI~III~ 111 lllc I~IIC'T \I;I~cs, which I\ 

vey case sensiri\e E~cn iiliiiur \;~r~:il~u~i\ ul' Il~~iii~dily III:I~;I~~I~CIII 

re\~lltcd 111 clllicr

wilting or drylng of tl~e pliints. This prublci~i \L;I\ 

11eg;itct1 III tile I~>drul>oi~~cs syslcn~. 

However, a carckil cua~irinatioi~ US gro\\tli u1'1Iic \tc111,illti loor ~!~I~III. Ic;iS ~l~ospl~olugy 

should be dul~r ~hllc hordei~ing IIIC sooleil ~~I.IIII\ III at.itic II!~I.UPUIIIC~ I'uttii~g IIIC~IIIIII 

was one ot' tlic Inlportellll l;ictt11.\ 111;il 

,~l'Scct ?IIIc~\~~c;I II;I~~~CIIIII~ ,II:J 

~\I.~~~I\~IIIICIII. 

Chlckpca ge~lurdll) prckrs bl,ick soil liu\tc\cr. gio\\lli 01' S~ILII r!\Iclll \\.I, g~c,i~ly 

111hib1ttd by tll~s pou~~lg IIIC~ILIIII 111 :111 ilic tli~cc SI;I~CS tIc\crlbcd 111 IIIC I~l>u\~ WCIIOII\ 

Sand \\as used :it Ihiglic~ ps~~~~itiui~s IU 

I)SU\ iilc ~CIICI JCI;I~IOII 

ii~~d ~~I;III\cI) lcs. llllitlllll~ 

of bi;~ck soil \\as ;~ddctl lu I~~,IIIII;III~ ~IIC \\.I~cI 11ultli1ig C,II);ICI~~ 01I1cr I;ICIOS~ l~kc 

temperature, liglit 111te11bity. pIioto/)1'11t1tl, IIII~;I~IUII :illil \\CIC 

cilw \t,~i~tI~i~tli/~d OI)~IIII;I/ 

green huusc or LIY 

~tl~er li;iraI~ ~IIYI~UIIIII~III C,III OC ~CIIIII~CIII;I~ tu IIIC ILI~C~CIIIII~ 01' 

chlcl\lx". OII~ li~iliti~t~un ~1'111~ CIIICI\I)C;I L~\I;I~~I\~III~CIII btucIl~\ 111 I/I;II IIIC IC~IIIICIIICIII (11' 

walk-ln type gro\vtli cIi;~i~~bsr, ~VIIICII 111;1l,c\ II,I~~~CIIIII~ III~ICC\ 

\OIIIC~~.~I,I~ I.CSIIICIC(/ 10 

Seiv lnstitiiteb t11:lt c;111 iit'ii~~~l tile iiiciIil> Sliit~c II~~~OI)OIIIC\ \)\IC.III 15 LIII cfIi~,ie111 \y\t~111 

that can \harden 111 LIIIO 

prod~i~td ~IIIc~~)c;I ~~I,IIII\ 111 illc inori~~,iI CIIIILI~C 1.00111 CUI~~I~I~II\ 

without conil)sol~~i/iiig tile 111tqr11y ,iil~l v~l,~li~j. 01' IIIC pl:iill. 111 ~ilro ~ICIILC~ cl~ickl~ei~ 

pla~~ts sl>o!\ed :ill cii~ly tlt~\~crii~g ;iiltl II~:IIIISII~ 111 \OI~IC III\~;IIIC,C\ 

IIIC ~I;IIII> Ilo~vcrcd 111 

culture tubes while they \rere rootliig. 1111s siiggc\l\ III,II il~iltl cu~~tl~tioii~ of lcnlpclalurc, 

light intensity iind l~~iil~icl~ty ~>r~vidctl 11) tlic CIIIILII~ roo111 111ig11t ci~tii.it~ ~C~II~CLIVL: 

ge11c11clemc~~ts Sur Ilo\+cr~~lg ;ind III,I~LI~II) 'I III\ r,ltc ul'i~~~~t~irit~ \\a\ I~IUII~ 10 be bluib'cr 

In case of tjeld-so\\il sci'~ll111g~. I

stem and lravca Secol~d;lrq ~'IIICII~~ \\err .~lso ~pru~~~otcd b! 1111s IIC;IIIIICIII 1'111s sort 01' 

pron~otion of veget~~t~vc gru\vtl ~:IIIIS 

~I~IIII~C~III~C 

\\IIIIc I~~~IIIII;I~IIIII~ :IIILI 1proccss111g of 

l~ivaluable transgenic pli~~lts. 

5.1.3 Histological slutlica ol'i~~ultiplc slioul tlr\rlol)i~iel~t I'I.UIII .\A14 c\pl:~l~t: 

L)c\cluj>~i~c~~t of ~n~~lt~plc ~Iluurh i1t1111 :\\I4 C\~I~,IIII \\:I, 

iti111~1 I,I I~c ;IC~\CIIIIIILI~I\ 

[~Ilcllulyl~~;~ll! I-~o\\c\cs. IIICII. 

