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 tr ansformation of broccoli. This procedure uses compounds that enhance the vi rulence of A. rhizogenes and a Brassica campestris feeder cell layer. Leaf explants or intact cotyledons of three broccoli cultivars: Green Beauty. Sh ogun and Green Belt, were co-cultivated with A. rhizogenes strain A4T harbo uring the binary vector pLN35. The T-DNA of this binary vector contains gen ts encoding antisense 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase ( 35S-ACC-5'7') and neomycin phosphotransferase II (NOS-NPTII-NOS). Two culti vars were successfully transformed, Shogun and Green Beauty, with a transfo rmation efficiency of 35% and 17%, respectively. Fertile plants were regene rated from kanamycin-resistant hairy roots by transfer to hormone-containin g media. Integration of the T-DNA was confirmed by the polymerase chain rea ction (PCR) and Southern analyses. Analysis of ethylene production by fully open flowers of three transgenic lines of Shogun demonstrated the feasibil ity of down-regulating ethylene biosynthesis using an antisense ACC oxidase gene. One transgenic line, Sh/2, showed a 91% reduction in ethylene produc tion after 96 h in comparison to the non-transgenic control. (C) 1999 Elsev ier Science Ireland Ltd. AI rights reserved.