Tissue Culture And Establishment Of Hairy Root Culture System

Scrphularia buergeriana belongs to Scrophularia ningpoensis Hemsl herb, which is an important traditional medicinal herbs. There has been a long history to use Scrphularia buergeriana replace buergeriana as the medicine in the northern of China.This study has used different methods to do the disinfection treatment on Scrphularia buergeriana seeds, obtain aseptic seedlings, do data statistics on the rate of seed germination and pollution rate, established the aseptic system of Scrphularia buergeriana. Using the stem segment, leafstalk, leaves as explants, based on orthogonal design, we have done some research on Scrphularia buergeriana callus, adventitious buds and rooting culture with culture medium of different plant growth regulator combinations, screening the optimum portion of culture medium at each stage of Scrphularia buergeriana tissue culture, establishing high frequency regeneration system of Scrphularia buergeriana. This study has also carried out some research on genetic transformation conditions of Scrphularia buergeriana with 4 kinds of Agrobacterium rhizogenes, establishing hairy root culture system of Scrphularia buergeriana. Through the above research, the results are as follows:1. Scrphularia buergeriana seed is quite small, it is difficult to finish disinfection treatment. This Study shows that sterilization 6 min directly with 0.1%Hg Cl2 can achieve good disinfection efficacy, pollution rate is 4.95%, the seed germination rate was 81.49%; the germination of the seeds evenly, seedling growth is normal, good growth state.2 with the stem segment, petiole, leaves of Scrphularia buergeriana aseptic seedling as experimental material,inducing them to produce callus and the adventitious buds,then inducing differentiation of adventitious root,finally establishing high frequency regeneration system of Scrphularia buergeriana. Through orthogonal design analysis, the suitable sequence for the Scrphularia buergeriana in vitro regeneration explants were leaf, petiole and stem segment explants. The optical culture medium to induce adventitious shoot regeneration is MS+6-BA 0.5mg.L-1+NAA 0.2 mg.L-1, the induction rate could reach 100%, The optical culture medium to induce bud proliferation is MS+6-BA 0.2mg.L-1+NAA 0.2 mg.L-1, the proliferation multiples can reach 9.13; The optical culture medium to induce rooting is 1/4 MS+IBA 0.2mg.L-1, the rooting rate is 100%, the average number of rooting is 11.33; Regenerated Plantlet Transplanting Survival rate can reach 86.96%.3 with Agrobacterium rhizogenes A4, R1601, C58C1 and ATCC15834 strains, through the process of infection, getting Scrphularia buergeriana hairy root, comparing the induction rate, the most suitable Agrobacterium rhizogenes to induce Scrphularia buergeriana hairy roots are A4 and R1601 stains. Screening the Explants(leaf, stem segment, petiole), we found that the tender and mature leaves are suitable to be taken as the experimental material. the induced condition of Scrphularia buergeriana hairy roots: pre cultured 2-3D,explants infected, time 10-12 min, co cultured 2-3D days, the highest induction rate could reach 90.01%. Acetosyringone can promote the transformation of Agrobacterium rhizogenes, improving the induction rate of Scrphularia buergeriana hairy roots. When the concentration of AS is 100μmol/L, the highest induction rate is 90.01%.but the higher the concentration is, the bigger damage to explant leaf is. Lately, explants become browning easily and reduce the rate of induction of hairy roots. 1/2MS liquid culture medium is favorable to the growth of Scrphularia buergeriana hairy root, the increased fresh weight to culture medium may reach by 13.5 times. PCR test results on hairy root prove that T-DNA of the Ri plasmid has been integrated into the genome of hairy roots of Scrophularia buergeriana.4 Scrphularia buergeriana hairy roots in culture medium MS + 6-BA0.5mg/L+NAA0.2mg/L can form the callus, after subculture, a large number of buds emerging from the callus buds put into hormone free MS medium, buds gradually grow up, and then cut them, putting them into the culture medium 1/2MS+IBA0.5mg/L for about 2 weeks, there will be a large number of roots, transplanting.