不同培养条件对悬浮培养茶叶细胞生长及茶氨酸合成的影响

Effects of Different Culture Conditions on Cell Growth and Theanine Biosynthesis in Suspension Cells of Camellia sinensis (Theaceae)

婺源绿茶嫩叶用MS培养基(加IBA2mgL,6BA4mgL)进行茶叶愈伤组织悬浮培养,研究了不同培养条件对茶叶细胞悬浮培养过程中细胞生长与茶氨酸合成的影响。结果显示,NH4+NO3-1.060.0mmolL、K+100.0mmolL、Mg2+3.0mmolL、H2PO4-3.0mmolL、蔗糖30.0gL、水解酪蛋白2.0gL条件下,茶叶细胞生长量和茶氨酸积累量均达到最高值;提高培养基中蔗糖和水解酪蛋白浓度可使细胞对数生长期和稳定期得到延长,从而有利于茶氨酸积累;H2PO4-浓度主要影响细胞生长速率和茶氨酸积累速率的同步性,低H2PO4-浓度环境中茶氨酸积累速率峰值滞后于细胞增长速率峰值,高H2PO4-浓度环境中早于细胞生长速率峰值出现时间;K+和Mg2+对细胞生长的影响不明显,但影响细胞茶氨酸合成酶活性,维持适量的K+和Mg2+有利于茶氨酸积累。添加盐酸乙胺可大幅度提高茶氨酸积累量,并且先加入一定量盐酸乙胺再每天进行少量补充,茶氨酸合成量比一次性加入的效果要好。茶叶细胞生长和茶氨酸积累高峰期在整个培养过程的第19～22天出现,从生产效率考虑,培养周期以19～22天为宜。

The suspension culture of callus obtained from tender leave of Camellia sinensis was conducted with MS medium plus IBA 2 mg/L and 6-BA 4 mg/L in a fermenter. The effects of different factors on the growth of tea cells and the amount of synthesized theanine were analyzed. The results showed that both the tea-cell growth and theanine accumulation reached their peak period from 19 to 22 days after culture. Both growth of the ceils and the amount of theanine reached optimal values when the medium contained NH4^＋/NO3^- 1.0/60.0 mmol/L, K^＋ 100.0 mmol/L, Mg^2＋ 3.0 mmol/L, H2PO4^- 3.0 mmol/L, sucrose 30.0 g/L and protein 2.0 g/L. Both the logarithmic and stationary phases were enhanced when the amount of sucrose and protein were raised in the medium. Alteration of H2PO4^- concentration in the medium had an effect on the simultaneity of the cell-growth and theanine accumulation. The activity of theanine-synthatase （L-glutamate： ethylamine lingase） can be affected by K^＋ and Mg^2＋. Accumulation of theanine could be greatly raised when ethyl-amine was supplemented in the medium. The efficiency of ethyl-amine on the accumulation of theanine is related to the addition method i.e. sequential supplement of ethyl-amine into the medium was better than single addition of that.