Development Of Rapid Propagation System Of Cut Flower Chrysanthemum 'Wanohakusen'by Open Culture In Vitro

'Iwanohakusen' with pure-white color and elegant pattern introduced from Japan are white chrysanthemum cultivars for cut flowers,and used in sacrificial and funeral and interment in recent decades.They have huge market demand and are exported to Korea,Japan,the Netherlands,etc.At present,most seedlings of the chrysanthemums mainly from cutting are uneven in flowering period,and propagation rate of cutting is low;conventional rapid propogation in vitro can produce seedlings with uniform flowering period,but it needs more investments including expensive equipment such as superclean bench,autoclave,etc.which will increase cost and be not benificial to large-scale industrialized seedling production.To overcome disvantages of conventional rapid propogation in vitro,Technological system for open rapid propogation in vitro of'Iwanohakusen' was developed using stem segments with the axillary buds of the original seedings as explants,and using sodium hypochlorite as disinfectant,by adding different kinds and concentrations of antimicrobial agents such as mancozeb,potassium sorbate and sodium hypochlorite to the primary culture medium,proliferation medium and rooting medium to find the optimal disinfection conditions of explants,to selected the optimal combination of antimicrobial agents added in primary culture medium,secondary proliferation medium and rooting medium,and optimal ratio of transplanting media for rooting of rootless seedlings in and ex vitro,with 'Iwanohakusen' mother plants as control,the genetic differences between multiplifed seedlings and mother plants of original seedlings was compared using IS SR to detect genetic stability of seedlings obtained by open rapid propogation in vitro.The research results will provide theoretical bases and technological support for industrialized production of cut chrysanthemum 'Iwanohakusen' seedlings,and have important theoretical significant and practical value,The main results are as follows:1.The optimal disinfection conditions of explants were determined:Stem segments with axillary buds form cut-flower chrysanthemum'Iwanohakusen's cuttings of the original seedings were used as explants and treated with different concentrations of sodium hypochlorite,the results showed that the optimal disinfection conditions for explants were 0.1%sodium hypochlorite treating for 15 minutes,whose contaimination and survival rates were 53.3%and 46.7%,respectively.2.The optimal amount of agar added in media for open rapid propogation in vitro was determined:Different concentrations of agar powder were added into the cooked media,and the media were mixed evenly by stirring and adjusted pH,then were directly dispensed into culture flasks,after cooling solidification status of media was observed,the results showed that the optimal amount of agar added in the media for open rapid propogation in vitro was 4.5 g/L,which was much lower than the amount of agar added in conventional media for culture in vitro(7.0 g/L).3.The optimal concentration combination of antimicrobial compounds added into media in open primary culture was selected:Different kinds and concentrations of antimicrobial compound were to conventional optimal primary culture medium e.g.MS medium addition to 1.0 mg/L of 6-BA and 0.1 mg/L of NAA,rate of contaimination and induction of adventitious buds and quality of resulted seedlings were compared,the results showed that the optimal combination of antimicrobial compound in open primary culture was mixture adding 50 mg/L of mancozeb,0.010%(v/v)of sodium hypochlorite,and 5 mg/L of potassium sorbate,whose containation and survival rates were 36.7%and 63.3%,respectively,whose resulted buds and seedlings grew well.4.The optimal combination of antimicrobial compunds added into media for open proliferation culture were selected:Different kinds and concentrations of bacteriostatic compunds in conventional proliferation medium,e.g.MS medium adding 1.5 mg/L of 6-BA and 0.1 mg/L of NAA.the stem segments with axillary bud from aseptic seedlings induced in open primary culture were inoculated on open proliferation media,contaimination rate,proliferation coefficient and quality of resulted seedlings "were compared,the results showed that the optimal concentration combination of antimicrobial compunds in open proliferation medium was mixture including 40 mg/L of mancozeb,0.010%(v/v)of sodium hypochlorite,and 5 mg/L of potassium sorbate,whose average contaimination rate and proliferation coefficient were 10%and 5.93,respectively,the leaves at the bottom of the resulted seedlings were partially withered and yellow,quality of seedlings was slightly less than that of those resulted in conventional proliferation culture.5.The optimal combination of antimicrobial compunds added into media for open rooting culture in viro were selected:Different kinds and concentrations of antimicrobial compunds into conventional rooting medium e.g.1/2MS medium adding 0.1 mg/L of NAA,3 cm rootless seedlings resulted from open proliferation culture were inoculated to induce adventitious roots,contamination rate,rooting rate arid quality were compared,The results showed that the optimal concentration combination of antimicrobial compunds in open rooting culture was mixture including 40 mg/L of mancozeb,0.010%(v/v)of sodium hypochlorite,and 5 mg/L of potassium sorbate,the average rooting rate and root length were 80.0%and 2.93 cm,respectively,the average number of roots was 8.68,and the rooting quality was better.6.The optimal rooting and transplanting conditions of seedlings resulted from open rapid propogation in vitro were selected:after acclimatization,rooted seedlings were transplanted to different ratios of media,the results showed that the optimal time of acclimatization was 3 days,the optimal transplanting medium was mixture(peat soil:vermiculite:perlite = 2:1:1,the survival rate was 100%,and the plantlets grew vigorously.7.The optimal conditions of rooting ex vitro was selected:3 days after acclimatization,rootless planlets resulted from proliferation culture were taken out and cut,and dipped into 60 mg/L of NAA solution for different time,and then were transplanted to different rooting media,the results showed that the optimal dipping time was 10 minutes,and the survival rate was 94%,the optimal rooting medium was sand,and the survival rate was 100%.,the rooting time was 6 d.8.The genetic stability of seedlings resulted from open rapid propogation in vitro was detected by ISSR:the genetic difference between seedlings resulted from open rapid propogation in vitro and mother plants was compared by IS SR,the results showed that:105 bands were amplified in 15 seedlings from multiplicated 3 times,27 bands were polymorphic,mutation rate was 23.8%.