Genetic transformation of broccoli and promoter tagging in brassica species
Lee, Sun Kyo
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The genus Brassica includes many economically important vegetable, condiment and oilseed crops. Within this genus, B. oleracea includes many major vegetable crops, broccoli, cauliflower, Brussels sprout, kohlrabi, kale and cabbage. The key objective of this work was to develop an efficient Agrobacterium-mediated transformation system for broccoli (Brassica oleracea var. italica) and related crops to facilitate crop improvement through genetic engineering. Five broccoli (B. oleracea var. italica) cultivars and one breeding line were evaluated for in vitro shoot regeneration. Hypocotyl and cotyledonary petiole explants taken from five-day-old seedlings were plated on media consisting of MS salts with B5 vitamins (MSBS) and conditions for obtaining direct shoot regeneration from explants were optimized by testing a range of growth regulator combinations. The optimal growth regulator combination varied with cultivar. The highest percentage of shoot formation (100%) and greatest average number of shoots per explant (28 for hypocotyls and 12 for cotyledonary petioles) were observed with the cultivar 'Cruiser'. Shoots rooted with up to 100% frequency after four weeks of culture on growth regulator free MSBS medium. Shoot regeneration consistently occurred after two weeks of culture and plants with roots were obtained within six weeks. Using the optimal shoot regeneration condition, Agrobacterium-mediated transformation methodology was developed. Cotyledonary petiole explants from the cultivars 'Cruiser' and 'Green Valiant' were co-cultivated with Agrobacterium tumefaciens carrying a plasmid coding for β-glucuronidase (GUS), neomycin phosphotransferase (NPTII) and phosphinothricin acetyl transferase (PAT) which confers herbicide (phosphinothricin) tolerance under the independent regulation of the cauliflower mosaic virus (CaMV) 35S promoter. Plants resistant to 20 mg/L kanamycin were regenerated at a frequency of 1-10% from cotyledonary petiole explants and were shown to be transformed by GUS staining, NPTII and PAT assays, PCR analysis, Southern blotting and genetic segregation analysis. An efficient Agrobacterium-mediated transformation method was developed, with significant modifications subsequently made, to facilitate handling of large numbers of explants in a short period of time. Transformation of other B. oleracea varieties such as cauliflower, kale, kohlrabi, cabbage and Brussels sprout was also achieved with this method. Agrobacterium-mediated transformation technology was employed to tag promoters in broccoli and B. napus by employing a gus::nptII promoterless fused gene construct. Seven promoter-tagged transgenic lines were produced by this method and these lines were analyzed for GUS expression in different tissues and at various developmental stages. Promoters tagged in this study included: constitutive, root predominant, phloem predominant and shoot specific promoters. Novel promoters, once isolated and characterized, could have important applications in crop genetic engineering strategies.