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GENETIC TRANSFORMATION OF Cicer arietinum, L<br />
FOR INSECT RESISTANCE<br />
I&44Lhah!b<br />
SRI VENKATESWARA UNIVERSITY<br />
~~~<br />
G . t ~ ~ ~ 4 ~ + 4<br />
DOCTOR OF PHILOSOPHY<br />
IN<br />
BOTANY<br />
GJ<br />
B. JAYANAND<br />
DEPARMTENT OF BOTANY<br />
SCHOOL OF BIOLOGICAL AND EARTH SCIENCES<br />
SRI VENKATESWARA UNIVERSITY<br />
TlRUPATl - 517 502<br />
INDIA<br />
JANUARY, 2003
CERTIFICATE<br />
Certified that the entire work embodied in this thesis entitled<br />
"Genetic Transformation of Cicer arietinum L. for Insect Resistance"<br />
has been carried out by B. Jayanand under my guidance in the Genetic<br />
Transformation Laboratory, International Crops Research Institute for the<br />
Semi Arid Tropics (ICRISAT), Patancheru, Hyderabad, India and that no<br />
part of it has been submitted elsewhere for any degree or diploma.<br />
I(\( ,L, ,%<br />
' 1 1 r 1<br />
Dr K. K SHARMA<br />
Research Supervisor<br />
Hyderabad, Inha<br />
January, 2003<br />
DR. KIK4RI '( St-14RM4<br />
Cen-'icT ' . .OlVtO v<br />
ICRIS"' . L<br />
Andhla FrdJ,sh 502323, INUIA.
Certificate<br />
Ger/$ed /La/ /Ae eldire morL emGoded ;I,<br />
/B>s /Aesis en/i//ed<br />
"Genetic Transformation of Cicer arietinum, L, for Insect<br />
Resistance" LUJ Gee21 curried ou/ 6j 8. Jayanand unr/er my<br />
yuidance ill /Le ~e,var/men/ of'~o/uny, 3'ri ve~~nh/erruuru Ulioesiiy, .<br />
1'<br />
Xrupu/i, 9ndLru %odes4 Yn;rJlu a11 J /La/ no par/ of i/ As 6ea1<br />
suln2i//edel;euLere/br a ~ deyree y or d+/otno.<br />
Tirupati<br />
January, 2003<br />
Dr, G. SUDARSANAM<br />
Sii,lleI'~~~O!'<br />
,>,,,* P - . ., /<br />
p, , ,, '<br />
5 I<br />
('<br />
7, , -;.:,..;, ., ;, \ , ,, I,,,. .,
DECLARATION<br />
9 hereby aeclare that the bis~eratiorr entitlob "GENETIC<br />
TRANSFORMATION OF Cicer arietinum, L. FOR INSECT<br />
RESISTANCE" is an oriuinal and inbepenbent recorb o$ research work<br />
unbertaken by #re during the periob 06 my study nt S ri Uenkateswarn<br />
Mniuer~ity, qirupati, unbar the superuisidn 06 Dr. G. SUDARSANAM,<br />
Departuient 06 Sotany, 5.3.2.5., 5.3. Mniuersity, Ti+upafi,<br />
and that it ha$ nof prooiourly been subwritfed $09 the awarb o$ any<br />
other begree or biplorna 06 any Mniuersity.<br />
Tirupati - 517 502<br />
Date : Jan. 2003
I deem il as a great pleastire to exprcss my IicartSelr grat~tutie and profouiid<br />
respect to lily Researcli Supervisor, Dr. G. Sudarsanam, Associali: Professor, Dep:, or<br />
Botany, SBES, Sri Ve~ikateswara University, l'irupati. His colistant, ilispiril~g guidaiice.<br />
dedicntio~i, patieiicc, iinliri~ig, incessant e~icouragenient, eve~.last~~ig smile and unfl~gg~ny<br />
interest shown tliroughout my rcscntch is really i~nme~iior;ible for tile complclion oTrhis<br />
~esearcli ~\orl
"I'm convinced biotecllr~ology is going to help us. There's fear,<br />
but biotecllnology has been going on since the beginning of' time. Mother<br />
Nature was crossing plant genes loug before scierltific man and<br />
agricultural lrlall began doing it. If you like to eat spaghetti, you are<br />
eating a GlIO that Mother Nature made."
CONTENTS<br />
ABBREVIAI'IONS<br />
SUMMARY<br />
LIST OF TABLES<br />
LIST 01; FIGURES<br />
I. INTIIODUCI'ION<br />
2. lIE\'IEW OF LII'EIIAI'URE<br />
2.1 Gclietic trnnslorrnation<br />
2.1 1 Varioi~s liictl~ods olgclielic tralislbrinatio~i in 1pi:iliia<br />
2.1.1.1 Gcneric tr;~nslbnii;l~io~i by the biol~slic psoccss<br />
2.1 1.2 .AIlern;~tlve ~iietliutls olgullcl~c I~alislbl-iiial~o~l 11110 pI,:nl cells 12<br />
2 1 hl~laclc riiic~oorg,~~i~s~ii: ,,!gi.ohiir,/r,i.i~iii 17<br />
2 1 2 1 TI i~lasmitl ;111d IS C / ~ ~ I ~ ~ C ~ C I . ~ ~ ~ I C S 1 j<br />
2.1 2.2 ioleculiir ~~icclia~iisiii olT-DNA ~r:i~isicr ~nto pllillls 15<br />
2 1.2.3 Tools ofycnct~c ilansi'or~~il;il~o~~ 15
2.2.2.1 Organogenesis<br />
2.2 2.2 Sonlatic enibryogellcsis<br />
2 2.2.3 Ollier ~iietliods<br />
2.2 2.4 tic~~ullc 11,111albril1~1l1w<br />
2.3 Insect rcsistaiice ~~~a~iageiiicnt<br />
2 3.1 Pvls~liotls of co~iirol olhci. 1l1,iii b~olccllriolog)<br />
2.3 1 . I Ecological control<br />
2.3.1.2 Pliysicnl colltrol<br />
2.3.1.3 Chemical control<br />
1.3.1.4 B~ological conirol<br />
2.3.2 Biotechnology for insect resistance<br />
7_,3,?.1 BI: An amazing concept<br />
2.3.2.2 Genes employed [or 1nscc1 reslstnncc 01I1er tli~li DL<br />
3, hlATEKlALS AND METHODS<br />
3.1 Pli~li~ ~iiilteri;ll and culture co~id~l~o~ls<br />
3.2 Rcgelieration<br />
3.2. I SOII~YL~C e~iibryogeliesis<br />
3.2 I I Prepar,itio~~ of various csplants<br />
3.2.1.2 [~ltlilction of solilatic e~iibryos<br />
3.2. I .3 Maturation of sumaric eiilbryos<br />
3.2.2 Discct d11i1 Indi~ect org;inoyznssis<br />
3.2.2.1 Prcparntiorl orexplalits<br />
3.2.2.2 Inducr~ol~ of mi~lliple sllools
3.2.2.3 Shoot elongat~on<br />
3.2.2.4 Rooting oi'slioo~s<br />
3.2.2.5 Hardening ,ind trCi~isplaniatio~i orrootcd planti<br />
3.3 liisioloy~cal studies on 111~1it1plc SIIOOI<br />
i~~~iiaiioii !'ro~li tZM4 ehplant:<br />
3 4 Cic~ietic ~IXI~~~~IIIJIIOII<br />
3.4.1 'Irn~~sibr~iint~oi~ by b~oiistics ~iielliod<br />
3.4 I. I L,I~~ii-prcl):~r,~l~u~i<br />
~I'~II;I~IIIIcI DNA (AII
4.1.2.2 Eloiig;itio~~ ofllie siloor buds<br />
4.1.2.3 Rooti~ig of the elo~iyatcd sliools<br />
4.1.2.4 H:~rde~l~ng and \ra~isplanla~~o~i of rooted pla~its<br />
4.2 Histological slud~es of multiple shoo! development from AM4 expla~it<br />
4.3 Gc~ie~ic transfoniiat~on<br />
3.3.1 Biolistics ri~clliod of gc~ietic tra1lsfo1.1linr1o11<br />
3.3.2 T~arisforniatio~l by il,qi~oh~~c~~ei~iiiirr ~ilerliod<br />
5. DISCUSSION<br />
4 3.2.1 GUS liistocllem~cal assay<br />
4.3.2.2 Molecular analysis of To gelieratio~l pi~tative transgenic<br />
5 1 l'issi~c culti~rc suidics<br />
platlts ~sarisSorriied wilh pHS723.Bl and pHS737:SBI'I<br />
b~nary vectors conlaining B/Cr~i/lh and SBTI genes respect~vcly<br />
5 1.1 So~natic siubr)ogenes~s<br />
5. I .2 Ol.ga~iogencsis<br />
5 1.3 Histological stud~es of iiiuliipli. slioo~ dc~elop~netli<br />
from AM4 explanl<br />
j,2 Genetic t~.ansSor~ii~tioil<br />
5.3 Co~icliisio~is<br />
6. REFERENCES<br />
AI'IiENI)IN
ABBREVIATIONS<br />
2-il'<br />
2-isopci~~cnpl ;tdc~iillc'<br />
2,4-0 - 2,4-Dicl~loroncclic acid<br />
2,4,5-'I' ~,~.~-TI-I~I~I~I~~:IC~I~C<br />
acid<br />
AD ..lsc,oc~/~~~/cc I311gl11<br />
AUA<br />
BAP<br />
~\bsc~ssic ac~d<br />
Bcnzql Alllilio PLII-III~<br />
BGM - UO/I:I /is Grey Molt1<br />
'<br />
Bt - ~trcrli~rs ililii-i~rgiel~srs<br />
Bti - L~~~ccli~r~<br />
/~IIII~III~~~~IIAI.S-;A~~IL~~LJII.~~S<br />
CaMV 35s - C'alillo\ver Mos;iic V~ri~s 35s proniolcr<br />
CAT - Chlora~npl~iliical ,\cciyl Trallsferasc<br />
DNA - Ueosqr~bonucleic ticid<br />
FA0 -- Food anti A ~~IcLII~II~~<br />
Orgnni~at~ol:<br />
GA - Gibbzl-cllic aciW<br />
IAA - Indolc-3-ncclic ac~il<br />
IBA - Illdole-3-bulyric iicid-.<br />
ICRISA'I' - Internaiio~l~ll Crop Rescnrcll I~~s~iti~tc. Tor Seini-Ar~d fropics<br />
ICAKDA - Illicl-ilatio~~:il C'cnlel- for Ay~.iculu~r,ll Kcse,lrch ill [lie Dry Arzai<br />
IPhl - Inrcprnled Pcsr Matlnscine~ll<br />
111111 - iiiill~iics '<br />
MS medillm - Murnsllige and Skoog ~nediiini/<br />
NAA - Naplitl~alene Acer~c Acid<br />
I - Nsonlyciil Pl~osplio Tsa~lsks~sc I1<br />
PCR - Polyllicrasc Clinill RsCic.tion<br />
Pls - Protz'isc Illhibilors<br />
QTL - Quo~itit~itive Tra~t Loci<br />
RNA - Iiibonucleic acid<br />
SB1'1 - Soybeall Trypsin lilliibilor<br />
SCRl<br />
Scottisll Crul) Rubi.;~rcl~ l~lblll~ilc<br />
sec - seconds<br />
1'-DNA Tr:ii:sSe~. DNA<br />
TDZ -- Thidinzuro~i '<br />
'Ti plasnlid - l'i~rnol. i~itiuci~lg pl,~slllid<br />
iirtiA - (3-Gluciiro~ii~l~isc
SUMMARY<br />
Agriculture con~ributes major part of world's food especially i i ~ the developing<br />
world. Over 200 plant species are cultrvable ou~ of whicli rlce, wheat and maize make<br />
70% ofthc total output. Legunles have very important attributes such as protein arid lipid<br />
rich seeds and symbiotic nltrogen fixat~on, which qualifies them as best alternatii'e food<br />
crops. Legumes lire broadly divided Into food and forage legumes out of wli~ch forage<br />
legumes for111 major part. Chickpea is one of the most important leguminous, cool season.<br />
alternative food crop cultivated prevalently in Asin I'acific region. Even though it lias<br />
co~ivincing nutritional importance, its area ot'cult~vatio~l has been low anti i~iipro\ement<br />
virtually stagnant. Conve~itional breeding lias not been an effective crop improvement<br />
strateyp Ibr i.iiickpea and recclit advances in biotech~~ology s~rcli ;is plant t~ssue culture<br />
and gc~lctic irailsl'ormatiun, paved tlie way for aliernntive crop Iniprovcment<br />
metllodologies. 1'111h work \\as carr~ed out with principal object~ves of optimr~ation of<br />
tissue culturc iariables n11d tra~ihfo~~lldt~or~ of selected explo~its by ujlilg genes fro111<br />
Bacillirs 11iio.irigietisi~ Ui('ryL,lb ;itid soybeall trypsi11 inliib~tor (SUTI) geilrs io co11ti.r<br />
resistance to ~l~licovctprr cu.~~go.(i or tile legli~iie pod borer. The work can be broadly<br />
divided into tllree parts, I. So~liatic rnibryoge~lcsis, 2. 0rg;inogenrsis and 3. Genetic<br />
transforniation.<br />
S~iri(~iic e~~ibr):oge~~esis: Differo~it explnnts like mature embryo axis, parrs of<br />
nlatilre embryo axls, leaflets, stell1 seg~~ie~its anti root segrnci~ts \\ere derived bum 111<br />
vitro growl1 seedlings of different ages. Soinntic e~ubryos were induced by ilslng ?.1,5-T<br />
and 2,4-D as principal growtli rcguiarors ill co~~lbinntioli wit11 Iiorn~oncs like hllletiil,<br />
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<br />
maturation and rege~icration of embryos induced were unsuccessl'ul though good number<br />
of media coinbi~iatio~is involving ABA, zeatin, BAP and TDZ were testcd. Hence fu~.tlier<br />
work on the somatic embryogenic pathway of regeneration war not carried fonvard.<br />
Oqtriioget~csi~: Vnr~ous cxl~l;inh such as nlature e~itbryo axis, shoot tip, Icalleta.<br />
leaf' base, sten] seyn~enta, l~ypocotyl, epicotyl, root segmclits, root tip, ;~xillary bud.<br />
cotyledo~iary node and axillary nleriste~n explants (AMI, AM2, AM3 and AM4) werc<br />
prepared l?om 111 vitro grown sectllings and their multiple slioot regencrati~~g eflicicncy<br />
was tested via direct and indirect orgaliogcnlc pathways. Multiple shoot inductioli<br />
frequencies \\ere tested with slloot ~nduction mediilm (SIM) that consisted of4 p.LI TDZ.<br />
10 pM 2-iP and 2 pM klnetin. Explallts that do not co~itai~i ally traccs of pre-esishg<br />
meristerlis, si~cl~ ns liypocotyl, epicotyl, leaflets were also uscd so as to achieve<br />
rege~irratioii via callui phase. It was observed that i~tdirect orgauogenesis via callus pllasc<br />
cannot he acliievcd fro111 tlie abo\e-mentioiicd esplants with any of the tcsted ~nedia<br />
comb~nnt~oiis It \\as also observed that asyncliro~ious iiiultiple shoot regenerdtion could<br />
be achieved II-OIII<br />
pre-ex~sti~ig il~eriste~ns of expla~ita likc sliuot tip, axillary bud. Verq<br />
low frequency oisl~oot iliductio~i was obwrved wit11 l~ypocolyl and ep~cotyl and it was<br />
found th;i: k\v slioots tIi;it originateti werc fro111 traces of nieriste~~is associated n,~th sonle<br />
of the expl;~nls. Diised on ll~rsc resul,rs it \vas apparent that :~dve~ititious regeneration is<br />
very difficult wit11 o no st of the explants and inod~licario~i of the tested esplants. Four<br />
axilla~y meristem expla~its iianirly Akll, AM2, AM3 and AM4 were prepared so as to<br />
achieve adventit~ous rcgclielxtion 2nd tlirce of ~IICIII AM], AM2 and 1\hl4 were found to<br />
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<br />
experiments where negation of thc aprcal dominance of axillary bud and shoot t ~p was the<br />
salient feature of this explant. Removal of axillary bud followed by regeneration of<br />
multiple shoot buds ga\e bctter adventitlous regcneratlon and multiple wounding sites.<br />
Multiple shoot induction was found to bc betrur on liled~uin containirlg TDZ, when<br />
compared ro the medium contaiiiing BAP. However, it \+as observed that prolonged<br />
cul~c~rc uf llic cl~l;inls o~i 'l'D%-co~l~:~~~iiiig liicd~~~~ii ~ieg:~livcly i~it~rfcrcd wit11 I11rtI1cr<br />
elongation. Ilc~ice, [lie inclus~on of TDZ was restricted to gerriiination and first pliasc of<br />
induction. Sevcl.al other factors played an iniportant role 111 shoot bud induction. For<br />
example, acidic pH sliowed efficient ~~iductiuii of niultiple shoots. Age of the seedling<br />
was optimzed for all axillary meristeni explants wliere AM4 preparation required 5 to 6<br />
days for the rcn~oval of axillary bud and another week for removal of multiplc shoot<br />
buds. Inclus~on of cotyledo~i with AM4 explant was found to enhance multiple shoot<br />
regeneration frequency. Two step elo~igation us~ng shoot elongat1011 medium (SEMI; MS<br />
with 5 pM 2-iP and 2 1M klnct~n) in the first step followcd by 2 to 3 passages of sub-<br />
cultures on SEM2 (MS wit11 2 pbl GA,) resulted a better elongation frequency. A novel<br />
rooting sysrem was developed by employing filter paper br~dge tccli~iique aid tile liquid<br />
root inductloll mediuni (RIM) consisted of MS will1 5 pM IDA. This ~rietliod resulted in<br />
high rooring as ~vsll as transplantation frequelic~es. .All clficient liardeniiig nild<br />
transplaiiration method was standardized by optimizing variables such as potting<br />
medium, temperature, humidity, irrigation and phoroperiod. ~onviron~" growth chamber<br />
was found to be better Fac~lity for Iiardening arid i~i~tial phase of tnnspla~itation while pod<br />
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<br />
nutrie~it solutio~l.<br />
heric,iic irn~i~joj~nriuiio~i. Genetic tri~~isSor~~~;~tion ol' cli~ckpcu ivas achicvcd by<br />
using Ah14 eupla~its as ll~u startlllg plo~~t ii~atrrial and butli biol~st~c illid Agr~~h~icii~riii~)~-<br />
~iied~ated ~iietliods wcrc employed. A vector, ~RTO(I:GUS-III~ nla~i~tn~~led 111 E, cull was<br />
used 111 b~olisric process. 'fh~s vector li:rd irpill pc~ic ;IS ~lic aclcct;~blc ininrkcr and r~itli\<br />
gem nb the rsporrcr. Prcpar;~t~on of thc cxpl,i~ils, niicrocarricr<br />
preparation and<br />
bombard~ncnr of tlie expl:i~~ts was ci~n-~cd out by al;~nd;lrd j~roccd~~rcs. Largc SIX 01' llic<br />
AM3 cxpla~~t was a li~liitatio~~ in 1111s process ns olily fi.w cxplu~ils could be grouped st tile<br />
center of the petri plate. l'ra~lsl'or~iiation by tliis ~lietliod d ~d ~iot result in creatio~~ of any<br />
transgenlcs as some of the selected slioots d~ed at tlie stri~~gerit seluction bkp.<br />
J~grobatie~.i~~~ri-~~lediated transfor~i~otion rcsuited in relatively higli IYeque~icy of<br />
transgenies as far as 111s TO results wcrc considered. Two binary veclors ~ia~iicly pl-IS<br />
723:Ut and pHS 737:SBTl based 111 stra~n C 58, wcrc used for co-cultivarions. Tl~c former<br />
has 81C'ryIAb as agrono~iiic;illy important gcne, ~lptll as thc seiectio~~ 111;lrker and iridA<br />
gclie as ~.cportcr \\l~ere i~s tllc later binary vcclor liad soybcc~~ t~-ypsin i~illib~to~ (.SljTi) as<br />
tlie gcne of agrono~n~c ~ntercst, ~ipill as tllc re1ect;iblc marker arid ilitlA gcnc as reporter.<br />
In both ~hc vectors ripill ;)lid iiidA genea ivc~c I't~hed ~nto ;I singlc unit tl~ougli their<br />
products exli~bit indepe~ident activities. AM4 ehpla~its ivc~c co-cult~vatcd with<br />
Agrobucicriirr~l stralli of lrltercst follow~rig tlie standard procedures. Ccfotaxiiiie was used<br />
to terminate tlie growl1 of Agrobucieriu~~i and kanamyein was used for select~un of the<br />
putative rransfornia~~ts. Starldtlrd~zed protocol of icgeneralio~l was used fu~ ~ege~ieration<br />
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<br />
designed for growlng rra~isgi.nics. A total of I I plants w~lli UiC,?;lAb ,ind 9 pla~its w1t11<br />
SB77 genes were obta~iicd. Molec~~lar analysis of tlic>e pi~ti~t~ve tr,ins!'o~nia~its was done<br />
irlitinlly by GUS hisrocliemicnl assay followed by conlir~untion by PCR and Souther11<br />
blot analy~is. Soutlicrn blotting \\as done by 11o1i-rad~o~ict~vc ~iicthod by us~ng tile<br />
coniniercially available no~l-rnd~o;ict~ve AlklJlios clirect 1nbtl111g iir fro111 A~ncrsl~a~n<br />
(USA). PCll a~iiplificat~o~l of tlpill, uiiiA, B/Ci),lilD and SB1'1 gc~ics \r;;is carried out for<br />
preliminary screening of the putatlvc transformar~ts, About (10% of [lie Iputall\e<br />
transformants sho\ved positivc reaction for PCR for ~rpiil, 70% for ~iidi\ genes, 30% for<br />
BtCrylAb and 10% for SET1 gciic. Factors affecting restriction of geno~nic DNA such as<br />
enzyme conci'ntration, watcr and BSA were opt~~ilizcd. Southern blot analysis of<br />
B~CrylAb piarits sliowsd 70% ofpla~~ts ivill~ /lp/Il and BIC/yMb gene integrat~ons.<br />
In co~iclus~on tlic rcgencratio~i protocol developed during tlic course of this study,<br />
was tbi111d to be very eflic~cnt slncc culli~re coiid~r~ons for all tile stages of rcge~~cration<br />
and recovery of 111 vim grown plants tllat incl~ded seed geriiiirlatio~~, rnultiple shoot<br />
induction, elongatio~~, rooting hardening ;ind tra~isplantatio~i were optinlized, l'h~s<br />
protocol w:is effect~\,cly i~scd for succeasl'i~l geilctic trn~~ifor~~iatiu~~ of cllickpca w1t11<br />
insecticidal genes like BiCrjIAb and .SUTI genes. Follow~~ig tliis protocol, 40% of the<br />
sclccted putat~\c transgenic plants can be obtn111cd 111 per~ud of 90 to 100 days. In the<br />
present study, over 30tr;1nsgen1c plants carrqing U/C',),IAb wcrc obtained. Eight of these<br />
pl:iiits arc being 11in111taincd Sor cspcri~~ic~ir;~l iuorl< 011 illsect b~oassayi and field trials.
LIST OF TABLES<br />
Table 3.1<br />
Pr~~iier composlllons of all the Sour gellcs used Ibr penruc trdndirlwt~un. tlluir rr>pcr.t~vc<br />
annealing tempclatures. 2nd s~zc oSIhe rehpectlve liaglilellts umplliieti.<br />
Table 4.1<br />
Inductlu~l ol' aom;ltic clnblyuh oli MS ~UIII~IIIIII~ cu~lib~~i;lliulia ui 2.4.5- l (2.U aid 5 0<br />
IIM) with 'TDZ. Uhl', hlnelln or zearln, l lie results \\c~c recorded at tllc end 01 4 \vcch\ slid<br />
represent mcans of thee repllcalions.<br />
Table 4.2<br />
lnductlun of cmbryos on MS contnlnlng conlb~n:~t~ons oi' 2.4.5-'I' (10.0 o~id I5 0 phl)<br />
with 'I'DZ, BAP, klnetln or zealin. Tile results ac~c ~ecorded at l!ic elid or3 weks and rcproc~il<br />
means ofrhrev rcpl~cat~ons<br />
l~iduct~on of e1i1bryos 011 MS coninlnlng con~b~~lations of 2,4-11 (5.0 and 10.0 pM) wltll<br />
TDZ, BAP, klnetln or zearin. 'The result, were recorded at Ilir end of 4 wccks and rcprcaenl<br />
liicans oi'rluei. ~epl~cat~ona<br />
lnduct~uli oSenibryos on .CIS contalnlng cumb~narions 01 2,J-1) (15.0 and 20 0 phi) wllh<br />
TDZ, BAP, kllietln or zealin The resulls wc~c reco~dcd at thc c11d oi' 4 wceks and represent<br />
lneons ufthrec rc]~l~cal~uns.
I~iduct~o~; of e~nbryos from varlous ciplanis 'llic ehpla~its \\crc. culli~rcd 011 JEhl?!,<br />
~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'<br />
thrcc rcililctlte5<br />
Table 4.6<br />
Table 4.7<br />
lnduct~on of ~liult~ple shoots from nialure embryo axla cxplatita on 111cd1a cuntalnllig HAP<br />
aa [he pr~ncipal giowlli rsgulalur. I\ total or36 exl)la~its pcr trealmclll acre cultured and lliere ivas<br />
100% response 111 terms of nu~iiber uCcxplants rcapond~np. I'hu rcsul~s wcrc rccordcd at tlie IIII;~<br />
of 2"" and 4"' weeks. All llie results are the niean of t1;rc.c. repl~catca.<br />
'Sable 4.8<br />
Effect oi"I'DL, 2-11' a11d k111cl11i 011 slloot rCgCl;erdllo~i ho11i the c~~IJI;~ der~\cd Iio111<br />
ax111ary rner~steli>i uf cI11ckpe3. 'I he results here recorded at the end US 2"\11id 4"' \\,eeks and tl;c<br />
\.olues ore nirans of 1l11ee ~~eplicales.<br />
Effect oi pi1 oC [he culture ~i;ed~urii on r~iultiple sl~ool rcgclicrJllon li)r~ll llic ax~llary<br />
mer~stcm cxplants ot'cli~clilxa. Rcaulra were recurdud at tllc elid of 3 \becks and tlic values are<br />
liltan from three repl~catcs.<br />
'Table 4.10<br />
Effect of ~nclus~o~i of cotyledon t~ssucs along w~th tlic rcgeneratlng ax~llary mer~stc~n on<br />
shoot form~ng capaclty of the ax~llary ~iier~itenl explal~ls Resulta were recorded from 1 lo 5<br />
weeks to show the plomotlun oi'rege~ierat~ny abil~t!' and rate oC rnult~plc aliout ~~lducllun by the<br />
~ncluded cotyledon
Table 4.1 1<br />
Effect ul'age uf lhe expI0111 donor srl'dl~~igs oil regerlrrdilull ciip~city ot'dlfire~it ehplanls<br />
der~ved from axlllary nierlstenis. Ilegeneratrrig multrple rhuuts i\'ele counted In ~hc tlirrd wrek<br />
and the \aIuer are ~iiea~is ~ftlirr~ repl1~a1c.s<br />
Tablc 4.12<br />
Inducriun of ~ilt~li~pli. blluul buds liulii \JIIULI~ se~dlllig i.\pla~i~r de~~vcd ii.oni ;lx~ll:~sy<br />
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<br />
tlic sliool clung~l~un 11leLi1~11il<br />
Tablc 4.13<br />
El'tec~ of mtd~a cornposltlons on elo~ig~t~on ol Ihe rugericrated slluols. Results scrc<br />
recordcd li-urn Ili~ei. ~epl~c~lc c\pcrrliicnla ~niol\~rlg shoots III~UCU~ UI~ JCI(13 rncd~llrn<br />
ch~ckpea.<br />
Eficct ui' iiiedln co1islllucnla on loullng ol' 111 i'ltro furmcd arid elong~trd allools ol'<br />
Table 4.15<br />
L'tlial du,c Icjllng ,rnd cl'fect of'l LIZ oil Ictll~l cficl of ka~larnyc~~i, Sumhcr ol'ddyb of<br />
expluiil suri~val w.13 couri~cd u1111l Ilie cxplun~ bleached 01 sliu\bcd nu rlgns of gro\i.111.
LIST OF FIGURES<br />
Figure 3.1<br />
Diagr:~mn~attc rcprcsct~t;~[to~i of ll~c cxplanls dorivcd li-0111 111ati1re ct~lbryo i~xls.<br />
plumule, radicle, s~de asins and m~ddle portio~~. (An.ows shows sites of surgery)<br />
Figure 3.2<br />
Preparation of ax~llary ~neristem explants AM), AM2, Ah13 and Ahl-l. t:i11;11<br />
stage is the stage of regelleratio~i of l~~lllltple SIIOOIS nlier otie \beck of c11I1ure 011 slioot<br />
~nduclion medium. Large arrows sho\b progressloll of prepamlion of expiants. Mcdiu~~~<br />
arrows show s~tes of surgcry and sniall ,isrows show sites of ~n~ultiple shoot rcgcncratior~.<br />
Figure 3.3 A - C<br />
Diagrammatic rcprescrllatioli Ibr preparallon oC nx~llary rnertsteni explant (AM2)<br />
showing the sites of surgery. A. 6-day old secdlitlg sl~owll~g pro~nincnt ;~xillary bud, B.<br />
Processing of the axilla1.y meristem explant (arrows shows tile sites of surgery), C.<br />
Axillary iilerls[cm sxpl,~nl (AM2) sl~owi~lg (lie ax~llary 111cris1e111 rsgioli w~th intacl<br />
cotyledoli.<br />
Figure 3.4<br />
Rcslrictiu~~ inlap of rhc plasmid pll'l'99:GUS-lnl used for biolistic-mediated ~CIIC<br />
transfer.<br />
Figure 3.5<br />
Figure 3.6<br />
Restriction map of the plas~u~d ptIS737:St31'1 used fo~ .4g1~obcic~eriiit11-1netI~od of<br />
transfomiatlon
Figure 4.1 A - D<br />
inducl~on of sonlatic enlbryos from mature embryo axis and leallei explants ;itler 6<br />
weeks of cullure on the 111edi1111l conla~ni~lg 2,4,j-T as pri~~c~pal growth regulator. A, B<br />
a ~ C. ~ d induction of enlbryos from Inlalure einbryo axis expla~il, D. Induction of soinatic<br />
embryos froin leaflet esplant (arrows iiidicotl: globular embryos).<br />
Figure 4.2 A, B<br />
lnductioil of sonlatic cmbryos SI-oin inlaiurc c111bryu asis CXPIOIII<br />
by uring 2.4-D as<br />
principal growth regi~lnlor. .A. Top \,icw of tlie einbryo axis sllowiiig inultiple globular<br />
embryos, B. Letefiil view of enibryo axis showing multiple globular embryos formed<br />
from plumiile region (arrows indicate globular embryos).<br />
Figure 4.3 A - E<br />
Effect of concentration of B.4P on ~nullrple shoot regeneration fro111 inalurc cinbryo<br />
axis explant after 15 d:iys of culture on slioot ~nduclion medi~lm. A. Multiplc slioot<br />
regeneration at 10 j~kl HAF concei~tralio~l, U. M~illipli: slloot rcgcncr~~ion will1 30 pM<br />
BAP, C. Miiltiple sliuot reguneration \$'it1140 pM BAP, D. Multiple sl~ool regeneralion<br />
with 50 pM BAP, E. Mult~plc slluot rcgenelntion w11Il I00 1tM BAP.<br />
Figure 4.4 A - F<br />
Multiple shoot regenel-ation fro111 dil'fcren~ cxplnnts al tile third week of culture 011<br />
sliooi iiiduclio~i ~iicdioni .A.<br />
Shoot lip sllowi~~g rn11111plc slioots cincrgitig fro111<br />
~neristeii~at~c region. B. Axillary bud showing nlultiple shools or~ginaling fro111 lip and<br />
basal regions, C. Mature enlbryo axis sliowing mulliple shoots cinerging rrom sllout tip<br />
as well as axillary bud regiu~is, D. AM1 explant showing ~liultiplc shoots from axillary<br />
region, E. AM2 explant showing swollen brisal region and multiple shoots emerging<br />
synchronously as cluster, F. AM4 explant show~ng synchroi~ous multiple shoots from<br />
different places oraxiIlai.y niel.istem region.
Figure 4.5 A - I;<br />
Stages of ~nultiple stloot regelleration from AM4 explant alter culture on the sl~oot<br />
induction medium A. Explant sl~owing the area of axillary bud re~noval (arrow sliows the<br />
sire of surgery), B. Muluple sl~oot buds oriyin;ltiiig fro111 the area loft by axillary bud<br />
removal (arrow sl~ows the s110ots buds e~ilcrging fru111 axillary ~nlcriste~l~ area). C. Esplii~~l<br />
showing swollen area after removal ofregcnc~at~~~g shoot buds (this stage of explant sas<br />
na~nsd 3s AM4 cxpla~it) akcr 7 days of culture OII shoot i~iduct~o~l rncdlulll, D.<br />
Regc~ierat~o~~ of ~il~iltiplc sl~oot biltls li.0111 tl~ cut ~po~tiul~s uftlic rcgclleratlng iiastiv after<br />
6 days ofci~lture on MS, E. Exubcra~lt nlultiplc sl~oot yrowtli Sron~ tile rcgcncrating tlssue<br />
after 10 to I? days of culture on MS, 1;. Mult~plc sl~oots originaiiny fro^^^ different p;~rts<br />
of the regellcrating area aCter 12 to 14 days of culture on MS.<br />
Figure 4.6 A - C<br />
Various stages of tlongatio~i of regenerating sl~oot buds on shoot elo~lgation 111edium;<br />
A. Elongut~on of young shoot 011 shoot clo~igatio~~ medic1111 1 (SEMI) ancr 1 week uf<br />
culture, B. Elol~gatio~i of sliool on shoot elollgation 111cdiu111 2 (SEM2) nlicr 2 wccks of<br />
culture, C. Elongallon of sl~oots 011 SEM2 after 3 wecks.<br />
Figure 4.7 A , B<br />
Rooting of eloiipaled slioots on root induction ~niedill~~~ co~~tailli~lg 5 pM IBA, A.<br />
Rooting 011 filter paper bridye i~lllrlcrsed ill liquid root ind~iclio~~ nicdium, 8. Rooting OII<br />
solid root induction ~nlcdii~n~ co~~iaininy 5 pM IBA.<br />
Figure 4.8<br />
Stage 1 Ilardening of tissue ci~lture gro\an cli~ckpea after transferring to 8 cnl pot<br />
containi~lg 2-4 mm sand.
Figure 4.9 A - C<br />
Alternative llletilods of hardening of rooted plantlets of chickpea prlor to<br />
transplantation; A. Hardening process by embedding !he root system ill the coarse sand<br />
wtth the cotton plug open. % Amon's solutlol~ \bas used for irrigation. B. Hardening<br />
process in static hydroponics system with root system immersed in tlie liquid mcdium.<br />
The Itquid med~i~ni co~llprlsed of % Arnotl's solution, C. A plant fron~ B showing prof~tsc<br />
groivth of root system 111 tile static hydropot~ics systel~~ after 15 days. Tllis plant is luady<br />
for transplantation illlo pols.<br />
Figure 4.10 A - C<br />
Hardened and transplallted chickpea planls growing in the glasshouse; A. Plant<br />
growing without any post-hardening treatment, U. Plant with multiple branches aftcr<br />
post-hardening treatment of terminal bud ampiltution and juvenile flower bud removing,<br />
C. Group of plants being ~nlaintained in 20 cm pots after Ilardening.<br />
Figure 4.1 1 A - I<br />
A con~plete sclleme of regeneration and recovery of wliole plailts tllrougl~ tissue<br />
culture lnetllod using axillary t~leristel~~ explant (AM2) obtained froln it! vi11.o grohn<br />
seedlings olchickpea. A. Axillary mcristem explitllt (AM2) on the first day of culture on<br />
shoot inductton rnedlum, U. Sl~oot buds regenerating froln the region left by thc rcnloval<br />
of axillary bud after 7 days, C. Cluster of 11i~lltip1~ slloot buds or~gli~ating fro111 region lcli<br />
by the removal of axillary bud after 12 days, D. A closer view of the liiiiltiple shoots<br />
regenerating from the axillary nleristem explant after 15 days of culture 011 shoot<br />
induction medium, E. Elongation of the slloot buds after 7 days of culture on slioot<br />
elongatiol~ medium, 1;.<br />
Rooting of the elorigated slloot bud on the filter paper bridge<br />
immersed In liquld rootlng medium after 8 days of cullure, G. Picture showing rx vlrro<br />
static hydroponics systenl for hardening of the rooted shoot after 15 days, H. An<br />
alternative method for hardening of the planllel oblaincd through tissue cullure ill which<br />
the root system was immersed in sand wltli the cutton plug of tlie crilture tube kept open,<br />
I. Hardened and transplanted plant sl~on,ing normal nlotphology.
Figure 4.1 2 A, B<br />
Histological sectio~lsliowing status of axlllary meristem A. Longitudinal sectloti of<br />
the growing axillary bud just before its re11iova1 froni 6-day-old seedling sliow~ng<br />
actively dividing merislenialic cells atid uti~for~i~ oon-ineristematic tissue in rlie basal<br />
region. B. LS of explant aner removal of ahillary bud ill thc process orprcparing axillary<br />
nieristem explant.<br />
Figure 4.13 A - H<br />
Hlslological studles ofdcvelopme~it of ~il~lltlple ~iicrisremolds from ax~ll:~ry mcristcm<br />
region of AM4 explailt alier tile renioval of ax~llary bud. A. Longitudi~ial scctioii of rlic<br />
axillary nieristcm area oli day-l aner axillary bud removal. U. Developmenr ol'<br />
rneristenioids at tliz basal poriion of axillary bud on day-2 (tlie stndll b:~d appearing is a<br />
shoot but1 emerging li-0111 tlie basal portion of the removed axillary bud), C. Appeara~~cc<br />
of rneristemo~ds 111 the axill;iry meristelii area on day-3, D. l~icrcnsed !lumber of<br />
nieristemoids 111 the ax~llary meristem area on day-4, E. Increase ln the nnnibcr and<br />
yrowtli of ~ncr~steino~ds 011 day-5, F. E~po~ic~it~;tl ilicre;~se ill thc ~nun~bcr and growl11 of<br />
merlsteniolds on day-6, C. Loligiludlnal section of the rnult~plc shoot butls as seen on<br />
day-6, H. Meristemalic activity of d~viding cclls at various places in tlie ax~lldry<br />
meristenr ;Ilea 111 d~l'fcren~ d~rccriun on day-7.<br />
Figure 4.14 A - F<br />
Closcr observation of dcveloptnc~it and growth of rncristenioids in tlic axlllary<br />
nieristem area of AM3 explant A. Closer view oftlie mer~stemo~ds on day-4 after the<br />
removal of ax~llary bud, U. Growth and divis~on oi'meristeniatic cells in the meristernoid<br />
region on day-5, C. Growtli and division of mcristem;~tic cells around merlstemoid region<br />
on day-6, D. Growth and division oi'mer~stematic cells ofnicr~stemoid reglon on day-7,<br />
E. Closer view (40X) of meristemoid region and ccll division activity, F. Hectic<br />
meristematlc activity oftlie dividing and growing ~ells.
Figure 4.15 A, B<br />
Restriction analysis of tlie plasniids used for tra~lsforniation. A. The plasmid<br />
pRTY9:GUS-lnt (6.9 kb) was tiscd In biolistic method ol'transfomiatio~~. Lane 1 sliows<br />
hDNA digested with UsrE I1 etlzynie as marker. Lanes 2 and 3 liave duplicate plasln~d<br />
un-restricted sample and lane 4 shows plas~iiid a!kr restr~ction with EcuRl showing Sour<br />
fragments. B. Two un-restr~cted plasmid constructs that were used in Agroboc.ierrii~,i-<br />
mzdtated transl'on~iation. Lane I shows hDNA d~gestcd w1i11 tiilid III ellzymc as 111arkcr.<br />
Lalies 2 and 3 llad pHS737:SBTI (14.3 kb) ~plasni~d wl~ile 1,111~s 4 and 5 llad pHS723:Bt<br />
(15 5 kb) plasm~d preparatluns.<br />
Figure 4.16 A - C<br />
GUS histochenlical assay of the leaflets Srum pl~tdtivcly transfornlcd plants 01'<br />
chickpea. A. 4 closer view ol'tlle leaflet sllowing GUS activity in [lie vclns, B, C. GUS<br />
activity as seen 111 tlie pcliole and veins of lallets.<br />
Figure 4.17 A, B<br />
PCK amplificat~on of 700 bp fragnlcnt of 1ipi11 gene fro111 \he gclio~~i~c DNA5 oSTu<br />
generation pla~its rratisSoni~ed w~th Brcr)>lAb and SBTl genes via Agrubricieriut~i-<br />
mediated transfonnalion. A. ~iplll-PCR of pli~nts lrallsforilled w~lli pMS723:Bt via<br />
Agroborieriu~~r-1n2dlatcd trallsfomiation. Lanes 1 to 10 lid transfomled samples and<br />
show the aniplification of ~tpfll gene. Lane I I negative co~ltrol and 12 lo 17 wcrc positive<br />
controls froni plasmid pHS723:Bt used for tralislbrniatio~~. Lane 18 IS - DNA and h<br />
DNA-BsiE I1 m:~rker was dddcd in the lane 19. Ll. ~rprll-PCR or plants lransfomicd \rill1<br />
pHS737:SBTI vector vln Agvobucieriu1?i-11ied1;itcd translorn~at~on. Lanes 1 to 9 had<br />
transformed samples CS5 to CS9. Lonc 10 L\,;IS negative coiitrol and l I<br />
to I5 were<br />
posltlve controls oSpHS737:SBTI vector tlial w;is iised In the transforni;ttion. ?, DNA-<br />
BsiE I1 marker was added in the lalie 16.
Figure 4.18 A, B<br />
PCR arnplificatio~l of 1.2 kb fragment of tridA gene from genomic DNA samples of<br />
To generation putative trat~sgenic plants tra~isfonl~ed with pHS723:Bt and pHS737:SBTI<br />
vectors via Agrobocferiir~~t-inediatcd transfor~~~at~o~~. A. GUS-PCR analysts of plants<br />
transfom~ed wit11 pHS723:Bt vector. Lanes I to I0 \\,ere added \vitIl putntivc tratisfor~ll;i~~t<br />
samples, while lane I I \\as positive control and lane 12 Ineg;ltlve control. Satllple addcd<br />
In lane 13 was -DNA, h DNA-UbfE II m;~rket was added in tlie lane 14. U. GUS-PC'K<br />
analysis or plants tra~~slbrlncd wttl~ pHS737:SB'l'l. Lanes I<br />
to 9 wcre putarivu<br />
transformant samples, wliile lane 10 and I1 were positive co~ltrols of plasmid<br />
pHS737:SBTI and lane 12 was negative conlrul. San111le added in lane 13 was -Dh'A. h<br />
DNA-UsfE II ninrket- was added ill llle lane 14.<br />
Figure 4.19 A, B<br />
PCR amplification of SBTI end BfCrj,I/IL gcncs fro111 ge110111ic DNhs of ptllative<br />
transforn~ants at To gene ratio^^ transformed vin Agrobacfn.ir~t~~-t~~cdi~~ted transforlnation.<br />
A. PCR analysis of plants rr:~nsfornied with pHS737:SBTI veclo~.. Ln~li: 1 tl~ruugh lane 9<br />
are putaive transfun~la~lt s;~tilples sliu~viiig an~pliiic;itio~~ ufJ97 bp fr:~gment. Ncgi~tive<br />
conrrol samples were added In llle lanies 10, 11 2nd 12 ptlS717,SBTI plasmid samples<br />
were positwe controls in the latles 13, 14, 15 atid 16. Lane 17 \+as -DNA and i, DNA-<br />
BslE I1 marker was added in lane 18. B. PCR allalys~s of pla~~ts trat~sformed wit11<br />
pHS723:Bt vector. Lane I<br />
rlirougli lane I I wcrc addcd with putative twnsforn~ants<br />
showing amplificat~on of 908 bp fragment. Lane 12, 13, 14 arid 15 had the negative<br />
control. Lanes 16, 17 and 18 were positlve controls of pHS723:Bt plasmid vector. Lane<br />
19 was -DNA and lane 20 was added wttll h DNA-U5tE I1 ~ilarker.
Figure 4.20 A, B<br />
DNA profile of genomic DNA isolated from putative transfonnants of chickpea. A.<br />
Purified DNA profile of genomic DNA of BlCrylAb plants. DNA was prepared in<br />
duplicate samples. Lanes 1 to 10 shows first set and lanes 11 to 20 shows the second set.<br />
B. Effect of amount of BSA, water and enzyme in the reaction mixture to digest the<br />
genomic DNA sample of chickpea. Unless otlienvise stated, the Lola1 volume of reactton<br />
mixture was maintained at 25 p1 with 10 p1 of genotnic DNA, 2 pI (I0 units) of clizpe,<br />
2.5 pl of reaction buffer and the rest being water. In case of volume variations arising due<br />
to tile changing enzyme and water collcenlratioils, tlie reaction buf'fcr volu~ile \\,as<br />
maintained accordingly with its working concentralion always kept at IX.<br />
Lane 1: Unrestricted genoliilc DNA (5 pg, was added to tile well)<br />
Lalie 2: 1 p g genomic DNA digested with I0 ltnils of enzyme<br />
Lane 3: 5 pg, gelionitc DNA digested willt I0 units of enzyme<br />
Lane 4: 5 pg. getlomic DNA digested with 15 units of etizynie and dot~blc llic<br />
quanlily of haler<br />
Lane 5: 5 pg, ge~iolilic DNA digested \z;~lIt 20 units of eti/.ylne with double llle<br />
quanttly ofwaler<br />
Lanc 6. 5 ~tg ~CIIOI~IC DNA digested will1 10 utllts of elizytiie and 0.1% BSA<br />
Lane 7: 5 pg, genornic DNA digested w~tll IO units ofcn~yme, 0.1% of BSA and<br />
double tlie quantity ofwnler<br />
Lane 8: 5 pg. getiomlc DNA digested \villi IO ~~iitls ofcnzynle, 0 356 of BSA<br />
Lane 0: 5 pg, getioniic DNA digested wit11 I0 ullils of enLytne, 0.3% of USA and<br />
double tlie cll~antity of water<br />
Lane 10. 5 pg. gellolnlc DNA digesicd will1 IO i~rlits of cnzyme, 0.5% of BSA<br />
Lane 11: 5 pg. genomic DNA digesled will1 10 units of enzyme, 0.5% of BSA<br />
atid double the quantity of water<br />
Lane 12: 5 pg, genonitc DNA digested with I0 unlts of enzyme, 1.0% of BSA<br />
Lane 13: 5 pg, genonltc DNA digested with 20 itnits of enzyme
Figure 4.21 A, B<br />
Southern blots of the To generation putatlvc lransfonna~~ts of chickpea transformed<br />
with i31CyIAb and SBTI genes via Agrobtrcleriior~-nlediated transfomiation. These blots<br />
show the signal accumulaled 011 tlie h~gher niolecular weiglit region due lo the li~liiled<br />
restrlctlon of genomlc DNA. Tll~s psoble~n was encountered wlie~i the factors affecting<br />
restriction of gelio~llic DNA, sucll as qu;~nl~ly of cnzyine, water and BSA were 1101<br />
standardized A. Southern blot will1 probe for BrCq~I/lb gene. Lanes I<br />
to 10 has<br />
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<br />
gene. Lanes 1 to 10 were added with put,it~ve tl.a~isfosrn;il~ls transConucd wit11 biC~:i.iilh<br />
gene. Lanc I1 lias ~legativc co~ilrol, whilu Idlie 12 lias lllc pos~live control. Rcslricted<br />
genoltiic DNA samples from putative transgcnlc trs~lsfornled with SBTI gene wcre added<br />
in the lanes 13 to 2 1<br />
Figure 4.22<br />
Soutiicni analys~s of tlic To genelntio~i of BrC',ylAb p~~lalive transgclilc plants of<br />
cliickpea triinsibniied via Agruhtrcreri~~rrr-medlated lransfur~nat~on. Ge~iom~c DNA was<br />
restricted wit11 EcoRl enrynie. A~ialysis of copy nuntber w~lh respccl to the ~rplll gene.<br />
Lanes 1 to 11 were added ~v~tli transfonnant salliples CB I to CB I I. La~le 1: CBI, Lane 2:<br />
CB2, Lane 3: CB3, Lane4: CB4, Lanc 5: CB5, Lalie 6: CB6, Lane 7: CB7, Lane 8: CB8,<br />
Latie 9: CB9, L:tne 10: CBIO and Lalie I I. CBI I. Lane I2 11as the negotlve co~ltrol and<br />
lane 13 has the pos~tive con1101 (Rcslricled plasliiid pMS723,Bi that was used in gc~iel~c<br />
transformatio~i)<br />
Figure 4.23<br />
Southern a~ialysis of theTo generalion of br('ry1Ab pla~its. Genomic DNA was<br />
restricted with EcoRl eilzyllie that has two rcstricl~on sites within the RlCryiAh gciic.<br />
This double cut releases tile 3 lib fragnielit of UrCryIAb pc~lc. Analysis of the tra~lsgenics<br />
for the U~CrylAb gciic. Lanes I<br />
to I I were addcd w~lll Il-ansforman1 samples, Lane I:<br />
CBI, Lane 2: CB2, Lane 3: CB3, Lane 4: CB4, Lane 5: CB6, Lane 6: CB5, Lane 7: CB7,<br />
Lane 8: CB8, Lane 9: CB9, Lane 10: CBIO and Lane 11: CBI I. Lane 12 has the negative<br />
control and lane 13 has the posit~ve control Restricted plasmid pHS723:Bt with EcoRl<br />
enzyme).
