Genetic Transformation for Fibre Quality Improvement in Cotton (Gossypium spp.)
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Date
2016-01
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University of Agricultural Science, Dharwad
Abstract
A study was conducted during 2014-15 in Agriculture Research Station, Dharwad Farm, University of Agricultural Sciences, Dharwad to validate different concentrations of growth regulators for callogenesis from cotyledon and hypocotyl explants of Coker-312 cotton. Between explants, hypocotyls were found most prominent in callus growth. MS medium supplemented with 0.1 mgl-12, 4-D + 0.05 mgl-1 kinetin was found useful with respect to days of callus initiation (9.5 days), per cent callus induction (99 per cent) and callus weight (0.85g) in hypocotyls. Creamish friable callus observed higher in callus induction (94 per cent) in three per cent glucose. The per cent somatic embryogenesis was significantly higher (71 per cent) in MS medium with reduced concentration of 0.01 mg/l kinetin + 0. 1mg/l 2, 4-D followed by 0.1 mg/l kinetin (63 per cent) and 1.0 mg/l kinetin (58 per cent). Embryo maturation and plantlets development was highest in media devoid of PGR. MS with 4 week incubation followed by one week incubation in growth chamber showed significantly higher number of root and shoot growth.
Agrobacterium strain EHA-105 carrying pCAMBIA2300 construct having E6, Expansion, Annexin+cry1Ac and Arabinogalactan genes were used for genetic transformation in Coker-312, BN and Sahana. Explants without Agrobacterium were highest when the colonization period of 10 minutes with 24 hours of co-cultivation. Cefotaxime at 1000 mg/l showed complete elimination of excess Agrobacterium from explant surface, Kanamycin at 100 mg/l showed complete death of untransformed explants. Pre-culture of 48 hours prior to transformation resulted in highest number of kanamycin resistant calli. Confirmation of putative transformants obtained via in planta method was done by PCR. Significant variation for halo disc length confirmed the expression in these transformed plants. Further study should be carried out with RT-PCR/qRT-PCR for expression analysis and then such plants from these events can thus be used in introgression breeding.
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