| Dioscorea composita tubers contain extremely rich dioscin.Dioscin is an important raw material for the synthesis of many steroids,named as "medicinal gold".However,with the wide application of steroid hormones and the predatory exploitation of the wild resources of Dioscorea composita,the regeneration speed of Dioscorea composita was far behind the demand of the market and dioscin had became one of the most scarce drug raw materials at home and abroad.Therefore,to improve the planting yield of Dioscorea composita and the content of dioscin have become urgent problems to be solved.This paper intended to achieve the rapid propagation of Dioscorea composita through the formation of embryogenic callus to regenerate plants.The embryogenic callus were used as explants to establish the hairy root system of Dioscorea composita,which provided an excellent transformation system for the functional studies of key genes in dioscin synthesis pathway.At the same time,by screening the reference genes at different developmental stages of Dioscorea composita tubers,laid a foundation for the molecular mechanism study of dioscin synthesis pathway in Dioscorea composita.The experimental results obtained were as follows:(1)Callus were induced by different explants of Dioscorea composita.It was found that the optimum medium for inducing callus of Dioscorea composita seeds was MS+1 mg/L 2,4-D+2 mg/L 6-BA+2 mg/L KT+2 mg/L NAA,the induction rate was 55.6%;the optimum medium for leaves of the tissue culture seedlings of Dioscorea composita was MS+2 mg/L 2,4-D+2 mg/L 6-BA+1 mg/L NAA,the induction rate was 58.3%.When the stem segments of the seedlings,field leaves and stem segments of Dioscorea composita were used as explants to induce callus,they were not sensitive to the expression of all hormone combinations and had no significant difference in the induction.The optimal subculture medium of leaf callus was MS+2 mg/L 2,4-D+2 mg/L 6-BA+1 mg/L NAA.The optimal subculture medium of stem callus was MS+2 mg/L 2,4-D.In summary,the callus induction system of Dioscorea composita was initially established.(2)The hairy roots was induced by Dioscorea composita stem-inducing callus and rootremoving plantlets of Dioscorea composita.It was found that K599 had the best inducing effect on the callus of stem segments of Dioscorea composita,and the induction rate was the highest 18.92%;C58C1 had the best induction effect on root-removing plantlets of Dioscorea composita,and the highest induction rate was 42.86%.Used root-removing plantlets of Dioscorea composita as explants,the infection rate of K599 increased significantly with the increase of AS concentration,the highest induction rate was 40.91% at a concentration of 400 μmol/L AS.The hairy root induction system of Dioscorea composita was preliminarily established.The explants were root-removing plantlets of Dioscorea composita;the Agrobacterium tumefaciens K599 was inoculated for 20 min,and then co-cultured in dark on MS solid medium with AS(400 μmol/L);after 2 days co-cultured,the explants were sterilized in sterile water with Cephalosporin(Cephalosporin,Cef,250 mg/L)and finally inoculated on Cef(250 mg/L)MS solid medium at a constant temperature of 25°C in the incubator to induces hairy roots.(3)We analyzed Dioscorea composita transcriptome and selected eight candidate genes for expression stability analysis.Statistical analysis of quantitative real-time PCR(qPCR)results using GeNorm,NormFinder and BestKeeper showed that the expression of TUB7 and H2A2 are themost stable in tubers,and thus can be used as reference genes for qPCR analysis in Dioscorea composita.This paper lays a foundation for the establishment and improvement of the callus induction system and the establishment of hairy root system of Dioscorea composita,and provides valuable reference for callus induction and hairy root induction of dioscorea plants;The basis of the molecular mechanism of the dioscin synthesis pathway is laid,and it also provides clues for the screening of the reference genes of the dioscorea plants. |
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