In Vitro Induction And Identification Of Autotetraploid And Research On Genetic Variation Of Atractyloides Lancea (Thunb.) DC.

This study induced the autotetraploid using colchicine in vitro on the basis of the technology system of rapid propagation and combined with variety of identification methods to screen out mutant strains,in addition,the research on the genetic diversity between parents and autotetraploids of Atractylodes lancea(Thunb.)DC.were carried out.The specific contents and research results were as follows:1.In this experiment,Sterile system was established after the wild seeds of Atractylodes lancea DC.were disinfected with 0.1%HgCl2,the optimization system of proliferation and rooting medium were studied through the orthogonal design,and transplantation medium of tube seedlings were selected.The optimal disinfection method for screening was described as follows:The results showed that the rate of seeds germinating was 87%after pretreatment with alcohol of 75%for 30s and 0.1%HgCl2 for 4min;The best culture medium suitable for the proliferation of Atractylodes lancea DC.was MS+6-BA 2.0 mg·L-1+NAA 0.2mg.L-1,and the average multiplication factor was 9.2±1.1 every 30 days;The optimum medium for rooting was 1/2MS+NAA0.2mg.L-1,root thickness and were easy to survival,the average rate of rooting was about 82.6%;The highest survival rate of the tube seedlings was 91%in vermiculite and they were very strong.This research established the technical system of rapid propagation and created new autotetraploid germplasm of Atractylodes lancea(Thunb.)DC..2.Tetraploid induced with buds in vitro and the identification of autotetraploid plants of Atractylodes lancea(Thunb.)DC.based on morphological characteristics,observation of stomatal,Chromosome tablet and flow cytometry.The buds soaked in 0.2%colchicine solution for 12h was ideal conditions to induce autotetraploid of Atractylodes lancea(Thunb.)DC.with induction rate of 36%;Morphological indexes such as leaf area index、leaf length and width、the density of stomas and the number of chloroplast of the suspected tetraploid after colchicine mutagenizing were distinctly different from diploid.The experiment obtained 23 autotetraploid lines and laid a foundation for the further study on mechanism Atractylodes lancea(Thunb.)DC..3.Diploid and tetraploid plantlets of Atractylodes lancea(Thunb.)DC.were used in this study and MSAP(Methylation-Sensitive Amplification Polymorphism)analysis was conducted to explore the level and pattern of DNA methylation in them.520 and 527 loci were detected in diploid and autotetraploid by 20 pairs of selective primers,and the corresponding total methylation rate were 56.92%and 55.03%.Compared with diploid,the genomic DNA total methylated rate and full methylated rate of autotetraploid were decreased,but hemimethylated rate was increased,and the DNA methylation patterns adjusted by 52.53%.This research laid a foundation for the further study on genetic epigenetic mechanism of autotetraploid of Atractylodes lancea(Thunb.)DC..4.One diploid strain and five autotetraploids strains of Atractylodes lancea(Thunb.)DC.were used in this study.The results showed that 6 strains were detected 34 alleles and 19 fragments were polymorphic by 5 IS SR primers,percentage of polymorphic band swas 55.6%,the bands of ISSR amplification and chromosome number to be unrelated,some autotetraploids amplified extra bands and some autotetraploids with missing bands;Diploid and tetraploid of Atractylodes lancea(Thunb.)DC.had large differences in genetic distances and genetic similarity coefficient.