Culture Of Solanum Lyratum Thunb Hairy Root And The Obtainment Of Diosengin

Solanum lyratum Thunb, is often used as Chinese herbal medicine to treat cancer.Steroidal saponins and steroidal alkaloid are the primary active pharmaceutical ingredient in the whole plant of Solanum lyratum Thunb. Diosengin is a significant medicinal component of steroidal saponins synthesis by Solanum lyratum Thunb,which is an important material to product steroid contraceptives and steroid hormones medicine. As the material of steroidal drugs industrial production, Dioscorea resource was huge consumpted, yield of diosengin cannot satisfy the market requirement.Therefore, how to develop new resource of diosengin become to a urgent problem to tackle.This study has established the plant isolated culture system and rapid propagation system of Solanum lyratum Thunb, which provide the technical support for quickly obtain aseptic seedling of Solanum lyratum Thunb in laboratory. The study also established the genetic transformation and culture system of Solanum lyratum Thunb hairy root. Moreover, explorations was maked to dynamic of its diosgenin content,which provide new source for industrialized process of diosgenin. In addition, the plant regeneration system of Solanum lyratum Thunb hairy root has been established which lay the foundation for obtain genetic stability and diogennin high yield plant.1. This experiment use sterilized Solanum lyratum Thunb leaf as explant to inducethe regeneration plant of Solanum lyratum Thunb, and the optimal culture of callus induction and differentiation was obtained. Experimental results show that the optimal disinfecting proposal of Solanum lyratum Thunb explant is 75% ethyl alcohol sterilize 10 s and 0.1% HgCl2 sterilize 6 min; the optimal culture of callus induction is MS+1.0 mg?L-1 6-BA+ 0.15 mg?L-1 NAA; the optimal culture of callus differentiation is MS+2.0 mg?L-1 6-BA+0.1 mg?L-1 NAA.2. In this experiment, we established the hairy root system by the transformation mediated by agrobacterium rhizogenes C58C1 and the explant was Solanum lyratum Thunb leaves. The transformation system was optimized. What’s more, the growth curve of hairy root and the dynamic change of diosengin was detected. HPLC was used to detect the content of diosengin in hairy root. Experimental results show that, the best infect time was 10 min, the best co-culture time was 4 d, the best antibiotic(Cef)concentration was 500 mg?mL-1. The results indicated that when culture hairy root to36 d, the content of diosengin will reach the maximal level. Detection of the content of diosengin show that, content of diosengin in hairy root significantly higher than in control group. Moreover, diosengin high yield hairy root was obtained in the experiment, the content and accumulation was 4.526 and 10.043 times than wild type root of Solanum lyratum Thunb respectively.3. Results of hairy root liquid culture shows that, adventitious bud was emerged at10 days cultured hairy root, which can grow into entire plants after single cultured,they were proved to be hairy root regeneration plant by PCR. In this experiment,Solanum lyratum Thunb hairy root was used as plant material to induce the regeneration plant by medium with different concentration ratio hormones. One-step method was used in the experiment, results shows that, the best medium to induce multiple shoots was 2.0 mg?L-16-BA+ 0.1 mg?L-1NAA+MS, the inductivity was93.33%. HPLC detection shows that content of diosengin in hairy root regeneration plants was significant higher than wild type Solanum lyratum Thunb. Diosengin content was 3.430 mg?g-1、2.993 mg?g-1、5.429 mg?g-1 in the root、stem、leaf of diosengin highest regeneration plant respectively, which was 3.060、3.127、3.117 times of wild type plant. Furthermore, the regeneration plant was survived and growing fast when transferred it to the soil.This study suggests that, hairy root of Solanum lyratum Thunb mediated by agrobacterium rhizogenes C58C1 and regeneration plant induced by Solanum lyratumThunb hairy root can both improve the content of diosengin, which provide new resource to commercial process of diosengin.