关键词: 茶树, 幼嫩叶, 幼嫩胚根, 原生质体分离, 原生质体融合

Abstract:

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%.

Key words: Camellia sinensis, young leaf, young root, protoplast isolation, protoplast fusion