Hairy Root Induced From Tetrastigma Hemsleyanum And Its Propagation And Analysis

Tetrastigma hemsleyanum Diels et Gilg is a medicinal plant only in China. The mainly medicine ingredient is belongs to underground napiform root. However, it need grow in special environment and its growth rate is very low under natural conditions. It can not meet the demand of medical market. On the other hand, hairy root is adventitious root which is T-DNA of Agrobacterium rhizogenes inserted into plant cell and integrated and expressing. Hairy root can be used in the synthesis and accumulation of plant secondary metabolism substance.In the study, the hairy root was induced from explant of Tetrastigma hemsleyanum by cucumopine type Agrobacterium rhizogenes strain K599and the hairy root was normally propagation. Analysis on the medical ingredient of hairy root was conducted. The results are as follows:(1) The hairy root was induced by Agrobacterium rhizogenes K599(with endogenous plasmid pRi2659and exogenous plasmid pRI101-AN-GFP) on leaf of natural plant and aseptic plant in vivo. As induction medium, MS with0.4mg/L NAA is the best, in which the induction rate of hairy root is as high as93.7%. While in the subculture culture, B5medium is better than MS medium, and the medium of B5+1.0mg/L KT+1.0mg/L IBA is the best. The survival rate of hairy root could reach90%on subculture medium during the hairy root subculture. During suspension culture of hairy root, the flat growth period is from0to15day, the rapid growth period is from15to28day, while from28to35day returned the flat growth period. After the35day, the growth of hairy root is almost stagnation.(2) The hairy root was identified by PCR, Western Blot, fluorescence microscope and laser confocal microscope. According to the results of PCR detection, among the12hairy root lines, the GFPgene from the exogenous plasmid pRI101-AN-GFP were all amplified, but the some of11genes on the endogenous plasmids pRi2659T-DNA were failed to be amplified. It indicated that several genes of11genes on the pRi2659T-DNA were deleted in some hairy roots. Besides, the fluorescent protein (GFP) localized on the cell nucleus, wall and membranes by laser confocal microscopy. Compared with natural napiform root and common root, histocytological observations showed that hairy root is lack of starch grains. The xylem structure of hairy root is simplified and only has the primary structure.(3) Using the method of UV spectrophotometry. The results shown that the content of total flavonoids in napiform root was the highest (140.210mg/g.DW), and there was differences among hairy root lines. The hairy root line1(64.167mg/g.DW) and5(58.542mg/g.DW) were higher than those of common root, stem and leaf. In addition, quercetin and kaempferol (belong to the flavonoid) were determined by liquid chromatography. The quercetin content from high to low are leaves, mapiform root, hairy root lines2,1and5, stem, common root, hairy root lines4. In the6hairy root lines, there are some differences in the content of kaempferol in hairy root lines, and all were significantly higher than that of napiform root, common root, stems and leaves.The above-mentioned results lay a foundation to the use of hairy root as bioreactors for the production of medicinal component.