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Lec!/i?is: Young anci juve~iile leallers, semi-iiiaturi. leatlets ti.oni thc ni~ddle portion of the seedli~ig and ~ii;~tiire leallcts ti.otn tlie bilsal p;itls of7-day-old scedli~ig were sep;lr;~ted and cultured oil SIM. Lrq Dii~e. Tlie dr-co:itcd scuds wcrc pcr~ii~~i,~tcd u~i hlS lbr 7 d.~yb 11r1or to obt;111111ig tile leal'cxplti~it. Lcal'b;~sc (pct~olc base) II~;I ~iic~~stircd ;~but~t 3 111i1i W;I~ C\CI~C~ ;111il c~~lttircd on tiit SIM. //ip~~~o(~/: The orcr~i~giit so;iked ;il~ dc-co;rlcd jccds \vcrc eriii~~i;i!cd oil MS and 4 to 5-day-old seedlings were bclcclcd for dcr~v~iig tlic ~~I~;IIII 'Tlic stelii 11;11t that was just abo\e tile cotylcdon,iry liotlc jt~~iclio~i iv~~h csc~vtl a~~tl 111s sliout 111) rugion was rcniovcd. Tlic explant tha ~iieasurcd :iboui 4 iiim \v\.;ls cululred on SIM. Epicu~l: Tile ove~.nigIit soohctl and dc-co;~tcd scccis \+ere pcr~iii~i:ilctI on MS and 4 to 5-day-old sccds \\ere stlccted ib~. obl;ri~iil~g tlic c\pl;~~it Tlic ~~ly)er niost Iparr ol'lhc tap root, just below the cotyledoii:rry iiodc jt~~ictiu~i \+;I\ exc14 and tlie cxpl;i~it ~iicasuring 4 mm devoid ol'roor 1111 rcglon was cult~~rcd on the ~~iductioti ~ncdiu~ii. Roo, A?~I?I~,II/.Y "1111 I.UIJI /I/). Root \egliielilb ;111tl root lip MCSC ehcibcd fro111 7-day-old sccdlingr and cultiired ~epa~.ately on tlie i~itlt~ctio~i iiicd~ii~ii. A.~rIlory bi11i. T\io types axillary bud uxpla~ila wcrc prcparcd. Tlie ~iia~jor v:iriatioii for preparat~on of these cxpla~its wt~s their gsl-~iii~lation paltcrli. Tlic first onu, dcsignatud aa ABI, was prepared by excisi~ig tlie avillary bud from a 7-day-old secdli~ip hat was gerniiliated on hlS medium. Tlils nx~llary bud ~iieoaured around I to 1.5 mm In leiigtll. Tile second one. 57
des~gnated as AB2, !\.as prepared by excising the ax~lla~y bud ti0111 a 6 to 'i-J;~y-old seedlings tliat were ger~~linated on MS conr;~ining 5 pM tliid~i~/~~ron (TDZ), This nxllla~y bud nieasured approx~niatcly ? to 3 nil11 ('r~iilc~~li~~ilr~~i~ liori, Tlle oicr111g11t ~ooLed ,~nd tic-co,itcd 5ci.d. \\ere gcr~i~~~i;~tcd on 11s. 3-dnyold scedl~~ig was t:1ke11 ;\lid tlie slioul t~p, root tip u~ltl cotylsdo~~s ucrc re~uovcd. Tlie cotylcdonasy node ju~ic~iu~i th,it ine;~surcd 3 lo 4 lii~ii \\';IS CIIIIII~C(I 011 Slhl A.v~//oI~I~ 1111~1~1~1~~111 L'Y/J/~III/\' ~\~>~;llll ~~~CpLIRlll~ll ;llld tl~l~lll~lllOll 011 111~ ~Lll~~lr~ mediuni Is cr11cl:l~ tbr o~~lIl11lllll U1'g,lIIoQeI11~ I.L!S])OllhC. O\\lll@ I0 I~IC d~lksc~~~cs ill t l l ~ gesmlllatloli lp:~ttcrli, age ol'tlic hcetll~ng ;inti proccasllip. four typch of lllc ;~xlll:~ry l~~cr~slclii cuplal~ts were prcparcd anti they \+ere n;~mctl ;IS 11M I. Ahl?, AM3 ;111d Ah14 (see 171g 3.2 for diagr;i~i~lii;~~~c 1relxe%nt:1t1011 oi'e~l)l;~~n ~)scl~:~~tiuli). 'rliclr I~~L!~;II';IIIUH ii ;I\ li~llow\. AM1 explant: Seed coiit ol' the sust~ce stcsili~cti a~itl oicr~iiglit su;lhed seeds W;IS re~iioved nlid tlic dc-coatcil aced\ \+ere gcrni111;ltctl 0111pl;iiii MS. 2-d;iy-old scedli~ig~ were selected slid roo! portloli \baa 1c111ovcd by 1cav111g suliie oi'tlie Ih~~)ucu~~l rcyiol~ 'l'l~cn two cuts here 111:ide tl~roi~gh tlic ax~ll~sy ~ncr~stc~i~, 'I'his rcaultcd 111 1l1ri.e cxl,1;111ts 111;1I 111cludcd two axlllilry nier~steni, one hiloot tip witli cpicotyl. 'lliu iix~lli~ry ~~icr~hle~ii cxpli~~ils ~vllli cotylcdo~intact was na~lied as AM1 i~nd itas cilltllred on thc lnduct~oli ~iiediuni. AM2 explant: Tlic dc-coated secds were gsriiu~~;~ted 011 SIM al~tl ;ifIcr pruniine~~l appe;il.ancc ul'axlllary bi~d, in ,iboul 4 ro 5 tl,~ys. tlley were c;~reli~lly runloved and two cuts were given through rhe ;~x~ll;isy Iiieslstem, dlhcasd~ng rlic root ponlon, rlic rcsulta~it axillary mcristem explants w~th co~yledons i~itacl (A.112) wcse cult~~rcd 011 tile above-mcnt~oncd SIM again.
