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桃组织培养及遗传转化的研究

Studies on Tissue Culture and Genetic Transformation of Peach

【作者】 吴延军

【导师】 张上隆;

【作者基本信息】 浙江大学 , 果树学, 2004, 博士

【摘要】 桃是世界性重要果树,我国是桃主产国,产量位居世界各国之首,浙江省是桃主产区之一,平均年产量在10万吨以上。桃果色、香、味、质俱佳,很受消费者欢迎,在果品市场占据重要地位。但桃是乙烯跃变型果实,采后软化进程迅速,大多数品种成熟季节又适逢高温高湿,果实极不耐贮运,严重影响其销售与外运,这是长期制约桃生产的主要问题之一。 延长桃贮藏期常采用低温冷藏,但它易造成桃果冷害,结果并不十分理想。桃贮藏保鲜的现状表明延长桃贮减时间、提高贮藏品质,延长货架期,减少采后腐烂等目标难以靠常规措施实现。随着现代生物技术的发展,植物转基因技术已得到广泛应用,国际上番茄转ACC合成酶和ACC氧化酶反义基因的成功为其他作物解决贮藏难问题提供了范例和良好前景,因此本研究也运用这一思路开展桃组培与转基因研究。 本研究以我国南方主栽品种玉露桃和湖景蜜露桃为试材,建立了桃幼胚和幼胚子叶再生体系,运用基因枪介导法和根癌农杆菌介导法转化桃幼胚子叶获得了转ACO反义基因的转基因植株,并对转基因材料进行了分子鉴定。主要研究结果如下: 1.以奉化玉露和湖景蜜露桃为试材,分别在盛花后45 d和55 d采集桃幼果并取其幼胚接种,置于正交设计的16种培养基上,26℃±1℃暗培养,研究基本培养基类型、激素等因素对桃幼胚脱分化的影响,筛选出适合愈伤组织分化的激素组合。结果表明:1)培养基是影响桃幼胚愈伤组织诱导的最重要因素,MS适合45 d、55 d玉露和55 d湖景蜜露幼胚诱导愈伤组织。2)激素诱导愈伤组织因品种而异,NAA 1.0 mg/L对45 d、0.5 mg/L对55 d玉露,BA 0.5 mg/L对55 d湖景蜜露效果较好,在试验浓度范围内2,4-D对二品种幼胚发育45 d、55 d均无明显效果。3)幼胚的发育状态是影响诱导愈伤组织的另一因素。盛花后45 d的幼胚愈伤组织诱导率最高达96.6%,而盛花后55 d的幼胚愈伤组织诱导率为81.6%。桃幼胚愈伤组织可在MS+0.05 mg/L NAA+1.0 mg/L BA的培养基上分化成芽,再生芽可在1/2 Ms+1.0 mg/L IBA培养基上生根。 2.盛花后45 d、55 d玉露和55 d湖景蜜露桃幼胚子叶,置于MS+2%蔗浙江大学博:卜学位论文:桃组织培养及遗传转化的研究(2 0()4)搪+0.75%琼脂并加入不同浓度配比激素的培养基上,于26℃士l℃,光照强度2000一3000 lux,16h/8h光暗周期的环境下培养,研究激素配比、品种、取样时间、损伤方式对桃子叶再生能力的影响,并对不定芽发生部位进行了观察。结果表明:NAA 0.50 mg/L+BA一0.0 mg/L、NAA 0.05 mg/L+ToZ 3.0 mg/L二组激素配比有利于桃子叶再生:玉露的再生能力较高于湖景蜜露,盛花后55d的幼胚子叶比花后45d的幼胚子叶稍易再生;带胚芽子叶纵向刻伤再生率高。桃幼胚子叶可通过愈伤组织分化和器官直接分化二条途径再生,不定芽可产生于子叶的背茎面、向茎面等各部位。再生不定梢(芽)在1/2MS+2.岛蔗搪+0.75%琼脂+IBA 1.0 mg/L+Ad 20 mg/L的培养基上可以生根。 3.以玉露桃幼胚作为根癌农杆菌介导转化的受体,通过对GUS基因瞬时表达率的分析,研究此转化体系的最佳实验参数。实验结果表明,预培养时间、感染时间、共培养时间和根癌农杆菌诱导物AS等对转化效率都有一定的影响。预培养ld、感染巧min、共培养42h和不添加AS时GUS基因的瞬时表达率最高。 4.通过基因枪轰击盛花后55d玉露桃幼胚带胚芽的单瓣子叶转化带有桃ACO反义基因的PBIO.8载体,在363个外植体中最终获得了5个抗性芽和l个抗性植株。 5.利用根癌农杆菌GV3101将ACO反义基因导入桃幼胚子叶,获得了卡那霉素抗性植株。在供试的1379个玉露桃幼胚子叶中获得了39个抗性芽,湖景蜜露桃614个幼胚子叶中获得了11个抗性芽。抗性芽通过微芽嫁接培养有可能成株。 6.对基因枪和农杆菌介导获得的桃转ACO反义基因的部分抗性材料进行PCR、Southern blot和GUS基因表达等分子检测,结果表明外源反义ACO基因己经整合到桃的基因组中。

