Keen Patricia, Hastings Amy Picard, Agrawal Anurag A, Van Eck Joyce
The Boyce Thompson Institute, Ithaca, New York.
Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, New York.
Curr Protoc Plant Biol. 2020 Mar;5(1):e20105. doi: 10.1002/cppb.20105.
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.
马利筋对于依赖它们作为生命周期一部分或全部宿主的食草昆虫具有生态意义。随着时间的推移，已经演化出了一些有趣的相互作用，其中一些尚未完全被理解。为了将这些物种开发成阐明与食草昆虫相互作用的模型，我们建立了根癌农杆菌介导的转化方法，用于处理哈氏马利筋（Hall's milkweed）、阔叶马利筋（common milkweed）和块根马利筋（butterflyweed）。该方法基于对茎节间外植体的感染，茎节间外植体比叶片外植体更易于转化。我们发现添加新制备的二硫苏糖醇对于防止茎外植体褐变至关重要。根据物种不同，从感染到转基因株系再生的时间为2至4个月。哈氏马利筋的转化效率为9%，而阔叶马利筋和块根马利筋的转化效率分别为6%和13%。© 2020约翰威立父子公司。基本方案1：根癌农杆菌介导的马利筋茎节间外植体转化 基本方案2：制备含有基因构建体的根癌农杆菌甘油菌液
