| Polygonatum cyrtonema is a perennial herb of the genus Polygonatum in Liliaceae family.Its rhizome is used as medicine.In this study,a high-efficiency in vitro propagation system was established for tuber formation of P.cyrtonema,and the effect of stress tissue culture on its growth and development was studied,and the phenotypes of P.cyrtonema in vitro cultured plantlet under dark stress and salt stress were analyzed.The transcriptome of P.cyrtonema in vitro cultured plantlet in response to salt stress was preliminarily analyzed,and its salt resistance mechanism at the molecular level was discussed.Based on the salt stress transcriptome data of P.cyrtonema,the stress resistance genes and tuber morphogenesis genes of P.cyrtonema were analyzed and verified by and real-time quantitative PCR.In addition,based on the high-efficiency in vitro propagation system developed with the tubers,a production model of P.cyrtonema plantlet was established and its benefits were analyzed.The main research conclusions are as follows:(1)In this work,the medium of in vitro cultured of P.cyrtonema was optimized,and the optimal medium for rooting of P.cyrtonema was1/2MS + 1mg/L NAA + 0.6wt% agar.Tuber formation induction medium was MS + 5mg/L NAA + 0.5mg/L 6-BA + 8wt% sucrose + 0.6wt% agar.At the same time,a new P.cyrtonema microtuber propagation mode was established.Under these conditions,all intrinsic buds of P.cyrtonema could form microtubers,avoiding the formation of adventitious buds originating from callus,and developing an effective microtuber formation pathway for the tuber plant P.cyrtonema,which is helpful for easier storage,transplantation and tuber seeding,higher genetic stability can be obtained without callus formation.Through the section staining observation of P.cyrtonema,it was found that the cytological morphology of each part of P.cyrtonema was significantly different,which provided a basis for judging the growth and development stage of P.cyrtonema.(2)Through stress-induced in vitro cultured,it was found that the dark culture conditions are conducive to the proliferation of P.cyrtonema tubers,which not only provides a new idea for tissue culture of underground rosettes,but also conducive to large-scale industrial production,energy saving and environmental protection.The results of salt stress on the in vitro cultured plantlets of P.cyrtonema showed that P.cyrtonema was not a salt-tolerant plant and could not survive under the condition of NaCl concentration above 300 m M.In addition,the electrical conductivity under salt stress of P.cyrtonema was measured,and the calculation formula of the conductivity corresponding to the salt content of P.cyrtonema plants was obtained,which provided a basis for quantitative detection of the salt content in plants.(3)In this study,6 c DNA libraries were sequenced before and after500 m M NaCl salt stress treatment of P.cyrtonema,and the results showed that a total of 163,617 new genes were obtained.Based on the difference analysis results,it was found that there were a total of 4473 significantly expressed genes after NaCl treatment of P.cyrtonema plantlet,of which 1609 genes were up-regulated and 2864 genes were down-regulated,and the number of down-regulated genes was significantly greater than that of up-regulated genes.It indicated that the gene expression of P.cyrtonema in vitro cultured plantlet was significantly inhibited under high salt concentration.(4)The relative expression levels of related genes at different time points and different NaCl concentrations were analyzed by q RT-PCR,it was concluded that Pc-CIPK,Pc-WRKY,Pc-MYB and Pc-PLD were positive regulators under salt stress of P.cyrtonema Hua.Pc-LOX,Pc-NPF,Pc-SPX,and Pc-PER showed an overall downward trend under salt stress,Pc-Fd,Pc-PETH,Pc-LOX,and Pc-NPF were negatively correlated with NaCl concentration.Pc-PLP was highly expressed at the initiation stage of P.cyrtonema tuber.(5)The technical route of industrialized production of P.cyrtonema established in this study mainly includes the following steps:establishment of aseptic system of P.cyrtonema → inducing seed germination → proliferation of clump buds → rooting → inducing endogenous buds to form tubers → tuber proliferation → hardening and transplanting → seedling management → mature harvesting → market sales.Through cost calculation and sales forecast analysis,it is concluded that the five-year investment profit of P.cyrtonema factory is 236.9%.The payback period of P.cyrtonema test tube seedling factory is 2 years and 3 months.On this basis,the micro tuber propagation method can save two months of manpower and electricity for the factory nursery.To sum up,the dark culture mode of P.cyrtonema microtubers is beneficial to greatly improve the industrial production efficiency of P.cyrtonema.Darkness and salt,as factors affecting the tuber development of P.cyrtonema,are convenient and quick to simulate and screen under In vitro cultured conditions,and provide a new method for fast propagation and transplanting of underground tuber crops.At the same time,the phenomenon that P.cyrtonema can grow and develop normally to form micro-tubers under the condition of low salt concentration also provides the possibility for the planting and cultivation of P.cyrtonema Chinese medicinal materials industry in coastal areas. |
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