Optimization Of Vitro Regeneration System And Establishment Of Genetic Transformation System Of Peony

Peony have a very vast market foreground,however,Seedling propagation of peony cannot keep up with demand of flower and medical market for peony seedlings.In order to solve the problems of the rapid production of peony seedlings and accelerate the process of breeding new varieties,it is of great significance to research regeneration system and genetic transformation system of peony.Peony cultivars'Fengdan' was used as the experimental materials,the experiment builded and optimized the regeneration system from mature embryos,immature embryos,callus,cotyledon nodes,cotyledon leaves,soil buds in vitro in peony.And genetic transformation system of peony were firstly developed using cotyledon nodes.In vitro regeneration phase,the effects of disinfection methods,different species,concentration and compatibility of plant honnones on the regeneration process of peony were studied,including adventitious bud induction and elongation,adventitious root induction.The results are as follows.1)The optimum medium for the adventitious buds induction from different explants was WPM+2 mg/L 6-BA+0.2mg/L NAA?mature embryos?,WPM+0.4 mg/L 6-BA+2 mg/L GA₃?immature embryos?,WPM+3 mg/L 2,4-D+1.5 mg/L NAA+0.5 mg/L Inositol?callus?,WPM+0.3 mg/L 6-BA+2 mg/L GA₃?cotyledon nodes?,WPM+1 mg/L 6-BA+0.3 mg/L NAA?cotyledon leaves?,WPM+1 mg/L 6-BA+0.1 mg/L NAA?soil buds?;2)The suitable medium for adventitious bud elongation was WPM+0.3 mg/L 6-BA+1.5 mg/L GA₃.The elongation efficiency was about 81.12%,and the average shoot length was 1.93 cm;3)In addition the best rooting method is grafting,and the survival rate was 66.54%after transplanting.Using hypocotyl as foreign genes transferring target,the effects of Agrobacterium strain type,acetosyringone concentration,broth concentration,immersion time,co-culture time,pH value and temperature on the genetic transformation system of peony were studied.The results showed that it is appropriate to choose EHA105?agropine type?as engineering bacteria used to import foreign genes,and optimal concentration of acetosyringone(200??mol/L?,bacteria concentration?OD600=0.8?,time of elution?30 min?,Co-culture time?3 d?,co-culture medium pH value?5.4?and co-culture temperature?22 ??could effectively improve the transient expression frequency of the peony cotyledon node transformation system.Transgenic resistant seedlings of Peony are obtained successfully by using this transformation system,the transformation efficiency was 1.33%.