| Manchurian ash(Fraxinus mandshurica) is an important valuable broad-leaved species in Northeast China, belonging to the genus of Oleaceae Fraxinus. The research was to establish a plant regeneration system through somatic embryogenesis of Manchurian ash with mature zygotic embryos as explants. Morphological observation and biochemical detection in the process of somatic embryogenesis, had made to clear whether the existence of the phenomenon of programmed cell death in the process of somatic embryogenesis in Manchurian ash, meanwhile lay the foundation for the subsequent biochemical mechanism and molecular mechanism of somatic embryogenesis of Manchurian ash research. The conclusions were as follows:(1) The Optimal induction medium was MS1/2(MS in which all components were reduced by half), with the addition of5mg·L-1NAA and2mg·L-’16-BA400mg·L-1acid casein hydrolyzate (CH),75g·L-1sucrose,6.5g·L-1agar, pH to5.8. Somatic embryo induction rate was67.5%. mainly occurred directly, which occurred mostly in the cotyledon surfaces and edges; approximately81.5%of the somatic embryo occurred on the browning explants.(2) The cotyledon embryos were cultured on maturation medium adding10mM ABA. training17days developed physical maturity, the highest somatic embryo maturation rate was90.0%, while browning were also less likely. The best secondary somatic embryogenesis medium was MS1/2, supplement with0.05mg·L-1NAA,400mg·L-1acid hydrolyzed casein CH, adding25g·L-1sucrose and6.5g·L-1agar. Somatic embryo proliferation rate was93.3%. Germination medium was MS1/2medium, adding0.2mg·L-16-BA,20g·L-1sucrose,200mg·L-1acid hydrolysis of casein CH and6.5g·L-1agar. After cultured30days, somatic embryo germination rate was92.0%. rooting rate was27.1%. Rooting medium was1/3MS adding0.01mg·L-1NAA, and94.4%transformed into regenerated plants. Seedlings were transferred into sterilized peat soil, vermiculite, perlite ratio of5:3:2, with MS (sugar-free and hormone) nutrient solution mixed with matrix, and ultimately survival rate of transplant was85%.(3) Morphological observation and histological analysis of somatic embryos induced by mature zygotic cotyledons part, directly originated in the epidermal cells, as single cell origin. The occurrence process of somatic embryos and zygotic embryogenesis process was similar. Mature cotyledons embryo had a "Y"-shaped vascular tissue, and differentiated cotyledon, hypocotyl, radicle. There was a structure similar to the suspensor in globular embryo, heart-shaped embryo, torpedo-shaped embryos and early cotyledonary embryo stage, disappeared after degradation. The formed somatic embryos with the surrounding cells had clear boundaries, and could be easily separated from the surrounding tissue. Secondary somatic embryos occurred in the the radicle end surface of the original somatic embryos; also experienced by spherical, heart-shaped, torpedo-shaped, cotyledonary stages, and did not occur synchronously deformity cotyledon.(4) TUNEL apoptosis in situ testing and DAPI nuclear staining resulted when cultured13d, cells had been occurred PCD, the period of17d to19d occurred the peak of PCD.(5) H2O2content when inoculated0d was the minimum; and the maximum peak was first found in18d, when the period was at the stage of embryonic cells into somatic embryogenesis. No significant changes in the content of superoxide anion. The Cell death percentage was in the15d, the27d, the40d found the peak. The increase in H2O2content could induce PCD. |
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