Establishment Of Cell Suspension Cultures Of Elaeagnus Angustifolia L. And Primary Study On Metabolic Regulation Of Condensed Taninins

Young leaf and stem of Elaeagnus angustifolia L.were taken as culture material for suspension culture.On the basis of optimal suspension condition,kinetics parameters during the cell culture period and the effect of Salicylic acid on cell growth and condensed tannin accumulation were studied.This study would provide theory and technology basis for industrialization production of condensed tannin by suspension cell culture.The results are as follows:1.Callus induction and establishment of Elaeagnus angustifolia L.suspension culture systemThe callus was induced on the NT culture medium supplemented with 0.1 mg.L^-1 TDZ+0.2 mg.L^-1 6-BA using Elaeagnus angustifolia young stem as explants.The rate of callus induction was as high as 100.00%.During the subculture,the frible and rapid growth calli were obtained on the NT medium with the 0.1 mgL^-1 TDZ and 0.1 mgL^-1 6-BA.The best medium for suspension culture was NT+ 6-BA0.1 mgL^-1+TDZ0.1 mgL^-1.2.Optimization of Suspension Culture Conditions of Elaeagnus angustifolia L.The best suspension culture system for cell growth and condensed tannin accumulation were obtained on the NT medium supplemented with 0.1 mgL^-1 6-BA and 0.1 mgL^-1 TDZ under light,sucrose concentration was 30 g.L^-1,inoculation weight was 30 g.L^-1,ferric salts concentration was 27.8 mg.L^-1,pH was 5.8.Comparison of effects of fructose,sucrose and glucose on suspension culture,the results showed the optimum carbon source for cell growth of Elaeagnus angustifolia L.and the production of condensed tannins was sucrose.The most optimum concentration for cell growth and production of condensed tannins were respectively 20 g.L^-1 and 40 g.L^-1.The results showed that major element contents apeared positive correlation with cell growth,the condensed tannins was 5.82 mg.g^-1 DW under half-dose major element.The inoculation weight suitable for accumulation of condensed tannins was 30 g.L^-1,content of condensed tannin achieved 4.39 mg.gDW^-1.27.8 mg.L^-1 Ferric salt and light was not only suitable for suspension cell growth,but also beneficial to accumulate the condensed tannins.3.Kinetic Study on Suspension Cultures of Elaeagnus angustifolia L.After 24 days' culture,the total biomass reached 11.56 g.DWL^-1.The content of condensed tannin began to accumulate from 9th day and reached its maximum(77.71 mg.L^-1) on 21^th day.So,Condensed tannin synthesis in Elaeagnus angustifolia L.cell was growth-associated and mainly accumulation in exponential phase and stationary phase.On 21^th day, sucrose and ammonium salts of culture medium were consumed and concentrations were 0.35 g.L^-1 and 0.05 mg.L^-1,respectively.Phosphate concentrations was 0.049 mg.L^-1 in the 18th day, which was consumed about 94%.Compared with ammonium,the absorption of nitrate was much more slowly.The nitrate concentrations was 706.42 mg.L^-1 on the 15th day,which was consumed only about 42%.4.Effect of Salicylic acid induction on cell growth and condensed tannin accumulation in the cell of Elaeagn us angustifolia L.The condensed tannin content in the Elaeagnus angustifolia L.cell was maximum(14.39 mg.gDW^-1) in the sixth day after induced by 0.5 mgL^-1 SA solutions,which was 2.79 times than that of the control.And the best SA induced time was on the 18th day,the content and yield of condensed tannin reached to 20.37 mg.gDW^-1 and 188.32 mg.L^-1,respectively.The optimal times of adding SA was twices.And the yield of condensed tannin reached up 237.46 mg.L^-1,which was 2.58 times than that of control.