Exploratory Study Onhairy Roots Of Taxus Media And The Establishment Of The Hairy Roots Of Cassia Obtusifolia L.Induced By Ri Plasmid

The hairy roots from the medicine plants transformed by the Root inducing plasmid(Ri plasmid)Agrobacterium rhizogenes have unique advantages which include fast growing and genetic stability,large scale production of secondary metabolites of many kinds of medicinal plants which provide a scale up solution to product active ingredients of medicinal plants in the industrial fermentation of bioreactor technology.Taxus media is the raw material for producing taxol.The successful establishment of hairy root of the Taxus media may be used as an alternative source for the viable production of taxol.As a kind of traditional Chinese medicine,Cassia obtusifolia L.has the effect to lower blood pressure,fight cancer,boost immunity,improve eyesight and so an,the major medicinal components are anthraquinones,contain aurantio-obtusifolin,chrysophanol and so on.The establishment of the hairy root cultures of Cassia obtusifolia L.will lay the foundation for the large-scale production of the medicinal ingredients.This paper was to establish the hairy roots both of Taxus media,infecting the stems and leaves of 10-years-old Taxus media,and Cassia obtusifolia L.,infecting the cotyledons of asepsis seedlings.The main contents and results of this paper are as following.1.Hairy root cultures of Taxus media were established by the strain ATCC 15834,the optimal sterilization conditions of the branches and the leaves were determined.For the optimum levels,the branches were cultured hydroponically for 6 days,sterilizated by mercury for 30 min and 75%ethanol for 30s,compaired with branches,the leaves were sterilizated by mercury for 20 min.Result shown that,only one sterm differentiated into hairy root after infecting 5 months,the branches as explants for infecting was better than the leaves.For the optimum levels,establishment of the hairy roots of Taxus media infected for 5min by the strain ATCC 15834,co-cultured for 2 days and achieved 2.5%induction rate,the hairy roots were identified by PCR analysis.2.Agrobacterium rhizogenes strains LB A 9402,ATCC 15834,ACCC 10060 were compared for the hairy roots induction from Cassia obtusifolia L.in combination with different induction condition.All of these strains successfully transformed thecotyledons to induce the hairy roots,however,the optimum levels were infected for 10min by the strain LBA 9402,co-cultured for 1 day and achieved 87.5%,and PCR comfirmed the integration of rol B and rol C.Through observation and paraffin section method,the cells of cambium and vascular tissues arranged more closely and have thinner cambium and thicker vascular tissues.3.On the comparison of the anthraquinones content among the Cassia semens hairy roots and seedling roots,the yield were 0.078mg/g,0.772mg/g,5.417mg/g respectively.4.Basing on the results of this study,we might found that the infection time of the woody,Taxus media,is much longer than the herbaceous plant,Cassia obtusifolia L.For the establishment of hairy roots,Taxus media’s need five months and Cassia obtusifolia L.just need one week.What’s more,the hairy roots of Cassia obtusifolia L.was thinner,grew faster,went brown more hardly than the hairy roots of Taxus media.