Induced Of Hairy Roots Of Panax Quinquefolium By Ri Plasmid And Research On Suitable Culture Condition Of Panax Quinquefolium Hairy Roots

Panax quinquefolium L. is a valuable medicinal plant with ginsenosides as its main effective components. Ri plasmid of Agrobacterium rhizogenes can lead to the excessive formation of adventitious roots at the site of infection in plant, and we called them hairy roots. The hairy roots induced by the infection of Ri plasmid are characterized by rapid growth, growing without hormones and stable secondary metabolic products. The hairy roots will be formed at the site of infection after induced by Ri plasmid of Agrobacterium rhizogenes. The hairy roots cultured on the culture medium without hormones can increase by 20~200 times after one month.Our country paid lots of money on the Panax quinquefolium L.,and the price of Panax quinquefolium L. is much higher than the ginseng. Panax quinquefolium L. is a perennial plant and has a very strict demend for soil condition, and the produce of Panax quinquefolium L. cannot satifated the need of people.We can induce the Panax quinquefolium L hairy roots by the Agrobacterium rhizogenes. That has opened a new way for the industrial production of secondary metabolic products of Panax quinquefolium L. in the production of plant secondary metabolic products for the hairy roots are characterized by rapid growth and stable hereditary and biochemical traits.in this thesis, hairy roots of Panax quinquefolium L were induced from the root explants of two-year-old Panax quinquefolium L by Agrobacterium rhizogenes A4 with directly inoculating. The hairy roots of Panax quinquefolium L were acquired on MS medium without hormones. The cultured clones of the hairy roots were established after large-scale cultivation. We found the the suitable culture conditions for Panax quinquefolium L.hairy roots.In order to differ the hairy roots of Panax quinquefolium L. from them of ginseng, we analyszed the protein of them using SDS-PAGE. Transformation was confirmed by PCR amplification of rolC genes from the hairy roots of Panaxquinquefolium L. At the last, the content of total quinquenoside of Panax quinquefolium L. was determined by UV-spectrophotometry. The main contents and conclusions are as follows:(1)In this paper, Panax quinquefolium L hairy roots were induced from the root expants of Panax quinquefolium L by the co-culture of Agrobacterium rhizogens A4 and ginseng roots with the method of direct inoculation. The rate of the induction is 15.9%. The different explants infuenced the inducing of hairy root. In this experiment, we used the roots of Panax quinquefolium L as explant. Before it, we haven`t obtained hairy roots used stems and leaves. It may becaused the roots have response of wound. The spot for medicinal purposes is the roots, so it is significant to induce hairy roots from the root explant of Panax quinquefolium L. We induced hairy roots with directly inoculating, and it prevented the massive reproductions of Agrobacterium rhizogens.(2) After lots of experiment, we found the suitable culture conditions for Panax quinquefolium L hairy roots growing, included culture medium, temperature and the rate of the shaker. the suitable culture conditions for Panax quinquefolium L hairy roots growing were 1/2 B5 liquid medium, 25℃, in a shaker at 100rpm, changing the culture solution at 4 weeks. Growth rate of hairy roots on liquid medium much faster then that on the solid medium. and the growth rate of hariy on B5 medium was much better then that on the MS medium. the medium with low density is better for growth and branchingof Panax quinquefolium L.(3) We analyszed the protein of hairy roots of Panax quinquefolium L. and ginseng using SDS-PAGE. It was a simple, fast and exact method. Panax quinquefolium L. hairy roots and ginseng hairy roots had their unique brand respectly, so we can distinguish them each other. The experiment proved that the SDS-PAGE was the effective method for distinguishing the Panax quinquefolium L. and ginseng.(4) We drowed the gene from Panax quinquefolium L. with CTAB method. A pairof primers which were designed by Zhao Shoujing and Li Changyu et al.were synthesized . 872bp fragment of rolC gene was amplified by PCRvia primer of rolC gene. PCR analys is confirmed the in tegration of T-DNA including 872bp rolC sequence in the transformed plants.(5)After the determination of UV-spectrophotometry, The content of ginsenosides in Panax quinquefolium L hairy roots after 4 weeks is 3.88%, The content reached the level of that in 3-year-old cultivated Panax quinquefolium L roots. It may becaused the experimental material was the roots of two-year-old Panax quinquefolium L. It indicated that the content of ginsenosides in Panax quinquefolium L was more than before. We enhanced the efficiency and Reduced the cost With this method.In this paper, hairy roots of Panax quinquefolium L. were induced from the root explants of Panax quinquefolium L. by Agrobacterium rhizogenes A4. the hariy root had the characters of short growth cycle, exuberant growth and high content of ginsenosides. It avoided shortcoming of planter of Panax quinquefolium L., such as long growth cycle and high cost. The spot for medicinal purposes is the roots, so it is significant to induce hairy roots from the root explant of Panax quinquefolium L. for producing the effective component.The conclusions in this thesis will play a positive role in the uptream engineering of producing the medicine with the hairy root.which provided the basis of production and promotion of the ingredients of the P.quinquefolium L. At the same time, they offered certain reference for making the quinquefolium L. hairy roots and their chemical compound preparation on a large scale and realizing the real traditional Chinese medicine modernization.