Tissue Culture-Cultivation And Podophyllotoxin Determination Of Medicine Plants, Dysosma Versipellis And D.Pleiantha (Berberidaceae)

Dysosma versipellis and Dysosma pleiantha are both perennial herbs that belong to Dysosma Woods of Berberidaceae.And they are traditional folk herbs especially used in the treatment of snakebite,rheumatism and other diseases.Antitumor effect of an active compound of these two species,podophyllotoxin,has been reported in recent years,and anti-cancer drugs made from podophyllotoxin precursor have been successfully produced and extensively used in clinical.However,D.versipellis is on the edge of extinction because of destructive overexploitation,and now it has been included in the list of national endangered plants.In present study,we investigated the wild habitat of these two species,developed tissue culture system for them,and observed the growth of both the wild and cultured plants after transplanting. Furthermore,the content of podophyllotoxin was determined by using HPLC technology.Our work provides a scientific basis for the protection and recourse-exploration of these two species.The main results are as follows:In the transplanting experiments of wild D.versipellis,the phenophase showed some difference in green house,both flowering and fruiting stages were advanced, while dormant period of some plants disappeared.We found that the flowers and fruits of D.versipellis from different populations existed morphological differences,some of which can be used as characteristics to distinguish different source of D.versipellis. On the other hand,in the transplanting experiments of cultured plants,we found that light played a critical role in plant growth;strong light could hurt plants and lead to earlier wither of the plants.Under the trees with canopy density of 0.7,the plants grow well.But in the condition when the plants were exposed directly under the sunshine,the plants would get burning-hurt at the beginning of summer and wither later.Vitro culture experiments of D.versipellis and D.pleiantha indicated that embryos could germinate in proper mediums.For example,the medium MS+IBA0.5+BA1.0+GA₃4.0 could induce the germination of cotyledons and roots embryos;callus-like tissue would occurred on MS+BA1.0 mg/L+TDZ 0.2 mg/L and MS+BA0.2 mg/L+NAA0.5 mg/L;and clumpy buds and roots can be successfully induced from embryoes of D.versipellis of JFS on MS+BA0.2 mg/L+NAA0.5 mg/L and MS+IBA0.5 mg/L+BA1.0 mg/L + GA₃4 mg/L.Moreover,if we put buds explants of D.pleiantha in mediums with some hormones,the buds could generate whole plantlets.We determined the content of podophyllotoxin for both wild and cultured plants materials,the results illustrated that:1) significant difference of podophyllotoxin content was detected between the populations of D.versipellis from Eastern,Central or Western China,and it is similar to the pattern of phylogeographic divergency revealed by molecular data.Plants from TPS(Central China) had the highest content of podophyllotoxin(1.54%),while plants from EMS(western China) had the lowest content;2) High content of podophyllotoxin was detected in the stage of flowering and fruiting of D.versipellis,and it reached the top in July(1.02%);3) Generally speaking,D.pleiantha had lower content of podophyllotoxin than D.versipellis,(less than 0.2%averagely),and is not suitable for further production development;4) The accumulation of podophyllotoxin was not increase as the increase of growing years after transplanting,cultured plants from EMS attained 0.58%of podophyllotoxin after 2-years transplanting,which is nearly 2-flod more than wild ones.The result revealed that the content can be enhanced by bionic cultivation.We suggested cultured plants can be harvested after 2-3 years growing.