Physiological Characteristic And Genetic Variation Research On Diploid And Autotetraploid Of Platycodon Grandiflorum A.DC.

In this study,We in vitro induced autopolyploid plants by using Plarycodon grandiflorm A.DC.adventitious buds and adopt variety of identification methods to screen out mutant strains.In addition,the preliminary research on physiological characteristics,genetic variation and the level and pattern of DNA methylation between diploid and autotetraploids of Platycodon grandiflorum A.DC.were carried out.The main contents and research results were as follows:1?In.this experiment,Sterile system was established after the Platycodon grandiflorum seeds from chifeng city,Inner Mongolia were disinfected with 0.1%HgCl2,and the optimization system of proliferation and rooting medium were studied.Autotetraploid plants was induced by immpersing adventitious buds with 0.05%,0.10%and 0.20%colchicines solution.Then autotetraploid plants were identified based on morphological characters,chromosome determination and Flow cytometry analysis.The results showed that the optimal medium for proliferation and rooting was MS+6-BA 1.5 mg L-'+ NAA 0.3 mg·L-1 and 1/2MS+ NAA 0.6 mg·L-1,respectively.The adventitious buds soaked in o.2%colchicine solution for 12 h was ideal conditions to autotetraploid of Platycodon grandiflorum A.DC.,The rate of induction reached 32.0%.2.Growth and physiological characteristics were compared between diploid and tetraploid plantlets of Platycodon grandiflorum A.DC.The results showed that the autotetraploid plants displayed thicker and deeper green leaves,stomatal length and width was significantly higher than diploid plants.The contents of chlorophyll a,chlorophyll b,carotenoids and total chlorophyll on unit leaf area of autotetraploid were respectively increased by 36.11%,69.23%,31.71%and 38.85%;The activities of SOD,POD and the content of soluble protein and soluble sugar of autotetraploid are also higher than those of diploid,which increased by 92.91%,12.09%,27.69%and 60.06%,respectively.While the MDA content of autotetraploid was less than diploid;In the aspect of endogenous hormones,the content of Part of the endogenous hormones was increase with increasing ploidy.3.Diploid and autotetraploid plantlets of Platycodon grandiflorum A.DC.were used in this study and SRAP(Sequence-related Amplified Polymorphism)and AFLP(Amplified fragment length polymorphism)analysis was conducted to explore the varaiation of genomic sequence in different ploidy of Platycodon grandiflorum A.DC..SRAP results showed that 206 bands were amplified with 12 pairs of primers and 23 bands were polymorphic.Among them,there were 16 specific bands in diploids and 7 specific bands in tetraploid.The rate of polymorphism was 11.17%.AFLP results showed that 477 bands ranging with 80-500 bp were amplified with 24 pairs of primers,23 bands were polymorphic and the polymorphic rate was 2.73%.the results generally indicated that genetic diversity revealed by the SRAP and AFLP among the diploid and autotetraploid of Platycodon grandiflorum A.DC.was low.After chromosome doubling,most genome nucleotide sequences remain stable,only a tiny part of the DNA base sequence was changed.4.The technology of methylation sensitive amplified polymorphism(MSAP)were used to explore the level and pattern of DNA methylation on the base of acquisition of Platycodon grandiflorum A.DC.autotetraploid.Totally,1586 bands were amplified by 20 pairs of selective primers,of which 764 and 822 bands were detected in diploid and autotetraploid respectively.The total methylation ratio,full methylation ratio and hemimethylated ratio were 91.25%,61.25%and 30.65%in diploid of Platycodon grandiflorum A.DC.,respectively.However,the total methylation ratio,full methylation ratio and hemimethylated ratio of autotetraploid of Platycodon grandiflorum A.DC.were 86.13%,54.38%and 31.75%.Compared with diploid,the genomic DNA total methylate ratio and full methylation ratio of autotetration plants decresed by 6.02%and 7.14%.But the hemimethylated ratio of autotetraploid are higher than diploid,which more than 1.6%.All this results indicated that DNA methylation patterns have adjusted during the polyploidy process.