1'1.0 INTRODUCTION<br />
L~fe is the ter~in uscd to explaiii the to-ordinate i~itegrity oi defitned nintter energy<br />
transacttons manifested in tlie living organisms. These tlansacho~ns hove been made<br />
possible with the ava~lnbility of fixed energy. Early life derived energy tiom anaerob~c<br />
environme~it and evolutio~i ofautotropliic orgoiiisms divcrsificd course of evolutio~i. Pla~its<br />
liavc bee11 thc I'irgcat source of lised energy, ,IS tiicy trd~isl'urm tile 1portio11 of solar ci~c~g!<br />
illto clietn~cal energy, ivliicli ~nakes theill cligiblc tbr tlic title of'thc Producers'. ' hs liked<br />
ericrgy is being used at vaiious levels of hod cliitin III v.iriuus Ibrrni. In platlts. lhis cncrgy<br />
has bccn uscd for various metiibolic oct~vit~cs 2nd stored in nialcrial fornis of tliocl-o<br />
molecules siicli as carboliydrates, protein itnd I~pids, collccrively struclured 111to orgalls like<br />
seeds, graltis, l'ri~~ts, vtgetobles ctc., to bc uscd at tlnc ti~iic of titcli, 'l'lic cot~ccpt ul<br />
agriculture was born \\lie11 11i:t11 rcltlizcd tlie ~il~tritio~iitl sig~nificii~itc 01' tlnesc plat11 parts.<br />
Agriculture brought about r~voli~tio~~iiry socii~l ~Iia~igcs by Ifiiiisfor~iiing no~iiad~c ir~bcb<br />
into inore adlanced static civiltzatiot~s, 2nd nictliods of:igricul~ur;il pr:~cticcs li,tvc been tliu<br />
principal inleasure to clsscss tlie ad\anceliiciit of'ctvilii;~t~o~is<br />
Abulid,nntly water supplyi~ig rcgioris and ailst,~ini~i'lc rainied areas l~ave becorrne tlic<br />
Inavens for settling and advancement of svutic civiliriittons. Agriculture gained sign~ticance<br />
priiicipally by ilourislii~lg llie Iiunian world dnd ll~ei~ dotncat~catcd aii~mals. Consistent<br />
discovery of plii~its with diversified ~outisliinp \:dues expanded tile span of :igriculture<br />
while one Faculty of agriculture concent~.ated oil d~scovery and cultivation of plants with<br />
added benefits siicll as niediciiial Iiiipottancc, titiiber, orn:imenlals etc., that have varted<br />
degrees of economic Ilnportance. Vi~ricty of' plutlt organs such as seeds, tiuits, leaves etc.<br />
were identitied io be nutr~ttonnlly very sign~:ica~it. A ~rritritious hod is identilied as a<br />
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'<br />
energy while llp~ds and some protelns are structurally s~gntficant. Another class of<br />
proteins, v~tamins, carbohydrates and various small organic and 1norg:lnic nlolecules arc<br />
function;~lly very important. Most of tllcsc org;lnlc n~acromoleculcs In conibinat~o~~ wttll<br />
several lnorganlc molecules must be supplied estcr~ially in the form of diet though tl~crc IS<br />
synthesizing machinery wlthin the body for sotlie of the molecules. Most of tlle countrlcs<br />
depend upon meat, poultry, eggs ctc, fo~ these d~ctary supplements. Ucvcloped world<br />
derives liiuch of its protein (46%) and cnrboliydrates (20%) from antnlal products llkc<br />
meat, poultry, eggs and fish and otlier seafoods, while developing coutitries der~ve thc<br />
protetli (64%) and energy source (65%) primarily iio~l~ agrl-based foods. Ilenceforth,<br />
economies of the later are directly deperide~>t on agriculture.<br />
Agricultural practices and crop selectiol~ dates back to ovcr ten tilousand ycura and<br />
ancient agriculture \+as mostly characterized by few crop v;lr~etics that nourislled relatively<br />
few peoplc and thcir domesticated anitnals. Tlic early moderate popul:~l~ona demanded<br />
equally tnodcrate agricultural outputs. Howcver, static civilizations stiniulnted wide array<br />
of social and cultural tratisfoiiiiat~ons tliat ste;ldlly increased the population as well, which<br />
in :urn nccc,s~tated better yield froni the selected crops. It was oi~ly stncc 19'~ century that<br />
dramatic advances took place in agriculture in terms a!' quality and quziotity i\,llicll was<br />
made posslble by well-concerted efforts of sc~cl~tists lion1 all ovcr tile world co~nmitted fbr<br />
a common czluse of food-for-all.<br />
Presently around 200 plant species with tliousnnds of cultivars address the issue of<br />
human diet. Alllong these only 15 species col~tribute 9OU/o of diet where rice, wheat and<br />
corn contribute up to 70%. Though these three crops arc kliown as staple food crops of thc<br />
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,<br />
with only 1% of water available for ci~lt~vat~o~i and wit11 inevitable population outburst<br />
there is an ii~creasiilg concern of how to feed people as cautioned by enliile~lt economist of<br />
yesteryears. Malthus. When domarid was n~orc than the supply, 1960a witnessed mqor<br />
cllailgss by revolutionary agricultural outpiils, nil put 1ogetll~'r under the iilsj)iring phrase<br />
'Greei~ Revolution' that was carefully crafcd by cini~le~~t brccdcr Nonnan Borlaug, who<br />
was riglltly awa~ded ll~e Noble Prize for pence. lilcepted \vilh wlleat and cxtendcd to<br />
almost all the crops, green rsvolution paved the way to light up smiles OII the faces of the<br />
hungry tl~ougll it shot down the world food prices. 'This led to a more sustainable<br />
agriculture, land use efficiency and opened tile doors to nlany hopeful avenues, wh~ch<br />
could address various other imminent problenls. These results came wllcn delllographic<br />
realities were seiiding ripplcs throiigh the scientific community about thc ways and means<br />
to manage {lie yioblcms of starvation and n~alnutr~tioii. With over 6 billion pcolllc on<br />
board, an est~inated increase of 1.7 bill~ons inore by 2020 and wit11 around 800 million<br />
people still statrving, tbcre are more questiuns t11ni1 answers. Nevenheless, all the efforrs<br />
put togetlier will record an est~mated increase in the oniu~al growl11 rate of 2.6% food grain<br />
productloti wllile popiilat~oii increase rate ii l:iUA. Uuto~.tunatcly evcii hit11 these results<br />
the iiunger atill rcmalns sii:ce the outputs are not reaching the nccdy because oi'tl~e low<br />
purchasing power and standard of living. Tllesc concerns led to cultivation of alternative<br />
food suppleincnts such as leguines other than the popular cereals.<br />
Pulses are dry seed legumes that h;i\e relatively lesser nutritional popularity.<br />
Nutritionally i~nportant pods bearing seeds and herbaceous naturc ci~aracterize these crops.<br />
More thaii 50% of the world pulse y~eld 1s being co~ltrlbuted by Asia-Pacific region. The<br />
proteii~ and oil rich seeds of these plants liave an ~nd~spcnsable coiisidcratiou in the human<br />
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<br />
these plants is that they tix atmospl~eric nitrogen 111 symb~osis with Rhirobiunt, which<br />
qualifies them to be used in Inter cropping and rotation cropping practices in contblnation<br />
with the cereals to enhance the product~v~ty of tile later. However, despite their protein rich<br />
and n~troge~l fix~ng attr~butes, the production of pulses lids increased at nluch slower pace.<br />
This pace could have been the result of Pictors like rice-whcat rotation, illter croppirig w ~tl~<br />
few other cereals and lesser ava~lub~iity of ~~u~rition;~lly dcpcildable Icgumcs in hot xa,oll.<br />
Most of the Important legumcs are cool season crops where the cereals relatively domi~~;itc<br />
the cool season. These hcts pushed the areas ot'lsgu~ne cnltivat~o~~ to ~nargitlal regions that<br />
are mostly ramfed. Sigiiiiicai~i crop losscs are observed due to varlous b~otic 2nd ab~ot~c<br />
stresses, as farnmrs of the developing countries are not inclined to use expensive remedial<br />
inputs such a5 fertilizers and pesticides. This lias resulted in a wide gap betwceil thc yiuld<br />
ratios of pulses to cereals tit 1:32 111 I990 (l'aroda, 1995). Howevcr, various scie~ltific.<br />
social and nutrition concerns 111dicate that the legu~lies must be given duc impo~lance in the<br />
regular agricultural practices which would be Illore beneficial to thc third world countries.<br />
In accordance with this pointer, various govcr~i~nental and 11o1l-goveriin1e11~;~l researcl~<br />
institutes including ICRISAT, India; ICARDA, Syria; IATA, Nigeria etc., embarked onto<br />
understand~ng the metabolic and agrono~nic ~ntricacies of various food and pasture<br />
legumes and organize maneuvers for Improvelncnt and preiervatio~~ of the el~tc germplasn~<br />
of peas, beans, lentils, grain legumes and various other pulses.<br />
During early i~griculture, selectio~~ of ci~ltivable varieties was principally based<br />
upon natur:~l selection. Iluuvever, ~t was in I:~ter par1 of 19"' century that dcti~al plant<br />
breeding programmes started. Varieties with elite phenotyp~c troits wele aelected and<br />
ciossed for co~i~bi~i:jt~on ~f neccssaly cIiar.icici,, in lhu otl'spr~ng. blendel'a famous pca<br />
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<br />
resulted III an exponential Increase in brcedlng programs, which w~s in turn expanded to<br />
countless nlembers of cultivable variet~es. Socio-eco~lomic Importance of these progranla<br />
gave birth to thousands of varieties initially In cereals and later in otl~rr dol~~esticated<br />
crops. However, breeding is a highly t~~r~c consuming process and labor intensive as the<br />
crossing means polll~lat~ng every plant mailually. Crops wit11 lo~lg duration life cycles pose<br />
variety oT problcms in co~~ve~ltio~lal breeding. Another I~~liltat~o~l 01' the cunventio~i:~l<br />
breed~ng lies in the susu;~l ~ncompatib~lity of the cultivated varieties with their wild<br />
relatives, where the ancestral wild varieties were proven to tie the reservoirs of several<br />
agronomically important traits. Tools of modern biotechtlology have come to the rescue for<br />
some of the difficult constraints to crop improvelncnt by understand~ng their molecul;ir<br />
basis and providing ren~edles at the n~olec~~lar level itself. ldc~lt~ficalio~l of physical basis<br />
of life and niolzcul.ir cl~;iractcri~atlo~~ of i~ll~critn~~ce pattenls ~nads brecd~ng a more<br />
syste~llatic and mcani~~gfui science of crop improvement. Stat~stical appropriat~on ot<br />
polymorphisms ttlllcrlri'd with the help of techn~qucs l~lte Southern blott~ng and<br />
polymerase chn111 reaction (PCII) gavc birth to a new faculty of'rnode1i1 agriculture that is,<br />
marker assistcd selection (MAS). This was not only uscful ill identificat~on of quantitat~ve<br />
tra~t loc~ (QTLs) but also uscful III isolation of gencs for a specific trait. ldentificat~on and<br />
isolation of agronomically important gencs fro111 different prokejotes and eukaryotes<br />
ignited a desperate wisl~ ill sc~entists to ~ntroducr: Illem into plants and observe tiieir effect<br />
In the new env~ronnie~lt. Tile existing bacterial transformation systems (Mandel and Higa,<br />
1970) gave some logistic support to this idea ofplant transgenes~s by recombination events<br />
in the genetic material. Discovery of the ab~l~ly of a crown gall induc~ng soil bacterium,<br />
.4grobacfrriton f~rr~~ejiiciert~ to introduce the genetic material Into the plants (Drummond,<br />
1979), in late 70s revolutionized the genetic transfurn~ation research. Agrobacteriurn
tumefaciens, popularly kl~own as a 'natural genetic engineer' transfers genetic material<br />
with the help of the tumor inducing (Ti) plasmid (Chilton, 1983). The native genes of Ti<br />
plasmid perform the function of cancerous establishnient of the Agrobactrrium in the<br />
infected plant cells. Among various attributes of' this plasmid, the niost important one is<br />
that it transfers virtually any genetic sequence present between the left and right borders of<br />
the famous T-DNA. Tliougll this organihm's ilifectlvity 1s restricted to dicois, tcchnologlcal<br />
advancements made [his organism useful to tr,lnsfer genetic ~llatcrial to monocots (Slemcl~h<br />
and Schieder, 1996) atid even to animal and hun~an cell cultures as well according to rccclit<br />
reports (Kuriik et al., 2001). Various otlirr techniques evolved with the inspiration liol~l<br />
this organism, but tliey arc host and teclin~que spccilic monocots (Siemens and Scliiedrr,<br />
1996). Biolistics or part~cle bombardment teclinique is onc potential technique (Saliio~d,<br />
1993) that could solve tile problem ofliost incolnpnl~b~llty.<br />
\/Chickpea<br />
(211-16) is one of tile important food legumes prevalently cultivated in<br />
Asia-Pacific region \+liere large portiun oi' the yield is contr~butcd by the Indian sub-<br />
continent. Broadly [here are two vorletlcs of cl~lckpea, Desi arid Kabuli. It is a cool season<br />
pod bearing crop, seeds of wh~ch are excellent source of protclns rlch with nitrogenous<br />
amino acids espec~,llly lyaiiir and arglnlne 111;iklng the products of 1111s crop, very good<br />
food aildilivcs. Ue>ldrs, it 1s also ktlo\vn to Il111)rove soil fertility with the Iielp of'sy~i~biotic<br />
nitrogen fixation. It contributes 15% of the world's pulse Iialvest of about 58 rnill~on tons,<br />
anllually. Deap~te sign~iicant gains In world pulse production during the last two decades<br />
with an annual growtli rate of 1.9% cllickpeli production growtli has been slow. Ch~ckpca<br />
yields worldw~de have risen by 0.6% annually which :mounts to 800 kgha, and thc area of<br />
cult~vatioll has reniaiiled v~~iunlly stagn;lllt. Tll~slower p;lce has bee11 the result ol'vu~ lous<br />
refractory biotic aiid abiotic constrdlnts such as ,lrL~oc/iylu bl~gllt (AB), Liurrj'li~ Grey hloid<br />
(BGM), dry roo!jz,<br />
- - -<br />
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<br />
over 20% of the total crop loss (Vyas et al, 1983). The enhancement of insect and disease<br />
resistance in chickpea could increase its yield potential by as much as three times. The<br />
available chickpea germplasm also lacks effective resistance sources for use in developing<br />
pest resistant genotypes. An attractive option is to introduce genes for insect resistance<br />
from other sources to chickpea by the iise of transgenic technology that has shown a great<br />
promise (Sharma and Ortiz, 2000).<br />
Attempts to create transgenic chickpea to combat tlie above constraints have been<br />
the short cut strategy adopted by various groups working for creation of el~te germplasm of<br />
chickpea. However, reliable regeneration and transfor~nntio~i protocols lmve been evaduig<br />
sucli efforts due to tlie perce~ved recalcitranl nature of chickpea towards tissue culture.<br />
Several regeneration protocols involving somatic ernb~yogenesis and orgallogenesis have<br />
been published during past one and half decade olily to show the difficulty in regelierati~ig<br />
chickpea in the in vltro cnvlroiiment. Micropropagation has inot been a serious problem and<br />
it can be ;ichieved using explant co~itai~iit~g prc-cxisting Illerlstema such as shoot tip and<br />
cotyledonary nodes (Rao and Chopra, 1987; Riazuddin et a]., 1988; Rao and Chopra,<br />
1989). Coiisiderable work has been done for regenerating wliole plants via sonlatic<br />
embryogenesis from mature leaflets (Rao and Chopra, 1989) and imn~ature leatlets (Barna<br />
and Wakiiulu, 19931, niaturc emrbryo axis (Sul~as~~ii ct ol., 1994) and initnature embryo<br />
axis (Sagare et a]., 1993) or cell suspension cultures (Prakasli et a]., 1994). Ilowever, the<br />
success rates on the niatiirat~on of indilced embryos into fully differentiated plants have<br />
been very low (
Wakhulu, 1994). However, regeneration of quiet a few shoots from traces of pre-existlng<br />
meristem with that of the explants such as hypocotyl and epicotyl were mistaken for<br />
indirect regeneration from callus phase. To date, effective chickpea regeneration has been<br />
possible only through the use of explants based on cotyledonary nodes or shoot apices<br />
derived from seedling explants. In most of the cases the shoot buds originated<br />
asyncbroiiously making these systems inefficient for genetic transformation studies.<br />
However, rooting and transplai~tation of (he in vitro recovered plants lias remained a major<br />
bottleneck in the meaningful application of this technology for serious crop improven~ent<br />
programmes. Such systems have been used to genetically transform chickpea and the<br />
transfostnation frequencies reportrd were very low (Fontana et a]., 1993; Kar et al., 1997;<br />
Krishnamurthy et al., 2000).<br />
J I ~ the ongoil~g efforts at ICRISAT to develop suitable tissue culture and<br />
trai~sfosn~;~tlo~~ protocols for cl~ickpea, the preseilr work was ain~cd at the improvenient of<br />
cxisting protocols for ;ill the atagcs or regenefi~tioii and transformation. The work was<br />
carried out with the ibllo\\ing object~ves.<br />
I. Development of efficient protocola for plant rcgcneration in tlssue cultures oi<br />
cliickpen.<br />
2. Optimization of varlous factors aficting legeneration via orgdnogenic<br />
pathway<br />
3. Optimization of factors i~ffecting liardenlog and transplantation of in vitro<br />
regenerated plantlets.<br />
4. Genetic transformation and successful recovery of chickpea transget~ic plants<br />
by using agronon~ically impoitant BlCryiAb and SLIT1 geiles.<br />
5. Molecular cl~aracterization of putative transgenic plants or ch~ckpea for future<br />
use.
jf.0 REVIEW OF LITERATURE<br />
Large part of the world's food is bring coi~tributrd by ogriculturc though nlost of<br />
the developed countries derive their food BOIII animal source. World food grain yruducl~o~l<br />
toucl~ed 2 billion ton mark at the turn of the last n~illet~niurn. The econoniic, con~niercial<br />
and social realities resulted in an uneven distribution of the food grains amply ava~lable<br />
that left 800 million people still undernourished (FAO, 2001). Tlie surpluses are used for<br />
exports and tl~e costs were dearer for the developiiig coul~tries that kept the number of<br />
undernourished people still alarming. Alter~iative food supple~i~e~~ts froin pulse crops<br />
gained all iiicreasii~g significance in tlic recent times that prompted developed cou~~try I~ke<br />
United States to include the lentils, broad beans atid cliickpeas in their 1:drnl bill 2002 so as<br />
to encourage the exports in this area (Food Outlook, FAO, 2002). Pulses are tile cd~ble dry<br />
seeds of legum~nous plants. They are of special liutr~tioi~al and economic i~nportance due<br />
to tlieir contribution to the diets of ~~~ill~ons ofpcople worldwide. Tlie nialn importnncc of<br />
pulses lies primar~ly 111 their liigli proteni content (two to three t~~iies higher than most<br />
cereals) as %ell as in being a valuable source ofenergy. In addlrion, pulses contai~i good<br />
anioulits of r~utritionally essential minerals sucli as calcium and iron. The use of pulses as<br />
-.<br />
hod is concetitrated in developing countries, accounting for about 90% of global l~uman<br />
pulsc consump~ioi~. In i~lost low-income countries, pulses contribute about 10% of the<br />
daily protein and about 5% of energy intakes 111 tllc diets of people (Paroda, 1995). Dur~ng<br />
the initial years of legume cult~vat~on brecd~ng was an important means of crop<br />
improvement. These efforts niet wit11 considcrablo succesa that resulted in iniproved yield<br />
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<br />
biotechnology may prove to be an effective altert~ative.<br />
Evolution is a process of natural creation of var~ations in the genetic ~~iake-up of<br />
organisms so that ~t firs into the variable conditions of the changing environnlent. This<br />
process takes ~iiillio~is of years :ind 11 goes 011. As the requ~rcmct~ts doubled and tripled.<br />
there is a ~necess~ty to induce val.iatiolls dellberatcly to lileet the 11ecd. This fart enierylnp<br />
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<br />
genetlc recomb~nat~on by meatis of con\cntional breeditig. Thougll this tccliriiquc is bung<br />
applied to many urganisnis, ~ncluding ni~crobes atid animals, the scope of [his thcs~s 1s<br />
restricted to deal with plants only. Moreover, nlosl of tlie publislicd and on goilig \wrh IS<br />
on platits whcn compared to the animals. Microbe transfonnatio~l is rather a procedure Tor<br />
basic research and only a part of preparative tcchnique for the higher organibm<br />
tratlsforniation.<br />
/Genetic transformatio~i, 111 principle is ~ntegrntion of alien genes into the foreign<br />
organisn~. Stable ~ntcgratioll arid lriheritalicc of ubcl'ul gcocs ib the main object~ve of gene<br />
transfer enperlmentsy"&e<br />
concept of genetic transt'ornlat~oli started with Avery et al.<br />
(1944). In angiosperms, gene transfer, however, IS a regular process where paternal<br />
chromosomes iioin the explod~iig sperm arc tr:insfcrred to the egg cell of tlie fen~,~le<br />
gametopllyte (Frankel and Galun, 1977). So, the problem 1s to transfer tlie gencs<br />
deliberately. Advent of plant tissue culture espec~ally the protoplast sola at ion (Cocking,<br />
1960) and cybrid formation gave an encouragi~lg impetus to the concept of gene transfer.<br />
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<br />
were of Hess (1969; 1970) when no tools for transfer and characterization of transformed<br />
plants were available. Discovery of ability of the soil n~icroorgan~sm Agroboc!erhoii<br />
iu~iieJucie~ls to trensfer its T-DNA to the pinlit genollie revolutio~~ized tliis arca and crcated<br />
altogether a differcnt faculty of science.<br />
2.1.1 Various lnetl~ods of gel~etic tr:r~~sfo~.~~~atiol~ in plnnts<br />
Various neth hods of gene Iralisler ~nlo pl:ints have been designed w~tll<br />
Agrobacierii~nl-mediated transfer as the principal ~netliod. Its miraculous ability to transfer<br />
part of its ge~ietic material, T-DNA into the plan1 genonle for its own beliefit was one of<br />
the significa~lt discoveries of 20Ih century. V;ist literature accumulated in describing tl~e<br />
whole mcclianisnl and ~PP;II.:I~IIS used by ilic microbe Tor gene transfer. llence, this nietl~od<br />
will be dealt in detail in tile foilow~~~g sccrloll. Metliods oliier tl1a11 those relying 011 the<br />
Agrohiicie~~ii~~ii sucli ;IS b~olis~ics, n~icroi~ijcct~vii elc. ir~ll bc dsacribcd ill 1111s aection.<br />
"li~oiisuc" IS n short term for b~ological b;lIlistics; the proccss is one by whicli<br />
biological r~iolccule>, sucli as DNA and RNA, are accclcrated (usually on microcarriers,<br />
termed microprojcctiles) by gullpowder, con~prcsscd gas or other means. The biological<br />
molecules are driven nt liigh velocity inlo the torgct, i~i this contcxt, tile plant cells. A team<br />
of nanoiibr~car~on fac~lity of Col-11el1 Uii~vers~ty developed this tccl~nique, and Sanford<br />
(1988) gave early descriptio~~s. Tlils 1s b;~sically si~nplc device wllere the genetic lrlaterial<br />
is coated on to the tungstei~ or gold pal-ticlcs illid accelerated with 11igI1 pressures into the<br />
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<br />
replaced by safer cot~lpressed hel~uni system. A different acceleration system was also<br />
developed based on the spark di,cliarge cllaniber 111 wliicl~ a water droplet was placed<br />
between two electrodes and a h~gh voltage capacitor caused all instant vaporization of the<br />
water, creatlng a shock wave. This sl~ock wave ;~cceltrates DNA coated particles illto the<br />
target plant cells (Christou et al., 1990). Several labs tried their owti honlemade particle<br />
guns (Per1 el a]., 1992). Var~ous dev~ces illid ilitr~ci~cics of b1011stic I>I.OCCSSSS IVC~C<br />
reviewed by Potrykiis ;ind Spa~igenberg (1995). Tlic firat application uf tlic biolistic<br />
process was niadr: by its inventors uallig chlora~~~plienicol acetyl transferase (CAT), a<br />
riiarker gelie (Klein et al, 1987). Later ye;~rs saw [lie two i~~ipo~til~~t j)ublica~iolis ot'<br />
successful transforrriatio~~ of chloroplasts ill Cliln~tiydo~rio~~us and niitochondria in yeilsl<br />
(Jolinston et iil., 1988).<br />
Tills protocol becotiles significant w11eii tlic host cells are not complacent w~th t!lc<br />
methods llhe /Igrobuc~er~iorl-t~iediatcd and direct protoplast transfornialion. Especially<br />
Ayrobuc~o.i~i~i~-li~ediatcd transforniation is, to some cxtent restricted to dicots and it also<br />
requires wounding. Helice, several rzports appear~d using mctliod to obtain transforn~ed<br />
platits in rice (Dntta ct a]., 1990), soybcat~ (Cliristou el al., 1990), niai~c (Fronint el dl.,<br />
1990, Kozicl et al., 1993) 2nd barley (Wan ;ind I.ciiiuux, 1994).<br />
2.1.1.2 Alternative nletl~ods ofgci~etic tra~~sl'orm:itio~~ into plant cells:<br />
Alternative nictl~ods can be div~ded inlo two types: direct physical i~~troduction of<br />
DNA and tr;insnilssion of gellettc tnaterlal by nlodlfied pl;lnt viruses. Viral gene transfer<br />
can also involve physical transmissioli to tlic plant (e.g., nib inoculation). Most iniportant<br />
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<br />
was later replaced with calcium phosphateipolyethylene glycol (PEG) (Krens et al., 1982).<br />
However, the success of protoplast transformation lies in tlie successful regeneration of the<br />
whole plants from them. Most of the direct DNA transfoni~ation involves usage of E.coli<br />
plasniids such as pBR 322 and pUC derivativea. Both plant DNA 2nd RNA viruses offer<br />
possibilities as plant transi'ormntio~ vectors.<br />
~'2.1.~ Miracle microorgaois~~l: Agrobacferirr,rr<br />
Early ~n tweiit~eth century, S~ilitlillid Townsend (1907) studled these crow11 gall<br />
tumors of cultivated Paris daisy and for the first tinie astablishcd that this "plant tumor" is<br />
of bacterial origin. The ineffective bdcterid isolated fro111 these samples produced tumors<br />
011 tlie stems of other crops. This bacteriuni clia~iged its lialiie nialiy times froni Batferi~int<br />
riiniefucien~ through Pliyioiiroilo~ rtiiiirfiiciri~s, Uucillir~ ir~i~iejiicieris and finally settled at<br />
Agrubocrrrii~iri riiiiieJ~icie~is. lllkcr (1923) and 1'111ck;ird (1035) st~ldted llially i~itrlcacies of<br />
-.-.<br />
the plant-niicrobe ~l~teractiolis that resulted in varlous cell stiniulations in plants. The<br />
-.<br />
opines were dctccted 111 the tumors (Pctit, 1970) and tlic vttnl role of these opines in the<br />
establishine~~t of "genetic colonization" was revealed by Shell (1979). lliitially it was<br />
perce~ved that the genes for opme sy~itliesis is pltlnt borne n~id only after rigorous studies<br />
the source of opines was confirmed to be fro111 the ~nfecti~ig bacteriuni (Montoya et al.,<br />
1977). Much earlier to that Kerr (1971) fouiid that the virulence could be transferred from<br />
Agrobncicriiriii to sapropliytic bacteria tlirougli DNA transforn~ation. Persistence of<br />
bacterial DNA in bacterial free tu~nor cells was observed by Johnson et al., (1974) and<br />
DrLica and Kado (1974) with the less efficient techniques of their ti~nes. A large plastnid<br />
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<br />
---<br />
genes of TI plasrn~d were responsible for the synthes~s of op~nes which were necessary for<br />
the progression of turiior w~tliout Ilie additton of growth liorniones They also found that<br />
only 5% of the plarniid DNA war responsible for virulelicc (Chilton, 1978). Since then<br />
many groups embarked upon exploring the lntr~cacies of the Agrobocieriuni and its abil~ty<br />
to transforni the host cells.<br />
2.1.2.1 Ti plnsltlid a ~ its ~ cllarncterislics<br />
d<br />
Many scientific groups in early 70s found that tlie TI plasmid, precisely part of it's<br />
DNA is responsible for tlie tumorigenic nature of the infecting bacterium. Tile process of<br />
identification of causal sequence for the tumorigenic activity was many folds expedited by<br />
the d~scovery of Southern blot (Southern, 1975) and DNA sequencing metllods (Maxam<br />
and Gilbert, 1977). Hooykaas, Schilperoot anti their associates found additional evidence<br />
for the role of Ti plasmid III<br />
tu~iior induction (Hooykaas et al., 1977). A detailed.<br />
d~scription of eah-events In crown gall researcl! was provided by Shell et al., (1979). It<br />
a? becatlie evident fiom Cliilton's experimelits thnt part of Ti DNA (termed as T-DNA)<br />
- -.<br />
was transferred t ~ l k !$I. Transcriptio~i of tlie T-DNA was confirnied by northern<br />
. ---<br />
blot experiments (Drunimond ct a]., 1977). All tile T-DNAs were found to be similar and<br />
has a length of 23 kb flanked by alniost idelltical borders (Zambryski et al., 1980).<br />
Spontaneous deletions studies rcvenled the gclietic componelits of tlie T-DNA and their<br />
oncogcliic tiatuse was confirnied (Gelvin et al., 1981). Schilperoot atid colleagues revealed<br />
many aspects of the T-DNA by inducing mutations and tracking tl~em down in the host<br />
plant cells by transfortiling tile tobacco protoplasts (Hockcnia et al., 1984). By this time it<br />
was also evident that a set of vir genes was involveti ill the transfer of T-DNA. It became<br />
...<br />
14
evident by 1983 that the Agrobacteriuni IS a sure candidate for genetic trallsformation<br />
(Chilton, 1983; Herrera-Estrella et al., 1983). Where T-DNA of the Ti plasm~d call be<br />
--. . .<br />
transferred to the plant cells and that can servr. as an cxcelle~lt tool for the genetic<br />
transformation of plants by dlsarnming the T-DNA and introducing the genes of interest into<br />
that area.<br />
2.1.2.2 Molecl~lnr ~nccl~a~~is~~~<br />
of T-DNA Ir:~~~sfcr illto pla~~ts<br />
Tlis dlrtails of tliesc ~nechanisms can be obta~ned from scveral ~revicws (Hooykans<br />
and Schilperoot, 1992; Zambryski, 1992; Grcene and Zambryski, 1993; Zupan and<br />
Zambtysk~, 1995). Wirlatls (1992) provided a critical review of chenlical signaling between<br />
Agrobacirriu~~i and plants cells. .<br />
2.1.2.3 Tools for ge~letic tl.ansformatio~~ /<br />
Understanding the plant gene structure and its essential components is n<br />
prerequisite for dcsig~~iny the tools for gellctic transformation. By the time the coilcept~~nl<br />
basis fbr the genctlc transfonnation was ready using Aprubaclei~iu~~i, the components of a<br />
complete plant gclie to be effcctivcly exl~resscd was also ready. The ainalgamation of<br />
various niolecular biology tech~iiques led to the dcs~gning of vectors for transforming the<br />
plant cells.<br />
i<br />
Tru~s/orrnuiio~~ vectors: Most widely used vectors are binary vectors (Bevan,<br />
1984) and cointegrate type vectors (Draper el al., 1988; Rogers ct al., 1987; Deblaere et al.,<br />
1987). The co~ntegrate type vectors have bccoiile less popular since they are more difficult<br />
to engineer than binary type ones and are less efticiel~t.
~ -<br />
/'-<br />
Binury vectors: A binary vector should contain essentially at least one of the<br />
borders, it should have the ability to replicate in E.coli and it should contain a selectable<br />
marker (Armitage et al., 1988; Hood et al., 1993). In principle, a binary vector consists of<br />
two plasmid; a plasmid that is transferred and a helper plasln~d. In the initial years it was<br />
pBI101, and later Inany verslot?s were constructed by Beckcr et a!., (1992). Additional<br />
information on binary vectors was provided by several authors (Jones et al., 1992; Futterer,<br />
1995). Rcccnlly it lias been suggehted to use binary vectors that conla~~l two acparale '1'-<br />
DNAs (Crameri el al., 1996). The log~c of tlie authors is that the antibiotic resistance<br />
marker will be lost during subsequent generations. A helper plasmid contains the vir gene<br />
complements that are essential for transfer of T-DNA.<br />
Protfioiers.. Futterer (1995) reviewed the subject of promoters for genetic<br />
. .<br />
transformation of plants. In the early years of gcnetic transformation of plants,<br />
~nvestigalors were merely interested ill showing thal integration and expression of<br />
transgenes is a rcality 111 plants. So, initially promoters endogenous to thc T-DNA were<br />
used. Soon 11 was observed that the promoters ibr opine synthesis were weak. Cliua and his<br />
--..<br />
collaborators (Odcll cl al., 1985) isola~ed tbc CaMV 35s promoter from turnip leaves<br />
~nfected w~rh the Caul~flower nlosaic virus (C;IMV). This promoter was found to be many<br />
folds stronger and resulted In constitutive expressior~ of tlie introduced genes. However.<br />
/<br />
sub-domalns of this promoter were found to be exelling tissue specific expression (Benfey<br />
and Chua, 1989). Since then this proliloter became an attractive candidate for plant<br />
molecular biology research. Its fusion wit11 part of mannopine synthase (MAS) promoter<br />
- -- .<br />
increased the potency of this promoter (Kay et al., 1987). Valuable information can be<br />
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<br />
.- -<br />
distinguished interest for finding out the promoters for monocots. In the early studies with<br />
/-<br />
rice (Shimanloto et al., 1989) the CaMV 35s promoter was used to activate tile selective<br />
7<br />
and repotter genes. However, it was found that this promoter was more efficient in dicots.<br />
Conlbination of this promoter with other pro:lioter segments and introns were even tried.<br />
This concept was followed by the wage of ccieul alcol~ul dehydrogenw I (Adlil) gene<br />
- +...- .-<br />
(Callis et dl., 1987; Kyozuka ct al., 1990). A silliilar approacll to integrate the first intron of<br />
the 3hru1iki.11 I gene of maize was also followed in cereal tratlsformat~on (Mass et al.,<br />
1991), but I: becatue less popular in the subsequent years. Rice actin gene pro~l~oter (Acll)<br />
- .<br />
was found to be even nlo1.e potent than the above two (Zliang ct al., 1991). This pronioter<br />
showed more or less similar potency as that of Ettiu pron~otcr tllat is a recombii~allt<br />
promoter cotltaining a ti-ul~cated Adlil~n?_oter with other elements (Last et a]., 1991). The<br />
--<br />
curretlt most effective promoter is tile Ubiyuiiiii I (Ubii) of n~aize (Clrrislcnseit et al.,<br />
1992). Tii~a proilioter was used successfully to tlnnsfornl !\,\.heat (Weeds el al., 1993)<br />
barley (Wan and Lemaux, 1994) and rice (Toki et nl., 1992). Another pronloter of tile rice<br />
Aldviure P (Aidl') gene was ibund to be one of tile bctter alternat~ve (Kagaya et al., 1995).<br />
Tcnrii~zrrlars: Knowledge of the ele~lleiits for geilc expression 1s as important as the<br />
promoters. It is considered that fundamentally n1RN.A is stable unless destabilizing motifs<br />
are involved. Specific examplcs of the studles that handled the polyadenylation signals in<br />
plants are investigations of Mogen ct al. (1990). 1:otiinie et al. (1994) studied the essence<br />
of the terminator regions and impact of 3'-end ~.cgions on the level of gene expression of<br />
octopine syntliasc gene and otllcr gene constructs was studied by lngelbrecht et al. (1989).<br />
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<br />
the respective chimeric gene.<br />
Selectablr O I I ~ rryorriilg nrarkers: These are essential comporlents of the total<br />
cassette that is to be tr:rrlsferrcd lo llle pla~ii cells, These genes select ihe transfor~lled cells<br />
from that of the untransibrnled. Sorlle of the popular selectable markers arc ant~b~ot~c<br />
markers such as kanamycin, hygromycin, strepto~nycin etc. Other groups of' selcctablc<br />
nlarkers are the ones that co~lfer resistu~lce to herbicides, such as phopbinohtrici~~.<br />
biolophos, glyphosate. dalapllon etc. As noted above this group of selectable nrarkers can<br />
serve a dual pull,ose: to select transfom~i~nts and to render crops resistant lo respective<br />
herbicides (D'Halluin et al., 1992). The third group is ti~verse, including gencs that cause<br />
resistance to high nitrate, 111gIl a~lli~~o ac~d levels (lys~ne or tl~rconine) or amino ac~d<br />
analogues (Schroit, 1995). Most commonly used selectable genc IS the kanamyc~n<br />
rcsista~~ce gcnc ineo~~lyci~l pl~ospl~otra~~sfcrase (~rpfll). This gene producl dctuxilics<br />
an~inoglycoside ;~~~ub~otics such as ka~~alnyci~~, neonlycin, gencticin and paromomyci~i<br />
(Vurdi et al., 1990). The gene Iipt was isoloied f'rom licoli. It codes for hygrornyc~n<br />
phosphotransfrrasc that deroxilies antibiotic hygronlyci~~.<br />
Kcporter ge~~cs are coding sequences that, upon expressloll in the transgenic plant,<br />
provide a clear 111dicat1011 tlliit gc~letic transfor~llat~o~l did take place. They are useful also<br />
for transient expressio~~ experimenis, in wlirch tl~c iransgene is not necessarily integrated<br />
into the lhost gcnolne. Scllrott (1995) rcvicwcd a review of genes used and tllcir assay<br />
methods. blost co~ll~no~lly used genes used, as ~cporters arc the ones that code for CAT,<br />
GUS, Luciferase and Glee11 1:luorescent I'rotein (GFP). Thc assay for the riidA that codcs<br />
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<br />
without extrnctiiig llie tissue. The luoiferase reporter gelie bas developed by de Wet a1111<br />
associates (De Wet el al., 1985) and was reviewed by Luelirsrn el al. (1992). The<br />
gene<br />
that codes for green fluorescelit prote~~i (GFP) *as the recellt one isolated from jcllytish by<br />
Chalfie et al. (1994). Many reports apprarcd In support snd against tlie usage of GFP<br />
(Ilaseloffai~d Amos, 1995).<br />
f<br />
2.2 Legume tissue culture and tra~~sfor~iiatio~~<br />
Leguminosae is a very i~i~portant falllily of angiospcrnls comprising of ~nany<br />
species in relation to Iiuman nutr~tion, pasture and tbdder needs. Important protein rich<br />
seed bear~ng plants, n~ostly lierbaceous, such as pros, lentils, beans collectively known ss<br />
pulses are members of this filmily, They rank next to cereals in tenns of human nutrition.<br />
In qua~ititative significance they are far bchind the cereals, however, gaining some due<br />
inlportancc as food additives ill tlie recent yc,irs. Domi~~ot~ol~ of tlic cereals in tlie food<br />
sector allowed oi~ly miirpinal ~~icreascs in tile overall yield of pulses. I
2.2.1 Food legumes<br />
There are several apecles and subspec~es classified as food legumes. But, only few<br />
(15 to 20) genera are vely important, Hundreds of cultivnrs witl~i~i these genera ore<br />
included in the agricultural practices, each having sorue selected attributes. Most ilnportaut<br />
of tliese species are Glycitie irrar, Amchis /i)y~ugoeo, CI~CL'~<br />
(~ri~iiir~iiri. L~IIS (1111i11~rris.<br />
Pisurri ~crfivu~ri, Lor/r)v.irs saiivirs. C(ijnrrres ccijair, Yigiiri ruiiiirio, Vig~in rriu~rgo, Yi,qiitr<br />
ocoirirfo/icr, Viyrrci rtr~ibcllntc~, Vigiia rirrgrricril~i/ir, Plroseulir~ vrrlgriris, Mrrrro~~~lorrrri<br />
nuj7onrr11 etc. These species col~stitute over 80% of total food legume output. 1111t1;1lly<br />
many of these specles were thougl~t to be recalcitlant ill<br />
tissue culture and latcr<br />
advancements ol' biotcchnological tecliiiiqlles gr;rdunlly e;ised the technical difficult~cs.<br />
Micropropagation was relatively easier wl~cli colnpared to adve~~titious shoot ~~rgenerauon,<br />
where the shoots originate fro111 pre-existing nieristems. Advelitilious regeneration is a pre-<br />
requisite for a successful ge~iet~c transforniation. Each species responded differently In<br />
tissue culture and some of tlie protocols were successf't~lly used for gcnctic transforniation.<br />
Organogenesis is a widely used tissue culture strategy for rcge~icration of whole<br />
plants via direct and indirect illduction of varlous plant organs such as sl~oots atid roots.<br />
Generally shoots are induced ~~iitially fiom selcc~ed expla~its followed by roots. Mult~ple<br />
shoots are il~duced e~ther directly or through callus phase where the techniques are termed<br />
as direct and ind~rect ol.gal~ogenesis respectively.<br />
Direcr organogeriesis: Following are some selected repons where organogenesis<br />
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<br />
developed for soybean (Glycine niar) using 2,4,5-T for embryogenic callus induction.<br />
Somatic embryogenesis was best on SE (soyabeen embryo) medium supplemented with<br />
BAP and best regeneration of shoots was found on hormone free medium or on medium<br />
/<br />
with IBA (Cho et al., 1992). Multiple shoot regeneration was obtained from leaf and<br />
hypocotyls explants of Glycine wigtii. 3-4 day old seedlings cultured on NAA and IBA<br />
containing medium gave rise to multiple shoot buds (Pandey and Bansal, 1992). A wild<br />
relative of soybean, Glycine clandeslina, was induced with brown, compact and nodular<br />
callus and plants were regenerated from it (Sharma and Kothari, 1993). Culturing of<br />
zygotic embryos and nlultiple shoot regeneration was studied envisaging their use for<br />
micro projectile bombardment in Arachis l~ypogaea (Schnall and Weissinger, 1993).<br />
Effects of auxins (IAA, NAA, IBA and 2,4-D) and cytokinins (kinetin and BAP) were<br />
studied for multiple shoot regeneration from cotyledons and cotyledonary node explants of<br />
Arackis liypogaea (Venkatachalam and Jayabalan, 1997). Various concentrations of BAP,<br />
2-iP, chloropyridylphenylurea (4-PU), TDZ and zcatin in co~nb~nation with NAA were<br />
used to i~~lprovise the regeneration from cultured leaf segments in Aruchis hypugueu<br />
(Akasaka et al., 2000). Explants such aa petioMes, ep~cotyl sections and other seedling<br />
explants were used for regeneration of AI.uL.~~J liypoguea (Cheng et al., 1992).<br />
Regeneration via caulogenes~s (shoot organogenesis) was achieved in Arachis /~ypaguru,<br />
from plumular explants. Tile shoot buds rcgeilerated on medium containing brass~n, BAP<br />
and P-naphthoxy acetic acid (Ponsamuel et al., 1998). In vitro regeneration of Arachis<br />
