DNA Methylation Alterations Of Chrysanthemum (Dendranthema Grandiflorum) During In Vitro Growth By Artificial Inducement

DNA methylation is critical for regulation of plant gene expression, and plays an essential role in plant development. DNA methylation inhibitor (5-azaC) has been used in the studies of plant epigenetics that based on DNA methylation. 5-azaC could substitute the low temperature and promoted plant flowering, ahead of flowering time. Methylation-sensitive amplification polymorphism (MSAP) technique was used to study the variation of DNA methylation during continued generation of tissue culture in the'moqilin'variety of Chrysanthemum(Dendranthema grandiflorum). Also, the effect of 5-azaC to the morphogenesis and flowering time has been evaluated by developmental phenotypes and DNA molecular marker techniques. The main results are as follows:1. The DNA methylation status during the successive transfer culture of Chrysanthemum was analysised by MSAP technique. The results indicated that three single shoot system of the tissue culture seedling had distinct DNA hypermethylation and demethylation compared to the field materials, the mutation rate were 8.929%,8.902%,8.986% respectively. All the successive transfer materials had DNA methylation variety compared to the parent materials. During the variation, demethylation were more than hypermethylation. Along with the successive transfer culture, the diversity between the two variety pattern was smaller, and the proportion allmost similar.Meanwhile, the different individual of each single shoot system also existed DNA variation phenomenon.2. The development of the samples with 5-azaC treatment and the controls were observed. The results indicated that, The number of multiple buds differentiated from treated shoot-tips was fewer than control's. The concentration of 5-azaC higher than 100μmol/L also affect root's morphogenesis. Research indicated that, the inhibiting effect of 5-azaC has time and dose adding domino offect. Meanwhile, the role of 5-azaC to the inhibition of multiple bud was stability. During the successive transfer culture after removed 5-azaC, the number of multiple buds differentiation of all treatment were ascend, but also less than control. Meanwhile, 5-azaC even affected the flowering time of Chrysanthemum, of all low concentration 5-azaC treatment promoted flowering. while the time that aheaded was correlated to the concentration, and these effect to the flowering time were stability when treated materials growth in the field.3. The genome DNA of treatments and controls were analyzed by amplified fragment lenth polymorphism (AFLP) technique. The DNA were distilled from the leaves of treatments treated by different concentration and controls after growthed 30 days. The DNA were digested by EcoRⅠand MseⅠ, and 1267 bands were scored using 25 pairs of primers combination. No variation of bands was found during the samples, which suggested that no nucleotide sequence had altered during 5-azaC treatment.4. Methylation-sensitive amplification polymorphism (MSAP) technique was used to investigate the DNA methylation status of the treated samples, the materials after removed 5-azaC and the control. 5-azaC caused the genomic DNA methylation decrease. The decrease was increased when 5-azaC concentration was added, and when the materials treated by the same concentration of 5-azaC, the decrease was increased with the treated time extend. The demethylation status was stability after 5-azaC was removed. The materials treated by four kinds lowness 5-azaC had 3 sites DNA hypermethylation and 8 sites DNA demethylation when they were growth in field, and the patterns were consistent.