Induction And Liquid Culture Of Psammosilene Tunicoides Hairy Roots

Psammosilene tunicoides, endemic to western region of China, is an endangered speciesand has long been used as a precious medicine in treating pain-relieving, anti-inflammatory,blood-stopping, and immune-adjustment and so on. Compared to other cultures, it has manymerits such as high biomass, strong accumulation of active compounds, and stable production,using the method of liquid culture of hairy root from the Agrobacterium rhizogenes-infectedexplants. Hence, it has become a hot point to obtain natural active compounds by hairy rootculture in the field of medicinal plant cell engineering.In this study, Agrobacterium rhizogenes (ACCC10060) was employed to induce hairyroot from the young leaves and stem segments of Psammosilene tunicoides plantlets, and thena culture system for hairy root was further established through optimization of several culturefactors including selection of explants, infection time, co-culture time, removal of extralbacteria, and so on. Subsequently, the hairy roots were transferred to hormone-free media forfurther culture, and hairy root growth-related parameters and changes in morphology wereanalyzed. The main results are as follows:1. According to the induction rate of hairy roots after48h co-culture of explants with Ri,it was found that young leaves were more suitable to produce hairy roots than stems. Thehairy roots exhibited abilities of kanamycin resistance and hormone-free growth, andsynthesis of opines, all confirmed the transfection of P. tunicoides explants by ACCC10060strains.2. During the co-culture of explants with ACCC10060, addition of AS to the media couldincrease the induction of hairy roots, and it reached the maximum induction rate of69%at the20mg L-1of AS.3. In the stage of removal of extra ACCC10060from the transfected explants, it couldgain better removal rate of extra ACCC10060when500mg L-1Cefotaxime was added intoB5with a removal rate of83%.4.It was transferred to the hormone-free1/2MS for further culture when the hairy root was2-3cm long on the solid B5. After35d culture, the biomass of hairy roots increased14.11times (fresh weight) and8.39times (dry weight), respectively, and the content of totalsaponins in hairy roots reached to0.857%(DW), by contrast, it’s only0.388%and0.217%in callus and seedlings respectively.5. The obtained hairy roots exhibited some significant properties of hormone-free growth,fast growth, and more branches being suitable for large-scale production of hairy roots.