Search Results
Ethylene is a known causal factor in the decay and senescence of fruits and vegetables. The aim of the present study was to incorporate a gene for control of ethylene biosynthesis in order to prevent or delay the senescence of the cauliflower curds. We first developed a reproducible transformation system using marker genes for beta glucuronidase (GUS) and antibiotic resistance. Brassica oleraceae L. var. botrytis was transformed by inoculating hypocotyl explants with the Agrobacterium tumefaciens strains C58 or EHA101 containing plasmids pAG5110, pAG5420, or pAG5520. The plasmid pAG5110 contains the genes for neomycin phosphotransferase II (NPTII) and GUS. The plasmids pAG5420 and pAG5520 contain a functional gene for S-adenosylmethionine hydrolase (SAMase) under an ethylene or wound inducible promoter, respectively. Hypocotyl explants were screened on regeneration medium with kanamycin for selection of transformants. Shoot regeneration occured within 4-6 weeks and morphologically normal plants developed within 3-4 months. The transgenic nature of the plants was confirmed by histochemical GUS assay, an ELISA based NPTII assay and Southern blot analysis. Transgenic plants outplanted in the greenhouse are being evaluated and selfed to study expression and inheritance pattern of the introduced trait.
Broccoli and cauliflower are among the most regeneratively intractable genotypes found in the brassicaceae. To develop a method for transfer of the gene encoding S-adenosylmethionine hydrolase (SAMase) into inbred broccoli and cauliflower germplasm, we investigated the morphogenic competence and Agrobacterium susceptibility of a wide range of tissues of varied source. Appropriately controlled expression of the SAMase gene should, theoretically, reduce the plant's capacity for ethylene biosynthesis and extend the post harvest shelf life of the flower head.
Through examination of the in vitro response of a wide range of tissues we identified procedures which support caulogenesis from 100% of explants, each producing more than 30 shoots which readily convert to plantlets. Studies with several wild type and disarmed Agrobacterium strains, and utilization of the binary vector system and appropriate marker and reporter genes, led to the identification of methods for high frequency T-DNA transfer to explant tissues and the flow frequency of transgenic plants containing SAMase gene.