Selection Of Hairy Root Lines And Establishment Culture System On Hairy Roots Of Psammosilene Tunicoides

Psammosilene tunicoides, an endemic and endangered species in China, is a traditional precious Chinese medicine. It plays an important role in traditional medicinal development for pain-relieving, anti-inflammatory, blood-stopping, immune-adjustment and so on. A long-term uncontrolled usage has caused a reduction in P. tunicoides geographic range and population size, and now the species is in a great risk of extinction. It was listed in China Plant Red Data Book as a rare and endangered species. In addition, growth cycle of P. tunicoides is long, thus comeback of yield could not act immediately. So it is necessary to find a new medicine resource.Hairy root cultures have been proposed as an alternative method of producing plant secondary metabolites because of genetic and biochemical stability, rapid growth rate and a capability to synthesize secondary products at levels comparable to that of original plants. This work is concerned with the establishment of transformed root systems, selection of hairy root lines and establishment culture system of hairy roots. So lay the foundation for industrial production on active ingredients in medicinal plant for the future use for genetic transformation.The main studies and results of this paper are indicated.1Establishment of transformation system in Psammosilene tunicoidesEffects of different explants, pre-culture time, co-culture time, bacterial concentration, infecting time and AS concentration on the transformation frequency were studied by A. rhizogenes strains15834. Inducing conditions of hairy roots were optimized. Then transformation of T-DNA was examined by PCR assay.The results showed that the hairy roots appeared after infecting for10days by ATCC15834. The optimal transformation conditions were follows:2days pre-culture and co-culture, OD6000.8for10minutes, additional acetosyringone10mg/L. The transformation of T-DNA was confirmed by PCR to determine the presence of a T-DNA sequence in their genomes. The percentage of stem explants was up to76.7%.2Selection of hairy root linesThe transferred hairy roots were cultured in vitro. Variations in growth and saponin content of36rooting clones were examined to evaluate the effect of clonal selection on saponin accumulation. The result showed that clone A-33grew fastest.The biomass yield was up to0.673gram (g, DW), and the content of total saponins was0.64%. Clone A-23presented the significantly higher content of saponin. The production of total saponins was0.68%, the biomass yield was0.513g (DW). Taking into account both growth and the content of total saponins, clone A-23, which had the highest yield of saponins, was selected for future experimentation.3Establishment culture system of hairy rootsIn this paper, we described the influence of media, carbon and nitrogen materials, sucrose and plant growth regulator’s concentrations, elicitor and additional liquid media on growth and saponins production in hairy root of P. tunicoides.The results showed that it was favorable to the accumulation of total saponins in1/2MS medium, the content of the dry weight and total saponins reached0.601g and0.69%, respectively. Among three carbon source, sucrose was the best, especially,3%sucrose concentration in liquid medium was the best for accumulation of saponins in the hairy root, the biomass was0.606g, the content of total saponins was0.69%; The addition of lactoalbumin hydrolysate to the hairy roots promoted accumulation of growth and saponins in the hairy root. LH at a concentration of250mg/L increased hairy roots growth and saponins by23.0%and38.6%comparable to control, respectively. The addition of0.5mg/L IBA to the hairy roots promoted the growth, the content of dry weight and saponins increased24.9%and24.3%comparable to control, respectively; YE at a concentration of0.5mg/L stimulated accumulation of total saponins, but no obvious influence on the production of the hairy root. The addition of Ca2+and Co2+to the hairy roots had significantly inhibitory effect on root growth, but promoted the saponin accumulation. The elicitor at3mM Ca2+and100μM Co2+increased by33.8%and29.6%comparable to control, respectively; Additional liquid medium could significantly promote the growth of the hairy root, but had no much effect on the accumulations of total saponins. The dry weight increased31.6%compared with hairy root cultured for20days.