:I~\CIIII~IU~I> 11~11111.~ !\.I\ LOII~~III~C~ .II'ICI IIIC I1i~tolt1~1c;il 

obscr\;~t~u~li c;~rr~ctI utit .II d1ll2~11t sliigc\ 01' gr~\\tIi uI' 111c C\~~~.IIII 11r~i.1tllv IC ,\hl-l 

ehplal~t LIC\CIUJ)III~III oc~,~irj 111 t\\u ~I;I~L', I. 111) 10 .I\III;II! but1 ICIIILIV,~~ ,111~12. 1111 ti) 111c 

Stiipe ufrt1110\~1l 111'111~1lll~1lc JIIUOI buli UII~III~I~III~ fio111 ~C~CIICI.;I~III~ .I~C:I 

I'~C\CIICC 

01' 

ax11l;lry bud c\crlh I)IC\\LI~C 

01' ,II)IC;I~ ~UII~III~IIICC OII IC II~\LIC 01' CIII)ICLI~III;I~~ IIO~C 

regloll. Ile~icc. ;IS 

c\l~cctcd I~CIII~IV;I~ 01';1\1ll;iry hut1 IIC~.IIC\ t11c ;I~)IC;I~ ~~IIIIIII,IIICC :IC/ 

d~i'l'trc~~~ regiulib III rlrc LIIII~I 111 IIOII-III~I.I\ICIII;~IIC C ~ ~ IC\LII~\ ~ i 11 C~III l)c 

suggested that re~lloval ot'rnt~lt~plc >Iiuur bud, 011 t1.1)-5 UI d,~y-c~ ;III~ C~-LLIIIII;I~I~III lilr 72 

~OLIS~ COLII~ giic bcttcr 1icqt1c11cy ~~'~CIICIIL, ~I;III~~~IIII;I~I~II~ 

5.2 Genetic tra~~sl'urnlatioa: 

TransSirni~ng ~IC ALll c\l)lallt\ 

1t11 111c b111;iry co~i\lri~ct\ contalnlllg Ili('~:ylAb 

and SBTI genes, p~uduced cll~ckpca Irdll>gvlllcs. 111 ;lccurdancc IVIIII 

lllc convci~l~onal

molecul~ir analysis of tlic I'III~~~I~IC 

~I,!III~, 111c lpIa111s \vcrc 111111;illy scrcc~icd for GUS 

histochemical assay and tiboul 6U"u 

oi tlie ~pl~i~ilr LIIO\ICJ 

PU~III\C ~e;iitii)ii tlii/i\ gelit 

act~vity has clearly demo~~sti.atetl it! v;iactil~r tishlic, l'llc 1cr1111n;11 !o!111g lc;~~lcts hiit,\vcd 

a clear blue color ;lctl\ ity oi'tlic i~icurpol.;i~ed III,/:\ 

gcilc Ilu\\c\cr. tile 1.11cr >lager ol'llie 

n~olccul~ir ~II~:II)SIS hlio\!cd ~OI~IC \a!r~,~blc ~cholt\ III ICYIII\ 

co~itir~ii;il~on iiiid Soulliein ;~il,ilyaih. 

,)I'IIIII~~I)L~V 

OI'~~),III\~~ 111 Itc'l< 

111col.pol.nied gcllc 111\;ir1;1bly cu~ilir~llb tli~ I~YCICIICC 01'ilie IC\I)C~II\C gelic III IC ge11v111e 

ui llie lraiisgeilic plalil. Ho\vc\s~, tluc lo suliic \,irl;niulia 

1111l\iio\\11 tllc l)i'l< rc\~~Its 

showed home variable results 111 llic J~III~IIILL' I~,III\I~III~~,IIII~ ' 1 1 1 ~ j~l,i~it\ \VIIII Ui('rl,ll/h 

gene sllowcd n 50% ~l.;in~lbr~~i;iriw ilctlucncy \\1111 ~c\l,cct lo llic ~ipill kclie In lllc I'CI< 

conlir~ii:il~o~i ;ili;ilyals 

I:lic )11;1111\ 0111 01' 10 >IIO\VC~ ;IIII~I~I~~C;III~III UI'IIIC 700 bl) Ii~;ig11ic111 

tlinl \vu\ y~cc~lic to illc ~i/i/lI gclic 511111;11.1) tiic ~I~II til'0 tc\lc~I lpl;~iili \\ 1111 \/)I/ \IIII\VUII 

amplllicaiiu~l ot'llle Ical)ccII\c li,ig~iic~it, \/l~lIi IC\I)CCI lo I.! L11 II;I~I~ICII~ :~~i~pl~lic;~l~~ii 

IY01ii tile 111i1.k S~II~, 111erc \\.IS 

\l~gliIly IIICYC;I~~(I lrct~~~c~~cy 

X 0111 01 I0 /~il'r~'lA/~ lpl:1111s 

slio\ved a~npl~lii.;ii~v~~ iind 5 11111 ul' 0 .S/j77 I~I~IIII\ >ll~i+ctl ~IIC ~CII)~III\U lil~~l~l~li~d 

Srag~~icnt It c;i11 be 5~1ggcslctI lic~c lI1,il ioiiic 111111111 IIICI II~CLII~I~CII~ 111 II~c rc,!cIIoII liilxlurc 

ni~ght be 

inlerfcr~ii~ \VI~II 11ic ii~i~~)l~Iic;~t~tl~~, 

llc~)cc, tieqt~ci~c! ul co~ilirr~ied 

transfor~nat~o~~ trltli respccl IJ ilic 11ii11 kcr ge~lcb e;111 Ibc fixed III 1111. ra~lgc 01'50 10 70% 01' 

the piitatlve tra~isfor~n;~n~.r. ~~tuc11 lo~er lictluclicy ui tr;~~isiirrii~;~l~u~i was ol)servud w~tli 

respect lo llle sgronumicall!, 

II~I~IO~~~III~ ;III~ i113ec1 ~~>I~I;II~CC gc11l.i U/( rj,lilb and SBTI. 

