【作者】 毕静华；

【导师】 刘永立；

【作者基本信息】 浙江大学 ， 果树学， 2005， 硕士

【摘要】 猕猴桃作为新兴水果,营养丰富,被誉为“水果之王”,以高于其他水果数倍甚至几十倍的维生素C含量而被人们所青睐(黄宏文,2000)。阔叶猕猴桃(Actinidia latifolia)又名多花猕猴桃,生长在云南、广西、湖南、四川和贵州等地,果实可加工食用,适合造果汁和果酒,其鲜果中维生素C含量高达939.8～2140mg/100g,是猕猴桃属中发现的维生素C含量最高的种(崔致学,1993)。阔叶猕猴桃作为珍贵的猕猴桃种质资源,具有广阔的市场前景和开发利用价值。 本文以阔叶猕猴桃叶片和叶柄为材料,建立了高效可重复的再生体系,并在此基础上通过根癌农杆菌介导法对阔叶猕猴桃叶柄进行基因转化研究。经过抗生素筛选培养和植株再生,获得了转基因抗性芽,并通过GUS组织化学染色、PCR等技术对转基因材料进行了检测。本文首次研究了阔叶猕猴桃的器官形成和植株再生,并建立了遗传转化体系,为阔叶猕猴桃遗传资源的利用和分子育种提供了技术参数,为通过基因工程有目的的改良猕猴桃某些性状奠定了基础。主要研究结果如下: 1.以阔叶猕猴桃叶片和叶柄为外植体,通过器官发生途径诱导形成不定芽,建立了高效的再生体系。探讨了不同激素组合、暗培养时间以及蔗糖浓度对叶片和叶柄离体再生的影响。结果表明,叶片外植体在I/2MS(Murashige and Skoog,1962)+0.1μM NAA+5μM Zeatin附加20g/L蔗糖的培养基上,进行21d暗处理后转到光下培养效果最好,6周后不定芽再生率达91.7%,平均再生芽数6.9个/外植体。而叶柄外植体在1/2MS+0.1μM NAA+5μM Zeatin附加20g/L蔗糖,进行14d暗处理后转到光下培养效果最好,5周后不定芽再生率达到100%,平均再生芽数18.2个/外植体。同时发现,高浓度的BA不仅抑制不定芽的形成也会导致玻璃化现象的产生;光对不定芽的再生至关重要。再生不定芽生根良好,并被成功驯化后移植大田。 2.以阔叶猕猴桃叶片和叶柄为外植体,探讨了各种抗生素对愈伤组织和不定芽形成以及不定芽生根的影响。结果发现,卡那霉素(Kanamycin,Kan)明显抑制叶片和叶柄的分化。当Kan浓度达到20mg/L时叶片的分化被完全抑制,当达到25mg/L时,叶柄的分化也被完全抑制。在愈伤组织和不定芽诱导过程中,头孢霉素(Ceftomine,Cef)对不定芽分化的效果明显好于羧苄青霉素(Carbenicillin,Carb)。Cef浓度为300mg/L时叶片和叶柄的再生频率最高,均达到100%,叶片平均再生芽数为9.8个/外植体,叶柄平均再生芽数为12.4个/外植体。高浓度的Cef(≥200mg/L)对不定芽生根有明显的抑制作用,无论生根率还是平均根数、根更多还原

【Abstract】 Kiwifruit with rich nourishment especially high content of Vitamin C finds favor in human’s eyes in recent decades (Huang, 2000). China is rich in kiwifruit resources with more than 59 species out of total 66. Among these 59 species, most grow naturally and haven’t been exploited, even some are at the brink of extinction. It is imperative to exploit and use the germplasm of these wild species while maintaining their genotypes. Actinidia latifolia generally known as "Duo Hua", grows naturally in Yunnan, Guangxi, Hunan, Sichuan and Guizhou provinces of China and bears edible fruits. Its fruits are best for making fruit juice and wine. Vitamin C content in its fruits of 939.8~2140mg/100g is higher than all other kiwifruit species (Cui, 1993). To our knowledge, this is the first study on an in vitro regeneration method and genetic transformation for A. latifolia. The results of this study will help facilitate the application of genetic engineering methods to improve the commercial traits in A. latifolia. The main results are summarized as follows:1. An efficient plant regeneration protocol in vitro for A. latifolia was achieved via organogenesis from leaf and petiole explants. In this paper, effects of explant types, medium formulation, different combinations of growth regulators, dark treatments and sucrose concentrations on the organogenesis are examined. For leaf explants, after 6 weeks of culture maximum shoot regeneration frequency of 91.7% with 6.9 shoots per explant was obtained on half-strength MS medium (Murashige and Skoog, 1962) containing 5μM Zeatin, 0.1μMα -naphthaleneacetic acid and 20g/L sucrose, incubated in the dark for 21 days then exposure to light. For petiole explants, after 5 weeks of culture maximum shoot regeneration frequency of 100% with 18.2 shoots per explant was obtained on half-strength MS medium containing 0.1μ Mα-naphthaleneacetic acid, 5μM Zeatin and 20g/L sucrose, incubated in the dark for 14 days then exposure to light. High concentration of 6-benzyladenine (10μM) inhibited shoot formation, and also resulted in vitrification. We proposed that light is essential for shoot regeneration for A. latifolia. Regenerated shoots were transfered on BW rooting media (Sugawara et al., 1994) supplemented with different combinations of auxins and 6-benzyladenine. The highest rate of root formation reached 100% associated with maximum roots per explant (17.5). Plantlets with good root system were successfully acclimatized to thefield conditions and produced healthy plants.2. Effects of antibiotics on in-vitro regeneration and organogenesis of A. latifolia from leaf and petiole explants were examined. Kanamycin inhibited significantly in-vitro regeneration and organogenesis. Callus and shoot formation from leaf explants was completely inhibited in presence of 20 mg/L kanamycin, and that from petiole explants was in presence of 25 mg/L kanamycin. Results also showed that cefotaxime is superior to carbenicillin for shoot differentiation. When 300mg/L cefotaxime was present in the regeneration medium, both shoot formation rate and mean number of shoots per explant reached the highest levels, 100% and 9.8 shoots/explant from leaf explants, 100% and 12.4 shoots/explant from petiole explants. However, a negative effect of cefotaxime (≥200mg/L) on rooting of regenerated shoots was also observed. Carbenicillin had no marked influence on rooting. In the treatments of different combinations or alone of kanamycin and carbenicillin on rooting, carbenicillin had no marked influence on rooting but kanamycin inhibited markedly rooting. With the increase of kanamycin concentration, the rooting rate decreased sharply. No root appeared on rooting medium with 50mg/L kanamycin. It was considered that cefotaxime 300mg/L was suitable for A. latifolia to elimination of Agrobacterium tumefaciens during transgenic shoot induction; kanamycin 20mg/L was suitable in the selection of transgenic versus false-positive shoots from leaf explants and 25mg/L was suitable for petiole explants; during rooting phase kanam更多还原