| Phellodendron amurense Rupr. is the deciduous broad-leaved trees which from Phellodendron Genus of Rutaceae family. It is valuable and famous resources of lignum andharmacal plant in Chinese traditional medicine. Its main secondary metabolic producterberine has been developed clinical application for many years, jatrorrhizine and palmatineare main alkaloids focused by the investigators. In this paper, an effective tissue culture regeneration system was established, by which plantlets can be regeneration from eitherillary bud way or callus way. Hormones and environment factors on the callus growth and alkaloids formation in callus tissue of P. amurense were systematically studied also. The study will be conducted to train a large number of cells suspended rapid production berberine provideinformation and research methods, and helpful to solve the problems of berberine provisionsn the whole country. The main contents and results as follows:1,After sterilized shoots were obtained from axillary bud, a series of factors that influenced P. amurense tissue culture were studied. In the way of the plantlets regeneration from either axillary bud, the results showed that taking antisepsis for stem segment with axillary's bud with 10% javel water eight minutes were the best. MS medium supplementedith 3% (w/v) sucrose, 7%agar, 0.8mg/1BA produced the highest shoot regenerating rate2.8%. The average number of regenerated shoots was 10 per axillary bud.2,In the way of the plantlets regeneration from callus, The optimum medium for theallus induction is MS+BA0.5mg/L+NAA 1.0mg/L+suc30g/1+agar7g/1. Callus induction rate was up to 98% . In medium of MS+6-BA 1.0mg/L+NAA 0.1mg/L+suc30g/1+agar7g/1 the callus differentiation rate of P. amurense was heighest, 66.5%. And the optimum medium for rooting of the regenerated plantlets was 1/2MS supplemented with 0.8mg/1 IBA, 3% (w/v) sucrose and agar 7.0g/1 .Root frequencies were 46% with high root number, 4. Transplanting rate is over 80%.3,Using the axenic leaf of P. amurense as explants. The paper firstly researched the effects of 6- Benzylaminopurine, 1-Naphthalene acetic acid, 2,4-Dichloro phenoxyacetic acid and different culture condition (Basic medium, Sucrose concentrations, pH and. lightintensity).on callus inducing of P. amurense The results were as follows: 6-BA using solelyouldn't induce the callus of P. amurense; the effect of callus inducing were bad when usedAA and 2,4-D solely. There effect as: 2,4-D＞NAA＞6-BA. The optimum plant hormoneoncentration that promotes leaf to callus inducement and growth were BA1.5 mg/1+2,4-D 2.0/1 callus bloomy fissions and callus appeared green. MS was the optimum medium type.ucrose concentrations 20g/L, pH 5.8 and light intensity 40μmolm-2s-1 were the optimal culture condition for leaf callus inducing of P. amurense.4,HPLC was applied to determination of the three kinds of alkaloids including berberine, jatrorrhizine, palmatine in the leaf of P. amurense Rupr..The results showed thatthe content of berberine among different explants,basic medium,concentrationof hormones,sucrose concentrations,pH and light intensity. The effects of environment factors on formation in callus tissue jatrorrhizine and palmatine are small. Explants is leaf, B5 isbasic medium, higher 6-BA, 2,4-D and sucrose concentrations, pH 5.8 and dark conditionare suited to culture the berberine of P. amurense. |
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