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1、广西师范大学 硕士学位论文 地宝兰无菌播种快繁技术及原球茎的形态发生研究 姓名：胡琦敏 申请学位级别：硕士 专业：野生动植物保护与利用 指导教师：王任翔 20070401 I 地宝兰无菌播种快繁技术及原球茎的形态发生研究 地宝兰无菌播种快繁技术及原球茎的形态发生研究 姓名：胡琦敏 导师：王任翔 副教授 年级：2004 级 专业：野生动植物保护与利用 研究方向：保护生物学 摘摘 要要 兰花在植物分类学上属于兰科(Orchidaceae)植物，是单子叶植物，为多年生草本，附 生、地生、或腐生。兰科植物按生态习性可分为地生兰和气生兰两种，都具有较高的观赏 价值和经济价值。兰科是有花植物中最大的科之一
2、，估计有 800 个属，3 万3.5 万个原 生种。但随着人们对兰花的热爱，不断的从野外挖掘，以致很多兰花濒临灭绝。由于兰花 传统上靠分株繁殖，周期长、繁殖率低，不能满足国内外市场的需求。随着现代生物技术 的发展， 以组织培养为主要手段的生物技术逐步应用到兰花的繁殖过程中， 大幅度的提高 了兰花的繁殖系数，促进兰花的工厂化生产从而提高其经济价值。地宝兰Geodorum densiflorum (Lam.) Schltr是一种很有开发价值的野生兰，具有绿化、美化、药用等多种用 途。 本研究以地宝兰（G.densiflorum）种子为外植体进行无菌播种快速繁殖技术体系的研 究及对地宝兰原球茎的形成
3、及分化过程进行了形态解剖学研究， 重点研究了地宝兰的种子 萌发、原球茎增殖及分化、生根培养的最佳培养基及激素水平，并对试管苗的移栽基质进 行了选择，结果表明：(1) 适宜地宝兰荚果灭菌的方法：用自来水冲洗 5 分钟，75%的酒 精表面灭菌 30 秒，无菌水冲洗 23 次，再用 0.1%的升汞溶液灭菌 1015 分钟，无菌水 冲洗 45 次。(2) 种子的处理：用灭过菌的解剖刀划开果荚，取出种子，作三种处理比 较，结果发现用 0.1mol/L 的 KOH 溶液浸泡 10 分钟，无菌水冲洗 56 次，后播种，此处 理组的种子萌发率高达 45%，转入光照培养后可原球茎易转绿且原球茎生长健壮。(3)
4、在 原球茎诱导时，通过采用先暗培养，后光照培养的方法进行原球茎的诱导。(4) 适宜原球 茎的诱导培养基为：1/2MS+6-BA 1.0 mg/L+NAA 0.5 mg/L，诱导率为 95%。(5) 适宜原球 茎的增殖培养基为：1/2MS+6-BA 0.5 mg/L +NAA 0.2 mg/L +1 gL -1 活性碳，1/2MS+KT 1.0 mg/L +NAA 0.1 mg/L。原球茎的增殖倍数分别为 2.5、3.0。(6) 适宜原球茎的分化培养基 为：MS+6-BA 3.0 mg/L +NAA 1.5 mg/L，芽的增殖倍数为 2.8。(7) 适宜不定芽的生根壮 苗培养基为：MS+6-BA
5、 0.2 mg/L +NAA 0.5 mg/L10%香蕉汁。生根率为 100%。(8) 移 栽基质为：苔藓、沙、泥土(1：1：1)，成活率为 80%。 对地宝兰的种子进行解剖学观察,可以看到种皮由一层透明的细胞组成。地宝兰种子 试管培养条件下，细胞分裂，胚体积增大，胀破种皮，形成原球茎。其顶端分生组织细胞 继续分裂，形成根状茎。在激素诱导下，根状茎的表皮和皮层细胞可分裂形成侧生分生组 织， 然后顶生和侧生分生组织分化出叶原基。 本论文认为地宝兰的芽顶端分生组织符合原 II 套和原体学说，以后原套分裂形成表皮层，以后原体形成茎的皮层和维管柱。在芽中发现 有含晶细胞， 其结晶物主要是由草酸钙所形成
6、的针晶。 这些草酸钙结晶一般都是在液泡中 发生，当液泡中出现一种包裹的腔室后，便在其中形成针晶。 本试验通过地宝兰的种子进行无菌播种来实现地宝兰的快速繁殖，以提高其繁殖系 数，满足国内外消费者的需求，并筛选出各个阶段较优的基本培养基、激素种类、浓度及 配比、 附加物等一系列技术参数组合， 为今后实现野生兰工厂化和商业化生产奠定技术基 础。本实验试图从组织培养入手，探讨原球茎的结构特征，发现了原球茎的形成过程，有 利于进一步提高地宝兰的繁殖系数，为地宝兰的快速繁殖和试管苗移栽提供形态学依据。 地宝兰无菌播种及原球茎形态发生尚未见报道。 关键词：地宝兰 无菌播种 原球茎 形态发生 III Abst
7、ract Author: Hu Qi-min Supervisor: Prof.Wang Ren-xiang Grade: 2004 Speciality: Protection and utilization of wild plant and animal Orchid which is the Orchidaceae plant of monocotyledon is perennial herbage in Botany-taxonomy. Orchid has three growth styles, which is adnascent、 terrestrial or saprop
8、hytic. Orchidaceae plant can be disparted to terrestrial orchid and aerial orchid. There have upper ronamental value and economic value. Orchidaceae plant is one of the largest groups of flowering plants with as many as 3000035000 species in nearly 800 genera. But with the passion for orchid, people