hypogaea was achieved via organogenesis by employing BAP as the principal multiple<br />
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<br />
responsive in tissue culture. However, differentiation in tissue culture using mature leaf<br />
expiants was first reported i~i Aracltis vil/os~r/ic.orpu (Johnson atid Pittman, 1986). A<br />
protocol for tlssue culture based regeneration of Artichis pitrrui, a wtld perennial peanut.<br />
was developed. Day length and media effects were directly correlated with variations In<br />
regeneration (Ngo and Quesenberry, 2000). Effect of aluminium on the tissue cultures of<br />
Piiuseoliis t'iilgirvis was studlcd (Esp~tio et ol., 1994). Direct plant regeneration and<br />
multiplicat~on was obtatiicd ftom the embryo and cotyledons of cominon bean Pkusc.olus<br />
vlugaris that were inibrbed for 3 days atid cultured on a soil rnediun~ for 7 days (Mohamed.<br />
1990). A small portion of split embryonic axis sliowed a genotype dependent multiple<br />
shoot regeneratioti in Pliuseolirs vulgaris and P, cocci~~rus (Santalla ct al., 1998). Various<br />
factors sucli as l~ght intensity and duralion plant growtli regulators etc, were studied whilc<br />
regeneratitig multiple al~oots from mature en~bryonal axes of Cujanus Cajar~ (Franklin et<br />
al., 2000). Epicotyl, hypocotyl, leaf, and cotylcdo~iary nodal explants were shown to<br />
regenerate a h~gli frequency of multiple shoots with high BAP and kinetin of pigeon pea<br />
(Cajoirus caja~~) (Gretha et al., 1998). Multiple shoots %ere regenerated from distal ends of<br />
cotyiedo~~ary segments of. Caja~rirs cajan. This was achieved using conibinations of BAP.<br />
kinetin with adenine sulfate (Molian and Krislinarnurthy, 1998). Cotyledonary node culture<br />
using BAP and IAA was reported in pigeonpea, Cajirtlus cujut~. A mass of~nultiple shoot-<br />
initials formed at the axillary bud region of. tlrc cotyledonary node of the seedlings within<br />
two weeks. The cotyledotia~y node along witli the mass of shoot-init~als excised from the<br />
seedling, continued to form new shoot-initials on MS medium containing 6-<br />
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<br />
done. Mult~ple sliuots were induced fro111 the cotyledoliary node explants of FI hybrids<br />
(Aven~do et al., 1991). Effect of culti~re ~iledi~i~n o~i plat11 rcge~~eratioii fron~ cotyledons of<br />
Yigrrcr radiaia was studled. Genotypc size, oriclitation 2nd age of thc explant showed very<br />
sign~ficant effects on plant regeneration (Gulati and Jaiwal, 1990). Diftkrsnccs 111 siioot<br />
regeneratio11 liolii coryledo~iaiy llodc expla~its 111 Asiatic Vigrtci apccies were used for<br />
gelioliiic group~~ig wtth~n subgei~us ('rro/oiro/~/rb(Avcnido and Ilattor~, 1099). Shoot lip<br />
cultures were establislied for plant regeneration of n~u~igboa~i, Vigtzucirliolci Complete<br />
plants njere regenerated d~rectly without an intervening callus phase from shoot tips on<br />
basal nlediuril (MS salts -. I35 vitamins). Regeneration frequeilcy var~ed with cultivara,<br />
explant slze and growth regulator conib~natioii; 111 [lie ~ncdiu~n. Additio~l of cytok~~lins<br />
induced a var~able atnount of callus at the base of tlie slioot tip, followed by multiple shoot<br />
formation. BAP, kiilrtili and zeatiii each ~nduccd i~~ulliplc slioors ill 100% of the cxplants<br />
but thc liigliest ~luiuber of regenerntits per explants (9) was produced with BAP. (Gulati<br />
and Jaiwal, 1992). Regeneration was achieved using cotylcdo~laiy nodes giving rise to<br />
axillary shoots of I'ignu<br />
i~tuttgo (black gram). Regz~ierntion lias been acli~eved through<br />
organogcnesis usiiig explilnts from axillary slioota or~ginatiilg froni the nodes of seedlirigs<br />
ger~n~~iated in cytoki~li~i co~i~ai~lillg rnedluni. Seeds gerulinated in tllidiazuron (TDZ)<br />
supplemeilted MS produced 11 axillary shoots/cotyledonary node. Stern and petiole<br />
explants derived froin these axillary-shoots produced callus along with shoot-buds after 2<br />
weeks of culture on half stl-engtll MS supple~nentcd with NAA. Shoot-buds were also<br />
produced from varlous sites of injury caused by ~ncisions on the stem explants. Full<br />
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<br />
high percentage (96%) of expli~nts prod~icilig .n least 30 slioot bud per expinlit (Iletre~ c~<br />
al., ~YY~J.<br />
Irii111~~c.l o~~flriogcvw~i~: Regcncri~tioli ~III(I ~IIJI~SIS ur c;~llus li.olii Ilu\v-sorted<br />
heterokaryolls of soybeall (Gli,c,~ric 1rici.1) mid 6' c.ciir~,sc,c.irs was donc (Haliiluatt ct a1 .<br />
I9S8). I'lcces<br />
of 1e:rvcs li-o~n sectll~ligs ot' ,liii~,/ii\ jirii/oi ivclc IC~L'IICI;IIC~ \I;I<br />
orgaliogelieals a~ld so~li;~tlc e~iibryogcncsis path\\.;lys. Plant rcgelleratioli was obtaillrd<br />
I;I<br />
two developmentnl path\vays o,-ganogenesls and soniatic eniblyogenesis. Orgaliogcnic<br />
callus cultures were i~illiatcd Sroni pleces of leaf oli MS niediunl suppleliirlitcd wltli NAA<br />
or 2,1-D ill culiiblliatiuli \villi BAP, kinetill or 2-IP. Tlie niust sui~able co~~ibi~iat~u~i for<br />
plant rege~ieratioii tliruugl~ orgn~iuge~icsis was a11 iliitial lliediuli~ cornposed ol' NAA and<br />
BAP fbliowed by transfer ot'the callus to a slioot inductioli medium (MS+ BAP) Routing<br />
of regenerated shoots was read~ly acli~cved by culture on MS w~tli NAA. Embryogenic<br />
callus cultures were inltialcd froni plcces of leaf on MS n~ediurn supplenlented with<br />
picloram ~n coliib~lietio~l w~ih kitletin, Aeatlil, BAP or 2-iP, alid the iiioat suitable<br />
comblnat~ons wcre picloram, BAP or 2-il'.<br />
(Iky ct al., 2000j. A comparative study of<br />
callus t"ormation and pl;~nt regelicratlon was dolie using d~fttrciit explants of Phuseolus<br />
vulgaris and P. coccitieus (Ruiz et al., 1986). A different sylithctic auxin 2,3,5-<br />
trilodobenzoic acid was sllowii to ~nduce callus atid roots on stem cuttings of niungbeali,<br />
Phaseul~is aureus (Ali and Jarvis, 1988). A novel liietliotl of culturing lcaf disc explnnts of<br />
pigeon pea (Cojrirruv ccijtrri) on ~~iulril)lc slioot ~liduction niediuni with IAA and BAP where<br />
the shoots originated via callus phase (Itathore ct al., 2000). Plants were regenerated fro111<br />
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<br />
solidified w1tl1 0.8% ag;ir ,lnd colitallnlig NAA 311d b~~i~ylilde~i~ne. Slloot regeneration was<br />
readily ach~eved and roots were induced when shoots were transferred to medium devoid<br />
of growth regulators or w~lh NAA. Callus from A. jnlcciicl failed to regenerate shoots.<br />
Explants froni leaflets of A. jluri~i~~ei~sis d~d not produce callus w11e11 cultured ill vltro. (Rey<br />
and Mroginskl, 1996). Pre-soaked sccds of Viglici rtrtliiiici showed variable callus growth<br />
wile11 exposed to varioua liite~lsit~es of light whcrc tho slloot regenciatlon was also vnr~ablc<br />
under dlferellt intens~tics of light. Hormonal supple~nents of tlii: culture rnediun~ liad<br />
some proniotive effect on regeneration under various light intensities (Narciso et al., 19L)7).<br />
Analysis of tissue culture borne genetic variations (somaclonal variations) was done in<br />
Pislo11 sotiviirii (Griga et al., 1995). One hundred and forty six son~aclo~ies were generated<br />
that were resistant to the puritied toxin of Cc.rcuipuru cciilesctvii (Kaush;~l et al., 1997).<br />
Somatic cnibryugcnesis is anuther el'ticient strategy where regenerating tissue<br />
Initially attalns some dctined globul'ir, ell~pt~ct~l etc, shapes and .tilose units gradually<br />
regenerate illto whole planls. Usually tile globular proenlbryos split into torpedo shaped<br />
ones and shoot primordium regenerate from tile axillary portion. Relatively regeneratloll<br />
via somatlc embryogeilesis pathway consumes more ti~ne than organogenic pathway. A<br />
fast and efficient regeneratloti system vla somatlc enibryogei~esis was developed uslng<br />
BAP and NAA on cotyledo~l explailts of Glycirie rliux. Plants were ready w~thin six weeks<br />
from explant stage (Fu et al., 1995). Ail efiic~cnt regeneratton system was developed for<br />
Glycirre io~r~ertrc.ito via sonlatic embryogenesis pathway. Effect of plant growth regulators<br />
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<br />
from seedl~ng explants of Arochis l~ypogaeii (Gill and Saxena, 1992). Direct somatic<br />
embryogenesls from zygot~c embryos derived from 40-day-old immature pods of Arachi~<br />
i~ypoguea using 2,4-D were studied where various factors like growth regulators, sucrose,<br />
genotypes and length of elnbryo~iic axis influenced frequency (Reddy and Reddy, 1993).<br />
2,4-D-induced somatic elilbryoge~iesis was obtained from embryogenic calluses derived<br />
from hypocotyls explants of Arucilis iiypoguea. High concentration of 2,4-D decreased the<br />
frequency of somatic embryogencsis (Venkataclialam et al., 1997). Synthetic seeds oS<br />
Arucl~is iijpogueu were obtained by encapsulatilig 5 to 30-day-old somitlc embryos and<br />
geminated by on medium with various concentrations of sucrose, maltose, BAP and NAA.<br />
25% of these embryos were filially converted into plantlets (Padmaja et al., 1995). A<br />
refinement of embryo rescue technique to improve plant recovery from early heart shaped<br />
embryos of interspecific hybrids of Pi~ascolur poiyutlrtlus and P. viilgaris was reported<br />
(Geerts et al., 1999). Rates of ethyle~ic production were determined in highly embryogenic<br />
and virtually non-embryogenic tissue cultures of Aledicago sarivu ssp. faicata during a 10-<br />
day induction period on medium containing 2,4-D and kinetin, and during the first 10 d of<br />
somatic embryo ibrmation on growth regulator-frcc medium. It was concluded from thesc<br />
experiments that the high rates of ethylene product~on during embryo iilduction are not<br />
essential for subsequent enibryo differentiation (Meijer, 1989). Efiicient plant regeneration<br />
via somatic enibryogenesis has been developed in pigeonpea (Cajanvs cajan). Cotyledon<br />
and leaf explants from 10-day-old seedlings produced embryogenic callus and somatic<br />
embryos when cultured on MS supplenlented with 10 pM thidiazuron (TDZ). Subsequent<br />
withdrawal of TDZ from the induction medium resulted in the maturation and growth of
'<br />
the ernblyos into plantlets on MS basal rnediun~. (Sreenivas et al , 1998). Distal ends of<br />
cotyledons were used to induce somatic embryogenesis in Cajanus cujan by applying<br />
BAP, kinetin and adeilitie sulfate atid whole plants were regenerated (Patel et al., 1994).<br />
f<br />
Direct somatic embryogerlesls was induced from excised seedling leaf segments of<br />
/<br />
vegetable legume, Psophocnrp~is reirngo~iolobiis by using NAA atid BAP and the<br />
coliversioll frequency of cotyledonary embryos was 53.3% upon culture on MS medium<br />
cor;taiili~lg ADA for 7 days followed by transfer to MS medlum supplemented with 1BA<br />
and BAP. (Dutta Gupta et al., 1997).<br />
2.2.1.3 Other methods<br />
Besides orgal~ogellesis and sotllatic enlbryogenesis the techlliqucs such as<br />
regeneration of platits from isolated protoplasts, microspore, anther and ovule cultures etc.<br />
are less frequetltly applied in the tissue culture studies. Excellent ylelds and quality were<br />
achieved for soyabean (Giyci~ie ~llax) protoplasts and l~latits regenerated from agitation-<br />
derived protoplast preparations had a higher cllance of being derived from intact cells.<br />
(Zaghnlout et al., 1990). I1rotopl;~st isolation in Araciiis iiypogu~a is relatively rare<br />
phenomenon and there is one repor1 for isolatioll and regeneration of plantlets through this<br />
method and tiis method was effectively used Ibr elcctroporatioll mediated tralisforniatioll<br />
(Li et al., 1995). A different method us111g 1hi11 cell layer tech~i~que and transverse thin cell<br />
layer (iTCL), where the ffCLs were cultured on TDZ for Piiaseolus vulguris was<br />
employed. Shoot multiplication was enhanced uslng BAP with silver nitrate (Cawallio et<br />
al., 2000). A reproducible protocol for plant regeneration from seedling hypocotyls<br />
protoplasts using varlous growth regulators such as zeatin riboside, GA, and 1Bh was<br />
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.,<br />
1980). Protoplasts were isolated from leaf tissue of Lens culinaris by using cellulase,<br />
macerozyme dissolved in 0.5 M mannitol witit pental salts. However, the callus cultures<br />
could not be regenerated into plantlets (Stiff et al., 1991). Genetically variable plants were<br />
obtained from anther derived callus cultures obtained from microspore cultures of Cuja~ius<br />
cajun (Kaur and Bhalla, 1998). Cryopreservation is an excellent method for germplasrn<br />
conservation provided an efficient method is abailable for regeneration. Methods for pollen<br />
embryo cryopreservation and conservation of germplasm of Arachis, Rrossica and<br />
Triticunl sp. were explicitly reviewed (Bajaj, 1983).<br />
2.2.1A Genetic transforniatio~i<br />
h/<br />
Aerobacierii~m-~nediated frat~sJ?~r~t~urio~i: Direct crown galls were induced by<br />
infecting stem explants of Lentil (Le11s a~lbiuris) with four strains of Agrobacieriuni<br />
lurne/iucie~is. Opines were detected in the crown gall and Southern analysis showed that T-<br />
DNA was transfersed (Warkentin and MctIughen, 1991), inclusion of potato suspension<br />
culture in the culturc niediuni cnilanced the trailsformation frequency of the callus obtained<br />
from In vitro grown seedlings ofthe Glyci~~e 1lia.r (Chang and Chan, 1991). An efficient<br />
protocol for Agrobacreriuiii-n~ed~ated transfornlatio~l of cotyledon explants from in vitro<br />
grown seedlings of Amchis 1typogoc.u that resulled in a very h~gh frequency of<br />
transforniation (55%) was reported. The expla~lt, were transformed with binary vectors<br />
pBI12I and pROK1I:IPCVcp that consisted oC1ipt11 as seleclable marker gene and lndlan<br />
Peanur Clump Vlrus coat protein gene as the agrono~llically important gene (Sharma and<br />
Anjiah, 2000). Co-cultivation of cotyledonary node explants of Ari~cir~s Iiypuguea was<br />
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<br />
(Venkatachalam et al., 1998). Transformation of A. hjyogaea was also done using the<br />
somatic embryogenesis pathway. The cotyledons were co-cultivated with A. rumefaciens<br />
strain LBA4404 containing uidA and 11pill genes. Somatic en~bryos were ~nduced with<br />
NAA and BAP and later regenerated into whole plants with a transformation frequency of<br />
47% (Vcnkatachalam et al., 2000). Embryo axis explants were uaed for thc<br />
Agro6ucie1~iiii1i-ri1ed1ated transform;~tion in /lriiciri;, I~ypogcieu (McKenlly et al., 1995). In<br />
vitro grown seedlings of Crijoirrrs crijait were inoculated with three types of wild strains of<br />
Agrobncirriu~ir A281, A6 and T37 and cultivar-Agrobacleriiiir~ strarn specilic induct~on<br />
tumors was found (Rathore and Chaod, 1997). Lcaf disks of pigeon pea cv. ICPI5164 were<br />
transfor~iied by il iur~iejiciens strain LBA4404 plasniid pBAL2 carrying kanumycin<br />
resistance and GUS reporter genes under tile control of tile 35s promoter. The optimul~i<br />
period of coc~ilt~v;i[ion was 4 days, giving 47.8% tronsfor~ned calluscs (Arundhati, 1999).<br />
Tr;~nslbrn~alion of pigconpea wila acli~evcd LISII~~<br />
A. r~i~rlefu!/a(.if~t~ strain GV2260,<br />
containing the construct of isolated cowpea proteinase inhib~tor gene, pCPI. The gene was<br />
dr~ven by CaMV 35s promoter containing kanamycin resistance as plant selection marker.<br />
hlolecular analysis of tile putative transibrniatits was done by Northern blotting technique<br />
(Lawrence and Kuundal, 2001). /Igrobocleriiii11-n1ed1ated transformation of Vigi~u<br />
sesquipeiiuiis was achieved using cotyledonary node explant, where 2% of the shoots<br />
showed integration of riprII, phosphinothr~cin acetyl transferase bur) and uidA genes.<br />
Integration was confirmed using GUS histo-chemical assay and Southern blot analys~s<br />
(Ignasimuthu, 2000). Hypocotyl and primary Icaves excised from 2-day-old in vltro grown<br />
mungbean (Vigrla rudiata) were used for tlle transfomiation studies. This particular gram
legume was considered to be liighly recalcitrant. A convincing transforniation frequency<br />
was obtained and tlie frequency was confir~ned using GUS li!stochelliical assay and<br />
Southem blot analysis (Jaiwal et al., 2001). Culture and co-cultivation of priinary leaves<br />
of Vig~a mungo resulted In transfor~~ied calluaes tllat dld not regenerate into whole plants<br />
althougli the selected calluses exhibited positivr NPTll assay (Karthikeyan et al., 1996). A<br />
genomtc fragment e~lcodiny Plraseolrrs vulgaris arcelin-5a protein that confers resistance to<br />
an insect pest Zalrrores sirbjisciatus, along with 11pil1 and ~ridA genes were constructed<br />
into binary vector which was used to tmnsfon~i Pliasrolur ucrtri/o!ius<br />
where bud explants<br />
were used froiii genotypes (Dillen et al., 1997a). Callus of Phasrolus acutifolius var.<br />
acut~alius, the Lepary bean, was co-cultivated with Agrobacterium tumefaciens strain<br />
C58ClRif. Due to the high regeneration co~llpetence of P, ~curijolius, transfonned plants<br />
could be raised and transformed seed was obtn~ned. It is suggested that by interspecific<br />
hybridization of rransfbrnied I-', acurijoliirs witli tlie regeneratioil recalcitrant P, vulgclris,<br />
inrrogress~oii of desirable genes into P. virlycrris could be achieved. The relevance of thia<br />
approach with reference to alternative techniques aimed at reducing or omitting the ~ieed<br />
for in vitro regeneration (e.g. pollen transforniation, mcristem transfoniiation) is assesscd<br />
(Dillen et al., 3997b). Epicotyli and iiodal explants of' Pisii~~r surivlrm were transformed<br />
using binary and co-integrate vectors. The transformntion frequency was found to be the<br />
funct~on of explant source, A, tu~~~rfaciens straio, pea gellotype and duration of co-<br />
cultivatiori (Katlien et al. 1990). Sonx co~nniercial breeding lines of Medicago sutiva (alfa<br />
alfa) were transfornied by Agrohacteriuni method. Stable transgenic material was screened<br />
with nptII speclfic PCIl amplification and Soulliern hybridization (Desgagnes et al., 1995).<br />
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<br />
protoplasts (Kuchuk et a]., 1990). A. lui?teJ'ucieils med~ated transformation was performed<br />
with some members of a population of Medicogo sii/ivu into wli~ch a trait of somatic<br />
elnbtyogenes~s was ~ncotporated vla breeding and the transge~iic pla~its were analyzed by<br />
PCR (Du el al., 1994). A. i~oiie/i:cieilr mrdia~ed tra~isformetian of Liipiiitrs riiuiubilis was<br />
done using shoot apical explants. A first report in tliis liarticular platit, tratisforman~s werc<br />
contirlned with 11oi1-'idionctive Soutlicri~ iiybi.~dtfiition (U;iboogln et al., 2000) AII clllc<br />
accession, ClAT 184, of an important paslure legume, Sly/osutilh~'~ guiuiie~is, was<br />
transformed by Agrubac./eriiiiii-111ediated oictl~od with binary vector harboring itpill and<br />
11idA genes (Sarria et al., 1994). A rapid and reproducible protocol for l'orfoliirr~i<br />
subterrar~eiiin, a subterranean clover, was standardized uslng itpill, uidA and an alplia-<br />
amylase i~iliibitor gene. Tlie protocol shows that glucose and acctosyringo~~e was required<br />
in thc co-cult~vation medium. Four cointiiercial cultivars were successfully traiisfomed<br />
(Khan et al., 1994). Genetic tral~sfortnation of tllc broad bean, Vicici jhbu, was done using<br />
.4, tu~?iejircie~is and A, rhizogencs. Ttiree cultivars and mutants wcre used for the<br />
transforniai~on stud~es (Jelenic et a]., 2000).<br />
,,&lisiics:<br />
Biolistic trnnsibrniation of Gl),ciiie iiiclx was done using a bovine milk<br />
protein, p-casein cloned under seed specitic lectin promoter (hlaughan et al., 1999). The<br />
method of electroporatlon was used to transform the protoplasts of Glycbie urgvreu.<br />
Protoplast colonies developed into callus and 78% develop into transformed shoots that<br />
showed nplII activity (Jones and Davey, 1991). Elnbtyogenic callus tissue from various<br />
cultivars of Aracliis liypogaeu was used for bioiistic-mediated transformation. Callus from<br />
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<br />
(Livingstone and Birch, 1999). A novel method for transfor~natiol~ of Cajo~lus cajatt via<br />
biolistic bombardment was developed uslng a vector contailling heterologous oat arginine<br />
decarboxylase cDNA that is an Important gene ill polyami~ie metabolisni. An increase of<br />
putrescine levels was found in the transgeliic lines (Sivarnant et a]., 2001). Genetic<br />
transforn~ation of Pi~aseoius corcineris and P. v~ilgiiris was done using novel nylon micro<br />
projectiles. Ethanol co-prec~piration niethod for DNNpartlcle preparatio~l was superlor to<br />
that of ~a'*/s~ermidine (Genga ct al., 1992). A ~iovel method of electric discharge partlcle<br />
acceleration was used to transform seed l~ieristenl explants of Phusrolus vlgaris. 0.03% of<br />
confirmed transformed plants were recovered (Russell et al., 1993).<br />
Orher ntetho[ls. Electroporation, microinjection etc. are sonic methods of genetic<br />
transforn~ation of plants that were used less oftcn. Mesophyll protoplests of Cajutius cuju~i<br />
were elctroporated wit11 pias111id construct conta~ning 11p1ll gene as selectable ~narker and<br />
the transgenic callus was produced. Tlie transforn~ation freql~e~~cy was observed to be 30%<br />
(S;ira~lg~ ct :I\ , 1991),<br />
2.2.2 Forage atid pasture legumes<br />
Legunlinoceae is famlly of over 18,000 species, which has a distinct economic<br />
importance by virtue of processing protein rich seeds and ability to fix atmospheric<br />
nitrogen through symbiotic nitrogen fixation. Over 70% of the legumes are forage and<br />
pasture legumes (NAS, 1979). Many domesticated and wild animals feed on these forage<br />
legumes deriving most of their nitrogen require~nents from this class of pasture legumes.<br />
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<br />
donlesticated anltnals.<br />
2.2.2.1 Orga~~ogencsis<br />
Llirecr orgai~age~iesis: A complete in vitro plant regeneration syste111 was<br />
developed for nzuki bean, Vigils migiiiuris wllerc advent~tious regeneration was observed<br />
from hypocotyls segments of cotyledonary node explants by using BAP (Avenido and<br />
Hattori, 2000). Elhylciie inl~ibnurs such :IS sliver nitate. 2,5-i1orbun1ad1ene 2nd cobitlt<br />
chloride were shown to enllailce the orgallogenic frcqucncy from the cotyledon explants of<br />
cowpea, Vigila ur~yuicuiulo (Brar et al, 1999). Regeneration wus achieved uslng<br />
hypocotyls and co:ylcdonary explants exc~ssd from green ~nln~ature pods of cowpea<br />
(Vigi~rc uiiytiicuiuru). A primary polyani~ne putrescine was also used and some son~aclonal<br />
variation was also observed (Pellegrineschi, 1997). Morphogencc potentials of shoot<br />
regeneration from root explants of Loius coriricuiurus were exploited to regenerate whole<br />
plants. Explants were ~soli~ted fro111 3 different parts of the root from 4, 8 and 16-day-old<br />
seedlings and buds were ibr~ned on the proxinlal end of explants, and roots on the distal<br />
end. Explaiits located proximally regenerated mow shoots than those orig~nat~r~g from the<br />
distal end. In the presence of BAP the ~nu~llbcr of rcgcneratcd plants was higher because<br />
numerous meristi'matic zones formed in the secondary cortcx. In contrast to the explant<br />
response oil horoione-fret: medium, disturbance of explant polarity wcre observed in the<br />
presence of BAP. (Rybczynsk~ et al., 1995). The pie-lrealrne~lt of immature inflorescence<br />
with phytohornlones, especially BAP, and culturing on horrnor~e free medium, resulted in<br />
shoot bud induction and shoot dlfferentiat~on, of an inlportant pasture legume Medicago<br />
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<br />
nlultiple shoot frequency was obtained on ~ilrdiuili contain~ng 50 pM BAll (Malik et al.,<br />
1992). Mulliple shoots were induced born slioot tip explan~s of iii vitro grown seedlings of<br />
iWacror)~io~~~ii ii~iijluriri~i. Effects of ;tdclllne sulfatc. BAP and IBA were studied<br />
(Varisaimoliamcd ct al., 1999). High frequency regeneratio11 of adventitious shoot buds<br />
was observed in Pi~~ilit suti~~ir~ii. Silver nitrate did not sllow :my promotive effect on<br />
nlultiple slioots but resulted in slloots \vttli ucll-dcvelopsd tetldrtls and large stipulcs<br />
(Ozcan et al., 1992). Young i~lflorzscenccs of Cerutu~iic~ silic/uu were cultured 011 MS<br />
mediuni supplenlerited with BAP with various concentrations of casein liydrolysate fbr<br />
obtaining multiple shoots (Bl~alerao and Chinchanikar, 1992). Luthyrlis sj~lvestris (flatpea)<br />
is an important forage legume especially in acid~c soils. Hypocotyl derived multiple shoot<br />
systeni was developed for the clonal propagation of this species (Foster et al., 1991). All<br />
efficient micropropagation metllod was developed for Scsbo~licr rostru~u by opt~~li~z~ng<br />
parameters 11ke variations in the basal mcdiulli and pilotoperiod changes (Pellegrtneschi<br />
and Tepfer, 1993). Mult~ple shoot rcgenvrurion was observed fro111 Immature seedl~ng<br />
explants of Lupiriu~ ~riu~ubilis by using TDZ with modified Schenk and Hildebrandt<br />
lnediulrl (Rah~m et a]., 1999). Complete plants of Lupi~rus iutetrs were regenerated from<br />
hypocotyls segments and were eficielitly ~nodulated by Brudyrliizobiu~n sp. (Dam and<br />
Chamber, 1993). A~~tityilis cytisoides, a legulne shrub used for aforestation and reclamatiori<br />
was successfully regenerated fro111 both juvenlle (cotyledonary nodes and apical buds) and<br />
mature (axillary buds) explants (Gavidia et al., 1997).<br />
Indirect orgunoge~~esis: Plant regeneration was achieved via callus phase using<br />
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<br />
NAA were sl~own to br crit~cal factors for both the frequency and \he type of callus<br />
format~on as well as for the potential of callus differentiati011 (Luo and Jia, 1998). Petiole,<br />
stem, lzaf and cotylcdonaty explants of Medicoyo saliva werc used to induce callus by<br />
ustng 2,4-D and whole plants were regenerated (Moursy et al.. 1995).<br />
NAA in<br />
combi~iation w~th BAP was used to regenerate plants via callus phase by using stem, rach~s<br />
and leaf explants of beach pea, Lurliyr~rs jcipuriicrrs (Debnath et al., 2001). Callus,<br />
organogenesis and plantlet fornution was observed from seeds of Clltoriu renlutru in the<br />
presence of high kinetin and IAA levels (Lakshmanan and Dllanalakshmi, 1990).<br />
Canavanine and canaline were detected in the callus cultures induced with BAP and IAA<br />
in Cariavuliu lirieum (Hwang et al., 1996). Shoot buds were regenerated either directly or<br />
through callus phase from leaf explants of fodder legume Vignu ocorti~i$olin and various<br />
factors affecting regeneration were also studied (Bhargava and Chandra, 1989). Four<br />
species of Srsburiiu, S brspirrosa, S, caniiubiria, S. l;'oniiosu and S, se~buii were<br />
regenerated in vitru by using root, hypocotyls and cotyledon explants. Callus was induced<br />
with 2,4-D and shoots regenerated with BAP (Zhao et al., 1993). Callus was obtained from<br />
mature leaves, stems, petioles and roots of young seedl~ngs of Psoruleu corylifolia and<br />
regenerated to whole plants on BAP contai~ling n~edium (Saxena et al., 1997). Hairy rools<br />
that were induced by infection wltll A.rl~izoger~es were used for the regeneration of<br />
rnulr~plc slloots In Crotolureu juilcea followed by colrlplete plant regeneration and<br />
confirmation of their transgenic nature through Soutllcrn blot analysis (Ohara et al., 2000).<br />
Seventeen accessions of Medicagopolyr~iorplicl were screened for their capacity to produce<br />
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<br />
the winged bean, Psophocorprrs tetrugo~~olohris. Shoot tip, epicotyl, hypocotyl and<br />
internode explants were cultured on Ms rnedia supplelnented wit11 different concentrations<br />
and combinations of BAP, NAA and IAA. Plant regeneratton was acllleved from internode<br />
and hypocotyl explants via direct orga~iogenesis and fronl internode and ep~cotyl explants<br />
via indirect orgallogenesis drpend~ng on tllc gsuwti~ regulators (A~~julll;iilara et sl., IYc)X).<br />
Callus derived shoot regeneration was achieved from root explall~s of Lutilyr~a sor~vus by<br />
using various concentrations of kinetin and rootillg was done on IBA (Roy et al., 1992).<br />
Macrufiiiurn uirupurpureuni is a ~uodei plant wit11 broad synlbiont range fbr ~lodulatlon.<br />
First report of in vitro plant regeneration 111 th~species by using hypocotyls expiants was<br />
reported by Ezura et al. (2000). A novel rnethod of ind~rect organogenic regeneration was<br />
developed for Pisuni soiivirn~ using thin cell layer segments of nodal explants I'roni which<br />
leaflets and axillary buds were removed (h'aue1.b~ et al., 1991). Indirect regeneration of<br />
shoots via callus phase was obtained In paature legu~lles Lolo~~urlls Darrie~ii (Bovo et al.,<br />
1986), Cer~iruse~rla brusilionurr~ (Angclo~u et al., 1YY2), Desr~totliur~i uJJine and D.<br />
unciirofu~r~ (Iley and Mroginski, 1997). Various explants like hypocotyls, root and leaves<br />
of pasture legume Slyloso~rfhrs g~lyane~~~is were used to regenerate shoots via callus pha~e<br />
(Meijer and Broughton, 198 1).<br />
2.2.2.2 So~natic enlbryogr~~esis<br />
Five species of Metiicagu, M. ciliuris, M. ~r~erc.~, M. orbicuiaris, M. polyniorpha<br />
and M, truncuiulo were assessed for somatic en~bryo induction with 2,4-D. Incorporation<br />
of PEG resulted in better maturation of the embryos (lantcheva et al., 2001). Callus was<br />
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<br />
somatic embryogenesis and plant regeneration was obtained from protoplasts of red clover,<br />
Trifoliitm pruretrse (Radionenko et al., 1994). Plants were regenerated by the direct sonlatic<br />
embryogenesis from the cultured embryos of genus Trijoliu~i~ (Repkova, 1991). High<br />
kequency somalic enibryogenes~s and plant regeneration was achieved from the callus<br />
cultures of Asrrugulus udsurge~ls (Luo el al.. 1999). A protocol for regeneration vla<br />
somatic erlibryogenesis pathway in the pasture legume Cliloria iertiuieri was developed.<br />
Manipulation of kinerill in combi11at~o11 with IAA was found to be usethl (Dhanalaskhnl~<br />
and Lakslimanan, 1992). Methyl jasmonate and Abscisic acid were used generally for the<br />
maturation of induced soniatic embryos. However, their application in the soniatic embryo<br />
induction medium was tested and they were found to be inllibitoly to tlie embryo<br />
induction. Somat~c enibryo induction in Metliccigo sarrvu was also fol111d to be Inhibited by<br />
ami~ioetlioxyvi~iylglycine, amino-oxyacetic<br />
acid, 2,4-dintrophenoi and salicylic acid<br />
(Meijer and Brown, 1988). With a view of obtaining plailts free lion1 neuroloxin, a qulck<br />
and efricielit system was developed for r.egeticrating plants by using explents from wide<br />
range of tissues of Lailyrur sciiiviis. An embryo rcscue method was also described to<br />
fiicil;tate inter-spec~tic hybridizations (Misra et al., 1994). Immature cotyledons of Vignu<br />
sinensir gave rise to somatic embryos on 2,4-D and BAP-containing medium, which<br />
eventually developed into whole plants (Li et al., 1995). Treatment with 2,4-D followed by<br />
BAP treat~llclil reger~ct;itcd whule plants vla ;III exuberant sonlatic embryogenesis from<br />
leaf sections of h.letliccigo ~ujjrrtricosn (LI and Denlarly, 1996).
~$2.3 Other metltods<br />
Protoplasts were isolated from immature co~yledons of G/yciile soja. These<br />
protoplasts started to divide after 3 days of culture and the division freqtle~lcy of<br />
protoplasi-der~ved cells counted at 12 days was 36.8%. Slioot buds were regencrated on tl~e<br />
surface of the nodular celluses with a frequency of25% \\hen tlic cnlluses werc placed OII<br />
MSB medium with IAA and BAP. Wliole plants were regencrated uiio~i transfcr of 3-4 cm<br />
shoots to 50% MS mediu~ni witli IDA (\\lei n11d Xu, 1990). A brcnhtl~rougli In the pl,lnt<br />
regencratlon from tile protoplasts isolated from Vicia jrrba and V. tror6oire1r~i.s. I'rotupl;i,i~<br />
of 10 cult~vars were isolated l'rom etiolated slloot lips and tested for their regcnc~~lt~o~~<br />
capacity. After purification, protoplasts were embedded in sodiun~ algitiatc and cultured 111<br />
the medium contait~ing 2,4-D, NAA and BAP. Division frequencies of up to 40% werc<br />
obtained. Six weeks afier cmbcdding, protoplast-dcrivcd calluscs were transfcrrcd to<br />
Gelrite-aoiidified mcdin wit11 different colnbinationa of groivtli rcgulalors (Tegeder et al..<br />
1995). Low voltage treatment and nurse cells from Medicngo ~crriva were used to<br />
regenerate callus from protoplasts isolated from seedling aiid suspcnsio~n cultures of<br />
7i.i/olirii11 slrb~errri~~eii~ir (Li et al., 1990). A protocol Tor the isolatiorl of root proloplasts<br />
from Vigi~ri rridiara and leaf nlesopl~yll protoplasts of ~niotlibean, V cico~ritijolia gave a<br />
plating efficiency of 1.3% and 2.81% respectively. Shoot mcristemoida devcloped o~lly<br />
from mothbean, into shoots and later lrlto ivholc piants (Avenidu et al., 1993). Genotype<br />
dependent protoplast ~regencration into wlloli: plants was observed in red clover (Trfiliioli<br />
pruiense).<br />
Protoplasts were derived from leaf and suspelision cultures of the culi~var<br />
(Myers et al., 1989). Cull suspension cultures were grown and plantlets were regenerated In<br />
Indigofera<br />
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<br />
optimized (.4rcioni et a., 1985). Protoplasts were ~soiated tkoni ~~icsopi~ylls of Mcdicago<br />
sari\,a and various phytohormoncs tested for a better frequency of regeneratloll (KIIII and<br />
Cho~, 1989). Frotoplasts of Lolus coniiculurus were ~solated us~t~g pre-plasn~olysaiio~i of<br />
green cotyledo~i, in CI'W salts containing 13% iliann~tol. Plantleta were also regeiicrated<br />
w~tll two lines being somaclo~ial varlanta (Vessabulr atld Grant, 1905). Callus and<br />
protoplast CUI~LI~SS were used to Iregenerote ~)ln~lilcts ol"Wedinigu and leaves of hleilicagu<br />
Iirroralb, an anlllial legunic reslstat~t to li~llgl~s l's~'i~i/op~'~i~n ~tii'di~~giiris. I'lalltlets wcrc<br />
regeiicrated 011 niedium co~ltai~ii~ig 2-IP cornbi~icd wit11 IAA, andlor DAP w~th NAA (ZaSar<br />
et al., 1995). Pla~lt regoleration fium cotyledo~l p~oloplasls was achlcvcd in Meiiicagu<br />
saiiva cv. Krnsnovodopadskaya 8 by culturing cell aggregates on agar tiiedium aftcr tlic<br />
immobilizat~on of protoplasts in agarose. Regeneration was also achieved from tissue<br />
cultures of tile wild species M. prosrrara, hi, urbiculuris, M. rruurverleri, M. borealis, M.<br />
cueriilea, M. rigiduila and M fulcnrn (Svanbaev, 199 1) Cotyledon protoplasts served as<br />
useful tools for regeneration of Sesbatiia bispi~losa. 111 a liquid-over-agar culture systetn<br />
with MS ~nediutii supplemented with 2,4-D, BAI', glutanline and mannitol, 84% of these<br />
protoplasts divided and formed callus. Callus fosn~ed from the protoplasts differet~tiated<br />
shoots on MS medium supplemented wit11 IBA arid BAP. These slioots developed into<br />
complete platitlets when excised and cultured on MS containing IBA (Zhao et al., 1995).<br />
Protoplasts der~vcd froin 3 species of Siylosarrrl~es were cultured in K81' medium at<br />
densities of 5 X 1041t1iI and I X 1051tiil I'rotoplast-derived colonies tratisferred to MS<br />
tiiediu~n suppleme~ited wit11 cot~ibitiatiot~s of NAA and BAP formed compact, green<br />
microcalluses. Shoot regeneration, which occurred after 28-56 days of culture, was by
organogenesis rather than somatic embryogenesis, with leaves and stems developing<br />
directly on the callus surface (Vie~ra et al., 1990). Protoplasts were isolated from epicotyls<br />
and shoot tips of Vicin ~tiirboriersis etiolated seedl~ngs. They developed into plantlets vla<br />
somatic e~nbryogenic pathway when cultured on less auxin medium, arid via organogenic<br />
pathway when cultured on TDZ contai1111ig medium (Tegeder et al., 1996).<br />
Cotylcdotlary explants of Sesba~~~o grci~itlifilicr were irrad~ated with ganlnia rays for<br />
callus growth and regeneralion. Cytoge~iet~c studies showed dist~ilct cliro~~iosomal<br />
aberrations (Sinlia and Mall~ck, 1993). Enhanced shoot regeneration was observed us~ng<br />
homogenized callus oELoius cort~iculaius (Orsbinsky et al., 1983).<br />
2.2.2.4 Genetic translormatioo<br />
4'<br />
Agrobocieriir~n-mrdiated traiistbrniation: A rapid and reproduc~ble protocol for<br />
transformat~on of Lorirs cornicirlaius by using cinilamyl-alcolid dehydroge~iase (CAD)<br />
was developed for the purpose of Iignin reduct~on. The transgenics were confirnled by llie<br />
polymerase chain reaction (PCR) and CAD activity. The gene was derived fro111 Amliu<br />
cordura (Akashi et al., 1998). Tllrec cultivars of Medicc~go surivu and one cultivar of<br />
Onobtycl~is viciijoliu were evaluated for their response to inoculation witli A, rliizoget~es<br />
strain A4T (containing pRiA4b). A cultivar-dependent response was observed in M. suiivu<br />
with 94%, 25%, and 4% of infected stem explants producirlg transfornled roots in the<br />
cultivars Vertus, Rcgen-S, and Rangelander, respect~vely. In 0, vrciqoliu cv. Hampshire<br />
Giant, an cxplant-dependent respolise was observed with 78% and 50% of seedling<br />
cotyledon and hypocotyl explants responding, respectively (Golds et al., 1991). An<br />
accession-dependent genetic transfornlation was observed when Glycine cunescens and<br />
Glycine clarrdes~it~u were transformed with A, rllizogenes (Rech et al., 1988).<br />
The
transformants were identified by sylitllesis of opines in the shoots. An important fodder<br />
legume, Astrogalus sinicus was transformed by A rhizogrr~es atid the uidA gene activity<br />
was confimied (Cho et al., 1998). The regenerants exhibited Ri-plasln~d syndron~e (sn~all<br />
thin lcaves and short internodes) arid 55% of tlle seedlit~gs diowed GUS activity and<br />
positlve gene integrations. Genetic transforniation of tile pasture legurne and the model<br />
plant for study~ng Rllizobilrn~ legume symbiosis, Medicugo truncorulu was reliably<br />
transforti~ed wtth b~nary vector harboring ~iptll and rriclA and NOS atid CaMV 35s<br />
pronioters respectively. The TZ geileration of the platits showed Mendelian inheritance of<br />
the integrated transgenes (Wang et al., 1996). A rapid regeneration system from<br />
cotyledotiary node explants was used for stabbing and injection co-cultivation of<br />
Agroboclcri~r~rr, In Pisu~ir sutivunr (Jordan and Hobbs, 1993). Genetic transibnnatron via<br />
somatic embryogenesis pathway with immature leaflets and l~odal explants of pasture<br />
legume Lutlryr~rs sarivus was reported by Barlia and Mehta (1995). Mature de-<br />
enibryonated cotyledons with intact proximal end of Yigrto rorgrritulutu were used for<br />
Agrobacreriu~?~ transfortnation. Over 15% of the shoots were selected on selecttoti<br />
medium.<br />
Iritegration of Itpt gene was detected by usi~ig Southerti blot analysis<br />
(Muthukumar el a]., 1996). Ri plasmid atid dlszir~iied 1 i plasmids were used to transfonn<br />
Mediccigo trnnccrttrlo. Genes of Ri plas~iild negatively ~nterf'ercd with<br />
soinatlc<br />
embryogenesis. Only Ri plasmid with an inactivated rol A gene regenerated transgenic<br />
plants (Thomas et a]., 1992). Loitis jupoilicris is a ~iiodel plant for Rhirobium host<br />
interactions. A. rhizogenes mediated transformation was perfomied and hairy root<br />
formation was observed. Most virulenl strains A, rliizogenes for this species were also<br />
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<br />
A. rhizogerres transformation was developed for Lorus attguslissimus. This was receded by<br />
an efficient regeneration systerli of indlrect organogenesls using hypocotyl, leal; stem,<br />
cotyledon and root explants (Nenz et al., 1996). Bean yellow rnosalc virus rcsistenr<br />
transgenics were obtalned by tr;~~lsforiiia~~oi~ of 7i.ijoiiiiiri subterru~rerirrt with vorlous<br />
segmellts of virus coar protein (Chu et al., 1999). Rapid and efficient trdnsfomlation of<br />
.I4etl1~.cigo ii.ri~rctririh~ i~nd ,M. ~ci/i~,n w;i> tlo~lc \\,ill1 twu ciiily iiodulin and latc nodul~n<br />
genes where pl;ilit rcgrncratlon occ~irred tlirougl1 somatic e~nbryogelies~s (Trinli et al..<br />
1998). Two different regeneratioll systems were used to obtain transibr~iled plants of<br />
~Medicagu fulcaia. Tlie A. turnefacierrs ilioculated plants were regellcrated via direct and<br />
indirect somatic embryogeliesis pathways (Sllao et al., 2000). Mrdicago trtiricatulu was<br />
tralisfoniled with A, iiirr~cjhciens using put selection marker. Trailsforn~ants could be<br />
obtained In just 2.5 months (Trieu atid Harrisoii, 1996).<br />
Biolisfics. Plasmids containing 11ptI1 gclie under CaivlV35S promoter were used for<br />
particle bombardment experiments into Lucerne calluses derivcd from petiole and stem<br />
sectiolis of Med~ccigu srrlivn. Analysis of Irailsge~~ic plallts showed ~ntegrntion while<br />
progeny showed I: I Mendelian segregatioli ratio (Pereira and Ericksoli, 1995)<br />
