Establishment Of Cotyledon Tissue Culture System Of American Ginseng

American ginseng（Panax quinquefolius L.）,belonging to Araliaceae ginseng species and a traditional precious herb,which has the functions of enhancing immunity and lowering blood lipid levels.In recent years,as a medicinal and edible plant,American ginseng has become more and more appeared in pepole’s daily consumption,which leads to the market demand increasing year by year.However,there is a serious problem of continuous cropping obstacles in ginseng cultivation.And,American ginseng is a perennial root and often cross-pollinated plant,which cause the difficulty of conventional hybrid breeding.Using tissue culture micropropagation technology can quickly obtain sterile tissue culture seedlings,which is an effective approach to solve these problems.In this study,the cotyledon of the American ginseng aseptic seedling was used as explant in tissue culture to study the effects of basal medium,different test types and concentrations of hormones,and culture conditions on the callus induction and differentiation,and on the adventitious buds differentiate directly.Consquently,American gensing tissue culture system was established initially,and the main research results are as follows:1.The induction effect of the basic medium on embryo seedlings was significantly different in American ginseng.On SH medium,the embryo seedlings grew best with the highest induction rate.On B5 Medium,the seedlings grew slowly with much lower seedlings induction rate than SH medium.MS medium had the lowest induction rate of embryo seedlings,and the growth of seedlings was slow and weak.GA₃ has a significant promoting effect on the embryo seedling induction and its growth.Adding a lower concentration（5.0 mg/L）of GA₃ into MS medium can significantly improve the germination rate of seedlings.But,with the increasing of GA₃ concentration,there was no significant difference in the germination rate of seed embryos.Adding GA₃ into SH medium could significantly improve the germination rate of seed embryos and the seedlings grew strongly and fastly.When the concentration of GA₃ reached 10.0 mg/L,the germination rate of seed embryos was the highest,reaching 84.4%.Adding a certain concentration of TDZ into medium can significantly improve the germination rate and seedlings grew well.Athough,with the increasing of TDZ concentration,there was no significant difference of the seed embryos germination rate in MS medium,the germination rate also reached the maximum at 92.3%and the seedlings grew strongly when adding 1.0 mg/L TDZ into SH medium.In general,the best medium for American ginseng seed embryo culture was SH+GA₃ 10.0 mg/L+TDZ 1.0 mg/L+3.0%sucrose+0.5%agar with the highest embryo seedling induction rate of 92.3%.2.The basal mediums and phytohormone had significant effect on the induction of cotyledon callus in American ginseng.The callus induction rate was significantly higher in SH medium than in MS or B5 medium.2,4-D can significantly promote the induction of cotyledon callus.Within the tested concentration range,with the increasing of 2,4-D concentration,the induction rate increased at first,then decreased,and the peak appeared at2.0 mg/L with the rate of 58.4%.Adding 0.5 mg/L 6-BA or 0.25 mg/L TDZ into the medium containing 2.0 mg/L 2,4-D could significantly improve the induction rate.The combination of the three hormones could be further improve the callus induction rate.In addition,light-dark culture had a significant effect on the induction of callus.Besides,hydrolyzed milk protein and activated carbon had certain effect on callus quality but on the induction rate.Overall,the optimal media for cotyledon callus induction of American ginseng is:SH+2,4-D 2.0 mg/L+TDZ 0.25 mg/L+6BA 0.25 mg/L+0.05%hydrolyzed milk protein+0.05%activated carbon+3.0%sucrose+0.5%agar,with the highest induction rate of96.7%.3.The combination of 2,4-D and 6-BA can significantly promote the differentiation of cotyledon callus in American ginseng.There was significantly difference of callus differentiation rate among different concentration ratios of hormones.When the concentration of 2,4-D was constant,with the increasing of 6-BA concentration,the induction rate increased at first,then decreased.The highest callus differentiation rate was16.1%when 2,4-D and 6-BA were both 1.0 mg/L.TDZ had the same trend as 6-BA in promoting the callus differentiation,but the effect of TDZ was stronger.When the concentration of TDZ reached 1.0 mg/L,the callus differentiated was the highest rate,reached 19.4%.Overall,the optimal media for American ginseng callus differentiation is SH medium+2,4-D 1.0 mg/L+TDZ 1.0 mg/L+0.05%hydrolyzed milk protein+0.05%activated carbon+3.0%Sucrose+0.5%agar.4.The concentration of cytokinin had significant difference on the adventitious bud differentiation in American ginseng cotyledons.Both 6-BA and TDZ can significantly increase the induction rate of cotyledon adventitious buds.Within the tested concentration range,with the increasing of hormone 6-BA and TDZ concentrations,the induction rate increased at first,then decreased.However,the differentiation rate of TDZ on the adventitious bud induction in American ginseng cotyledons was significantly higher than that of 6-BA.When the concentration of TDZ reached 1.0 mg/L,the callus differentiated was the highest rate,reached 28.3%.The optimal medium formula for adventitious bud induction in American ginseng cotyledons is SH medium+TDZ 1.0 mg/L+NAA 0.5 mg/L+0.05%hydrolyzed milk protein+0.05%activated carbon+3.0%sucrose+0.5%agar.