Of Pokeweed Regeneration And Genetic Transformation System

Phytolacca americana is a kind of perennial herb, belong to Phytolaccaceae. Its wild resources are rich in China. It has been widely used in medicine industry, pest controlling, food industry and phytoremediation and etc. However, the long growth cycle of P. americana obstructs its application for the industrial production of a variety of important secondary metabolites, the cloning the drug-related genes, the study on the Mn hyperaccumulating mechanism and Mn-enrichment related genes of P.americana, the establishment of rapid propagation system and genetic transformation system of P. americana are necessary. In this paper, we tried to establish an efficient regeneration system and a genetic transformation system of P. americana by using Agrobacterium tumefaciens. In addition, the effects of MnSO4 on the growth and manganese accumulation of P. americana aseptic seedling were studied in this paper. The major results were as follows:1. The optimum culture condition for seed germination and aseptic seeding cultivation of P. americana are as follows:the seeds were first put into concentrated sulphuric acid for 12 minutes, then inoculated in MS basic medium supplemented with sucrose 20g/L and agar 7g/L and cultured in dark at 26±1℃for 3-4days; finally, they were cultured in 2000 Lux with a photoperiod of 14h/10h at 26±1℃for 2-3 weeks. Under such conditions, all seeds can grow into seeding.2. Leaves, stems and nodes of P. americana aseptic seeding were used as explants to select the optimal explants, optimal culture medium and optimal culture condition. The results indicated that the optimal explant was node, the highest differentiation rate of adventitious buds reached 12.7 on the culture medium of MS+6-BA3.0mg/L +NAA0.01mg/L+TDZ0.02mg/L+sucrose30mg/L+agar7g/L; the optimized medium for adventitious buds elongation was MS+TDZ0.02mg/L+6-BA3.0mg/L +NAA0.01mg/L+GA32.0mg/L+sucrose30mg/L+agar7g/L; the optimized medium for rooting was 1/2MS+NAA0.4mg/L+sucrose25mg/L+agar7g/L, and the rooting rate could reach 90%.3. The node of P. americana was used as transformation receptor. It was first pre-cultured for 5 days, then co-cultured with Agrobacterium culture fluid with an optimized OD value of 0.6 and supplemented with acetosyringone for 2-3days; Cef with a concentration of 250mg/L was used to inhibit the growth of Agrobacterium until plantlets formed.4. With the increase of MnSO4 concentration, stem height, fresh weight and dry weight etc. of P. americana aseptic seedings were all decreased; pigment contents changed little when MnSO4 concentration was less than 5mM and decreased with MnSO4 concentration increased further; MDA contents increased with the increasing of MnS04 concentration. Under MnSO4 treatment, roots and leaves of P. americana aseptic seeding could accumulate Mn, but its degree did not reach the standard of hyperaccumulators. So, P. americana aseptic seedings are not suitable to be used as materials to study the Mn-hyperaccumulating mechanism of P. americana.wild plants.