Studies On Regeneration In Vitro And Disease-resistant Gene Transformation Of Strawberry

Shoot in vitro regeneration of strawberry cultivars Fengxiang, Moh, Lst and Nor were investigated. The result showed that several factors affected regeneration rate of strawberry including cultivars, homone concentrations and combinations, the type of the nutrient medium, sucrose and agar con-centration, explants types, leaf age and dark treatment. A rapid and efficient regeneration system of strawberry was established. By using leaf discs co-cultivation with Agrobacterium, an optimal transformation system was obtained. Putative transgenic Moh. Strawberry plants containing the nptll, gus and Rs-AFP, genes were obtained based on this system. The incorporation of the transgene was confirmed by PCR with Rs-AFP,-specific primers and histochemical assay of beta-glucuronidase activity.The regeneration result showed that the shoot regeneration frequency of strawberry cultivar "Moh." was the highest. And the efficiency of regeneration depended on the combination of hormones and the type of the nutrient medium. MS minerals and B5 vitamins supplemented with 2.0 mg TDZ and 0.1 mg IB A/liter produced the greatest shoot regeneration rate(97.5%). MS, N6 and WPM medium decreased shoot regeneration. Shoot regeneration frequency was not changed when TDZ was replaced by BA, however the regenerated shoot grew less vigorously. The concentration of sucrose and agar, pH value, dark treatment and leaf age had a significant effect on shoot regeneration. With 35-40 days old leaf discs and seven days dark treatment, the highest shoot regeneration frequency was obtained on medium of pH 5.8 supplemented with sucrose 3% and agar 0.75%. Leaf discs showed a much higher regeneration rate than that of petiole segments. There was no evident difference of shoot regeneration frequency between ways that leaf discs touched medium. Regenerated shoots rooted on MS medium supplemented with 0.2mg BA and O.lmg IBA/liter.On the basis of regeneration system of "Moh." leaf discs, investigation was conducted on screening transformation condition with GUS assay. The result showed that the GUS transient expression of explants without pre-culture on regeneration medium was higher than that with pre-culture. IBA, AS and three days of co-culture with Agrobacterium increased GUS transient expression. Bacteria concentration (OD550nm) for inoculation ranging from 0.3 to 0.5 and inoculation time from 30 to 60 minutes were suitable for transformation. Higher concentration of Kanamycin inhibited shoot regeneration and the explants were not sensitive to Carb. Thus the 20mg/L Kan and 300mg/L Carb were used in the experiment. By GUS and PCR assays, three transgenic plants were obtained. Analysis of transgenic plants will be further persisted.