LM111sect resistance nlallagement<br />
World-wide crop losses without the use of pesticides and otl~er non-chemical<br />
control strategies is estlniated to be about 70% of crop pruduction, amountillg to US S 400<br />
billion. The world-wide pre-harvest losses due to insect pests, despite, the use of<br />
insecticides is 15% of total production representing over US $ 100 b~llion (Krattiger,<br />
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<br />
years were high yielders, but had poor storage characteristics (Kerin, 1994), Insect pests<br />
are capable of evolving to biotypes that can adapt to new situations; for instance, they<br />
overcome the effect of toxic materials or bypass iiatural or artific~al plant reslstance, w111ch<br />
fu~ther confounds the problem (Rousli and McKennc. 1987). An iiltegrated pest<br />
nianagenient (IPM) prograni, con~pris~ng a combination of practices including tlie<br />
judicious use of pcstic~des, crop rot~t~on, lield sanitation :ind abovc all ex]>loitation oi'<br />
~nherently resistaiit plant varletles would prov~de the best option (Meiners arid Eldcn,<br />
1978). The last option includes the use of transgenic crops, expressing foreign insecticidal<br />
genes, which could ~iiake a significant contribi~tion to sustailinble agriculture and tllus<br />
could be an impo~tant coniponent of IPM.<br />
Insect reslstance nlanagelilent is iiot a concept evolved from a single method of<br />
operation, but a11 integrated approach, as opined by niany others. Classification of<br />
methodologies has taken a different turn with advcnt of bioteclinology. Biotechnological<br />
control conceptually is a part of biological control nietliod, however, separated itself from<br />
other methods owing to vast literature and logical base it has acquired. Hence this sectlon<br />
describes all the tliethods other than biotechnology, which have been an integral part of<br />
Integrated Pest Manage~nent<br />
2.3.lJEcological<br />
control:<br />
This is a strategy in which various methods are employed. It is somewhat similar to<br />
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<br />
primarily based on ultderstandilig the interaction of pest with i(s environment, in the<br />
present context, agro-ecosystem, defined as the effective environment at the crop level<br />
(Altieri, 1994), or at thc level of local lalidscape (Duelli, 1997; Collins and Qualset, 1999).<br />
It is defined that an insect become5 a pest when general equlllbrium populatioll exceeds<br />
econonlic lnjury level (EIL) (Higley and Pedigo, 1996). Exploitat~on of species divers~ty to<br />
decrease Ihe herbivore population was done et'li.ctively (Altier~, 1994). Biodiiersily<br />
increase in agroaosystems results ill di.\~cloplirent of tilore inter~ial links with111 the food<br />
webs resulting in fewer pest oulbreaks (Altieri and Niclrolls, 1999). These understandings<br />
led to some practical approaches such as poly harvesting (Pral~cis, 1990) and interplanting<br />
(Alticri and Whitcoinb, 1980) are potent~al eco-control mcnsurss.<br />
2.3.1.2 Pliysicnl control:<br />
J<br />
Tile methods in lliis strategy are relatively ancicnt, but stood for ayes, as they are<br />
farmer and eco-friendly while bcing easy-to-use. They requ~re leaser scientific kriowledge<br />
and are based on physical destructioli of inscct populations. These havc attracted<br />
considerable attention In the recent past because of the development of effective food and<br />
visual attractants (Lindgren and Fraser, 1994). Understanding the insect behavior is a<br />
crucial prerequisite for making traps (Alm et al, 1994). Usage of baits such as pheromones<br />
and novel trap designs made insect trapping devices attractive tools in the industrial point<br />
of view (Dowdy and Mullen, 1998). Usage of some sort of banding material especially<br />
arourid the trunks of the trees had some degree of antagonist effect on insects (Raupp et al,<br />
1992). Live stock insect traps (Tozer and Sutherst, 1996), colored traps (Vernon and<br />
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<br />
Thimijan, 1986), electrocuting traps (Gilben, 1984) micro ii~adiatiolls (Biron ct al., 1996),<br />
gamma irradiation (Metcalf atid Metcalf, 1993) and otlier irradiations, temperature,<br />
coiltrolled atmospheres with COi ~n combination w~th other gasses and varlous othcr<br />
physical methods designed at the coliverilelice of the user and (ype of insect pest are being<br />
widely used In various industr~ol and domestic envll.onmcnts.<br />
2.3.1.3 Cl~e~~~icill colitrol:<br />
., '<br />
Use of chemicals to fight Insects, dates back to 1200 BC. This is thc most popular<br />
but cco-unfr~tndly strategy. Global market for insect~cides was estilnated to be over 20<br />
billion in 1989 (Environnlent Protection Agency, 1989). Wide varietics of i~isecticidcs<br />
were discovered due to the~r instant resul~s despite their adverse effects on thc ~ntegrity of<br />
the ellviron~iient. To name a few, orga~ioclilorilles (DDT, BHC), orgaliopliosphatcs<br />
(Carbophenothion), Carbarnates (aldicarb, carburyl), pyretliroids (nllethrin, cyalotllrill),<br />
averniectins are some of the widely used insecticldes. Many plant based insecticides have<br />
been used for ages, such as pyrethrin, rotenone, azadirachtin etc. Keeping as~de their<br />
popularity, ~nsect~c~des usage is highly alarnling as they negat~vely affect many of the<br />
living things living in their respective ecosystems. These concerns have bccn published by<br />
many authors, emphasizing the adverse eficts of the insecticide use (Rand, 1995; Edwards<br />
et al., 1996). Their effect on soil microorganisms (Domsch, 1983) microorganisms of the<br />
aquatic environments (Parr, 1974; Curlley and Robinson,<br />
1989), soil inhabiting<br />
invertebrates (Edwards acid Thompson, 1973), liematodes (Yardim and Edwards, 1998),<br />
alukatic invertebrates (Brown, 1978), fish, amphibians, birds (Hardy, 1990) and many<br />
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<br />
over 15,000 eventually die.<br />
2.3.1.4 Biological co~:trol:<br />
Various pest control strategies llavc bceu developed where each one provldlng<br />
partial answer to the singular or a sct of pest problems. 1540s witnessed d~scovery and<br />
application of wide array of chemical pesticides and people thought a "panacea" lias<br />
evolved for all pest problems. Th~seemed true till it was ioglcally d~sproved firstly by<br />
Cars011 (1562) and was repeatedly e~npllasized by various authors In the follow~~~g years.<br />
For example, an estimated 7% of crops were destroyed by inscct pests pr~or to 1940s, and<br />
by late 1980s crop destructio~i has risen to 13% despite a 12-fold increase in the pesticide<br />
use (Environmental Protection Agcncy, 1985). These alarming statistics divertcd tile peat<br />
management strategies to opt Ibr age-old control practices such as phys~cal and biolog~cal<br />
meiliods.<br />
Definit~ons of biolog~cal control var~ed through ages, and one of the latest itlcluded<br />
the use of predators, parasitoids, pathogens, pheromones, and natural plant producta.<br />
According to this defi~litiorl biotechnological approacli becot~les an integral part of<br />
biological con~rol. Oldest known method was colonizaiio~~ of ants in china and Yemcn<br />
(Coul$on et al., 1982). There are three bn51c types of biological control, na~nely,<br />
conservation, i~ltroduction and augmentation (Waage and Mills, 1992). introduction of<br />
natural enemies beco~lles a necessity when tllere 1s a sudden outburst of foreign insect<br />
pests, which are tnot native to ;I particular region. In such cases their natural enemies are<br />
imported, multiplied and introduced (Waage, 1996). Augmentation is increasing the<br />
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<br />
control include bacteria, fungi, viruses, protozoaus and nematodes. Mass production ant1<br />
marketing of these agents tire relatively easy co~npared to tlie otller two (Cook et al., 1996).<br />
Over 700 species of fungi were bel~eved to infect Illsects (Jaronski, 1997); for exan~ple.<br />
Beauveria bussianu is a potentla1 biological cotitrol ~igcnt of many arthropod insects<br />
(Maddos. 1987; Georg~s, 1992). The mctliod of lis~~~g bacteria for b~ological co~itrol I><br />
relat~vely donlin;~ted by Uncillii~ //iir~.i~igie~isis speclcs and tlie S-endotoxins twlenacd by<br />
them, molecular genetlcs of the resistance ~n~eclla~iis~n lias bee11 the order of the day 111 tile<br />
recent bioteclinoiogicai approaches.<br />
2.3.2 Biotechnology for illsect resistance<br />
1<br />
One ofthe methods of b~otechnology, m;~rker assisted selectio~l could not cater to<br />
the inirnincnt needs of the IPM specialists, as resistance phenotypes and their QTLs could<br />
not be conclusively ide~ltitied. Hence there was a need of short cut methods to combat the<br />
worst biotic constraint, lnsecl pests. Discovery of 6-endotoxins produced by the famous<br />
Bucillus liiuringiettsis gained considerable sig~nficance in the recent past. Along with U<br />
thuringiensis tnany other genes conferring resistoncc partially have bccn discovered out of<br />
which protease inhibitors are of significant importance.<br />
>:3.2.1 Ut: All A~nazi~ig Concept: Pcrhaps n~ost widcly used entomopathogen is the<br />
bacterium BuciU~~s thiirinyiensi~ Berliner. Many sub-species of th~s bacter~u~n release w~de<br />
array of larvicidal S-endotoxins that confer resistance to various insects. Other species of<br />
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<br />
properties.<br />
Bacillrrs ihuringiel~sis subsp. isrocliet~sis (Bti) was the first subspec~es of Bt that was<br />
found to be toxic to Dcptcra larvae (Margalith, 1990). It was also foulid to be effcctive<br />
against many species of mosquitocs and black fly larvae. Extensive srud~es wcre crimed<br />
out to demonstrate the toxicity of these orga~iisnis towards nianinials and it was not foulid<br />
to be highly host specific thus ensuritig its s;~fcty (Murtliy, 1997). 1r was also Iprovcli to<br />
have an expanded host range sucii as snails, sonie liunia~l ;~nd avian parasites and few othcr<br />
insect pests (Saraswati and Ranganathan, 1996).<br />
Tlie toxin pruteins rind ilreir ge~~e.s: Thc fanlily of irlsccticldal crysral proteins (ICPs) is<br />
iiomally associated with larger plasnllds of various sizes rangllig Ilct\ceeil 5 to 210<br />
kilobase pairs that liave been broadly class~fied as Cry atid Cyt 6-c~idotox~ns(Lereclus et<br />
al., 1993). Though these toxins are liot related structurally, tliey arc fi~nctlonally related in<br />
their membrane permeating activities. All<br />
insccticidol 6-cndotoxiiis are initially<br />
syntliesized as a larger protoxin that is eventually cleaved at specific s~tcs to form the<br />
actual toxin. In total there are seven classes of Bt insi.:ticidal proteins, but four proteins<br />
and few accessory proteins and their molecular genetics associatcd hitb Bti are well<br />
characterized. Tlic major four proteins arc locallzcd in pnrasporal crystlillinc cndotox~iis<br />
sy~itlies~zed during sporul;itio~i and tliey arc sylltllesized by rcspcctivc genes: B~Ct~l,lo,<br />
BfC~jllA, BiCry4B ant1 HtCry4A (Fcder~ci et al., 1990). Much of the ~nosquitocidal<br />
properties is attributed to the synergistic i~iteract~oils of these four proteins, but the wh0k<br />
crystal was found to be niucl~ niore 10x1~ (Crickmore et al., 1995). Vi'ealtil of Information<br />
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;<br />
Guerchicoff et al, 1997; Purcell and Ellar, 1997). Two accessory proteins ~iarncly PI9 and<br />
P20 that arc requ~red ill tile usse~nbly ol'a~i i~~clus~o~i body linvc also been cliaracter~zsd<br />
(Wu and Federici, 1993; Tlliery et nl, 1997; M;~~i;isiierob et al. 1997).<br />
Mode oJuctio~~: Early studlcs showed that the prunary target of Bti toxicity is tlie<br />
niidgut epithelium, where e~izynialic systems tra~isfornis the protoxin tiito an active toxin<br />
under alkaline coi~ditions [Al-yahyaee 2nd ElI;ir, 1995) Tliese toxins act coordinately and<br />
synergistically to distupt tlie epithelial cells of tlie larval gut where niidgut cells vacuolisc<br />
and lyse (Lahkim-Tsror et al., 1983). Tliese sym)~tonis are more or less snnie for toxins of'<br />
all Bt strallis other than Bti. Though Cry and Cyt toxi~is are structurally dissi~n~lar, tiley<br />
have the similal. ti~embrane-pem~eatirlg ab~lity, where Cry toxi~is bind to nicmbranal<br />
protcilis and B~Cvtiilu binds to ulisatt~ralcd pliospholip~ds acting as "bind~ng sites"<br />
(Feldriiann et ai., 1995; Gill ct al., 1992).<br />
Mode of action takes place in two steps; binding to a cell reccplor and subseque~it<br />
pore formation (Knowlcs and Ellar, 1987). Soon after conversio~i of proto-toxin into active<br />
toxin by gul proteases, the toxin is distiliguished illto two doiliains (Donlain I and Donio~n<br />
11) wherc Doiiia~~i I1 bt~tds to a brusli border membrane receptor, acts as an anchor, while<br />
Domain I inserts ~tsclf illto the ~iiembra~ie for~ning ;I pore (Deal1 et al., 1996; Flores et al.,<br />
1997). Binding of the toxin beco~iies irreversible alier llie i~isertion of Donlain 1 illto<br />
mernbra~ie (Che~i et al., 1995). ~4 and =5 liel~ces 1nscl.t into the membrane and a7 serves<br />
as sensor to initiate tlie structural rearrangelllent of the inserting dornaln (Gut and Shai,<br />
1998). Many otlier fi~~ictio~is of seglnents of the toxin have been elucidated. It was found<br />
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<br />
1Aa was studied ill detail. It was found tllnt plasma membrane liposonles contain~ng<br />
phospholipids arc the target of this loxi11 (Tl~onlas ilnd Ellnr, 1983). Tox~n binding leads to<br />
a deterge~~t-l~ke rsarraligenieilr of the bound I~pids, re,ult~ng in hypcrtroplly disruptio~t of<br />
the membrane ~ntegrity and eventually cytolys~s. I'ore ibrmatio~l was observed prior to<br />
cytolys~s (Gill st al., 1992). The 24 kDA active toxi11 \+as lbund to be three times actlve<br />
tlla~l the protoxin (Butko ct al., 1996). Anotllcr ~nlcrest~ilg aspect of the ~ilode of:~c[~o~~ 1s<br />
that thc tux111 monolllrrs display a synel-gist~c aclio11 towards spec~fic i~isecls. D~ffcrc~~t<br />
synerg~st~cally exert~ng some host specific action (Crickmore et al.. 1995) and tllis ,was<br />
dcmonatl.;lted by cloning a conlbinatlon of cry 4A and cry I IA of Bti into E.coli (Ben-Uov<br />
CI :I\., 1995).<br />
2.3.2.2 Genes employed l'or illsect resislancc otller t11a11 Bt:<br />
Protease Inllibitors:<br />
The p~.oduction of transgenic crops has seen rapid c~dvnnces during the last decade w11h the<br />
commerc~al ~ntroductio~i of Br tra~isgenics However, the n~ajor concern with tllese crops<br />
has been the developmellt of resistance by pest and public acceptabil~ty, Hence, there has<br />
been a need to discover new effective plant genes, which would offer resistance and<br />
protectio~l against these pests. Protease i~ihibitora (Pls) are one of the pritne candidates<br />
with highly proven inllibitory acrivity against insect pests.<br />
Pla111 proteitse bshibilors: Tlle possible role of protease inh~bitors (Pls) In plaill<br />
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.<br />
Subsequently the trypslti inhibitors present 111 soybean uere shown to be toxic to the larv;ie<br />
of tlour beetle, Tri6oiilr111 co~rfirslr~~~ (Llpke et al.. 1954). Following tliese early studies.<br />
there I~JYC been riiany exa~i~ples of protcase ~~~l~ibrrors active against certalli inscct species.<br />
both in ~n vltro assays agalnst insect gut proteases (Panneticr et al. 1907: Koiwa et al.<br />
1998) and in in vivo artificial diet bioassays (Urwi~i ct al. 1997; Vain ct al. 1998). The tcrrn<br />
"protcase" includes both "endopeptidases" and "exopcptidases" wlierc:is, thc tcrlii<br />
"prote~nabe" 1s used to describc only "endopeptidases" (Ryan, 1900). Several lion-<br />
lloniologoi~s farnilres of proteinase<br />
inhibitors arc recognized anlong tlie animal,<br />
microorgaliisriis and plant kingdom. Maijority of protei11;ise rnhibitors studicd in plarlt<br />
kingdom originates fro111 three main fiimllies liarnely Ieguminosae, solanoceae and<br />
graniineae (Ricliardsoli, 199 I).<br />
A Iiirgr 11u111ber of protease rnliib~tor gc11i.s wit11 distilict niodes ofactioli liavc beeti<br />
isolated from a wide range of crop species. Drvelop~ne~it of tra~isgenic crops lia\,e coriic ;I<br />
long way from tlie tirst transgeliic developed by H~ldcr et al. (1987). Considering tlic I11gl1<br />
cornplex~ty of proteaselinhibitor interactions ill host pest systems and the d~versity of<br />
proteolytic elizyliies used by pests and palllogens ro liydrolyre dietary protelns or to cleave<br />
peptide bonds in more specific processes (Graham et al. 1997), the choice of an appropilate<br />
proreinane inhtb~tor (1'1) or set of PIS represents a primary determ~rl;int In the succcsa or<br />
failure of any pest control strategy relying 011 protease inliibition. Firstly, tlle choice of<br />
su~table PIS should be based oil a detailed understanding of the biological system assessed.<br />
Resistant biotypes of insects may evolve aAer prolonged exposure to selection pressure<br />
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<br />
will be controlled by using inducible plolnoiers, sucl~ as !liosu of PI-1185 al~d TobRB7, tllat<br />
are act~vated at tlle site of lnvaslon by pests, pa!lioge~l atid nematodes, respectively<br />
(Oppemian et a1.1904). An ideal promoter should be highly responsive to ~nv;~sion of tlic<br />
lios! plallt by ;I pest, or rcglilated by i11ducel.s just prlor to pest attack. Tlic promoter should<br />
be sufficiently active to mediate 8 sub~tal~t~al defenbc, spscinlly loculiiecl to tile site of],cat<br />
usefulness of reconlbinont Pls in plant protection still re~l~ains to be dcmonstriltcd<br />
111sect resista~~ce genes other tllnn Cry cl:tss of genes transferred to crop species<br />
Bean<br />
I ----- 4<br />
Snowdrop lect~n-GNA<br />
I<br />
Cereal<br />
I<br />
Pea leclin<br />
I<br />
1 1<br />
I<br />
I<br />
Protra~e ii~/ribi/ors<br />
Wllrat germ ;lgglutinin-WGA<br />
Soybean (serine protease) Jacalill 1<br />
I<br />
I<br />
F<br />
Barley (tryps~n)<br />
- --. - --<br />
lllcc Iectin<br />
1 _<br />
1<br />
. . '<br />
I<br />
Cowpca (trypsin) Bcan cliit~r~nse !<br />
I<br />
I i .-I<br />
I Mustard (ser~nc protease)<br />
Tobacco peroxitlase<br />
~<br />
I<br />
i Rice (cystelnc proteuse)<br />
Tomato chitinase<br />
I<br />
I<br />
Potato (protease inl~ibitoss 1 2nd ,I) Tlyptophan decarboxylase i<br />
I<br />
Soybean (Kunitz trypsin inhibitor)<br />
Atibi~ul gcties<br />
'I I<br />
Torneto (protease ~nllibltors I and 11)<br />
Various enzyme inh~b~tors
3.1 Pl:~nt 111nteri:il sllrl culture col~ditiul~a:<br />
hfatorc hccdh ut'cliiclq~c~~ (('I~~,I,~IIC,/IIIIIIII 1.) CLI~II\,I~ C.235, ,I !\~cIcly gro\\~i ~LIIIIV;IS<br />
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<br />
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<br />
5ecda \rere Lcpt iir~ gcniii~l,~~~oii 011 hli~~;lzll~gi' ,111d SI\OU;'> (IOO?) I~IC~ILIIII (\IS: we<br />
;~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,<br />
liqul(I or ;IS<br />
scln~-,olld Iho\111g 0.8'io (\v \) I)ilku-lj;~cto :I:I<br />
:I rcq~~~r~d<br />
dilcl 1pII \\;I\ ;iiljusted<br />
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<br />
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<br />
\verc soaked uicril~glit ;iiid tile sscd co;it \\,is ic~ilo\cd IC<br />
tile O-do)-oltl accdlilig. Thc seeds<br />
i'illo\\ III~ I~IU~I~III~. Tlic eo~yledo~~~<br />
\+ere bplit ope11 JII~ IC CIII~I:)~ ;I\I~\!i~\<br />
ici~~o\c~l c:~~cliill~ ,111cl CLIIILII:~~ 011 11ic ciiibryo<br />
~~~cilictiu~i 1iicd1i1111 (Elhli 111:11 cunsiatcd of I0 11hl 2.4.T-1 :ind 2 1111 killc1111<br />
Synthetic ourci~is. 2.4.5-T and 2,4-0 iirc lllc ba\ic co~iipoi~cii~~ ui' il~e enlbryoge~lic<br />
indiiur~uii media. All tile 111cdi:i were re,ted<br />
un ~lintilrc cniblyo akis eul>lalil li~r opli~i~izatio~~.<br />
The ~~iducrio~i ilicdi;~ were tiiu type!, 01ic 2.4.5-l' based cilitl llie olhcr 2.4-U based. Tllc<br />
lornicr was iiariied ;is JEJI scrics niid llic lalcs \\a> ilamcd na JIIM serlcs. 111 JLIhl series tlic<br />
2,4.5-T \+\.as :idded ;it 2. 5, IO ;ind 15 phl :~nd in cach tscntniclil ~ar~able COIICCII~~B~IOI~S or<br />
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<br />
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.<br />
zcat11i ;it 0.5, 2,0 ;iiid 5,O pM iii~d LIIICIIII<br />
;it 0 5, 2,0 ;IIILI 5.0 pbl ucsc ;iddcd,<br />
Bsst iiied111r11 tior III~LIC~IOII of I~I;IYIII~LIII~ 11ii11lbcr 01' C\PI;IIIIS \V:IS JtS \\irIi I0 hihl<br />
2.4.5-'r :iilil ? lib1 hiticti11 (J1:Jl 20: T~blc 42) !\I1 tllc c\~)~,II~Iz ~ C Z C ~ I ~ ;IIJO\C<br />
C I ~ \$CI.C<br />
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<br />
~~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<br />
induction w~th liquid n~ctl~i~ni \r.:~s dolie 111 cullilrc ~iibc\ I:or<br />
tlie Iincr ;I \lcr~lc lilter p;lpcr<br />
bridgc was 1111l11cr\cd III tile Iiq~lid illcdi~~il~ :iiid 1\11' CYI;I<br />
\\ere pI:~ced 011 1111' brlilgc.<br />
Sitice moat of~l~r cl'fons lor inaturatloli and coil\crsloii of tile iirduccd cl~ib~yus were<br />
iinsucccssful soille dddl~iun;~l ~nccIion~c;il ~iictliods \\ere ;ilbo rcstcd.<br />
55
Cold shock: Tlir explants benr~ng the eli~bryos \\.ere pl;iceci on MS containing 5 pM<br />
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<br />
~iicdiu~ii \\ it11 5d11ie coiilpo~~c~i~s.<br />
Heo! .\/rock. The expl;~l~tbcar111g c11lbr)oz \vcrc pl;iccd o~i 1111. II~~~I~III~<br />
;12 I~ICIII~OIIC~<br />
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<br />
IIIIIILI~CI<br />
fiill~\\i'~i by 111~1s II.;III~~;.I 10 fi.esI1 II~C~ILIIII iil~d IIICII~~IIIOI~ 111 IC<br />
CLI~III~~ 1.00111.<br />
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<br />
gc11e11e ir~~~isl~r~~i,~t~u~i<br />
~Y]>c~II~~~III><br />
and proccsslllg cond~t~ons were P~CI);ISC~<br />
\',I~I~LI~<br />
~XIII;II~I~ IYo~ii >cc(ll~~ig~<br />
01 il111k1e111 ,igc, c~~lturt<br />
;iltriiir.c o~rbl.i o ir.uii Thc scctls \vc~c ~oahed ui.cr~ugl~[ ‘inti llic sccil cui~t iraa rclnovcd<br />
rile follo\+,~lig nior11111p 'Tile cutylcdo~ir \vcsc j)I11 opeli :111d the cll~bryo :IAI\ W;IS silrgically<br />
exc~sed ;ind ci~l~urcd OII 111~1li1pli: IUOI<br />
II~~LIC~IUII lilcd~~~~ii tlii11 Ilrid hlS iv~lli BAP wltlliti il<br />
mnge of 5 lu IOU pM III co111b11!;111ufl \vllh ? ul 5 jlL1 klllcl~ii.<br />
Sirool tip Tile dc-coaletl seed> \rere pcsl~i~~i;~lcd un L1S fur 2 dtiys, [he slloot tip (2<br />
mm) \\as tiie~i cxclscd susgicolly 2nd culti~rcd on 111c shoot III~U~IIOII<br />
~ni~diulii (SIM) that<br />
co~lslsted of hlS UIIII 4 pV TDZ, IU KM 2-IP and ? ubl kinellli<br />
56
Lec!/i?is: Young anci juve~iile leallers, semi-iiiaturi. leatlets ti.oni thc ni~ddle portion of<br />
the seedli~ig and ~ii;~tiire leallcts ti.otn tlie bilsal p;itls of7-day-old scedli~ig were sep;lr;~ted and<br />
cultured oil SIM.<br />
Lrq Dii~e.<br />
Tlie dr-co:itcd scuds wcrc pcr~ii~~i,~tcd u~i hlS lbr 7 d.~yb 11r1or to obt;111111ig<br />
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<br />
on tiit SIM.<br />
//ip~~~o(~/: The orcr~i~giit so;iked ;il~ dc-co;rlcd jccds \vcrc eriii~~i;i!cd oil MS and 4<br />
to 5-day-old seedlings were bclcclcd for dcr~v~iig tlic ~~I~;IIII 'Tlic stelii 11;11t that was just<br />
abo\e tile cotylcdon,iry liotlc jt~~iclio~i iv~~h csc~vtl a~~tl 111s sliout 111) rugion was rcniovcd. Tlic<br />
explant tha ~iieasurcd :iboui 4 iiim \v\.;ls cululred on SIM.<br />
Epicu~l: Tile ove~.nigIit soohctl and dc-co;~tcd scccis \+ere pcr~iii~i:ilctI on MS and 4 to<br />
5-day-old sccds \\ere stlccted ib~. obl;ri~iil~g tlic c\pl;~~it Tlic ~~ly)er niost Iparr ol'lhc tap root,<br />
just below the cotyledoii:rry iiodc jt~~ictiu~i \+;I\ exc14 and tlie cxpl;i~it ~iicasuring 4 mm<br />
devoid ol'roor 1111 rcglon was cult~~rcd on the ~~iductioti ~ncdiu~ii.<br />
Roo, A?~I?I~,II/.Y "1111 I.UIJI /I/). Root \egliielilb ;111tl root lip MCSC ehcibcd fro111 7-day-old<br />
sccdlingr and cultiired ~epa~.ately on tlie i~itlt~ctio~i iiicd~ii~ii.<br />
A.~rIlory bi11i. T\io types axillary bud uxpla~ila wcrc prcparcd. Tlie ~iia~jor v:iriatioii for<br />
preparat~on of these cxpla~its wt~s their gsl-~iii~lation paltcrli. Tlic first onu, dcsignatud aa ABI,<br />
was prepared by excisi~ig tlie avillary bud from a 7-day-old secdli~ip hat was gerniiliated on<br />
hlS medium. Tlils nx~llary bud ~iieoaured around I to 1.5 mm In leiigtll. Tile second one.<br />
57
des~gnated as AB2, !\.as prepared by excising the ax~lla~y bud ti0111 a 6 to 'i-J;~y-old seedlings<br />
tliat were ger~~linated on MS conr;~ining 5 pM tliid~i~/~~ron (TDZ), This nxllla~y bud nieasured<br />
approx~niatcly ? to 3 nil11<br />
('r~iilc~~li~~ilr~~i~<br />
liori, Tlle oicr111g11t ~ooLed ,~nd tic-co,itcd 5ci.d.<br />
\\ere gcr~i~~~i;~tcd on<br />
11s. 3-dnyold scedl~~ig was t:1ke11 ;\lid tlie slioul t~p, root tip u~ltl cotylsdo~~s ucrc re~uovcd.<br />
Tlie cotylcdonasy node ju~ic~iu~i th,it ine;~surcd 3 lo 4 lii~ii \\';IS CIIIIII~C(I<br />
011 Slhl<br />
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~<br />
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 ~<br />
gesmlllatloli lp:~ttcrli, age ol'tlic hcetll~ng ;inti proccasllip. four typch of lllc ;~xlll:~ry l~~cr~slclii<br />
cuplal~ts were prcparcd anti they \+ere n;~mctl ;IS 11M I. Ahl?, AM3 ;111d Ah14 (see 171g 3.2 for<br />
diagr;i~i~lii;~~~c 1relxe%nt:1t1011 oi'e~l)l;~~n ~)scl~:~~tiuli). 'rliclr I~~L!~;II';IIIUH ii ;I\ li~llow\.<br />
AM1 explant: Seed coiit ol' the sust~ce stcsili~cti a~itl oicr~iiglit su;lhed seeds W;IS<br />
re~iioved nlid tlic dc-coatcil aced\ \+ere gcrni111;ltctl 0111pl;iiii MS. 2-d;iy-old scedli~ig~ were<br />
selected slid roo! portloli \baa 1c111ovcd by 1cav111g suliie oi'tlie Ih~~)ucu~~l rcyiol~ 'l'l~cn two<br />
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<br />
two axlllilry nier~steni, one hiloot tip witli cpicotyl. 'lliu iix~lli~ry ~~icr~hle~ii cxpli~~ils ~vllli<br />
cotylcdo~intact was na~lied as AM1 i~nd itas cilltllred on thc lnduct~oli ~iiediuni.<br />
AM2 explant: Tlic dc-coated secds were gsriiu~~;~ted 011 SIM al~tl ;ifIcr pruniine~~l<br />
appe;il.ancc ul'axlllary bi~d, in ,iboul 4 ro 5 tl,~ys. tlley were c;~reli~lly runloved and two cuts<br />
were given through rhe ;~x~ll;isy Iiieslstem, dlhcasd~ng rlic root ponlon, rlic rcsulta~it axillary<br />
mcristem explants w~th co~yledons i~itacl (A.112) wcse cult~~rcd 011 tile above-mcnt~oncd SIM<br />
again.
AM3 explant: This was a by-product during the preparation of AM2 explant as<br />
indicated above. Removal of AM2 explants resulted in shoot tip with eplcotyl region. Further<br />
removal of shoot tip provided [lie AM3 explont that contalned au~llnry nieristeni on either side<br />
of the eplcotyl and was cultured on SIM.<br />
AM4 explant: The de-coated seeds were gerlti~n;itcd OII SIM at ;I dclisity of 10 to 15<br />
per plate. They were grown for about a week ~tlit~l :inlllaiy bud was prollilnent. Then the<br />
axiilary bud \\as rcltioved up to tlic base two cuts \\,ere give11 as 111 the casc oTprt.pol.;~tioll of<br />
AM2 explont. The resultant axillary nieristeiti explant, were sub cillturcd on tlic same<br />
niediuni for another 6-7 days. Tile b,lsc ofaxillary bud enlarged and sotiic ~ii~~ltiplc shoot buds<br />
cnierged. Tlie emerglng shoot buds were reniovcd apolil by canli~lly acr,lpping the shoot buds<br />
irith ;I sharp blade Tlir res~ilt;inr cxplnlit corit:i~li~tig cotyledoli uitli ;I bulge on \he<br />
cotyiedu~iaty ~iode rcg~oli \\CIS cultttrcd 011 IIU~IIIUII~ lice MS.<br />
The ~iirdia tbr itiductiott ul'~iiult~ple ~Iiou!) liutil tlie givc~i cx~luttt~ ciili be d~v~ded in<br />
rive m;~jur cl;isscs. I. TDZ-ba~cd ;tiiil 2. DAl'-bt~scd. Thc\c conslats oScltllcl- 'TDZ or UAI' as<br />
pril~cipnl niultlplc slioot iliti~~ctilig cytuk~n~ii ;III~ a111c acc~s\ory C ~ ~ O ~ I I ;ilid ~ I I ~UXIIIS ~ S werc<br />
i~icludcd ~n tile tnedi;] ;is per tlis rcquircn~ciit. Tl~c orhcr cyt~kilillls illid d~~xillj ilddcd were 2-<br />
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<br />
BAP-bnscd illsd~;~ conib~n.itio~is). Optinliutlon of'tllc i~~tiuct~oli nicdi~~m was dolie initially<br />
w~tlt tnaturt. elnbtyu 3x1, and I;iter w~tli ,~xillnr) meristem explant, Ah12. Unless otherwise<br />
mentioncd. the explants were cultiiled 011 tlic 111duc1iori iiiedium Sor abuut IX to 20 days alicr<br />
wliicli they werc tri~nsverred to the clongat~on ~nediulti. An average of 6 to X expl;l~lts werc<br />
c~llt~lrcd per plate and the re,puliies werc atudlcd.
EJJkcr<br />
ofpH on 1~111l11ple slloor rtlditcrio~r: The shoot induction nlediuni (SIM) was<br />
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,<br />
Solidificatio~~ of the ~iiedii~~~i \\as corufully ~ilon~torrd and the prepared AM2 cxpla~lts were<br />
culturud on [lie SIM with abo~e-nlcntio~ied pi4 v;iriatio~is.<br />
Role of<br />
co(vletlui~ni~' iis~~~r: Co~iiplete or parti;il inclusioil of co~yledon w~tll the<br />
regenerating expla~lt. 011 mult~ple slioot i~ldiictiun ficcjilcncy wiis slud~ed. Tlie AM2 explallts<br />
were prepared accord~~ig tu tlie proci.durc nisntiuncd abovc and illc cotyledon portion was<br />
surgically exc~sed ci~lier co~iiplstcly or pa~.ti;~lly (I~~ilt) and one set ol'c~pI,iilts were cultured<br />
witli inclusion of co~nple~u cotyledo~l. 'The rcsulti~ig cxpl;inta ivcrc ci~ltured on SIM witli a<br />
tlclis~ty of6 to 8 sxpl;~~its per 1)1;1tc.<br />
CUIII~)OI.II~I~~L,<br />
IIII~/~I/I/~, ,/iooi iiiilirti~o~~ liviii i/~/]e~.r~~ii ci.i~/coio M:il;irc c111Oryo axis.<br />
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<br />
procedul-es rncntlo~icd III tllc 5ccti{1113.2 2.1 ii~ld cu1111:cd UII Slhl. Nu111bi.r uf rcslio~iding<br />
exl~la~lts and inunlbcr oi' I~ILIIUIIIC<br />
before their trunslir to tlic ~lioul clu~lp:~iiun mctliii~ii<br />
$110uts ]per C.X~~;IIII bere rcc~r~lud 111 lllc third ivcck just<br />
Age o/ [lie \cctl/iiiy: I3as11ig UII tllc ~[>li111t I>~UI):I~;I~IUI~ sIr:~Iegy, d~lli'rc~il ehplillits<br />
\sere derived boll1 hecdllngs of iarluu ;I~A. Ahll. Ahl!,<br />
AM3 a~id Ah14 cnplanls were<br />
prepared accord~~lg to tl~eirc,pectivc proccdi~rca ~ncn~ioncd abow konr 2.1. 6. X, 10, 12, 14<br />
and I6 day-old seedlings. Tlic rcsuli~iig cxpl;ints werc culiiired o11 SlM.<br />
3.2.2.3 Shoot elongatioll:<br />
The hiloot elo~igatio~i 11iedi11m (SEM) bnslcully coiis~stcd ol'lo\rcrcd co~~cc~~tration of<br />
plant growth regulators, %lie11 compared to tlie r~tdi~ction rncd~~~ln Various plant growth<br />
regulators ~ ~sed for this pnrposc werc 2-il' (2 and 5 pM), BAP (2 i111d 5 pM), kiiiet~n (2 and 5<br />
60
PM) 2nd GAJ (2<br />
5 PM), eltller singly or ill conibilinlion with c;sh otlier. Tlie induced<br />
nlultiple shoot buds Here caretiilly excised ti.0111 llic e\pl:in~a ;IS<br />
;I bu11211, rhoill ill ;11ly extn<br />
gro\rtlis of c;lllus or elobulnr structl~rcs ;IIILI CLIIILIIC~ 011 I~IC C/OII~:IIIOII<br />
111ciliii111111tl:illy ti)r<br />
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.<br />
During each passazc k\v alioo~a clo~~g,rtctI ;)lid ~lic u~~-clu~~g;itctI butis \\cw sub culu~rcd OII<br />
tlic lroh 1ncdit1111. Tlie bu~lch ol'~ili~l~iplc \11uo1 but15 gl.c\\ rl1i11 \\ 1111 ~cspccl 111 1111. I~IIIII~C~ OS<br />
\Iiuut btlds thruugll c:lcii p,iss:lgc I1rckr,1iil). tilt sli~llg;llillg sliutllr ,111il a/lt)ol l~~idr li'~lll1 lllc<br />
,ecuiid pass;igs \\ere cul~urcd 011 b1S \\i\li tiA; (2 1111) I.lo~~g;~lctl rliuols tu :ihuul 5 c111 In<br />
Tile I~~C~~IIIIII<br />
used \\;I CIIIIS~ III sei~i~-r(~l~il lb1111 01. li(l~~i(l li~rl~i, l'lis clo~igilcil s11oots<br />
\lerc ~~sctl to optinli~c lie rouliny 1lcdi;i \;I~I;IIIVII~<br />
\verc IIIC~III tlic litll~id ~i~ed~tilli iib lie<br />
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<br />
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<br />
ilic phase 3 Dark grceil<br />
lic:~ltlly \llooir ol'oroilnd 5 ciii lcilylli \\c~.s 1dc;il lor ruotlnp, TIC<br />
rootable slioots wcrc ci~ltiircd ii~ cult~irc ti~bcs (!Sx!OO<br />
IIIII?) ~~I~~~IIIIIII~<br />
lil~cr 1);1j)er bndges<br />
immerscd I liquid roo( ~nductlon mcditlm (Itlhl) lliat consi\tcd of MS MI<br />
~nod~licd lcvcls<br />
of KNOl (9.4 ~IM: 11,lll'of the CUIIC~I~II.~IIIOII<br />
111 \IS) Il3A \v;I~ lillcr ~~c~III/c~ a11d added at 5<br />
pM. Wliilc 60 to 805; ~1'111s cloi~garcd >tloo15 roo~ctl In thc pli;iac I tllc slioc~i, (>8 clii) devoid<br />
of roots were canied on to phase 2. ljiicli shoots were briclly dippsd ill filter stcrili~cd<br />
solution of 100 pM IBA and placeti 011 lilter paper bridge in cuhure lubes contain~ng<br />
hormone-free liquid MS. Effect of half MS and MS devold of ;~ny growth regulalors and<br />
61
effect of varlous co~iccntriirio~is of NAA (5 31iJ 10 ~IM) a11d IBA (5 and 10 yM). Effect of<br />
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<br />
llic other co~lstit~~c~its ol'RIM<br />
groivlli rrguliitor 111 lllehe ~IL'~I;I \\;IS<br />
~ii;ii~~tii~~~cd S:III~L' :IS II~CIIIIOII~~ ;~bo\,c. Tlic root lliducing<br />
IBA illitled ;it 5 li\l cullcellrrntlon.<br />
tllis sl~p<br />
IS :I<br />
Rootirig 111 ilii, lii~(liu/~oiiii.\ .\I \/ciii Tll~s aptciil 15 Icl~llcti ;I> pliiihc 3 oi' ruut111g ; i~~d<br />
O~IIUII;II o~lc .\boil1 10 lu 2O"o ui'tlic rooi;~blc al~oota. \\l~icl~ tlid ~iot root cveli<br />
ii1ii.r ! lo 3 aub-ci~ltliri.\ un IKILI ncrs C,I~~ICL~ to 111~ II~~IO~)UIIIC \)\ICIII tll,it \\;IS ge11e1.11lIy<br />
~ised fir 11ardv1111ig i111ri11g tile I~,III~~II,IIII~III~I~ lproce\a. 75 strc~~gtli I~~IIOII's \u11111oii \V;IS<br />
it1 8 CI~I Miigeiit,~ 1,it<br />
lillcci<br />
;III(I tlic 511001 \\;is s~is~)c~icIcd \v1111 ji~pliort h11cli !list I el11 or 1I1e sl~out<br />
basc \$ah imnlcrscd III tlic bulutiu~i 111;1t coiltn~~lctl 3 ~IM ILL\, Tlic IIIC(~IIIII~<br />
\&;I> c11;111gcd every<br />
H:~lilci~iiig ;ind ~I~I~\~~,IIII;I~IUII proccja \+,is blu;~dli dib~dcd illlo tlllcc atagcs: akge I:<br />
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<br />
wliicl~ the plniils ucrc gi;iduoIly cul)oacd to tlic a~~lb~cncc by ~plticli~ng l~ulcs and rcniovel of<br />
the corncrs oi'the cover (I5 to 20 days): stage 3: tl.ill~\ti.r oI'l11c ~pliilita 10 tllc 20 CI<br />
(d~a) puts<br />
and mainteliance in glnsahousc lbr fi~lllier growlli.<br />
I-lnrdeliinp a1:111cd u 1111<br />
tlic rcmurnl ofconu~~ 11lug.i ol'tllc culturc lubo for I lo 2 days.<br />
Tlie plants wcre careliilly t;iken ULIT ofthe liibe and rhe roo15 ivcrc tlioroiiglily washed, dipped<br />
ill diluted tliiram"'<br />
(fi~nglclde) solutiun nild tr:in\ferred to 8 cni (dia) cunr;ilnlng co;irse rand<br />
(2 to 4 nlm dia) as the potting 1iicdi11111. Tllcy wcre completely covcrcd will1 1r;insparent<br />
polypropylene bags and allo\ced to grow fur 7 to IU days. Coi~densarioi~ on tlie inner surface<br />
62
of the plastic bag was removed rwlce di111y. 'The plallts were cxposcd to ~lic anibiclit<br />
conditions gnduall) by pi~i~liilig lhules aid cu~ti~~g the cor~icss of tlic polyprol~ylelie bags.<br />
F~lially after 10 &)a tlic cater \\ns upelled on lop slid nllo\\ed to SI;I~ lilr ;ihout a \beck<br />
Ibllo\\i~ig \\llicli Ilii. plallt \\as c:irci'~~lI) lr;~~ral;.rrctlo 20 CII~ (dlii) 1put II,I\III~ tlic 1p11111119 ~riix.<br />
'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:<br />
Preparation of Ahl4 cuplant 1s described 111 tile srctio~~ 3.2.2.1. The liistulogical<br />
studlea \\ere carried out ti.oni llic z~;~gc ~~llcil the ax~ll,ll.y bud \\;15<br />
~ciiloved. Tlic clay of<br />
a\illai) bud rctiiu\;il \\:\a<br />
co~is~dercd ;IS d~y- I The dilielopii~e~i~ ~1'hl\ll4 c\l)I;ltit \.,IS<br />
st~idied<br />
111)to Y days ;ilier the rc~~lo\.~l oS;ixill;~s) b~lcl i.c . 111) IU d,i!-X<br />
Pre/~iii'~~iou. linrriotr iiiid ileln~~irociu~~ ul ii,\\i~r, .\,i~rrp/c.s'Tile c\pIa~llr \vcrc ~prcp.lrud<br />
,111d buli)re !i\3tio11 CUI~IC~OII<br />
\\JS re~lioied D:I\J/ IIS~IIC of IC r~gc~icl.;~ti~ig arc.1 \\,la ,iIau<br />
rc~nuicd ni'ikl~ig 1l1c 5;11nple Into ;I bluck of1 to 5 II~III bide. TIIC 'i~ilpli' \.,IS<br />
~~i~t~~cd~;itely<br />
plilccd In 1ix;hiie so1~1tlo11 (see ilppc~iiii~ Ibi. co~ilpus~t~ul~) i111d ~~osccl OVCIIII~I~I ill 4 'C', l'he<br />
IisaIiic u;I:, d~sc;lrded ,111d IC s;~lllpIcb \\e~.e del~ydr'ilcd \$llli IO"',i, 30U'~i. 4006, 50%. ;1111170%<br />
alcohol scq~lentl:~lly for % Iiui~r c;icli and 1iii;illy 111~) wcle storeil 11170% ;~lcul~ul.<br />
Tlir S~CCIIII~II<br />
\r;l m;lrkctl \villi ;III ~clc~nil)i~ig nllilibcr 1'111s ~iuiiibcs W~IS kept wit11<br />
1Iie t~ssire block tlirouglioul procv\lng. Tlic li,we bloclc i\ilb co~ivcycd IIIIOLI~II<br />
a acrio of<br />
Ibllo\r ing aolients :I, pcs tlic sclicdulc Ibs ileliytl~,~~iu~i, clu,lrlng ;III~I p;~s;ll'li~~ ~i~liltr;~tioii.
- . --<br />
laopropyl alcoliol ; Absolulc. I hour<br />
During ttic process 01'<br />
e~i~bctltl~~ig, tt~e II~SLIC block~ here or~c~ilctl so tli;~l sectiolia<br />
\+ere cut in the dcsiscd pl;i~ie ul' llic II,~IIC.<br />
'Tiru L-~liapcd nletal nluultis wcrc laid on nlctol<br />
plate so as lo cnclohc 3 rectangular or squ;lre yyacu. l'li~s IS<br />
paraftfill (58 lo 60 'C ~iieltrd p,~~.iiSti~i \\;I)<br />
tlicl~ parlly lilled with ~iiellcd<br />
LIJCL~) iind tlie tls>lle w;is plt~ccti i~i the deslrcd<br />
posltio~i. Tlir colitalncr xas rlicri tilled w~th nieltud p;iraffin and allowed to cool untll<br />
rcaso~iably tinil so that tlie set block of paraffin wilh 111e tissue car1 be rcli~ovcd f'rotn tlie<br />
moulds. The block was tlic tr~nirned to a ,u~tabte size atid fixed on a ~liclal ubjrct liolder. Thc<br />
65
lock was fiirtlier trimmed so that paraftill o~erlyrng the piece ol'tissue was excluded and an<br />
adequete area of the tlssile thc111g the knlfe was exposcd. Tl~e block \\:la the11 kept I'or coo1111g<br />
at 0 "C.<br />
"C'. Tllc bectiolls \\ere cut u\ilip Lclc,~ 11h1 !155'",<br />
111illi1 I~I~C~OIUIIIC (ICVICC. 'I.11~ ~UCIIUIIS<br />
ii.om the water \\err niou~ircd 011 clc,111 513s~ AIIIIC~, \\IlicI~ II;I\C<br />
bcc11 ari~e~~rcd \~IIII a droll 01'<br />
Strii~ii~~g u/ tlrt, sii111ple~ 011 r/iili\. S~~ii~iilig ol'tl~e slide5 \+:19<br />
~OIIC ~15illg tic~~idtoxyliri-<br />
Losin btairi (see Al)li~'l~iii~ lbr COII~IIOSII~OI~) 'I.11~ sl~dc COII~;IIIIII~~ IC ~CCIIOII \$;I\ s~.r~aIIy<br />
processed as ibllo~rs.<br />
x}lul I 3 111111<br />
Xylol I1 3 111111<br />
Ruliriing \$;lter 3 111111<br />
tlclll~llo~yllll \l~llll I? 111111.<br />
Wash 111 I~UI~I~III~ t;lp u:itcr I? 111111.<br />
Los111 working solut~un<br />
I niili.<br />
95% alcoliol 2 to 3 dip5<br />
9SU/" ;ilcol~ol - 2 cI1~111ges<br />
Acetone - 2 clia~iges<br />
Xylol - 2 changes<br />
3 m111 each<br />
I to 2 min cecli<br />
3 rnln each
The sl~des were Illell muiaited and b'icued ulldcr ~ni~croacclpc. The nuclei stained<br />
bluish violet and cytoplasm in ranous sh;ides of piilk.<br />
Ge~ietlc transfiir~~i;ii~o~i of rllc AM4 c\p1;1111> \v,i:,<br />
dtil~c \in b~ul~a~ic :I> \i'cll :IS<br />
.~~~O~~IC/~I~~~IJII-III~~I;II~~<br />
111et11otls. BI~II~IIC IKII~\~U~III;ILIOII \\;I> LIOIIC liy LI\III~ 111c ~>l~~s~ii~d<br />
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)<br />
;is the reporter allel ;IIII~)~CI~~II~ IC~I~~CIIICC pe11e I~II. OIi~tcr~~~I >CICCIIOII. 11~11111/1/11 i111e1 iiidA<br />
genes \\ere under lllc rcgiilat~o~~ ul'C;1I\1V-355 proiliotcr. I\ \;illel) (11'<br />
\111glc ;lntI n~t~lt~plc<br />
cluiiil~g altes wcrc prcaclit ;it diili.relit luc;~~ioiis 011 tlic pl,i>lnid 'Tlic ; ~,~Iu~~I~I~~~IIIII<br />
nletllod ir,ls ~>crhri~ied by 11ai11g t\io iiii~,ir) \~CIOII,<br />
ii;i~i~cIj, pI1S 723:131 (I,ig 3.4) and pIIS<br />
737:SUTl (Flp 3.5) (bull1 the \cc~ora\vcl.c kl~itily ~)rov~clcil hy 111.<br />
(; Scli;lr;~j, I'I;IIII<br />
131otecl111ology li~~l~~utc.<br />
Siiaka~uon. C'ii~~id,iJ \~'IIICII II;II~OIC~ U/('~:~l:ll) ;tiid SU77 ;la<br />
;~grono~~~~c;illy inlpurtal~t genes real~e~tivcly. Uutl~ rlic vscton 1i;lic 111iiA r ~ 11/1/11 ~ ~ gc11es d<br />
ll~sed Intu ailigle UIIII<br />
tlrlveli by C'aMV 35s prolilotcr. Ui0.1.l:lh gcnc \v;li ilr~vei~ by doublc<br />
35s proniotcr where ;la SUl7 gciic ir;ia dr~vcii by \~iiglc 35s Iproilll~c;~lio~~ t11;il C~II<br />
operate both III E. (oii as i\cill aa ilgrubot/ei.~ii~i~ ;III~ otl1e1. at111b~1tcs aucI1 ;IS ~~i~~ltiple C~OII~II~<br />
SllCS.<br />
In borl~ biolistic ;is \$ell as dyrohot /eri~i~ii-~?lciIinlcd procesw procedi~res tl~e putolive<br />
transforn~iints iverz obt;i~~~cil b) ~elccling llle t~-;iiirliir~i~n~n, using 111~111 as tile sclcctlng gcnc<br />
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<br />
(5, 10, 15, 20,25 and 30 111glL) iiiid with viiryillg COIIC~IIIMI~OIIY ofTDZ (0. 2. 4. 10 pM) 6 to<br />
8 explants xerc cultured per plat<br />
3.4.1 Tra~~sfornintio~~ by biolistics nlvtl~otl:<br />
C:I~I~FIC<br />
111:1s111id ~sol;it~t>~~ i111d I~LI~I~~C~III~II 13 IIIC Ii13t '11~1) LI I>IO~I~II~'I<br />
111vt11od of<br />
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<br />
uf tile c.xpIo111s wit11 tl~c n~icruc~~s~icrs. DOIII~;I~~II~CIII C\CI~I\<br />
\\c~c I)CI~~IIIIIC~<br />
by I~IuI(:I~<br />
1000/llc PIIS systcln (IjioI~~s~)il~~dcd III IUO 111. (; 1'1:<br />
EITrA) bi~l'lkr ;111d\\ah I,cpl VII lee l'or 5 111i1i<br />
(Cjlucow-Tris-<br />
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<br />
lcli for 5 nlin on Ice.<br />
3. 150 ~ IL 5 31 polu,s1~1111 ~cciolc u;~i otl~lcd, \crrlc\ccI ant1 lcli UII I ~ lilr C 5 111111.<br />
5. The seaelion m ~xt~~re cenrrifilped Ibr ilhuut 5 111111 at 14000 rpnl ~III~ the supernata~ll<br />
wo, traiisli.~red to n ft.csli tube (care \$as 1;1kc11 nut to c;lsry okcr tlic prccipilate or lloatl~lg<br />
ll~,llcrlal).
