大规模悬浮培养茶叶细胞合成茶氨酸培养基组成优化研究

婺源绿茶嫩叶用MS培养基（加IBA 2βmg/L,6-BA 4βmg/L,盐酸乙胺25βmmol/L）进行茶叶愈伤组织悬浮培养,采用正交试验设计研究了培养基不同组成条件对茶叶细胞大规模悬浮培养过程中细胞生长与茶氨酸合成的影响。结果显示,整个培养周期中,细胞收获量和茶氨酸积累量峰值出现时间为培养的第19~22βd;在NH₄⁺/NO₃^- 1.0/60.0βmmol/L、K⁺ 100.0βmmol/L、Mg²⁺ 3.0βmmol/L、H₂PO₄^- 3.0βmmol/L、蔗糖30.0βg/L、水解酪蛋白2.0βg/L条件下,茶叶细胞生长量和茶氨酸积累量分别可达到16.33βg/100βml培养液和3.357βg/100βml培养液;提高培养基中水解酪蛋白浓度可使细胞对数生长期和稳定期得到延长,并有利于茶氨酸积累;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⁺ 和蔗糖对细胞生长的影响均不明显;Mg²⁺对细胞生长产生明显的影响;NH₄⁺/NO₃^-对茶氨酸合成具有非常显著的影响。从生产效率考虑,培养周期以19~22βd为宜。

Study on Optimum Medium for Theanine Biosynthesis in Suspension Cultures of Camellia sinensis

The suspension culture of callus obtained from tender leaves of Camellia sinensis were conducted with MS added to IBA 2 mg/L, 6-BA 4 mg/L and ethyl-amine 25 mmol/L in a fermenter. The effects of different medium composition on the growth of tea cell and the amount of synthesized theanine were analyzed. The orthogonal design was utilized in the experiment. Results showed: both the growth of tea cell and the theanine accumulation reached its peak at19th~20th in the whole cultrue cycle. Both growth of the cells and the amount of theanine would present an optimal increment (16.33 g/100 ml and 3.357 g/100 ml), when the medium mixed with NH4+/NO3- 1.0/60.0 mmol/L, K+ 100.0 mmol/L, Mg2+ 3.0 mmol/L, H2PO4- 3.0 mmol/L, sucrose 30.0 g/L and protein 2.0 g/L. The logarithmic phase and the stationary phase were enhanced and the theanine accumulation was increased, when the amount of protein hydrolysate was raised in the medium. The increasing rate of tea cell growth and theanine accumulation was influenced by the different amount of H2PO4- in the medium. the growth of tea-cell was not significantly effected by both K+ and sucrose, but it was significantly effected by Mg2+. The activity of theanine synthetase(L-glutamate: ethylamine lingase)was significantly effected by NH4+/NO3-. The optimal culture-time should be ranged from 19~22 days in a culture cycle.