农杆菌介导的三种乳草(夹竹桃科)的转化

Q1 Agricultural and Biological Sciences Current protocols in plant biology Pub Date : 2020-03-09 DOI:10.1002/cppb.20105

Patricia Keen, Amy Picard Hastings, Anurag A. Agrawal, Joyce Van Eck

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引用次数: 1

摘要

乳草对依赖它们作为部分或全部生命周期宿主的食草昆虫具有重要的生态意义。有趣的相互作用，其中一些还没有完全理解，随着时间的推移而进化。为了开发这些物种作为模型来阐明与昆虫食草动物的相互作用，我们建立了根癌农杆菌介导的Asclepias hallii (Hall’s马利筋)、A. syriaca(普通马利筋)和A. tuberosa(蝴蝶草)的转化方法。该方法基于茎节间外植体的侵染，茎节间外植体比叶外植体更容易转化。我们发现添加新鲜制备的二硫苏糖醇对防止茎外植体褐变至关重要。根据品种的不同，从感染到转基因株系再生的时间为2至4个月。鸢尾草的转化效率为9%，而叙利亚蒿和晚熟鸢尾草的转化效率分别为6%和13%。©2020 by John Wiley &基本方案1:农杆菌介导的Asclepias结间茎外植体的转化基本方案2:制备含有基因构建物的农杆菌甘油储备

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Agrobacterium tumefaciens–Mediated Transformation of Three Milkweed Species (Asclepias hallii, A. syriaca, and A. tuberosa: Apocynaceae)

Milkweeds have ecological significance for insect herbivores that rely on them as hosts for either part of or the entirety of their life cycles. Interesting interactions, some of which are not completely understood, have evolved over time. To develop these species as models to elucidate the interplay with insect herbivores, we established Agrobacterium tumefaciens–mediated transformation approaches for Asclepias hallii (Hall's milkweed), A. syriaca (common milkweed), and A. tuberosa (butterflyweed). The method is based on infection of stem internodal explants, which were more amenable to transformation than leaf explants. We found that addition of freshly prepared dithiothreitol was critical to prevent browning of stem explants. Depending on the species, the time from infection to the regeneration of transgenic lines ranges from 2 to 4 months. Transformation efficiency for A. hallii was 9%, whereas efficiencies for A. syriaca and A. tuberosa were 6% and 13%, respectively. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Agrobacterium tumefaciens–mediated transformation of Asclepias internodal stem explants

Basic Protocol 2: Preparation of Agrobacterium glycerol stocks containing gene constructs

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来源期刊

Current protocols in plant biology Agricultural and Biological Sciences-Plant Science

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. Current Protocols in Plant Biology provides reproducible step-by-step instructions for protocols relevant to plant research. Furthermore, Current Protocols content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols in Plant Biology to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Plant Biology is the comprehensive source for protocols in the multidisciplinary field of plant biology, providing an extensive range of protocols from basic to cutting edge. Coverage includes: Extraction and analysis of DNA, RNA, proteins Chromosome analysis Transcriptional analysis Protein expression Metabolites Plant enzymology Epigenetics Plant genetic transformation Mutagenesis Arabidopsis, Maize, Poplar, Rice, and Soybean, and more.