The Study On Genetic Transformation System Of Paeonia Ostii.

Paeonia ostii has high ornamental,pharmaceutical and oil value.The breeding of new paeonia ostii.varieties is restricted by the traditional method of breeding due to the long reproductive cycle and the lower propagation coefficient.The establishment of regeneration system in paeonia ostii.is fundamental to seeding rapid propagation and genetic transformation.In recent years,there has been a great advancement in the vitro culture of paeonia ostii.,but there still exist some problems in the genetic transformation in the propagation system and the transformation coefficient.The mature embryo and cotyledon of paeonia ostii.used as explants for callus induction and bud differentiation in this experiments.The purpose of this research is to construct a embryonic binary vector that was induced by Glucocorticoid.The factors in fluencing the frequency of Paeonia ostii.transformation mediated by Agrobacterium have been investigated by using the mature embryo and cotyledon.The research contents and results were listed:1.The germination rate of mature embryo is 92%in this research.Adding putrescine can both promote growth of root and seeding.The result of experiment showed that the best method to break the dormancy of seeds is soak in the solution of500mg/L gibberellin for 48h.Then pick out mature embryo to put on the germination medium.The germination medium is:modified MS+6-BA 0.5mg/L+GA₃0.5mg/L+Put 1.0mg/L;The growth medium is:modified MS+6-BA 0.5mg/L+GA₃0.2mg/L+Put 1.0mg/L.2.The induction frequency of callus from cotyledon and mature embryo was68.7%and 95%respectively.For cotyledon,the best induction medium is:modified MS?sucrose 100g/L?+6-BA 0.5mg/L+2,4-D 2.0mg/L;For mature embryo,the best induction medium is:modified MS?sucrose 100g/L?+6-BA 0.5mg/L+PIC 4.0mg/L.3.The callus are light yellow and white pallet after cultured for 25-30 days in the mature medium.Using the mature embryo as explants,the differentiation rate of callus was about 54.5%.Meanwhile,there are a few multiple shoot were induced on cotyledon.The 0.5mg/L zeatin could promote the differentiation of the callus,whereas the higher concentration of zeatin inhibition differentiation.The mature medium is:modified MS?sucrose 30g/L?+6-BA2.0mg/L+NAA 0.2mg/L+TDZ 0.3mg/L+ZT0.5mg/L;The differentiation medium is:modified MS?sucrose 30g/L?+6-BA 0.5mg/L+NAA 0.2mg/L+ZT 0.5mg/L.4.In this experiment,a plant expression vector which containing a gene of betacyanin was used for transformation of paeonia ostii.There are 29.1%and 5.8%resistant callus respectively that expressed red character come from cotyledon and mature embryo.The results showed the objective gene sequence was expressed successful in host cells.5.In this study,a embryonic binary vector pYB9664 that induced by DEX was constructed;And a small amount of resistant embryos were obtained after agrobacterium infection,which was proved the vector could induce embryogenesis preliminarily.