Study on Enhancing the Growth Velocity of Roselle Cell by Protoplast Fusion

Firstly, callus induction and subculture for Mikania micrantha were studied systematically. The research on callus induction showed that: Shoot tip has higher induction rate than other explants ; At 25℃ and dark regime, the callus was better cultivated on Murashige and Skoog （MS） medium （pH5.8） containing 2.0mg/L 2,4-D and 1.0mg/L KT. Browning control is the key problem in subculture of callus and optimum subculture condition is that culturing in light 16h/d with light intensity 20001x and the addition of coconut juice 15 percent of medium, V_C 5.0mg/L, 2,4-D0.5mg/L+NAA2.0mg/l+KT0.5mg/L+BA0.5mg/L, subculture during cell log phase. And the conditions in cell suspension culture of Mikania micrantha were studied. The results showed that sucrose was the compatible carbon sucrose, and 30g/L sucrose concentration can satisfy the growth of Mikania micrantha cell; ammonium was absorbed under different sucrose concentration that haven’t demonstrated significant specificity, and was completely absorbed on the lag phase and the early logarithmic phase; while nitrate was mainly absorbed on logarithmic phase. The density-dependent of Mikania micrantha cell starting to grow and density-inhibited of cell growth were proposed, the fittest inoculating quantity of Mikania micrantha in cell suspension culture was 40g/L. Growth of Mikania micrantha cell in suspension culture was characterized by a typical sigmoid pattern during a culture course. The logarithmic phase of Mikania micrantha cell in vitro Roselle cell’s was longer for two days. And Roselle cell was faster up to dying after logarithmic phase while Mikania micrantha cell had been growth vigorously during the later retarding growth period.Secondly, the preparation and regeneration conditions of protoplasts of Mikania micrantha and Roselle were systematically studied in this paper. The optimal condition shown was that the two parent cell material was pretreated procedures and digested by mixed enzymes （2% Celluase + 0.5% Hemicellulase + 0.2%Pectinase） for 4 hours , and the mixed enzymes are dissolved in CPW solution with 0.65 mol/L mannitol and 0.1% MES as osmotic stabilizers and 10mmol/L CaCl₂,pH5.7. Effect factors on protoplast culture were discussed. The result showed that protoplasts embedded in low melting temperature agarose could be easily culture. The optimal density of protoplast culture is 5 × 10⁵ cell/mL. the optimal hormone combination was