Studies on plant regeneration and Agrobacterium-mediated genetic transformation in cotton
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Date
2005
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CCSHAU
Abstract
Experiments were conducted to optimize conditions for callus induction,
somatic embryogensis and shoot tip proliferation in four varieties i.e. H-1117,
H-777, H-1098 and HS-6 of cotton (G.hirsutumn L.). Efforts were made to explore
the possibility of Agribacterium mediated genetic transformation in cotton.
Seeds were cultured on ST (Steward's) medium for germination. Callus
were initiated in cultured hypocotyl explants from 10 to 12 days old seedling by
culturing on seven MS based callus induction media containing glucose as carbon
source and auxins (NAA, 2,4-D) along with cytokinin (Kinetin, Zeatin). Callus
induction percentage varied between 43.57 to 97.26 per cent using hypocotyl
explants depending upon genotype used. Out of four cultivars studied, H-1117
gave maximum frequency of callus induction (97.26%) on MS1 medium
containing NAA (2.0 mg/l) and 2,4-D (0.1 mg/l). Two facterial CRD revealed that
variance due to genotype, medium and their interaction was significant at 1 per
cent level. The induced callus generated from hypocotyl explants was cultured on
eleven media for somatic embryogenesis. H-777 gave maximum (50.26%)
embryogenesis among the four cultivars used for study. Various additive like
Putrescine, Spermidine, TDZ, Glutamine etc. were used along with MS basal
medium for somatic embryogenesis. Medium containing TDZ (2.0 mg/l) and 2,4-D
(0.1 mg/l) showed maximum embryogenesis in all the four cultivars. For shoot tip
proliferation, excised shoot tips of above four genotypes were cultured on various
media containing different concentration of auxin (NAA), cytokinin (BAP and
kinetin) and gibbrellin (GA3). Per cent shoot proliferation on different media
varied between 37.81 to 78.76 per cent. Maximum being on medium containing
BAP (2.5 mg/l) and kinetin (2.5 mg/l) in genotype H-777 Agrobacterium
tumefaciens strain EHA 105 harbouring plasmid pCAMBIA 1301 (Gus, KanR) was
used to optimize agrobacterium mediated transformation in four selected
genotypes. Hypocotyl and cotyledon explants were co-cultivated with the strain
EHA-105 containing plasmid pCAMBIA 1301. Per cent gus expression ranged
between 6.82 to 25.55 percent depending on tissue type and genotype.
Cocultivation time was 72 hours and acetosyringone concentration used was 100
M. Variety H-1117 gave maximum gus expression and was found to be the best
genotype for agrobacterium mediated transformaton. Hypocotyls were found to
show maximum percent of transient gus expression.