【Abstract】 Peach (Primus persica (L.) Batsch) is one of the world most important fruit and a famous crop of Zhejiang Province of China. Excellent quality, attractive color, good taste and marvelous fragrance make it a popular fruit in the market. Peach is a climacteric fruit and ripened in summer season which is associated with high temperature and humidity and all these factors make it difficult to store for longer periods. Storage under low temperature causes chilling injuries and makes it exposed to degradation. Much work has been done to control this problem by manipulating the storage conditions, but couldn’t solve this problem.To keep pace with the global market, a year-round supply of produce is required. Fruit varieties need to be evaluated for appropriate post harvest characteristics that allow longer periods of storage without losing quality or experiencing internal breakdown. New methods are to be explored to control the ripening process. In peach there are genes such as stony hard and slow ripening that control ethylene production and maturation similar as in found in tomato. The fruit maturation and post harvest physiology studied in the model plants as tomato should be followed to identify new genes in peach that control ripening. To achieve this objective we extensively studied trait for the further studied.This research study describes peach tissue culture and gene transformation technique in detail. Plants were regenerated from the immature embryos and cotyledons of Peach cv. ’Yulu’ and ’Hujingmilu’. Immature cotyledons were infected with particle bombardment and Agrobacterium tumefaciens, which carries ACC oxidase antisense gene. Kanamycin-resistant (KanR) shoots were regenerated from the cotyledons. The results of PCR, PCR-southern and genomes southern hybridization analysis showed that the peach ACC oxidase antisenes gene was integrated into peach genomes. The results were summarized below.1. The main composition of culture media such as the basic medium and hormones were tested for their effects on peach callus frequency from immature embryos (which picked after full flower 45 days and 55 days respectively) under 26?’C without light in an orthogonal experiment. The result showed that: 1) The basal medium was the most important factor for inducing callus from embryos. MS was adapted to ’Yulu’ (45 d and 55 d) and ’Hujingmilu’ (55 d). 2) NAA 1.0 mg/L and 0.5 mg/L were better for Yulu (45 d and 55 d), BA 0.5 mg/L was better for Hujingmilu (55 d), but 2,4-D hadn’t any important effect on callus inducing from the examined material. 3) Days after fecundation is another factor effect on callus induction. The highest callus frequency from 45d immature embryos was 96.6% and it was 81.6% from 55d embryos. Shoots regeneration occurred when callus was transferred to a medium in which the NAA concentration was 0.05 mg/L and BA was 1.0 mg/L. Shoot regenerated roots in the medium of 1/2 MS +1.0 mg/L IB A.2. A method for plant regeneration from immature cotyledons of peach has been developed. The immature cotyledons (45 d and 55 d) of peach were cultured on MS media supplemented with 2% sucrose and 0.75% agar and hormone in various combinations. Cultures were incubated at (26 + 1)℃ with 16h/8h light/dark cycles of approximately 2000-3000 Lux. The result showed that 2 types of hormone combinations (NAA 0.50 mg/L + BA 10.0 mg/L, NAA 0.05 mg/L + TDZ 3.0 mg/L) were favorable to shoots differentiation. The regeneration ability of ’Yulu’ peach was higher than that of ’Hujingmilu’, and the regeneration frequencies of 55-days immature cotyledons (’Yulu’) were slightly higher than that of 45-days. The regeneration was changed when cotyledons were wounded horizontally by a razor blade. The best results were obtained when cotyledons with embryonic axis and wounded in the surface. Shoots could regenerate from every part of cotyledons, such as the abaxial side and adaxial side. Adventitious shoot could regenerate on the 1/2MS medium with IBA 1.0 mg/L and Ad 20 mg/L.3. Using transient expressio

  • 【网络出版投稿人】 浙江大学
  • 【网络出版年期】2004年 03期
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