Establishment Of In Vitro Plant Regeneration And SSR And AFLP Reaction System Of Chimonanthus Praecox Link.Var. Concolor

Chimonanthus is the only representative species in China in the genus. It isendemic to China and dates to Tertiary. In China it is a widespread floricultural species andis a2.5³.0m tall deciduous shrub. Chimonanthus is a rare ornamental species that flowersduring winter in the Northern Hemisphere and the bright and scented flowers appear fromNovember to February, usually in mid-January. Chimonanthus is frost hardy, survivingshort periods of frosts with temperatures reaching as low as18°C. Chimonanthus is theunique flowering plants for China, many varieties, has a high ornamental value, thedevelopment of Chimonanthus for improveing the ecological environment conditions,improving people’s living standards and increasing farmers’ income has importantecological, economic and social significance.This article use Chimonanthus praecox Link.var.concolor as the materials, developthe establishment of in vitro plant regeneration system and SSR and AFLP technologysystem and primers screening. Now results are summarized as follows:（1）Establish the in vitro plant regeneration system of Chimonanthus praecoxLink.var.concolor. The best medium for Chimonanthus callus induction is MS+2.0mg.L^-16-BA+0.9mg.L^-1NAA, the induction rate is93.5%.The best medium for adventitiousbuds induction is WPM+2.37mg.L^-1TDZ+1.48mg.L^-1NAA, the highest induction rate is71.33%, the most suitable rooting medium is using1/2MS+1.5mg.L^-1IBA pretreatment30days, the rooting rate is80%.（2）Establish SSR reaction system. Through the selection of template DNA, dNTP,primer concentration, Taq enzyme and annealing temperature for the impact of SSRamplification, establishing appropriate molecular markers reaction system ofChimonanthus SSR, also screening suitable SSR primers of Chimonanthus. The resultsshowed that the appropriate annealing temperature of Chimonanthus SSR amplificationwas55℃. In20μL reaction system,Chimonanthus template DNA, dNTP, primerconcentration was5mg.L^-1、0.2mmol.L^-1and0.5μmol.L^-1, Taq enzyme and10×Taqenzyme buffer was2U and2.5μL. Using the reaction system,41pairs of SSR primers forChimonanthus SSR amplified were screened from417different woody plants primers.（3）Establish AFLP reaction system. Through study on the main factors whichaffected the AFLP system quality, the technology system of AFLP which was suitable forChimonanthus was established. The results indicated that the AFLP optimum conditions for restriction enzyme digestion of the Chimonanthus were as follows:600ng DNAtemplate,3U Pst I and Mse I respectively in20μL reaction volume, at37℃for2h; Theligation system （20μL） with15μL the digestion product,3U T4ligation enzyme,0.25μmol·L^-1Pst I adapter,2.5μmolL^-1Mse I adapter,1μL10×T4Buffer, at22℃for12h wasthe optimum condition; The pre amplification reaction （20μL） with5μL the ligationfragment which was diluted10times,2.0mmol·L^-1Mg2+,2U Taq enzyme,300μmol·L^-1dNTP,0.5μmol·L^-1Pst I and Mse I primer （P+AGA/M+ATC） was the optimum condition;The selective amplification reaction （20μL） with5μL preamplification product which wasdiluted20times,2.0mmol·L^-1Mg2+,2U Taq enzyme,300μmol·L^-1dNTP,0.5μmol·L^-1Pst I and Mse I primer （P+AGA/M+ATC）. Finally,96pairs of primers were chosen for theAFLP analysis of Chimonanthus plants.