Establishment Of Culture System Of Peanut Hairy Roots And Its Applications

Peanut is one of important economic crops in the world. Peanut contains not only a plenty of oil an protein, but also an phenolic compound-resveratrol which has possess a wide range of biological and pharmacological activities including antioxidant, anti-inflammatory, anti-mutagenic, and anti-carcinogenic effects. Its production need a large number of raw materials for purification,and purification rates is very lower.Hairy roots can exhibit properties of rapid growth, auxin independence and high differentiation, as well as hereditary stability of high synthesis of natural compound. After induction conditions optimization of peanut hairy roots, optimization of culture medium composition conditions and hairy roots transformation of resveratrol synthase gene were carried out in this thesis. All results are as following:First, it is prerequisite to optimize factors of hairy roots induction of peanut. Effects of different Agrobacterium rhizogenes strains, different explants, different bacteria concentration, different co-culture time, different infection time and different AS concentration were studied on frequency of hairy root induction.Agrobacterium rhizogenes ACCC10060had higher induction rate of hairy roots than strains ATCC11325. Leaflet had higher induction rate of hairy roots than cotyledon, the optimal bacteria concentration was0.2～0.4, the optimal co-culture time was2days, the best infection time was10～15min, adding of75μmol/L acetosyringone (AS) could improve induction rate of hairy roots. Hairy roots could grow independently on MS medium without hormone, and presence of opines from hairy roots of peanut confirmed that Ri plasmid of Agrobacterium rhizogenes was integrated into the genome of peanut. Soluble proteins of common root and hairy roots of peanut had difference by SDS-PAGE electrophoresis.Second, effects of phytohormone, carbon source, nitrogen source, phosphate, initial pH value of medium on growth of hairy roots of peanut variety Luhua No.11were studied using single factor and orthogonal experiment. Results showed that the optimal medium was MS liquid medium without phytohormone. Adding of NAA,6BA, IBA in medium restrained growth of hairy roots. The optimal liquid medium for hairy roots growth was as follows:1.5%of sucrose, IMS of nitrogen source,3.75mmol/L phosphate, initial pH6.1. Salicylic acid could improve production and secretion of resveratrol glucoside and improve production of resveratrol in hairy roots of peanut.In order to improve resveratrol synthesis ability of peanut hairy roots, a plant expression vector with resveratrol synthase(Rs) gene was transferred into Agrobacterium rhizogenes ATCC11325by electronic pulse.Recombinant clone was confired by kana screening in the medium and PCR assay of Rs gene. Hairy roots from different genotypes were induced by recombinant Agrobacterium rhizogenes. DNA from hairy roots were used to PCR assay of gus gene and amplified a band specific to gus gene,which indicating that gus and Rs gene were integrated into the genome of peanut.