Only four oul of 10 tcs~ed 5;1111pich iroln llic pla~its 1~111ilbl.111cd w~rl~ Iir('i1IA0 showed

arnplitication of the sopccIi\i. 905 bp lkigincni. Co~iil):ir,i~~\sly lligli OC COIIISI~I 

01'1hc 

primer and rhc gciie could be tlic sc;~son for 1111s luircr li.eijucnc) cuinp;ised to 1l1c 111;isker 

genes. 01ily oiir snii~ple isoin the plaiila tl-oiislorii~c~l \i 1111 SHI'I pcili, uut 01'9 ~ca[cd pl;iiiir 

showed the a~iiplitic~~r~o~~ 

01' rcil)cc~~~c 497 bl) ti..iy~iclit >iiggc>~i~ig SOIIIC 

II~CIIII~~CIII 

problcml \\ 1111 tlic I)~IIIICS 

c~i111110\1t1011 

Suuthesii an;lly~ls li;i\ bcc~~ IIIC 111ust ;i~ilI~c~nii. I~L~~IIIIL~IIS IU c0iili1111 1111' I)~II;III\C 

Iranslbrni;liit~ cvcr riiicc llic gc11i.11~I~:II~S~~)IIII.I~I~II ~~c\c,ircli I)cg,iii '1'111 IC~IIIIIIIIIC I\ 

\\iidcly uaed to ii~i.~l>/e ill? IIICU~I)I)I,III~~II ,iiiti cop) II~IIII~CI 

ul' tllc IIIIC~I~IIC~I gctii' 111 tlic 

genomc of tlic ~r.iii~~c~~ic ~1~1111. I1C'I( ~i~i~l)Iilic~l I~.I~IIIu~~I~ 01' ill ,lnil Hit '1.1 i:lh gllc 

\vcrc iiscd to probe tlic iiitcgs~i~c~l gc11c III llic ISCIII~~~III~ 1pl,111t\ C'111cklic;i \\:I> IL~LIIICI 111 hi. 

Iiiglily sccalc~~r;iiit 111 lhc t~h\~ic CLIIILISC syh1c111 due 10 ~!IIICII ll~c lr;il~~Ii)r~i~,il~w II.CIILICIIC~ 

rcporlcd to d;itc II;I~ bcci~ 

lo\\ ;I\ ;iiuu~itl?'$,I (K,Is ct ,II . 1007: KSI~~III,III~~ISI~I~ ci ill.. 

2000). Uiol~a~ic-iiiciIi.~~c~I IS,II~~~'~IIII,I~ILIII \\;I\ LIU~IC III~U /Y~OIIC CIIII~S)~LI$ ,111tl IIIC II~II~~ICI~ 

negnting tl~e ap1c;il tluniiii;iiicc ul' 5u111c \I~CILII OLI~. regciicl;i~ii~g ii~ni;illy, g;ivc IIIIIC~I 

bct~cr rcs~il~h 111 tcs~iih 01' I~;II~~S~SIII;I~IUII l'rct~~~c~~cy. I,, gc11c1;1110ii 111 111c IILII:II~VC 

~s~nrl'or~iiaiita~ \\a> tc~~cd ii~ ~~~euipor;~teil ~CIIC, 

:III~:IIO!III~ 7iJ''0 01' 

slio\rcd iiicorporarioii of 1111. IS;II~,~~II~ III iliclr gimiio~~ic~ IIIIII,II 

11ic ~c>tc~l pIzi111h 

CY~)CIIIIICII~~ i\lic~i the 

cotid~t~oiis for upt~n~al re,trictiuii of111c ycriuiriic [IN,\ ivcic 11o1 \t;~~idurd~~utl, shuwcd tlic 

sign" Ifor [he trallagencs ;n a no~i-apccilic icg~u~):, ul'llic blots and >l;~litia~d~/nt~oli of Ihc 

condi~io~is ,~icli ;IS 

qu;iiilirles of cn/y~ilc, i3S.4 .iiitl \r;i(cr \$,I\ tloi~ 

oild 111c ;iclual sc.;ulls 

were obtained. A lolal of 7 ~)ln~iI$ oil1 ul' Ill tr.$rcd pl;iills \llo\$cd inlcgs,illuii ol llic iii~ill

with a variable copy 11~11nber CBI slio\\ed bur cop~cs oi'intcgrated ~ipill gc~ics. S:imples 

CB2, CB3, CB7 and CBI I slio~ed tao copies each and sa~nples CB4 ,ind CB5 showed 

single copy Irisertiolis W~th respect to Ut('r~,lAh gene i~itegr;ition tile ge~~o~ii~c DNA of 

putatice transfor~ilants \\;is d~gcsted \r.itli E

Explant Preparation 

-Geminate seeds on SILI [MSG p? TDZt? pM kinet~n] for I wk 

-Prepare AME explant by removlng ax~ll:iry bud from Ihc cotyledonary notlc 

Shoot bud'intluction 

Step I: Culture AME explant on Slkl medium for 2 wk 

Step 2: Sub-culture explants with shoots buds to MS basal medium for 5 d 

Shoot elongation 

Step I: Transfer shoots to SEMl [MS+S pM 2-iP+2 pM kinetin] for 10 d 

Step 2: Transfer unelongated shoots to SEM2 [MS+2 VM GA,] for 2 to 3 wk 

Rooting of shoots 

Phase I: Transfer shoots toliquid RIM [ MS+9.4 mM KN0,+2% sucrose+5 

@I IBA] on filter paper bridge for 2 wk [60-70% shoots rooted] 

Phase 2: Pulse treatment of shoots with 100 @I IBA and culture on filter 

paper bridge in liquid MS for 2 wk [lo-20% shoots rooted] 

Phase 3: Transfer unrooted shoots to static hydroponic system containing 114 

Arnon's nutrient solution+3 ,&I 1BA for 2-3 wk [lo-15% shoots rooted] 

Transplantation 

Step 1:Transfer rooted shoots to 8 cm (dia) pots containing 2-4 mm sand. Cover 

the plants with polypropylene bags and gridually open the covers over 7-10 d 

period. 

