Crony Levin Genetic Transformation Model To Establish

A genetic transformation model for the brown seaweed Undaria pinnatifida has been primarily set up by using micro-particle bombardment as the method, female or male gametophytes as the gene recipients, hybridization as the regeneration route and chloramphenicol, hygromycin or basta as selective reagents. Several vegetative clones of male or female gametophytes of U. pinnatifida has been induced and parthenogenesis of female gametophytes has been observed. 90% of the gametophytes can grow into young sporophytes one month after fertilization. No staining background of either LacZ or GUS was found in different tissues of U. pinnatifida. Transient expression of LacZ gene driven by SV40 promoter has been carried out in rhizoids, blades and sori of U. pinnatifida 48h after transformation. Afterwards stable expression of SV40-lacZ was observed in vegetative growing male gametophytes and regenerated diploid sporophytes (by sterilizing) 6 months after transformation. 30% of male gametophytes in the vegetative growth clones showed blue color after staining. About 5% of regenerated young sporophytes showed blue spots of which the area was about 0.1% to 60% of total surface area. By using primers designated for LacZ gene, PCR result suggested an integration of lacZ into U. pinnatifida genome. Nine reagents including Penicillin G, Kanamycin, G418, Teicoplanin, Zeocin, Chloramphinicol, Hygromycin, Basta and Methotrexate were tested as selective reagents for selection of transgenic sporophytes. The results showed that young sporophytes of U. pinnatifida were sensitive to Chloramphinicol and Hygromycin and very sensitive to Basta which suggest the potential of using the resistance genes as selectable markers.