| Pseudostellaria heterophylla is a perennial herbal medicinal plant belonging to the family Caryophyllaceae,and is used as a medicine by tuberous roots.It is a genuine medicinal material in Fujian Province,with the effects of supplementing qi,strengthening the spleen,promoting fluid production,and moistening the lungs.The cultivation of Pseudostellaria heterophylla is mostly carried out by asexual propagation of root tubers,which accumulate a variety of mosaic viruses,leading to a degradation trend of germplasm,mainly manifested as short plants,leaves appear chlorotic yellow spot,shrinkage,curl and other symptoms,the finally harvested Pseudostellaria heterophylla has small tuberous roots,low yield,and poor quality,which seriously affects the planting efficiency and medicinal quality of Pseudostellaria heterophylla,becoming a bottleneck problem in the development of the national Pseudostellaria heterophylla industry,seriously affecting the planting efficiency and medicinal quality of Pseudostellaria heterophylla,and becoming a bottleneck issue in the development of the national Pseudostellaria heterophylla industry.Using plant virus-free methods to cultivate virus-free seedlings for production is currently the most effective way to solve the virus disease of Pseudostellaria heterophylla.In this project,methods such as plant virus-free culture,virus molecular detection,plant factory breeding technology,and field block control cultivation were used to carry out field investigations on the mosaic disease of Pseudostellaria heterophylla and research on the virus-free breeding of seedlings.To select the most suitable virus-free method and rapid virus detection technology,to breed virus-free seedlings of Pseudostellaria heterophylla,and to establish the virus-free seed root propagation technology system,to provide theoretical guidance and technical support for the large-scale production and application of virus-free seedlings of Pseudostellaria heterophylla.The main research results are as follows:(1)A field investigation and detection of the mosaic disease of Pseudostellaria heterophylla was conducted in multiple townships in Zherong County,Ningde City.The diseased leaf rate of Pseudostellaria heterophylla plants was 82%-93.3%,and the disease index was 81-93.3.Multi point sampling was carried out at each survey site.A total of 30 samples of Pseudostellaria heterophylla were collected.After RT-PCR molecular detection,30 samples carried turnip mosaic virus(Tu MV),16 samples carried broad bean wilt virus(BBWV),and 2 samples carried cucumber mosaic virus(CMV),tobacco mosaic virus(TMV)was not detected.The Pseudostellaria heterophylla cultivated in Zherong County is all infected by the mosaic virus,and there is a phenomenon of multiple virus compound infections,turnip mosaic virus was first found to be the main virus infecting Pseudostellaria heterophylla.(2)A triple RT-PCR molecular detection technique was established for the detection of Pseudostellaria heterophylla virus,which can quickly and accurately detect Tu MV,BBWV and CMV of Pseudostellaria heterophylla at the same time.The triple RT-PCR amplification conditions were as follows: 94 ℃ pre denaturation for 4 minutes,94 ℃denaturation for 30 seconds,53 ℃ annealing for 30 seconds,72 ℃extension for 1 minutes and 25 seconds,a total of 33 cycles,with the final72 ℃ extension for 10 minutes.(3)The method of virus-free of Pseudostellaria heterophylla was established by low temperature treatment combined with microshoot tip culture and embryo culture.The four methods of microstem tip culture,low-temperature treatment combined with microstem tip culture,ultra-low temperature culture,and seed embryo culture were used to carry out virus free culture of Pseudostellaria heterophylla.The survival rates were 33.3%,64.7%,16.2% and 56.7%,respectively.The virus free rates of BBWV were 28.6%,68.2%,33.3%,and 100%,respectively;The detoxification rates of Tu MV were 14.3%,31.8%,33.3%,and 100%,respectively.From the perspective of maintaining the excellent characteristics of Pseudostellaria heterophylla varieties,low temperature treatment combined with micro stem tip culture is the preferred choice for the antiviral method of Pseudostellaria heterophylla;If genetic variation of the seeds of the Pseudostellaria heterophylla is not considered,embryo culture is best choice for antiviral methods of the Pseudostellaria heterophylla.Tu MV was found to be more difficult to remove than BBWV for the first time.(4)A technical system for virus-free seed and seedling breeding of Pseudostellaria heterophylla was established and applied to production.The system includes:(1)Selection of typical plants with variety characteristics of Pseudostellaria heterophylla;(2)Obtaining,sterilizing and cultivating materials;(3)Inducing the formation of microtuber roots in tissue culture seedlings;(4)Low temperature induced germination of microtuber roots;(5)Peeling and culturing of micro stem tips;(6)Virus detection of microstem tip tissue culture seedlings;(7)Expansion and culture of tissue culture seedlings of virus-free original seeds;(8)Breeding of Microtuberous Roots of Virus-free Original Species;(9)Breeding of virus-free original species;(10)Breeding of virus-free production seeds.Based on this technical system,the "Technical Regulations for Improved Seed Breeding of Pseudostellaria heterophylla"(enterprise standard)has been formulated to guide enterprises in the industrialized production and application of virus-free seedlings of Pseudostellaria heterophylla.(5)The effects of different viruses on the growth,yield,and quality of Pseudostellaria heterophylla were studied.Individuals carrying only one common virus(BB)of BBWV,two common viruses(TB)of Tu MV and BBWV,and no virus(CK)of Pseudostellaria heterophylla were screened out.After expansion,they were planted in the field using a randomized block method.Compared with CK,the total chlorophyll content,net photosynthetic rate,and shoot height of TB plants decreased by 24%,32% and 26%,respectively.The root tuber yield,extract content,and polysaccharide content per unit area decreased by 77.5%,76.2%,and87.3%,respectively;There were no significant differences in photosynthesis,plant height,root tuber yield,extract content,and polysaccharide content between BB and CK plants.The results showed that Tu MV significantly inhibited the growth of Pseudostellaria heterophylla,decreased the yield and quality of Pseudostellaria heterophylla,while BBWV had no significant effect on Pseudostellaria heterophylla.Therefore,Tu MV should be removed firstly in the process of Pseudostellaria heterophylla virus-free culture. |
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