DNA Methylation Of Zygotic Cotyledons At Different Collecting Time And The Somatic Embryogenesis Of Fraxinus Mandshurica

Manchrian ash (Fraxinus mandshurica Rupr.) is one of the three valuable hardwood species in Northeast China, and is important for timber production. By using cotyledons of zygotic embryo at different collecting time as explants, to investigate somatic embryogenesis; and by adding 5-azacytidine into the medium to investigate somatic embryogenesis; at the same time, measured genomic DNA methylation level of cotyledons of zygotic embryo in different collecting periods and explants by MSAP, to study the relationship between the DNA methylation level and somatic embryogenesis of F. mandshurica. The study makes the foundation for in-depth analysis the relationship between the somatic embryogenesis of F. mandshurica and genomic DNA methylation status of explant, and provides the regulation of scientific basis for somatic embryogenesis of F.mandshurica. The conclutions are as follows:(1) Because of different temperature, the optimal collecting stages is different for somatic embryogenesis of F. mandshurica. Using cotyledons of zygotic embryo in different collecting periods as explants, the highest rate of somatic embryo investigated happened in late July; the highest investigate rate of callus appeared in mid-July and late July, and were significant difference with other periods (p<0.05). Therefore, to determine the material is carried out in late of July, is the best collecting periods for somatic embryogenesis.(2) By using MSAP, we established the detection system, which was used to detect the level of genomic DNA methylation of cotyledons of zygotic embryo of F. mandshurica. By detecting the CCGG sites of cytosine methylation of genomic DNA in zygotic cotyledons of F.mandshurica at different developmental periods. According to the different periods, maps showed out 933～939 clearly identified PAGE bands respectively. The least bands appeared in late July, methylation of each period was different, but the difference was not significant. The lowest DNA methylation level apeared in late July, and the highest rate of somatic embryo investigated happened in the same period.(3) Medium supplemented with 5-azacytidine for somatic embryogenesis of F.mandshurica, it influenced the rate of the explant states, but did not affect the type of explants. The effect of 5-azacytidine on the number of somatic embryogenesis was not significant, however, it was significant difference on the number of abnormal embryogenesis. And 20.0μmol/L of 5-azacytidine significantly promoted the number of abnormal embryogenesis.5-azacytidine can inhibited browning of explants, callus induction and somatic embryogenesis, it was different inhibitory effect by each concentrations. Of which,0.5μmol/L of 5-azacytidine inhibition intensity.(4) Added 5-azacytidine with 20.0μmol/L during different periods of culture, the number of somatic embryogenesis and the number of abnormal embryogenesis were evidently improved by the addition during the first day of culture and they were the significant difference with the other treatments(P<0.05), at the same time, evidently suppressed the browning of explants but significantly promoted the rate of callus induction. It was not significantly impact with the other treatment. Added 5-azacytidine in 20.0μmol/L during 14 to 21 days of culture could significantly promot somatic embryogenesis of F.mandshurica.(5) The influence patterns of 5-azacytidine on explant browning was almost same with that on somatic embryogenesis proved our former discovery of "somatic embryogenesis of F. mandshurica companied by explant browning". There was no obvious effect of supplementing 5-azacytidine into medium on the DNA methylation of explants.