Agrobacterium rhizogenes- mediated transformation of broccoli (Brassica oleracea L. var. italica) with an antisense 1-aminocyclopropane-1-carboxylic acid oxidase gene

An improved protocol was developed for Agrobacterium rhizogenes-mediated transformation of broccoli. This procedure uses compounds that enhance the virulence of A. rhizogenes and a Brassica campestris feeder cell layer. Leaf explants or intact cotyledons of three broccoli cultivars: Green Beauty, Shogun and Green Belt, were co-cultivated with A. rhizogenes strain A4T harbouring the binary vector pLN35. The T-DNA of this binary vector contains genes encoding antisense 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (35S-ACC-5'7') and neomycin phosphotransferase II (NOS-NPTII-NOS). Two cultivars were successfully transformed, Shogun and Green Beauty, with a transformation efficiency of 35% and 17%, respectively. Fertile plants were regenerated from kanamycin-resistant hairy roots by transfer to hormone- containing media. Integration of the T-DNA was confirmed by the polymerase chain reaction (PCR) and Southern analyses. Analysis of ethylene production by fully open flowers of three transgenic lines of Shogun demonstrated the feasibility of down-regulating ethylene biosynthesis using an antisense ACC oxidase gene. One transgenic line, Sh/2, showed a 91% reduction in ethylene production after 96 h in comparison to the non-transgenic control.