The Study On Primary Factors Influencing The Protoplast Culture And Isolation In Sugarcane

The primary factors influencing protoplast isolation and culture were investigated. Five genotypes of sugarcane included two cultivated varieties (Mintang 70/611, Xingtaitang 15), Gesoumi(S. Spontanewn L.), Badila(S. Offlcinarwn L.) and Songxizuze(S. Sinense Roxb.) were selected as materials. First, callus were induced from young leaf explant of sugarcane. Through several subcultures embryogenic calli were selected and transferred to liquid MS1 medium for .zspension culture. When small round cell riched in the suspension cultures, they could be selected for protoplast isolation. The protoplast yield and vigor were investigated under the condition of pretreatment, different constitution of cellulase (Onozuka R- 10), and isolation time. Difference of protoplast isolation and culture among five different sugarcane genotypes, a comparison on protoplast isolation and culture between main-stem and tiller with given material, effect of different exogenous hormone constituents and different culture methods on division rates of protoplast and the of cell-regenerated, investigation of subculture and differentiation culture on callus regeneration from protoplasts of different sugarcane genotypes etc were also carried out. The results were as follows: 1) The concentration of 1 O/~ 1.5% cellulase was best for protoplast isolation of embryogenic cell suspension culture, at which both the protoplast yield and its vigor were high. It was not good for protoplast isolation at too high or low concentration of cellulase. 2) Bodi the protoplast yield and vigor increased first and then decreased with the time on, but the pmtopiast yield and vigor reached its highest level at different time. The protoplast vigor reached its high peaks I or 2 hours earlier than its yield. Investigation on the variations of protoplast yield and vigor with isolation time at the concentration of 1.0% cellulase showed that 1.0% cellulase with 7桽 hours for enzymolysis was best for cultivated varieties, while 9桰 1 hours for wild sugarcane resources, which were benefit for protoplast yield and vigor. 3) Di&rences of protoplast yield and vigor isolated from cell suspension cultures of 39 different sugarcane~enotypes were significant. Both the protoplast yield and vigor of Mintang 70/611 weY~抙ighest ...i ~ ~ , while the Gesouxnins were the lowest among them. 4) Monnitor pretreatment with given concentration and time could increase the protoplast yield and shorten the time for enzymolysis. With the pretreatment of 0.85M monnitor for 15 minutes, 74 X iO~ live protoplasts were isolated and obtained from embryogenic cell suspension cultures per gram induced from Xingtantangl 5. 5) Difference of five exogenous hormone constituents on cell division frequency and callus formation were significant. Treatment with NAA+KT was the primary factor influencing the commence of cell division, and the treatment of NAA+2,4-D was benefit for callus formation and growth. 6) The effect of agar-pack culture after liquid-thin layer culture for 6 days was the best among five culture methods of protoplast, at which both the cell division frequency and production of callus formation were high. 7) Among these genotypes, all materials occurred division and formed cell colonies, these cell colonies occurred sustaining division and formed callus except Gesoumi, the quality of callus regenerated from the protoplast of Xintantang 15 was the be...