Studies On Establishment And Optimization Of Loquat Cell Suspension Culture

In order to establish good callus induction and subculture system. The different varieties ofloquat as test materials, Ursolic Acid and Oleanic acid content and callus state as index,selecting the suitable culture, explant and hormone ratio for loquat cell suspension culture. Thecallus was transferred to the liquid medium, optimized the initial training system, established thesuitable training system for loquat cell growth and UA、OA accumulation. The main resultswere as follows:1Induction and subculture of quality loquat callusResearch on the loquat varieties, explant, hormone, microscopic observation to developsuitable callus for loquat cell suspension culture. The results showed: the most suitable loquatvarieties for cell suspension culture and UA、OA accumulation was Zaozhong six, while UA, OAcontent containing were low (6.898,2.343mg/g.DW), but its induction rate was high (83.697%),the callus was yellow, loose, granular.The most suitable explant was immature embryo,induction rate (83.17%) higher than filaments、leaf、stem tip. Filaments induction rate was high(nearly100%), but callus texture was hard. and in the callus induced from immature embryo,theUA and OA (5.099,1.617mg/g.DW) was significantly higher than that of filaments, leaf, stemtip. So the immature embryo was the best explants. The most suitable induction medium wasMS+1.0mg/L2,4-D+0.5mg/LKT, subculture medium was MS+0.5mg/L2,4-D+0.25mg/LKT.Microscopical observation also indicated that the induction of immature embryos was mostsuitable for loquat cell suspension culture. At the same time, in view of the superiority of thefilaments induced callus, research on the regulation of filaments callus, two generation later, thecallus turned soft, bright yellow, but after two or three times subculture, the subculture callusturned white、water stains、poor state, the regulation of filaments callus need further study. 2Establishment of cell suspension culture system of LoquatThe growth curves of loquat cell suspension culture showed typical "S" curve,18d as aculture period. The increase in FW and UA, OA growth rate as the index, optimized thesuspension culture system.The results showed that the optimum suspension culture medium was:MS+NAA0.5mg/L, loquat cell fresh weight25.863g.FW, was3.577times to the initial, UA andOA content(36.490and6.975mg/g.DW)were4.744times,2.881times to the initial, respectively.The optimal culture conditions were as follows(inoculation quantity was80g/L, pH6.0, carbonsource concentration of30g/L, temperature25℃, light400Lux, speed of130r/min, the volumeof liquid130mL/250mL). Study on the hormone, NAA>2,4-D>IBA, KT>BA, and the separateroles of auxin, cytokinin were more effect than combination, perhap it was the antagonismbetween the hormone. At the same time, study the influence of elicitors on cell growth and UA、OA accumulation, results showed that the addition of100mg/LMJ was advantageous to loquatcell suspension culture, the fresh weight of34.65g.FW, UA and OA content of33.089,6.496mg/g.DW, respectively, were1.080,2.540,2.590times to the control, respectively.3Extraction and determination of UA and OA contentThe modified method based on UA and OA ultrasonic extraction experiment from loquatleaf, using95%ethanol as solvent, ultrasonic for UA, OA extraction method was used tooptimize extraction method, obtained optimum: ultrasonic power50W, ultrasonic time20min,1:20ratio of material to liquid, ultrasonic frequency of2times. In this method, ultrasonicextraction rate of UA and OA was99.3%,98.4%respectively. The experimental results alsoindicated that the HPLC method reliable, high accuracy. UA have a good separating degree fromOA, the HPLC method was suitable for extraction and determination of UA and OA content.To sum up, the test had established good callus induction and subculture method, obtainedthe suitable callus for loquat suspension(light yellow, loose, granular obvious). And the test hadestablished suitable culture system for loquat cell growth and UA、OA accumulation.