6. The bupcniatant \\,I,, t,ikcli illid [IIC IINA \\;IS prcc~pit~~~cd \\1t110.8 iol. (400 IIL) of<br />
isopropanol. The I ~!I~~LII~ \$:I$ l~ll~\~tld to slatid ;it roo111 tcnip. Ibr ? Inln and ce~itrili~ged :~t<br />
12.000 1q>t11 for 10 illill :I! routii te~t~j>cs,~t~~rc<br />
7. The pellet \\:11 u.i,lied \ill11 icc-cold 70"'. ctll:~nol. LtIi,~~iul \\;IS<br />
rc111o\cc1 (by cIc~;~~lt~ltio~i<br />
or ~S~IKI~IOII) ;111cl 111~ pcllct \\,I,<br />
:III-~~I~L~,<br />
8. Tllu pcllel N;I~ dls~olicd 111 3U-50 ~IL \\,IIc~ ur Sl: S h~1111.1 (11 \Iniulil ~111110111<br />
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'~<br />
\\ere J~I'IIL'IICLI by SI)IIIIIII~S 1;1r 5 \C~OII~\ III ,i ~lilcr~li~ge<br />
5, The IIC]LIIC~ \\:I> rc1110icd ~11111 ~Ih\c;i~dc(l.<br />
6. The l'ulloi\~l~g slcph \\~rc<br />
re~)c,~led 3 111111's:<br />
;\, i ml ofs~crllc iv,llcr \\,IS<br />
b. Vor~cscil Ibr 1 111111.<br />
.~ddctl<br />
c. The p;lrllclch i\cvi' :lll~liictI 111 hclllc hr I lllll1lllC<br />
ti.<br />
Mic~opnrrlclcs iicri. pcllcltcd by spln~li~~g lor 2 ,ccu~id~ III ;I 1111cr~)111gc<br />
e. Llijiiid waz rcmovc~l and dl\i.:inicd<br />
7. 1 1111 sterile 50"iu ~I~CCIOI ivi~s ,iddcd lo bs111g llle 1111ero IS:ICIC CUICL' I.,IOI 10 00 111gl1.<br />
8 Tllc ~n~sropa~l~clcs v,el.c aiorcd ,II roclnl tcl!lpcr'ul~lre li)r 1111 lo ? wccRs.<br />
3.4.3.2 C'o:~ti~lg I)S.\ u~lttr ~i~icror;~rricl"is<br />
Cuat111g uf UNII onto Inlcrocarrlcrh \\a\ dune by u~111g tl~c pt.o~ucoi ticicloped by<br />
5:111ford ( 1993).<br />
I The ~lllcrucarlieliic~c \u~lc\ctl li~<br />
d~\rupr ;~gylo~iicr,~~ctl P~IIIICIC~.<br />
5 IIIII~LIIL'~ (111 'I I)/;IIIUII~~<br />
IUIICYC~<br />
2 50 p1. (3 nlg) ul IIIIC~II~LI~~ICI? iicrc 1;1j\e11 11110 ;I I 5 1111 IIIIC~O~~I~L: lube<br />
3 lVl11lc vol~ex~ng \~goruusly, lkc IbIloi:i~~g \+c~c ;iddcd III oidc~<br />
I. 5 1iL DNA (I LI~}II.)<br />
11. 50 111. C;ICI 2 (2.5 Mj<br />
I~I. 20 111. apunliidlne (0.1 Mj<br />
4, Vo~tcx~~~g<br />
wa> cunl~nuctl for 2-3 ~ilirlulsz<br />
111 rcs~~\[)elld il~ld
5. Microcal~lers were allowed to settle for 1 111111.<br />
6. Microcarriers \\ere pellcttcd by sp~iili~iig hr 2 sccuiids ill ii IIIIC~UI'U~I' 111be.<br />
7. Liquid was relllo\ed and dihc;lrded.<br />
S I-IU 111. 01 70'; ctI~;r~~ol \ins ;iddcd \\II~ULII dl\t~irht~lg tll~ pellcl<br />
9. Llquid \\;ij renloicil ;III~ di\cilrticd<br />
lU. l4U 1iL of IOU";<br />
etIl,lilol \\;is ,idilcd \r~tliuut d15turb111g 11111 ~pcllci<br />
1 I, L~ilu~d \+;15 re111oi cci ;111d tl~s~;~rclc~l.<br />
12, 48 } ll~ 01 IOU0" c1l1.111c1l \\$IS ~lllll~~ll.<br />
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<br />
by vorlcxlilg ;it low apecd lbr!-3<br />
\CCOIIL~~.<br />
Si\ allquuta 01.6 pi. c,lcli ul' ~n~croc;irricrs \\ere collcclcd a~rd triln\li.rrcd<br />
to tile center ol'a<br />
~ii;icroc:~rrlcr. Equ,il ;III~O~II~I~(500 big) ol' ~nicriic;irr~crs c;icli IIIII~ ;iiiiI 10 rpl.cad cicllly over<br />
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<br />
hood consislillg ol'll~e biollsl~cs gene g(111 ibil\ 1l1i110~1gIlly CIC~IIIC~I \~ilIr IOUUh ethnnol. The<br />
petri plate colltaii~ing tile cxpla111\ w;is placed ;II ;In ciplxupri;lte d~at:~nce li.oln tl~e device<br />
consistllig of rbe macrocarricr,<br />
The explat)ls iwrc bo~ilbardcd with the lnicrocarriers by<br />
applyllig appropriate pressure ill tlie r,ingc of000 lo I 100 psi. Sprcadilig oftlie iniicrocarriers<br />
was ensured and tlic eupl;l~its \\ere sepa~ited atid \\ere culrurcd 011 shoot induction nlediuin<br />
(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<br />
about two weeks. The rcgcncratlllg ahuot buda \$ere clilti~ird on tIu slioot clo~igst~on iiiedium<br />
(StMI j. I~t;~tetl cells \bere s~~apc~~dccl 111 25 1111. 01'~1crilc 12 MS. 1'111s CIIIIUI.~ was<br />
dcbcribed ill tiit prcviuii~ \ectlun (ace 3.2 1.1) Tllc cxl)la~ita 14c1.c bl~elly dipped llito tlic<br />
dprobiic~rc~riioli culture poured 111 rlie petri pl;~lc Ibr 1 to 2 xc. 'fhcy wcrc cultured in MS<br />
niedlunl co~it~llii~lg 4 /IM TDZ. I0 phl 2-11' ,ind 2 pM kitletin (SIM). The crpl;~lits wcre co-<br />
cultl\ated iiitli llie bacrcrl;~ lor 18 IU~I,<br />
;III~I Mcle CLII~LI~C~ VI MS ~ncd~uni cunt;~~ni~ig 250<br />
mg'L ceibtaxlme<br />
Tlic ontib~ouu celbti~n~~ilc bras uacd to tcrlnlniitc Ihe growth of tlie<br />
agrobscterial cells, ll~clusio~~ of cctbtax~nic in thc culturc nicdla co~it~nued fbr I lo 2 passages<br />
72
till the growtli of bacterial cells \Val complet~ly tcrn~in~ted. Tlic cultl~red explaits were<br />
maintamed in the inon-select~on induction nlrdli~nl tllal d~d 1101 co~lsibt of kanil~nycin, for<br />
clbout 3 to 4 diiys. TIIC l'\jild~~ts \\ere tlle~l trG~nsl;.rrcd lo tile MS IIIC~IIIII~<br />
co~lti~i~ling 25 III~IL<br />
k;~noniyc~n i111d III~II~~~IIIIL'~~ tbr OIIC \\eck. TIICII the c\l>l;i~~t> \\ere CLIIIII~C~ 1111hlS III~~~~IIII<br />
COIII;IIIIIII~<br />
50 1113 L /\~II~~III~!C~II ;III~\\ere I~I;IIIII;IIIIC~ liir ;10o11t 7 10 lU d,iy\. 'rile slloot buds<br />
~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<br />
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<br />
pb1 ~III~IIII (SEhll) i111ti 75 1n1g'L Lc~~~,~~li!<br />
CII~ Ali~~r LILI{I IU ~I,I)\ 111~ICIII~;IIC~ :IS \$,ell 21s 111c<br />
~~~i-clo~l@,~tcd sllootb iicre tr;~~~sli.rretI to tile slioot clongatioll iniedii~n~ 111.it ~olltaincd MS \vilh<br />
2 pM GA, (SFhl2) ,~nd IUU m$L L;~I~;~III~~III. 'She ~~n-lr;~~~slurn~cd >11oo1~ICLICIIC~ i11 every<br />
sli~gc oi'tllc III~IIC~I[III<br />
,III~ ~ICIII~~II~UII Tile ~CICCIC~ \IIUUI)<br />
III~II \\CIC g111\1111g II~;II~IIII~ \vcrc<br />
carci'i~lly separntcd liom lllc elotipotii~g slioot builcll :lilti irere cuhl~rcd ill the root inducrlon<br />
II~~~~IIIIII \\IIIc~~\\;I,<br />
~~re~i,~red \VII~IOLII<br />
;I~~III~ tl~c \CICCIIUII ;IIII~~~~IIC ~~III~IIII~CII~ i~\<br />
kil~~ili~iycin<br />
\ 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<br />
malntenalice, l'heac pl;llits ,Ire lr.rii!ed ;IS<br />
p~~t,~ti\cly tr,i~~sibr~~~ed pl;i~irs.<br />
200 ILL (or autliclcllt to d~p 111s iia>iic) of assay Iiilxtlirc w;ih acldcd to tlbcuc aaniple<br />
(sections or dlscs 01. strips) and vaciluni ~nlilttated for 3-5 ~iii~iurcs. Tlic {anilile was iricubated<br />
74
;I! 37 'C for 3-24 Iiours 111 d;irL. Tlie iias,iy 1111~t111.c \\.;I\<br />
rc~ii~\ed ;i~id tlie ~ I~SUC \\;IS<br />
cleared<br />
otT chloropli)Il by accji~c~lt~:il cli;~~~ge~ III 70-Ili(i"~ ctl1;111ol LII~II~ llsallz Iiod 110 cliloropliyll.<br />
Allcnioli\eIy, for ditticillt tu clear tissuc ;~tid 75"" I;ictic i~cld \vnr iltidcd .ind ,111tocl;i\cd tbr 15<br />
mlllutcs. This gl\c> ;I \cry good 111idge Cur ~)IiuIugr~~~Iiy Tlie tia5~1~ \\'I> IIIULIII~C~ III gIyce1.01<br />
illid ob\er\ed ~lnJer a illlcroacope<br />
~rail.dcrreti Ilno 30 1111 ILI~S<br />
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<br />
;i11d lilt lubes werc i~icub~tcil a1 05 degreca Ibr I0 111111<br />
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<br />
llllll<br />
1. hlo\t Iprutclllh il~i~l lpolyiic~Ili~ride> ~CIIIU\C~ iiltli llie IIIIOI~IIJIC li-tluilecyl ,ultiitc ppl.<br />
5 Tlic snnlplc \\:15 ce~lt~~~i'~~gctl ;it 25000g ibr 20 111111 ;III~ lilte~cd lllru Miril cIol11 illlo 30<br />
nll tube contalllilig 10 ~ nl ~supropn~iol, ~ll~xcd aiid IIICII~~IICLI ill -20Y(' for 30 111111.<br />
0. U\A \\;la precip~t;~led '11 2OUUiJy Sur I5 111111 illid ~IIVCI.III~@ tlie lubes 011 piipcr towels<br />
tbr 10 111111 dried pellct.<br />
7 Tllr pcllct was rcauspended ill 5UU ~IL 'I'L 8 111id IU ILL of' IKU;lse A solution was<br />
added (5 mdnil).<br />
8. The samplc w;ls i~icubated n\ 37'C for 3U 111111. I vu1 (500 jiL) PI~uIIoI: ~hlorohr~n<br />
nllxture \\as added, lii~xcd \~goruuhl) ;III~ cc~ilr~S~~gcd ;it 12,000 I~II~I /br 2 mill. Tliu<br />
7s
SII~)~I~XI~VI~~ \\,la t;~hen (;I~~UCOIIS plldst) alij I 101 (COO 111.) ~I'cIIIo~~I~~)sII~ \\as added, mixed<br />
and cc~itnf~~gcd ;11 I!.OUU Il1m lor! nili1. 0.1 yo1 (50 111.) of3hl soclil~iii :lccl;itc (pll 5.2) \,;IS<br />
nddsd to tlic rllpeni;ltelir ;111d lllc DhA \\,I> prcc~p~tnrcci \r111i U.S ~ o(400 l 111.) ~soplops~iol.<br />
Tlie s;~~ilple \\:IS<br />
nllo\\d lo slalid ;II roo111 te1111) 1;)s ! IIIIII<br />
;111d cc~i~r~li~gcd $11 12.000 ~11111 hr<br />
10.15 111111. Tlie supcr~~.~lc~ir \\;IS rc~lio\tti ,111cl IC<br />
I~CIICI \\,ir \\;~\l~cd b11cl1) \\ill> ice-cold<br />
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;.<br />
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<br />
denaturation of 94 'C for 3 111111 (one cycle) ;111d c:icIl cycle o1';111iplili~;1t1011 \villi steps of<br />
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<br />
~~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<br />
ions c!i'lc).<br />
/'('I? lo!. ijf( !.I,/.I/I gi2iii' I'CII lor Ui('r.\!.l/i CIIC<br />
\\ah tlul~c \\1l11 rc.~ctioi~ cu~iil~t~o~i\ ol'111111;1l<br />
ilci~.ilur.~~~o~i oI'04 -i' lur 3 111111 ((IIIL'<br />
C)CISI $111il i':~~li cycle or ~~~i~lililic~t~o~l<br />
\v~tIi >ISI)S 01'<br />
~~II,I~LI~.;I~IUII (94 'C'<br />
lii~ I III~II), ;IIIII~~I~II~~ ((13.1 'C' I'CX I 111ilij. ~IIICI C\~CI~\IUII (72 "C' livr 1.5<br />
IIIIII),<br />
;III~ C\ICIISIOI~<br />
(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<br />
(Olli. c)clC).<br />
P('N /vr SUT! gc~rc. I'CI< Ibr .S'/~iI gule ":I\ d011s \\IIII<br />
rc;iitlon ianiI~lioli\ ol' in1ti:ll<br />
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<br />
\LIIII steps OS<br />
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<br />
C\~~II\~UII<br />
(72 'C' lur 1.5<br />
III~I~). ;III~ CX~CIISIOI~ (72'C 101. I 5 111111) li)r 30 CYCIC~ i111d ;I li~li~l CXICIISIOII<br />
;II 72 "C' Ibr 5 mln<br />
(ulie c)clv).<br />
3.4.4.4 Soutller~l blot 11ybritlir;ltion:<br />
Thc ertractcd ;~nd pu~~licil I)NA ~111ckl)ea I~III;III\C<br />
II-:III\SU~II~~III~S used ior Suuthcrn<br />
blot lhybr~diz;ltiol~s \\IIII<br />
a specilic p~obc lulloir~rip tllc 111c11lod glieli by Soutllcrn ct al.,<br />
(15175).<br />
Th~s tcclln~que w;i, i~scd to ldcntitj rlic ililcgratlon of ~iprll dnd UtC'rviAb gencs<br />
from plants translbr~~isd \v111i agronon?icaIly Important UiCi;ililh 81ld YU7'1 ~CIICS.<br />
11piII
gclle was probed uirh rlie PCll ti.ng~iic~lt 01'700 bp ,111ii Bi('r~,l:lb gclii' \\,;is probed with<br />
908 bp fiagmenr I'CK '1fnii.111. Tlic prube \\;IS lnbclcd w~rh tlie co~~i~ncrci;~lIy available<br />
:\I~PII~~s' i11rcc 1'1bel111g AII pro\ ~ddd b! :\~i~c~\li~i~ii (L!Sj\i<br />
Hc,\ii.ir~iioi~ o/ go~uitlil /),\',I<br />
ciiiil c~ii~c~o~ripIir~i~!~\i\<br />
I<br />
Tlie ge1iu1111c DNA \\;I> ~ II~c~Ic~<br />
\\IIII LIII,IL)IC<br />
I~!,I~~CII~II c11/y1iic<br />
;I, rollo\\b:<br />
!U 111. cliru~iiosu~ii.~l L)NA (15-20 11g1<br />
5 IL IOX rcalncnon bul'l'cr<br />
5 111. rcblr1c11011 cll/~lllc (50 \lllll>i<br />
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<br />
211 nr 37 C.<br />
2. 2 ILL oi' gel loud~ng but'li.r \.,IS ddcd lo eocii rotriciet! DNA belbre<br />
clccrropliorc~~s Tlic rc~tricictl DNA \\;I> ~i/c-li;~ct~u~i.ircci ill O Xu" ;~g.~rusc gcl prcparcd III<br />
IX TDE buffer nlollg \\111i<br />
5 111, pl.~s~iiitl DNA rc>rr~crcd ~1111 I:colll ;IS \I;i~ld;ird 11i:irkcr<br />
;III~ IS clcc~~opliu~c~ed o\e~-n~gIi~ ;I( 30 volt\ III IN TDI: bufir<br />
3. Tlie gels wcrc i1;1111cd tiur 20 iil~ll UIIII<br />
~lli~iiii~~~i br~ili~ilc ;it 1 11g;riiI bull;.r<br />
and tlie<br />
rcbtr~crcd DNA ii.ngmcnt b:inds wcre v~suali/cd on ;I UV I~o~is~ll~~i~ii~i~lur<br />
:illd plio~oglaphcd.<br />
Prui r,\.siiig titi!/ i rii~~~iltrri hloiiiiiq o/ ihc g1.i<br />
1 Tlic sue fi.ecrioriatcd gcrio~il~c DNA In !lie gel bvi15 covered LII~~I 25U n~bl 11CI and<br />
nglrated ~lii~il lie b~~oiiiupl~c~i~~l blue lurlib lo ycllo~v color (5-15 n~inutcs) tu depurintilc<br />
DNA.<br />
2. The gel was asslicd three ~inici w~rli dcnll~iefi~lized ukircr.<br />
3. Tlic gel was ~ncubated w~th dcnzirurarion solur~o~i (l0X gel volumc. I .5M NaCl + 0.5 M<br />
NaOH) for 15 miiiiltes twlcc, c;ich at room ~ciiipcraturc on a sliaker
4. Tliu pel \\as \r;~alicd 3 ti~iirs \VIIII<br />
dc1ii11icrali7cd \\:lter ;111d co\urud \vltli ~ieut~-dlizi~i,g<br />
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<br />
carefully ;~ddsd Tlic ~ ~~~-I~!~~I~IL~I~IUII<br />
\\,15 tIo11c ~LII ,111t1111 I IICILI~ t~t 0UL'C,<br />
3. Whlli. rlic prc-hybr~iil/;~t~w is 111 prugrcss. ~lic ~irobs \\*I\ builcd ~ br tibout IO ~iii~i. :It<br />
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<br />
\\ere ;idded to t11c probe.<br />
rcp:~rctl I~cslily L~IIII 2 111 c,ruhh.li~ikcr \VIIII 8 111<br />
i\atcr suppl~sd b) tiis colilp;~~~! 1.<br />
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<br />
liyl)r~di/;~t~o~i Ix~ttlc,<br />
5 Tile liybrldl/;~t~(i~~ \\;I> dolic o~cr~iiglit<br />
6 The ~nc~nbl.;ilic \\;I> lal,e11 o~it o~i tlic Ibllosring niornllig ,111tl\rab \v:~slicd w~tli ~)rilil;~i.y<br />
iv;~sli b11t'ii.r (1;~ biii'f'cr colilpo>itio~i. rcc Appcndlu) twlcc krr I0 111i1l. C;ICI<br />
;III~ \villi<br />
sccu~itl,lry ua4i bull21 (fur bullbr cunipo,iliu~i, see ;'\~>~~IIL~IAJ luicc li~r 5 nii~i. c:lcll.<br />
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<br />
d,:\ e1o1111ic1it C;I>SCII'.<br />
8. AII X-ray fill11 wni kepi oil tile blot :III~ CXIIUSII~C ivi~, ~UIIC ;IS I~CI. ~IIC SI~II~I~ recorded.<br />
9. The X-ra) film \vas \v;~sIistl ill tile folluw~~ig o~tler.<br />
;I. Developer for ? 1ri111<br />
b. V4;lti.r ior 30 scc.<br />
c. Fixer for 2 tii~n.<br />
d. Wiltcr for 2- 3 mln.<br />
10. The developed X-r;ly filni \\'as dricd and v~cwed on the sl~de-viewer
4.0 RESULTS<br />
$loaf of tllc c\l~cr~~llc~lls UII ~UIII;I~IC snlbryogc~ic>~s ncre cio~~c II~III~ llic c111bryo<br />
;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<br />
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<br />
oI'c11111r)us I'ru111 III~IIIII~ c111h1)u ,1ii5 c~p1~11il rill 11511ig 2.4.5-T '15 I~IIIII;II~ $~o\\lli regulator<br />
and 1:1g 3.2 clio\vr llic ~ndi~ct~tr~i ol' enibr)u* 11s11ig 2.-1-1). Add~l~il~i~~l ~~OLCIII reg~~li~tors<br />
s~lch ;is TDZ. DAI'. /t.,~titi.:III~ ki1icI111 \I.CI.C 01>0 119cd 111 co~iibi~i;it~i~~i UII~ llie 2,4.5-T illid<br />
2,4-D. ?,4,5-T co~~la~~i~ig ~iiudi;~ (JEM ssrics) T:~ble 4.1 sliowa Ihc cfict of 2 and 5 pM<br />
?,4,5-'1' in co~iibrii,~t~on wltli TDZ, MI', ~III~IIII ur /c,~ti~l uli su~ii;it~c~iibryu ~nductioii.<br />
Comb~~l;n~una colitainllig TDL ,111d UAI' sllowc~l \cr) luw liciji~c~icy ul'c~iibryoye~icais 111<br />
terms of the nu~iibcr oI'rxpl;inrs respo~ldl~ig ;111d average ~ii~nlbcr oi'c~iibryoa per explant.<br />
- -\C__-<br />
The expltinta on tliese ~iied~a<br />
--<br />
con~binat~olis prodi~ccd awdl yrcc~i niass of callus on which<br />
two to four embryo$ ;ippt,lrcd Tlic cnibryos ;~ppc:ircd ;IS I;lrge ylubular entiricb and lliey<br />
-.<br />
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<br />
the number oienibryos per espl.~nt \\as verb lo\\. Eliibryo axis gre\\ 11i1o a sa.ollell Inass<br />
on wli~cli embryos appc;ircd I~nlc I;lrgsr \\1tI1\\sll-delilied globul;~r ali.~lie. Irilcdio with<br />
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<br />
used. ,411 ;lveragc of 7 6 eliibryoa \\'crs i~lditccd \per c\l>l~~lt flo~ll tile bcat rcal)utidl~lg<br />
COI~~~IIIJIICII~<br />
I e., 2 lib1 2.4.5-1' ;1101ig\VIIII 2 pkl h111cri11, 'l';~blc 4.2 ~lio\vs i~icrs,ise 111 tllc<br />
concelitrJtioti ot'2.4.5-'I' to IO ;~nd 15 [~hl \\IIIIP :IPI)J~III~<br />
tlic otlicr Ii~r~iii~i~ss ;I\ 111 tile c;ise<br />
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<br />
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<br />
tilorpliolo~y ofcali11~ ;i~id cti~hryos \\;IS<br />
li1111li1 to be s ~~il~li~r ill ill1 111s ~LII. XIS<br />
~~'I~CLIII~~CIIIS<br />
I.c., lo\% Ireq11c11~) III~UCIIOI~<br />
;111d grcc~i callus \vitIi 'I'D/. ~III~ lj/1IJ c~~iit;~iiii~ig<br />
co~~ib~nat~un~, al~glitly iiicrcaaed t'rcilr~ency ,ind irlliie c;illu~ \v~tIi rc;~rii ;III~ besl 111ductio11<br />
iicqucnc! iritl~ broivii c;illu in tllc c;~hc oi' h~~ist~ii CUIII;IIIIII~~ 111c(/ii1, Aiiiu~~gst ill1 tlie<br />
conib~~i;tt~ui~~ LI~CII. CUIII~III;IIIUII<br />
01' IU lib1 2,J.S-'1 \villi 2 lihl ~IIIC~III iilduced all ;Ivcrage<br />
ut' 19.0 cillbryol per cspl;int. Tllcre W;I\<br />
cnl;lrgc~licnt of tlic e~nbryu ;]xi\ ~n 2.4.5-T and<br />
kinct111 c~~nib~~i;iiro~is \\llcrc (lie cliibryos or~g~~iiitcd di~cctly li.0111 tlic ,II)IC;II<br />
;itid ax~llary<br />
reglolls Tli~ set of embryos :lppcarcd crcaliiy ~Iiltc w~th well-delinuti globular littad and a<br />
stt~lk. All the otlici. co~iib~ii;~tions co~it;ii~iing 2.4,5-T \villi 'I'D%. UAI' ;ind zcatlli showed<br />
embryos wltli no st;ilks aiid looked litrls Imrgcr tiinn rile fornlcr. I'rolongcd c~ilturr of~he<br />
etiibryogcnlc explants fol. 6 to X wceka slio%ed indtiction of sccoiidary embryos. Tlie<br />
secondary en~bryos or~ginnted from the surlilce of tlie globular licad ol'priliiary eiiibryoa.<br />
Tablcs 4 3 ;~nd 4.4 sho\\s tlle inductio~i li~eq~iencics on tllc liledia cuntaitiing 2,4-D<br />
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<br />
the embryos originated liul~i tl~ callus III~IS~CII~. TIic iii~~iibcr ~I'expla~~ts respo~idi~~p was<br />
s~m~lar but tllc aicrage I~LIIII~C~ ut'e~~ibryu~ per exl>Ialit \\.;IS<br />
cu~iil);~~.;~~i\eI! Io\ver tl1i111 the<br />
abo\e-nient1u11cd JELl aeiies ol'~~icd~;~. Tlic c~iib~qus :~l>l>c;~rcd I~~rgcr tliii~~ tlic UII~S III JEM<br />
scrle,<br />
;III~ tiley l;~cbctl ;illy atalhs. Fig. 4.2 sllu\rs 111~luction ol' e~~~bryos fro111 111;lturc<br />
e111bri.o ;ial:, cul)l;~~ir. Ucs~ rcsl)o~idlng COII~~III~I~IUII \\,IS<br />
?O 11hl 2,411 w~tli 2 1lM kkieti~i<br />
tli;ir ilidi~ced 9.3 c~iibryos per c\pl;iiit No sccontl;~ry enlbryos ~ c ubscrvetl ~ e e\en VII<br />
prolonged CLIIILISC ul' tlic c\pl;i~it\ 111 11ic b~~~lie 1iiec11111ii \\li~Ic tl~c 111d11ccd c~iibryos<br />
gfiidunlly entered c;~lli~s pli;isc<br />
V;il.~oua otllcr ~ hpl~~iti s~lcli :la 1);1r1:,<br />
o1'111:1lurc e~iibryu ~1x15 (~>lu~iii~l~, radicle. side<br />
;irlns ;lntl ~ii~tltllc purrion). Icaflcl>, stelii aeglnclits ;uid root acg~ilcl~t\ wcrc ci~ll~~rctl on bcst<br />
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<br />
results ;ire allo\\li 111 rlic 1;lblc 3.5. I'lu~nc~le iiolil 1ii;iture c~iibryo ;IXIS<br />
;111tlcaflel:, g;~vc bcst<br />
c~iibryi~ ~iiJui.t~o~i li.cij~lcllc) ol'70 ;IIILI JO'% I~II~SCII\CI~. All IC UI~ICI CSPI;II~~~ CKCCP~<br />
r.id~clc ti.uni c~iibryu ,isls sliu\rccl luglicr iiccltlc~icy of II~~LICII~II \+liere ;I t~~iic tlepc~idc~~t<br />
~nducuo~i \ids obhcric~l. 111 ge~icl.;il, clilbryu ~~~~luctioli \V,I~ observed u~tliin 4 to 5 weeks<br />
and s~icli phcnornc~lui~ \\;I, obser\cd ii~tli iiinlllre enibryu ;ix~s dc~.~vcd cxpla~its and<br />
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<br />
:,;i~iic 1nedi~1111 Iur ;~buut X lo 9 \\ccks. Tlic cnibryo\ ;~ppcared a~ni~l:ir tu tllc ones on cliibryo<br />
nsls and leaflet explnl~ls. Tile epldernial I;iyer ol' SI~III<br />
and root scgmcnts broke opcn into<br />
crevasses and embryos apl)c:~rcd orig~~ii~ti~ig fro111 tlic ililicr tissue.
'I'lle c.;pesllilclith<br />
011 slluol osg;llloycnc>lb wusc Il~u;~tlly d~\~dcd illlo two types<br />
namely dlrvct ;111ti ~nillrcct ~nle~liods B;ISIII~ on lliis co~iccpt tllc expl;ll~ts used were<br />
6<br />
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.<br />
Euplants that gave liltllrect slioo~ org;inopenesls. E.\pla~ll\ 1i;iving prc-cx~stllig ~nicsistenis or<br />
any traces ol'mes~s~cms come u~lticr lis,~ gloul) i111i1 llic olhcri like Ic;~ilc~~, Ic;ll'basc, stel11<br />
segments, ep~cotyl, liypocutyl, root segnients n11d root tip tllel were 11o1 ;lssociated w~tll any<br />
mrl.istemat~c tissue conles ulidcr tllc seco~id clabi. Tile ubscrvnt~o~is slioued that ~ndirect
egeneratloll \!;IS<br />
dli'licull ;~nti could 1101 be ocl~icvctl n.itl~ 111s tcsted 111cdiu. M;lture elnbryo<br />
axli, shout tip. ;lxlIl;iry bud, cot)li'llo~i.~ry node .ind d\~li;lr) III~~I\~~III (AM) explnllts<br />
sl~u~vcd sl~uut rcge~~cr;~l~u~~<br />
;IIIIUII~~I \\ IIICII ;1\111;1ry II~~~I\I~III c\p1;111ts ,AM2 :III(I<br />
AM4 were<br />
~CICCICJ ,IS [lie best UIICL OL'C F I J ~ 4).<br />
Sla~~d:~rdi~:~tio~~<br />
ol' il~~ll~cliu~~ ni~di11111: SI,III~~:I~~I/;IIIUII oi';111 ;I~I)~UPII,IIC I~I~L~I~IIII<br />
ii~r III(~LIC~I~~II LII'II~LII~I~)~~ hl~out\lio\\h II~IIIIII)/C hliuut II~~UCIIUII I~UIII III~IIIIIC<br />
CIII~~)O iixih by<br />
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<br />
~~iy~~cliru~ioti~l~<br />
;it IIIC \IIUUI 111) ;IIICI iixlll;ir) bud rcg1011~ 01' I~I,IIIIIC eillbryo :IXCS. Thc<br />
~spli~~it gre~i I~CC<br />
IU ~ULII t1111es III sl/e ;illil Iiicri~tcIii;iLIc IC~IUIIS br,~~~cli~d IU 111uIt1ple<br />
\tiout\ 'rllc otllul cullib~~~;~l~u~l~<br />
~ulll;llllitly 2-11' ;III~~\I\;'+<br />
\4llll Di\l' 111 ~CIICI.;II sI10i~ed<br />
ciicuuraylilg results, h1;11iy ~I:IIII gru~,th rcgl~l;i~or\ lilw LC~IIII~, ~ICIU~~IIII, lAA iilld IDA<br />
ucse<br />
citlicl- i~liIividu,illy or 111 combtnolion \LIIII UAI' sliowcd soinc ~rcdundiint rcsults<br />
of lo\+ li.eqiic~~cy 111ul:iplc illout ~~iduct~oil and llrcy were IUI<br />
tzibulalcd due to ltlclr<br />
~iiiig~iiticant rcsponic I'ur ilioot i~lduct~o~i<br />
Separate crperime~us for ~ hc sla~~d;~rdi~,itiut~ oi'sliout illduct~o~~ mcdlum were done<br />
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<br />
s11i11lar ~iuiiibcr ot' ~nilliiplc rIiou~\ per e\l)l.ii~r (l',~blc 4 X i rI)L \\;IS 11act1 ;II various<br />
conccntr,itioii, ralig111g liutu 2 lo IOU phi. Tile 111rdln C~III~III:UI~IIS \\ere 11;1111cd 11s JCR<br />
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<br />
\\;la done hccpll~g tile I,IIU st,i~cs ui ~IUII~:IIIOII ;IIIJ suo1111g 111to ~,o~is~tIcl.;i~~ol~ :is<br />
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<br />
ctf'cc~ UII<br />
a!~bsci]t~c~~t diuul cIo~ig:it~o~~. IIIC~\ISIUI~ 01' !.il' i111tI L~IICIII~ \\;I< :IISO t'o~111ci<br />
easc1111~11 ;IS 111c) \\ere ;IISO IIIcI~I~I~~I 111 ~IIC SIOUI cIoi~g,itio~~ 111c~li~11i1 [SEM) ;I(<br />
Iu~~cr<br />
~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<br />
clollg:~ted \+ell 011 ~lic ~II~IUI ~I~I~~;IIIUII 1i1c111111ii<br />
Effect of cot!Icdollary<br />
liswe 011 rlluot ilnluctiul~: l'hc et'tet,t uf ~liclusion of the<br />
Intact co~yledun or .I pur11011 of it :~lolig\villi 111c rcgclicr.nllig II~~IIC IS alio\\~i 111 tlio Toble<br />
1 10. Iliclusio~i ol' c~I)Icdo~i Ibis :I co~lr~~l~r:~liIc ;I~\;II~I,I~~ l'or ~~~CIIC'IIIOII<br />
of ~ii~~l~iplc<br />
alluulr ti.oiii :l\~lI~r! 11ltc111 ~ C ~ I ~IIICC U I ~ COIPICIS C\C~II\IOI~ of IIIC co~).Icdc)~i ?~Iio\ved<br />
dcl;~ycd rel)u~isc ;III~ ,I \~gli~lic;i~it ~Iccrc.~~~ 111 IC 11l111iher 01' SI~UUIZ 1)cr rcsl>o~id~~ig<br />
explant. E\cIi~sio~l of col~lctlo~i 1111>:11.1\<br />
I c . CII~III~III~ oI';I'LIII;I~~ IIICI.I\ICIII IISSLIC \VIIII h;lIl'<br />
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.<br />
cu~nl~;~~cil ru tllc e\~il;llir a11l7 11.11l' cu~ylctlo~~ Ibllv\~ecl by /cru i.otylctio11. Iluwever,<br />
\\lrIi rcjpect to tile nll~iihcl. of siioo~s per expl;lnt in c.cl~l:lli!\ WIIII<br />
fill1 ;III(I lin1fcutyIcdo11.<br />
Sub-culturiiig oftlic rcgciicratllig hoot butis In clu\lera, Iio\rcver, rho\\'cd a11 cxpolientlal<br />
II~C~C,I~C 111111111ibcr<br />
01'11111111~11~ \11001\ 1pcs UY~I;IIII\ Tl11\ ~ I~CIC:I~ W~IS li11111d 10 be two to<br />
tlirer 111i>o5 al~licr~ur 111 !lie cspl:1111i \+IIII III!;ICI<br />
cot~lcdu~i.<br />
Effect ol'expl;~~it dul~ur scctlling .nge: i\gc oftlie accdllllg lj.0111 ivl11cIi the CXPIBII~S<br />
ucre derived, playa 'III 11iil)ortn111 role 111 rcgclicl-;itiun oI'1ii~1l11plc slioota (Table 4.1 1). All<br />
i~x~llnry Illerlatelii rliItint\ slluuetl ;lgc tlcpel~tlc~it rcspoiirc>. A.11 cxp1:11it difircd ti.oln<br />
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<br />
of rnerirlei~~;ltlc /olics 11irougl1 \rliicl~ dircct rcgencr;ltioil \\,IS<br />
;~cliicvcd. Fig 4.4 shows<br />
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<br />
of \;iriuils cspI;i~its, .All tile c\pI;inis e\pccl ,4113 al1uni.d IOO",, li.c~luc11i.y of IC\~,UI~~III~<br />
eupl;ints,<br />
\\it11 vdried 1i~111lbcr 01' SII~UIS 1pcr c\plil~~t, E111bry0 ;IXIS<br />
c\~)~;II~I ~II;II \\:IS<br />
liciliicllily iiscd 111 rcge~~er.~liu~i CXI)C~IIII~'I~IS \l~uiii'cl :I\)IIC/I~~~IIOII\ II~LIIII~~IC s11u1)ts<br />
origiii,it~i~g ~ZCILICIIII~III) li.u~il ~ UUI 111) ;iliil l~\~/I,~r) bud I~UIIILIII~ 'foi;il 11111nhcr of zlioo~s<br />
origin.~llng li.u~il 5hou1 111) ,111d t\$o ;~\ilI:ir) bilil I)U~~I~UII~ NCIC<br />
C~ILIIII~~ ,111d ~~OIIIIC(/<br />
luge[lier 111 recordil~g !he ~caults A ~icor \)~icliruny ot'~cgc~ici:~~~~~p<br />
II~~IIII~)/C I~OOIS ti.0111<br />
sliuot t~p \us obscrvcd. I'rolongctl cului~c 01' 111c cujil,~~~~.; 1.11 SIM ici~~l~cil 111 de~rciiscd<br />
I'rcil~lc~~c) oi'tllc \llool clu11&1t1u11, i\x~ll:iry bull \\,IS<br />
I,llic11 a\ a bl-limdi~ct ol' AM1 311d<br />
,'tL12 r\p1;111ts Yc~r<br />
~~IIC~IIUII~I~I\ 111~111iplc \l~uuti \vcrc l)rucl~~~ccl by IIII~ e\/~Iii~~t. N~i~ilhcr<br />
u1' 11i11ltil1lc \/IUUI~ jpcr ~A~~;IIII \i.'ia<br />
1)cltcr tl1:111 cllibryo :Ihla ;III~ \Iioot III) IIolvcvct~. llle<br />
;~x~ll;lry bud cxpl,~~it ubi,~incil ;I:, ;I byp~utl~lct ul' AZll c\pl;1111 hilowed IIIIIC~I<br />
Ivivrr<br />
li-cilucllc) uf' III~IIIII)IC ~IIuI~I~. 1 IC 11i1111ber o!' 1li11111l)lc >IIou~~ \lit19 Iu\\cI. tIl;~li cll~bryo :IXI~<br />
allti aliuoi 111). Abuw OOYI ol'ilic ;i\~lli~r! Ih\;ds aIiu\vcd III~IIII~IC \11uuta OSI~II~~~III~ Ii-0111 llie<br />
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<br />
tr:~~isl'ur~i~;~tiu~i, Tllc axill;~~,y bud 1purtiu11 c~~l;irgccl ;IINI tlic ii1111111)lc sI1ou15 origi11;itcd fi.o~~i<br />
siiollcn portion oi'AL1 I cuplatli. AM2 cxl11,iili tI1,1i g:~i,c riic lo ~nii~ltiplc ,Iioots lio~ii olie<br />
oi boll1 sldcs ot'illr ;ire;! ol tllc ;IIII~)LI~;II~~ ;~rlll;iry IILI~. 111 ~uo\t ol'tlic caho ~iii~ll~plc sllools<br />
\+ere produced Crolli ollc sldc oi' illc ;ixlllar) bud ;irc;i wlicrc syiichronoua mtrltlplc slic~ots<br />
were observed from ll~is espl;liit. 111 AM3 expl;lni llie rcgcncr;llioli I'retjuc~~cy \+as vcry low<br />
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<br />
bc cons~dcred as itic:ll tbr gene IV,III~(~.I CK~~~IIIICIIIS yve risc to lllultiplc s11oots 3s<br />
nllllt~ple cl~iatcra \rl~cre tllc hiloot.; ;II.C<br />
lbr~~~sd 1rci111 TIII<br />
1p0r11011~ 011 tl~c si+ollc~~ 1p;ltt of'<br />
,~k~llilr! 11icr1>lt111. I:IC 1.5 ~IIu\\~ regi'~icr,~l~~~g IIIIIIII~>IC ~IIUI)I\ litllli 4\11 ~\~)I,IIII \+l~cre<br />
III:III~<br />
~Iloutb U~I~IIIJIC~ li.0111 tllc ~;Is;II I~U~~IUII:, ui 1I1c ~CIIII)\C~<br />
SI~~I~II h~ld\ I'r~lu~~gcd<br />
CLIIILIIC ul' ill1 1llc ;~bu~c-~~~c~~tiu~~ccl<br />
L'\~~:IIII> 011 sl~oul ~t~d~~eliti!> III~~IIIIN 1~h111tetl ill<br />
L'\~~ 11er C\JI~~IIII \\~lli C:ICI<br />
sl~uut but1 ll~\ 1118 tlic ab1111) tu tlc\clul) 111tu CUIII~~~CIS ~)I:IIII<br />
4.1.2.2 Elol~g;~tiul~ ul'tlle slloot buds:<br />
I'lilllt gro\\tll r~g~ll~ltorh \bere elll~lloyccl :It IU\V cullcclltr,ltloll:, 10 lllcrc~lhe tI1c<br />
~nunlbcr ut'sllooti elo~~giling ~ C cul,l;1111 I ('l:~blc 4 13) Ijhl'. 2-11], ~IIICIIII<br />
;III~ GAi kcre<br />
IC<br />
key COIII~)~~I~CII~> 1;ir b11iiot C~OII~;IIICIII,<br />
Tllc rlluul cl~~lg~ltio~l 111eil1,1 H~I.C I; IC~ iis C'I:L<br />
scrio koln \\111cli Cl:l.? iuln;lllrlllg 5 11hl 2-11' ,111d 2 lib1 ktiieull ;111tl CI:L? collrallllng 2<br />
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).<br />
EIollg;~tion 011 ~~IIC~III \+,IS<br />
iu1111d IU be I~~IIIIIII;I~, E~UII~,I~IUII W:I\<br />
ill50 tcated 011 basal MS,<br />
bur ll~c rcsillts were 11ot s~~coui.ng~~~g irlic~i cu~lil);~rcd tu tlic ullicr II.U;I~IIICIIIS. IIowcv~~,<br />
prolo~lgcd cul~ure UII MS ioultcd III ille ruuul~g 01.5 lu 10% ul'tlic clo~igallilg shoola. All<br />
lilt tested conib111,111o11 sliu\rcd elollg;~t~un uS0.1 to 3 sl~uuls per uxpl,lnl 111 the illirlul step<br />
(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<br />
explat11 bas placed 011 fl-csli clo~ig;itiun II~C~ILIII~. Ucat results IVC~C ubralncd ~vl1e11 tile<br />
elollgatlon \+us done \\~rli CkL? 111 the clulig,it~o~~ I ;III~ CLL7 111 tlic I;ltcr stilgca. The<br />
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<br />
application showed lncre;~sed internodal Ic~~ptli illid bctlc~ Icat' ~~~orliliulupy. Tile slioots<br />
cluriil;ited In cocI1 p;ias,l$e \vcrc tr~~~ist>rrctl lu tile louti~~p III~~IILIIII.<br />
I cupu\ud to tllc ~uutlng incd~;~ dr;~b~lcally di'crc;~actl the rootilig<br />
Srequency. Late roots rnent~oncd abobe, sliowcd \low growth illid they ~uritil~r~ed several<br />
root hairs. Sliools, wll~ch did 11o1 lout, ~ crc;~rrlcd lo the P!I;I~C 2 III HIIICII they were pulsc<br />
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<br />
de~d llllll. roots \\we t'urll~ed 111 buncl~cs.<br />
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\<br />
S~CIIUII), Sucrose<br />
I 5':" ~III~ ?",, ~110i\ctl Oc\l rc.;t1115 III ICIIII\ 01 Iicq~~c~icy ,IC~ r ~ t<br />
~~~orpliolog) L~trlc or ~io rc\po~~\c !\;I\ obscr!c~l III cull~lrc?, ilc\uid crl'\ucru\c<br />
Effect of ~lletliul~~ on rooting: Ll'lkcl ut'v;~riuu\ cu~~cc~~lr;~t~u~rs ol' IDA ,111tl NAA.<br />
\IS 1111\ ;IIY >IIU\VII III 111c I',~t?lc 4 18 lI3tl ;I\ 5 pM<br />
\\;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).<br />
Coniblnationa of procesbed org;llilc m:iltcr SICI<br />
Cell l
humidity uf 50% 111 the dayrinie a11d uier 80Uh 111 the 111gh1 'rllc pl:1111s ivrre inltlally<br />
exposed to humidity as lilgli as over 90% b) ~I~ILIC 01. cuver~~ig 1lic111 w~tll PI'ISIIC<br />
b;igs.<br />
Tliry were sloicly i~cclltilnti~cd to tllc i~~iib~clil IIIIII~I~II~ b) grililu;illy c~l~cliilig [lie cover.<br />
Prolonged coierlng. 1.c.. oicr c\liohi~rv 111 li~gl~ I~III~II~II) c:ii~~cd \\IIIIII~ ;111d dccrc;~sed<br />
exposure caused preinillllrc dl.).lllg, b011i ~.~il~ll~iig III dciilli of Illc ~llii~il. IICIIC~ llic iili~11~<br />
hu~nidlty accliniat~?at~ol~ procesh rcqi~~retl ;1bou1:I ~iiunlli ;III~ i11111c1\1 C;IK<br />
bc1i)rc c~itcril~g<br />
illto the stage 3.<br />
lhc pl:ilil. Iicl;ce, tllc e1illl.e II;I~~CI;III~ I)IOCC~\ \v;I\<br />
duli~ ;II ,I II~III IIIICII>II~ OS 10.000 lo<br />
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<br />
house caused dry111g 01' 111 ~I:IIII. lY1111 ~C~~ICCI IU IIIC I~IIOIO~)CI IU~. \I;I~U<br />
I W:I> lii;~~~itillll~d<br />
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<br />
1011 1101.11011 01'11111 plilllt<br />
was exposed to tllc ULII side COI~~I~IUI~~<br />
,111d hlo\\ly c ~ e e l ~ \\IIIIS ~ l gro\\illg ~ ~ ~ ~ I:igure ~ ~ 4.10 ~ d<br />
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<br />
completely 111111i~'racd<br />
[lie 4 a11e11g111 A~'IIOII'\<br />
~O~~IIIOII. 111 1111, 111crI10~\. IC<br />
I~~,IIII \\I18<br />
direclly cxpoaed 10 1I1e ;III!~ICIII ~UII~IIIUII$ IYVIII ~IIC lir>r cI,I\ ll~c\er, (1ry111g\\;I><br />
pl.evented bccauac ol' Ihc Il(l111d 11;11i1rc 01' 111c IIULISII~IIII~ IIIC~IIIIII. 'Illla ~i~e~li~d<br />
CZIII<br />
supple~ne~ll rile atagc 1 ;111d 2 ~Isacr~be~l ,~bi)\c;111d<br />
ll!~ plil~il\ CJII<br />
dlrc CIIIPII))C~I ~~I~CCIIIII~ \\ I~CII IIICIC \\:la 110 g~u!\lli ol'lli~ 10ti1 \)\~cI~I. \\IIICII<br />
dirsctlj i11111blti11g lllc g10\\111 of I11e ~I;IIII. ' ~III~ IIICI~IU~ \\;la<br />
IIII~~UI~,III\ III ~II~IJIICIII~ tllc<br />
survi\;il ticq~~u~lcy by irouble>llout~ng In 1Iic IIJI~CIIII!~ I)IUCCI\ IIICII~IOIIC~ 111 llie III;I~C~~~I~S<br />
:~nd methods section. C;ro\vlh of llic i'uor jysli.~!!, wliicl! 1s .I Ley I:lctor Ibr Ills II;I~~CIIIII~<br />
appeared III nbou~ 15 ti;lys alicr IC I~.III~~)~:IIII,I~I~~II i1!10 ?O CIII 1po1.;. lllc Ilu\~ei':, kcre<br />
renloved aa sooll tiley ;ippcorcil ior ;lboi~t 3 \vi.cL\ >u ;I$<br />
10 II~CI~.I\~<br />
llic icgelanvc grow111<br />
and lliiis ilici'ease the ~ii~mbci' ol' accda per ~I,IIII I~CIIIU\,II<br />
ul' Il~i\\'cr\ :II\u rcs~111cd III IIIU<br />
111creabe of br:111cl1111g ;III~ IC<br />
~I;IIII~ ;i5$11111e~l
4.2 Histological studies ~II nlultlple slloot de\tloplllel~t I'I.~III Ahl.1 espl~l~~t:<br />
As described 111 secliull 4.1.2,l. Ah14 elplants alio\\.cd ;~dvcn~~t~uus niul~iple shoot<br />
induction ir~lll alloul bud> OII~II~;I~III~ (i.0111 dilji'reiit 11;1r1\ uI' IC ~C~CIICI.,IIIII~ ;I~c;I.<br />
Histological slut11i.a ol' ~iitlltiplc slloo~ III~IICIIUII 1j.oi11 ~llebe c\lll;ii~ts \\ere co~iductctl 10<br />
colitirm tile for~ii;lt~oli oT~iit~ltiple ~~dvc~~ti~iou~<br />
~ilet~iate~ili~~ila ,ilicr the i.s~iiuv:~l ol',~\~llo~~y<br />
bud. 'I'hc dey \+Iicii ;ixill,~ry but1 \+';is rc~iiuvcd \\;IS<br />
eu~isiilercd as d.ly-I ;\lid tile slud~cs<br />
were coiiduclcd 1111 7 diiyb. Fig 4. I3 A tu H ~ll~\\'b 11c\cIu~i111~1it ol' I~~C~I~ICIIIUI~~ ;II<br />
dlff~~~lll ~ 1 ~ ~~;l.\l~~;l~)<br />
~ 1 ~ ~ 5<br />
lll~~l\l~lll<br />
;lrC;l \\ 11~1C 15 10 ?(I lll~Vl\lClll~llt~~ ~~L'\c~u~JL'~~<br />
~1~1\~~!~11<br />
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<br />
axillary bud rc~iiaii~iiig cvcci alicr ~lic cvcislu~l of tile gruw~lig ,~\~llary but1 and<br />
dcvelopmeii~ of rnerlstelllo~d\ ;I[ the base of 11. .fliis rcsiilt alluiv\ l~islolt~g~c;~l c\ldcncc lor<br />
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<br />
: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:,<br />
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<br />
I{).<br />
Tliese tIi\isio~ib rcullcd 111 [lie Ibr~ii,~r~o~i ol' II~C~I~ICII~~IIII. /OIICI<br />
IIl;it ~C\LIIIC~ ill llic<br />
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<br />
nicrlstcniatic ;icti\wl! ol'd~i i~l~~ig ecI1 iIi;~t C\CIIIII;III~ ~c~cIuI~c~I ill10 ~ ~iiilt~lll~ sliouts.<br />
Ccnetic tl~;insfuriii;it~on iraa carr~cd uiir by LI,III~ b ~ul~\ti~ ;IS IVCI 3s Afirohoi!i,~.ili~~~-<br />
lncdiilted lllelhoda In boll) llie proccd\ire> llic p~~l,~l~\c rr:iii~l'orniaiira \vcrc obtalllcd by<br />
selccti~lg the trallhl'urlllallt,<br />
liilllg il/illI ii\ lll~<br />
s~~c~l~lble lll~.l.k~~. gCI1S iilld k:~naillycill 2s llle<br />
antjblouc br selcc[lon Conlrul cxplanls \r.crc ubcd lu tesl thc Ictl1,il tluse (LD 50) by<br />
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<br />
(Table 4.15). TDZ \\;is eniployed so .IS<br />
lo Itst tile clli.c~ ol' TI)% ti11 c~lli;~~~ci~ig survibal of<br />
thecultured explants 011 k;~~ianiyc~ii COI~I:IIIIIII~<br />
III~LIIIIII~ hi~r\l\:~I \\.I, IC IIIC;I\II~L' tlf ;III~<br />
collcel~~lble gro\\lll 01' tI1c ~\l~I~llll ~1lcI~lLllllg llllllll~llc \lltlcrhl>lellcc of<br />
chlorophyll plgliicnt In llic eul~l,1111. Tl~oi~yl~ llic c\l~lLl~~r p~o\\~l~ \\;I.; 111li11111cd c\cn ;it 20<br />
11i9'1 ka~ia~n)ci~~ COI~C~IIII~II~~II. cliloro~~l~yll 1pig111c11t 11crh1\1etI (or re\\ ti~~irc cI;iy,<br />
,~ttcr<br />
wli~cli expl;l~~ta blcuclictl. F.\~>I~IIII sro~111 ;111d aI1tlo1 b11tI II~~~~ICI~OII \\;I\ COII~PICICI~<br />
lnliibitcd :11it1 CX~I~IIII ~OI~II~ICICI) blisc~cl~cil 111c C~JI~~CIIII.;IIIIJII<br />
LII' 30 111s I. I\;III;IIII)ICIII .A<br />
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<br />
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<br />
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<br />
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<br />
11onnal gro\\,tli ;lnd ~iior~iliull;~gy cu~lll>,ircJ tu tile coinrul\ they \bcsc I~I;III~I;IIIIC~ 111 tlic<br />
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<br />
the tra11splentatioli illto tlic 20 el11 put,<br />
The 1)1d111r MCS~ pr~1)~18;11cl;I ~cgctilt~vcly Sor ill1<br />
extended limo b) alill,ut.itllig<br />
llle IC~I~~III,II bud> ol' ,oiiie<br />
bl-alichc ;III~ rc~liuvi~ig llle<br />
enlerglng flo\vera. Thii nrc~liutl could plu\ itlc rLiSlic~cnl IcaS tlrs~ic ~\II. ~ln)lccul;~r ,~iialys~s.<br />
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<br />
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<br />
BICI:~.,I/J<br />
9c11e \\c~e i1;1111eel ;IS CB (CB I, ClQ, .,, 111 series ,111~1111c O I I ~ IV,II~\VO~IIICL~ ~<br />
\vitl~<br />
T I I c I:III S ' I 2 . . 11) \er~c\ .I 1u1;1101 1 I ('13 llh1111\ :IIIL~ 0 C(S<br />
~)lants \\.ere obt;i~~ictI ;111,I llicy \\CI.C L ~LI~~C~~IICI~I~! ;111;1ly/i,t1,II IC<br />
III~IICZLII;II le\cl<br />
IIIIII,I/ >el 01' II~IIIS~UIIII;IIIUII CX~ICIIIIICIII~<br />
1\41 i1011e \\1111 111iS723 131 2nd pIIS<br />
737,SBTI bln;ll-y vcclurs CUII~;I~IIIII~ Ull'i~./,lh ;III~I .S'li/7 ~CIICS ~csl)ecl~iely. I'u~;~livcly<br />
rranslbrmed ~planlr w1t11 Ui(',~l,.lh<br />
\rere ,i~~al!/ctI by 11it11g IICI< ;III~ Suulller~i blulli~ig<br />
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<br />
Molscul;ir ;in;~lya~s irnh dune 11l1t1~11ly by do111g 1'('1<<br />
lollowcd by Soulhcr~~ blorl~ngs.
