【作者】 李莹；

【导师】 彭方仁；

【作者基本信息】 南京林业大学 ， 森林培育， 2012， 硕士

【摘要】 论文以铁皮石斛无菌苗幼嫩组织（茎段、叶片、茎尖和幼芽）为外植体，经过对不同组培方式、激素种类与浓度、最适环境条件优化等系列研究，最终建立了经丛生芽诱导途径和拟原球茎诱导途径两套较为完整的组织培养再生体系。并针对在不同光照、不同培养方式和不同有机添加物条件下拟原球茎的次生代谢产物含量变化规律进行了初步研究。主要试验结果如下：1.铁皮石斛快繁技术（1）丛生芽途径铁皮石斛组培快繁的最佳基本培养基为MS，以铁皮石斛无菌苗的带腋芽茎段为材料，诱导丛生芽，最佳激素浓度配比为NAA0.1mg/L+6-BA2.0mg/L，30d增殖系数为2.82，60d增殖系数为4.8。（2）PLBs途径分别以铁皮石斛幼嫩茎段、叶片、茎尖和幼芽为外植体进行拟原球茎（PLBs）诱导，结果显示，茎段和幼芽的诱导率较高。以茎段为外植体，PLBs最佳诱导培养基为MS+6-BA0.5mg/L+NAA1.0mg/L+2,4-D2.0mg/L，以幼芽为外植体，PLBs的最佳诱导培养基为MS+6-BA1.0mg/L+NAA1.0mg/L+2,4-D2.0mg/L。PLBs增殖最佳培养基为MS+NAA0.1mg/L; PLBs分化最佳培养基为MS+NAA0.1mg/L+6-BA1.0mg/L。（3）壮苗生根铁皮石斛幼苗壮苗的最佳培养基为MS+0.5mg/LIBA+0.5mg/L NAA。同时，单独使用0.5mg/L NAA的培养基中小苗生根快，根数多。香蕉泥能促进幼苗根系的生长。组培苗移栽到纯树皮的基质中，成活率最高。2.拟原球茎次生代谢产物积累规律研究对PLBs的生长规律及其次生代谢产物（总生物碱含量和多糖含量）积累动态进行了研究。结果表明，PLBs的生长大致呈“S”曲线，在第40d左右达到了生长高峰。此时，总生物碱含量和多糖含量也达到了最大值。在PLBs的分化过程中，当PLBs分化出叶时，PLBs的次生代谢产量均达到分化期最高，之后的幼苗期次生代谢产物含量均略有下降。黑暗条件下PLBs鲜重增殖率明显低于光照条件下，且铁皮石斛PLBs的次生代谢产物积累受到抑制。液体培养下的PLBs生长曲线均大致呈“S”型曲线，液体培养下PLBs的鲜重增殖率高于固体培养方式。液体培养中多糖含量和生物碱含量均高于固体培养。不同有机添加物对PLBs次生代谢产物积累产生不同的影响。其中土豆培养基的PLBs鲜重增殖率最大，香蕉汁培养基中的PLBs生物碱含量最高，而苹果汁培养基中的的多糖含量最高。更多还原

【Abstract】 An effective method for rapid propagation of Dendrobium officinale was established toinduct cluster shoots after a series of culture study. took young tissue of aseptic seedling wereexplants, through Stem axillary bud induction pathway and Young tissue intended protocorminduction pathway, two more complete tissue culture regeneration system were established. Apreliminary study for the proposed protocorm in a different light, different training methods anddifferent natural additives medium conditions secondary metabolites content were couducted.1.Tissue culture and rapid propagation of Dendrobium officinale(1).Buds waysDendrobium officinale plantlet grew best in MS medium. The proliferation rate of clustershoots reached the highest in MS medium with NAA0.1mg/L and6-BA2.0mg/L．30dproliferation coefficient is2.82,60d proliferation coefficient is4.8.(2).PLBs waysRespectively to Dendrobium officinale tender stem segments, leaf, stem tip and buds asexplants of PLBs induced, the results showed, the stem segments and bud induction rate wasbest.The stems as explants, the best PLBs induction culture medium for MS+6-BA0.5mg/L+NAA1.0mg/L+2,4-D2.0mg/L, with buds as explants, PLBs optimal induction medium wasMS+6-BA1.0mg/L+NAA1mg/L+2,4-D2.0mg/L. The best medium for proliferation of PLBsMS+NAA0.1mg/L, PLBs differentiation optimum medium for MS+NAA0.1mg/L+6-BA1.0mg/L.(3). Rooting and transplantationThe most suitable medium for strengthening plantlet and taking roots was MS+0.5mg/LIBA+0.5mg/LNAA.The plantlet rooted rapidly and got more roots with0.5mg/L NAA alonein addition. The banana juice could promote growth of plantlet roots．The highest survival ratewere raised, when Seedlings were transplanted into the matrix of pure bark.2. Studies on Induction of Protocorm-like Bodies and Accumulation of Production ofSecondary Metabolites of Dendrobium officinaleThe Protocorm-like bodies were inducted with the young stem section of Dendrobiumofficinale as explant using the method of orthogonal test of three factors at three different levels.The relationship of growth rhythm and its secondary metabolites (total alkaloid content and polysaccharide) accumulation were researched. The results showed that the best inducedmedium was MS+6-BA0.5mg/L+NAA1.0mg/L+2,4-D2.0mg/L.Simultaneously the growth ofProtocorm-like bodies appeard in "S" curve in one semester (60d),reached the growth peak at the40d around. At that time, the total alkaloid content and polysaccharide also reached the peakvalue. The results also showed that the correlation coefficients between the total alkaloid contentand polysaccharide and the growth of Protocorm-like bodies were highly significant. When theleaf were divided from Protocorm-like bodies,the Secondary metabolism production ofProtocorm-like bodies were highest in differentiation phase, and then declined slightly in periodof young seedling.Fresh weight of the proliferation rate of PLBs were significant lower exposed in lightconditions than in dark conditions. The accumulation of Secondary metabolites of Dendrobiumofficinale candidum PLBs were inhibited in various degree.Simultaneously the growth of Protocorm-like bodies appeard in "S" curve in dark conditions.Fresh weight of the proliferation rate of PLBs were significant lower exposed in Solid culturethan in Liquid culture. Polysaccharide content and alkaloid content were much higer in Liquidculture.The accumulationof PLBs secondary metabolite were influencede by different naturaladditives. PLBs fresh weight of the proliferation rate got largest by Potato medium, alkaloid ofPLBs got highest by Banana juice medium. polysaccharide content got largest by apple juicemedium.更多还原