9、 digs uninterrupted orchid from wild, so as to many orchids on the verge of dying out. Because the propagation of orchid by means of offshoots trationally, which has long periods and has the low propagate rate, so it can not satisfy the internal and external demand. With the development of modern bi
10、ological technology, tissue culture can be gradually applied to orchids propagation, which can greatly increase its propagation coefficient and accelerate its mass production which can increase the economic value. Geodorum densiflorum which is the plant of Geodorum of Orchidaceae of monocotyledon is
11、 a wild orchid which has valuable in exploitation and has many uses ,for example: virescence、 beautification、officinal, and so on. The seeds of Geodorum densiflorum were used for explant in the study on the techniques system of aseptic sowing and rapid propagation and morphogenesis of protocorms. Th
12、e main objects of the study were seeds bourgeon and protocorm-like bodies（PLBs）and adventitious bud induction, proliferation, plantlet rooting and translanting. The results were as follows: (1)The best way of sterilizing for pod was as follows: rinsing 5 min with tap water immersing in 75% alcohol f
13、or 30srinsing 23 times with sterilized waterdisinfecting in 0.1% mercurichloride solution for 1015 minrinsing 45 times with sterilized water.(2)The disposal of seeds: lacerated the pod using sterilized scalpel and took out the seedsimmersing in 0.1mol/L KOH 10minrinsing 56 times with sterilized wate
14、r.(3)During the PLB induction,the induction could be through the method of dark culture at the first and light culture following.(4)The optimal culture medium for PLB induction was 1/2MS+6-BA 1.0 mg/L+NAA 0.5 mg/L,in which the induction rate was 95%.(5)The optimal culture medium for PLB proliferatio
15、n was 1/2MS+6-BA 0.5 mg/L +NAA 0.2 mg/L +1 gL-1 AC,1/2MS+KT 1.0 mg/L +NAA 0.1 mg/L,in which the rate of PLB proliferation could reach 2.5 and 3.0.(6) The suitable culture medium for PLB differentiation was MS+6-BA 3.0 mg/L +NAA 1.5 mg/L,in which the rate of buds could reach 2.8.(7)The best rooting a
16、nd strengthening culture medium for IV adventitious shoots was MS+6-BA 0.2 mg/L +NAA 0.5 mg/L,in which rooting rate was 100%. (8)The transplanting survival rate of plantlets could reach 80% when using lichen, sand, earth (1:1:1) planting medium. Anatomy observation on seeds of Geodorum densiflorum was undertaken. Seed coat was made up of one layer transparent cells. Moreover, morphogenesis during the period of seed germination in vitro was studied. At fi
