Adaptation of cotton shoot apex culture to agrobacterium mediated transformation (Gossypium Spp)
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Date
2013-08-19
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Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani
Abstract
Cotton (Gossypium spp.) is an excellent natural source of textile fibre. It is a
crop of significant value throughout the world because it is not only a source of natural
fibre, but also an oilseed crop. Because of its high economic importance, considerable
attention has been paid to improving cotton plants against biotic and abiotic stress
through transgenic technology. The present investigation was carried out with an aim to
study fire regeneration potential and standardize the transformation protocol for cotton
genotypes NH-615 and NH-635, which are the straight cultivars ,so that the farmers can
conserve the germplasm and get seeds available at low cost. The results of regeneration
were statistically analysed using FRBD under three replications. And the results of
transformation were analysed using RBD under four replications. The shoot apex
explants were used for regeneration of cotton. The explants (shoot apices) showed
highest response for multiple shoot induction on MS media supplemented with BAP
and NAA. Similarly the elongation of shoots were reported on MS media supplemented
with GA3 and BAP, also the media MS with DBA was found significant for induction of
roots to the growing explants. Simultaneously an Agrobacterium mediated
transformation protocol was optimized in the same cultivar NH-615 using the disarmed
Agrobacterium strain EHA-105 harboring binary vector pCAMBIA, carrying CrylAc
gene. The seedlings with just emerging plumule were infected with a separate
Agrobacterium strain carrying the gene construct pBI-121 made up from engineered
reporter gene, p-glucuronidase (Gus) and a selectable marker gene neomycin
phosphotransferasell to confirm the transformability of the cultivar. Gus histochemical
analysis of the young leaves and hypocotyledonary node revealed that the gene was
successfully integrated into cotton genome and expressed in the transgenic plants.
Through statistical analysis is proved that NH-615 has better results for transformation
and regeneration than NH-635 using the shoot apices as explantf
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