Page 1:
GENETIC TRANSFORMATION OF Cicer ari
Page 4 and 5:
Certificate Ger/$ed /La/ /Ae eldire
Page 6 and 7:
I deem il as a great pleastire to e
Page 8 and 9:
CONTENTS ABBREVIAI'IONS SUMMARY LIS
Page 10 and 11:
3.2.2.3 Shoot elongat~on 3.2.2.4 Ro
Page 12 and 13:
ABBREVIATIONS 2-il' 2-isopci~~cnpl
Page 14 and 15:
frequency was observed wit11 2,4,5-
Page 16 and 17:
in vitro grown plants w;is ocli~eve
Page 18 and 19:
LIST OF TABLES Table 3.1 Pr~~iier c
Page 20 and 21:
Table 4.1 1 Effect ul'age uf lhe ex
Page 22 and 23:
Figure 4.1 A - D inducl~on of sonla
Page 24 and 25:
Figure 4.9 A - C Alternative llleti
Page 26 and 27:
Figure 4.15 A, B Restriction analys
Page 28 and 29:
Figure 4.20 A, B DNA profile of gen
Page 30 and 31:
1'1.0 INTRODUCTION L~fe is the ter~
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them. About 10% of the Earth's land
Page 34 and 35:
observations GkI Shull's experiment
Page 36 and 37: (Nene and Haware, 1980). The legume
Page 38 and 39: jf.0 REVIEW OF LITERATURE Large par
Page 40 and 41: plants from them enlhuscd scientist
Page 42 and 43: introduction into protoplasts using
Page 44 and 45: evident by 1983 that the Agrobacter
Page 46 and 47: The above promoters were found to b
Page 48 and 49: gained an illstant popi~larity owi~
Page 50 and 51: legume crop species. Cotyledon expl
Page 52 and 53: 1994). Clonal propagation of FI int
Page 54 and 55: were induced to for111 callus when
Page 56 and 57: ' the ernblyos into plantlets on MS
Page 58 and 59: and nprII for selection. PCR and So
Page 60 and 61: Agrobacleriuiii and ill otlier expe
Page 62 and 63: ioteclinologicsl i~iiprovemel~t of
Page 64 and 65: combinat~ons and concentrdtions of
Page 66 and 67: egenerated via somatic eliibryogene
Page 68 and 69: egenerat~on from protoplasts and ca
Page 70 and 71: transformants were identified by sy
Page 72 and 73: urgent econoniical control measures
Page 74 and 75: applications of traps. Technically
Page 76 and 77: in the laboratory and releasing tl~
Page 78 and 79: have been exhaustively reviewd (Dai
Page 80 and 81: larvae of certaln Insects were unab
Page 82 and 83: 3.1 Pl:~nt 111nteri:il sllrl cultur
Page 84 and 85: 0.5. 2.0 and 5.0 pM were added. 111
Page 88 and 89: AM3 explant: This was a by-product
Page 90 and 91: PM) 2nd GAJ (2 5 PM), eltller singl
Page 92 and 93: of the plastic bag was removed rwlc
Page 94 and 95: - . -- laopropyl alcoliol ; Absolul
Page 96 and 97: The sl~des were Illell muiaited and
Page 98 and 99: 6. The bupcniatant \\,I,, t,ikcli i
Page 100 and 101: 5. Microcal~lers were allowed to se
Page 102 and 103: till the growtli of bacterial cells
Page 104 and 105: ;I! 37 'C for 3-24 Iiours 111 d;irL
Page 106 and 107: PCN for 111cl.4 ge!iiJ: I'C'I< tbr
Page 108 and 109: 4. Tliu pel \\as \r;~alicd 3 ti~iir
Page 110 and 111: 4.0 RESULTS $loaf of tllc c\l~cr~~l
Page 112 and 113: These combi~iatiori sl~owed liiorc
Page 114 and 115: egeneratloll \!;IS dli'licull ;~nti
Page 116 and 117: decreased a[ cllkalinc pH \+li~le 1
Page 118 and 119: either side of'bws;tl portiol~ oill
