Studies On Direct Somatic Embryogenesis And Embryogenic Callus Induction And Differentiation Of Paeonia Sect.Moutan

Tree peony(Paeonia sect.Moutan)is a well-known traditional flower in China with important values in ornamental,medical and oil use.However,the traditional propagation methods severely limited its breeding and industrial development.The existing tissue culture protocols can not meet the practical needs of mass propagation and genetic engineering breeding of tree peonies.Somatic embryogenesis(SE)have many unique advantages but it is still in groping stage in tree peony.In the present study,both direct and indirect SE have been investigated to established a SE protocol,moreover,histological and cytological analysis were carried out,this study will be benificial to the propagation and breeding of tree peony.The mainly results are as follow:1.By application of the zygotic embryos(ZEs)of P.rockii and P.ostii‘Feng Dan',9.39%-37.8%direct SE was obtained.A protocol of direct SE of P.rockii‘Jing Hong'was established firstly.The mature ZEs collected at 95d.a.a of P.rockii‘Jing Hong'was the optimal explants for direct SE;the best induction medium was MS+0.5mg/L BA+80g/L sucrose,Cu²⁺can promote the direct SE,the highest frequency of SE and No.of somatic embryos per explant was47.62%and 5.00,respectively;the optimal proliferation medium is WPM+0.5mg/L BA+80mg/L sucrose,with 4.76 somatic embryos per explant;the germination rate of somatic embryo was44.44%.2.Both SE and SO of tree peony originated from the hypocotyl epidermis and subepidermis,with unicellular and multicellular origin,respectively.Starches is the main energy sources for in vitro morphogenesis of tree peony and provide energy reserves for the conversation from somatic embryo into seedling;the ultrastructural observation indicate that the hypocotyl explant cells after10d of culture were characterized by low metabolic activity of reserve cells;the cells of globular somatic embryo after 30d of culture exhibited enhanced synthesis and metabolism;the cells of cotyledonaty somatic embryos after 50d of culture exhibited characteristics of meristematic,with the strong synthesis and metabolism and intense intercellular communication.3.The filaments with translucent or white appearances and anthers in tetrad stage of P.ostii‘Feng Dan'were optimal explants for embryogenic callus(ECs)induction,the optimal medium is MS+1mg/L 2,4-D+1mg/L NAA+0.5mg/L BA,with 96.66%and 100%ECs induction rate,respectively,the ECs were yellow,loose and mucilaginous;the optimal ECs induction medium for immature ZEs was MS+2mg/L PIC+BA 0.5mg/L,with 31.66% induction rate;the optimal ECs induction medium for hypocotyl was MS+2mg/L NAA+0.5mg/L CPPU,with 53.38%induction rate,the ECs from immature ZEs and hypocotyl were white or translucent and loose.4.The optimal ECs maintainence medium for filament was MS+0.5mg/L NAA+0.5mg/L TDZ,with 2.83 proliferation rate,few differentiation of globular or torpedo-shaped somatic embryos were obtained from medium of MS+0.5mg/L PIC+0.5mg/L BA;the ECs from hypocotyl produced globular somatic embryos from various medium;the surface of somatic embryos were covered by a thin film of extracellular matrix,which may be one of the marker of early embryonic development.