Brassica Chinensis Blue (isatis Indigotica Of Fort.) And Analysis From Cultured System And Its Secondary Metabolites

Isatis indigotica Fort. was taken as the material in this dissertation. The callus culture system, suspension cell culture system and transgenic baby-root culture system were established and the yield of secondary metabolites, indigotin and indinibin, were detected in onier to do some basic work for production active secondai-y metabolites industrially by the biotechnological methods. Callus culture system was established. Different sorts, concentrations and combinatione of plant growth regulators were very important to induction of callus as well as parts and physiological status of ex-plants. Conditions of I. indigotica callus induction were optimized through the orthogonal test of [³⁴?The best medium was MS+4 mg.L?NAA+O.5 mg.L?6-BA +1 mg.L?2,4-D, and the best part of the ex-plants was hypocotyl. Under this condition, rate of the callus induction was 1000/0. The best medium for callus growth was screened out as MS+l mg.L?2,4-D+ 0.5 mg.L?6-BA+ 4 mg.L?NAA. The growth rate of callus was measured within 30 days every five days. The curve of the growth shaped ?? About 25 days later. bio-mass yield of the callus was 21.3 g.L扗W and the yeilds of indigotin was 29.14 mg.L) and indirubin ,20. 63 mg.L? The suspension cell culture system was established and inoculum densities, caiton sources and nitrogen sources were discusseii The yield of bio-mass and yield of indigotin and indirubin were highest in following conditions. The inoculum density was 55 g.L扚W; the caibon source added in the MS medium was sucrose and its concentration was 50 g.L? total nitrogen concentration in the medium was 1.5 times as much as that in MS medium and the ratio of ammonium and nitrate NI-I4 N03/KNO3 3 was 1856.25 mg.LP/3562.5 mg.L? and harvested at 15k?day. Conditions of transferred gene via Agrobacferiwn rhiwgenes were optimized Compared ^th strain R1000, A4 had stronger ability to induce haiiy roots when it抯 0D6(0 was about 0.7. Among Different parts of ex-plants, such as calli, leaves, petioles, cotyledons and hypocotyls, cotyledons had the highest inducing rate as 65.63%. Ex-plants sprouted out for 4桽 days, pre-cultured for 2? days and co- cultw^d with Agrobacterium rhizogenes for 2? days had the highest inducing rate. Added SOmg.U? AS in the medium also promoted the transferring rate. I-Iaiiy roots were identified by detection of opine. It indicated that T-DNA in Ri plasmid was transferred and integrated into I indigotica genome successfully. The transferred haiiy roots were cultured in vitm. Four hairy root clones were cultured and the result showed that clone FIR4 grew fastest and the yield of indigotin and indirubin were highest. The bio-mass yield of the clone HR4 was 16.68 gJJ1DW, and the yield of indigotin and indirubin were 138.28 mg.L?and 103.58 rng.L?respectively. Hairy roots grew fastest.in 1/2 MS medium without plant growth regulators among different basic media. And growth rate of the hairy root was detected In 20th day, the bio-mass ,yield of indigoiin and indirubin were highest as 15.36 g.L扗W 124.42 mg.L? and 91.85 mg.L?respectively. lndigotin and indirubin in different tissues, such as hairy roots, calli, suapension cells, roots and leaves of normal plants had been detected by I-IPLC. The result showed that the yield of indigotin and indin.tbin in hairy roots were highest. Yield of indigotin and indirubin in normal leaves were lower than that in hairy roots but higher than that in normal roots.