Factors that influence Agrobacterium rhizogenes-mediated transformation ofbroccoli (Brassica oleracea L. var. italica)

An improved broccoli transformation system was developed by optimising seve ral factors that affect the rate of effective Agrobacterium-mediated transf ormation. Leaf explants of cultivar Shogun were co-cultivated with Agrobact erium rhizogenes strain A4T harbouring the binary vector pART278. The T-DNA of this binary vector contains a neomycin phosphotransferase II (NOS-NPTII -NOS) gene for kanamycin resistance and a beta -glucuronidase (35S-GUS-OCS) gene. Several media and factors were evaluated including combinations of a rginine, mannopine, acetosyringone and the use of feeder cell layers. The n ew protocol includes the use of 200 muM acetosyringone in LB medium for bac terial growth, the use of a Brassica campestris feeder cell layer, 10 mM ma nnopine and 50 muM acetosyringone in the co-cultivation medium and 1 mM arg inine in the selection medium. The use of this optimised protocol produced transformation rates of 33% in preliminary experiments transforming broccol i with the antisense 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase ge ne from pTOM13.