高效的农杆菌介导紫花苜蓿遗传转化体系的建立
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摘要
紫花苜蓿是世界上栽培最早、分布最广的优质豆科牧草之一,其干草产量高、营养成分完全,堪称“牧草之王”,在畜牧业中占有不容忽视的重要地位。近年来,我国越来越严重的土壤盐碱化、荒漠化、干旱缺水以及病虫害的频繁发生都极大程度地影响了苜蓿的产量和品质,南方地区标志性的酸性土壤也限制了苜蓿的引种栽培。此外,紫花苜蓿一直面临着营养品质问题如干物质可消化性低、家畜采食苜蓿后易患膨胀病等。这都要求人们培育出抗逆性强、品质好的苜蓿新品种。
传统育种方法复杂、周期长、且育种资源少。近年来,组织培养和遗传转化技术的迅速发展为人们提供了一种方便快捷的紫花苜蓿新品种培育方法。自从1986年的首例苜蓿转基因成功以来,苜蓿遗传转化的研究已取得一定的进展。但现有的紫花苜蓿遗传转化体系转化效率很低,限制了优良外源基因对苜蓿的转化,从而限制了优良苜蓿新品种的获得。
本研究首次以农艺性状优良的紫花苜蓿引进栽培种“德宝”为转化受体,将兼具选择标记和目的基因双重功能的bar基因,采用目前苜蓿遗传转化中使用最多、效果最好、转化机理最清楚的农杆菌介导法转化到受体中,从外植体选择、bialaphos筛选、共培养时间、农杆菌侵染浓度等因素进行探索,优化苜蓿遗传转化体系,最终建立了高效的苜蓿再生体系和农杆菌介导的转基因体系。主要研究结果如下：
1.子叶是德宝苜蓿最好的外植体,其愈伤诱导率最高,可达97%,最佳bialaphos筛选浓度为2.5mg/L,子叶最佳预培养时间为3天,最适农杆菌菌液侵染浓度为OD600值0.5-0.7,8-10min是子叶的最佳农杆菌侵染时间,2-3天为子叶的最佳共培养时间,抗性愈伤率最高可达30.5%。
2.在含有bialaphos培养基上经过反复筛选共计获得100余株T0代独立的转化植株。
3.利用PCR方法对随机选取的50株T0代转基因植株的bar基因进行扩增,再生植株全部为阳性转基因植株。
4.对PCR阳性植株和野生型植株叶片喷施5‰除草剂Basta溶液,阳性植株全部表现为除草剂抗性,进一步证实了我们建立的苜蓿遗传转化体系是高效的、可靠的,这将为苜蓿新材料创新打下坚实的基础。
Alfalfa (Medicago sativa) is one of most important legume forage. Because of the high forage yield and comprehensive nutrition, it is called the "the king among forages" and acts a very important role in animal husbandry all over the world, as well as in China. However, in current, China increasingly serious soil salinization, desertification, drought and the frequent pests and diseases have greatly affected the yield and quality of alfalfa, the acid soils in South china also limit alfalfa introduction and cultivation, at the same time, many nutritional quality problems of alfalfa such as low dry matter digestibility and animal tympanites yet not be improved. Therefore, to cultivate well resistance and high quality new varieties of alfalfa has an important significance.
The traditional breeding methods need long period, and are lack of breeding resources. In recent years, tissue culture and genetic transformation technology have provided a convenient method of cultivating new varieties of alfalfa. Since the first success of genetically modified alfalfa in 1986, many alfalfa genetic transformations have been reported. However, the existing system of genetic transformation of alfalfa showed lower transformation efficiency, limiting the quality of foreign gene in the transformation of alfalfa, thus limiting access to superior new varieties of alfalfa.
In this study, an alfalfa cultivar "Derby" with important agronomic traits is used as the recipient for transformation, the selectable marker and target genes both dual function bar gene, using the Agrobacterium-mediated methods which is commonly used in genetic transformation of alfalfa. The explant selection, antibiotic screening, culture time, the concentration of Agrobacterium infection solution and other factors were tested. Finally, an efficient alfalfa regeneration and transformation system was established. The major findings are as follows:
1). Cotyledon was the optimal transformation explant, which has the highest callus induction rate, up to 97%. The best bialaphos screening concentration is 2.5mg/L, the best pre-culture time is 3 days, the optimal Agrobacterium strain OD6oo value of liquid infection is 0.5-0.7,8-10 min is the best time for Agrobacterium infection to cotyledon, and reasonable co-culture time is 2-3 days. From above system, the highest rate of resistant callus is up to 30.5%.
2). After repeated screening in the medium containing bialaphos, about 100 lines of To resistant plants was obtained.
