Establishment Of Regenerative System And Chromosome Doubling Of Wild Lycium Ruthenicum Murr

Lycium ruthenicum Murr is a small deciduous shrub of lycium in nightshade family.In China,it is mainly distributed in Xinjiang Province,Ningxia Province,Tibet,Gansu Province,Qinghai Province,Inner Mongolia and other regions.It is a wild plant in northwest China.To solve the difficulty of harvest,the fruit production low of Chinese wolfberry industry development conditions,at the same time for our country agriculture and environmental problems,provide valuable data,this study respectively to golmud,Lycium ruthenicum Murr no vaccine as the explant,annual branches,and seeds are established in vitro culture regeneration system,compared the two kinds of explant suitable choice,which use regeneration system to complete the process of building a stable factory breeding new technology,for the Lycium ruthenicum Murr in vitro culture technology direction of development train of thought,also aims at providing a Lycium ruthenicum Murr germplasm resources mining and provide a reference basis.In the research of Lycium ruthenicum Murr factory nursery culture techniques at the same time,with the strain seeds were used as material,using colchicine for chromosome doubling experiment,in order to obtain polyploid(tetraploid)plants,and uses the morphology,cytology,and Flow Cytometry,and other chromosome ploidy identification method of chromosome(tetraploid),to the strains of Lycium ruthenicum Murr germplasm initiative,reasonable utilization of resources to establish on the basis of theory and practice.The main research results are as follows:(1)The best way to sterilize the annual stem segments of lycium rubra as explants is:soak them with 75%ethanol solution for lmin～2min,then soak them with 8%sodium hypochlorite solution for 12min.(2)The callus medium is MS+6-BA 0.5mg/L+IBA 0.5mg/L,and the callus formation probability is 94.89%;The culture medium formula for callus proliferation is:MS+IAA 0.1mg/L+IBA 0.5mg/L;The growth and differentiation medium of callus is MS+6-BA 1.0mg/L+IAA 0.1mg/L;The culture medium of bud is:MS+NAA 1.0mg/L:Cluster bud proliferation medium is:MS+NAA 0.1mg/L+IBA 0.5mg/L;Regeneration system establishment rooting medium is MS+IBA 0.5mg/L.(3)The survival rate of transplanted seedlings was 71.6%in the mixed substrate with loam:nutritive soil:vermiculite ratio of 3:1:1.(4)Seed culture of sterile seedlings was used to establish a regeneration system for explants.Seeds were treated with 75%ethanol for 1min+8%NaCIO solution for 8min.The germination rate of seeds in MS basic medium was 94.00%.The establishment medium of the regeneration system using sterile seedlings as explants was MS+IBA 0.5mg/L.(5)The diploid karyotype formula was:2n=2x-=24=12sm+12m(3SAT),and the karyotype was type 2B.The concentration of colchicine was 0.3%,the treatment time was 24h,and the tetraploid karyotype formula was obtained:2n=4x=48=24sm+24m(3SAT),and the karyotype was type 2B.(6)The authenticity of chromosome double polyploid(tetraploid)was identified by means of morphology,cytology and DNA content identification by flow cytometry at the molecular level.