Conlirmntioll of plllati!e<br />
t~.~ll~afor~~~a~lts II~~II~ ~)ulyll~crasr rl~ai~~ re;~ctioa<br />
(PCK): PCR wi15 fbu~~d to be 011s ol'111c II~\ISI<br />
e1'1;.ct1\c ;III~ ~ULIIIIICI~ IISCJ tecll111q~1~1 hr<br />
Initial co~~lirfiii~l~u~i ui' rrI,IIIII \\'~tIl rcqlcc~ ICI L'lj (/11('r1,/..1/1) ~I;IIIIS.<br />
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<br />
oi' 10 ICSI~CI pl;ll~la. (~CIIOII~I~.<br />
I)\ \ \,1111lili.\ 111 ('111. C'I12, C131. C111 i~~ld ('117 >II(I\vc~<br />
proliilncllt t ~ ~ ~ ~ ~ ~ I .2 l LO ~ :1111pI11ictl<br />
l i c ~ ~ l ~ K ~ o g spcc~l.~~,<br />
~ ~ b ~ 1i)s c ~ iii,/,\ ~ ~ gc11e \\,I> ob~crvctl<br />
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.<br />
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<br />
;IC~<br />
5 li't1111 0 ]I~.II~!\(L h2, C'S?, C S5, C Sf1 ~11tl L'S')) jI~t)\\cd 1c\pcc11\e I 2 Mi I~;I~III~III 01<br />
iiii/A pclic i~~iipl~licd ,\ 1111111 b,tlliI ,llhu :~l)lic:~secI III ('SX II~~ uI'I3r ,111il 5131'1 I)~~IIII\ ;IIC<br />
I~~I\\II 111 IIIC 1.1014. I4 ,111d<br />
re>~~ltb vl't11c l'Cl< ;III~]IIII~IC,III~II~ vl 1.2 kb ~I~I~I~ICIII \pcc~lic tu III~/I~ ge11c ;~rc SIIU~VII III 111c<br />
1,'1g 4 15. \YI~II sty~ccl 10 /~I('I.I/,~~/I gc11e ,II~I~III~~.,IIIUII~ livv \ct\ ~I~~II~~IIILIcI~~I~cI~<br />
1pri111crs<br />
\b3iiI~(hi. tiil~cr he1 ol' I)I.IIIIC~~ I~c~IIII~ ul' IIIC I'CI< :III~~~III~C;I~I~III\ 01' I~,I~I~IUIII rl)cclIic to<br />
I~I('I:I/~~!J gc11e CI~C 5Iiu\\11 111 ~IIC 1'1g 4 I6 (L3) 4.10 (I\) \II{I\\\ ~C\LIII\ oI',S/1/Y gc11c<br />
~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<br />
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~~<br />
01'<br />
genomlc DNA 15 ;I prcrcqu~\~tc lus CI<br />
cl'lkcuv~ Suurllcrn II~~I.I~I/~IIIUII :III~ IIIC COIIS~~~UCII~S<br />
ui' 111c ~C~I~ICIIUII<br />
~C'ICIIUI~ II~II~LII~ 111,iy CI ~11rcct role III 111c lprucc~~. llo~cvcr, dur111g tile<br />
cuur\c of rl115 atlid). II \$
~~IIOII~IC<br />
DYA ror SOLIIII~SII L~~~:~ly,~> ~CIIOI~IIC 1)'4,\\ uf IIIC p111;111\cly II;III~U~III~~<br />
ch~ckpea pl;1111s \\ere dlgeali.~l enl~cs \\ 1111 I.'< oIIIu\\~~ III;II USA IS ri.q~~~rc(I III IC<br />
I~;~CIIOII<br />
1111xture. ~\tId~riol~.il ~IICIIIIII~ or l\lllllr \ill11 I.tbll~CCII~C 111obc 01' 1i011-rildiuil~l1~~
5.0 DISCUSSION<br />
During tile la31 dccadc, btu~cci;~;ulug~ I1i1, 111~1(lc i~ill)~.e\hivc ;I~V;II~CCIIIL.II~S.<br />
Genetic ~ranstbrrn~r~o~; oi'crop l)la~il\ I\ 11, \:ilil,~blc ,I~J)cc~. Ie;1d111g tu~~irdb \lie h~ildt~ig<br />
of an organized and hcallliy ,ig~~culti~re \).>IL.III<br />
ficc li.ulii 111c u>c ol'pullr~l~ng i~~acc~icidca<br />
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<br />
agronoln~cally 1lnport:lnt tr;l~ts lilto othcr crup pl.iilr\ tu dcvclup cllte c~ilt~\ars (Hirch.<br />
19971. Thc trcliliology c~llpll.~t~c~illy aupport\ lllc ir,l~~sl;.l, III~~.~~.III~II ;III~ c\l)rcsaloll ol'<br />
foreign gsries 111 l~etcrologou> orga~iisiiis. I~~IUIIUIIIIC;I~~~ IIII~)O~I,IIII ~CIIC:, 10 1111l1ru\c tll~<br />
crop ylrld qualirativcly ;IS<br />
\\,ell as qu:~i~tiiat~\~cIy II:I\~ beell ~;ul.~tcd ;III~I TIOIIVLI 1'1.0111<br />
vartous sourccs. TI~csc 111clu~lc gclics Ibr li~l~giil r~h~st;i~lcc- C~II~III:I~C~ :ICI<br />
~IUC~III;I~CS:<br />
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-<br />
a~iiylasc ~~il~ib~torr, ~irutci~~.i\e i~ili~b~tvir ;IIIJ 111 ~O\III gcilca ,111c1 ~CIIL,~ hr IIII!II~ILIII;I~<br />
improvenicnl- ?S iilbi1111111 ~CIIC<br />
B~ulceI~~iuIog) c~~~o~iip,~~~lllg<br />
gc11e11c<br />
CII~IIICC~III~ 011;'r:,<br />
ave~it~es to o ~crco~~~c tlle b;lrr~cr> 111 ~c111e\ III~ I~IJ\IIIILIIII<br />
cl~~chl~c;~ ~~I~ILILICIIUII<br />
5.1 'l'issuc r~lllure atlldirs<br />
p~.otocol.; aplx,iscd 111 il~ickl)c;i rutlr,lllc cnlbr)o~c~lcs~\ 111:it (lid IIO~ \;I~I>I) Ihc<br />
~ ~ 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<br />
ii>cii IU iildiice soiii;nic ciilbryos<br />
from inimature uotyletlo~~s \'cry liigli ti.cql~elicy ofpI;i~itlc~~ \\ere regcner;~lcd tio111 tllcse<br />
embryos (Hila el al. 1997) Hunc\e~. .ill l / l ~ 6 I)~I~IOL,U~\ ~ re~t~r,~l~~d<br />
111' II~~I~CIIIIY 01<br />
rnaturat~on and regcliclniioli ol' nliole p1~11tb ii.1 \olii,nic c~i~br~o~ei~c~~~<br />
~p;iili\\i~~, rliis<br />
was rc-co11firmt.d 111 our c\perlliitIi1\ A tcr! good ~~.CLIIICIIC! ul' ~II~~~ICIIOII \\;IS obhcr\ed<br />
tion1 m;iturc eiiibryo ;I\IS :iiit! Ic;itl~.i c\l)l;iii!s I:~iibryopi'l~li II~\IIC<br />
\\.,IS ~hsci\ed to<br />
iiiducc bcticr e~iibryugcnc.;ia 1Llc~c1l~l1)ll liarlle ol')utliig lc.~Ile~\ \\.;I\<br />
;II\ 1'11~ p~ihli~llcd prolocolh<br />
were repeatedly ~ricd \rill1 Iirilc ur 110 ~LICCC\A. I'ICIC<br />
r~i1111> s~~ggesI 111~11 ~C~CII~~:IIIO<br />
protocol vla soiiinlic ei~ibryugciics~h calilior be ;I rcll.ibls t11ic lirr gci~cl~c ~~~iislbrin;lt~oli<br />
exper~iiicnts<br />
5.1.2 Ori~g~~~~ogc~~csis:<br />
~!IC~O~)~U~);I~~IIIOII ,iiitI ~~~~I~V'IIIUII \),I tlirtc~ or t11~11rcc1<br />
organogclie>ls 1i;ive beell e\le~~\~\cli ~itlti~cd :III~\CVCI:II 01<br />
ICI~UIIZ \\ere ~p~lbl~~~l~rd.<br />
liiclus~o~i of I~~L'~I~ICI~I:IIIC IUIICI<br />
ii~lli IC c\I)I,II~I\ \L,I\ liltl~id ti OC cruci;iI for<br />
regcncr;ltiuil of slluoi butla .ind i\liulc 1)1;111t\ 111 CIIIC~~)C:I. I.;II.I) rcj)ort\ ~III cli~ckpca<br />
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<br />
I;irgc-sc,~le<br />
multipl~catio~i of a speclea is leqilircd 111 oilier \\orti> IIIIS c\pI;ili~:, IS USC~III tbr<br />
ni1croprupng;itloli :1101ic ;ilid i)gutic cliibr)o b;lsctl c11Ilu1i.a \\ere IIOI coliaitlcrcd :IS 1dc;ll<br />
for l~~~isfur~ii~~t~o~i<br />
stiid~eb, Ullicr e\pl,1111h II~S<br />
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~<br />
medlum sho\ved n better elongi~tio~~ cu~~~p:~red to 111c ones nn\li 011 '1'1)L<br />
Ilu\\c\er.<br />
~iu~iiber ofsl~oors per e\p1,11l1 UII TIIZ II~C~I~IIII<br />
\\.I\ I i 10 ? IIIII~\ bct~cr III:III<br />
l3:ll' IIIC~I;I.<br />
As IS III~IIIIOI~CL~<br />
,~bo\c.:I~~IIIOII;II C;ISC<br />
~IIOLII~ be c\crc~,ct but<br />
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<br />
nlodifical~on over euls~lng protocols<br />
cotylcdo~inry node ere. ilu\ri.\cl., sclcct~u~i ,11111 ~irocc\\ll~g ul' ~p~ol)cl s\l~l:lnl I:,<br />
other. Rcnioval ol' a\ill,iry<br />
b~~il. CLII<br />
111ro~1gl1 tl~c ;i\iIl,~~j IIICII\ICIII ,iiid ~IIC~U\IIIII 111'<br />
-- -<br />
cotyledon .- -~ were tile s ~y~i~lic:~~~l v:~ry!ms~:l!~~j:~ 111 llic prcsc111 brudy. '\ I~OIIIP~C~ICII\~\.C<br />
----<br />
~irolocol for rcyci1cr;iiluii ontl ~sccuvcry ol' \\llulc ~IJIII\ oI'cli~cl\l~c;i Ii) II\III~ :i\~ll:iry<br />
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~<br />
\%,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<br />
oC;IS~ t 01' s ;l\ill;lry .<br />
-- .--<br />
meristen~, removal of axillas) bud liiilllliea tile .~pic;il ~OIIIIII~IIIC~<br />
111 :idtlilit111 to tllis. IC<br />
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~<br />
shoots. Et'rcct of age ur tllc >CCLIIIII~ I~UIII \\IIICII llic ckl11~1111\ \\ere dcri\cd sIIu\\\ 111,it<br />
expl:~nts \\.ill be derived. Uoci;ll .II~L~;i\lll.iiy mcrlhicilla II.I\~ bee11 I~~LIIIII\II~~ II~\IIC~ lili<br />
regeneraling eapacily \+IIII cul~\i~~ci~~gly ;IC~~~.IIIIIIULI> 11:ilurc ul'lhc rcgci~ctill~~ig IIILIIIII~IC<br />
rrgellerarloil protocol, ol' v.il.lou> 111,1111\. I\\I/~;II) IIICII>IL'III<br />
\\.I\ ~LIIIIIIC~I IIII~C~ \:IIIOLI\<br />
IISSU~ has a direct correlation \\IIII<br />
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<br />
- -<br />
it afkctr upt:ikc of~:~r~uuh I~LI~WICI~I 11111>. 1'111.1Lc 111' 111li~i1~<br />
;III~,1111111~111~1111<br />
%ire I~~IIII:III~~<br />
affected by the mediiilii 111-1 (Ilclirc~itl ;illti \l:!ti.l~\. lCi:5. l(,i\sn. ICjSi)i 11 \\;is ubscl~\cd<br />
that nltr;lte 11pr.ike la 1:1\uurrd :!I lo\\ 1111 :1nd ~1111111011111111 .II III~IICI 1111 .\\lll,~r! 011d<br />
111ult1pli~a11011 III shoot CII~~~IIC:, UI' C'i~il~~llc:~ \\:I\ II~UII<br />
\i~~~\l';~~~~r~<br />
\\IIcI~ 111~' 1111 (11' hlS<br />
\\,as red~~ccti to 4 (Cllsvrc el :I]., IOh'3). lC~\C~ \\IIII cIiich11c:1 tll~t \I~.C~C~I\C ill 111~<br />
C~IIICCIII~,IIIOII ~i IIIIKIIC\<br />
in the ibrm ol'KY01 \\':IS t;~~or.~blc Ibr luul~iig Ths III~~~I~C<br />
LIIII;II\C<br />
I\ I;I\II~CCI :I( IL)IYCI pII<br />
and iininio~iiulii at IllgIle~ plI. it c.111 bc li)putl~c\i/ud LII:II l ~ ~ loll\ ~ IIII~III ~ I~c ~ ~<br />
lil\orlng routing at li~glicr pli ;111d III~~,I~~S 111v~~r111g SIOI<br />
III(~UCIIOII<br />
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<br />
rcg~1ier;ilIoli \i~cIi ;IS<br />
clo1ig:111011 'III~ ~OOIII!~ 1 IS\II~, lo\\s\l 11111 sli2ct1\ c COIIL~II~~;I~IOII ul'<br />
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<br />
~ ~ 1 ~ ~<br />
ol'TDZ negati\cI) ~llrcrli.~c\ \r 1111 tl~c IILIIII~CI 01 >IIUUI bull\ ,111~1 IIICI~ ~IOII~;I~IUI!, 4 ILM<br />
was conaidered 45 u1111111:11 iiorh~llg COIICCIII~~IIIOII<br />
\LII~I~CI ui'\I~uut hi~ds ;~lro(ICC~C;I\C~<br />
~vitli illcrease 111 TL)% CUIICCII~I,III~II<br />
L'cI~ 111gIi CUII~CIIII~III~JII<br />
III'IIIC TI)% M:I\ ~bher~cd lo<br />
gibe stunted slio~~t biiil, \\IIII l~ttlc or no ~IUII~~IIIUI~ In atldlt~on to tills exlcndcti I111ic of<br />
culturc on TUL ,ilsu 11;1a ,umc ncg;ltl\r cllka on lllc I;llcr at;lgo 'l'llougl! 4 pM 'I'D% was<br />
optlmnl work~ng cunceilrratloli. CLII~LI~III~ ills C\PI~IIL~ UI<br />
Lllc s~ille IIIC~IUIII Sur 5 tu 6<br />
necks dccrcabed tlic clu~ig~t~uti l'ic~~ucrlc~ !\~II~IL,IIILJII<br />
uI';i~iy gru~tli rvg~11;11urs :iI'tcr lllc
prololigcd culture on TUL sho\\~d 110 pru~llot~\c cii~ct UI<br />
~IUII~;III~II. Iicr~cc, illc<br />
lnductloii tlnir \\as re:,trlcted tu ! to 3 uccLa \\'nil ~~SI>L.CI to :\hl4 ~\~)I,IIII, tlie \VIIUIC<br />
preparation \\as dollr OII<br />
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<br />
----..<br />
i'otllalrlitig 5 lib1 2-11' ;iliil! 11Cl LII~C~III IIIC~~IIIIII<br />
('I:[.!<br />
CIUII~~IIC~I lilil I),IIC/I 111 ~11oot~ 111 iibut~i ?<br />
shoo15 \\ere CIUII~~III'II.<br />
I Io!\c\cI. ~LI~-CLI~ILI~III~ l11c il~oota UII C L1.7 CLIIII.IIIIIII~<br />
2 IIM (i,41<br />
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<br />
\veeks cunsldcrably ~iicrc:~icd ~IOII~;IIIUII<br />
~'~CCIIICIIC~. (;,Ii ~~ICICII~ III ~IIC 1'1.1.7 IIIC~IUIII<br />
is<br />
5l1o&1i to pi;]) :III~ 1111purla111 rule 111 CII~I~IIICIII~ IIIC ~IOII~;IIIUII ~ IC~ILICIIC~ I or UII~IIU\VII<br />
reasons, frequency of >houla clu~lgattd 011 CIA, cullt.llnllly II~C~ILIII~ rrglll lro111 llic lirst<br />
pliaac was 1101 ;IS goud tllc li.cilui.~icy U~I,IIIICL! III \lcp\v~>c m;iliiier 1\11 ~hc Ialcl 5ub-<br />
cultures oil CEL7 rc5ul1cd III nur.~blc IIICSC,I\C 111 ilic 11uinber of alloola clongalcd. Sub-<br />
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<br />
medlu~n drled earl~er ilia11 tile o11cs culli~red UII ('EL7<br />
Hs~icc, clonyst~uii \\;is dolie<br />
~n~tially tbr about 2 \veeka o ~i C'EL? fullu\red b) CELi 111 ,111 I.lrc~. p;ih\;lcca.<br />
p,<br />
(13t<br />
.<br />
1ccort1<br />
.<br />
or rc10111ig ol' clu11g~11ctl \I~~UI,<br />
cIl~cLpe:~ \\ 1111 ;II,~(11'<br />
llic ~>CI~>III;I~<br />
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<br />
\el-) lllgh freq~iu~lcy ul'rootii~g. ;I 111c1l1od \\ l11cIi \\;I\ 11o1 rc1~ru~l1ic1blc. !\ IIII\C~ ~~~IIIIIL~IIC<br />
of ruotlllg on filter p;~pcr b~~tlgus III;II<br />
\rc~c 1111111craud III l~tli~iil ruuflnp nictli~~~n \v.I~<br />
developed. Root~ng n:lr .~lau tlo~~c UII \cll~~-wlltl I~IC~~IIIII~ i111t1 ;I e111111).11;111\~ b~utly \\:is<br />
dolie. Rooling li~cq~~c~~c~ III IILIIII~ ii1cd1i1111 \\%I\ 5 10 S III~IC~ ~CIICS 111~111111e ru0t111g<br />
frequency 111 se~ii~-sulid 11icd1t11ii. I~~C~~LICIIC~<br />
\\;IS \cry li~gh III I~q~iiil IIIC~ILIII~ ;I[ 70 IU<br />
90% cu~npared lo 5 to Ill?',, 011 ac~l~~-aul~tl 1iiet11111il IIIC ~ic\\Iy CIC~CIUI)C~ ~~~clliud 1101<br />
only clisurcd better li.cql~cncy but ;ilsu sIio\vcd bc~ter iiiur~>I~olc~g ;111tl IIICI.C;I\V~<br />
I;LI~\I\'~I~<br />
tili~c ol' tlie pl;1111s. 111 u11ie1 \+t~rcl\. e:irI) ~I~\I~~;IIICIII uf ~IIUC)I\ ei111111cd (111 ~~I~II.\~II~<br />
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~.<br />
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<br />
shout, acqu~rcd liypcrliydric~ty u11 \crl;ll ~LI~-CII/III~III~ ,ICI<br />
\CCOIICII~, IIIC 1111ots blio~cd<br />
;In ~ncrc;~scd grow~li ralc '1'11~ Iiy~~crli~tlr~c~~y 1problc111 \\.I, i.eairlc~~~I 10 111c aI1001\ OIII~ 111<br />
the sub-culruring slags, .III~ 1per51a1111g /)robIc111 CULII~ be III~III'I~C~ by pl.1c111g 1l1c I.LIUIIII~<br />
CUIILI~~ tubes 111 the srcr~le clivilonlliclir (I~III~II~I llu\v) hill1 CUIIOII plugs O~CII Ibr bout<br />
kw hours. The second problcln cuuld bc 111:111dgcd by usltig lu~~gcr culrurc tiibcs (25x200<br />
mm) and reltluvlng llic C~CC>>I\C Iuv,cr lll~lt US tll~ \lci11 diir~tlg thc >II~-CLII~LI~CS.<br />
Addltlona[ care was exercised to ;i~htc\c the high li.cq~lclicy of routing. I:irstly, dark<br />
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 ~<br />
in the culture tubes. Shoots III;II<br />
d~d IIUI<br />
rout aliould 1101 st.i) ior lollger riil~c 111111s aanie<br />
culture rnediiiill and be tKlnsli.rrcd tu 111c licrl~ IIIC~IIII~~ ,ilicr II~C tirht 0;11cl1 II;I~ IULIC~.<br />
Care should be riikrii iiut lo ;illon tlic aliouta 10 II~L~~III~<br />
IIYIIL'I./I!~~II~II!<br />
bi'c;iii~c SLICII<br />
shoots do no1 rout \\ell a~lci Ii.i\c tu be c,iriicd IU IC ~)II~IZS ! ul'r~ot~~~g.<br />
tli~rdcn~iig iiiiil trC~~is~>li~~it~it~o~i<br />
(11' cliick~~c:~ li:i\ I)cc11 ,I ,L~~IL!LIS IJILIIIICIII 1 ~C IVI<br />
10 ~~~cl'licic~it<br />
111 \ 11ro $ri~(iiiig<br />
by utilizing tlic roo1 sysle111 I'rolii tile prc-gcr~i~in;~~e~l sccd1111gs (Ki~~liii;i~~~~~rll~y<br />
CI ill.<br />
~iiiiiiber ~t'Iic~~~~le~ic~l<br />
I>~;IIII\. 'l'lic co~ii~ircIie~~\~\ c I)IOIO~OIi)i l~;i~.clc~i~~ig ,IIII,IIIIS ivctc l;ick or<br />
gro\+~h of roo ti^^^ ~~,ICIII<br />
t~r~cl 1i~1111icli1y ;I~~~III~,III/;III~~II (~IIIE 10 111c cllic~c~~cy ~~f\I;itic<br />
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<br />
nicthod for hardcn~np. I'lii. ollicr 111c1Iiud ui II;I~L~SIIIII~ ,111d II;III\~)~~I~~I;I~IOII III~OLI~II tlircc<br />
different stages \baa al,u<br />
cific~~~it ~ n rc11;lblc. d Ihu\rci'cr, \ ii~~pl~c~lj, ~i~ti. ;III~ III 1ot;iI tllc<br />
efficiency of Ilydropoilic\ a)slenii\ si~pcr~(~t In ;I~~I~IUII. Ilic 3-bI;igc n~ctliod cil>plu~s<br />
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\<br />
vey case sensiri\e E~cn iiliiiur \;~r~:il~u~i\ ul' Il~~iii~dily III:I~;I~~I~CIII<br />
re\~lltcd 111 clllicr
wilting or drylng of tl~e pliints. This prublci~i \L;I\<br />
11eg;itct1 III tile I~>drul>oi~~cs syslcn~.<br />
However, a carckil cua~irinatioi~ US gro\\tli u1'1Iic \tc111,illti loor ~!~I~III. Ic;iS ~l~ospl~olugy<br />
should be dul~r ~hllc hordei~ing IIIC sooleil ~~I.IIII\ III at.itic II!~I.UPUIIIC~ I'uttii~g IIIC~IIIIII<br />
was one ot' tlic Inlportellll l;ictt11.\ 111;il<br />
,~l'Scct ?IIIc~\~~c;I II;I~~~CIIIII~ ,II:J<br />
~\I.~~~I\~IIIICIII.<br />
Chlckpca ge~lurdll) prckrs bl,ick soil liu\tc\cr. gio\\lli 01' S~ILII r!\Iclll \\.I, g~c,i~ly<br />
111hib1ttd by tll~s pou~~lg IIIC~ILIIII 111 :111 ilic tli~cc SI;I~CS tIc\crlbcd 111 IIIC I~l>u\~ WCIIOII\<br />
Sand \\as used :it Ihiglic~ ps~~~~itiui~s IU<br />
I)SU\ iilc ~CIICI JCI;I~IOII<br />
ii~~d ~~I;III\cI) lcs. llllitlllll~<br />
of bi;~ck soil \\as ;~ddctl lu I~~,IIIII;III~ ~IIC \\.I~cI 11ultli1ig C,II);ICI~~ 01I1cr I;ICIOS~ l~kc<br />
temperature, liglit 111te11bity. pIioto/)1'11t1tl, IIII~;I~IUII :illil \\CIC<br />
cilw \t,~i~tI~i~tli/~d OI)~IIII;I/<br />
green huusc or LIY<br />
~tl~er li;iraI~ ~IIYI~UIIIII~III C,III OC ~CIIIII~CIII;I~ tu IIIC ILI~C~CIIIII~ 01'<br />
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'<br />
walk-ln type gro\vtli cIi;~i~~bsr, ~VIIICII 111;1l,c\ II,I~~~CIIIII~ III~ICC\<br />
\OIIIC~~.~I,I~ I.CSIIICIC(/ 10<br />
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<br />
that can \harden 111 LIIIO<br />
prod~i~td ~IIIc~~)c;I ~~I,IIII\ 111 illc inori~~,iI CIIIILI~C 1.00111 CUI~~I~I~II\<br />
without conil)sol~~i/iiig tile 111tqr11y ,iil~l v~l,~li~j. 01' IIIC pl:iill. 111 ~ilro ~ICIILC~ cl~ickl~ei~<br />
pla~~ts sl>o!\ed :ill cii~ly tlt~\~crii~g ;iiltl II~:IIIISII~ 111 \OI~IC III\~;IIIC,C\<br />
IIIC ~I;IIII> Ilo~vcrcd 111<br />
culture tubes while they \rere rootliig. 1111s siiggc\l\ III,II il~iltl cu~~tl~tioii~ of lcnlpclalurc,<br />
light intensity iind l~~iil~icl~ty ~>r~vidctl 11) tlic CIIIILII~ roo111 111ig11t ci~tii.it~ ~C~II~CLIVL:<br />
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<br />
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'<br />
pron~otion of veget~~t~vc gru\vtl ~:IIIIS<br />
~I~IIII~C~III~C<br />
\\IIIIc I~~~IIIII;I~IIIII~ :IIILI 1proccss111g of<br />
l~ivaluable transgenic pli~~lts.<br />
5.1.3 Histological slutlica ol'i~~ultiplc slioul tlr\rlol)i~iel~t I'I.UIII .\A14 c\pl:~l~t:<br />
L)c\cluj>~i~c~~t of ~n~~lt~plc ~Iluurh i1t1111 :\\I4 C\~I~,IIII \\:I,<br />
iti111~1 I,I I~c ;IC~\CIIIIIILI~I\<br />
[~Ilcllulyl~~;~ll! I-~o\\c\cs. IIICII.<br />
:I~\CIIII~IU~I> 11~11111.~ !\.I\ LOII~~III~C~ .II'ICI IIIC I1i~tolt1~1c;il<br />
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<br />
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<br />
Stiipe ufrt1110\~1l 111'111~1lll~1lc JIIUOI buli UII~III~I~III~ fio111 ~C~CIICI.;I~III~ .I~C:I<br />
I'~C\CIICC<br />
01'<br />
ax11l;lry bud c\crlh I)IC\\LI~C<br />
01' ,II)IC;I~ ~UII~III~IIICC OII IC II~\LIC 01' CIII)ICLI~III;I~~ IIO~C<br />
regloll. Ile~icc. ;IS<br />
c\l~cctcd I~CIII~IV;I~ 01';1\1ll;iry hut1 IIC~.IIC\ t11c ;I~)IC;I~ ~~IIIIIII,IIICC :IC/<br />
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<br />
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<br />
~OLIS~ COLII~ giic bcttcr 1icqt1c11cy ~~'~CIICIIL, ~I;III~~~IIII;I~I~II~<br />
5.2 Genetic tra~~sl'urnlatioa:<br />
TransSirni~ng ~IC ALll c\l)lallt\<br />
1t11 111c b111;iry co~i\lri~ct\ contalnlllg Ili('~:ylAb<br />
and SBTI genes, p~uduced cll~ckpca Irdll>gvlllcs. 111 ;lccurdancc IVIIII<br />
lllc convci~l~onal
molecul~ir analysis of tlic I'III~~~I~IC<br />
~I,!III~, 111c lpIa111s \vcrc 111111;illy scrcc~icd for GUS<br />
histochemical assay and tiboul 6U"u<br />
oi tlie ~pl~i~ilr LIIO\ICJ<br />
PU~III\C ~e;iitii)ii tlii/i\ gelit<br />
act~vity has clearly demo~~sti.atetl it! v;iactil~r tishlic, l'llc 1cr1111n;11 !o!111g lc;~~lcts hiit,\vcd<br />
a clear blue color ;lctl\ ity oi'tlic i~icurpol.;i~ed III,/:\<br />
gcilc Ilu\\c\cr. tile 1.11cr >lager ol'llie<br />
n~olccul~ir ~II~:II)SIS hlio\!cd ~OI~IC \a!r~,~blc ~cholt\ III ICYIII\<br />
co~itir~ii;il~on iiiid Soulliein ;~il,ilyaih.<br />
,)I'IIIII~~I)L~V<br />
OI'~~),III\~~ 111 Itc'l<<br />
111col.pol.nied gcllc 111\;ir1;1bly cu~ilir~llb tli~ I~YCICIICC 01'ilie IC\I)C~II\C gelic III IC ge11v111e<br />
ui llie lraiisgeilic plalil. Ho\vc\s~, tluc lo suliic \,irl;niulia<br />
1111l\iio\\11 tllc l)i'l< rc\~~Its<br />
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<br />
gene sllowcd n 50% ~l.;in~lbr~~i;iriw ilctlucncy \\1111 ~c\l,cct lo llic ~ipill kclie In lllc I'CI<<br />
conlir~ii:il~o~i ;ili;ilyals<br />
I:lic )11;1111\ 0111 01' 10 >IIO\VC~ ;IIII~I~I~~C;III~III UI'IIIC 700 bl) Ii~;ig11ic111<br />
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<br />
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<br />
IY01ii tile 111i1.k S~II~, 111erc \\.IS<br />
\l~gliIly IIICYC;I~~(I lrct~~~c~~cy<br />
X 0111 01 I0 /~il'r~'lA/~ lpl:1111s<br />
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<br />
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<br />
ni~ght be<br />
inlerfcr~ii~ \VI~II 11ic ii~i~~)l~Iic;~t~tl~~,<br />
llc~)cc, tieqt~ci~c! ul co~ilirr~ied<br />
transfor~nat~o~~ trltli respccl IJ ilic 11ii11 kcr ge~lcb e;111 Ibc fixed III 1111. ra~lgc 01'50 10 70% 01'<br />
the piitatlve tra~isfor~n;~n~.r. ~~tuc11 lo~er lictluclicy ui tr;~~isiirrii~;~l~u~i was ol)servud w~tli<br />
respect lo llle sgronumicall!,<br />
II~I~IO~~~III~ ;III~ i113ec1 ~~>I~I;II~CC gc11l.i U/( rj,lilb and SBTI.<br />
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<br />
01'1hc<br />
primer and rhc gciie could be tlic sc;~son for 1111s luircr li.eijucnc) cuinp;ised to 1l1c 111;isker<br />
genes. 01ily oiir snii~ple isoin the plaiila tl-oiislorii~c~l \i 1111 SHI'I pcili, uut 01'9 ~ca[cd pl;iiiir<br />
showed the a~iiplitic~~r~o~~<br />
01' rcil)cc~~~c 497 bl) ti..iy~iclit >iiggc>~i~ig SOIIIC<br />
II~CIIII~~CIII<br />
problcml \\ 1111 tlic I)~IIIICS<br />
c~i111110\1t1011<br />
Suuthesii an;lly~ls li;i\ bcc~~ IIIC 111ust ;i~ilI~c~nii. I~L~~IIIIL~IIS IU c0iili1111 1111' I)~II;III\C<br />
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\<br />
\\iidcly uaed to ii~i.~l>/e ill? IIICU~I)I)I,III~~II ,iiiti cop) II~IIII~CI<br />
ul' tllc IIIIC~I~IIC~I gctii' 111 tlic<br />
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<br />
\vcrc iiscd to probe tlic iiitcgs~i~c~l gc11c III llic ISCIII~~~III~ 1pl,111t\ C'111cklic;i \\:I> IL~LIIICI 111 hi.<br />
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~<br />
rcporlcd to d;itc II;I~ bcci~<br />
lo\\ ;I\ ;iiuu~itl?'$,I (K,Is ct ,II . 1007: KSI~~III,III~~ISI~I~ ci ill..<br />
2000). Uiol~a~ic-iiiciIi.~~c~I IS,II~~~'~IIII,I~ILIII \\;I\ LIU~IC III~U /Y~OIIC CIIII~S)~LI$ ,111tl IIIC II~II~~ICI~<br />
negnting tl~e ap1c;il tluniiii;iiicc ul' 5u111c \I~CILII OLI~. regciicl;i~ii~g ii~ni;illy, g;ivc IIIIIC~I<br />
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<br />
~s~nrl'or~iiaiita~ \\a> tc~~cd ii~ ~~~euipor;~teil ~CIIC,<br />
:III~:IIO!III~ 7iJ''0 01'<br />
slio\rcd iiicorporarioii of 1111. IS;II~,~~II~ III iliclr gimiio~~ic~ IIIIII,II<br />
11ic ~c>tc~l pIzi111h<br />
CY~)CIIIIICII~~ i\lic~i the<br />
cotid~t~oiis for upt~n~al re,trictiuii of111c ycriuiriic [IN,\ ivcic 11o1 \t;~~idurd~~utl, shuwcd tlic<br />
sign" Ifor [he trallagencs ;n a no~i-apccilic icg~u~):, ul'llic blots and >l;~litia~d~/nt~oli of Ihc<br />
condi~io~is ,~icli ;IS<br />
qu;iiilirles of cn/y~ilc, i3S.4 .iiitl \r;i(cr \$,I\ tloi~<br />
oild 111c ;iclual sc.;ulls<br />
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<br />
CB2, CB3, CB7 and CBI I slio~ed tao copies each and sa~nples CB4 ,ind CB5 showed<br />
single copy Irisertiolis W~th respect to Ut('r~,lAh gene i~itegr;ition tile ge~~o~ii~c DNA of<br />
putatice transfor~ilants \\;is d~gcsted \r.itli E
Explant Preparation<br />
-Geminate seeds on SILI [MSG p? TDZt? pM kinet~n] for I wk<br />
-Prepare AME explant by removlng ax~ll:iry bud from Ihc cotyledonary notlc<br />
Shoot bud'intluction<br />
Step I: Culture AME explant on Slkl medium for 2 wk<br />
Step 2: Sub-culture explants with shoots buds to MS basal medium for 5 d<br />
Shoot elongation<br />
Step I: Transfer shoots to SEMl [MS+S pM 2-iP+2 pM kinetin] for 10 d<br />
Step 2: Transfer unelongated shoots to SEM2 [MS+2 VM GA,] for 2 to 3 wk<br />
Rooting of shoots<br />
Phase I: Transfer shoots toliquid RIM [ MS+9.4 mM KN0,+2% sucrose+5<br />
@I IBA] on filter paper bridge for 2 wk [60-70% shoots rooted]<br />
Phase 2: Pulse treatment of shoots with 100 @I IBA and culture on filter<br />
paper bridge in liquid MS for 2 wk [lo-20% shoots rooted]<br />
Phase 3: Transfer unrooted shoots to static hydroponic system containing 114<br />
Arnon's nutrient solution+3 ,&I 1BA for 2-3 wk [lo-15% shoots rooted]<br />
Transplantation<br />
Step 1:Transfer rooted shoots to 8 cm (dia) pots containing 2-4 mm sand. Cover<br />
the plants with polypropylene bags and gridually open the covers over 7-10 d<br />
period.<br />
OR<br />
Suspend the rooted shoots in Magenta jars containing 114 Amon's nutrient<br />
solution.<br />
Step 2: Transfer the hardened plants to 20 cm (dia) pots containing sand:black soil<br />
(3:2)+Cell Rich (5%) +rice straw compost (5%).<br />
Schematic representation of the protocol for in vitro regeneration of whole<br />
plants from axillary meristem explant (AME) of chickpea [Jayanand &<br />
Sham, 20031
Explant Preparation<br />
-Germmate seeds on S141 [MS& llh.1 TI)Z+2 pM kinetin] ~OI- I wk<br />
-Prepare AME explant by removing nxill;~ry bud from thc cotylcdo~i;~~.y ~iode<br />
Shoot bud intluction<br />
Step I: Culture AME e\plant on Slbl nictl~uni for 2 wk<br />
Step 2: Sub-culture explants with shoots huds to MS basal ~ncdiuni for 5 d<br />
Shoot elongation<br />
Step I: Transfer shoots to SEMI [MS+5 pM 2-iP+2 pM kinetin] for 10 d<br />
Step 2: Transfer unelongated shoots to SEM2 [MS+2 pM GA,] for 2 to 3 wk<br />
Rooting of shoots<br />
Phase 1: Transfer shoots toliquid RIM [ MS+9.4 mM KN0,+2% sucrose+S<br />
pM IBA] on filter paper bridge for 2 wk 160-70% shoots rooted]<br />
Phase 2: Pulse treatment of shoots with 100 pM IBA and culture on filter<br />
paper bridge in liquid MS for 2 wk [lo-20% shoots rooted]<br />
Phase 3: Transfer unrooted shoots to static hydroponic system containing 114<br />
Arnon's nutrient solutiont3 pM IBA for 2-3 wk [lo-15% shoots rooted]<br />
Transplantation<br />
Step 1:Transfer rooted shoots to 8 cm (din) pots containing 2-4 mm sand. Cover<br />
the plants with polypropylene bags and gradually open the covers over 7-10 d<br />
period.<br />
OR<br />
Suspend the rooted shoots in Magenta jars containing 114 Amon's nutrient<br />
solution.<br />
Step 2: Tmnsfer the hardened plants to 20 cm (dia) pots containing sand:black so11<br />
(3:2)+Cell Rich (5%) +rice straw conipost (5%).<br />
Schematic representation of the protocol for in vitro regeneration of whole<br />
plants from axillary meristem explant (AME) of chickpea [Jayanand &<br />
Sham, 20031
6.0 REFERENCES<br />
Adkins, A.L.. Godwin, I.D., Adkins, S.W., (1995) All efictent regeneration system for<br />
Australian grown chickpea. (Cicer arielirrr~~~i) cultivars. A~islr(rlia~r Jo~irnal 01<br />
Boiany, 43:49 1 - 497.<br />
Akasaka, Y., Daimon, H., Mii, M., (2000) l~iiproved plant regeneration fioni culturcd<br />
leaf segments in peanut (Arocl~is irypogcirci L.) by limited exposure to tlltdinzuron.<br />
Plunl Sciellcr. 156: 169- 175.<br />
Akashi, R.. Kawamuro, O., Hoffi~iall~l, F., (1998) Tlic advance of transfur~i~alloii ill<br />
Lotus conliculalus: to\vards low-lig~liti pns1Lii.e tllrougli ailtiscnsr I1NA. Utilization<br />
of transgenic plant aiid getlonie analysis in forage crops. Proceedi~lgs of<br />
ci~i<br />
b~ierriulio~~al u~orksliop Arid 01 lire "Vuliu~~crl C;r.ussltri~d Keseu~.ck I~rsriru~e,<br />
Nisiibrus~i~~o, Tocl~igi, Jripulr, 57-65.<br />
Ali, A.H.N., Jarvis, U.C.. (1988) Eff'ects of 2,3,5-triiodube~~zuic acid on tllc<br />
regeneration of callus and adve~ltttious roots on sten1 cuttitlgs of iiiu~ig bean,<br />
Phaseoilis alrreris Roxb. Biuche~~r Plrysiol PfIatiz 183:509-5 13.<br />
Alm, S.R., Yeh, T., Canipo, M.L., Dawson, C.G., Jenkins, E.B., Simeoni, A.E., (1994)<br />
Modified Trap Designs and He~ghts for lncreascd Capture of Japanese Beetlc<br />
Adults (Coleoptera: Scarabacidae), Jourriui oj"Ecor~o~~iic E~iiorrluloyv, 87: 775-780<br />
Altieri, M.A., (1994) Biodiversi~y aad Pe~t Ma~~rrge~neni in Ayroecosyste~ris. hod<br />
Products Press, Newyork, pp. 185<br />
Altieri, M.A., Nicholls, C.I., (1999) Uiodiverslty, Ecosystem function, and pest<br />
management in agricultural systems. In: Biodiversity in Agroecosy.sle~trs. CKC<br />
Press, Boca Haran, 69-84.
Altieri, M.A., Whitcomb., (1980) Weed n~anipulation for insect pest n~nnagen~cnt III<br />
corn, Environt~~et~mi Mrinnget~iett/ 4: 483-489.<br />
Al-yahyaee, S.A.S., Ellar, D.J.. (1995) Maximal tonicity of clo~ied CytA S-endotoxill<br />
froill Uricillirs rlirrri~iyioiesi~ subsp. i.srricle~isis requires proteulytic processi~~g lioll~<br />
both thc N- and C- tcrnllnl. ,Vficrobiologl~, 14 1 : 3 14 1-3 148.<br />
Angeloni, P.N., Rey. H.Y., Mroginsk~, L.A., (1992) Regenernti011 of plants fron~ callus<br />
t~sauc ul'll~c p;~st~~sc ICSLIIII~<br />
521.<br />
('i~~il~~o.~orirr 61rr.~ilitr1i11r1i, l'l~r~i~ C'i,il I{t,/)ilo~./.>, I 1. 5 1')-<br />
Al~jun~an:il.a, K., Il:~dillzz:~lnan, S., Jallat~, hl.A.tZ., Hossnin, M.T., Khatull, A,,<br />
Hadiuzzaman, S., (1998) In vitro direct and indirect ~ui~ltiple shoot regeneration ill<br />
PsopIioccr,piir t~.irngo~ioloDrrs (L). DC, wil~ged bean. Bri~~gla(lesli Joirrnul uj<br />
Scietlrijic (inti Itld~isrriai Research, 33: 449-453.<br />
Arcioni, S., Mariotti, D., Pezzotti. M., (1985) Hedy~rir~r~~~ corotiuriuni L. in vitro<br />
conditions for plant scgcr~eration fr0111 pro~oplasts :III~ callus of vi~rious explnnts.<br />
Joirrnol oj'Platii Pii~rioiog~, 12 1: 141 - 148.<br />
Armitage, P., {L'i~lden, R., Draper, J., (1988) Vcctors for transformation ot' plant cclls<br />
using dgrob~irirriirt~r. In: Plutri Gelicric Triinsjbrt~rn!iott (ittd Gore Erpresvioti<br />
(Orupel., J., Scoii, II , An~riirige, P. Eds.) Bliick~vell Sci. PirD., O?rJurd, pp. 1-67.<br />
Arnon, D.I., (1938) Microelements in culture sulutioli experiment with higher plants.<br />
A~iiericat~ Jourtiul ofBotniiy, 25:322 - 325.<br />
Arundhati, A,, Agrobocreriirnl-mediated trunsfurnlation of pigeo~lpea (Ciljrmus ccljun L.<br />
-.<br />
Millsp.) by using luaf d~sks. Inierticitio~~ai Ciiickpcu rind Pigeo~~pcu New.slcller.<br />
(1999) 6: 62-64
Avenido, R.A., Hattori, I
Proceedings of rhr Natiorlul Acaderrry oj'Scieilces Urliled S/n/es of' America, 94:<br />
12722-12727.<br />
Bajaj, Y.P.S., (1983) Regclieratioli of plallts from pollen-embryos of Arachis. Brassica<br />
and Trilicoiu sp. cryoprcscrved for one year, Ci~rrorr Scie~ice. 52: 184-386.<br />
Barloy, F., Lccader, M.M., Delecluse, A., (1998) Distribution of clostr~dlal cry-l~ke<br />
genes among Bucillus liiuri~igietrrsis and C'losrridiiitr~ strains, Currrrrr<br />
Microbioiogj~, 36: 232.237.<br />
Banla, K.S., Mehta, S.L., (1995) Gcnctic tra~lsibrn~ation and somatic cmbryogcncsis in<br />
,,$&a,<br />
K. S, W~klil~, A. I., (1993) Somatic en~bryogencsis and plant regeneratio11<br />
. -.<br />
-<br />
froni callus cultusci ~!'chickpea (Cice~, cir~clirirrir~ L.) Plntlr Cell Reporls, 12: 521 -<br />
. - . .-.<br />
krna, K. S., Wakhlu, A. K., (1994) Wl~ole plant regeneration from Cicer orie~~tiiur~<br />
-<br />
from callus cultures via organogenesis, Pluiir Cell Reporis, 13: 5 13 - 5 13.<br />
_r.-~<br />
- -.<br />
Becker, D.. Kaniper, E., Schell, J., Masterson, I
Benfey, P.N., Chua, N.1-I., Ilcgiiiiltcd gcllcs 111 trn~isgcnic pl;lllts. (1989) Scie~ice, 244:<br />
174-181.<br />
Bevan. M., (1984) B~liary Agroborreriir~~r vectors tbr plallt t~l~lslbr~ii;ltio~is. ,Vii(~/~,i~<br />
Acids Reseuxh 12: 871 1-8721.<br />
Bhadra, S.K., Hammatt, N., Powcr, J.B., Davey, M.R., (1994) A rcproduciblc<br />
procedure for plant regcilrration from sredi~ng llypocotyl protopl;~sts of Vig~rri<br />
subiubaio L. Pitriir Cell l
Biron, D., Vincent, C., Giroux, M.. Maire, A.. (1996) Let1i;il effects of mlcrowavu<br />
exposure on cggs and pupae of the cabbage nieggot atid cabbage plants, Jo~iriitrl o/<br />
Microit five POII'E~ (ii~d Eleclro iriagrrrrrc Eirergy. 31 : 228-237.<br />
Bovo, O.A.. Mroginski. L.A., Rey, H.Y., (1986) Rcgencration ut' plants iron1 callus<br />
tissue of llie pasture legunle Loloiior~is bairiesii, Plorrr Cell Repro, 5: 295-297.<br />
Brar. M.S., Moore, M.J.. Al Klinyri, J.M., Morelock, T.E.. Alidcrson, E.J., (1999)<br />
Ethylene ~nhtbltors protnote in vttro regeneratio11 of cowpea (Vig~zo trr~giricrrloiu L.).<br />
In Vilro Ccllirlar atrcl Urvelopirlrri!nl Biology -Plor~r, 35: 222-225.<br />
Briggs, B.A.. McCulloch, S.M., Edick. L.A., (1988) Micropropngation ofa~alc.as using<br />
thidiazurot~, Acrcr Horriculrzrrae 226: 205 - 208.<br />
Brown, A.W.A., (1978) Ecolog~ oj'Pcslicities IVile)~ uiitl SUIIS. Neiv York, Ci~ichestrr,<br />
Bris6011e. Torcinro, pp. 525.<br />
Butko, P..<br />
Haung, I:., Pusztai-Carey, M.. Surewicz, W.K., (1996) Merilbralle<br />
portncab~lizat~w 111duccd by cylolylic 6-cndotoxt~i CytA fi'otli I~'tit~i1l11.s<br />
rhtiringic.irsis var, isi'aeloisis. Uiocheiriisrry, 35: 11355-1 1360.<br />
Callis, I., Fro~iim, M., Wnlbut, V., (1987) Itltron increase gene expresston In culturcd<br />
maize cells. Ge~res aiitl Developrtieilr, I: 1 183- 1200.<br />
Cambecedes, J., Duron, J., Decouriye, L., (1991) Adve~ltittoi~s bud regeneration fioni<br />
leaf explatlrs of the sllrubby ornamental hoticysuckle, Lolricerc~ azirdi~ Wtls. Cv<br />
Maigrun: elTect of th~dlazuron and 2,4,5-tr~~odobenzoic acid, Pltrnr Cell IIeporls,<br />
10: 471-474.<br />
(hrson, R., (1962) Silriii Spri~ig, Hamish Hamilton, London, pp. 304.
