ECO-FRIENDLY DOSE OF ANTIBIOTICS FOR SUPPRESSION OF AGROBACTERIUM TUMEFACEINSJ. RASTOGI, N.N. TIWARI, S. GUPTA, P. BUBBER AND R.K. SINGH
One of the major obstacles in sugarcane transformed tissue is the persistence of Agrobacterium tumefaciens after co-cultivation. Many antibiotics are used to eliminate the bacteria. Antibiotics interrupt the synthesis of bacterial cell wall and also suppressing the growth of co-cultivating tissues at high concentration. An assay was performed to fix the minimal eco-friendly lethal dose of the eliminating agent that was precisely efficient against A. tumefaciens. The disk diffusion assay and co-cultivation of leaf disc explants were performed by using the A. tumefaciens strain LBA 4404 P-35 GUS Intron having the plasmid pCAMBIA1301, which carries the selectable marker gene (npt II) for kanamycin. It was also correlated whether these antibiotics at high concentration inhibit growth of sugarcane callus and shoot formation. In this study five antibiotics i.e. Cefotaxime, Erythromycin, Chloramphenicol, Amoxicillin and Ciprofloxacin were tested with seven different concentrations @ 50, 100, 150, 200, 300, 400 & 500 mg/l along with control. The antibiotics which showed maximum zones of inhibition were assayed to determine their effects on shoot growth and elimination of A. tumefaciens. Maximum inhibition was observed with Chloramphenicol @ 50 mg/l against A. tumefaciens LBA 4404 P-35 GUS Intron without any adverse effect on regenerated shoots through young leaf disc. The Cefotaxime @ 200 mg/l was found effective to eliminate A. tumefaciens and promote growth of regenerated sugarcane plantlet. This is the first report to eliminate A. tumefaciens LBA 4404 P-35 GUS Intron eco-friendly, an efficient and cost-effective transformation system in sugarcane.
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