Study On Establishment Of In Vitro Culture System And Hairy Roots Of Podophyllum Hexandrum Royle

Sinopodophyllum hexandrum Royle (P.hexandrum) is a medical perennial herb,which is one of the Berberidaceae Podophylloideae subfamily. Podophyllotoxin in the roots and rhizomes higher than other podophyllotoxin class plants, synthetic anti-cancer drugs is an important source of raw materials. With people's living standards and quality improved continuously, the demand on anti-cancer drugs are increased, the demand on Sinopodophyllum resources are also increased.But the characteristic of long growth cycle,low germination, narrow distribution area, the scarcity of wild resources made the market tight. Combined in recent years, Sinopodophyllum has become an endangered resources, has been included in the "Rare and Endangered Plants", national levels to protect plants.Therefore,it has important theoretical and practical significance to artificial propagation Sinopodophyllum and a "podophyllotoxin" industrial production with plant cell engineering technology principles and methods,which is widely considered an effective protection to Sinopodophyllum endangered scarce resources, alleviate the "podophyllotoxin" market demand for an important new approach.In this experiment, Sinopodophyllum embryos as material, through non-sterile method of induction and training, we established a highly efficient and stable embryo culture system. Sinopodophyllum embryos induced hairy root with Agrobacterium rhizogenes 2100 to establish Sinopodophyllum hairy root culture system and the high content screening "podophyllotoxin" Train Lines. Main achieved the following results:1. Sinopodophyllum seven embryos in vitro culture establishment and optimization.The best medium in vitro embryo culture of Sinopodophyllum MS+IAA0.5mg/L+KT 0.2mg/L+GA30.3mg/L,the induction rate reached 98.34%, and growing the best. 22℃temperature and clock light most suitable for the induction of aseptic Sinopodophyllum, The best medium for subculture MS+hydroquinone0.5mg/l +AC0.5 mg/l,Baowo bulbs increased, root growth and gradually thicken, effective resoved thin, rooting hard questions. 1-5mg/L hydroquinone and 0.5mg/L AC on Sinopodophyllum sterile rooting are significantly facilitated. Add appropriate concentration of AC reduced the browning rate of embryo, effectively improved the induction rate; to create the dark environment is conductive to root induction and root growth.2. Induction of hairy root in SinopodophyllumThe experiment with Agrobacterium rhizogenes R1000 and 2100 for the transformation lasted one year, Receptor on the transformation, culture conditions, the conversion method for multiple comparison debugging. Each way inoculated 80 embryo for four times repeated. Ultimately infected embryo receptor, pre-cultured for 1 day, bacteria levels between 0.2 and 0.4 for 15 minutes, the infection were cultured for 1 days , Off bacteria culture with 500mg / Lcef, accessed to traditional root were obtained under these conditions. The light is beneficial Sinopodophyllum hairy root induction.Sinopodophyllum hairy root with Agrobacterium rhizogenes 2100 was no geotropic but growth slowly, no more branches, did not have all of the typical features of hairy roots, and the induction rate is very low, unable to PCR, which can not be determined the Agrobacterium rhizogenes induced hairy root.3. Grape hairy roots were induced and detected.To prove the difficulties of Sinopodophyllum hairy root induction and verify the reliability of the method used in transformation. Agrobacterium rhizogenes 2100 pairs induced single-bud stem grapes for the control hairy root, A large number of hairy roots have the typical characteristics produced in the base of the stem segment, which were white, with no gravitropism, more branches, rapid growth and so on. After PCR, Agrobacterium rhizogenes containing the RiT-DNA of the rolC gene was integrated into the roots of grape genome of hairy roots, that the conversion used in this experiment feasible.Considering the necessary of podophyllotoxin industrial product need to further explore the transformed condition by Agrobacterium rhizogenes on Sinopodophyllum The next step could be considered by the method of physical and chemical to improve the explants sensitivity with Agrobacterium rhizogenes.To explored the off bacteria method to improved effectiveness while reducing the sterilization of explants of poison.