Suspension Culture Of Tea Cell And Biosynthesis Of Catechin

Using the tender tea leaves of camellia sinesis Yunnan Dayezhong growing at spring as explant,the tea callus was induced with B5 culture medium adding 0.5mg/L 2,4-D and 0.1mg/L KT.After several subcultures, soft callus was obtained and used to suspension culure.Suspension cell was developed by optimizing culture condition. The effects of some physical and chemical factors and elicitors on the growth of suspension cell as well as contents and components of catechins were investigated.1. The effects of different factors on tea callus induction were studied.The results showed that there was a difference between MS and B5 culture medium only at early stage of formation of tea callus. B5 culture medium was suitable to the formation of tea callus compared to MS culture medium after one week. In term of tea explants, the inducing rate of tea callus from seeds was high up to 80%. The inducing rate of tea callus from tea leaves was higher than those from autumn . The illumination time for 12 hours could decrease the browning of tea callus, while long illumination time inhibited the formation of tea callus. However callus was cultured loose under the dark condition relatively which was suitable to cell suspension culture.2. With orthogonal experimental design, the effects of rotational speed, inoculation and medium amount on the growth of suspension cell the production of catechins were investigated. The results indicated that the optimum conditions suitable for tea cell suspension culture and catechins production were considered as below: 80rpm of rotational speed, 30ml of the amount of medium and 1g of the amount of inoculation. At the optimized conditions the growth rate of cell ,catechin accumulation in cell and total sugar consume were calculated. The dried weight of cell multiple reached 3.77 time, and the catechin content in cell reached at maximum to57.83 mg/g dry weight and the catechin production in medium was 501.39 g/dm3.But there existed EGC,EC and ECG in catechins.3.The effects of illumination and sucrose on the cell growth and catechin cumulation in suspension cell were studied. The results showed that both fresh weight and dried weight of cell descended to 32.73% and 25.61% respectively of control when suspension cell was irradiated during the logarithm grow period, but the catechin increaced by 61.45% of control. HPLC analysis showed that illumination treatment stimulated the synthesis of +C, EGC and EC. The content of +C increased by 76.91% compared to that in fresh tea leaves. The content of EC and EGC increased by 20.98% and 259.04% , respectively compared with control. 8 % of sucrose had a positive effect on the growth and catechin content in tea cell. Fresh weight, dried weight and catechin content in tea cell suspension increased by 29.31%,19.38% and 19.80%, respectively,compared to that in control. HPLC analysis showed that the content of +C increased by 6.33% of that in the fresh leaf. The content of EC increased up to 1.25% of that in control. The content of EGC increased by 207.93% of that in control. However, neither ECG nor EGCG was detected in tea cell suspension by both treatments.4. The effects of salicylic acid(SA),methyl jasmonate(MJ) and yeast extract(YE) on the cell growth and catechin accumulation in suspension cell were studied. 1mg/L of MJ and 5mg/L of YE could slightly inhibit the growth of tea cell, but promote the catechin production. The catechin contents in tea cell suspension treated by MJ and YE increased 78.02% and 71.10%, respectively, compared to control. The content of +C in tea cell suspension treated by MJ increased by 103% of that in the fresh tea leaves. The content of EC was 44.41% of that in CK. But the content of EGC increased 189 % of that in the fresh leaf. It suggested that EC might be gallated. The content of EGC increased 1175% of that in CK and was 81.75% of that in the fresh tea leaf. But EGCG were not detected. In 5mg/L of YE suspension cell, the content of +C increased 156% of that in the fresh leaf.But EC and EGC were not detected. The content of +C increased 653.76% compared to that in control and increased 48.08% compared with fresh tea leaf. It also suggested that EC might be gallated. SA had showed no effects on the growth and the catechin production.