| Pinellia ternata is a traditional Chinese medicine. It has multiple chemicals, and alkaloids are regarded as its main pharmacological component. Among the single alkaloids isolated, trigonelline can be used to investigate the quality control of P. ternata. Guanosine is the index component of P. ternata. Inosine is regarded as the authenticated component of P. ternata. Most researches have focused on isolation and detection alkaloids of P. ternata, whereas there has no report on study the secondary metabolism of Pinellia alkaloids. In this study, two systems were set up to study secondary metabolism in P. ternata, eg. callus aggregate suspension culture and protocorm-like body (PLB) suspension culture. The endophytes of P. ternata were isolated. Both biotic and abiotic elicitors were used to elicit the biosynthesize of Pinellia alkaloids in PLB suspension cultures. The effect of metabolize substrates on the activity of key enzymes involved in purine alkaloids metabolism pathway were also studied. The main results are as follows:(1) Three callus types, which varied in growth rate, color, texture and alkaloid content, were induced from tuber explants with various hormone combinations. Total alkaloid contents were determined. Callus cultured on MS + 2.0 mg/L 2,4-D + 1.0 mg/L Kin had an alkaloid content of 0.0293%. Callus cultured on MS + 0.2 mg/L 2,4-D + 2.0 mg/L 6-BA had an alkaloid content of 0.0234%. Callus cultured on MS + 0.5 mg/L 2,4-D + 1.0 mg/L Kin had an alkaloid content of 0.0175%. Alkaloid content of all three calli were higher that that of field-grown tuber, which was 0.0072%. Compared the microstructures of calli, it may be concluded that 2,4-D could improve the alkaloid content in Pinellia calli.(2) Callus cultured on MS + 2.0 mg/L 2,4-D + 1.0 mg/L Kin had the highest alkaloid content. This kind of callus was used to set up cell suspension culture of P. ternata. It indicated that callus aggregate suspension culture could be obtained in liquid MS containing 1.0 mg/L IBA and 3.0 mg/L 6-BA. With methodological experiment, the detection condition of guanosine, inosine and trigonelline was set up. Guanosine, inosine and trigonelline in both tubers and cultured cells could be detected by HPLC. Alkaloid contents of callus aggregate in suspension culture were determined. Trigonelline content was 412μg/g, which was 6.8 times higher than that in field-grown tuber. Inosine content was 69μg/g, which was 8% lower than that in field-grown tuber. No guanosine was detected in callus aggregate.(3) PLBs of P. ternata are a kind of intermediate before the formation of tissue cultured seedlings. PLBs are similar to field-grown tubers morphologically and histologically. It was observed that MS medium containing 0.5 mg/L NAA and 1.0 mg/L BA induced the highest frequency of undifferentiated PLBs from tuber explants; whereas, a combination of 0.2 mg/L NAA and 1.0 mg/L BA was best suited for inducing undifferentiated PLBs from leaf and petiole explants. When these PLBs were subcultured on solid MS medium containing 0.6 or 1.2 mg/L abscisic acid (ABA), ABA promoted proliferation of PLBs, but inhibited their germination. To elicit alkaloid biosynthesis, suspension cultures of PLBs were established in half-strength MS (1/2 MS) liquid medium supplemented with 0.6 mg/L ABA. Total alkaloid contents of two kinds of PLBs were higher than that of field-grown tubers. Guanosine content of PLBs derived from field-grown tuber was 857μg/g. Inosine content of that was 27μg/g, Trigonelline content of that was 155μg/g. Guanosine content of PLBs derived from leaf and petiole was 83μg/g, Inosine content of that was 21μg/g. Trigonelline content of that was 117μg/g. Results indicated that PLBs could be used as a model material for study alkaloids metabolism in P. ternata.(4) Eighty seven entophytes were isolated by spot-planting method from P. ternata collected from three differernt habitations. Endophytic fungi were 48. Endophytic bacteria were 34. Endophytic fungi were morphologically identified belonging to 15 genera, and 4 families by three methods. The results suggested that entophytes were abundance in different organs of P. ternata. With HPLC, it was found that 12 endophytes can produce the alkaloids of P. ternata. The strains may be used to produce alkaloid in fermentation industry.(5) Two endophytic fungi, Penicillium sp.and Ozonium sp., could elicit the biosynthesize of alkaloids in PLB suspension cultures of P. ternata. Extracts from mycelium of Penicillium sp. increased guanosine content in PLBs by 13-115%, inosine content by 2-21%, trigonelline content by 1-153%, compared to control. In contrast, extracts from mycelium of Ozonium sp. increased guanosine content in PLBs by 7-35%, inosine content by 4-41%, wheras trigonelline synthesis was restrained, compared to control. The elicitation effects were better when elicitors were added to PLB suspension culture at the beginning. Trigonelline was detected in the fermentation broth of two strains of endophytic fungi, Penicillium sp.and Rhizoctonia sp..(6) Two strains of endophytic bacteria, Pseudomonas sp. and Enterobacter sp. were isolated and identified. Both bacterial strains increased the growth rate of P. ternata PLBs. Pseudomonas sp. promoted the differentiation of the PLBs, whereas Enterobacter sp. inhibited PLB differentiation. The bacterial strains increased guanosine production in PLBs by 9 to 166%, inosine production by 2 to 33%, and trigonelline production by 114 to 1140% compared to the control. For Pseudomonas sp., guanosine and trigonelline production was greater when bacterial extracts were added to the PLB suspension cultures rather than living cells (co-culture treatment). Inosine production was similar in both the bacterial extract and co-culture treatments. For the Enterobacter sp., guanosine, inosine, and trigonelline production tended to be greatest when living cells were added to the PLB suspension cultures rather than bacterial extracts. We also observed that Pseudomonas sp. and Enterobacter sp. produced some of the same alkaloids as their host plants.(7) The effect of abiotic elicitors on the biosynthesize of alkaloids in PLB suspension cultures of P. ternata was investigated. These elicitors were CaCl2, inositol, salicylate, chitosan, nicotinic acid, glycin, aspartic acid, IMP. Results indicated that CaCl2 and inositol restrained the accumulation of guanosine in PLBs. As to other elicitors, they could stimulate the accumulation of guanosine, inosine and trigonelline in PLBs. Two substrates, glycin and IMP, were investigate their effects on the specific activity of two key enzymes, IMP dehydrogenase and sAMP synzyme. It indicated that IMP could increase the specific activity of both the two enzymes slightly, compared to control. Glycin could increase the specific activity of the two enzymes notablely compared to control, usually a increase of more than 10 times.
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