Optimization of Agrobacterium-mediated genetic transformation in gherkin (Cucumis anguria L.)

摘要

The present work was aimed to study various factors influencing Agrobacterium tumefaciens mediated genetic transformation of gherkin (Cucumis anguria L). Agrobacterium strain LBA4404 harboring binary vector pBAL2 carrying the reporter gene ?-glucuronidase intron (gus) and the marker gene neomycin phosphotransferase (nptII) was used for transformation. Factors affecting transformation efficiency, such as Agrobacterium concentration, effect of acetosyringone, pre-cultivation, infection and co-cultivation time of Agrobacterium were studied. After co-cultivation, explants were transferred into MS medium plus B5 vitamins (MSB5) containing 1.5 μM benzylaminopurine (BAP) with 0.5 μM naphthalene acetic acid (NAA), 100 mg L-1 kanamycin and 300 mg L-1 carbenicillin for callus induction. Regeneration of adventitious shoots from callus was achieved on MSB5 medium containing 3.0 μM BAP, 100 mg L-1 kanamycin and 300 mg L-1 carbenicillin. Transgenic shoots were elongated in MSB5 medium fortified with 2.0 μM gibberellic acid (GA3), 100 mg L-1 kanamycin and 300 mg L-1 carbenicillin. The transgenic elongated shoots were rooted in MSB5 medium supplemented with 3.0 μM indole 3-butyric acid (IBA) and 100 mg L-1 kanamycin. The putative transgenic plants were acclimatized in the greenhouse. A strong ?-glucuronidase activity was detected in the transformed plants by histochemical assay. Integration of T-DNA into the nuclear genome of transgenic plants was confirmed by polymerase chain reaction and southern hybridization. The nptII gene expression in transgenic plants was confirmed by RT-PCR. A transformation efficiency of 15% was obtained. This protocol allows effective transformation and direct regeneration of C. anguria. Pages 231-239 |