Relationship Between PCD And ROS Metabolism And NO Synthesis In Somatic Embryogenesis Of Fraxinus Mandshurica

Fraxinus mandshurica is a hardwood species in the genus Fraxinus in the Oleaceae,which has important wood utilization value and ornamental value.This research studied the morphology and function changes of mitochondria in cultured cells during somatic embryogenesis by adjusting the osmotic pressure and the concentration of plant growth regulators in the medium,and combining with intracellular reactive oxygen species and nitric oxide metabolism and programmed cell death analysis,in order to demonstrate the relationship between programmed cell death,reactive oxygen species and nitric oxide in early somatic embryogenesis and reveal the mechanism of osmotic pressure and plant growth regulators regulating somatic embryogenesis and programmed cell death.Main research results were as fellows:(1)During somatic embryogenesis in F.mandshurica,at 60th day,adding the plant growth regulator could promote the browning of explant,which was higher than the treatment of without plant growth regulators about 20.45%on the medium of adding 20 g·L-1 sucrose.On the 75 g·L-1 sucrose sucrose medium,the browning percentage of the explants treated with without plant growth regulators was 94.15%,and browning percentage of explants treated with plant growth regulators was 100%.In the absence of plant growth regulators,there were no somatic embryos on the explant.In the addition of plant growth regulators,somatic embryogenesis percentage and the number of somatic embryogenesis were 58.55%and 20.02 per explant on the meduim supplemented with the 75 g·L-1 sucrose,and on the medium supplemented with the 20 g·L-1 sucrose,the somatic embryogenesis percentage and the number of somatic embryogenesis were 11.43%and 11.56 per explant.The results showed that plant growth regulators played a decisive role in somatic embryogenesis and promoted the browning of explants,high concentration of sucrose could promote somatic embryogenesis and explant browning.(2)During somatic embryogenesis of F.mandshurica,with the cultured time extended,chromatin condensation inside the nucleus and the internal structure of mitochondria became blurred and showed a spherical shape,and the Caspase-3 enzyme activity increased.It was demonstrated that explant cells had programmed cell death.Mitochondrial membran absorbance,mitochondrial membrane potential and the absorbance of Cyt c/a decreased.The higher osmotic pressure and plant growth regulators as an environmental stress that could induce H2O2,and the H2O2 could change the mitochondrial structure,which caused changes in mitochondrial membrane potential and the mitochondrial membrane permeability increased,and released cytochrome c into the cytoplasm.Finally,Caspase-3 enzyme activity increased and caused irreversible programmed cell death.(3)During somatic embryogenesis of F.mandshurica,programmed cell death occurred in explant cells,and the content of intracellular H2O2 and NO increased.The activity of SOD,POD,and CAT associated with H2O2 metabolism increased,in order to maintain the homeostasis of H2O2 in the cells.The correlations between NOS activity,NR activity and intracellular NO content were not significant.The intracellular NO synthesis in somatic embryogenesis of F.mandshurica might be the result of non-enzymatic reaction.In summary,plant growth regulators play a decisive role in somatic embryogenesis of F.mandshurica,and higher osmotic pressure promotes somatic embryogenesis.The somatic embryogenesis of F.mandshurica involves H2O2 signal and NO signal.H2O2 as an induction factor is involved in PCD of explant cell and NO signal synthesis is involved in this process.