Optimization Of Plant Regeneration System And Studies On Agrobacterium-mediated Transformantion Of PSAG12-IPT Gene In Zoysia Japonica

The P_SAG12-IPT gene was transformed into the mature seeds-induced calli of Zoysia japonica Steud. through Agrobacterium-mediated transformation on the base of o ptimization of zoysiagrass regeneration system, a Agrobacterium-mediated genetic transformation system of zoysiagrass was established, which will be base of a new variety of zoysiagrass. The major results as following:1. Optimization of zoysiagrass regeneration system. Frequency of differential Calli induced from Zoysia japonica seeds was obviously improved;A callus line,high-frequently regenerated at random and similar in the status, was triumphantly subcultured until today.2. The Agrobacterium-mediated high-efficiency genetic transformation system of Zoysia japonica was established using long-term subcultured calli as infectious material,the frequency of resistant-callis reaching 71% and transformation frequency of resistant -plants 45%.Main factors were studied through PSAG12-IPT gene transformation using the frequency of resistant caUi as indication of genetic transformation.The results was showed:The calli that had been pre-cultured for six days were immersed in the Agrobacterium suspention (OD₆₀₀0.4) for 36h and then co-cultured for 6 days.After that, the calli were transferred to selected medium containing 400mg/LCn and 50mg/L hyg, and then subcultured every 4 weeks. After 8 weeks, the resistant calli were transferred into differential medium supplemented with 50mg/L Cn and 20mg/L hyg ,and then rooted in 1/2MS basal medium with free-hormone.The PCR results preliminarily indicated that foreign gene had been integrated into 7 of 15 resistant plantlets ,which is choosen completely from 45 hyg resistant plantlets at random.3. Another long-green gene transformation...