橡胶树热研8-79原生质体培养再生植株

Plant regeneration from protoplast culture of Reyan 8-79(Hevea brasiliensis Müll. Arg.)

【目的】优化橡胶树热研8-79原生质体分离和培养条件,建立橡胶树原生质体培养植株高效再生体系,为橡胶树体细胞杂交育种奠定基础。【方法】以橡胶树热研8-79花药愈伤组织建立的胚性细胞悬浮系为试验材料,设计不同酶组合、酶解时间和悬浮细胞继代时间进行原生质体分离,采用不同培养基、看护细胞浓度和接种密度进行原生质体培养,对原生质体分裂发育的小愈伤组织进行体细胞胚诱导及植株再生培养,统计原生质体的产量、分裂频率、体胚数及体胚萌发率。【结果】酶组合为纤维素酶1.50%+果胶酶0.15%+离析酶0.50%、酶解时间为12 h时,继代培养第5 d的胚性悬浮细胞达最高产量(3.6×107个/mL PCV);用酶解细胞和看护细胞继代培养基分别作为原生质体液体培养基和看护培养基,比使用KPR培养基更有利于原生质体的分裂,当看护细胞浓度为5%、接种密度为5×105个/mL时原生质体的分裂频率最高,达54%。原生质体持续分裂形成肉眼可见的小愈伤组织增殖后经体胚发生途径发育成完整的小植株。【结论】通过优化橡胶树热研8-79胚性悬浮细胞原生质体分离的酶组合、酶解时间、继代培养时间及看护培养过程中培养基组成、看护细胞浓度和接种密度等影响因素,可以建立橡胶树原生质体培养植株高效再生体系。

【Objective】The effect factors on protoplast isolation and culture of Reyan 8-79 were optimized to establish an efficient protocol for plant regeneration from protoplast culture, which was the premise for somatic hybridization in rubber. 【Method】The embryogenic cell suspension derived from anther calli of Reyan 8-79 was used as experimental materials. Different enzyme compositions, exposure time and time after subculture of suspension cell were designed for protoplast isolation, and different medium compositions, concentration of nurse cells and planting density were used for protoplast culture. The microcalli formed from protoplasts were induced for somatic embryogenesis and subsequently plant regeneration. Protoplast yield and its division frequency, number of somatic embryo and germination rate were counted. 【Result】 The results showed the highest yield of 3.6 × 107 protoplasts/mL packed cell volume （PCV） was isolated from the suspension cells 5 days after subculture with 12 h of exposure time in the enzyme solution containing 1.5% cellulase R-10, 0.15% pectolyase y-23 and 0.5% macerozyme R-10. The modified media derived from subculture media of embryogenic cell suspensions used for protoplast isolation and nurse cells were superior to KPR medium during nurse culture. The highest division frequency of protoplasts （54%） was obtained when protoplasts at a density of 5×105/mL were cultured on feeder layer containing 5% nurse cell. The visible microcalli derived from protoplasts proliferated and regenerated into plantlets via somatic embryogenesis. 【Conclusion】A routine and relatively efficient protocol for plant regeneration from rubber protoplast culture was established by optimizing the effect factors on protoplast isolation from embryogenic cell suspension of Reyan 8-79 and nurse culture, including enzyme compositions, exposure time, time after subculture, medium compositions, and concentration of nurse cells and planting density.