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目的利用高山红景天组培苗叶片分离得到原生质体,经培养后获得再生植株。方法研究了外植体前期预处理、外植体大小、酶液配比、酶液中甘露醇浓度等影响原生质分离的相关因素,确定最优分离条件。结果用于原生质体分离的外植体无需黑暗预培养便可进行原生质体分离,外植体叶片长度大于1.5cm为宜。获得原生质体的酶液组成为:1.0%纤维素酶Onzuka R-10+0.5%果胶酶Macerozyme R-10+10mmol/L CaCl2.2H2O+0.1%MES+0.7mmol/L KH2PO4+0.5mol/L甘露醇,在25℃条件下酶解4h,原生质体最高产量为39.43×106个/g鲜质量,原生质体活力为78.6%。原生质体培养基为1/2MS+1mg/L2,4-D+0.5mg/L ZT+0.5mol/L甘露醇+500mg/L水解酪蛋白。浅层培养40d时形成小愈伤组织。愈伤组织在MS+1mg/L6-BA+0.1mg/L NAA诱导产生不定芽,不定芽转入1/2MS基本培养基可获完整再生植株。结论本研究为高山红景天多倍体育种提供了科学依据。
OBJECTIVE To isolate and obtain protoplasts from leaves of Tissue Culture of Rhodiola sachalinensis, and to obtain regenerated plants after culture. Methods The factors related to the separation of protoplasts, such as the pre-treatment of explants, the size of explants, the ratio of enzyme solution and the mannitol concentration in the enzyme solution, were studied to determine the optimum conditions for the separation. Results Protoplasts used for protoplast isolation could be protoplast isolated without dark pre-culture. It is advisable that explant leaf length should be longer than 1.5cm. The protoplasts obtained were: 1.0% cellulase Onzuka R-10 + 0.5% pectinase Macerozyme R-10 + 10 mmol / L CaCl2.2H2O + 0.1% MES + 0.7 mmol / L KH2PO4 + 0.5 mol / L Mannitol, enzymatic hydrolysis at 25 ℃ for 4h, the highest yield of protoplasts 39.43 × 106 个 / g fresh mass, protoplast activity was 78.6%. Protoplast culture medium was 1 / 2MS + 1 mg / L, 4-D + 0.5 mg / L ZT + 0.5 mol / L mannitol + 500 mg / L hydrolyzed casein. Shallow culture 40d formed small callus. Adventitious buds were induced by MS + 1mg / L 6-BA + 0.1mg / L NAA in callus, and intact regenerated plants could be obtained by transferring adventitious buds into 1/2 MS basal medium. Conclusion This study provided a scientific basis for P. polyclinia breeding.