| As one of the important native tree species in northeast China,Fraxinus mandshurica,with fast growing and excellent timber properities,has great developmental prospects.Somatic embryogenesis can multiply a large number of limited genetically modified materials in a short period,and give full play to its excellent qualities.In this study,we screened the best ascorbic acid(ASA)treatment concentrations and the best quality mother trees,and explored the differences between mother trees from different sources,for the perpose of obtaining effective proliferation pathways of Fraxinus mandshurica,we analyzed the physiological and biochemical reactions in the process explant browning and somatic embryogenesis of Fraxinus mandshurica though adding ASA and hydrogen peroxide(H2O2)exogenously,and explained the internal biological mechanisms of ASA and H2O2 regulating the browning and somatic embryogenesis of Fraxinus mandshurica in different culture stages.The main results are as follows:(1)Eigher low concentration(0～20 mg·L-1)or high concentration(above 150 mg·L-1)of ASA could inhibit the somatic embryogenesis of explants,90～120 mg·L-1 of ASA would promote somatic embryogenesis of explants,110 mg·L-1 ASA was the best treatment concentration for somatic embryogenesis of mature zygotic cotyledon explants of Fraxinus mandshurica.Low concentrations(0～50 mg·L-1)ASA would slow down the occurrence of browning and somatic embryogenesis,high concentrations(90～120 mg·L-1)of ASA would accelerate the occurrence of browning and somatic embryogenesis.110 mg·L-1ASA has the best effect on accelerating the occurrence of browning and somatic embryogenesis.(2)There were significant differences in the incidence of somatic embryos between different mother trees from the same forest farm,with a maximum difference of 4.08 times.In the three forest farms where the mother trees were collected in this experiment,the comprehensive order of all aspects was Northeast Forestry University Experimental Forest Farm>Qingshan Seed Orchard in Weihe>Jinshan Seed Orchard in Linjiang.The mother trees of Fraxinus mandshurica in the experimental forest farm of Northeast Forestry University had the most somatic embryo induction rates,and most of them appeared on single cotyledon explants.The mother trees of Qingshan Seed Orchard in Weihe had the most callus regeneration rates.(3)The non-browning explants hardly had somatic embryogenesis,and most of the somatic embryos occured on 3/4 browned explants.The browning rates of all of the living explants were almost all reaching 100%.Along with the degree of browning deepened,the activities of polyphenols,PPO,PAL,SOD and POD all increased.(4)ASA would be oxidized to DHA in the early stage of explant culture.After degradation,a large amount of H2O2 would be generated,which will promote the increase of polyphenol content and PPO activity.In the culture after 21 days,ASA would participate in the metabolic process of explants,which lead to the increase of T-ASA content,ASA content and APX activity,and reached the maximum at the time point of mass somatic embryogenesis(42～49 days).The externally added oxidant H2O2 would reduce the content of ASA and the activity of APX in the explants.(5)The addition of ASA or H2O2 could significantly increase the browning area and the incidence of somatic embryos,but when the two were added together,it would significantly reduce the browning area and the incidence of somatic embryos,and the survival rate of explants was the lowest.ASA and H2O2 did not neutralize each other in the early stage of culture,but in the early stage of somatic embryogenesis(28 days).The effects of ASA and H2O2 began to cancel each other out,they would still play their roles steadily,but the efficiency was greatly reduced.(6)In the process of somatic embryogenesis,ASA and GSH mainly exist in a reduced state.Within 0～21 days,due to the sugar stress in the medium,the ASA-GSH circulatory system in the explants had a strong antioxidant capacity. |
PDF Full Text Request