Establishment Of Culture System Of Silybum Marianum (L.) Gaertn Hairy Roots And Assessment Of Its Active Ingredients

Silybum marianum (L.) Gaertn, a.k.a. silymarin, milk thistle, mouse tendon, or silybum,belongs to silybum genus, Asteraceae family. In traditional Chinese medicine, the dried ripefruit of the plant is used. Originating in Western Europe and North Africa, it was brought fromGermany to China in1972. After being successfully planted, it is cultivated in most areas inChina because it is not highly dependent on the environment. After analyzing different factors,this research determines the condition under which hairy roots are induced with sixAgrobacterium rhizogenes. The good hairy root system is chosen and put into Proliferationculture. Meanwhile, the conditions of the Proliferation culture are optimized. The transferredroots are evaluated through PCR and the content of silybin in the hairy roots is determinedthrough HPLC.This research cultivates aseptic seedlings with ripe silybum seeds to determine thecondition under which the seeds are processed to get the seedlings. On this basis, the sixAgrobacterium rhizogenes1025, R1, R15834, R1601, R1000, A4are used induce the threeexplants–roots, stems and leaves–to grow hairy roots. Among the six rhizogenes, therhizogene A4is the best in inducing the roots when infecting for4minutes on the silybum root.The induction rate reaches84.83%. When the OD600of bacterial concentration is0.5, theinduction capacity of the strains is the highest. The pre-culturiing time of the explant is36hoursand the co-culturing time is12hours. The pH value of the medium is6.50.Using PCR detection and identification methods on the successfully induced transformedroots of silymarin roots, leaves and stems with the positive control strain A4, it is proved thatthe T-DNA segments in strain A4is integrated into the cell genome of the transformed roots ofthe roots, providing molecular biology basis.In silymarin proliferation culture, different physical and chemical factors are studied: typesof the media, different carbon origins and their concentration, different pH values and theeffects of Methyl jasmonate of different concentration on the growth and proliferation times ofsilymarin hairy roots. Test the growth cycle of hairy roots and optimal-screen the conditionsunder which the hairy roots proliferate quickly. The finally determined proliferation culturingcondition: cut0.15g of the break-induced hairy roots; put it into the MS liquid media withMethyl jasmonate of15μmol·L-1, saccharose3%, pH value of liquid MS6.0and culturingcycle21d.Extract the silybin from successfully induced hairy roots and testa and roots of naturallygrown seeds, use HPLC method to test the volume, and compare the contents in the three. Meanwhile, compare the silybin contents between the hairy roots with and without Methyljasmonate. From the research, it can be seen that the mass fraction of silybin is2.197%in thetesta of silymarin seeds,0.117%in the root-induced hairy roots, and0.046%in the silymarinplant. Although the silybin content in the hairy roots is far lower than that in the testa, it is2.5%that in the original plant. Furthermore, when Methyl jasmonate is added in culturing, the silybincontent reaches0.699%,5.97times that in the media without Methyl jasmonate. From the result,it can be seen that Methyl jasmonate plays a positive role in the production and enrichment ofsilybin.