Vitro Culture And Genetic Stability Of Rubus Sachalinensis Leveille

Objective:The clinical use of Rubi Ramulus all from the field collection of Rubus sachalinensis Leveille.Wild resources were not only limited,but also the components and quality of Rubi Ramulus were different in different areas.A large number of high quality seedlings were obtained in a short time by establishing the rapid propagation system of Rubus sachalinensis Leveille.It is not affected by seasonal,environmental and other factors.This technology effectively solves the problem of scarce wild resources.Finally,the genetic stability of tissue culture seedlings was determined by ISSR molecular marker technique.It not only expands the medicinal materials resources,but also protects the rare wild resources.In particular,the genetic characteristics of medicinal materials have been stabilized and improved.Methods and results:?1?Establishment of rapid propagation system of Rubus sachalinensis Leveille.The explant is the adventitious bud of Rubus sachalinensis Leveille.To investigate the effect of PGR species and concentration on explants proliferation culture,strong seedling culture,and rooting culture.Results show,The explants were sterilized with 75%ethanol for 40 s,followed by sterilization with0.1%Hgcl₂ for 8 min,which showed the best germination rate of 78.34%.The optimal proliferation medium was MS+6-BA 2.0 mg/L+KT 0.5 mg/L+NAA 0.1mg/L+IBA 0.1 mg/L+GA₃ 0.5 mg/L.The multiplication factor was 7.71.The multiple shoots were green and their stems were thin and weak.The optimal strong seedling medium was MS+6-BA 2.0 mg/L+KT 0.5 mg/L+NAA 0.1 mg/L+IBA0.1 mg/L+GA₃ 0.5 mg/L.The multiplication factor was 6.60.The multiple shoots were green and their stems were thick and strong.The optimal rooting medium was1/2 MS+IBA 1.5 mg/L.The rooting rate was 97.04%.The average rooting number was 8.05 and the average root length was 3.05 cm.The roots grow fast,long and strong.After hardening-seedling,the survival rate was 100%when the seedlings were transplanted to the humus-perlite-vermiculite?2:1:1,V:V?matrix.?2?Establishment of ISSR-PCR Reaction System.The whole genome of leaves and stem segments were extracted by centrifugal column method.The results showed,the quality and concentration of DNA in leaves were higher than that in stem segments.Therefore,leaf is the best extraction site.The optimal system for ISSR-PCR reaction was 20?L total volume,0.3 moL/L primer,30 ng DNA template,11?L Mix enzyme,0.75%deionized formamide,3?g/mL bovine serum albumin.Best amplification program for 94?modified 2 min,after 94?modified 30 s,30 s according to the different primer annealing temperature annealing,72?extension 2 min,a total of 30cycle,72?extending 30 s,save at the 4?.The mutation rate of tissue culture seedling was 0.63%.?3?To determine the genetic stability of tissue cultured seedling.In this paper,an ISSR-PCR reaction system was established to analyze the genomic DNA of tissue seedlings and wild seedlings.A total of 475 clear strips were obtained,with only 3 variation bands and a variability rate of 0.63%.Conclusion:Rubi Ramulus is the dried stem of Rubus sachalinensis Leveille.It is used to treat cold,immature fever,stroke fever,cough,“heyi”fever and other diseases.Since the twentieth century,with the rapid development of Mongolian medicine industry and the increasing amount of Mongolian medicinal materials,the wild resources are decreasing day by day,It severely restricts the healthy development of Mongolian medicine industry.In order to protect the wild resources,this study established the tissue rapid propagation system for the first time.Using adventitious buds as explants,it can cultivate high quality seedlings in a short period of time.It overcomes the scarcity of wild Rubus sachalinensis Leveille and protects the ecological environment.At the same time,the tissue culture seedlings with good reproducibility,low mutation rate and high genetic stability were obtained by PCR amplification with simple repeat interval.It not only opened up a new way for the rapid propagation of raspberry tissue,but also laid a foundation for the preservation of fine characters and the protection of resources.