Cnrvalho. IC1.H C dc , Uui Vat1 LC . Zully I:odil, Y., 'flii, A.T.I'.. Klstn Tran TIi;1111i<br />
Van., dr Carvallio, M.H.C., (2000) ESfic~rnt wliolr pl;lnt rcgenrr.~tiotl of co~iiilioti<br />
be;~n (Pl~nsroiirs viiigiiri~ L.) us111g t1i111-cell-l;iyer cultu~~c and silbcr iiitr,itc, /'/~III/<br />
Scieiitr. 159. 223.232.<br />
Chalfic, M.. Tu. Y.. Euskircheii, (2.. Wood, W.1\'.,<br />
l'raslicr. DC.. (1094) (irceii<br />
tluoresceilt protc111 ;is :I nixkcr I'or ~SIIC c~~)rcs.\ioll. .SL.IL,II(.C~, 203. XU?-SUS<br />
Clia11dri1, I
Chilton, M.D.. Drummon, M.H., Merlo. D.J., Scic~ky, D. Montoyn. A.L.. Gordoli, kl.P.,<br />
Nester, E.W., (1977) Stable 1licorpor;ltloli of plas~iild DNA lnto Iiigllcr plnlit cells:<br />
tlie ~nolecular basts ofcrua,li g:111 tl~~iior~gc~icsis. C'L,//. I 1. 263-271.<br />
Cliilton, M.D. Molltoyo. A.L.. Mc~lu. D J.. Dru~il~iiond, hl.tl.. Nuttcr. I
Chu, P.W.G., Andersoli. B.J., Khan. M.R.I.. Sliukla, D., Higglils. T.J.V., (1999)<br />
Produclio~i ol' bra11 yellow mosn~c virus resistuot subtcrr~lieali clover (7i.,joliur1i<br />
slrb/err(rrtrlori) plaiits by tr;1nst'ormatlon will1 tllc virus co;~t protell1 gclic, ,l~i~rtils o/<br />
App11etI Broluy)', 135: -169-480,<br />
Cuckllig. E.C.. (1060) A iiietllod for tlic ~solatlon ui' pl;llit ~)rutupli~sts ;iiid Y;ISLIUIUS.<br />
:VNIIO~L,. 187: 962-763.<br />
Culllns, W M'.. Qu;~lsct, C.0.. (1999) Urodiir'~:\rr, 111 .4grr)c~~u.~y.sr.r~1r1.s, C'I(('p,u.\,\, Hl~(,ti<br />
I(cirori, pp. 334.<br />
Cook, K.J., Ur~~ckliiirt. \V.L.. COIIISOII,<br />
J.l/ii~igiorr,<br />
D. C.<br />
Crameri. C L.. Weisseiiborii. D L., 01sli1, K K.. tirab,lu, E A., Hennet, S., I'once, E..<br />
Grabowski, G.A., Radin, D.N., (1996) B~oproductiu~l of hunlan enzymcs ill<br />
transgenic tobacco. Anrrals of h'ew Yurk Acudertiy oJ .Tcie~rcr, 792: 62-71.<br />
Cr~ckmore, N., Bone, E.J., Williams, J.A., Ellat., D.J., (1995) Contributioii ol' the<br />
individual cornpolicntr of the 6 - endolox~n crystal to the inosqu~toc~dal act~vity of
Bucrllirs rhrrrmgre~iesis subsp. Isrcrelouis. FEMS Alicrobiology Lerrers, 131 : 249-<br />
254.<br />
Dur, S.M., Gill, S.S., (1993) In wro arid in vivo pro~culysis of 111s Hucillrrs<br />
tirirri~rgruirsis subsp. rsioelr~rs~s CrylVD protcirl by Crrler q~ri~rclrrejas~~~uritr la1-v;rl<br />
midgut prute;lscs. 11r~eci Uiucl~e~~ris/~:~~<br />
cr~itl hlolecrrirrr Hiologv. 23: 273-283.<br />
Dam~a~l~, F.. yen^. E., Pnolocci, F., Arc~olli, S., (1993) I~ltroduct~ori of Ihyproniyc~n<br />
reslstallce In Lor~rs spp. tlirougli /Igrohric/enir~rr ~II~~O~L'IIPA tra~~b~i)rn~:~tiw.<br />
li.rr~rsgt~~iic l~escrrrc~lr, 2: 330-335.<br />
Das, D.K , I'rnk:~sli, N.S , Blinlla S;~rin, N., (1998) An eflicrent ~cgcneratio~i systcill ol<br />
black graiii (YI~IIU II!UII~U L.) througli orga~iugcncsis, I'l~r~rr Sr.ie~rc?, 133: 109206.<br />
Datra, S.K., Petrrlians, A,, D;ltlu, K., I'otrykus, I., (1990) Gcnetic;~lly cng~~lccrcd lirt~lc<br />
~nd~ca-rice recovered lio111 protoplasts. Uiu~/i~cl~~ioiu~~~<br />
8: 010-029<br />
L);~\ey, bl,l
Deblaere, R., Reyneerts, A., Hotte, H., Hernaistce~~s. J.I'., Leerii;lns, J., Van Montagu,<br />
M., (1987) Vectors for cloni~ig in pla~il cells, Me/hu~is ill EII:~'IIIO/O~),. 153: 277-<br />
293.<br />
Dcbnatb, S.C.. McKenzie. D.B., McKac, K.B.. (2001) Callus itlductio~i slid sl~oo[<br />
rcgenrr;irioti li.0111 stem, rncliis and leaf expla~its ill beach pc;~ (Liirii\8riis jiri,o~iic.ri,s<br />
W~lld). Juiii.11111 oj Pla~ir Lliociie~~ii.sr~:i. iriiii I~ioit~cir~rulug~~, 10: 57-60.<br />
Dcll;i~)orl~i. S L , L$'vod, J , Il~cks, 1.13 , (1083) A ~?I:IIII IINA I I I I I ~ I ~ ~ ~ c \Jc~h~oti<br />
~ ? ; I ~ ~ ~ ~ I ~ I ~<br />
11, Plniit hlolec,iilrir Biuiug)' /(t,poi./er. 1.19-2 I<br />
Dcsgagnes, R , Laberge, S.,<br />
Allard, G., Klioudi, II., C:istonguay, T., Lapointc, J.,<br />
M~chaud, R., Vezin;~, L.P., (1995) Genetic tra~ist'or~llation ofcom~nerciel brccd~~ig<br />
li~ics of ;IILII?,I(~hlc~iiictigu<br />
~iiiii'ii), 1'Iii11/ C't,ii, I?.\.YI~LJ ii11i1 0rgii11 C'i~iiii~~c,, 42: 120-<br />
140.<br />
Dctrez, C.. Ndiaye, S., Dreyfus, B., (1994) In vilro rcgc~icr;itio~i of tlic Irop~col<br />
niultipurposc Icgu~iiit~ous trec Se.sbo~~ici ti~~ci~itii/lurir li.u~il cotylcdun cxlllatits. PIOIII<br />
Cell Re/~ur/r. 14: 87-93,<br />
Ue Wet, J.l
Dillen, W., Clercq, J. de., Goossens, A.. Montagu, M, van.. Allgenoli. G., (1997a)<br />
Agrobucrerirr~~~-nlediated trallstbt.nlarlon<br />
of Plitrx~~ulus r~crrtijolirrr A. Gray.<br />
Tl~eorrrrc~~l Ap/111eii Ge~reiics, 94: 15 1-158<br />
Dillen. W.. Clcrcq, J dc., Goossens. A., Za~llbrc. M., Molltngu, M v;~n.. Angclloll. G..<br />
De Clercq, J., (1907b) Exploiting lllc presence of' ~cgcncr~llioll copaclty 111 tllc<br />
Pltiiseol~rs gene pool for ;IgruDtrc/r~~~i~r~~~-llled~;~~ed<br />
gcllc tr.~~istkr tu 111c ~0111111011<br />
bc;tll<br />
~~
Drun~mond, M.H., Gordoil. M.P., Nester, E.W.. Clliltou, M.D., (1977) Foreign DNA ol.<br />
bacterial plasnlid orlgln is traliscribed in crow11 gdl tunluurs, Narui,r, 269: 535-536.<br />
Drummond, M.H., (1979) Crown gall discasc, Noriirc. 281: 343.347,<br />
Du, S., Er~ckson, L., Bowlzy, S., (1994) Effect of plant gellotype oil tllr tlnnslbl.n~;~tluli<br />
of cultlvared alt'~lSa (j41eilicugo srrrrvo) by ,lgrobiicrciiiri~i riiriic~ocic~ii.~. I1liiirr ('e~ll<br />
Hepora, 13:330-333.<br />
Duell~, P., (1997) U~ud~versity ev;ilu;~llo~i ill Apr~cultulnl L;~~idac.~pcs: ,111 ;~p])~o,~i.li ;I[<br />
two d~ffercnt scales, Agricul~iirul Ecus)~s/c~i~i.> nrlei Eii~ iroi~iii~~~if, 62. 163- 167.<br />
Dutta Gupta, S., Ahnied, R., Dc, D.N., (1997) Direct su~i~;~tic cri~bryogcnca~a and<br />
plantlet regeneration<br />
fium seedling lcovcs of wlngcd bean, Psup/iucii,pi~s<br />
re/royoiiulubii~ (L.) Plo~lr Cell Reporo, 16: 628-63 1<br />
pien, S., George, L., (1994) So~ilat~c embryo~e~icsia in ('icei. (~rierriirriir L: I~~lii~c~~cc<br />
of gel~otypes L I I auxins, ~ Uiologiil l'liiirrcirioii, 36: 343 -. 349.<br />
Edwards, C.A., K~l;~cl\er, T.T., I'oknrzl~zvski~, A.A., Subler, S., I';~rli~clcc, I
Cella, R., Falavigna, A., (eds.) Clrrrc~~rr P1ir11r Scinlcc cr~rli Biotech~rolugy ill<br />
Agriculrure. Klu~r'u Acode~~ric Publrsliers; Durilrrchr; Nrrherlu~itls, pp. 22.<br />
Ezura, H., Nukul, N., Yuhashi, K.I.. Mili;lmisawa, K., (2000) In vitro pln~it rcgci1er;lrion<br />
ir~ htucruprili~r~ri cr~ru~~irrpureiii~r. a leguilli. 1vit11 ;I broad sy~i~bioi~t ~111gc fir<br />
nodularlon, Plorii Scie~ice, 159: 21-27.<br />
Fnsolo, F.. Ziil~mcrm:~~~. R.lI., Fordli:ii~~. I.. (19SC)) Ad\ci~t~l~our hlioot ~~I~I~I~IIIUII 1111<br />
escissd Icnves of 111 villi gio\\ii sliooth o i ,~pple culti\;~ia, 1'1ii111 ('cll. Ii\\ric ri11i1<br />
Orguri Cirlritre, 16, 75-87.<br />
Federici, B.A., Luthy, P., Ibcrra, J.E., (1990) I'.il.aspornl body of lloc.illir.\ /liirrr~i,yit'~ii~.\r\<br />
isrnrlaisis. structure, proleln coinposition ;~iid loxicity, 111: U~rcl~'ri(~I ('01io.01 0/<br />
U~iiversity Press, New Bruilswick, NJ, pp. 16-44.<br />
Feldmanil, F.. Dullemaiis, A., Wanlwijk, C., (1905) Bi~~diilg of tlic CrylVD toxi11 of<br />
Applretl o~ici B~viro~~~~ie~ircrl Micruhrologv, 0 I. 260 I -2605.<br />
Flores. H., Soberori, X , S;lncliez, J., Bravo, A,, (1997) Isul;~tctl doliia~ii ll and I11 fioni<br />
Uocilllts rIluri~ryie~~e.sis CrylAb dclt;~-cndotoxiii brllda to Icpldoptcran midgut<br />
membranes, FEBS Latcrs, 4 14: 3 13-3 18.<br />
Food and Agr~cnlturc Organieat~on, (FAO), (200 I ) ~ l~~ir~~ol Nc,porr<br />
Food atid Agriculture Org;lnlzation, (FAO), (2002) Fuud Oirrlook, No 1<br />
Foiltena, G.S., Santlnl, L., Caretto, S, Frugis, G., Muriotti, D., (1995) Gcne~ic<br />
... - --<br />
/--<br />
legunle Cicer urieriiiu~n L. Plurir Scieni.6, 109: 207 -
Foster, J.G., Coulombe, B.A., Van Scoyoc, S.W., Veilleux, R.E., (1991) Intercrossing<br />
methods, mutagenesis treatments, and tissue culture techlliilues for expa~~dirig tlle<br />
gcnettc dlverslty of tlatpca (Luiir).riis syh,t,srrrs L.), I)L~I,L,/~/~III'III~I/ P~~IIII Soil<br />
Sciorce, 45: 1047- 1055.<br />
Francis, C.A., (1990) Sristcii~iable cigriciiltirri' i~i rrniperiirs :u~res Jol~li \Vtlcy& Solla,<br />
Nc\v York, pp. 187.<br />
Frat~kel, I
Gavidia, I., Zaragoza, C., Segura, I., Bernludez, P., (1997) Plant regenerat1011 from<br />
juvenile and adult A~irl~yllis cyrrsoidrs, a niultipurpose lcgu~~~i~ious shrub, Jouriiirl oj'<br />
Plont Pl~ysiolo$~. 150: 7 14-7 18.<br />
Gaz~t, E., Shai, Y., (1995) The asseiiibly and org,lnl/.;itiun of tllc u5 and u7 licliccs<br />
from the forfor~nilig domoin of Bociiirrs ririor~iyi~~isis 8-c1idotox111. Jo~iri~ol "1<br />
Bioiogi~oi C~IL~II~IAII:~, 270: 2571 -2578<br />
Geerts. P., Mergsn~. G . Buudoin. J.P., (IYi)O)<br />
llcscue of c;~rly 11c;irl-sli;~l>ed c~iibryoz<br />
and plant regciieratiun of IJii~.\coi~i> po!~a~rriitr.s Grccnin. :ind I'i~i~seol~r.~ \'~rlgiri.i,s L<br />
BASE. -B~urecl~~rologir, A~~UIIUIIIIL,, Suciece ($1 LIIVI~~IIII~I~L.,~~,<br />
3, I4 I - I48<br />
Geetha, N., Venkatacllalani, I)., Prdkasli, V., S~la, 6.L.. (1008) ll~gli li.cil~lelicy<br />
duction ion of ~ilultiplc slioots ;~nd 11l;lnt rege~lur;~tio~i iio~ii sccdlillg cnpl.~~its of'<br />
p~gcu~ipca (C'oj~iitir~ ccijir~i L.), C'ri~~rciir .St i~~~ict*, 75' 1036- IO4 l<br />
Gelvl~i, S.U.. Gordon. M I)., Nester, E.\\'. A~UII~OII, rZ. !I.. (1081) '1'1.:11ibcrij)t1011 01'<br />
.,~~I~U/JIIccl Mu~iugeilirr~lJor Food Sloruge<br />
& Processr~tg. American Assoclallon ofCcre;il Chemists, St. Paul, MN, pp. 89-108.
Gill, R., Saxstla, I'.K., (1992) Direct somatic rlltbryogencsis and regcileratloll of' plants<br />
from seedling expiants of peanut<br />
(Arr~cllL Il~pogtrcc~): Iprulllotlve rule ol'<br />
th~dla~uro~i, C'o~~utiicirr Jo1rr11~1 oj'tlo~ory., 70: 1 186- 1 192.<br />
GIII, S.S., Cowels, E.A., I1letrantotlio. P.V.. (1902) Tile mode of out~o~l ul' Briiiliir~<br />
tl~rrririgieriesis elldotoxnls, A~rrl~rcil i(r~~ie,i' of L~~currruIog~~. 37: 615-6.16.<br />
Golds, T.J , Lee, J,Y., Glluse, T,K., D;~\,cy, Ll.ltcl;~<br />
CLI~CU~IOIII~:I~), .,l~i~rtri~ oJ'il~~l~/~e~l fliulo~', 13 1 : 133- 139.<br />
Grce~li., LA., Z;l~nbry,k~, I'.C., (1993) Agrob;~ctcrln Illatu 111 up111c tlc~is, ('rrrrc~ri<br />
Biulog~ 33. 507-509.<br />
Ciriga, >I., SIq,L;il, J.. Bcbcl-, K , C,~baclla, rl.C., (1Y1J5) Analybla of t~ssuc cultu~c-<br />
dcrivcd vi1rl;it101111 pe;~ (P~JLOII ~u/1111111 l..) - ~~~~~~~~~~~~~~y rcsul~s, Lrq~/ry/ic(i, 85:<br />
335-339,<br />
Gucrclllcoi't', A,, Ug;~ldc, I
Gulati, A., Jaiwal, P K., (1990) Culture col~ditio~ls effccling pl;iilt regeneralion liolll<br />
cotyledons of Vigiiu rudiaru (L.) W~lczei, Plaiir Cell, 7issti~, oir~i Orgai~ C'rilrirre.<br />
23: 1-7.<br />
Gulati. A., J;llwal, P.K., (1992) In vitro induction ot' ~~l~~ltil~lc \11uots a~id pl;iiit<br />
reycneratioil tiom slloot tips of llluilg btail (l'igiicl riltli[ir[l (L ) \4'ilczeI\), lJ/(l~ir (;,I/.<br />
Ti.ssirc (11iil 01pri11 Culrrr~.e, 29: 199-205.<br />
Gurney, S.E, and Robinsoti, G.G.C.. (1080) Tlic ~nfluc~icc of two Ilcrb~cidcs UII tllc<br />
productivity, bion1;iss and cu~nnluility coillpositiorl of li.cslr \\;lter 11iars11 IICIIP~YIOII.<br />
Aqutlric Boru~iy. 36: 1-22.<br />
Ilnnlmatl, N., Davey, M.II., (1988) Apl)licnt~on of flow cytonielry to S~I~I;IIIC<br />
hybridization ol'soybenii wit11 Gi).ci~ir c,tiire~ce~is, Soybeci~i G'oiaiis Ne~+~slc~r~n. 15<br />
48-51.<br />
ll;irdy, A.R., (1990) Eslin1;iIing CX~OSU~C 1111: ~de~llificat~u~i o1'~~)t~i~s ;I[ risk atid ~OIIICI<br />
of exposure. In: Ex/~osrirc. oJ'lJc~ric~cics UI<br />
C.1-1 Wakes (Eds) Taylor ;~nd I:~ancia, Lu~iilui~. ~pp 81 -97<br />
7>rrc,slricil I.V1li/li/2. L. So~ilcrville ;ind<br />
Henry. K.J., l;oosllee, \V.C., (19Y0) Tllid~iiz~~ro~~<br />
S~IIIILI~~~CS bi~bal bud :~nd sllool<br />
formation in Alocusia X Chanrrier~ Aildrc, /iorririilr~~rci/ .Scie~~c,e, 25: 124.<br />
Herrera-Estrclla, L., Depicker, A,, Van Monti~gu, M., Scllell, J.. (1083) Express~o~i of'<br />
ch~emcric genes transferred into plant cells usiilg a Ti plasmid derived vcctur,<br />
Nrirure, 287: 654-656.<br />
Hess, D., (1969) Versueche zur tra~lsfornial~oil an Iiohcrc~l pfla~ize~l: Indukl~un u~id<br />
konstante we~tergabe der antllocyansynlhcse bci Perunicr hybridu. Zei/.~cl~riJ//iicr<br />
Pjla~tzeitp/~ysruluy~~~, 60. 348-358.
Hess, D., (1970) Versueche zur transforlnrition an I~olierell pl1;lnzen: Mogliche<br />
transplantation ei~les gens fur blatlt'orln bet Per~rrrici I~britlu. ZeirscilrVr jrr~,r<br />
Pflorr~orpi~ysiu/uyie, 63: 461-407.<br />
tligley, L.G., Pcdigo, L.P., (1996) Ecorru~rrii~ ~i~~~cshol~is /or Irrr~~griiir~cl 1'1,si<br />
ittirrragerrrr~rr. Univ. Nbreska I'ress, LIIICOIII. KE, pl). 327.<br />
Hildcr. V.A., Gatellouse. A.M.R., Shcernlall. S.E., I3;irhi.r, I< I:.. Buultcr. D.. (I%7) 11<br />
IIOVCI III~CII;IIIISIII 01' IIISCC~ ~S~~L~IIICC c~~gi~iccrsd IIIIV IO~~ICCO. ~'~IIIIIC,. 300 100-<br />
163.<br />
Ilit;~, O., Lafaiga. C., tiuerra, 11.. (1'107) So1n;ltrc c~l~bryogcl~uals fru111 clllukpsa (('1c1.r<br />
arietiriurri L.) lmmaturc co~yledolis: ll~effect of~cai~ll, g~bberell~c ac~d and 111dulc-<br />
3-butyr~c ncld, Acio Pl~~~siulogiti plti~~~iit~ro~r. 19: 333 - 338.<br />
llockenl;~, A,. Iiooyka;~~, PI., Sch~lperoort. I
Hooykaas, P.J.J., Klapwijk, P.M., Nuti, M.P.. Schilperool-1, R.A., Ilorbch. A,, (1977)<br />
Transfer of Agrobacreriu~n rii~~ieji~cie~is 'TI plasmid to Agrobaclerin and lo<br />
Hooykaas, P.J.J., Scllilperoorl, R.A., (1992) .Ayrobtrcr~~riir,,r ;ill11 pl;ttlt ~CIICIIC<br />
,'<br />
HU~&II, T.. Malik, T., Riazuddtn, S., tiu~dotl, hl.I1.. (1097) Stittltcs oti t11c C S ~ ~ C ~ ~ I O I I<br />
w - \-..-<br />
of marker genes ill tile clitckpea. Plo~rr Celi. 77,~ire t11ri1 01:y~iti ('~rltrr~r,. 40, i lo.<br />
tlwang, I.D., Kiln, S.G., Kwoti, Y.M., (1996) C;~~I;I\';\I~IIIC ~iiclilbolistll 111 ltssue c~~Ittrre\<br />
of Cur~uvuliu ii~ieara, i.'/a~~r Cell. Tissrre u11r1 01gu1i ('rilriir~., 45: 17-23<br />
lantcheva, A,, Vlahova, M., Trinh, 'I'.H., Bruwtl, S.C., Sl;itcr, A,, Elltutt, M.C..<br />
Atunassov, A,, (2001) Assessilie~l~ of pulysonlaly, ct~ibryu ~\)I.I~I~IIOII at~d<br />
regenet-atloll in Itquid nledia for vurlous spcc~cs ufdlplu~d ;t1111~;11 h/~,(/~i.ufio, l'li111r<br />
Scier~ce. I 60: 621-627.<br />
Ignactmulhu, S., (2000) Ay~~obuoeriir~~i 111edi;i~cti irdtl~Surt1i;t~iu11 of lli,q~iir ~c~y~ri~~c.tluii.\<br />
Koem (asparagus bean). 111dia11 Jo~rr~itii oj Erperr~ne~ilul fjiulu~, 38:493-498<br />
Ingelbrecht, I..<br />
8l-eync, I>., Vancomprrtlollc, K., K., Jacobs, A,, V;tn Monl;tgu, M.,<br />
Depicker, A.G., (1989) Dtll'crc~it 3' cnd regtotla slrugly inllucncc llic level ul'gclle<br />
expresston in plant cells, Pla~ir Cell. 1: 671-80.<br />
Jaiwal, P.K., Kumart, R., Ignac~muthu, S., Polrykus, I., Sautler, C., (2001)<br />
Agrobocreriti~~t rtintejriciens-nled~ated genetic ttansfornlat~o~l of ~ilungbean (Vig~iu<br />
ru~irarc~ L. Wilczek) -- a recalctlratll gratn Icgulnc, P/flrrl Science, 161: 239-247.<br />
Jaronski, S.T., (1997) Nerv l'uradig~~i.~ iir i;or~~r~rlul~ttg Mycuiri~ecticidrs, I'estitrd~,<br />
I;ormulorior~s utid Appijcatia~~ .SJSIPI?IX 17"' VUIUI~IC, ASTM STIi 1328, Guaa, Ci.l{.,
Hopkinson, M.J. ;md Collins, I1.M., Ed,<br />
An~er~cn~~ Society for Testing and<br />
Materials, pp. 99-1 12.<br />
/'<br />
Jayanand, B., Sudnrsa~lan~, G.. SIIO~IIIJ,<br />
-<br />
regeneratloll of bllole p13111s by LISIII~<br />
K.K., (2003) i\~~ eflic~el~t pro~ocol for tilt<br />
;~\lllary ~nler~alc~~l explnllrs der~ved ti.0111 111<br />
vitro ger~~~inated seedl~llgs of cIllcl\pc;~. Crct~r iirir~ri~~ri~rr L. 111 Llrw ('ell c i ~ i i l<br />
Ur,\,elol~i~ir~~iu/ Bio1ogi.-Plorrc (111 I'ress)<br />
I'<br />
Jcfkraol~. R.A., Ka\,a~lngl~, T.A., Dcvan, h1.W.. (10X7) (;(IS tilaion,, bc~;~-<br />
glucuron~duse as a scnsltlvu and vcrs;~t~le gelre fusion ~lli~rkcr III higl~cr ~I~IIIIS.<br />
EiLlBO Julrrtial6: 3901 - 3907.<br />
Jelenic, S , kl~tr~keskl, P.T., I'npes, D., Jcl~~ska, S., (2000) ,tgrobe~i~~eriio~r-~~~cdiatcd<br />
Ir~ll~i'~~lllall0ll of broad ~CLIII<br />
38 167.172.<br />
V~C~U jiib(1 L , /"uo~/ 7i'(./1~10/0,~ L.l1)1/ ~~~JIc~'/II~u/u,~).,<br />
Jolinholl, R., (iuderial~, I< H., II.H., f
~<br />
.-<br />
Jones, B., Davey, MR., (1991) Direct gent uptake atid rege~ieration of tralisgeliic<br />
shoots from protoplasts of Glycirre oryyrua Tilid, Sojbec~rl Gerreiics Nrwslerlrr, 18:<br />
183-186.<br />
Jones, J.D.G., Shlumukov. L., Carland. F.. English, J., Scolield, S.I
Karthikeyan, A.S., Sarina, K.S., Velutlianibr, K., (1996) Agrubucrerrrri~i rrr~iicyucinrs-<br />
mediated transfoni~ation of Vigna ~iiuiigo (L.) Hepper, Plo~ir Cell R~~ports, 15: 328-<br />
331.<br />
Kathen, A d., Jacobscl~. II.J., De Katlicri, A.. (1990) ,.Igrubrtc~rcrrtrni ru~ii~;/oci~~~rs-<br />
mediated transforn~atioii of Pisri~ii scicti,lr~ii L using b111;ll.y ;rrld coIntcgt.:Itc vc~tors.<br />
f/Ull/ CE// /?~!purIA, 9: 276-279.<br />
Kaur, P., Bh;illa, J.K., (1998) Rege~~cr:ltiori of Ii:ililoid pla~ils li-0111 ~i~icrospor-c cull~~rc<br />
of pigconpen (Cirju~rirs ccijci~i L.). I~iilirr~i .iorri.~rcil q/ L.~~-l,~~i~ii~rorrrrl Uioli~~)'. 30. 736-<br />
738.<br />
Kaushal, R.P., Katrshal, B., Rashmi, N., V;l~d, A,, S~ngl~, U.M., (1907) Induclio~i ol'<br />
callus cultures arid regenemtion of so~i~i~cloiies Srorri diffcrcnl cxpl;~rils VS urdbcari<br />
[Viyriu i~iir~rgo (L.) klcpper] and their cv;ilu;rtrori tbr rcslslaricc iu (i~r~~u.sporii<br />
cu~ie~ct.ri.s. P~~utcetli~rgs qf' lirr /~~rlirrrr ~Vrtr~oi~irl S~,ieiic,rJ ilc,uilciity<br />
/'cII~I-/~,<br />
Biologicril Scre~ice~, 63: 359-367.<br />
Kay, R., Cllari, A,, DJI~, M., Mcl'hcraori, J., (1987) Uuplicatiori US CaMV 35s<br />
pr01110ter ~C~IU~IIC~S CIC;IICS<br />
n slrong enh;iilccr for pl:ii~t ger~cb, Scie~rc~, 236: 1290-<br />
1302.<br />
Ker~n, J., (1994) Opcniiig address. In iJrutceti~rig,s of fiie Iiirerriurio~rul IVorkiir~<br />
Coii/ereiicc oil Srured-prodrrcr Prorecrtoti, Canberra, Australia Volunie I, p. xix-xx<br />
Kerr, A,, (1971) T~xnafcr of viiulcnce betwccn rsolalcs ul'Agrobucrcr~rrn~, ~Vuirire, 223:<br />
4117~:)7~110rh,<br />
I'D, (1984) Plantlet rcgclieriltiml Lo~n cutylnlunxy r~odis uf<br />
__.-- - -<br />
chickpea, i~~rer~iucio~~ul Cliickpeu iVeiewhIrrrer, I 1: 22 - 24.
Khan, M,R.l., L.M., Heath, L.C., Speiicer, D.. H~ggiils. T.J.v.. (1994)<br />
Agrobac~rriu~~i-mrdiated transfonixttlon of subtcrrjneall clo\er (yrgo/ililli<br />
subrenu~lelcl~i L.). Pln~irPliy~ioluy~~, 105: 8 1-88,<br />
Kim, D.M., Clioi, K.S., (1989) I~ldlictloii of ~ii~llti s11oots :IIIJ pl;111t ~C~CIIL'I.;IIIOII ti.0111<br />
protoplasts of allhliii (hledictigu siiiivci L.). 7711, Kurciii~ Jr~irririi! 01 1joi011\, 32. .I l3~<br />
322.<br />
Klcili. T.M.. Woll; E. D., LVu, 11.. S.111lurd. LC'. (1087) ll~gli \clucity ~i~~ciu~~~ojcct~lc<br />
for deli\eri~ig ~ii~clelc ;icids into li\ ing cells, Niiriii.~., 327: 70-73.<br />
Knowles, B., Ellar, D.J., (1987) Coilold-os~ilotlc lysis 1s a gcncr;ll k:ltilrc of the<br />
nlechanisni ofac~ioli ot'Uiicilliu t1r1,riiigioie~i.i 8-clidotoxins w1t11 drfl;.rent inaect<br />
specilicities, Bioclii~irict~ ei Biup11)~sicu Acrri, 924: 509-5 18.<br />
Koiwa, K , Sllade, R.E., Zliu-Snlz~nan, K., Si~b~.;~~iii~~iii~n, L., M~irdock, L.L.. N~clscli.<br />
S.S., Breasan, lI.A., llnsegnwa, I' M., (1998) I'hagc display sclecr~o~i villi<br />
differelltiate ~nsccuc~d:il ;ic[iv~ty of soybean cystall~is, ijicit~r Joio.1iii1, 1.1. 37 1-37'),<br />
Koziel, M.G.. beland, G,L , Bowmon, C., Cilrrorz~, N B., Crcnshnw, 11.. C'russlnl~d, L ,<br />
Dawson. I.. Uesai. N., Ilill, hl., Kudwell, S., Launis, K., Lcwis, K., Maddox, I).,<br />
McPl~er~un, K., tvlcgliji, M.II.. Merli~i, I:., I
Krens, F.A., Molendijk, L., L., Wullems, G.J., Schilperoort, K.A., (1982) In vitro<br />
transfosmatio~~ of pla~lt protoplasts w~th Ti-plasniid DNA, Ntiir~.~, 296. 72-71.<br />
.dhnaniurthy, K.V.. Sul@C.K.,<br />
/ - - . -- . -<br />
$;igare, A.P., Me~xnes. M., Katlie~~. A-de, P~ckardt.<br />
T.. Sclirider, 0 . (2000) Agrobucc~~riri~rr-~ned~;~Ird transfor~i~at~o~~ of chickpe;~ (('ice.<br />
urini~ru~rr L ) r~iibry axes, Plo~rr Cell I(e/~orts, 19: 235 - 2JU. ,.<br />
Kuchuk. N., Ko~narnitski, I., Sliakhovak~~. A., 6leb;l. Y., (1090) tic~~ct~c tr;~~~slbr~~~,n~o~<br />
of Mreircc~gu species by Ayrobircferi~o~i fii~~irfrici~,~~~ JIIJ clcctropo~it~o~~ ol<br />
/k+.<br />
PA. S~i~iini Uvrna, I'ii, 11.11,. Slii~r~i~i~, /
Last, D.I., Brettel, R.I.S., Chanibrrlalli. D.A., Cllaudhury, A.M., Larkill, P.J., Marsh,<br />
E.L., Peacock, W.J.. Dermis, E.S.. (1991) pEniu: Ail il~lorovcd proilioter for gcne<br />
expressron In cereal cells, Tlreorrticol c~irdrlpplictl C'eiieric~. 81: 581-588.<br />
Lawrence. P.K., Koundal, K.R., (2001) ..lyrobucrt~rtir,,i I~IIIC:/~IL~I~~~IS-I~IS~I;IIS~<br />
transibrliiatlon of plgeoli pea (Cii~u~ilis ccijli~i L. I\.lrllsp.) ;lrid 1iioIc~111;1r cil~;llys~s 01'<br />
regellcrated plants, ('irrwiri Sc,ie~rcc. 80: 142s-1432.<br />
Lcc. W.U., (1902) ltiip.~ct ofplant gro\r.tli rcgltl.~lors otid pl l on ,ollialic c~iibr)ugc~~cs~~<br />
and efticlelit plaiit regeneratloll In Glyciire iorrre~~rcllir (Hi~yata) origlnatcd li-olii<br />
Soutli Clilna, Suyberr~i Gt.rrerii,s :Veil.sIricn., 19: 46-5 1<br />
Lereclus, D., Uelecluse, A,, Leccidet, M.M., (1993) Dlvcrslty ufUuciliu.s llr~rri~~gi~~~~t~<br />
toxrns atid genes, in Bocillus rlll~ri~rgre~resrs, all knv~ronnieli(;~l U~opcsl~c~dc: 'I'licury<br />
and Practice, Enrwlstle, P.F., J.S.Cury, M.J. Uailcy, arid S.l
Li, Z.Y., Tanner, G.J., Larkin. P.J., (1990) Callus regenenrlon from Tr~$liut~r<br />
subrerrat~errtii protoplasts and rnli;lliced pl.otoplnst div~sion by low-voltage<br />
treatment and nurse cells, Pl(i~ir Ccli, 7iss1ie irtrtl Urgiilr C'iilrrr~.'~, 2 I : 67-73.<br />
Li X.U., Xu Z.H., Wei Z.M., (1995) Pli111l rcgc~~t's;~tion li.0111 protopl:~sls uf lnlrilnture<br />
Vig~~a sitietlsrs cotyledoils vla sonlatic cnibryogc~rcs~s, I'lirrrr ('ell H~,por.is. 15: 282-<br />
286<br />
Li, X.Q., Denial-ly. Y., (1995) Char;~ctcr~~;lrlu~~ ul' lilctura ;~l'rcct~lig 1pl;lnt rcgc~icr~~t~o~~<br />
frequency ofiliedinrgo iiil~itli~io L., Eri/~~'/ir,it. 86. 143-148.<br />
Li X.Q., Deniarly, Y., (1996) Sonlatic e~iibryoge~~csis and plant ~.cgcllclntion ill<br />
Medicago sujji~iricosu, Plu~ic Cell, Tissrre crrid Orgoti C'rilrrrrc, 44: 79-8 1.<br />
LI Z. J., Jarret, K.L., C'lie~ig, M., Dcmski, J.W.. (1905) l111p1.ovcd clcc~ropol.;ltioli buflbr<br />
enlia~lces translent gene exprcsslon in A~NCIIIS i~yj~ug(icii ~~rotol~lnla, (;e~rott~c, 38:<br />
858-863.<br />
Luehrsen, C.R., DO Wel. J.K., Walbot. V., (1992) 'I'rasle~it cxprcssion analys~h 111 pl;~nts<br />
usl~ig lirelly luciksase 1,cporter gcnc, ~Molioh 111 Gizy~~ioiogy, 2 I6 307-414.<br />
Luo, J.P., JI;I. J.F., (19")) Callus induct~u~i and pla~it legcncralio~i I'run~ hypucolyl<br />
explants of !lie Ibrage legunit' Aslrogciiu~ cicirirrgens. 1'1(1t11 ('ell Nepurls. 17: 567-<br />
570.<br />
Luo, J.P., JKI, J.F., till, Y.H., Liu, I., (IY9Y) Ii~gl~ licqucncy sonl;~lic cnibryogenesis<br />
and plant regeilcratlon In c;lllua cirlti~~ca ol' A.slrirgiilir.~ ud.~ii~:q~ir~ I'all. 1'1~111<br />
Scirrrcc, 143: 93-99.<br />
Maddox, J.V., (1987) Protozoan D~seases, in Epi~ootiology of lriscct Diseases, I'uxa.<br />
J.K and Tanada, Y., Eds. John Wlley & Sons, New York.
Malik, K.A., Khan, S.T.A., Saxella, P.K., (1992) Direct organoyttiesis atid plant<br />
regeneratloll tn precondittoned tlssuc cultures of Lu111yrirs cicl'ru L.. L uclirir.~ (L.)<br />
DC. atid L sarivus L., A~r~iols Botor!, , pp. 70.<br />
K. A., Saxeoa. P. K., (1992) TlitJ~;l~urotit1dui.i.s ltigli li.ciluency sltoot<br />
regeneration In Intact scedl~tigs of pea (Pisirir~ ~orrt,ii~~r), clilci\psa (C'i1,~'r ~~~,,IIIIIIIII),<br />
and Ientll [Lr~rb cirlr~iar~s). Arr~lrtr/ru~~ Jo~rr~ttrl (u 1'1ci11r Pltrsiu10~1~. 19: 73 1 740.<br />
Lli~nasliel.ob, R., Bell-Dov, E., LIU. Z.. Zttr~taky, A,, jlC)L)7) Tlie I9 kl).~ prutc~~i ol<br />
Bacillris rli~rro~yirr~esis subsp, i.srcre1~~11~i~ did 11ot protect Cc/ic.ricIiiu c,ulr cells li.0111<br />
lethal efl'ect of CytA, 111: Absrr(icrs o/ 29"' /I~t~trrtr/ hleerrr~g oj'rlrc Sucivry jur<br />
thurinnie~i'sis, Corciobu. Spoilt, pp. 5 1.<br />
Mandel, M., H~ga, A,, (1970) Colc~urn.depcndcnt bi~i:tcriul)l~,ige DNA ~~tl'ect~u~i.<br />
Joiir11t11 oj hlolecirlor Uiolug~l, 53: 159.<br />
Margaltth, Y . (199U) Discovery of Htr~~illiiv ~/rrrrirtgiorc',sr~ isrtir1cnii.v. It1<br />
Htrtreritrl<br />
Rutgers Uliivrrs~ty l'rcss, New Bru~iswtck, NJ, pp. 3-0.<br />
Mass, C., Laufs, I., C;r,i~it, S., Korfliage, C.J., Werr, W., [I'iYI) The conibitlaltoti of;^<br />
follow111g introri I<br />
enhancer-reporter gene expressloll up io 1OUO Sold, 1'1u111<br />
Maughnn, I]., Sagliat-Marool; M., Buss. G, Iluc~tis, Ci., (1996) Amplified fragment<br />
length polyniorpliism (AI'LP) tn soyabeon: specles dtvcrs~ty, inhcrit;ince, and tiear-
Maxan~, A.M., Gilbert, W., (1977) A new metliod of sequenci~ig DNA. Pruc,eerii~~gs qf'<br />
Nario~tal Acadenly oj'Scie~tce. US.4.. 71: 560.561.<br />
McKently, A.H., Moore, G.A., Doostdar. H., Neidr. 11.1'. (1095) ~lgroDe~r~c~~~~irr,,i-<br />
mediated transformation of peanut (.-li.ochi., li~pugrrerr L.) eilibryo ,~xcs ;111d lllc<br />
developl~isnr of 1ransgcnic pl~iils, Plci~ii ('rll Hc,por/r, 14: 690-703,<br />
Mcijer. E.G.M., Broughtoli, W.J., (1981) Ilegelier,ltlon ol'uholc plalics ti-0111 Ihyl>oco~yl.<br />
root, and IeaLdcrived tissuc cirltures ot'tlic pastLlre IC~LIIIIC S~/UAOII~/IL,A SIII,IIIIC,II.SI.\<br />
[phytohornione, platit rcgeneralloll], PI~)wiolug~ir ~~I~III~~I~IIIII,<br />
52: 280-284.<br />
Mcijer, E.G.M., Brown, D.C.W., (1968) l~lli~bitio~l of sullliit~c CI~I~~YU~CIICSIS ill IISSUS<br />
cultures of illrrlicrrgo ~u/ii,ci b) ,~~i~ii~ocliiosyv~~~ylgIyc~~ic,<br />
:illiilllr-oky;ic~~~~ dc111.<br />
2,J-dinitropheliol alid sal~cylic acid ;n conceiitl.:ltions wllicll do !lot ~lill~hil etliylclie<br />
biosy~itlicsls and growlli, Jorrrr~crl uj Ev~~c~~~~niei~roI<br />
Uoin~r).: 39: 263.270.<br />
Mcijer, E.G.M., (1989) Developlnentill oq1ccts ot'etliylc~ic btos)~itlics~a durilig .;oni;ll~c<br />
cnibryogel~e\~~ In t~ssue cultliwa of !\lcr/ir~iigo rriiiirr, Ju~rrr~iil i,j /:'~/~c~rri~~c~ito/<br />
Boiurry, 10:47'1-484<br />
Meiiiers, J.P., Elden, T.C., (1978) Rcaistallcc to Insccls illid dlscasca iii /'/lllAco/ll.~. 111:<br />
Advunces III 1cgio11c ~cie~lce. I~~lor~~c~lio~t(il Lrgrr~~le L'u~iji.i~errcc. Kuw Surrey. L'K.<br />
Summerfield, 1t.S and Bunting, A.H. eds., pp. 359-364.<br />
Mctcalf, R.L., hletcalf, ](.A,, (1993) Darrricrivc. rr~ltl U~eJiil 111secu: 7l1ei1, 1lobrr.s t111c1<br />
Control. 5"' Ed., McCir;~w-hlll, Inc., New Yolk.<br />
Meyer, H.J., van Stadell, J.,<br />
(1988) In vltro niult~plicat~on of 1x1~ jlcxuoscr,<br />
Horriculruriil Screirte, 23: 1070-107 l
Mickel, C.E , Standish, J.. (1947) Suscept~bility of processed soy tlour a ~ soy ~ d grits in<br />
storage to attack by Tribolruiii ciiArorieir, ti~iii,tr~irj of dhrirr~~,u[ii .~gi~rt~rrlirrrcii<br />
Ex/~c,ri~~irii~cil Sluliori Tecli~iicrrl Bitlle~iii. 178: 1-20,<br />
Misra, M., Adds, G.. Nari~yai~, K.K.J, (1994) Metl~ods tiur callus ~nduct~ull :111d<br />
differentiation of Loily,riir srirrvirs 2nd cil~bryo rescue 111 iii~ers~~cci~ic cruaaea by<br />
tishut c~~lt~ire, Juit~~~~111 (I/ [lit, ~Igrr[~~rl/r~i~~~l ,Sui,i~~i' tiiii~,c,i.si[,~ 01 II'III~~,~ (LIrii/~~d<br />
Kirigtlurir), 74: 129- 144.<br />
Mogen, B.D, MacDonald, M.H., Gray Uouch, I(.,<br />
Iiu~it, A.G., (1990) Upstrc;i~~i<br />
sequences other than AAUAAA are required for eflicicnt Ilicssenger llNA3' end<br />
fol.liiatioli $11 plants, IJluiri Cell, 2: 1261-1272.<br />
Muhamed, M.F., Rend. P.E.. Coyne, D.P., (1990) 111 vitro rcsI)oIisc 01. bean (lJlro.scolrr.~<br />
viilgaris L.) cotyledonary expla~its to be~izyl;lde~~~nc 111 tllC ~ii~di~~iil, I'ruc~~dii~gs 1!/<br />
lire Picirri Gruivllr I~c~gi~lcilu~~ Sucir(v ol'/li~iri.ictr. 17111 AIIII~I~I~ Meet~~ig, St I'il~il.<br />
Minne>ot,i. USA, pi) 134.<br />
Moha~~, M.L., Kri~ii~~;~~iiurth). K.V , (1098) I'ln~it rcgc~~cr;liiu~i in p~gcolipc;~ I('trjn~iir.\<br />
ciijirri (L.) M1llsp.1 by orgaliogeneais, 1'1c1ri1 (.'ell Rei,url.\, 17' 705-710<br />
Llontoya, A.L., Cli~llo~i, M.U, Gordoli. M.I', Scii~ky, I)., Ncjtc~, I:'&., (1077)<br />
Ocropiilu ,111d 1101)a111ie 1iietabol1~111 ill ~\grob;~c~erii~il~ ILIIIIC~CICIIS :iiid crow11 gall<br />
tuillor cells rule ol'l~lo>iiiid pelles, Jurtr~iul u/Uac/r~ rulogy, 129: 101 - 107.<br />
Morton, R.L., Scliroeder, H.E., Uatelii;~n, K S., Ch~ispccls, M.J., Ail~~strollg, I:.,<br />
Higg~iis, T.J.V., (2000) Bcaii a-;~mylnrc lnliibitor 1<br />
111 l~a~ihgcnic peas (l'rsur~i<br />
rurivur~r) provides cuinplett protection from pca wccvil (8rircltu.r /~i.soru~~i) under
field conditions. Procc.rdbigs o/Naiurionrrl Aarrlenry uj'Scic~ic~,s, USA., 97: 3x20 -<br />
3825.<br />
Moursy, H..4 , Haggag. M.E.A.. G~I~II~III, S.A., l
egeneration in excised cotyledon cultures of ~ilungbeaii (Yig111i rcidiucri (L.)<br />
Wilczek), Jupuiiest. Jour~iui oJ'Crup Soe~rct.. 66: 67-75.<br />
NAS (1979) Tropicui ieyiotte rtsoiircrs Jur //I') jiiriri.~, iVti1101rii1 ..lc.ii~i~,~ii~, P~CJA.Y.<br />
~t'u~iii~igroii UC, pp. 33 1<br />
Nauerby, B., Madssn, M., Clirisliansc~i, J.; 1Vynd;iele, I( , (199 I) A ~pld :iiid CSIICIL'II~<br />
rcgc~icratio~i systeni for peii (I1i.\i~ili ~11111 IOII), s111tabIc for tr~~i~~furi~~;itioi~, i'iiiii~ ('dl<br />
RL,/I I.\ 0: 676-070<br />
A,, Keddy, C.S., I
Ohara, A., Akasako, Y., Daimon, H., Mii. M.. (2000) lll;itit reyetierotion ti0111 hairy<br />
roots triduced by infectloll wttli Ayrohoc~tei~it~r~t r./ti:ugoit~~.~ it1 ('roralitric~ jtl~tc,cu L..<br />
Plattr Cell Kepurrs, 19. 563-56s.<br />
Oppertiiati, C.ll., Taylor, C G.. Co~ihling. MA.. (1994) Iloorltiot ncni;trode directed<br />
expression of a plat11 root spec~tic getic, Sct(~~tco. 203. 221-223<br />
Orshillsky. B.R., Swansoli, E.B., Totiies. I).l'.. (19s)) I:ttIi.~~icctI 511001 IC~CIICI;IIIOII<br />
froli) Iiotl~oge~itzed callus culf~tres of btrdsfoo~ trclbil (l.oiir.\ r~v~~rt~c~~t/r~/ti,<br />
L ) . I'liti~i<br />
C~.ll, 7i'sstrc oilti Olpari C'ttlrure, 2: 341-347.<br />
O~can, S., Burgliclii, M , Firek, S , Drd1)et, I .(1092) lltglt I'reil~~ettcy itd~~e~itt~to~~s ~ltuut<br />
regettrr;ition from t~nmaturs cotylcdo~ts of pea (Pist~i~~ sorii8ri~ir L.). Plriiti C'oll<br />
Hr/)or~rs, l l : 41-47.<br />
P;~dmatja, (2 . I
Parr, J.F., (1974) Effects of pesticides UI<br />
~il~croorga~iis~iis in soil ;~nd water. In:<br />
Pesricides iti Soil tl~ld 1Voler, W.D. GUCIIL~ (Ed.). Soil Sclellce So~eity ofAmrrlca,<br />
Madison. WI, 315.<br />
Parrot, W.A., Collins. G.B., (1983) Callus 2nd slioot-t~p CLIIILISC<br />
01' eight i;~ifol~ii~~i<br />
species 111 vitro will^ rcge~leratio~~ vi;! soin;ltlc c~iibl-yogc~lcsis of' li.ijuIirii~i r~tI~~~,rs<br />
[Clover], Pla~~r Sciorcc Lerre~lv. 28: 189- 191.<br />
I'atel. D.13 , Uarvc, U.hl.. Nilpr, N., Mclil;~. A.l(.. (1991) I~C~~II~I.,IIIUII uI' I)I~CIIIII)C;I.<br />
C~~UIIIIS ciqilri. througli so~iiot~c c~iibryopc~~cs~s, 111iiiii11 Joiii~~rcil oJ ~;Y/~L,I.I~II~II~~I~<br />
Bioloy)~, 32: 740-744.<br />
Pellegrinesclii, A,, (1997) 111 vitro plant rcgc~~clnt~u~i via org;1liogcllesla of' cowpca<br />
[Vig~tu ir~~yurciilc~~ci (L.) Walp.], Pliirrr ('ell l(cpuro, 17: 89-95.<br />
Pellegr~nescll~, A , 'fepfel.. D.. (1993) M~croprol):~garion ;III~ 1pI;11it ~C~CII~I.;I!IOII<br />
III<br />
S~,~Siitiiti n~srroio., Plor~r .%ICILL, 88. I 13-119.<br />
Pere~r;~. L.I:., I:r1cli5oii, L., (1995) Stable trunslbrnl;~l~on ul';lll'ulll~ (rlteilictigo .virlrvu I-.)<br />
by part~cle bonibard~iient. I'lii~rr Cell i(eljor15, 14:290-293.<br />
Pcrl, A,, Klcaa, [I., Ulumciilliul, A,, G;llill, (j., (ialun, E., (1992) I~iip~orc~~~cnt of~)l;~~rt<br />
regeneralloll and gus expressloll 111 scutell;~r \vileat calli by UI)IIIIIIIII~IUII culture<br />
conditions and DNA niicro projuct~le delivery p~.ocedurcs, Mt~lccrrlur tiencrrii<br />
Gerrerics. 235: 279-284.<br />
Pestana, M.C., Lacorte, C., Fre~tas, V.G. de., Oliveira, D.E. dc., Mansur, E., de Fre~las,<br />
V.G., de Olive~ra. D.E., (1999) 111 vitro regeneration of peanut (Arucitib irypogci~ci<br />
L.) through organogenesis: effect of culture tempcraturc and sliver nlllale, 111 Vilru<br />
Crlluiur ur~d Deveiopr~te~~lul Bioioy);-Plut11, 35: 2 14-216.
llechrnlics sur les prnidilies des<br />
relariu~i biocl~l~iiiqui. spec~tique<br />
et les ciliileurs qu'elles ~~lduiscnt.<br />
11ysioI. Veg 8: 205-2 13<br />
Plckel~s, L,G.. T~~III~IJ~III, l
' Potrykus, I., Spangenberg, G., (1995) C.c,/ir r,zi/r?f~,/, ro plii~rrs, Sprli~gcl- Vcrlny. Bcl.l~n,<br />
pp. 361<br />
,,kFh,<br />
S~,,~~l~~d~lury, I.B., hill, R,K., CI~o\\dliury, V.K., (1992) I.';IC~"~S ;ll'f'cctl11g<br />
plant rcgc~leratiori 111 cllickpca. C'ICL,~ LI~~C~~I~IIIIII<br />
L., 1110it11r JUII~IIIII<br />
uj J:.~/./)i~ri/~ir~/~i(Ij<br />
Jb;ak;iiil, S. Clu\rdliury, J. U.,<br />
-/<br />
Vadav, N. R. J~III, R. K. (.l~o\\dholy. \' K.<br />
Somalli' ei~lbryogenc,~~ ill suspcl~riol~ cul~urcs ol' cliichl~c;~, :~IIII~I/.\ r,j<br />
(I'II)J)<br />
Ilrr~/r~,qi,<br />
Prakasli, S.K., I'eiit;rl, D., Ullnlla S;irin, N., (1904) I
Rand, G.M. (Ed.) (1995) Ftordir~~rc~~r~ls q/'Aiitr'i/tc~ Tocivolog~: Wrcrs, E~rviro~r~~roltrri<br />
Fare o11d Risk Assess~rie~~r, pp. 1 125<br />
Rao, B.G., (1989) Effects of constiturtils 2nd tlieir coticc~i~r,~~iuiis ill Iiulrlclit 1iledi:l 011<br />
callustng lion1 chickpe;~ hypocotyls, Lc~gr111rc' Hnriiri11. I!: 186 18s.<br />
Rae, B.G., Cl~opra, V.L., (1987) Genotypic atid e\pl;itit diffcrrticea 111 c;illus 1111t1;1[ioti<br />
2nd ~niil~literla~~cr 111 chickpe;~, I~~rrr~~i~~~ro~iirl<br />
('iiic hprii ,Yr~~.\/~~rci~~.. 17 10- I?<br />
Icao, B G.. Choprtr, V.L , (1989) Rege~ieralioll ill cli~ckpe;~ (('i1.r~ irririi~i~r~~i L ) 111rougli<br />
somatic enibryogenesis, Jorrr~tul oJ Plir~tr PIri,~iolog,, 134: 637-638.<br />
11atliore, R.S., Sillgll, N.K., Garg, G.K., Clund, L., (2000) llegcr~eratiuti ol'~>l;lnts from<br />
callus tissue of AL'SCII~IIUIII~II~ spp. (Leg~~tiirtios;~~), JUII~IIII~ (I/ PI NruIo,~:y, 27.<br />
81-84.<br />
Rathore. R.S., Ch;lnd, L., (1997) In vitro tr;l~lsfbr~iiolio~i ol' piyco~ipc:~ gcnulypes by<br />
wild stralris of AgroDocrerirr~~i rrr~~icfiicie~rs, I~~/er~rcirto~rtrl Cl~ic'k/~cci o~ril /jrgro~rl)ro<br />
Neir~ic//el., 4:38-39.<br />
I?aupl), M.J., Koehler, C.S., Uacidsoli. J.A., (1992) Adv;rllccb 111 ~l~iplcn~cnt~ng<br />
integrated pest rnalingenient Ibr woody laiidacapc pl;~llls. A111rrtu1 /(ci'rci~' 1!/<br />
E~iro~~rulugy, 37: 56 1-585.<br />
Ila~en, J.A., (1986) B~ucllem~cal dispo\;ll ul' chccss Ilt 111 glowllig pl;ililb'!, New<br />
Ph)~roIogiii, 104. 175 - !U(I<br />
Kazdan, M.K., Cocking, E.C., Po~er, J.B., (IYXU) Callua rcgcrleratlurl li-otil nirsopliyll<br />
p~.otoplaita of sweet pca (Lor/r~~ru\ odorirru.~ L.), Zur.scltriji jiter<br />
PJla~~zr~~/i/~vsioiogir, 96: 18 1 - 183.
