Gentiana Tissue Culture, The Hair Root System To Establish The Changes Of Secondary Metabolites In The Hair Root

With the leaves and hypocotyls of Genetiana macrophylla Pall. As the explants, callus and regenerated plants were successfully induced. The optimal medium for callus induction was the MS medium with 2mg·L^-1 2,4-D and 0.5mg·L^-1 6-BA added, and its inductivity rate amounted to 100%. The induced callus was transferred to the MS medium with 2 mg·L^-1 2,4-D, 0.5 mg·L^-1 KT and 500 mg·L^-1 LH added for its secondary culture. The callus from the secondary culture was transferred to the MS medium with 0.1 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA added to differentiate, and its differentiation rate could reach 86.67%. The buds differentiated from the callus were transplanted to the MS medium without hormones and then developed into many differentiation seedlings.Transformed hairy roots were produced from the stem and leaf explants of Genetiana macrophylla Pall. in MS medium with 6mg·L^-1 IBA added, after infect by Agrobacterium rhizogenes strains A₄ and LBA9402Bin19 respectively. The best effective strains were screened. The highest infection rate ,i.e 87% ,was obtained frome A₄ ;63% from LBA9402Binl9,the transformation infection rate from lamina explants was higher than other explants. The hairy roots transformed by A4 and LBA9402Binl9 grew rapidly on MS medium without any phytohormones, and were characterized by strongly branching, clustering and non-geotropism,etc. The highest growth rate of hairy roots was observed on 1/2MS liquid medium (shaked at 120 r/min at 26±2℃ in darkness), and the fresh weight wes increased 95mg/g per day of A4 and 124mg/g per day of LBA9402Bin19. The reaction of PCR proved that the hairy roots were transformed by the strain A4 and LBA9402Binl9 , T-DNA of Ri plasmid of A4 and LBA9402Binl9 had been integrated into the DNA of hairy roots. Determine by HPLC, the content of gentiopcroside in hairy root was 0.099% of A4 and 0.102% of LBA9402 Bin19, higher than that cultured in 4 monns aseptic seedlings(0.058%) . Determine by thin layer chromatography, the content of other component in hairy root also had been increased.