OR 

Suspend the rooted shoots in Magenta jars containing 114 Amon's nutrient 

solution. 

Step 2: Transfer the hardened plants to 20 cm (dia) pots containing sand:black soil 

(3:2)+Cell Rich (5%) +rice straw compost (5%). 

Schematic representation of the protocol for in vitro regeneration of whole 

plants from axillary meristem explant (AME) of chickpea [Jayanand & 

Sham, 20031

Explant Preparation 

-Germmate seeds on S141 [MS& llh.1 TI)Z+2 pM kinetin] ~OI- I wk 

-Prepare AME explant by removing nxill;~ry bud from thc cotylcdo~i;~~.y ~iode 

Shoot bud intluction 

Step I: Culture AME e\plant on Slbl nictl~uni for 2 wk 

Step 2: Sub-culture explants with shoots huds to MS basal ~ncdiuni for 5 d 

Shoot elongation 

Step I: Transfer shoots to SEMI [MS+5 pM 2-iP+2 pM kinetin] for 10 d 

Step 2: Transfer unelongated shoots to SEM2 [MS+2 pM GA,] for 2 to 3 wk 

Rooting of shoots 

Phase 1: Transfer shoots toliquid RIM [ MS+9.4 mM KN0,+2% sucrose+S 

pM IBA] on filter paper bridge for 2 wk 160-70% shoots rooted] 

Phase 2: Pulse treatment of shoots with 100 pM IBA and culture on filter 

paper bridge in liquid MS for 2 wk [lo-20% shoots rooted] 

Phase 3: Transfer unrooted shoots to static hydroponic system containing 114 

Arnon's nutrient solutiont3 pM IBA for 2-3 wk [lo-15% shoots rooted] 

Transplantation 

Step 1:Transfer rooted shoots to 8 cm (din) pots containing 2-4 mm sand. Cover 

the plants with polypropylene bags and gradually open the covers over 7-10 d 

period. 

OR 

Suspend the rooted shoots in Magenta jars containing 114 Amon's nutrient 

solution. 

Step 2: Tmnsfer the hardened plants to 20 cm (dia) pots containing sand:black so11 

(3:2)+Cell Rich (5%) +rice straw conipost (5%). 

Schematic representation of the protocol for in vitro regeneration of whole 

plants from axillary meristem explant (AME) of chickpea [Jayanand & 

Sham, 20031

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17 

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" "- 

---- 

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e-- 

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1 

APPENDIX 

1. hIURASRICE AND SI(SO) 

--.- 

I 

0.025 

~- ~ 

OR 

Orgn~iics (X100) 

27 8 2.78 giL 

Glycine 200 mdL ~ 

10 1111 

h'icotir~ic acid ) 10 1111 

Thiamine HCI 

Pyridoxine HCI i 0.5 

100 myL 

111-lnositol ! 100 5.0 g5UU rill I I0 1i1i

2. Fixative used for fisntio~~ of tissue culture sun~l~lcs: 

Acetic acid and 95% Etllallol were ~ilixed at 1:3 proportions 

added. The co~ripo~ie~its wcrc sliakeri tint11 ill1 oftllelir ;~rc ill sol~~tlo~l. 'l'llu color oitl~c 

stain was redd~sll violct. Fol. Eosin stock sulutioli, I y of liosili (w:iIci. solilblc) \\as 

dissolved in 20 1111 ofdistilled water and 111;ldc lip to IUU rill \\it11 

05'i." ctl~;~~lol 

3. DNA exlrilctio~~ buffer (DELLAPOR'SA 111et11od) 

100 nlM Tris pH8 

4. Pri~nilry IV:I~~I buffer (I litre) (for Soutl~er~~ i~n:llysis) 

1 SDS 0.5% 1 

5. Secondary a'asl~ buffer (ZOS stock)(for Suutl~err~ c~~l:tlysis) 

Tris base I21 g. IM 

I'laCI 1 12 g. ?M 

Adjust pH to 10.0. M3ix i~pto 1 Iilrc wit11 water, l'h~s call be kept for up to 4 

months. 1 :20 I.e., 2 mlIL of lbi MsCl>

Table 4.1 

Induction of sorrlalic erllbryos OII hlS cor~t:~i~~i~tp eo~ttbi~~iltio~ts of 2,4,5-'1' (2.0 alld 

5.0 pM) wit11 TDZ, BAP, liilletir~ or zeatitr. Tile results rvere recorded at the elld of 4 r\eeks 

and represent meails of tlrrce replicn~iol~s.

Table 4.2 

Induction of elnbryos 011 hlS cont:lining con~binatio~~s of 2,4,5-'1' (10.0 and 15.0 phl) 

~ith TDZ, BAP, kinetin or zealin. l'l~e results were recorded at tile end of 4 \verks and 

represe~~t means of tllree replications.

Table 4.3 

111ducti011 of en~bryos OII hlS containing conibiaations of 2,4-D (5.0 and 10.0 pkl) 

with TDZ, BAP, kinetin or zealin. The results were recorded at the end of 4 weeks and 

represent means of tllree replicntions.

Table 4.4 

Induction of embryos 011 hlS co~~taini~lg colllbinstio~~s of 2,J-D (15.0 :III~ 20.0 pkl) 

with TDZ, BAP, ki~lrti~l or zealill. The res~~lts were recorded nt tllc c11d of 4 \kceks and 

represent ~~leans of t11rce replications.

