GENETIC TRANSFORMATION OF EMBRYOGENIC CALLUS CULTURES OF INDICA RICE USING PARTICLE BOMBARDMENT AND SELECTION OF STABLY TRANSFORMED HYGROMYCIN RESISTANT PLANTS.M. RAMESH AND ADITYA K. GUPTA
Rapidly growing regenerable embryogenic call derived from the scutellum region of mature seeds of local indica rice ( Oryza sativa L) cv. Rasi on Linsmarier and Skoog (LS) medium supplemented with 2.5 mg/ L 214-Dichlorophenoxy acetic acid (2,4-D) was biolistically transformed with plasmid pHx 4 containing hygromycin phosphotransferase gene (hph) under the control of CaMV 35S promoter fused with first exon and intron of shrunken 1 gene of barley, keeping rupture disc pressure as 1100pounds per square inch (psi), target distance of 9 cm and gap distance of 9mm. Transformed calli were selected using hygromycin B and analyzed histochemically by (3-D-glucuronidase enzyme assay. Particle bombardment parameters were optimized with pUCGUS. Randomly selected callus clumps after 24 hrs of bombardment exhibited blue stains distributed on the surface of the calli. Putatively transformed callus clusters, after 36 days of selection, when transferred to Murashige and Skoog (MS) medium supplemented with 0.05mg/ L Naphthalene Acetic Acid (NAA ), 0.5 mg/L Benzyl Amino Purine(BAP) and 50crig/L hygromycin-B regenerated shoots after 45days of transfer. Placement of regenerated shoots with callus clump on MS medium containing 0.5mg/L NAA regenerated shoots. Integration of introduced genes at plant level was confirmed by Southern hybridization. In five independent experiments, the average frequency of hygromycin resistant plants regenerated was 1.44%.
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