Establishment Of Suspension Culture And Isolation Of Protoplasts In Xanthoceras Sorbifolia Bunge

Xanthoceras sorbifolia Bunge is a plant belonged to Sapindaceae, which is one of the peculiar woody fuel and medicinal plants in China. This study dealt with the induction of embryogenic callus from mature embryos, establishment of suspension cell lines. Moreover, the leaves of tissue culture seedlings from Xanthoceras sorbifolia Bunge were used as experiment materials.Through selecting multi-impact factors of the separation of protoplasts, a good system of protoplast isolation was established. The results obtained as follows.The study used mature embryo as a test material and screened the optimal culture conditions of callus induction, callus subculture. In addition, the activities of antioxidant enzyme and contents of solubie protein in the callus from the seed of Xanthoceras Sorbifolia Bunge were investigated. The results showed that callus was induced from stem on B₅ supplemented with 2 mg/L 2,4-D + 20 g/L sucrose + 5.5 g/L Agar(pH5.8),at the temperature of (25±2)℃with darkness. B₅ + 1 mg/L 2,4-D + 1 mg/L 6-BA + 20 g/L Sucrose + 5.5 g/L Agar(pH5.8)could be the best culture conditions of callus subculture , with Low-light training, (25±2)℃. POD activity of white callus and yellow callus was significantly higher than transparent callus; CAT activity of white callus and transparent callus was extremely obviously higher than yellow callus, CAT activity of white callus was significantly higher than transparent callus; SOD activity of white callus was significantly higher than transparent callus and yellow callus; Soulble protein content of yellow callus was significantly higher than white callus and transparent callus, soulble protein content of white callus was significantly higher than transparent callus.The development of callus was related to the activities of antioxidant enzyme and contents of solubie protein.The experiment obtained callus from subculture culture for materials, established suspension cell line of Xanthoceras Sorbifolia Bunge and studied its growing characteristic. The results showed that the optimum initial inoculum amount was 2g in 40ml liquid culture medium, and the optimum hormone concentration were 0.5 mg/L 2,4-D and 0.5 mg/L 6-BA and 0.5 mg/L NAA. The highest cell mitotic index, cell activity and increased content of fresh weight appeared on cell suspension culture 10th day, 3th day and 14th day each other.The leaves of tissue culture seedlings from Xanthoceras Sorbifolia Bunge were used as experiment material and the influences of mannitol, cellulase, pectinase, and enzyme time on protoplast isolation from them were studied. The results showed that the highest yield and viability were obtained by treating the leaves with a mixture of cellulase 1.2﹪+pectinase 0.8﹪+10﹪mannitol + 2﹪MES + PBS for 4h at the temperature of (25±2)℃with the shaker speed of 40 rpm.The suspension cells were used as test material to study the effect of time, concentration of enzyme on protoplast isolation. The results showed the best combination of isolating large number of viable protoplasts were obtained by adding cellulase 2.5﹪+pectinase 1.5﹪+10﹪mannitol + 2﹪MES + PBS for 12h at the temperature of (25±2)℃with the shaker speed of 40 rpm.