| Podophyllum hexandrum Royle (P.hexandrum) is a medical perennial herb. Podophyllotoxin as well as its congeners and derivatives (VP16, VM26) exhibit pronounced biological activity mainly as antiviral agents, antineoplastic and anti-HIV drugs. Currently podophyllotoxin is extracted from the rhizomes of P.hexandrum, which are collected from the wild to meet the ever-increasing demand of the drugs. As a result, P.hexandrum has become an endangered species. In 1992, it had been collected and listed by"Chinses Directory of Endangered Plant"and"Chinese Plant Red Date Book". The production of podophyllotoxin by means of cell and seedling cultures may be better candidates for sustainability to protect P.hexandrum. But the characteristic of long growth cycle and low germination that made it difficult to tissue culture so that the studies were prevented. In this paper, the way of seed dormancy of P.hexandrum was studied firstly, by the basic of which the seedling and callus induction of P.hexandrum were conducted with the explant differentiation difficult and the brown avoiding, and then the system of tissue culture of seed embryo P.hexandrum and the technology of podophyllotoxin extraction by CO2-SFE and identification were both built up, which provided considerable theory to protect the endangerened plant of P.hexandrum and produce the podophyllotoxin by industrialization. The contents as following:1. GA3 and cold stratification were utilized to break the seeds dormancy of P.hexandrum. The results showed that seed coat and endosperm restriction, physiological after-ripping might be principal reasons which caused dormancy. Soaking the seed in 400mg/L GA3 solution for 24h or in GA3 following cold stratification could break the dormancy. The optimal method was soaking the seed in 500mg/L GA3 for 36h following with cold stratification for 90d, the germination percentage and power reached 81.11% and 50.00%, respectively.2. The characteristics of seed dormancy was studied with the seed doused by H2O, methanol and ethyl ether, the crude extract identified by cabbage seeds, the inhibition bioactive of the extraction was that the ethyl ether extract was more significant than that of methanol and H2O. The ethyl ether extract of seeds was then separated and identified by GC-MS, nineteen compounds were separated including fatty acid, Spirits, alkene and so on, most of which had been proved to have inhibition bioactive. Hydroquinone was firstly identified in the seed of P.hexandrum and has never been reported about its bio-activity in the reference recently. It had a strong inhibition in high concentration, but high bio-activity in low concentration. The percentage could reach 148.92% compared to the check with its low concentration 1×10-1 mg/L. In addition, Hydroquinone had a more significant bio-activity than other hormones such as IAA, NAA, 6-BA, GA3,which had similarity to promote plant development. But the mechanism of hydroquinone bio-activity needs further studying.3. Isolated maturated embryo of P.hexandrum were cultured on different medium (1/2MS, MS, B5, MB, and IMS) supplemented with different sugars, hormones and addictives and other treatments so as to optimize the cultural environment on induction of seedling and callus. The results showed that the MS medium supplemented 3.0% sucrose and 0.4% agar added 0.3mg/L GA3 under aseptic conditions at the low temperature of 22℃and 24 h full photoperiod was the best to induce the seedling, whose induction rate could reach 87.25%. The callus induction rate could be 36.67% on the same MS medium mentioned above supplemented with hormone interactions 1.0mg/L 2,4-D+0.4mg/L KT+0.3mg/L GA3. low 22℃temperature and 24h full photoperiod could be advantage of increasing the induction rate of seedlings and callus and avoid the brown effectively. Podophyllotoxin was extracted by CO2-SFE and detected after the seedling culturing 65 days with content of dry weight of rhizome and leaf 34.5337μg/g, 9.6088μg/g, respectively. |
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