Rech, E.L., Golds. T.J., Hammatt. N.. Mulliga~i, B.J., Dave), h.1.R.. (1988)<br />
Agrobocreriu~~l rhrroge~les lnediated trn~isfori~~atioii of the \\,~ld soybeans (;11,~,iri~~<br />
ca~iescens and G. cic~~iciesritio: producl~on of tmllsgenlc pluilts of (; c.o~resct~rts..<br />
Jocrrnni o/'E.rprri/~ti.~lrtrl Ilulo~t,,, 39: 1275- 12x5<br />
Reddy, L.R.. Reddy, G.M., (1993) Faclors :~ll;.ct~ng dlrccr soill;itlc cii~bryogeiles~s :111d<br />
p1a11t regc~~e~ilioii 111 grou11d11~11, .4r11ci1i.s Ii)pug~i~,c~ L, /III/~UII JOIII.IIII/ I,\<br />
EA/I~I.IIII~II/~I/ Uioiog): 3 1: 57-60<br />
Kepkov:~, J., Nedbalkova, B , I-iolrib, J . (1991) Ilegunel-;it~uil of pl,~nLa li.o~il /ygo~lc<br />
embryos after lnterspecilic<br />
Ihybr~dizalloll willli~~ tllc ~CIIUS I).!/uli~r~~i ;111d<br />
electrophoretic evaluario~i of hybrids. Scir~rlijic. S/iirii~,r OS6111 Koi,iirc.h I~i.\~iiiirc<br />
jbr Fu(1cier Pla~r/s, 12: 7-14,<br />
Iley, H.Y., Mroginski, L.A., (1996) Regc~lc'itio~l ol' ~pl:liils lioln c;~llur t~hrut~c ol<br />
IIJIIIIIIL spp. (Legurilillosne), i'lii~~t ('i,il, %~AIIL. IIIII~ 0rgo11 ('rtl/rir
Richardson, M.J., (199 I ) Seed storage proteins: The cnzynie inliibifors. 111: Mt~hods iir<br />
Plan/ Biochrrtrisrr),, R~chnrdson. M.J. ed Nc\v York. Ac;~dtm~c l'ress, pp. 259.305.<br />
Riker, A.J., (1923) Sollie relinlol~s of rlic croivli y
Ryan, Clarence A., (1990) Proteasr inhibitors ill plants: genes for i~~ipro~i~lg dcfc~ises<br />
Rybczynski, J.J., Dabrowski, L., Bndzlan, T., (1995) Cll;lngcs III ~lloiylioge~~~i. pote~~ti;il<br />
of shoot rcgc~lerat~on Sroln root explant of Lo~ur iur~~rc~r/u~us L, seedling. ..licii<br />
Physialogiue Pltr~rltrrior~, 17: 2 1 1-2 18.<br />
.A.-l!,<br />
'--<br />
St~l~:lhi~i~, K., KI~~SI~II~IIII~I~~II~,<br />
K Lf , (1093) I1l,tnt S~~~IICI-,I~IOII<br />
~,I;I<br />
S~II~;I!IC L'III~~~~~cI~c~I><br />
~IIIL'~.[Ic~I (C'ICC,I. ~III~~IIIIIIIII),<br />
655.<br />
/'It1111 ( ',/I I
Sarria, R., Calderon, A., Thro, A.M., Torres, E., Mayer, J.E., Roca, W.M., (1994)<br />
Agrobocierirr~~r-mediated tra~isfosmal~on ot'S~dostr~rilies gllici~ie~isis iiiid pr~ductio~l<br />
of transyenlc plants, Plti~rr Sciorce. 96: 119-127.<br />
Saxena, C.. pa la^, S.K., S;lili,lntaray, S., Rout, G.R., Das, IJ.. (1007) Pla~lt rcgc~ier,itioii<br />
from callus cuitures of Psoro1'i.r co,7'lijblio Linn.. Plri~ii Gro~i11 I~cgrilciiio~~. 22: 13-<br />
17<br />
Scarpa, G.M., I'upilli, I:., Darnliln~, I:., Arcio111, S, Vcro~lcsi, I:.. (ell.); Bulli[rn, S.. (cd.):<br />
Carcdda, S., ( 1992) 111 vitso culture and plnrlt scgslicratloii iii ~letlic.ogopolyrr1oip1~~1<br />
L. Ploidy and cliro~nosonie ma~l~pulation 111 tbragc brccdi~lg. 1'roceetling.s uj rile<br />
17111 irrcrtitrg oj llrc Fotltlcr Crops Scclioi~ oj L:LICilRI'Itl. Alphero. Italy, 177- 170.<br />
Schcli, J., Van Montagu. M., De Bcuckelecs, M., De Block. M., llc~~~ckcr. I\.. Dc<br />
Wiide, M., Engler, G., Genetello, C.. Ilcr~i~lstecns, J.I'., llolstcrs, M., SCUSIIIC~. J..<br />
Silva, U., Viin Vl~cl, I;., Villasscocl, I/i,r iu /)ltr~ii.s.<br />
" "-<br />
----<br />
Potrykus, I., Spangenbert, 1'., Eds., Springer Verlag. Berlin. 325-336.<br />
Scllnall, J.A., Weissinger, A.K., (1993) Culti~rrllg pcailut (Arlicliis Iry/)ogtieci L.) ~ygot~c<br />
embryos for tra~islbrn~atiun via ii~icsoprojcc~ilc bo~iibasd~llcnt, I'ltirir Cell Niq)urls.<br />
12: 316-319.<br />
Shade, R.E., Schrocder, H.E., Pueya, J.J., Tnbe, L.M., Murdock, L.L., Higgins, T.J.V..<br />
Chrispeels, M.J., (1994) Transgenic pea seeds expressing tlie u-amylasc inlilbitor of<br />
the cummon beall are resistant to brucli~d beetles, B~o/Tecl~~~olugy, 12: 793 - 796.
.'/'<br />
' Sh<br />
kar, S., Ram, H. Y. M., (1993) Induction of soinatis e~l~bryogc~lesis in tllr tissue<br />
cultures of chickpea (Cicer aririi~iurri L.), Procerdi~igs oj /lie III~IUJI Na/io~r~/<br />
Science Acudo~~)~. Part 8, Biologiccii Scte~ic~,~. 59: 629 - 632.<br />
Shao, C.Y., Russlnova, E., lantcheva, A., Ata~l:issoi, A.. klcCor~ilac, A,. Clicn. L).I,..<br />
Elliott, M.C., Slakr, A., (2000) Rap~d Irailsforil~at~on and regeIIcratiuI1 01. alli~lli~<br />
Sharma, L., Amla. D.V., (1998) D~rcct sliuut organuycncsla III ch~clipe;~ (('ice<br />
Shnrnla. K K., Ullojwulli, S.S., l'liorpc, ']'.A , (1901) llole oi'llic co~yIedu~~.~ry ~IS~LIC III<br />
thc in vltru d~t't'crc~~riat~un of shouts and roots from culyiedo~l explains ol'Drass~ca<br />
juncea (L.) Czern., i'lo~ii C'rll, ITsis,irt, cr~rd Orgtrti Qrllirrc, 24: 55 - 59<br />
Sliarnla, V.K., Kotl~or~, S.L., (1993) Iligh Srequc~icy ])la111 rcgencratlun 111 t~bsuc<br />
Shurma, K K., Orl~z, K., (2UU0) Yrogln~ll lor tile applicoilu~~ of ycnctlc t~a~isibrmal~o~~<br />
for crop ~rnprovemz~~t III thc sc~ll~-;lr~d tropics, IJI Virru Cell ~irill Ucvelup~~~eri/~~l<br />
Sheila, V.K., Mosa, J.P., Ciowda, C.L.L., I
Shri, P.V., Davls, T.M., (1992) Zentrii rnducod shoot regeneration from inilristurc<br />
e--<br />
chlckpea (Cicer arieiiiiiii~i L.) cotylrdoirs, i'1~11tr Cell, Tissue OII~ Oryon C~rlrure.<br />
28:45-51.<br />
Sierneiis, J., Scliirder, 0.. (1996) Tratrsgelilc plsl~ts: ticnetlc tr;rilforir~;~tioii.rccc~it<br />
developments and the stare oi' the art. PI;IIII T~SSLIC Cu1tu1.e and Diotrc1~11ology.<br />
IAPTC'~$'e~~~slrrrrr. 2' 66-75.<br />
Slnha, K.K., Malllck, I
Stiff, C.M., Leuba, V.. Sun. 9.. Le TOU~IIL':III, D., (1991) Isola1io1i. CLIIILIS~, and callus<br />
regeneratioti of ientll protoplasts from Is;if tissue [Lrrts ctilirrriris]. LEA'S-<br />
News/i.rrrr. 18: 30-33.<br />
St~ller, J., Mast~mn~. L., Tuppile, S.. Chian, It.1.. Chiusarzt, M.. Grcaslioll: P.M..<br />
(1997) Higll frequency traiisfor~nat~o~i and rsgcneratioti ot' trailagenlc planla in tlic<br />
model legutlie Lu/tis~ii~~or~icri.\., Jotintc11 u/'/:'\po.ir~i~~~i!(i/ Llorlrrrj: 48: 1357-1365.<br />
/<br />
Subliadra. Vasliisl~tli, IKK.. Clioi\dllury. J.U.. Si~igli, M., Silrcc~i. I'.K . (IOL)X) h1ulttl)lc<br />
shools from coly1ctIon:iry ]lode explt~tlrs of 1iu11-~iodulnt~~ig gelioiype (ICC4ISM) of<br />
cliickpe;~. Cicrr arieriirri~rr L.. Iridru~r Jui~r~ro/ i~/ L:x/~a.irire~r/ol Uiv/og\~. 36 1276<br />
-<br />
v- ..<br />
i K S I<br />
,<br />
1. Y,, Ks~~li~~;iiia~~l~~<br />
I(, V,, (1994) D~rcc! ~UIII;I!IC<br />
embryogrtieiia fro111 inlalure embryo xis 111 cIi~ckl~c;t (C'ic.1~ iirieiiriii~~r I..), i'lori!<br />
/Suliasini, K., Sag;~re, A.P., Sa~nkar, S.R, Krisli~iai~iurtlly, I(.\'.. (1997) Cumpar.~li~c<br />
/ .--. -=i.<br />
study of the dcvelopniellt of zygot~c and autli;iltc enibryub of cl~ickpc;~ (('i~er<br />
Svanbaev, E., (19")) IJlaiit regcticrat~o~i l'roti~ ~p~.oloplasts ~nd liasue cul[~iscs of' SUI~IC<br />
Tegeder, M., Gebliardr, D., Schieder, O., Picka~.dt. T., (1995) Thidiit~uron-induccd
Tegeder, M. Koli~i, I-I., Nibbbe, M., Scliiedcr. O., Pick;irdt, T., (1996) Plant<br />
regeneration from protoplasts of Viciu ~rurbut~e~isis via soulatlc e~~ibryoge~lesis and<br />
slioot orgliliogenesls. P~UIII Cell Repo~./~, 16: 22-25,<br />
Th~ery, I., Delecl~isc, A., T~~iiayo, M.C., Ordiu, S., (1997) lde~~t~li~iit~o~l ufi~ gi'lic liir<br />
CytA like lieniolysi~i fro111 Butiilus ihurOiyi~,~ie~~~ subap. nicdclii~~ i~nd expscs,loli 111<br />
a crystal ncgativc 8. Tl~uri~igie~~esis SI~,III~, ,4pj)/ie~l U I I ~ E~rvi~~o~i~~~oiirrl<br />
M~crobiolug\~, 63: 468-473.<br />
Thomas, W.E., Ellar, D.J., (1983) Mecli;i~i~s~li of actio~~ of Elr~cr/irrs ~Irrir~rig~r~~~rs \'at.<br />
isruele~r~rs i~isccticidal 6-endotoxi~i, FEBS L~,//e~lv. 154: 367-368.<br />
'Tilomas, M.K., Ilose, I
.-A.<br />
Uawithya, P , Tuntitippawan. T., Katzcnmeier. G.. Patiyitil, S.. A~~gs~~thil~i;~so~iibi~t, C.,<br />
(1998) Effects on larvicidal act~vity of single proli~ie substitutlotis In a3 or a4 of<br />
the Bacillrts rhttri~ryie~tsis Cry4B toxln, Biociie~~iisl~:~, trii~! Mol~.cri/ar Biolug~,<br />
In~en~iirio~rol, 14: 825-832,<br />
Urwin, P.E., Lilley, C J . Mcplierson, M.J., Arkillsoil, H.J , (1997) Rrsist;lnce to boll)<br />
cyst and rootnot iiematodes conferred by ~'IIIS~CIIIC .4robit!opris cxl,ressitlg ;I<br />
modified plati it cyst;ili~i, Plciiir Jurri~~riil, I?: 4.55-46 I.<br />
Va~n, l'., Worland, B., Clarke, M.C., Ilicli;~rd, (j., l3e;1vis, Rl., Liu, H., Kohli, A,, Lcccl~,<br />
M., Snape, J., Clir~sto~~. P.. (1998) Exprrssto~i of;lri c~ig~ncercd cyslclnc prutett1;rse<br />
inhibitor (Oryzacystatin-I delta DX6) for nenlatode resistal~cc i i ~ tm~isgcl~ic rice<br />
plallts, Tlreoreiicctl o11d Applied Ge~trtics, 96: 266-27 1<br />
Xani, A. K. S., Rcddy, V. D., (1996) Morpllogencsis fi.01~1 callus culturcs ofcli~ckpca.<br />
I .<br />
. ~<br />
Cicer ~irieii~~tl~ii L.. l~rdiaii Jurrr~rcil oj Er[~eri~riorrit/ Biolu,g,, 34: 285 - 2x7.<br />
Vardi, A,, Ble~ciiman. S., Avlv, D., (1900) Genet~c tratlslur~i~atioti uf Cttrrt, lprolul>l;~at\<br />
and regtrleration of~rat~sge~iic plants, Plitri Sr.ii,itce, 60: 199-206<br />
Var~sainioh;~~iied, S , Jayabal;in, N., Kislior, P.U.K., (1999) Plantlet regeticratluri fioli~<br />
cotyledo~i derived c;~ll~ of Ilorsegram (Mttc~rriry!ui~~u~rijlorir~i~ (Lam.) Verdc)].<br />
Venkatachala~n, P., Jayabalan, N.. (1997) Selectiol~ of groundnut plants wrtl~ c~iliar~crd<br />
reststancc to late leaf spot through In vlrro ~nut;~tion technique, I~~cur~rutiotirtl
Venkatachalam, P., Klshor, P.B.K., Jayabala~i. N., (1997) I11gh frequency sonlatic<br />
enibryoge~iesis and effic~e~it pla~~t regelisration<br />
tiom liypocotyl oxpla~~ts 01'<br />
groundiiut (Arucliis liy/)ogcirt~ L.), Cirrre~ir Sc,ie~icr, 72: 171.275.<br />
Venkatachalaiii. P.. Geetlia. N., J~yabalnii S;~~.ava~labobu. N., Sic;\. L.. (1')')X)<br />
Agrobac/eriii~~i-iiied~i~ted gelletic tnnsfur~il;~t~o~i of g~ou~idill~t (,Iror.l~i~ 1uy10gti~ci<br />
L.): All assessmelit of tl~ctors :~fli.cting regenc~.;ltioii ot't~.;~~isgc~lic ~I:III~s. Joiir~i~rl i!/<br />
Plri~rr I(aec1i~c11. I I I. 565.572.<br />
Venkatacliala~n, I'.,<br />
Geetlia, N., Ablia Kii;i~idelw;~l., Sliilila. MS., S~ta. G.L..<br />
Khandclwal, A., (2000) Agrobucieriui1i-111edii11cd genctlc ~ransl'or~iiniioi~ ;lnd<br />
regeneration of trolisgelilc ]pl;i~~ts from co~yledoii c~pl:1111s 01' grou~idii~~l (Ar~r~lii~<br />
11ypoyuc.u L.) vla so~iiatic e~iibrqogsncs~a, ('iirrC~ri Scie~rce, 78: 1 130-1 136<br />
Verno~i, R.S., Broalcli, J.S., (1996) I
__--<br />
Wa_a_s5JL(l1)96) "Yes. But Does It Work in tile Field'!" The cliallengc of tecll~lology<br />
-<br />
transfer In biologtcal conlrol, E~iiu~riuphiiy~i, 4 1 . 315.332.<br />
Waage, J.K.. Mills, D.J., (1992) B~oIog~c:~l CUIIISUI. 111 N;I~UTJ~ Eti~tllics: TIIC<br />
Popuiattoll Blology ol' Prrd:itors. I'nl-;rs~les, ittld Diseaacs. Cr;~\vley. M.J.. 1.d.<br />
Bl;lck\\ell Sctcnl~lic l'ubl~c~t~ons. Oxlbrd.<br />
W;III, Y.. Lc~naux. P.G.. (1904) tii.11cri111~11 of 1.11gc II~II~I~CI\<br />
oI' i~l~i~~iutldut~tly<br />
t'inst'ormed fcrt~lc. b;rrlcy p1;111ts. IJloiir IJi!)s~ulug~~, I04 37-4s<br />
Wang, 1-1.. Ciltler. A.J.. (1995) Proillulcrs ti0111 kin1 and cor6.6, I\\u ,.l~.iil~iili~~)sis<br />
ilicilic~~rci low tui~~pcrnturs 2nd ABA it~ductblc genca, d~rccl s[ro~~g ~-glt~ci~rot~itl;~a~<br />
exl)rcssloti in gt~iird CCIIS, pollen and youtig dcvelupitig scuds, I'ier~~i ,Ctolec,iricir<br />
Uioiug)! 2s: 6 19-634,<br />
Wol~g, J I1 , Rose, I< J.. Donaldso~~. U I .. (1'196) ,ly~.ob~ri,rc~.iii,,r-~~~ud~;~lctl<br />
cransfortn:~tiot~ 2nd cxprcsstotl ut' tbrcign gcncs ill Maliuigu rrir~~~oriilo, Au.srrciiir~rr<br />
Joiirricii it/ i'iri~rc Pig~siulug,: 23. 265-270.<br />
Warkctititl. T.D., McHugIlen, A,, (1091) Cro%n gall t~itlslurrnat~o~~ ul le11l11 (/.L,II.\<br />
L.N/IIINI.~A Med~k ) \bit11 v~rulent srrai~ls of Ayrubuc~eriii~~~ lu~ricjucie~r\, l'iuirl ('el/<br />
REPOI.IJ, 10: 489-493.<br />
Wet, ZM, Xu, Z.H., (1990) I'lanl<br />
regcncrauuil li'om prutoplusta US Itnnlalure<br />
cotyledons oi'L-'i)ci~ie ~ojo Sieb. et Succ., Plii~~tSi ir~ite, 70. 101-104.<br />
Weeds, J T., Anderson, O.D., Blcchl, A.E., (1993) Kapid Productton ol' niulttple<br />
independent Itnes offertile transgcnlc wheat (PIII~IIII ue\iivrrm), lJlunc l'iiy.sioluyv,<br />
102: 1077-1083.
Winans, S.C., (1992) Tiyo way chemical signaling in Ayrobncierilriir plant iliteractions.<br />
Microbioiogicrii Rrive~rs, 56. 12-3 l<br />
Wirth, M.C., Georgliiou. G.P., Federici, B.A.. (1997) CqtA cnablcs Cryl\' eiidotux~l~s<br />
of Bucillus iiiirri~igieiitsi.~ to overcome lhigli levels 01' CrylV resisf;~licc 111 thc<br />
Inosquitu, Cii1e.v cjiri~i~~ii~/irsc~rti/~i~, Procrct111iy.s oJ' Niirioirrrl<br />
riti~.ii~i~. (?/ .F~ic~iic~c~,<br />
b!SA, 94: 10536-10510.<br />
U'U, D.. Feder~ci, B.A., (1993) A 20-kiIod~11tu11 p~oteil~ prcs~'rves cell vinbtlity ;III~<br />
proriiotcs CytA ci-ystal forliiatlon durilig sl)uri~l;itio~i 111 I~'iic~~lliis ii~rrriiigicir,~is.<br />
Joiirircii oj'Boc.rerroioy)~, 175: 5276-5280.<br />
Yardim, EN.. Edw;irds, C.A., (1998) El'ticta ol' cheliiicul pcst d~seasc niid weed<br />
~~laiiilge~nciii 011 tlie tr~pliic sIri~cturc US tici~iiitod ~O~~U~;IIIOI~~ ill to~li;itu<br />
agroecosystci~is, r1pj)iirtlSoii Ecolog),, 7: 137- 147.<br />
Zaeneil, I., Van Larebckc, N., Teucl~y. H.. Vaii Muntagu, M., Sclicll, J., (1074)<br />
Superco~led circul;ir DNA 111 crown gall ~~itllic~~ig Agrvb(~cieri~iiir atrittiis, Juir~~itil oj<br />
:Llo/ecir/rir b'i~/og)~. 86: I U9- 127.<br />
Zaliir, Y., Ncn~, I!.,<br />
Doni~atli. I;., Pupilli. I:., Arctur~i, S., (11j05) I'lnnt rcgc~icr;~tloii<br />
iioni cxplnnt, and prutupiast derlved calli~scs of Medicugo Irr~oruii~, IJiuirl ('ell.<br />
l'issiic ciiici Orgciir C'~ilrr~rr. 4 1 : 4 1-48<br />
Zaglimout, 0.M.17., llollaiid, M A,, Torello, W.A., Polilcco, J.C., (1990) ,2 gclicral<br />
procedure for protoplast recovery, callus and plant regcncratiun in plants, 111 Virru<br />
Cellulur ri~id Devciopi~reiiierl Uio/ug~-i~iun/, 26:) 15-317.
Zambryski. P., Holsters, bl., Kruger, K.. DepicLer, A,. Scllrll, J., V.ln blontagu. M..<br />
Goodman, kI.M., (1980) Tti~iior DNA blrucrllrc in plant culls c~,~nsl'or~iicd by ,.I.<br />
lirn~clfacie~~s. Stie~icr, 209: 1385- 130 1<br />
Znnibryski, P.C., (1992) C'hrol~~cles from tllu .Agrobi~creriroir plat11 crll DNA rl.;insli.r<br />
Story. .~111ill~l/ fi('l'l~'\l
1<br />
APPENDIX<br />
1. hIURASRICE AND SI(SO)<br />
--.-<br />
I<br />
0.025<br />
~- ~<br />
OR<br />
Orgn~iics (X100)<br />
27 8 2.78 giL<br />
Glycine 200 mdL ~<br />
10 1111<br />
h'icotir~ic acid ) 10 1111<br />
Thiamine HCI<br />
Pyridoxine HCI i 0.5<br />
100 myL<br />
111-lnositol ! 100 5.0 g5UU rill I I0 1i1i
2. Fixative used for fisntio~~ of tissue culture sun~l~lcs:<br />
Acetic acid and 95% Etllallol were ~ilixed at 1:3 proportions<br />
added. The co~ripo~ie~its wcrc sliakeri tint11 ill1 oftllelir ;~rc ill sol~~tlo~l. 'l'llu color oitl~c<br />
stain was redd~sll violct. Fol. Eosin stock sulutioli, I y of liosili (w:iIci. solilblc) \\as<br />
dissolved in 20 1111 ofdistilled water and 111;ldc lip to IUU rill \\it11<br />
05'i." ctl~;~~lol<br />
3. DNA exlrilctio~~ buffer (DELLAPOR'SA 111et11od)<br />
100 nlM Tris pH8<br />
4. Pri~nilry IV:I~~I buffer (I litre) (for Soutl~er~~ i~n:llysis)<br />
1 SDS 0.5% 1<br />
5. Secondary a'asl~ buffer (ZOS stock)(for Suutl~err~ c~~l:tlysis)<br />
Tris base I21 g. IM<br />
I'laCI 1 12 g. ?M<br />
Adjust pH to 10.0. M3ix i~pto 1 Iilrc wit11 water, l'h~s call be kept for up to 4<br />
months. 1 :20 I.e., 2 mlIL of lbi MsCl>
Table 4.1<br />
Induction of sorrlalic erllbryos OII hlS cor~t:~i~~i~tp eo~ttbi~~iltio~ts of 2,4,5-'1' (2.0 alld<br />
5.0 pM) wit11 TDZ, BAP, liilletir~ or zeatitr. Tile results rvere recorded at the elld of 4 r\eeks<br />
and represent meails of tlrrce replicn~iol~s.
Table 4.2<br />
Induction of elnbryos 011 hlS cont:lining con~binatio~~s of 2,4,5-'1' (10.0 and 15.0 phl)<br />
~ith TDZ, BAP, kinetin or zealin. l'l~e results were recorded at tile end of 4 \verks and<br />
represe~~t means of tllree replications.
Table 4.3<br />
111ducti011 of en~bryos OII hlS containing conibiaations of 2,4-D (5.0 and 10.0 pkl)<br />
with TDZ, BAP, kinetin or zealin. The results were recorded at the end of 4 weeks and<br />
represent means of tllree replicntions.
Table 4.4<br />
Induction of embryos 011 hlS co~~taini~lg colllbinstio~~s of 2,J-D (15.0 :III~ 20.0 pkl)<br />
with TDZ, BAP, ki~lrti~l or zealill. The res~~lts were recorded nt tllc c11d of 4 \kceks and<br />
represent ~~leans of t11rce replications.
'Table 4.6<br />
Dificrent combinations of plant growth regulators used to acl~ievc maturation of induced somatic cmbr?.nids and responses observed
~<br />
Table 4.7<br />
lnductio~~ of multiple slloots from mature embryo axis esplu~~ts on lllediu<br />
containing BAP as the principal grotvtl~ regulator. A total of36 espls~~ts per<br />
treatment were cultured and there was 100% respo~lse in terms of ou~nber of<br />
expla~~ts responding. Tl~e results were recorded at the time of2"\nad 4"' weeks.<br />
All the results :Ire the nleall of three replic;~tes.<br />
NO. o1'shoo1s<br />
pcr c\l~l:~nt<br />
(nlicr 2 wcck)<br />
No, of shoots pcr<br />
cxpla111<br />
(;Ilk 4 wcck)<br />
~-<br />
.- -- --- - . .<br />
JBC' I 7 2 + 0.6 11.3+0.6 !<br />
-<br />
JBC ? T-7- 20.3 + 1.2 .<br />
-____-_t-<br />
- -----. .<br />
JBC 3 20 1 2 9.7 + 1.3 17.7 + 1.2<br />
-- -- . .-<br />
JBC 4 30 2 10.7 + 1.5 13.0 + 1.0<br />
-- -.<br />
. -.- -- .<br />
JBC 5 40 2 8 3i 1.4 11.3? IS<br />
-- -. . . .<br />
..-.-. --.<br />
JBC u 50 2 0 0 + 2.0 9.3 ? U.0<br />
-<br />
JBC 7 I00 2 37+ 3.5 5.1 i 2.0<br />
JBC S<br />
-.<br />
5 5 8021.0<br />
9.7 + 1.2<br />
JBC9 1 10<br />
5<br />
11.3 i 1.6<br />
17.0f 1.0<br />
1 1 .0 + 2.0<br />
JBC 12 40<br />
-.<br />
83i1.5 10.3 + 1.6
Table 4.8<br />
Effect oI'TDZ, 2-iP :~nd ki~~eti~~ 011 sllouf regcnel'atiu~~ fron~ tl~ esplar~ls<br />
derived fro111 asillary nieriste~~ls ofcl~ickpe:~. 'l'lle ~.esults were recorded :it the e ~ ~ d<br />
ol2"%nd 4"' \veeks and tl~c values :ire III~:IIIS oI't111.ee replicates.<br />
Media<br />
JCII ?<br />
I<br />
- I -<br />
JCR6 ( 20<br />
.<br />
JCR (I<br />
-- . -<br />
/<br />
3.3 f 0.6 4.7 i 0.6<br />
JCR l I 2 1 32 13.0 i 2.0 23 0 i 2.0<br />
1 ;; ~23,3?5";.6j<br />
JCR 12 4 5 1 4 1 32 1 15.3 i 1.5 20.3 $06 /<br />
1 JCRl3<br />
-- .<br />
JCR 14 14.7 12.5 25.3 i 2.5 1
Table 4.9<br />
Effect of pH of tile cllltare 111cdiu111 OII ~llultiple slluot rege~~craliu~~ Ibrn~ Hie<br />
anillory ~lleristem expl;ll~ts of ellickpen. Results !+ere ~.eror
Table 4.10<br />
Effect of i~lclusio~~ or cotyledo~l tissues along wit11 ll~e regeaeratisg axillar!<br />
1nerister11 on sl~oot for~l~i~~g cuplcity of tile ;~sill;lry ~neriste~n expli~~~ls. Results nerc<br />
recorded fro111 1 to 5 weeks to sl~on the pro~~~otio~~<br />
of rege~lerati~~g nbilit) and rate<br />
of multiple sllool illductioll b) the i~lcludcd cotyledoo.<br />
No of slloo~s per rcspondinp esplil~~ls (weclis)'
Table 4.11<br />
Effect of age of tllc espla~~t donor seedlings on rcgencration capacity of<br />
different espla~~ts derived fronl axillnry nleristenls. Regenerating multiple slloots<br />
were counted in the third \~ecli n ~ llle ~ d values arc mealis of three replic:~tes.<br />
Seedling<br />
age (4<br />
AM I<br />
No, of slioots per explants'<br />
AM2<br />
AM3<br />
AM4<br />
2<br />
17.0 i 2.0<br />
17.3 i2.1<br />
4.0 i 1.0<br />
17.3 i 1.5<br />
4<br />
15.7i 1.5<br />
18.3i 1.5<br />
6.7 i 1.5<br />
22.3 i 2.5<br />
1<br />
6 4,<br />
8<br />
10<br />
12<br />
10.7 i 1.5<br />
10.3 i2.1<br />
7.7 i 3.1<br />
10.0 i 1.0<br />
23.3 i 2.1<br />
24.0 i 2.6<br />
17.7 i 1.5<br />
12.7 i 2.1<br />
3.7 4 0.6<br />
7.3 i 1.5<br />
3.7 i 0.6<br />
2.7 i 0.6<br />
27.7 4 3.8<br />
22.0 t 3.0<br />
19.7 ?: 3.1<br />
~<br />
15.7?1.5<br />
I<br />
I<br />
14<br />
I I<br />
6.7 2 2.5<br />
6.3 f 1.5<br />
13.3 t 1.5 1.3 t0.6 11.7i 1.2<br />
10.3 t 1.5 1.0i 1.0 1 07il.5 1<br />
Mean f Standard error of tlirce replications
Table 4.12<br />
Induction of multiple shoot buds from various seedling explarlts derived<br />
from axillary meristems. 'The nun~bcr of multil)le sl~oots were counted in the tl~ird<br />
week prior to their transfer to the sl~oot elongatio~l medium.<br />
Embryo axis<br />
13.7 i 1.5<br />
I<br />
Shoot tip 40 36 (90.0) 10.3 i 2,lJ<br />
I<br />
Axillary bod<br />
40<br />
40 (100)<br />
17.3 i.2.5<br />
AM I<br />
AM? i.<br />
40<br />
40<br />
33 (82.5)<br />
37 (92.5)<br />
23.7 k 0.6<br />
30.3 i 2.9 ,*<br />
1<br />
AM3<br />
40<br />
23 (57.5)<br />
7.7 t 2.3<br />
AM4<br />
40<br />
40 (100)<br />
31.0i 1.7<br />
I<br />
Meal, i Standard error oC tllrec ~.eplicntioils
Table 4.13<br />
Effect of media compositions OII elo~igation of the regenerated sboots. Results<br />
were recorded from three replicate experiments involvillg sl~oots induced 011 JCR13<br />
medium (see Table 4.8)'<br />
CEL l<br />
2<br />
Plant yrowth regulators (yM)<br />
2<br />
1.2 1 2.3 1<br />
I<br />
I<br />
CEL3 1 -<br />
2<br />
2 1 -<br />
I.!<br />
CEL4 1 -<br />
4<br />
5 2<br />
1.7 3.1<br />
2,5<br />
CEL 5 2 0.3<br />
I<br />
1 I<br />
CEL 8 1 5 ill 4.8 I<br />
%ach replicate experimeih consisted ol'a total of 40 explar~ts with induced slioot<br />
buds at 18 to 23 per expla~~t and the ~~uniber ol'sl~oots elo~igati~rg per cxplant was<br />
the average of all the 40 esplants. Datr compiled from tliree replicated experiments.<br />
klongatio~i I: number of sl~oots elonfgated per esplant by the end ofsecorid weck;<br />
Elongntiorl 11: ilunlber of sl~outs elolignted on the next two or three subcultures.
Table 4.14<br />
Effect of media constituents on rooting of ill vitro formed aud elo~~gated<br />
shoots of cbickpea.<br />
CPR 2 (MS) 3.0<br />
CPR 3<br />
CPR? 1,,2.5 1<br />
i:: 1 ; 119.9<br />
1 5 , 7 164.00<br />
CPR 8 3.0 16.8 7.0 59 50<br />
-<br />
CPR 9 2.0 5.0 27.2 12.4 99 00<br />
CPR 10 j 2.0 1 10.0 I - 17.1 / 3.5 51.50<br />
CPR I1 2.0 I - 1 5.0 3,9 9.75<br />
- -<br />
'All the media aere used as liquid rontsi~~i~lg filter paper bridge. CPR3 to CPR8<br />
contained sucrose at various co~~ce~ltratior~s wit11 IBA as the rooting hormone added<br />
at 5 phf concentration. CPR1, CPR2, CPRII n ~ CPRl2 ~ d did not contain Phase 2 as<br />
there was no pulse treatment with IDA.<br />
b~esults were recorded at the end of 3 weeks for Pl~ase 1 and 5 weeks for Phase 2.<br />
Each combination tested uader three replication. Total umber of sl~oots per<br />
replicate was 40.
'Table 4.15<br />
1.etl1nl dose testing :III~ effect ol"SU% on Iclll:ll el'kct of I;:ll~:~myci~~. Nu~l~bcr<br />
of days of espla~~t survi\al was cou~~ccd ul~til tile chplal~t ble:~cl~ed or sllo~red II~<br />
signs of growtl~.<br />
No, ofdi~ys of expin111 surviv;~l<br />
25<br />
10<br />
-. . .-- -
Figure 3.1<br />
Dii~gralll~l~i~tic rcpresc~~tatioo of the cxplullts derived from llilture embryo<br />
;Iris, ~~lu~llulc, r:~tlirle, sitlc ;I~IIIS 811d ~llitltlle portion. (Arrows sllows sites of<br />
surgery)
I'relii~rutior~ ofi~xillary ~lleristen~ exl~lants AMI, AM2, Ah13 and AR.14.<br />
Final sti~ge is tllc stilge of regeneration of niultiple stloots after one week of<br />
culture 011 slloot i~~duclion 111ediu111. Large arrows sl~ow progression of<br />
prep~':~tion ol' cxl11;111ts. klediu~n arrows sl~ow sites of surgery and snlall<br />
arrows sllow silo ol'1n111tiple sl~oot regeneri~tiol~.
Figure 3.3 A - C<br />
Diagrn~nn~atic represc~~tatiu~~ ror preparation ul'axillary n~eristem erplant<br />
(AM2) showing the sites of surgery. A, 6-day old seedling sbo\ving prorl~iner~t<br />
i~xilli~ry butl, B. I'ruccssing of the i~xillary ~neristem explant (arrows shons<br />
the sitcs ol'surgery), C. Axillary n~eristc~n explant (AM2) sl~owi~~g the axillark<br />
~neristeln r~giun \vitl~ il~tilct cotyletlo~~.
Figure 3.4<br />
llestrictio~~ Innp of tl~e pl;ls~~~id pICI'99:GUS-Iet used for biolistic-<br />
nletlintcd gelle tr;il~sfer.
I<br />
. Nhu 1 51 ?C,%-<br />
.pal5125 &pa15125 1.-<br />
Iito 1 51 2 7<br />
-.T.i I,IPT I1<br />
ti710 by<br />
cu K\! 1640<br />
Ilcm, 1457:<br />
Act 12938<br />
___-/------<br />
Syh 12941
Itestrictiol~ III;I~) 01' the plasn~id pHS723:Bt used for ~igrobac~erirrin-<br />
llletllod of tr:r~~sforl~ri~lioll.
I
Induclio~~ of son~atic cn~bryos from mature embryo axis and leaflet explants<br />
after 6 weeks of culture on Il~c medium containing 2,4,5-T as principal growth<br />
rcgslntor. A, B and C. lntluctioi~ of embryos from illature embryo axis explant,<br />
I)) Induction of somatic cn~bryos from lcallct explal~t.
Induction of somatic embryos from mature embryo axis explant by using<br />
2,4-D as principal growth regulator. A. Top view of the embryo axis showing<br />
multiple globular embryos, B. Lateral view of embryo axis slioning multiple<br />
globulnr embryos formed from plumule region.
Effect uf co~~centrr~tio~t of BAP on mllltiple sltoot regeneration from mature<br />
embryo nxis erplal~t 8ftcr 15 days of culture on sl~oot inductiol~ medium. A.<br />
Multiple sl~oot regeneration at 10 pM BAP concentration, B, hlultiple sl~oot<br />
rcgeneri~tiot~ wit11 30 phl BAP, C. Multiple shoot regeneratio11 with 40 pM BAP, D.<br />
Multiile sl~oot reget~eratio~t wit11 50 pM BAP, D. Multiple shoot regeneration with<br />
100 pM DAP.
I.iy111. J.5<br />
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?<br />
\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~<br />
tiabuc<br />
t~r~llurc IIICIILI~LI<br />
r~bilrg :i\ill.~r) III~~~$I~III<br />
~AIJI:IIII (.AiIi?) ohI;~i~icLI fro111 ill t,i!?o Krn!\]]<br />
seedii~~gs of cl~icl,i~~:~. 4. .\\ili;rr! riier.iste~r~ cq~l;r~~t (.L%I2) UII llic lint (lily 01<br />
CIII~III.C oil \l~ii~t ir~ciuetiulr ir~edii~~~~, ii. S11oot buds r?g~r~er:i:ii~p ~'~OIII<br />
I)! Ill? r?i1111\.:1l 01 :1\il1;11.) b:111 :il'ler 7 d;r!s.<br />
ori:ii~:iriir~ II.~IIII<br />
\it.\!<br />
d:i!b<br />
:he 1.cgio11 icf't<br />
C. Cl~ibtcr 111' 111uI1i~~lc s11oot 1111db<br />
IC~~UII lei1 11) IIIL, I-CIJ\ :I! oi;r\iiI:tr? !iricl ;~ilcr 12 tla)s, I). .A clo\e~-<br />
ut llic 111ul1ipIr \I~oi)ta I~KC.IICI.~~~II::<br />
I'ru111 1I1c :i~ill:il!~ IIIE~~S~CIII<br />
e~pI:~~rt :rller 15<br />
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<br />
113)s yS CIII~UI<br />
L' OII b1101it elo~rg:~liu~i ii~ediri!ir, I:.<br />
Rooliirg ci1'llie eloi~g;rlccl slroot bnd<br />
UI<br />
the liilcr p:ilicr Ill-iilge ir~irnci~sc~i ill liquitl roolil~g lnediunr :~flrr X [I:r!s<br />
II~<br />
c~riturc~, (;. I'icli~rc blio!\il~g 0.r i';lro slxtic II~~I~U~IOII~C~<br />
hjhle~ii for- II:II-~?II~II~<br />
r(jtjte11 \IICII~I<br />
of tiiv<br />
;rlter I5 il,~j$, [I. ,411 :rlrcr~~:>li\c 111et1rod l'or li:tr(ierri~ig uf I11e ~II:~IIIIcI<br />
uhtai11~11irroug11 tissire ciiltrirc ir~ rvlricli ll~c IWJI ,!sten1<br />
tlrc cultu~r f~ikrg ol tire cr~ltirr.~~ 111be I,cpt UII~II,<br />
?IIO!V~II: IIU~III;~~ 1111~r1il1oiog~.<br />
>\,:I\ irir~nt~~-re(i ill air1111 rtilll<br />
1. H:i~.~lei~etl :rriil t~-;inbpli~~~lcti plai~l
IIistologic:~l stutlic\ of dcveioprllcl~t of<br />
~rluiiiplc tneribtcri~oitl, f'ronr<br />
;raill;rt.y 111eriatet11 rcgiou of hhI1 capla111 al'tci. ll~e re~~iov:tl of ;cxilI;t~-)' bud.<br />
.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)<br />
bud rcl~lov;il, 8. I)cl,eloprt~e~~i of i~rcriate~~roid, :I[ tlie I):rs;~l portiolt ol';~~ill;~r!<br />
botl or1 tl;ry-.? (il~' III:I~~ b ~ ;~pl~e:tri~~g<br />
~ d<br />
ih ;I sliuol hut1 ct~~el-gir~g l'rutn [Ire<br />
I);Is:II<br />
poriiot~ of the rc~~>n~e(l ;~xilI:~ry IIII~), C'. A~~IC~I~:IIICC of 111cris1~11roitis<br />
irr tht n\ili:~r! 11leri\Le111 arc;\ 011 d:~)-3, D. I~~cre:~scd 1111mber of IIIC~~'~ICIIIOI(IS<br />
iir L I I ~ :i\ill;lrj ~rlcri>ttrrl are;] oil tl;ry-4, I. 1ucl.c;rse ill rlrt 11ulnlie1. :LIIII<br />
yrul~t11 of rucristernoitlb oil d:l)-t;, 1'. l
C!osrr ol)sei~v:~~iuri<br />
tlc\~clo~)i~lei~l ai~d gru\vtli ol ~~~eristeii~oicls ill ille<br />
:~\iil:it.y rlicri\~i.ili ;irc:i US :\.\ll<br />
or1 tl:ij-4<br />
CY~I:III~. A. C105e1. \ ~c\Y of the ir~cri\teunoids<br />
:~I'lrr. tlic rc~t~oval of ;rrillar) blld, 8. C~.o\\tl~ :III~ di~isiol~ uf'<br />
i~ierislelli:~ticells iir<br />
tl~e i11cri5reri1oitl regiu~i ult day-5, C. (J1.urcil1 :rlrtl<br />
divi\ir~i: ui' iiie~.iste~l~;~!ic cells :rt.c~ur~tl ~iieristeiiloid region oil tl:~!..O,<br />
(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<br />
~ ~ s for ~I-:I~I\~U~III;I~~OII.<br />
.i. 'I'll? 11i;is111i(I<br />
[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<br />
\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<br />
[~l:t111s ol'i.11icl~pca. ;\. :I cloher. vien of'thc 1c:tllei sllo~vil~g GUS acliviiy ill<br />
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