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 protoplasts from tube plant leaves of Rhodiola sachalinensis and regenerate plantlets after protoplast culture.Methods Preculture treatment and size of explants,enzyme concentration,mannitol concentration in enzyme mixture related with protoplasts isolation were studied to determine the superior optimized conditions.Results Explants could be used to isolate protoplast without dark preculture,and leaf length should be longer than 1.5 cm.The best incubating enzyme solution contains 1.0% cellulase Onzuka R-10,0.5% Macerozyme R-10,10 mmol/L CaCl₂·2H₂O,0.1% MES,0.7 mmol/L KH₂PO₄,and 0.5 mol/L mannitol.The enzyme and explants mixture were shaken for 4 h at 25 ℃.The protoplasts yield and viability were 39.43×10⁶/g fresh weight and 78.6%,respectively.Purified protoplasts were cultured in medium 1/2 MS+1 mg/L 2,4-D+0.5 mg/L ZT+0.5 mol/L mannitol +500 mg/L hydrolysis of casein initially with shallow liquid layers,and calli formed within 40 d.After calli were transfered to MS+1 mg/L 6-BA+0.1 mg/L NAA,adventitious buds were induced from calli.Shoots longer than 2 cm rooted within 30 d when they were transfered to 1/2 MS medium.Conclusion The study provides the scientific base for protoplast fusion in polyploidy breeding of R.sachalinensis.

国家自然科学基金项目“药用植物高山红景天优良种质筛选与倍性创新研究”(30801516);吉林省教育厅“十一五”科学技术研究项目“高山红景天四倍体的诱导与鉴定(吉教科合字2007第367号)”