'Table 4.6 

Dificrent combinations of plant growth regulators used to acl~ievc maturation of induced somatic cmbr?.nids and responses observed

~ 

Table 4.7 

lnductio~~ of multiple slloots from mature embryo axis esplu~~ts on lllediu 

containing BAP as the principal grotvtl~ regulator. A total of36 espls~~ts per 

treatment were cultured and there was 100% respo~lse in terms of ou~nber of 

expla~~ts responding. Tl~e results were recorded at the time of2"\nad 4"' weeks. 

All the results :Ire the nleall of three replic;~tes. 

NO. o1'shoo1s 

pcr c\l~l:~nt 

(nlicr 2 wcck) 

No, of shoots pcr 

cxpla111 

(;Ilk 4 wcck) 

~- 

.- -- --- - . . 

JBC' I 7 2 + 0.6 11.3+0.6 ! 

- 

JBC ? T-7- 20.3 + 1.2 . 

-____-_t- 

- -----. . 

JBC 3 20 1 2 9.7 + 1.3 17.7 + 1.2 

-- -- . .- 

JBC 4 30 2 10.7 + 1.5 13.0 + 1.0 

-- -. 

. -.- -- . 

JBC 5 40 2 8 3i 1.4 11.3? IS 

-- -. . . . 

..-.-. --. 

JBC u 50 2 0 0 + 2.0 9.3 ? U.0 

- 

JBC 7 I00 2 37+ 3.5 5.1 i 2.0 

JBC S 

-. 

5 5 8021.0 

9.7 + 1.2 

JBC9 1 10 

5 

11.3 i 1.6 

17.0f 1.0 

1 1 .0 + 2.0 

JBC 12 40 

-. 

83i1.5 10.3 + 1.6

Table 4.8 

Effect oI'TDZ, 2-iP :~nd ki~~eti~~ 011 sllouf regcnel'atiu~~ fron~ tl~ esplar~ls 

derived fro111 asillary nieriste~~ls ofcl~ickpe:~. 'l'lle ~.esults were recorded :it the e ~ ~ d 

ol2"%nd 4"' \veeks and tl~c values :ire III~:IIIS oI't111.ee replicates. 

Media 

JCII ? 

I 

- I - 

JCR6 ( 20 

. 

JCR (I 

-- . - 

/ 

3.3 f 0.6 4.7 i 0.6 

JCR l I 2 1 32 13.0 i 2.0 23 0 i 2.0 

1 ;; ~23,3?5";.6j 

JCR 12 4 5 1 4 1 32 1 15.3 i 1.5 20.3 $06 / 

1 JCRl3 

-- . 

JCR 14 14.7 12.5 25.3 i 2.5 1

Table 4.9 

Effect of pH of tile cllltare 111cdiu111 OII ~llultiple slluot rege~~craliu~~ Ibrn~ Hie 

anillory ~lleristem expl;ll~ts of ellickpen. Results !+ere ~.eror

Table 4.10 

Effect of i~lclusio~~ or cotyledo~l tissues along wit11 ll~e regeaeratisg axillar! 

1nerister11 on sl~oot for~l~i~~g cuplcity of tile ;~sill;lry ~neriste~n expli~~~ls. Results nerc 

recorded fro111 1 to 5 weeks to sl~on the pro~~~otio~~ 

of rege~lerati~~g nbilit) and rate 

of multiple sllool illductioll b) the i~lcludcd cotyledoo. 

No of slloo~s per rcspondinp esplil~~ls (weclis)'

Table 4.11 

Effect of age of tllc espla~~t donor seedlings on rcgencration capacity of 

different espla~~ts derived fronl axillnry nleristenls. Regenerating multiple slloots 

were counted in the third \~ecli n ~ llle ~ d values arc mealis of three replic:~tes. 

Seedling 

age (4 

AM I 

No, of slioots per explants' 

AM2 

AM3 

AM4 

2 

17.0 i 2.0 

17.3 i2.1 

4.0 i 1.0 

17.3 i 1.5 

4 

15.7i 1.5 

18.3i 1.5 

6.7 i 1.5 

22.3 i 2.5 

1 

6 4, 

8 

10 

12 

10.7 i 1.5 

10.3 i2.1 

7.7 i 3.1 

10.0 i 1.0 

23.3 i 2.1 

24.0 i 2.6 

17.7 i 1.5 

12.7 i 2.1 

3.7 4 0.6 

7.3 i 1.5 

3.7 i 0.6 

2.7 i 0.6 

27.7 4 3.8 

22.0 t 3.0 

19.7 ?: 3.1 

~ 

15.7?1.5 

I 

I 

14 

I I 

6.7 2 2.5 

6.3 f 1.5 

13.3 t 1.5 1.3 t0.6 11.7i 1.2 

10.3 t 1.5 1.0i 1.0 1 07il.5 1 

Mean f Standard error of tlirce replications

Table 4.12 

Induction of multiple shoot buds from various seedling explarlts derived 

from axillary meristems. 'The nun~bcr of multil)le sl~oots were counted in the tl~ird 

week prior to their transfer to the sl~oot elongatio~l medium. 

Embryo axis 

13.7 i 1.5 

I 

Shoot tip 40 36 (90.0) 10.3 i 2,lJ 

I 

Axillary bod 

40 

40 (100) 

17.3 i.2.5 

AM I 

AM? i. 

