Tissue Culture And Enrichment Of Main Active Substances In Torreya Frandis

In this paper,the contents of total phenols,total flavonoids,paclitaxel,and baccatin III and their antioxidant capacity were studied in different organs（stems,leaves,buds,and arils）of Torreya Frandis.A large number of studies have shown that there are many advantages in the production of active products through cell culture.However,few attention has been paid to the research on the tissue culture techniques of T.Frandis.Therefore,the purpose of this study was to investigate the best program for callus induction and subculture of T.Frandis,and then to add biosynthetic inducers or precursors at the stage of calls suspension culture to enrich the target product.This provides a reference and experimental basis for the resources development of T.Frandis.The main achievements of our studies are as following:1.To study the active substances in the seed coats,stem segments,leaves and apical buds. The results showed that the contents of total flavonoids and total phenols were the highest in the aril,accounting for 15.637 mg/g and 20.727 mg/g,respectively,which were significantly different from the other three groups（P<0.05）.The content of paclitaxel in stem segments,leaves and arils was 0.0178%,0.0160%,and 0.0073%, respectively;baccatin III was found in stems and leaves,and the contents were 0.0034%and 0.0051%,respectively.2.The extracts were analyzed for their antioxidant capacity.The results showed that the antioxidant activity of the total flavonoid extract was stronger than that of paclitaxel. When the extract concentration was≧0.3 mg/ml,the clearance rate of OH·,DPPH·, and ABTS·was higher than 60%.However,the clearance rate was generally lower than that of VC.3.To explore the effects of exogenous hormones on callus induction.The test results showed that the callus induction rate of stem segments was up to 100%,followed by 82%of buds and less than 10%of leaves.The optimal medium was B5+2.0 mg/L NAA+0.5 mg/L KT.Orthogonal test results showed that the effects of exogenous hormones on callus induction were as follows:NAA>KT>6-BA>2.4-D;Analysis of variance showed that the effect of NAA on callus induction was significantly different（P<0.05）.4.There are many factors that affect the induction of stem callus.The orthogonal test results showed that the best induction conditions were:picking in March and lighting at 25℃for 10 h per day,with 20 g/L sucrose in the medium.Analysis of variance showed that the effect of picking season and temperature on callus induction was significantly different（P<0.05）.5.During subculture of stem segments,B5 medium supplement with 2.0 mg/L NAA was the optimal growth medium with a growth index of 4.064,and it was more suitable for callus subculture under dark condition.The stem callus growth stage showed an"S" curve,the biomass reached a maximum of 0.6324 g on the 28^th day.After subculturing for 5 times,the callus grew into a stable phase and tissue volume began to decrease.6.In order to inhibit the browning of the callus,ascorbic acid（VC）,hydrolyzed casein （CH）,activated charcoal（AC）,and polyvinylpyrrolidone（PVP）were added to the medium.The test results showed that there was no significant improvement in the degree of browning,the color of the callus turned yellow-brown,and the growth index did not increase.7.Effects of different inducers on the accumulation of paclitaxel in Plant Tissues.The results confirmed that the addition of 1 mmol/L,L-β-phenylalanine,10 mg/L,20 mg/L,and 30 mg/L methyl jasmonate increased the paclitaxel content by 1.9 times,2.14 times,2.54 times,and 1.28 times respectively,compared with the blank group. However,the addition of elicitors delayed or inhibited the growth of tissues,especially 10 mg/L MeJA.