Studies Of Isolation And Culture In Sainfoin(Onobrychis Viciaefolia) Protoplast

Protoplast could overcome sexual cell incompatibility and progressed distance Cell Hybridization. Protoplast was ideal recipient with studied on genetic transformation which could intake exogenous DNA, cell organ, virus and plasmid. The experiment used cotyledon and callus which induced from hypocotyls as explants, and studied the conditions of isolation of protoplast. The results are as followed.1. Established callus inducing system of hypocotyls: the medium of induction culture:MS + 2mg/L 2,4-D +0.6mg/L 6-BA, the callus ratio was 94.5%; The medium of multiplication culture: MS+2mg/L 2,4-D + 0.6mg/L 6-BA+ 400mg/L LH, the raise rate of callus is 1.72mm when it was cultured 10 days.2. Established protoplast isolation system of sainfoin: the experiment used callus as explant. The best enzyme solution was 2%Cellulase Onozuka R-10+ 0.5%Pectinase+ 0.5%Macerozyme R-10+ 5m mol/L MES+ 0.5mol/L mannitol, which dissolved into CPW(pH5.8). It was cultured in dark. The digestion condition was in cubated for 10 hours on a rotary shaker at 40 rpm. at 25℃in dark. And it used cotyledon as explant. The best enzyme solution was 1%Cellulase Onozuka R-10+ 0.8%Pectinase+ 0.5%Macerozyme R-10+5mmol/L MES+0.55mol/L mannitol, which dissolved into CPW(pH6.0). It was cultured in dark. The digestion condition was in cubated for 6 hours on a rotary shaker at 40 rpm. in dark conditions at 25℃.3. The explants were pre-treated before digestion in experiment, which could improve the yield and activity of protoplast. Pretreatment in 13% mannitol and low pressure could improve yield and activity of protoplas. But treatment of dark and low temperature was not much impact on yield and activity of protoplast.4. The best culture method is solid-liquid culture for callus protoplast. The protoplast culture medium is KM8p+2mg/L 2,4-D+ 0.5mg/L 6-BA. We can see the cell colony under the microscope after 30 days. But it can not grow to callus.