Page 120 and 121: IBA sl~owed bro\\~~i~~g i~lld de:~l
Page 122 and 123: humidity uf 50% 111 the dayrinie a1
Page 124 and 125: 4.2 Histological studies ~II nlultl
Page 126 and 127: sliowetl no liirtlie~ yrowlli Tllc
Page 128 and 129: Conlirmntioll of plllati!e t~.~ll~a
Page 131 and 132: 5.0 DISCUSSION During tile la31 dcc
Page 133 and 134: leatlets (Dilieshkuniar er al.. 109
Page 136 and 137:
used with an add111ou;ll care :u~d
Page 138 and 139:
tissue to regenerate illto lesser i
Page 140 and 141:
prololigcd culture on TUL sho\\~d 1
Page 142 and 143:
exceed 5 cni Icngtl~. Loiigcr shoot
Page 144 and 145:
stem and lravca Secol~d;lrq ~'IIICI
Page 146 and 147:
arnplitication of the sopccIi\i. 90
Page 148 and 149:
Explant Preparation -Geminate seeds
Page 150 and 151:
6.0 REFERENCES Adkins, A.L.. Godwin
Page 152 and 153:
Avenido, R.A., Hattori, I
Page 154 and 155:
Benfey, P.N., Chua, N.1-I., Ilcgiii
Page 156 and 157:
Cnrvalho. IC1.H C dc , Uui Vat1 LC
Page 158 and 159:
Chu, P.W.G., Andersoli. B.J., Khan.
Page 160 and 161:
Deblaere, R., Reyneerts, A., Hotte,
Page 162 and 163:
Drun~mond, M.H., Gordoil. M.P., Nes
Page 164 and 165:
Foster, J.G., Coulombe, B.A., Van S
Page 166 and 167:
Gill, R., Saxstla, I'.K., (1992) Di
Page 168 and 169:
Hess, D., (1970) Versueche zur tran
Page 170 and 171:
Hopkinson, M.J. ;md Collins, I1.M.,
Page 172 and 173:
Karthikeyan, A.S., Sarina, K.S., Ve
Page 174 and 175:
Krens, F.A., Molendijk, L., L., Wul
Page 176 and 177:
Li, Z.Y., Tanner, G.J., Larkin. P.J
Page 178 and 179:
Maxan~, A.M., Gilbert, W., (1977) A
Page 180 and 181:
field conditions. Procc.rdbigs o/Na
Page 182 and 183:
Ohara, A., Akasako, Y., Daimon, H.,
Page 184 and 185:
llechrnlics sur les prnidilies des
Page 186 and 187:
Rand, G.M. (Ed.) (1995) Ftordir~~rc
Page 188 and 189:
Richardson, M.J., (199 I ) Seed sto
Page 190 and 191:
Sarria, R., Calderon, A., Thro, A.M
Page 192 and 193:
Shri, P.V., Davls, T.M., (1992) Zen
Page 194 and 195:
Tegeder, M. Koli~i, I-I., Nibbbe, M
Page 196 and 197:
Venkatachalam, P., Klshor, P.B.K.,
Page 198 and 199:
Winans, S.C., (1992) Tiyo way chemi
Page 200 and 201:
1 APPENDIX 1. hIURASRICE AND SI(SO)
Page 203 and 204:
Table 4.1 Induction of sorrlalic er
Page 205 and 206:
Table 4.3 111ducti011 of en~bryos O
Page 208 and 209:
'Table 4.6 Dificrent combinations o
Page 210 and 211:
Table 4.8 Effect oI'TDZ, 2-iP :~nd
Page 212 and 213:
Table 4.10 Effect of i~lclusio~~ or
Page 214 and 215:
Table 4.12 Induction of multiple sh
Page 216 and 217:
Table 4.14 Effect of media constitu
Page 218:
Figure 3.1 Dii~gralll~l~i~tic rcpre
Page 222:
Figure 3.3 A - C Diagrn~nn~atic rep
Page 225 and 226:
I . Nhu 1 51 ?C,%- .pal5125 &pa1512
Page 228:
I
Page 232:
Induction of somatic embryos from m
Page 238:
I.iy111. J.5 SI:i;i.\ iil ~ ~ l ~ ~
Page 254:
IIistologic:~l stutlic\ of dcveiopr
Page 258:
Rc\[ricfiot~ ;111;1l)si\ ur ill? ~
Page 262:
I'C'R :~llipLiiir:iriuil 01'700 bl)
Page 266:
Fixi1r.c 17 \r:15 -l)K,.\ ;!lid J"
Page 272:
1:igurr 4.22 Sui~lllcrn :III:II!s~$
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