【作者】 张亚锋；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2007， 硕士

【摘要】 南瓜（Cucurbita moschata（Duoh．ex Lam．）Duch．cx Poiret）为葫芦科南瓜属中国南瓜种。目前，我国栽培的南瓜大多为长蔓品种，无蔓或者短蔓的矮化品种较少。南瓜的无蔓品种栽培中所需空间小，管理比较容易，适合密集种植，在生产中的优势很大。因此，无蔓性状对于南瓜的育种具有重要意义。在世界范围内南瓜属植物受病毒病的危害严重，尽管常规育种在其抗性育种上所占的比重仍很大，但由于周期长、遗传性状不稳定等原因，人们逐渐尝试通过转基因手段获得新品种。将相应病毒的外壳蛋白基因转入南瓜属植物体内，可以大大增加植株的抗病性，提高产量和质量。对南瓜无蔓突变体来说，还可将蔓性相关基因转入植株体内，来研究控制瓜类植物蔓伸长的机制。转基因技术的成功需要建立在高效再生体系基础之上。本研究对南瓜‘无蔓1号’自交后代中发现的无蔓性状纯合植株的蔓性和生物学性状进行观察和分析，以此研究控制南瓜无蔓性状基因的遗传背景。在构建南瓜‘无蔓1号’高频再生体系的基础上，采用酶联免疫吸附分析法（Enzyme-linked Immunosorbent Assays，ELISA）测定不同苗龄带柄子叶外植体中内源激素的含量。以此分析外植体中初始内源激素含量和比例与外植体再生频率之间的关系。目前获得以下，一些初步结果：（1）对南瓜‘无蔓1号’自交后代中发现的无蔓性状纯合植株作无蔓亲本，长蔓植株作长蔓亲本。对无蔓亲本、长蔓亲本、正反杂交一代（first filial generations，F₁s）、正反回交一代（first back crosses，BC₁s）和杂交二代（second filial generation，F₂）植株蔓性的观察结果表明，无蔓与长蔓性状在3片真叶时期可以分辨清楚，无蔓亲本全部为无蔓性状，长蔓亲本全部表现长蔓性状，正反杂交F₁s后代则全部是无蔓植株，正反回交BC₁s表现出无蔓和长蔓植株1：1的分离比例，F₂代植株中无蔓和长蔓植林符合3：1的分离比例。这说明南瓜蔓性是由一对等位基因所控制，控制无蔓性状的基因对长蔓性状基因表现为显性。无蔓性状的产生是控制长蔓性状的隐性基因发生显性突变的结果。对无蔓亲本和长蔓亲本生物学性状的观察结果表明，长蔓植株的雄花数明显多于丛生植株，但无蔓植株的花期比长蔓植株提前8d。长蔓植株与无蔓植株的叶和叶柄大小方面无显著差异。无蔓亲本和F₁植株在所观测的生物学性状上均无显著差异。（2）通过研究苗龄、激素浓度和种类、外植体来源等因素对再生过程的影响，建立了南瓜‘无蔓1号’高频再生体系。对上半子叶、带柄子叶、叶柄、根和真叶块5种来源外植体再生效果的研究结果表明，尽管5种外植体均可以高效地诱导愈伤组织，但只有带柄子叶有不定芽分化，并且不定芽不是由愈伤组织分化产生，而是直接由子叶基部产生。这说明我们的研究中所得的不定芽是直接器官发生途径，子叶基部是直接分化不定芽的关键部位。使用不同浓度的BAP进行不定芽诱导的结果表明，在所采用的四个浓度梯度中(0、0.5mg·L^-1、1.0mg·L^-1、2.0mg·L^-1)，当BAP不存在时没有不定芽产生，其他三个浓度对再生频率无显著的影响，说明BAP是诱导不定芽所必需的，但其浓度对再生频率并没有显著影响。对4～7d苗龄带柄子叶外植体再生过程的研究结果表明，单独使用BAP时，再生频率与外植体再生频率呈现规律性变化，即：4到5d时再生频率下降，之后上升并在7d增至最大值。（3）不同苗龄带柄子叶外植体的内源激素测定结果表明，ZR与IAA在4d苗龄时的含量最高，分别为0.41±0.069nmol·g^-1FW和0.96±0.223nmol·g^-1FW，之后随着苗龄的增加含量逐渐降低，到7d时降至最低点，分别为0.17±0.037nmol·g^-1FW和0.40±0.152nmol·g^-1FW，并且两者和再生频率之间无线性关系。iPA及IAA／（iPA+ZR）的变化与再生频率呈现出一种线性的关系。在7d苗龄时，带柄子叶外植体的再生频率最高，同时内源iPA含量及IAA／（iPA+ZR）值也最高；5d苗龄时，子叶外植体的再生频率最低，对应的内源iPA含量及IAA／（iPA+ZR）值也最低。内源GA₃的测定结果表明，其含量过高可能会降低外植体的再生频率。以上研究结果表明，本实验中无蔓性状的南瓜矮化突变体是因为控制长蔓性状的隐性基因发生显性突变所产生的。构建南瓜‘无蔓1号’高频再生体系的研究表明，最佳的外植体为7d苗龄带柄子叶，不同浓度BAP对再生影响不显著。内源激素测定的结果表明，带柄子叶外植体苗龄与再生频率之间的关系，可能是由于内源激素的含量和比例变化的结果。更多还原

【Abstract】 There are three economically important Cucurbita species, namely, C. pepo L., C. maxima Duch. and C. moschata Duch., which have different climatic adaptations and are widely distributed in agricultural regions worldwide. C. moschata plants possess long internodes and have trailing vines that may spread up to 15 m from the crown of the plants, thereby requiring wide planting distances. Some plants of developed cultivars of C. pepo L have short internodes. No vine plants in C. moschata have some features that make them valuable, i.e. they allow higher plant densities, facilitate cultivation, and have a more concentrated maturity than vine counterparts. This makes them value in pumpkin breeding. Curcubita is affected worldwide by several viral diseases that substantially reduce yield and fruit quality. Plant biotechnology appears to be a viable option for the improvement of the Cucurbita species by means of plant tissue culture and genetic transformation. The establishment of an efficient in vitro plant regeneration system suitable for genetic transformation is the key step in this approach. On the other hand, it is generally known that from the factors which influence organogenesis, the foremost are genotype, age and origin of the initial tissues, which, in turn, are related to their endogenous hormonal status. Moreover, the importance of the exogenous application of plant growth regulators is much discussed. In this wise, the organogenic potential of explants