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5. Microcal~lers were allowed to settle for 1 111111. 6. Microcarriers \\ere pellcttcd by sp~iili~iig hr 2 sccuiids ill ii IIIIC~UI'U~I' 111be. 7. Liquid was relllo\ed and dihc;lrded. S I-IU 111. 01 70'; ctI~;r~~ol \ins ;iddcd \\II~ULII dl\t~irht~lg tll~ pellcl 9. Llquid \\;ij renloicil ;III~ di\cilrticd lU. l4U 1iL of IOU"; etIl,lilol \\;is ,idilcd \r~tliuut d15turb111g 11111 ~pcllci 1 I, L~ilu~d \+;15 re111oi cci ;111d tl~s~;~rclc~l. 12, 48 } ll~ 01 IOU0" c1l1.111c1l \\$IS ~lllll~~ll. 13. Tile pcllcl \v.i, gellily r11~~1~j)cticIcd b) i;ij)/)i~~g tlie .\liIe 11I'tlic iubc \c\,cr;11 li~llcs, illid tllell by vorlcxlilg ;it low apecd lbr!-3 \CCOIIL~~. Si\ allquuta 01.6 pi. c,lcli ul' ~n~croc;irricrs \\ere collcclcd a~rd triln\li.rrcd to tile center ol'a ~ii;icroc:~rrlcr. Equ,il ;III~O~II~I~(500 big) ol' ~nicriic;irr~crs c;icli IIIII~ ;iiiiI 10 rpl.cad cicllly over tlic cc1itr~11 I el11 ctr~ 11li11c :I\ ;I g10i11) \\ IIIIIII ;I di;i~i~c~cr 01' I ,5 111~11 Thc la11iiii;ir hood consislillg ol'll~e biollsl~cs gene g(111 ibil\ 1l1i110~1gIlly CIC~IIIC~I \~ilIr IOUUh ethnnol. The petri plate colltaii~ing tile cxpla111\ w;is placed ;II ;In ciplxupri;lte d~at:~nce li.oln tl~e device consistllig of rbe macrocarricr, The explat)ls iwrc bo~ilbardcd with the lnicrocarriers by applyllig appropriate pressure ill tlie r,ingc of000 lo I 100 psi. Sprcadilig oftlie iniicrocarriers was ensured and tlic eupl;l~its \\ere sepa~ited atid \\ere culrurcd 011 shoot induction nlediuin (SIM) containing h.1S with 4 pM TIIZ, 10 pM 2-IP and 2 pM kinctili. They were incub;lted
for about 3 to J Jaya alid \\ere sub-ci~lturcd un the SIM consisti~~p 01'25 nl&'L kanaiiiyc~n for about two weeks. The rcgcncratlllg ahuot buda \$ere clilti~ird on tIu slioot clo~igst~on iiiedium (StMI j. I~t;~tetl cells \bere s~~apc~~dccl 111 25 1111. 01'~1crilc 12 MS. 1'111s CIIIIUI.~ was dcbcribed ill tiit prcviuii~ \ectlun (ace 3.2 1.1) Tllc cxl)la~ita 14c1.c bl~elly dipped llito tlic dprobiic~rc~riioli culture poured 111 rlie petri pl;~lc Ibr 1 to 2 xc. 'fhcy wcrc cultured in MS niedlunl co~it~llii~lg 4 /IM TDZ. I0 phl 2-11' ,ind 2 pM kitletin (SIM). The crpl;~lits wcre co- cultl\ated iiitli llie bacrcrl;~ lor 18 IU~I, ;III~I Mcle CLII~LI~C~ VI MS ~ncd~uni cunt;~~ni~ig 250 mg'L ceibtaxlme Tlic ontib~ouu celbti~n~~ilc bras uacd to tcrlnlniitc Ihe growth of tlie agrobscterial cells, ll~clusio~~ of cctbtax~nic in thc culturc nicdla co~it~nued fbr I lo 2 passages 72
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
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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 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 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
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Avenido, R.A., Hattori, I
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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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