3). The resistant plants were detected by PCR, the result suggests that all of the resistant plants were positive transgenic plants.
4). The regeneration plants and wild-type plants leaves were sprayed with herbicide Basta 5%o solution, all of the PCR positive plants showed resistance to herbicide, further indicating our alfalfa transformation system is efficient and reliable. This study laid a solid foundation for alfalfa materials innovation.
传统育种方法复杂、周期长、且育种资源少。近年来,组织培养和遗传转化技术的迅速发展为人们提供了一种方便快捷的紫花苜蓿新品种培育方法。自从1986年的首例苜蓿转基因成功以来,苜蓿遗传转化的研究已取得一定的进展。但现有的紫花苜蓿遗传转化体系转化效率很低,限制了优良外源基因对苜蓿的转化,从而限制了优良苜蓿新品种的获得。
本研究首次以农艺性状优良的紫花苜蓿引进栽培种“德宝”为转化受体,将兼具选择标记和目的基因双重功能的bar基因,采用目前苜蓿遗传转化中使用最多、效果最好、转化机理最清楚的农杆菌介导法转化到受体中,从外植体选择、bialaphos筛选、共培养时间、农杆菌侵染浓度等因素进行探索,优化苜蓿遗传转化体系,最终建立了高效的苜蓿再生体系和农杆菌介导的转基因体系。主要研究结果如下：
1.子叶是德宝苜蓿最好的外植体,其愈伤诱导率最高,可达97%,最佳bialaphos筛选浓度为2.5mg/L,子叶最佳预培养时间为3天,最适农杆菌菌液侵染浓度为OD600值0.5-0.7,8-10min是子叶的最佳农杆菌侵染时间,2-3天为子叶的最佳共培养时间,抗性愈伤率最高可达30.5%。
2.在含有bialaphos培养基上经过反复筛选共计获得100余株T0代独立的转化植株。
3.利用PCR方法对随机选取的50株T0代转基因植株的bar基因进行扩增,再生植株全部为阳性转基因植株。
4.对PCR阳性植株和野生型植株叶片喷施5‰除草剂Basta溶液,阳性植株全部表现为除草剂抗性,进一步证实了我们建立的苜蓿遗传转化体系是高效的、可靠的,这将为苜蓿新材料创新打下坚实的基础。
Alfalfa (Medicago sativa) is one of most important legume forage. Because of the high forage yield and comprehensive nutrition, it is called the "the king among forages" and acts a very important role in animal husbandry all over the world, as well as in China. However, in current, China increasingly serious soil salinization, desertification, drought and the frequent pests and diseases have greatly affected the yield and quality of alfalfa, the acid soils in South china also limit alfalfa introduction and cultivation, at the same time, many nutritional quality problems of alfalfa such as low dry matter digestibility and animal tympanites yet not be improved. Therefore, to cultivate well resistance and high quality new varieties of alfalfa has an important significance.
The traditional breeding methods need long period, and are lack of breeding resources. In recent years, tissue culture and genetic transformation technology have provided a convenient method of cultivating new varieties of alfalfa. Since the first success of genetically modified alfalfa in 1986, many alfalfa genetic transformations have been reported. However, the existing system of genetic transformation of alfalfa showed lower transformation efficiency, limiting the quality of foreign gene in the transformation of alfalfa, thus limiting access to superior new varieties of alfalfa.
In this study, an alfalfa cultivar "Derby" with important agronomic traits is used as the recipient for transformation, the selectable marker and target genes both dual function bar gene, using the Agrobacterium-mediated methods which is commonly used in genetic transformation of alfalfa. The explant selection, antibiotic screening, culture time, the concentration of Agrobacterium infection solution and other factors were tested. Finally, an efficient alfalfa regeneration and transformation system was established. The major findings are as follows:
1). Cotyledon was the optimal transformation explant, which has the highest callus induction rate, up to 97%. The best bialaphos screening concentration is 2.5mg/L, the best pre-culture time is 3 days, the optimal Agrobacterium strain OD6oo value of liquid infection is 0.5-0.7,8-10 min is the best time for Agrobacterium infection to cotyledon, and reasonable co-culture time is 2-3 days. From above system, the highest rate of resistant callus is up to 30.5%.
2). After repeated screening in the medium containing bialaphos, about 100 lines of To resistant plants was obtained.
3). The resistant plants were detected by PCR, the result suggests that all of the resistant plants were positive transgenic plants.
4). The regeneration plants and wild-type plants leaves were sprayed with herbicide Basta 5%o solution, all of the PCR positive plants showed resistance to herbicide, further indicating our alfalfa transformation system is efficient and reliable. This study laid a solid foundation for alfalfa materials innovation.
引文
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