Agrobacterium-Mediated Genetic Transformation of Wild Oryza Species Using Immature Embryos
Agrobacterium-Mediated Genetic Transformation of Wild Oryza Species Using Immature Embryos
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DOI：
10.1186/s12284-020-00394-4
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发表时间：
2020-06-03
期刊：
影响因子：
5.5
通讯作者：
Sato, Yutaka
中科院分区：
文献类型：
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作者：
Shimizu-Sato, Sae;Tsuda, Katsutoshi;Sato, Yutaka
遗传转化是揭示或调节基因功能的最重要技术之一。它广泛应用于水稻功能基因组学和分子育种。由于野生稻种的遗传多样性很高，对其使用的需求正在增加。鉴于远缘物种之间遗传杂交的困难，遗传转化提供了一种改变或转移野生稻种质遗传性状的方法。然而，使用从成熟种子中的胚盾片组织诱导的愈伤组织通过常规方法转化野生稻种质常常失败。在这里，我们报告了使用未成熟胚胎对多种稻物种进行遗传转化的方法。首先，我们研究了 20 个物种的 192 个种质的未成熟胚在多种培养条件下诱导愈伤组织和再生的能力。我们从 16 个物种的 90 个种质的未成熟胚中再生出植物。接下来，我们使用携带由玉米泛素启动子驱动的 GFP 基因的载体优化了农杆菌感染的条件。在 11 个物种的 51 个种质中观察到 GFP 信号。我们分析了 O. barthii、O. glumaepatula、O. rufipogon 和 O. brachyantha 转基因植物的生长和种子集。植物生长成熟并正常结籽。使用来自 T-0 植物的 DNA 进行 Southern 印迹分析表明，所有 GFP 植物均源自独立的转化事件。我们证实T-DNA通过T-1代中GFP信号的分离传递到下一代。这些结果表明许多稻种可以通过使用改良的未成熟胚方法进行转化。这将加速野生稻种质在分子遗传分析和分子育种中的应用。
Genetic transformation is one of the most important technologies for revealing or modulating gene function. It is used widely in both functional genomics and molecular breeding of rice. Demands on its use in wild Oryza species is increasing because of their high genetic diversity. Given the difficulties in genetic crosses between distantly related species, genetic transformation offers a way to alter or transfer genetic traits in wild rice accessions. However, transformation of wild Oryza accessions by conventional methods using calli induced from scutellum tissue of embryos in mature seeds often fails. Here, we report methods using immature embryos for the genetic transformation of a broad range of Oryza species. First, we investigated the ability of callus induction and regeneration from immature embryos of 192 accessions in 20 species under several culture conditions. We regenerated plants from immature embryos of 90 accessions in 16 species. Next, we optimized the conditions of Agrobacterium infection using a vector carrying the GFP gene driven by the maize ubiquitin promoter. GFP signals were observed in 51 accessions in 11 species. We analyzed the growth and seed set of transgenic plants of O. barthii, O. glumaepatula, O. rufipogon, and O. brachyantha. The plants grew to maturity and set seeds normally. Southern blot analyses using DNA from T-0 plants showed that all GFP plants were derived from independent transformation events. We confirmed that the T-DNAs were transmitted to the next generation through the segregation of GFP signals in the T-1 generation. These results show that many Oryza species can be transformed by using modified immature-embryo methods. This will accelerate the use of wild Oryza accessions in molecular genetic analyses and molecular breeding.