Studies On Somatic Embryogenesis Of Tree Peony

Tree peony was famous in the world and its wild species were originated in China. But its low propagation coeffidient and long breeding period have always been the big problems for the production and breeding .Somatic embryogenesis is the important approach for plant regeneration and it has been used successfully for many plants.But the research reports on tree peony were rare.So this research was carried out to set up a steady regeneration system and to provide a platform for the propagation in vitro or transgene breeding. Firstly, the suitable explant donator were filtrated from Paeonia rockii'Shu Sheng Peng Mo',P.ostii'Feng Dan Bai'and P.delavayi in vitro. Based on this,the developing stage and type of the explant to somatic embryogenesis were filtrated and decided, and the basal medium type and the concentration of BAP, NAA and Sucrose in the induction media on somatic embryogenesis were also explored. Secondly, the effect on induction rate of pretreating the explants with 1.0 M sucrose solution, as well as the light conditions and induction periods were experimentalized to improve the somatic embryos. Based on 416-B Optimized Mixed Design, the best suitable concentrations of BAP, Sucrose, CH and AgNO3 in induction medium were decided by DPS, so the somatic embryogenesis system was optimized.Thirdly, the secondary somatic embryogenesis from the abnormal and semitransparent somatic embryos, and the maintainability of continuance differentiation capability of somatic embryo from the original explants which had produced somatic embryos or the original somatic embryos which had produced secondary somatic embryo were experimented to get repetitive somatic embryogenesis. Fourthly, maturation and germination of the somatic embryos and the transformation from somatic embryo to plantlets were studied to explore the ways for more plants.At last, the research on indirect somatic embryogenesis from the green cotyledon of'Feng Dan Bai'was done for the first time. At the same time cytology researches on direct or indirect somatic embryogenesis were done.The main results were as follows: Among Pa rocki'iShu Sheng Peng Mo',P. osti'iFeng Dan Bai'and P. delavayi, P. rockii'Shu Sheng Peng Mo'was the most suitable one for inducing direct somatic embryogenesis, the better developing stage of embryo explants was 90 days after flowering; for the explants type, the whole embryo was best. The basal media and the concentration of BAP, NAA and Sucrose had the most significant effects on the induction rate of somatic embryos. The most suitable combination of the four factors was: MS or Revised MS medium+Sucrose 100.0g/L+BAP 0.5 mg/L+NAA0.0 mg/L, and the highest induction rate was up to 63.3%, which is 44.3% higher than the result 19% gained by the former researcher.Somatic embryogenesis in tree peony was promoted and the amount of fused somatic embryos was reduced by pretreating explants with 1.0 M sucrose solution. It was pretreating the young embryos 70 d after flowering with 1 M sucrose solution for 1d firstly and culturing them on the auxin free inducing medium, then subculturing them on the medium with BAP that had better effect on direct somatic embryogenesis in'Shu Sheng Peng Mo'. After 22 w cultured, the induction rate was up to 84.6%. There was an interaction between the pretreatment period and the induction medium for somatic embryogenesis. For the 3 induction media used in the experiment, it was better that pretreating the seed embryo explants 90d after flowering with 1.0 M sucrose solution for 2 or 3 d.Induction period and light had some effect on the somatic embryogenesis in tree peony. For the embryos 85 d after flowering of'Shu Sheng Peng Mo', it was better to be cultured for 60 days in dark and 25±2℃and then transferred to the hormone free medium.Based on 416-B Optimized Mixed Design,The main factors to affect the somatic embryogenesis of 85d embryo explants in'Shu Sheng Peng Mo'were BAP, Sucrose, CH, and AgNO3 And for the somatic embryo induction rate,the multiple quadratic regression equaton was:?=-1.5413-0.0824X1+1.0886X2-0.1532X3+0.0755X4+0.3720X12-0.1425X22+0.1377X32+0.4941X42+0.2816X1X2-0.1651X1X3-0.3375X1X4+0.2400X2X3(R2=0.9996).The main and interaction effects were analyzed. And the best levels of the four factors from the 500 combinations was gained by DPS ,they were BAP 0.1 mg/L, Sucrose 150.0g/L, CH600.0 mg/L and AgNO3 10.0 mg/L.For the embryo explants 85d after flowering in'Shu Sheng Peng Mo', the highest theoretical inducing ratio can up to 97.5% with Revised MS with this combination。Somatic embryos of'Shu Sheng Peng Mo'were proliferated vir secondary somatic embryogenesis. For somatic embryogenesis and secondary somatic embryogenesis, the inducing media in composing were similar, but the latter was with BAP in low concentration. The capability to differentiate somatic embryos continuously of donators including the initiated explants and the somatic embryos which had developed somatic ones were maintained in the media for preserving the capability.The somatic embryos of tree peony maturated on MS or SH solid hormone free medium, which was promoted by AC. When the somatic embryos developed to the cotyledon stage, it was necessary to separate them from the donators in time and culture to make them germinate, otherwise, they geminated poorly or even not. The medium with lower BAP or IAA concentration made the mature somatic embryos in shape germinate well but the media with higher BAP concentration (>0.5 mg/L) made the hypocotyls swelle strongly.While the medium with lower IAA concentration made the radicles elongated. It was not suitable to add NAA and GA3 in higher concentration to the germination medium, because NAA in high concentration made the hypocotyls callus highly and GA3 in high concentration made the somatic embryos germinate precociously. Just like the seed embryos, somatic embryos of tree peony have dormancy epicotyls, so it is required to treat the dormancy epicotyls with lower temperature or GA3 in order to make them elongate.The phenomenon of double embryos in the tree kinds of tree peonies used by this research was approved, and the ratio of double embryos was about 1.5%. Moreover, the development of the two embryos was synchronous or not, and both or one of them two grew up to seedling. And the abnormality in the embryos of tree peony was found firstly.Calli were induced from the green cotyledon pieces of'Feng Dan Bai'on SH basal media with 2,4-D and NAA in different concentration, and higher induction rate (100%) was gained.The globule embryos were found on the media with 2, 4-D after 80d induction.For direct somatic embryogenesis, somatic embryos were found originating on the protuberances from the superficial of the cotyledons or the hypocotyls, but for indirect one, they were originated from the superficial and inner cell of the calli.The somatic embryogenesis indirectly were improved by adding ABA in the media.