40 

40 

33 (82.5) 

37 (92.5) 

23.7 k 0.6 

30.3 i 2.9 ,* 

1 

AM3 

40 

23 (57.5) 

7.7 t 2.3 

AM4 

40 

40 (100) 

31.0i 1.7 

I 

Meal, i Standard error oC tllrec ~.eplicntioils

Table 4.13 

Effect of media compositions OII elo~igation of the regenerated sboots. Results 

were recorded from three replicate experiments involvillg sl~oots induced 011 JCR13 

medium (see Table 4.8)' 

CEL l 

2 

Plant yrowth regulators (yM) 

2 

1.2 1 2.3 1 

I 

I 

CEL3 1 - 

2 

2 1 - 

I.! 

CEL4 1 - 

4 

5 2 

1.7 3.1 

2,5 

CEL 5 2 0.3 

I 

1 I 

CEL 8 1 5 ill 4.8 I 

%ach replicate experimeih consisted ol'a total of 40 explar~ts with induced slioot 

buds at 18 to 23 per expla~~t and the ~~uniber ol'sl~oots elo~igati~rg per cxplant was 

the average of all the 40 esplants. Datr compiled from tliree replicated experiments. 

klongatio~i I: number of sl~oots elonfgated per esplant by the end ofsecorid weck; 

Elongntiorl 11: ilunlber of sl~outs elolignted on the next two or three subcultures.

Table 4.14 

Effect of media constituents on rooting of ill vitro formed aud elo~~gated 

shoots of cbickpea. 

CPR 2 (MS) 3.0 

CPR 3 

CPR? 1,,2.5 1 

i:: 1 ; 119.9 

1 5 , 7 164.00 

CPR 8 3.0 16.8 7.0 59 50 

- 

CPR 9 2.0 5.0 27.2 12.4 99 00 

CPR 10 j 2.0 1 10.0 I - 17.1 / 3.5 51.50 

CPR I1 2.0 I - 1 5.0 3,9 9.75 

- - 

'All the media aere used as liquid rontsi~~i~lg filter paper bridge. CPR3 to CPR8 

contained sucrose at various co~~ce~ltratior~s wit11 IBA as the rooting hormone added 

at 5 phf concentration. CPR1, CPR2, CPRII n ~ CPRl2 ~ d did not contain Phase 2 as 

there was no pulse treatment with IDA. 

b~esults were recorded at the end of 3 weeks for Pl~ase 1 and 5 weeks for Phase 2. 

Each combination tested uader three replication. Total umber of sl~oots per 

replicate was 40.

'Table 4.15 

1.etl1nl dose testing :III~ effect ol"SU% on Iclll:ll el'kct of I;:ll~:~myci~~. Nu~l~bcr 

of days of espla~~t survi\al was cou~~ccd ul~til tile chplal~t ble:~cl~ed or sllo~red II~ 

signs of growtl~. 

No, ofdi~ys of expin111 surviv;~l 

25 

10 

-. . .-- -

Figure 3.1 

Dii~gralll~l~i~tic rcpresc~~tatioo of the cxplullts derived from llilture embryo 

;Iris, ~~lu~llulc, r:~tlirle, sitlc ;I~IIIS 811d ~llitltlle portion. (Arrows sllows sites of 

surgery)

I'relii~rutior~ ofi~xillary ~lleristen~ exl~lants AMI, AM2, Ah13 and AR.14. 

Final sti~ge is tllc stilge of regeneration of niultiple stloots after one week of 

culture 011 slloot i~~duclion 111ediu111. Large arrows sl~ow progression of 

prep~':~tion ol' cxl11;111ts. klediu~n arrows sl~ow sites of surgery and snlall 

arrows sllow silo ol'1n111tiple sl~oot regeneri~tiol~.


Diagrn~nn~atic represc~~tatiu~~ ror preparation ul'axillary n~eristem erplant 

(AM2) showing the sites of surgery. A, 6-day old seedling sbo\ving prorl~iner~t 

i~xilli~ry butl, B. I'ruccssing of the i~xillary ~neristem explant (arrows shons 

the sitcs ol'surgery), C. Axillary n~eristc~n explant (AM2) sl~owi~~g the axillark 

~neristeln r~giun \vitl~ il~tilct cotyletlo~~.

Figure 3.4 

llestrictio~~ Innp of tl~e pl;ls~~~id pICI'99:GUS-Iet used for biolistic- 

nletlintcd gelle tr;il~sfer.

I 

. Nhu 1 51 ?C,%- 

.pal5125 &pa15125 1.- 

Iito 1 51 2 7 

-.T.i I,IPT I1 

ti710 by 

cu K\! 1640 

Ilcm, 1457: 

Act 12938 

___-/------ 

Syh 12941

Itestrictiol~ III;I~) 01' the plasn~id pHS723:Bt used for ~igrobac~erirrin- 

llletllod of tr:r~~sforl~ri~lioll.

I

Induclio~~ of son~atic cn~bryos from mature embryo axis and leaflet explants 

after 6 weeks of culture on Il~c medium containing 2,4,5-T as principal growth 

rcgslntor. A, B and C. lntluctioi~ of embryos from illature embryo axis explant, 

I)) Induction of somatic cn~bryos from lcallct explal~t.

Induction of somatic embryos from mature embryo axis explant by using 

2,4-D as principal growth regulator. A. Top view of the embryo axis showing 

multiple globular embryos, B. Lateral view of embryo axis slioning multiple 

globulnr embryos formed from plumule region.

Effect uf co~~centrr~tio~t of BAP on mllltiple sltoot regeneration from mature 

embryo nxis erplal~t 8ftcr 15 days of culture on sl~oot inductiol~ medium. A. 

Multiple sl~oot regeneration at 10 pM BAP concentration, B, hlultiple sl~oot 

rcgeneri~tiot~ wit11 30 phl BAP, C. Multiple shoot regeneratio11 with 40 pM BAP, D. 

