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Explant Preparation -Geminate seeds on SILI [MSG p? TDZt? pM kinet~n] for I wk -Prepare AME explant by removlng ax~ll:iry bud from Ihc cotyledonary notlc Shoot bud'intluction Step I: Culture AME explant on Slkl medium for 2 wk Step 2: Sub-culture explants with shoots buds to MS basal medium for 5 d Shoot elongation Step I: Transfer shoots to SEMl [MS+S pM 2-iP+2 pM kinetin] for 10 d Step 2: Transfer unelongated shoots to SEM2 [MS+2 VM GA,] for 2 to 3 wk Rooting of shoots Phase I: Transfer shoots toliquid RIM [ MS+9.4 mM KN0,+2% sucrose+5 @I IBA] on filter paper bridge for 2 wk [60-70% shoots rooted] Phase 2: Pulse treatment of shoots with 100 @I IBA and culture on filter paper bridge in liquid MS for 2 wk [lo-20% shoots rooted] Phase 3: Transfer unrooted shoots to static hydroponic system containing 114 Arnon's nutrient solution+3 ,&I 1BA for 2-3 wk [lo-15% shoots rooted] Transplantation Step 1:Transfer rooted shoots to 8 cm (dia) pots containing 2-4 mm sand. Cover the plants with polypropylene bags and gridually open the covers over 7-10 d period. OR Suspend the rooted shoots in Magenta jars containing 114 Amon's nutrient solution. Step 2: Transfer the hardened plants to 20 cm (dia) pots containing sand:black soil (3:2)+Cell Rich (5%) +rice straw compost (5%). Schematic representation of the protocol for in vitro regeneration of whole plants from axillary meristem explant (AME) of chickpea [Jayanand & Sham, 20031
Explant Preparation -Germmate seeds on S141 [MS& llh.1 TI)Z+2 pM kinetin] ~OI- I wk -Prepare AME explant by removing nxill;~ry bud from thc cotylcdo~i;~~.y ~iode Shoot bud intluction Step I: Culture AME e\plant on Slbl nictl~uni for 2 wk Step 2: Sub-culture explants with shoots huds to MS basal ~ncdiuni for 5 d Shoot elongation Step I: Transfer shoots to SEMI [MS+5 pM 2-iP+2 pM kinetin] for 10 d Step 2: Transfer unelongated shoots to SEM2 [MS+2 pM GA,] for 2 to 3 wk Rooting of shoots Phase 1: Transfer shoots toliquid RIM [ MS+9.4 mM KN0,+2% sucrose+S pM IBA] on filter paper bridge for 2 wk 160-70% shoots rooted] Phase 2: Pulse treatment of shoots with 100 pM IBA and culture on filter paper bridge in liquid MS for 2 wk [lo-20% shoots rooted] Phase 3: Transfer unrooted shoots to static hydroponic system containing 114 Arnon's nutrient solutiont3 pM IBA for 2-3 wk [lo-15% shoots rooted] Transplantation Step 1:Transfer rooted shoots to 8 cm (din) pots containing 2-4 mm sand. Cover the plants with polypropylene bags and gradually open the covers over 7-10 d period. OR Suspend the rooted shoots in Magenta jars containing 114 Amon's nutrient solution. Step 2: Tmnsfer the hardened plants to 20 cm (dia) pots containing sand:black so11 (3:2)+Cell Rich (5%) +rice straw conipost (5%). Schematic representation of the protocol for in vitro regeneration of whole plants from axillary meristem explant (AME) of chickpea [Jayanand & Sham, 20031
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GENETIC TRANSFORMATION OF Cicer ari
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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
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3.2.2.3 Shoot elongat~on 3.2.2.4 Ro
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ABBREVIATIONS 2-il' 2-isopci~~cnpl
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frequency was observed wit11 2,4,5-
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in vitro grown plants w;is ocli~eve
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LIST OF TABLES Table 3.1 Pr~~iier c
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Table 4.1 1 Effect ul'age uf lhe ex
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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
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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
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observations GkI Shull's experiment
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(Nene and Haware, 1980). The legume
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jf.0 REVIEW OF LITERATURE Large par
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plants from them enlhuscd scientist
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introduction into protoplasts using
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evident by 1983 that the Agrobacter
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The above promoters were found to b
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gained an illstant popi~larity owi~
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legume crop species. Cotyledon expl
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1994). Clonal propagation of FI int
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were induced to for111 callus when
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' the ernblyos into plantlets on MS
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and nprII for selection. PCR and So
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Agrobacleriuiii and ill otlier expe
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ioteclinologicsl i~iiprovemel~t of
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combinat~ons and concentrdtions of
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egenerated via somatic eliibryogene
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egenerat~on from protoplasts and ca
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transformants were identified by sy
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urgent econoniical control measures
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applications of traps. Technically
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in the laboratory and releasing tl~
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have been exhaustively reviewd (Dai
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larvae of certaln Insects were unab
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3.1 Pl:~nt 111nteri:il sllrl cultur
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0.5. 2.0 and 5.0 pM were added. 111
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Lec!/i?is: Young anci juve~iile lea
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AM3 explant: This was a by-product
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PM) 2nd GAJ (2 5 PM), eltller singl
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of the plastic bag was removed rwlc
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- . -- laopropyl alcoliol ; Absolul
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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 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
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1 APPENDIX 1. hIURASRICE AND SI(SO)
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Table 4.1 Induction of sorrlalic er
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Table 4.3 111ducti011 of en~bryos O
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'Table 4.6 Dificrent combinations o
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Table 4.8 Effect oI'TDZ, 2-iP :~nd
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Table 4.10 Effect of i~lclusio~~ or
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Table 4.12 Induction of multiple sh
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Table 4.14 Effect of media constitu
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Figure 3.1 Dii~gralll~l~i~tic rcpre
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Figure 3.3 A - C Diagrn~nn~atic rep
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I . Nhu 1 51 ?C,%- .pal5125 &pa1512
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I
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Induction of somatic embryos from m
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I.iy111. J.5 SI:i;i.\ iil ~ ~ l ~ ~
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IIistologic:~l stutlic\ of dcveiopr
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Rc\[ricfiot~ ;111;1l)si\ ur ill? ~
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I'C'R :~llipLiiir:iriuil 01'700 bl)
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Fixi1r.c 17 \r:15 -l)K,.\ ;!lid J"
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1:igurr 4.22 Sui~lllcrn :III:II!s~$
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