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;I! 37 'C for 3-24 Iiours 111 d;irL. Tlie iias,iy 1111~t111.c \\.;I\ rc~ii~\ed ;i~id tlie ~ I~SUC \\;IS cleared otT chloropli)Il by accji~c~lt~:il cli;~~~ge~ III 70-Ili(i"~ ctl1;111ol LII~II~ llsallz Iiod 110 cliloropliyll. Allcnioli\eIy, for ditticillt tu clear tissuc ;~tid 75"" I;ictic i~cld \vnr iltidcd .ind ,111tocl;i\cd tbr 15 mlllutcs. This gl\c> ;I \cry good 111idge Cur ~)IiuIugr~~~Iiy Tlie tia5~1~ \\'I> IIIULIII~C~ III gIyce1.01 illid ob\er\ed ~lnJer a illlcroacope ~rail.dcrreti Ilno 30 1111 ILI~S 2 I5 1111 ot'e\~rac!~o~i t~i~l'lbr :~iicI I ,0 1111 oS20!o SUS \vcrz ;i~l~Ic~l, hli,~kc~i n~cll for IU scc ;i11d lilt lubes werc i~icub~tcil a1 05 degreca Ibr I0 111111 3 5.0 1111 5hl I'OI~I\~ILII~~ ~~CCIJI~ \L;I~ ;~ddcd. ~ ur~c~etl :III~ IIICLI~~I~C~ :I( U degrees Ibr 2U llllll 1. hlo\t Iprutclllh il~i~l lpolyiic~Ili~ride> ~CIIIU\C~ iiltli llie IIIIOI~IIJIC li-tluilecyl ,ultiitc ppl. 5 Tlic snnlplc \\:15 ce~lt~~~i'~~gctl ;it 25000g ibr 20 111111 ;III~ lilte~cd lllru Miril cIol11 illlo 30 nll tube contalllilig 10 ~ nl ~supropn~iol, ~ll~xcd aiid IIICII~~IICLI ill -20Y(' for 30 111111. 0. U\A \\;la precip~t;~led '11 2OUUiJy Sur I5 111111 illid ~IIVCI.III~@ tlie lubes 011 piipcr towels tbr 10 111111 dried pellct. 7 Tllr pcllct was rcauspended ill 5UU ~IL 'I'L 8 111id IU ILL of' IKU;lse A solution was added (5 mdnil). 8. The samplc w;ls i~icubated n\ 37'C for 3U 111111. I vu1 (500 jiL) PI~uIIoI: ~hlorohr~n nllxture \\as added, lii~xcd \~goruuhl) ;III~ cc~ilr~S~~gcd ;it 12,000 I~II~I /br 2 mill. Tliu 7s
SII~)~I~XI~VI~~ \\,la t;~hen (;I~~UCOIIS plldst) alij I 101 (COO 111.) ~I'cIIIo~~I~~)sII~ \\as added, mixed and cc~itnf~~gcd ;11 I!.OUU Il1m lor! nili1. 0.1 yo1 (50 111.) of3hl soclil~iii :lccl;itc (pll 5.2) \,;IS nddsd to tlic rllpeni;ltelir ;111d lllc DhA \\,I> prcc~p~tnrcci \r111i U.S ~ o(400 l 111.) ~soplops~iol. Tlie s;~~ilple \\:IS nllo\\d lo slalid ;II roo111 te1111) 1;)s ! IIIIII ;111d cc~i~r~li~gcd $11 12.000 ~11111 hr 10.15 111111. Tlie supcr~~.~lc~ir \\;IS rc~lio\tti ,111cl IC I~CIICI \\,ir \\;~\l~cd b11cl1) \\ill> ice-cold XlUu CIII,II~UI. 'I Ilc lpcllc~ \\,I\ :~~s-elr~cci 11rio1 to cli\~ol\ ills IIIC l)h 1 III ?i!l!-.~OO 111, 1'1;. Gc~ion~lc [IN/\ of piir,ilivc ~r,~~islbrni;in~\ ol'cllicLpc:~ \
Page 1:
GENETIC TRANSFORMATION OF Cicer ari
Page 4 and 5:
Certificate Ger/$ed /La/ /Ae eldire
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I deem il as a great pleastire to e
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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
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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
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Figure 4.9 A - C Alternative llleti
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Figure 4.15 A, B Restriction analys
Page 28 and 29:
Figure 4.20 A, B DNA profile of gen
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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
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(Nene and Haware, 1980). The legume
Page 38 and 39:
jf.0 REVIEW OF LITERATURE Large par
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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~
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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 86 and 87: Lec!/i?is: Young anci juve~iile lea
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 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)
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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
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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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