Multiile sl~oot reget~eratio~t wit11 50 pM BAP, D. Multiple shoot regeneration with 

100 pM DAP.

I.iy111. J.5 

SI:i;i.\ iil ~ ~ l ~ ~ l i i\II(II)I j ~ l c , I(.~~.IICI:I~IIIII 11.0111 .\>I1 cq)1:111l :11lc1 ci~lllit.il UII Ill? 

\II~~III !II(~II~~~IIII IIICI~~III~I. \. IK\~I:IIII \IIII\!III~ il~il :II.C:I 111 ill:^!! l ~ ~ ~

.A cu~irj~lr~t~ scl~clrie 01' rcgelrerirliol~ 3!1d I-eco\'cry ol ~vliolc 11Ia11li Il~~uufil~ 

tiabuc 

t~r~llurc IIICIILI~LI 

r~bilrg :i\ill.~r) III~~~$I~III 

~AIJI:IIII (.AiIi?) ohI;~i~icLI fro111 ill t,i!?o Krn!\]] 

seedii~~gs of cl~icl,i~~:~. 4. .\\ili;rr! riier.iste~r~ cq~l;r~~t (.L%I2) UII llic lint (lily 01 

CIII~III.C oil \l~ii~t ir~ciuetiulr ir~edii~~~~, ii. S11oot buds r?g~r~er:i:ii~p ~'~OIII 

I)! Ill? r?i1111\.:1l 01 :1\il1;11.) b:111 :il'ler 7 d;r!s. 

ori:ii~:iriir~ II.~IIII 

\it.\! 

d:i!b 

:he 1.cgio11 icf't 

C. Cl~ibtcr 111' 111uI1i~~lc s11oot 1111db 

IC~~UII lei1 11) IIIL, I-CIJ\ :I! oi;r\iiI:tr? !iricl ;~ilcr 12 tla)s, I). .A clo\e~- 

ut llic 111ul1ipIr \I~oi)ta I~KC.IICI.~~~II:: 

I'ru111 1I1c :i~ill:il!~ IIIE~~S~CIII 

e~pI:~~rt :rller 15 

01 crill~!rc 1111 ~IIIJU~ iri~l~rctiai~ ~r~ccli~~~rr, 1;. i:i~jr~g:rli~~~~ 01' III? ~lrout i)ud> :11t?1- 7 

113)s yS CIII~UI 

L' OII b1101it elo~rg:~liu~i ii~ediri!ir, I:. 

Rooliirg ci1'llie eloi~g;rlccl slroot bnd 

UI 

the liilcr p:ilicr Ill-iilge ir~irnci~sc~i ill liquitl roolil~g lnediunr :~flrr X [I:r!s 

II~ 

c~riturc~, (;. I'icli~rc blio!\il~g 0.r i';lro slxtic II~~I~U~IOII~C~ 

hjhle~ii for- II:II-~?II~II~ 

r(jtjte11 \IICII~I 

of tiiv 

;rlter I5 il,~j$, [I. ,411 :rlrcr~~:>li\c 111et1rod l'or li:tr(ierri~ig uf I11e ~II:~IIIIcI 

uhtai11~11irroug11 tissire ciiltrirc ir~ rvlricli ll~c IWJI ,!sten1 

tlrc cultu~r f~ikrg ol tire cr~ltirr.~~ 111be I,cpt UII~II, 

?IIO!V~II: IIU~III;~~ 1111~r1il1oiog~. 

>\,:I\ irir~nt~~-re(i ill air1111 rtilll 

1. H:i~.~lei~etl :rriil t~-;inbpli~~~lcti plai~l

IIistologic:~l stutlic\ of dcveioprllcl~t of 

~rluiiiplc tneribtcri~oitl, f'ronr 

;raill;rt.y 111eriatet11 rcgiou of hhI1 capla111 al'tci. ll~e re~~iov:tl of ;cxilI;t~-)' bud. 

.I. l.~~i~;ii~~(liir;~l stctio~i 01' the :niIlary I I I ~ ~ ~ S 8re;1 ~ C ~ 011 I I tl:~y-1 ;tft?t. :txilIi~r) 

bud rcl~lov;il, 8. I)cl,eloprt~e~~i of i~rcriate~~roid, :I[ tlie I):rs;~l portiolt ol';~~ill;~r! 

botl or1 tl;ry-.? (il~' III:I~~ b ~ ;~pl~e:tri~~g 

~ d 

ih ;I sliuol hut1 ct~~el-gir~g l'rutn [Ire 

I);Is:II 

poriiot~ of the rc~~>n~e(l ;~xilI:~ry IIII~), C'. A~~IC~I~:IIICC of 111cris1~11roitis 

irr tht n\ili:~r! 11leri\Le111 arc;\ 011 d:~)-3, D. I~~cre:~scd 1111mber of IIIC~~'~ICIIIOI(IS 

iir L I I ~ :i\ill;lrj ~rlcri>ttrrl are;] oil tl;ry-4, I. 1ucl.c;rse ill rlrt 11ulnlie1. :LIIII 

yrul~t11 of rucristernoitlb oil d:l)-t;, 1'. l

C!osrr ol)sei~v:~~iuri 

tlc\~clo~)i~lei~l ai~d gru\vtli ol ~~~eristeii~oicls ill ille 

:~\iil:it.y rlicri\~i.ili ;irc:i US :\.\ll 

or1 tl:ij-4 

CY~I:III~. A. C105e1. \ ~c\Y of the ir~cri\teunoids 

:~I'lrr. tlic rc~t~oval of ;rrillar) blld, 8. C~.o\\tl~ :III~ di~isiol~ uf' 

i~ierislelli:~ticells iir 

tl~e i11cri5reri1oitl regiu~i ult day-5, C. (J1.urcil1 :rlrtl 

divi\ir~i: ui' iiie~.iste~l~;~!ic cells :rt.c~ur~tl ~iieristeiiloid region oil tl:~!..O, 

(21.ojr LIi ai~ti tii~itiol: US ~nerictc~~~:rtic cclls oS il~criste~l~uid region oil tl:iy-7, I

Rc\[ricfiot~ ;111;1l)si\ ur ill? ~ I : I ~ I I I 11$c(1 

~ ~ s for ~I-:I~I\~U~III;I~~OII. 

