茶树叶片和胚根原生质体的分离及PEG诱导融合

Protoplast Isolation and Fusion Induced by PEG with Leaves and Roots of Tea Plant(Camellia sinensis L. O. Kuntze)

植物原生质体是细胞培养和体细胞融合等细胞水平研究及植物遗传育种的重要材料。本研究用福鼎大白茶茶树的幼嫩叶片及胚根,分析了原生质体分离过程中的材料、酶解液组成及酶解时间、纯化方法等影响因子,建立了最佳原生质体分离体系,为茶树体细胞杂交等细胞水平的研究提供了高效获取大量高活力原生质体的方法。结果表明,23°C恒温黑暗或遮光培养的茶树实生苗的5周叶龄以内的幼嫩叶片是茶树原生质体分离的最佳材料,其次是茶树种子萌发后的幼嫩胚根;而以茶园健康生长的5周叶龄以内的幼嫩叶片为材料时,只能获得混有大量细胞碎片的少量具有活力的原生质体。以茶树幼嫩叶片为分离材料的酶解液组成为1.5%纤维素酶+0.1%离析酶+0.5%果胶酶+0.4 mol L–1甘露醇+20 mmol L–1 MES;以茶树幼嫩胚根为分离材料的酶解液组成为1.5%纤维素酶+0.3%离析酶+0.5%果胶酶+0.4 mol L–1甘露醇+20 mmol L–1 MES。分离茶树幼嫩叶和幼嫩胚根原生质体时,宜采用低速(分别为55 r min–1和50 r min–1)恒温(23°C)摇床振荡酶解培养,时间分别为7 h和8 h;最适宜采用15×g的转速,离心4 min可纯化获得高产量和活力的原生质体。用40%PEG-6000诱导20 min后可使茶树原生质体融合,融合率达10%。

Plant protoplasts are important materials for studies on plant cell culture,somatic cell fusion,genetics and breeding.This work established a stable and efficient method for getting a large amount of vital and intact protoplasts from young leaves and young roots of Fudingdabai tea plant.For protoplast isolation,the young leaves of tea seedlings grown in the condition of constant temperature(23°C)and dark or shading were the best materials,and the young radicle of tea plant was also the better,While the healthy young leaves of tea plants grown in tea plantation were used as materials,only a small quantity of viable protoplasts mixed with a large number of cell fragments were obtained.The optimal enzyme solution for protoplasts isolation from young leaves of tea seedlings contained 1.5%cellulase+0.1%macerozyme+0.5%pectolyase+0.4 mol L–1 mannitol+20 mmol L–1 MES,and that from radicle of tea seedlings contained 1.5%cellulase+0.3%macerozyme+0.5%pectolyase+0.4 mol L–1 mannitol+20 mmol L–1 MES.Protoplasts with high yield and viability were purified when incubated in a shaker with low speed(55 r min–1 and 50 r min–1 respectively)under constant temperature(23°C)for 7 h and 8 h,and then centrifuged at 15×g for 4 min.PEG-6000 was used to induce the fusion of protoplasts from young leaves and radicle of tea plants.The fusion time,the concentration of fusion agent PEG-6000 were discussed.The optimal protoplast infusion conditions were induction with 40%PEG-6000 for 20 min,and the fusion rate was about 10%.