Nuclear and plastid transformation of Brassica oleracea var. botrytis (cauliflower) using PEG-mediated uptake of DNA into protoplasts

Abstract

Most plastid transformation studies with tobacco, and all reports for other species (except tomato [G.D. Nugent, M. ten Have, A. van der Gulik, P.J. Dix, B.A. Uijtewaal, A.P. Mordhorst, Plastid transformants of tomato selected using mutations affecting ribosome structure. Plant Cell Rep. 24 (2005) 341–349]), have used biolistics for plastid transformation. However, nuclear transformation via biolistics has not been reported for any vegetable Brassica species so we used protoplast culture and PEG-mediated DNA uptake, to examine both nuclear and plastid transformation of cauliflower, an important vegetable Brassica. A vector containing genes for hygromycin resistance and β-glucuronidase activity (pGUS-HYG) was used for nuclear transformation, while plastid transformation utilised a vector (pZB1) containing accD– rbcL plastome targeting regions cloned from Brassica napus (oil seed rape), and the selectable marker gene aadA, conferring resistance to spectinomycin. Protoplasts were embedded in agarose and selected on media containing hygromycin or spectinomycin. From five experiments, a single plastid transformant of the commercial cultivar Thalassa was obtained, whereas nuclear transformants were obtained at an absolute transformation frequency up to 1.3 × 10 −5. No spontaneous spectinomycin resistant mutants were observed in any plastid transformation experiments. PCR and Southern blot analysis confirmed the transgenic status of plants regenerated from the protoplast-derived calli.