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丰香草莓高效再生的机理及遗传转化研究

Studies on Mechanism of a High-efficient Regeneration System and Genetic Transformation of Strawberry

【作者】 秦永华

【导师】 张上隆;

【作者基本信息】 浙江大学 , 果树学, 2005, 博士

【摘要】 草莓是重要的水果之一,在世界各地广泛分布。草莓栽培中存在不少问题,如耐寒、抗虫、抗病、抗病毒等优良品种缺乏,严重制约了草莓生产的发展。过去常规杂交育种在提高草莓品种的产量、品质等方面取得了一定进展,但草莓多为八倍体,遗传上高度杂合,给杂交育种带来很大困难,而且常规育种周期长,费时费力,给育种工作者带来了难度,因此需引进新的手段和方法进行育种。现代生物技术的飞速发展为草莓育种提供了新途径,它可以获得常规育种难以得到的新类型,从而创造出新种质。现代生物技术在植物育种中的应用是随植物组织培养技术的发展而兴起的。由于农杆菌介导的遗传转化主要依赖于受体材料的再生能力及农杆菌的转化效率,因此建立高效的受体再生体系是遗传转化成功的关键。本试验以草莓主栽品种丰香为试材,研究了再生过程中激素配比、有色膜、AgNO3、暗培养、抗生素等因素对草莓离体再生的影响,探讨了不同有色膜下丰香草莓再生差异的原因和AgNO3对草莓生长的影响及其机理,为建立高效、可重复的再生体系奠定理论基础。在分别构建ADP和INS基因植物表达载体基础上,通过根癌农杆菌介导分别将APD和INS基因导入草莓基因组,对影响农杆菌转化频率的各种主要因素进行筛选,得到一个最佳的转化体系,获得了转化植株。Kan筛选、PCR检测和Southern杂交检测证明目的ADP基因已经整合到草莓基因组中。对INS基因抗性愈伤组织PCR检测表明,INS基因已整合到草莓基因组中。主要研究结果如下: (一) 草莓高效离体再生体系的建立 (1) 不同激素组合对丰香草莓不定芽再生能力的研究表明:TDZ单独使用可以诱导出不定芽,随着TDZ浓度的增加,不定芽再生率逐渐提高,但诱导出的不定芽玻璃化现象也随之加剧。因此,TDZ不宜过高,以1.5mg.L-1为宜。TDZ 1.5mg.L-1与IBA配合使用,可显著提高丰香不定芽的再生率,当IBA的浓度为0.4mg.L-1时不定芽再生率最高(76.7%)。2,4-D不利于芽的分化,随着2,4-D浓度的提高,不定芽再生率逐渐降低,诱导出的愈伤组织由浅黄色逐渐变为灰白色,并且褐化加剧。 (2) 重金属元素Ag+对丰香草莓不定芽再生能力的影响表明:草莓外植体在MS+1.5mg.L-1TDZ+0.4mg.L-1IBA分化培养基附加1.0mg.L-1AgNO3的中预培养10 d后转入不含Ag+的分化培养基中培养,不定芽再生率(87.38%)和每个外植体再生的芽数(11.67)有显著提高,且始分化期可以提前5-7 d。 (3) 不同有色膜对丰香不定芽再生的影响很大,培养45 d后,不同有色膜条件下不定芽再生率以红膜和绿膜为最好,分别为100%和98.28%,其次为荧光(75.81%)、蓝膜