.i. 'I'll? 11i;is111i(I 

[IIU"N:(;L'~-~II~ (0.l) 1\11) )\;IS LI~C(! ~ I I I~ioliilic 111tt11ocI of ~I-~IIIS~~I-II~:I~~OII. L;IIIC> 1 

\l~u\r\ h1)Y.i tiigchictl willl hiC II ctl/yilr :I\ in:~rl

CliS I~i~locl~cl~tic;~l ;I,s;I) of the Ic:~flets I'ron~ putatively Lr:~~tsl'or~netl 

[~l:t111s ol'i.11icl~pca. ;\. :I cloher. vien of'thc 1c:tllei sllo~vil~g GUS acliviiy ill 

the I cirl~. 8, ('. GLiS ;~~tivity ;IS sccll it1 the pcliolc ;111d veilis ot'Ic:~Llzts.

I'C'R :~llipLiiir:iriuil 01'700 bl) S~;I~IIIPIII ul'iipi Ii FCII(. fro111 1111' g~ilolllic DN,\s 01' 

'Ic, p~l~ci~iiliuii 1)1:11ilb ~~:III~~'UFIIIC~ will^ lit cry1:U~ :III~ SU'I'i zeilc\ sia 

~~,~~o/~~I~~~~~~IIIJI-III~L~~:I~c~~ 

lr:i~i~lor~~~:iliu~~. \. II~J/II-I'C~< of 111:111ts tv;i115lor111cd ~ v k i ~ I ~ 

[)llS723:iit 

~~LIII~~~I~III~~I~ ~:IIII[IIC\ 

si.1 .~I~~~~b~i~~r~i.i~r~~~-rl~ctli~~tecL 

~I~;IIIS~'~I~III:I~~UII. 1,:tnes 1 cu 1U liad 

eoi~u.ui i111i1 I ? Iu I7 r\cl.c pu,ili!c 

;IIIII 5111ii\ IIIC ;~i~~])Iilici~cioi~ of i1~1tll XCIIC. ~.:IIIP I I ~icg:~~is' 

cu~ilvolb I'rolll pl:lsnlid 

1111SiZD:Iil 115cd I'or 

~I;I~I\~'UI-III:I~~OII, 1 :III(, I3 i\ -DNA;III(I 

h. L)\.-i-lJ.\tE 11 III:I~~;~I' \Y;I\ ;idiic

l:iyuris 4.18 

IY'lt ;~ri~l~Ijl'ic;lriu~~ oc 1.2 l

Fixi1r.c 17 \r:15 -l)K,.\ ;!lid J" l)>.-i-/J.s~li 11 111i1r1xr ir.15 :~~f(lril in I;IIIV 18. I

1:igitt.e 4.?O 

1)S.t 111.uIile 01' gc~t(~tt~ic 1)Y.i i\ol:~le(l ~'KOIII 1)111:11i\e tr:ti1si'o1-111;1111\ u1' c11icL~)c;t. 

\, 1'11rili?~l I)\ \ j)l.o!ik ui :ettol~iir l>h':i (11' I~I~~J:I~/~I~ 

[)l:tt~l~, 1)a.A \!;I\ III'C~);II~~~~ i ~ t 

tlttplic;~tc~ r;itt11jlcs, 1 :III~% I ((I I0 ~IIUIJ~ firs1 set :~tid l:tt~cs I I lu 2U slieins tl~c hecot111 

set. 13. I

1:igurr 4.22 

Sui~lllcrn :III:II!s~$ US tllc 16 ~ C I I C I ~ ~ ol'li/Oj~1/10 

~ ~ O I I 1)111:1ti\~' lri~i~sgei~ic ]I~;III(S 01 

GCIIUIII~C IjX,\ 

ci~icl,~~c:~ ir>i~slur~~~cd \i:i .~l,~~~~~/I~~cte~i~~~~~-~~~cili~~~il 

~~~IIIS~U~III;III~I~. 

\)a\ rcslricrctl r.iii11 licoltl ruzyilie. :\r~:~ly$ia ol cop! i~u~~il~c~. \\it11 respect ro Illc 

riprll ge11c. L;III?S I 

to I I \\ere ;icIcle(I \\it11 ~ ~:IIIS~O~II~;III~ 

\;111111le>( XI 10 Ci311. L.;IIIC 

I: C:I%I~ I.;I[IC 2: ('II:. I.:IIIC 3: (~13. L:IIIC 4: ('IN, Lme 5: CB5, L.~IIC 0: CB~. 

~.;IIIC 

7: CE7. I.:IIIC 8: L'US, I,;IIIC 0: CUY, I.:IIIC IU: Cl$lU : III~ L:III~ I I: Cl31 I. 1.a11c IZ IILIS 

Ill? ~~q;:~ti\c C U I I ~ I U :if10 ~ I;IIIC> 13 II:I\ t11c 11ubi[i\e cu1111~uI (l

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