| Pueraria lobata (Willd.) Ohwi, rich in puerarin, daidzein, daidzine and other isoflavones, has been wildly used in China and other East Asia countries as resources in curatorial, food and light industries. In this study, the P. lobata cell suspension culture system was constructed from callus of seedling leaves. The effects of some organic appendices, elicitors and organic solutions on cell growth and isoflavones production were investigated. The purpose of the study is to provide technical support for P. lobata cell engineering to produce puerarin and other isoflavones.Callus of P. lobata have been established on MS solid medium supplemented with 3% sucrose, 1.0 mg/L NAA, 2.0 mg/L BA using leaf explants of seedlings germinated from immature seeds. The callus grew fast and became yellowish and friable after subcultured on MS solid medium supplemented with 3% sucrose and 0.5 mg/L 4-PU. After these callus were inoculated and then subcultured for more than 8 times in B5 liquid medium containing 2% sucrose, 1.0 mg/L 2,4-D, 1.0 mg/L NAA, 0.5 mg/L KT and 0.2% CH, the cell cultures became homogeneous and grew fast. Hence the cell suspension culture system was established at (25?)癈, in 5.34 JJ mol ?m2 ?s"1 full day light, on gyratory shaker (110-130 r/min).The maximal biomass accumulation of P. lobata cell suspension cultures was obtained on day 8 for 12.58 mg DW/L. Cell productivity was 72.18%. The puerarin production was closely coupled with cell cultures' growth. It reached highest on day 8 both in cell cultures and medium with 7.98 mg/g DW and 3.46 mg/L, respectively. The total yield of puerarin reached peak (103.85 mg/L) on day 8. The time-courses of total isoflavones accumulation in P. lobata cell cultures and medium were different from those of puerarin. It coupled with cell cultures growth on the first 8 days and then became faster in cell stable growth phase. On day 10, it reached highest with 129.05 mg/g DW in cell cultures, and 1.59 g/L in cell cultures and medium, totaly. This content of total isoflavones in cell cultures surpassed the content in wild root of P. lobata that was 68.7 mg/g DW, while the content ofpuerarin was lower than that in wild root of P. lobata that was 12.6 mg/g DW. In P. lobata cell cultures and medium, the total content of puerarin occupied 9.22% of total isoflavones.The effect of coconut juice sterilized by autoclaving was different from that sterilized by filtration. 10% coconut juice restrained the growth of cell cultures and the accumulation of total isoflavones, while 0.2% casin acid hydrolysates (CH) promoted the growth of cell cultures and the accumulation and release of puerarin and total isoflavones. The total yield of puerarin and isoflavones were 34.05% and 40.76% highter than control, respectively. It was concluded that the optimizing medium for cell cultures in leaves of P. lobata seedlings was 85 liquid medium supplemented with 2% sucrose, 1.0 mg/L 2,4-D, 1.0 mg/L NAA, 0.5 mg/L KT and 0.2% CH.Both yeast extract (YE) and deacetylated chitin (DC) had no promotion effects on the growth of P. lobata cell cultures. 0.5 g/L DC restrained the cell growth when treated for 5 d and 8 d. YE could cause the cell brown slightly, and didn't benefit the production of puerarin. 0.01 g/L DC improved the total yield of puerarin over 28.40% after treatment of 8 days. YE and DC had no effects on production of total isoflavones.SA, MJ and ethrel had no promotion effects on the biomass accumulation of P. lobata cell cultures. SA and MJ both caused the cell brown, while ethylene had no negative effect on cell vitality. 0.1-5 mg/L SA and 0.1 mg/L MJ had not much effects on the production of puerarin. 1 mg/L MJ improved the yield of puerarin slightly after 8 d and 5 d of elicitation, while 0.1 mg/L ethrel had a better effect which improved 27.37% higher than control after treatment of 3 days. SAs MJ and ethrel had no effects on improving the yield of total isoflavones in cell cultures and medium.DMSO at concentration 1%~5% had no effect on cell vitality even treated for 5 d...
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