【Abstract】 Strawberry is one of most important fruit crop cultivated widely in world. In spite of its important, several factors such as its low resistance to chilling injury and its susceptibility to insect, disease and virus which limit the cultivation of strawberry fruit. Some progress has been made in strawberry yield and quality through conventional breeding techniques that has been limited due to several factors. Genetic limitations of strawberry associated with high heterozygosity and polyploidy are the main severe limiting factors that make it laborious, costly, and time-consuming to improve strawberry quality and yield by the conventional breeding techniques, which usually results in a low frequency. With the development of biotechnology, foreign genes could be incorporated into plant genome for desired agronomic traits by the new technologies of Agrobacterium-mediated transformation. An efficient regeneration method is one of the most important factors in determining the success of plants genetic transformation. Establishment of a high-efficient regeneration and transformation system in strawberry would make a significant contribution in the improvement of this fruit on gene engineering level. In this study, Fragaria ananassa Duch. cv. Toyonoka was used as source material, we studied the effects of plant growth regulator combination, plastic color film, AgNO3, dark treatment and antibiotics on shoot regeneration. Regeneration mechanism under color films and response of in vitro strawberry to AgNO3 were also studied. Using the EH 105 strain harboring pBI121-APD and pBI121-INS that we constructed respectively, leaf discs of strawberry were infected with the Agrobacterium, which carried the two genes. Kanamycin-resistant (Kan~R) shoots and calli were gotten from the explants. The results of PCR, Southern hybridization analysis showed that the APD gene was integrated into strawberry genomes. INS gene was integrated into strawberry genomes by PCR analysis. The results were summarized below:1. Establishment of a highly efficient system of shoot regeneration from leaf explants of strawberry1). Types and combinations of plant growth regulators had significant effects on shoot regeneration in strawberry. TDZ alone induced regeneration of adventitious buds. Maximum regeneration rate (47.45%) was achieved with higher concentration of TDZ (at 2.5 mg.L~-1), but it resulted in extended rates of vitrification. Therefore, TDZ above 1.5 mg.L~-1 on strawberry regeneration were not encouraging. 1.5 mg.L~-1 TDZ associated with 0.4 mg.L~-1 IBA significantly increased regeneration efficiency in terms of both percent of shoot regeneration and number of shoots per explant. The maximum percentage of shoot regeneration (76.67%) and the highest number of shoots per explant (5.78) were achieved by MS+1.5 mg.L~-1 TDZ+0.4 mg.L~-1 IBA. 2,4-D significantly decreased shoot regeneration with maximum browning and concentrations above 1.0 mg.L~-1 had adverse effect on shoot regeneration. 2). Significant difference was observed in percent shoot regeneration and number of shootsper explant when explants were exposed to AgNO3 for 10 d. With AgNC>3 in the medium, an earlier (5-7d) and stable shoot regeneration was observed, highest regeneration (87.38%) and numbers of shoots per explant (11.67) were achieved with the shoot regeneration media (MS+1.5 mg.L"1 TDZ+0.4 mg.L"1 IBA) containing 1.0 mg.L"1 AgNO3.3). Color films had significant effects on the shoot regeneration efficiency. Under red films, 100% shoot regeneration was achieved after 45 days in culture, closely followed by green (98.28%). Fluorescent light (Control) ranked the third (75. 81%), followed by blue (60.09%) and the lowest shoot regeneration was under yellow films (20.69%). Highest number of shoots (25.75) was recorded for green, followed by red (25.11) films. Calli under yellow plastic films started browning after 50 days in culture. Red and green films promoted while yellow inhibited regeneration of adventitious buds.4). Dark treatment could significantly affect shoot regeneration efficiency. Shoot regeneration percentage and number of shoots per explant were improved with increasing dark treatment weeks. 100% regeneration percentage and 9.05 shoots per explant were observed when a 4-week dark pretreatment was used. However, the regeneration percentage and number of shoots per explant declined when longer dark treatments were used.2. Regeneration mechanism of strawberry under different color plastic films1). A significant difference in the antioxidant enzyme activities was observed in culture of explants in vitro under color films. Changes in SOD, G-POD and CAT activities were positively correlated with shoot regeneration as compared to negative correlation between regeneration of adventitious bud and MDA. SOD, G-POD and CAT activities were highest under the red films, followed by green as compared to the lowest under yellow ones.2). Red and green plastic films induced a lower chlorophyll a/b ratio and a higher level of chlorophyll b in strawberry tissue culture. However, lowest content of chlorophyll highest chlorophyll a/b ratio was recorded for blue films.3). There was direct correlation between endogenous growth hormones contents and adventitious bud differentiation. After 15 days in culture, gibberellic acid (GA3) content was found highest under fluorescent light followed by red and green plastic films. Zeatin (ZT) and GA3 contents were highest under red plastic films; however, indole-3-acetic acid (IAA) content was highest under blue plastic films after 45 days in culture.3. Response of Toyonoka strawberry to AgN(>31). Half-strength MS containing 1.0 mg.L"1 AgNO3was an optimum medium for rooting. AgNO3 advanced root emergence and increased percent rooting, root length, dry weight and activity.2) AgNO3 had a significant stimulatory effect on chlorophyll, soluble protein contents and antioxidant enzyme activities. Chlorophyll and soluble protein contents, SOD, POD and CAT activities were increased in the presence of AgNO3 and reached maximum at 1.0 mg.L’1 AgNO3. Root water content, O2 ", MDA content, proline accumulation and

  • 【网络出版投稿人】 浙江大学
  • 【网络出版年期】2005年 06期
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