论文标题:长春花组织培养技术研究
Study on Technique System of the Tissue Culture in Catharanthus Roseus
论文作者
论文导师 刘兆普,论文学位 硕士,论文专业 植物营养学
论文单位 南京农业大学,点击次数 623,论文页数 66页File Size4535K
2007-06-01论文网 http://www.lw23.com/lunwen_5254902/
Callus;; Catharanthus roseus;; Tissue culture;; Multiplication;; Rooting
长春花(Catharanthus roseus(Linn．)G．Don)，为夹竹桃科多年生草本或亚灌木植物，其生长适应能力强，全年花期，并且体内含有多种生物碱类，因此在医学药用和园林绿化观赏等方面广泛应用。在转基因等方面的组织培养技术尚不完善，因此，研究长春花组织培养体系，具有较高的经济效益、生态效益和社会效益。 本试验选用本实验室海南基地长春花种子为试验材料，研究在不同外源激素配比及不同培养条件下再生植株的适宜培养基和培养条件，以及影响长春花分化、生长、生根的各种因子。实验结果如下： 1．长春花种子消毒处理，最佳方案是：肥皂水浸泡30 min，自来水冲洗36 h，在超净工作台上先用质量分数0．1％HgCl_2溶液浸泡10～15 min，无菌水冲洗4～6次，再用质量分数10％次氯酸钠消毒5～10 min，无菌水冲洗4～6次，再用体积分数70％乙醇浸泡60 s，无菌水冲洗4～6次。 2．以长春花幼嫩叶片、叶柄、下胚轴为外植体，不同种类、不同浓度的植物生长调节物质及其组合对愈伤组织诱导的效应均不相同。 3．6-BA、NAA组合中，以叶片为外植体时长春花愈伤组织诱导的最佳培养基是MS+6-BA 3．0 mg·L~(-1)+NAA 0．5 mg·L~(-1)+蔗糖30 g·L~(-1)+琼脂7 g·L~(-1)，诱导率达到95．3％；以叶柄为外植体时长春花愈伤组织诱导的最佳培养基是MS+6-BA 3．0 mg·L~(-1)+NAA 1．0 mg·L~(-1)+蔗糖30 g·L~(-1)+琼脂7 g·L~(-1)，诱导率达到86．9％；以下胚轴为外植体时长春花愈伤组织诱导的最佳培养基是MS+6-BA 3．0 mg·L~(-1)+NAA 0．5 mg·L~(-1)+蔗糖30 g·L~(-1)+琼脂7 g·L~(-1)和MS+6-BA 3．0 mg·L~(-1)+NAA 1．0 mg·L~(-1)+蔗糖30 g·L~(-1)+琼脂7 g·L~(-1)，诱导率达到88．6％和89．0％。 4．单独使用KT时，各浓度下叶片、叶柄和下胚轴均无愈伤组织产生。 5．再分化的研究表明：利用由叶片、叶柄和下胚轴诱导而来的愈伤组织能分化出芽点，虽最初芽分化率不高，但可以大量增殖。芽增殖的芽增值最佳培养基配方是：MS+6-BA 2．0 mg·L~(-1)(2．5 mg·L~(-1))+IBA 0．2 mg·L~(-1)+GA_3 1．0mg·L~(-1)，而1／4 MS+IBA 1．5 mg·L~(-1)+NAA 0．05 mg·L~(-1)+活性炭0．5 mg·L~(-1)是诱导生根的最佳组合。
Catharanthus roseus (Linn.) G Don is a kind of perennial herbaceous plant and subshrub of the catharanthus in Aocynaceae. Due to strong adaptability and year-round abloom, it can be used in many fields, such as in medicine and in landscape architecture. There is not a perfect tissue culture technique in transformation gene and other facets; therefore, there are many economic, zoology and society benefit to study the tissue culture technique of Catharanthus roseus. The techniques of test-tube seedlings tissue culture rapid propagation in seeds of Catharanthus roseus from Hai-nan Base were studied in the experiment. The optimal culture medium and conditions for plantlet regeneration had been selected under the variant phytohormones combinations and culture conditions. Moreover, the effects of various factors on Catharanthus roseus differentiation, growth and rooting culture were discussed in the study. The results are listed as followed: 1. The best method to sterilize the seeds of catharanthus roseus was marinated by suds for 30 min, washed by water for 36 h, marinated by 0.1% HgCl_2 for 10 to 15 min on the Situation of Bacterium Free, and then washed by asepsis water for 4 to 6 times; subsequently, marinated by 10 % NaClO for 5 to 10 min, washed by asepsis water for 4 to 6 times, marinated by 70 % CH_3CH_2OH for 60 s, and then washed by asepsis water again for 4 to 6 times. 2. When setting catharanthus roseus laminae, petiole, hypocotyl as explants, the effects of different kinds of homogeneity and concentrations of growth adjustive matter on inducing callus were different. 3. In the combination of 6-BA and NAA, the best culture medium to induce callus was MS+6-BA 3.0 mg·L~(-1)+NAA 0.5 mg·L~(-1)+sugar 30 g·L~(-1)+agar 7g·L~(-1); the callus induction rate reached to 95.3 %refering to laminae. The best culture medium to induce callus was MS+6-BA 3.0 mg·L~(-1)+NAA 1.0 mg·L~(-1) sugar 30g·L~(-1)+agar 7 g·L~(-1); the callus induction rate was to 86.9 % refering to petiole. The best culture medium to induce callus was MS+6-BA 3.0 mg·L~(-1)+NAA 0.5 mg·L~(-1)+sugar 30g·L~(-1)+agar 7g·L~(-1) and MS+6-BA 3.0 mg·L~(-1)+NAA 1.0 mg·L~(-1)+sugar 30 g·L~(-1)+agar 7 g·L~(-1); the callus induction rate reached to 88.6% and 89.0% repectively, refering to hypocotyl. 4. No matter what used laminae, petiole or hypocotyls of catharanthus roseus.as explant, callus could not be induced with KT. 5. The callus which was induced from young leaves could be used as rapid propagation materials. The optimum medium for buds multiplication was MS+6-BA 2.0 mg·L~(-1) (2.5 mg·L~(-1))+IBA 0.2 mg·L~(-1)+GA_31.0 mg·L~(-1)+sugar 30 g·L~(-1)+agar 7 g·L~(-1); the optimum medium for rooting was 1/4 MS+IBA 1.5 mg·L~(-1)+NAA 0.05 mg·L~(-1)+carbon 0.5 mg·L~(-1)+sugar 20 g·L~(-1)+agar 7g·L~(-1).
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