利用玉米 B104 未成熟胚胎的农杆菌介导快速转化法

Cold Spring Harbor protocols Pub Date : 2024-09-19 DOI:10.1101/pdb.prot108595

Minjeong Kang, Mercy K Azanu, Keunsub Lee, Kan Wang

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引用次数: 0

摘要

玉米基因转化是功能基因组学和作物改良的重要工具。然而，许多实验室在尝试实现玉米近交系基因型的常规遗传转化时仍然面临着多重挑战。在这里，我们介绍了一种使用未成熟胚胎作为外植体的快速、稳健的玉米 B104 转化方法。该方法使用农杆菌三元载体系统，其中包括一个传统的 T-DNA 二元载体（pCBL101-RUBY）和一个兼容的三元辅助质粒（pKL2299），后者携带额外拷贝的重要毒力基因。T-DNA 双元载体携带用于选择的新霉素磷酸转移酶 II (NptII) 基因和用于视觉筛选的甜菜碱生物合成标记 RUBY。我们提供了关于未成熟胚外植体制备、农杆菌感染、组织培养程序以及再生小植株温室适应性养护的逐步说明。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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A Rapid Agrobacterium-Mediated Transformation Method Using Maize B104 Immature Embryos.

Maize genetic transformation is a critical tool for functional genomics and crop improvement. Many laboratories, however, continue to face multiple challenges in attempting to achieve routine genetic transformation of maize inbred genotypes. Here, we describe a rapid and robust maize B104 transformation method using immature embryos as explants. This method uses an Agrobacterium ternary vector system, which includes a conventional T-DNA binary vector (pCBL101-RUBY) and a compatible ternary helper plasmid (pKL2299) that carries extra copies of essential virulence genes. The T-DNA binary vector carries the neomycin phosphotransferase II (NptII) gene for selection and a betalain biosynthesis marker, RUBY, for visual screening. We provide step-by-step instructions for immature embryo explant preparation, Agrobacterium infection, tissue culture procedures, and greenhouse care for acclimatization of regenerated plantlets.

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来源期刊

Cold Spring Harbor protocols Biochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (all)

CiteScore

3.00

自引率

0.00%

发文量

163

期刊介绍： Cold Spring Harbor Laboratory is renowned for its teaching of biomedical research techniques. For decades, participants in its celebrated, hands-on courses and users of its laboratory manuals have gained access to the most authoritative and reliable methods in molecular and cellular biology. Now that access has moved online. Cold Spring Harbor Protocols is an interdisciplinary journal providing a definitive source of research methods in cell, developmental and molecular biology, genetics, bioinformatics, protein science, computational biology, immunology, neuroscience and imaging. Each monthly issue details multiple essential methods—a mix of cutting-edge and well-established techniques.