An efficient Agrobacterium tumefaciens -mediated genetic transformation of bitter melon (Momordica charantia L.)

摘要

A simple and efficient protocol for Agrobacterium tumefaciens-mediated genetic transformation of bitter melon (Momordica charantia L.) has been developed. Pre-cultured leaf explants were transformed by co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBAL2 carrying the reporter gene β-glucuronidase intron (gus) and the marker gene neomycin phosphotransferase (nptII). After co-cultivation, explants were transferred in to a callus induction medium containing 7.7 μM naphthaleneacetic acid (NAA) with 2.2 μM thidiazuron (TDZ), 100 mg L-1 kanamycin and 300 mg L-1 carbenicillin. Regeneration of adventitious shoots from callus was achieved on MS medium containing 5.5 μM TDZ, 2.2 μM NAA, 3.3 μM silver nitrate (AgNO3), 100 mg L -1 kanamycin and 300 mg L-1 carbenicillin. Transgenic shoots were excised from callus and elongated in MS medium fortified with 3.5 μM, gibberellic acid (GA3), 100 mg L -1 kanamycin and 300 mg L-1 carbenicillin. The transgenic elongated shoots were rooted in MS medium supplemented with 4.0 μM indole 3-butyric acid (IBA) and 100 mg L-1 kanamycin. The putative transgenic plants were acclimatized in the greenhouse and seeds were subsequently collected from mature fruits. Further, the presence of acetosyringone (300 μM) in the co-cultivation medium, infection of explants for 30 min and 3 days of co-cultivation proved to be critical factors for greatly improving the transformation efficiency. Histochemical GUS assay and polymerase chain reaction of field-established transgenic plants and their offspring confirmed the presence of the gus and nptII genes, respectively. Integration of T-DNA into the genome of putative transgenics was further confirmed by Southern blot analysis. The nptII gene expression in transgenic plants was confirmed by RT-PCR. A transformation efficiency of 7% was obtained.