Plant regeneration and Agrobacterium-mediated gene transfer in brinjal (Solanum melongena L.)

Abstract

The research work was conducted to standardize a protocol for plant regeneration and genetic transformation in brinjal. Plant regeneration studies were carried out using two types of explants viz cotyledon and hypocotyl. The cotyledon explants showed high frequency of shoot regeneration (77.77%) on MS medium supplemented with 2.5 mg/l Kinetin and 0.4 mg/l Indole-3-acetic acid (IAA) as compared to hypocotyl (50 %) on Murashige and Skoog’s (MS; 1962) supplemented with 2.5 mg/l 6-Benzylaminopurine (BAP) and 0.5 mg/l IAA. MS medium supplemented with 0.10 mg/l IAA was found to be best for root regeneration (81.81%). The brinjal plantlets were able to regenerate within 6-7 weeks. Regenerated plantlets were acclimatized. For genetic transformation, disarmed Agrobacterium tumefaciens LBA 4404 strain containing a reporter âglucuronidase (gus) gene in binary vector pBI 121 system along with kanamycin resistance gene (npt-II) for selection in both bacteria and plant was used for co-cultivation experiment to transfer gus and npt-II genes in brinjal cells. The transformed cells were able to grow on selective shoot regeneration medium containing kanamycin and cefotaxime after co-cultivation only, whereas the control explants died on the selective medium. Transformation could be scored as early as 4 weeks after selection. Most of the calli obtained on the selective medium were GUS positive. Putative
transgenic shoots were obtained, which were able to grow on the selective medium containing 50 mg/l Kanamycin.