Changes In Methylation Of Genomic DNA During Dedifferentiation And Regeneration In Populus Nigra N46

DNA methylation was a principal form of epigenetic modification in eukaryotes which widely existed in all kinds of creatures and involved in the activities of a variety of life. DNA methylation easily influenced by the external environment conditions. The DNA that produce in the process of tissue culture was an important source of regeneration plant phenotypic variation. The cμLtivation in Populus nigra had an important position in the worldwide.This paper use Populus nigra N46 analysis the genomic DNA methylation changed in tissue culture plantlets, which suffered drought stress renewable generation and regeneration five generations of differentiation under normal conditions, by the using MSAP technology of capillary electrophoresis and fluorescence quantitative PCR detection method. Theresults were as follows:(1) A high efficient regeneration system of P.nigra N46 was established. Ourresults showed that the best differentiation medium of P.nigra N46 was MS+6-BA 0.5 mg·L-1 + NAA 0.01 mg·L-1+ sucrose 30 g·L-1+0.5% agar, and rooting medium was 1/2MS+IBA 0.01 mg·L-1+ NAA 0.01 mg·L-1+ sucrose 30 g·L-1+0.5% agar. The best temperature for differentiation and rooting was 27℃ and 25℃ respectively. The establishment of this regeneration system provides the reference for fast reproduction and genetic transformation of species in Aigeiros section.(2) This paper analysed the genomic DNA methylation changed in tissue culture plantlets in P.nigra N46, which suffered drought stress renewable generation and regeneration five generations of differentiation under normal conditions, by the using MSAP technology of capillary electrophoresis. The result showed that there were 17 pair of selective amplification primer, which were all obtained clear amplification band. Each pair of primer combination amplified fragment from 20 to 70, length was between 50 to 700 bp. There were a total of 725 amplification segments.(3) The result of MSAP technology of capillary electrophoresis showed that the methylation leval and total methylation and hemi-methylation level in primary generation somaclone was 23.17%, 16.52% and 4.76% respectively. The levels of methylation in tissue cultured seedling and after treated with drought stress was 22.59%、21.90%、20.86% 、 22.93% 、 22.34% 、 21.72% respectively. The result showed that the multi-generation did not affect the methylation leval of P.nigra N46.