is closely associated with their contents of natural and exogenous applied plant growth regulators.The purposes of the present work were to study the morphology and inheritance of the no vine pumpkin and establish a reproducible organogenic pathway for C. moschata. On this condition, we studied the correlation between the organogenic capacity of pumpkin cotyledon explants, as a function of their developmental stage, and endogenous hormonal contents. The main steps are as follows:（1） The no vine plants differ greatly in morphology from vine plants. No vine plants could readily be identified by its vine characteristics when three true leaves were present. No vine plants had short internodes, short vine and small number of internode compared with vine plants. Vine plants produced more male flowers （21.1±4.3） than no vine plants （13.7±3.3）. In contrast, no vine plants flowered eight days earlier than vine plants. The morphology of F₁ plants is also investigated. F₁ plants of crosses between no vine and vine resembled vegetatively the bush parent. Although no vine and F₁ plants had a higher rate of fruit abortion than vine plants, they had a larger total number of fruit than vine plants in a plant basis. All F₁ progeny were no vine phenotype regardless of the direction of crosses. The reciprocal BC₁ families were segregated approximately one no vine: one vine plant. In F₂ the distribution was 156 no vine to 49 vine-type, which fitted a 3:1 ratio.（2） To induce multiple shoots from pumpkin, cotyledon explants excised from various ages of seedlings after germination in vitro were cultured on MS medium augmented with different concentrations of BA. Highest frequency shoot regeneration （63.7%） was induced from 7-day-old cotyledon cultured on MS medium containing 0.5 mg·L^-1 BA. The frequency and time of shoot induction had closed correlation with donor seedling age. Multiple shoots elongated on MS medium with 0.1 mg·L^-1 BA and 5～7 shoots per explant were recovered. The number of shoot per explant had no relationship with donor seedling age or BA concentration of induction medium. Elongated shoots were rooted on MS medium easier than on 2/3MS, 1/2MS or MS supplemented with 0.1 mg·L^-1 NAA. The rooted shoots were then transferred to greenhouse where they grew and flowered normally.（3） Quantitative analysis of endogenous cytokinins （ZR and iPA） and auxin （IAA） in initial cotyledon explants of different ages showed that cotyledon explants with the highest caulogenic potential in each BAP concentration had the highest contents of iPA and Cks/IAA ratio, and that with the lowest caulogenic potential in each BA concentration had the lowest contents of iPA and Cks/IAA ratio. These results also suggested that the direct organogenesis of pumpkin was characterized by changes in hormone levels. A general principle that a high concentration of cytokinins and a low concentration of auxin promote the induction of shoot morphogenesis in vitro was confirmed in our studies.In conclusion, the morphology of vine and bush plants in C. moschata is compared in this report. Genetic analysis of plant habit shows that a monogenic inheritance is expressed whereas no vine is dominant to vine type. On the other hand, an effective regeneration protocol was constructed for C. moschata, though only one cultivar was tested. On the basis of endogenous analysis, we attributed a major role to iPA and the ration of IAA/Cks （ZR and iPA） in caulogenic response of pumpkin cotyledon explants.更多还原