【作者】 张良波；

【导师】 周朴华； 陈禹保；

【作者基本信息】 湖南农业大学 ， 植物学， 2004， 硕士

【摘要】 屋顶长生草（Sempervivum tectorum）系景天科长生草属多年生常绿草本观赏植物，主要分布在欧洲。其肉质叶有较高的药用价值和保健作用，提取液用于治疗咽喉溃疡，口腔疾病，支气管炎，以及烧伤、烫伤、蚊虫叮咬等皮肤疾病，其保健作用主要体现在其对肌肤的滋养、护肝作用以及泡茶饮用有提神的作用。为了建立屋顶长生草的快繁体系，本文以屋顶长生草的肉质叶为外植体，进行了离体培养、试管苗再生以及相关形态发生的组织学与生理生化特性的研究。主要研究结果如下： 1、建立了屋顶长生草离体培养体系，我们的研究显示植物激素对屋顶长生草形态发生类型起着重要的调控作用。 1．1 2，4-D、6-BA与2，4-D；6-BA与NAA加IAA配合使用均能诱导愈伤组织。但是，前者形成的愈伤组织比较疏松，难以分化芽，后者的愈伤组织较致密，易分化芽。 1．2 叶片培养在愈伤组织诱导培养基上，产生愈伤组织，转入6-BA1mg／l+NAA1mg／l或6-BA的培养基上，则分化不定芽，行器官发生型途径。 1．3 叶片培养在含一定浓度的6-BA或6-BA 2mg／L+NAA 0．2mg／L培养基上能直接再生芽，行器官型途径，加入GA₃能缩短诱导丛芽及成苗的时间。 1．4 屋顶长生草的生根以1／2MS为基本培养基，NAA与IAA配合使用效果好，活性炭有利于根的发生和生长。 2、屋顶长生草离体培养中出现的玻璃化苗现象与丛芽增殖速度过快有关。改善培养条件，控制丛芽过快增殖，如加强光照、减少培养基的用量（1／2MS培养基）、降低培养温度、提高培养基中蔗糖和琼脂浓度以及加入青霉素等措施能有效减少玻璃化苗。 3、屋顶长生草的叶具有肉质早生植物叶的特点，表皮细胞外有角质层，叶有较密的腺毛分布，气孔器由两个肾形的保卫细胞和两个镰刀形的护卫细胞组成，对控制蒸腾，减少水分损失有重要作用。叶肉细胞没有栅栏组织与海绵组织之分，细胞比较大，有贮水作用。维管束平行排列，导管和筛管分子都很小，为一圈维管束鞘所包围。4、屋顶长生草离体培养的叶片不定芽来源于近表皮细胞的叶肉细胞脱分化再生而成;器官发生型的愈伤组织来源于不同层次的叶肉细胞及维管束鞘细胞的脱分化，形成胚性细胞、胚性细胞团，进一步形成愈伤组织并突破表皮细胞而增大。大部分叶肉细胞的叶绿体、原生质体解体，初生壁上形成条纹增厚成为管胞。愈伤组织的芽分化为内起源。5、较系统的研究了屋顶长生草器官发生型形态发生与生理生化变化的相关性。结果表明:在细胞脱分化形成愈伤组织、愈伤组织增大突破叶表皮、愈伤组织分化芽、不定芽成苗等阶段，淀粉含量呈降低^增高^降低^再增高的变化趋势;蛋白质含量呈增高一降低一再增高一再降低的变化趋势;氨基酸与核酸含量的变化都呈增加一增加一降低一增加的变化趋势;可溶性糖含量的变化呈降低一增加^降低^降低的变化趋势;vc含量的变化呈降低一增加一增加一增加的变化趋势;过氧化物同工酶、脂酶同工酶、淀粉酶同工酶的酶谱的变化呈现增加一减少^增多一减少的变化趋势。但三种酶的同工酶条带的增加主要出现在愈伤组织的形成和不定芽的再生这两个标志性阶段。更多还原

【Abstract】 Sempervivum tectorum is a perennial succulent ornamental plant that spread widely in Europe. Its fleshy leaves have both pharmaceutical and health functions, the substance extracted from it can cure some skin diseases( such as faucitis, stomatitis, bronchitis, burn, scald, bite and sting, etc. ) and be helpful for skin nourishing, liver protection, furthermore, it can refresh oneself with it as tea. To establish the rapid propagation system, the fleshy leaves as explants were cultured in vitro. Researches on its in vitro culture, plantlets regeneration, characters of physiology and biochemistry and the histology of morphogenesis were conducted on the current paper. The results are as follows:1. Optimal mediums for Sempervivum tectorum in vitro propagation and the clone system were established. The research shows that the phytohormone is the key element for morphogenesis of in vitro culture.1.1 Callus can be induced on the medium added with 6-BA and 2,4-D and the medium added with 6-BA combined with NAA and IAA. However callus formed on the former medium is loosen than that on the latter one. The former is more difficult for bud differentiation whereas the latter is denser and it’s easier for bud differentiation.1.2 Plantlets formed from the calli, which is induced from the fleshy leaves cultured on the medium added 2,4-D combined with 6-BA and the medium added with 6-BA 1 mg/L, NAA 1 mg/L and IAA 1 mg/L, after that transferred to the medium added with 6-BA 1 mg/L + NAA 1 mg/L or 6-BA, in a organogeny-type morphogenesis way.1.3 Plantlets can be induced directly from the cultured explants on the medium added with 6-BA 2 ml/L + NAA 0.2 ml/L, in a organ-type morphogenesis way. The GA3 added addictively on medium can shorten the duration of buds formation.1.4 1/2MS medium combining with NAA and IAA was optimum for rooting of plantlets, the active charcoal was also propitious to that. And rapid propagation system with high reproductive coefficient and reproductive rate was established.2. Research on vitrification during the in vitro culture of Sempervivum tectorum was proceed.The results suggested that vitrification can be effectively controlled by strengthening the illumination, reducing the inorganic salt content (to use 1/2MS medium), decreasing the temperature, inducing the content of the saccharose and agar, even adding of antibiotic.3. Anatomic structures of Sempervivum tectorum leaf.The Sempervivum tectorum is a typical xerophil, its leaves are characterized with cutin, density gland hair, and the stoma was composed with two kidney safeguard cells and two hook convoy cells, which effectively control the evaporation. The leaf anatomic structure indicated that mesophyll was without the differentiation of palisade and sponge tissue. And the mesophyll cell is large and function as water storage. The vascular bundles distribute parallel; trachea and sieve tube were small and wrapped with vascular sheath.4. Characters of physiology and biochemistry of Sempervivum tectorum in vitro cultureDuring the morphogenesis in organogeny way, the explants expanded and turned green initially, and embryonic cell lump formed on the surface of explants, and then grew into plantlet. The results indicated that all the testing indexes were increased during the formation of the organ in culture and did not reduce until the organ formed. In terms of the curve, the content of protein, amino acid, nucleic acid, soluble sugar, amylum and the bioaction of proteinase, mylase, peroxidase both changed consistent with the morphogenesis of this approach.5. Close relationships between the change of the characters of physiology and biochemistry and different development phase of organogeny-type morphogenesis approaches.The calli may be induced when the contents of protein, amino acid, nucleic acid, soluble sugar(dry-weight), were relatively higher, whereas the contents of amylum, Vc were relatively lower. During callus accretion the contents of protein, were relatively lower, whereas the contents amino acid, nucleic ac更多还原