Induction And Culture For Hairy Roots Of Paris Polyphylla Var. Yunnanensis

Paris polyphylla var. yunnanensis, classified as Paris genera Trilliaceae Family, is an endemic and endangered medicinal plant species in the southwestern regions of China, and has long been used as major raw materials for some well-known TCM such as’Yunnan Baiyao’. For the past few years, the Paris increasing demand and reduced supply had led to price rise15times in the medicinal materials market. It possesses property of ’twice dormancy’ and lasts at least2years for its germination of seeds, especially its roots and stems grow slowly under natural conditions, which was the major limiting factor of Paris large-scale cultivation. From many previous researches, it has proven to be a potential and effective way to realize the sustainable utilization and protection of the medicinal plants resources, which by means of large-scale culture of hairy roots for the production of active ingredients of medicinal herbs. Therefore, to dissect the nature of seed germination and its hairy roots culture will be important for us to deal with the problem of Paris resources.Objective:To establish a protocol for the breaking of Paris seeds dormancy and for the induction of callus and hairy roots in vitro. Methods:To pretreat the dormant seeds with various solutions including distilled water,800mg/L GA3,20mg/L KT,19mmol/L KNO3and19mmol/LKNO3+800mg/L GA3for different duration of time (1d、3d、5d、7d、10d), and then analyses the germination rate. The culture bases and hormones combinations were optimized for the callus formation from different explants (seed, leaf, root, and stem), as well as a preliminary experiment to induce hairy roots was conducted through the infection of explants by agrobacterium rhizogene. The results are as follows:1.The dormant seeds of Paris polyphylla var. yunnanensis were stimulated significantly after a series of treatments including continuous storage under4℃for4to5months, irradiation by infrafred light for30minutes, treatment of fluctuating temperature twicely (4℃-40℃-20℃), kept in moisture in the complex liquid (19mmol/L KNO3+800mg/L GA3) for7days, and a germination rate as high as40%was achieved when these treated seeds were grown for60days. 2.In the.six materials(embryo,endosperm,rhizomes,lateral roots,stems and leaves), only seed embryos can induce callus under the circumstances of endosperm care. In the growth regulators, IAA has a significant effect on callus induction,6-BA has a obvious regulating action oncallus differentiation and the callus is induced best under such circumstances of MS added2mg·L-1IAA and0.02mg·L-16-BA, the induction rate was as high as53.33%.3.When the germinated seeds with2cm long radicles were sterilized with HgCl2for10min,it did not bring about high contamination rate nor a damage to the embryo buds. Cultured under conditions of12h illuminatio (10001x) and22℃for30days, a complete young seedling with one piece of leaf was developed from the germinated seeds without eddosperms on the MS medium supplemented with0.5mg6-BA and0.5mg IAA.4.The agrobacterium rhizogene in the growth logarithm period (bacterium concentration0.6-0.8) were selected to infect the different explants (endosperm, embryos, leaf, callus, stem, rhizome, fibrous roots) which were cultured in the non-phytohormone MS medium, and cultivated together and degerming culture, the result was showed:only leaves can be induced root, and grow slowlyConclusion:The important factor influencing Paris seed germination of is the bad testa permeability; The key of callus induction is the choice to explant of Paris polyphylla and hormones; The explant parts is the most important factor deciding Paris hairy root induction.