Establishment of an efficient Agrobacterium tumefaciens-mediated leaf disc transformation of spine gourd (Momordica dioica Roxb. ex Willd)

Abstract

Spine gourd (Momordica dioica Roxb. ex Willd) is a medicinally and economically important plant and also used as vegetable. In this study, we established an Agrobacterium tumefaciens-mediated transformation procedure for M. dioica. Leaf explants were incubated with A. tumefaciens strain LBA4404 containing a binary vector pBAL2 carrying the reporter gene β-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Following co-cultivation, leaf explants were cultured on Murashige and Skoog (MS) + Gamborg et al., (B₅) medium supplemented with 6.6 μM 2,4- dichlorophenoxyacetic acid (2,4-D) combined with 3.3 μM 6-benzylaminopurine (BAP) containing 100 mg L^–1 kanamycin and 300 mg L^-1 carbenicillin. Kanamycin-resistant calluses were induced from the leaf explants after three weeks. Shoot regeneration was achieved after transferring the calluses onto fresh selection medium 8.8 μM BAP and 2.2 μM 2,4-D. Transgenic shoots were excised from callus and elongated in MS medium fortified with 3.0 μM gibberellic acid (GA₃), 100 mg L^-1 kanamycin and 300 mg L^-1 carbenicillin. Finally, the shoots were rooted on MS basal medium supplemented with 3.0 μM indole 3-butyric acid (IBA) and 100 mg L^-1 kanamycin. High transformation frequency was achieved by using three-day-old precultured leaf explants. Furthermore, the presence of acetosyringone (200 μM), infection of explants for 30 min and three days of cultivation proved to be critical factors for greatly improving the transformation efficiency. Incorporation and expression of the transgenes confirmed by polymerase chain reaction (PCR), Southern blot analysis, reverse transcription (RT)-PCR and GUS histochemical assay. Using this protocol, transgenic M. dioica plants can be obtained in approximately three months with a high transformation frequency of 9%.

Key words: Agrobacterium tumefaciens, acetosyringone, growth regulators, GUS, genetic transformation, Momordica dioica.