Abstract

Waterhemp (Amaranthus tuberculatus (Moq.) Sauer) is one of the most problematic weeds in the Midwestern agricultural systems in the USA. As a consequence of extensive herbicide selection pressure, many waterhemp populations across the USA evolved resistance to multiple herbicides, leading to limited options for chemical control. Genome editing tools, including CRISPR/Cas9, have great potential in weed science, especially when coupled with a gene drive system for a sustainable weed management. The application of modern biotechnological tools requires availability of an efficient regeneration and genetic transformation system. The objectives of this research were to develop an efficient protocol for successful regeneration of waterhemp via callus culture and optimize an Agrobacterium-mediated transformation method for waterhemp. Hypocotyl explants cultured on callus induction medium (CIM), C6 containing 1.0 mg L⁻¹ kinetin and 4.0 mg L⁻¹ 2,4-D for 2 wk followed by subculture into shoot induction medium (SIM), S8 containing 3.0 mg L⁻¹ BAP, 0.05 mg L⁻¹ 2,4-D, and 0.3 mg L⁻¹ GA₃ for 4 to 6 wk was successful in shoot regeneration. Leaf explants failed to respond to any treatment combinations tested for regeneration. Using the hypocotyl explant–based regeneration protocol optimized in this research, Agrobacterium-mediated transformation was performed using uidA and hpt genes as a proof of concept. Although transgenic plants were not regenerated, molecular characterization of the transformed calluses confirmed the presence of transgenic genes, for example uidA and hpt. This is the first report of the tissue-cultured-based regeneration and Agrobacterium-mediated transformation of hypocotyl derived calluses in waterhemp, which is a step forward in exploring the possibilities of genome editing research in weed science.

中文翻译：

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水大麻（Amaranthus tuberculatus）基于下胚轴的再生和愈伤组织转化的第一个方案的建立

摘要

水麻（Amaranthus tuberculatus (Moq.) Sauer）是美国中西部农业系统中最有问题的杂草之一。由于广泛的除草剂选择压力，美国许多水麻种群对多种除草剂产生了抗药性，导致化学防治的选择有限。包括 CRISPR/Cas9 在内的基因组编辑工具在杂草科学领域具有巨大潜力，特别是与基因驱动系统结合使用以实现可持续杂草管理。现代生物技术工具的应用需要有效的再生和遗传转化系统。本研究的目的是开发一种通过愈伤组织培养成功再生水麻的有效方案，并优化农杆菌介导的水麻转化方法。下胚轴外植体在愈伤组织诱导培养基（CIM）上培养，C6含有1.0 mg L ^-1激动素和4.0 mg L ^-1 2,4-D 2周，然后传代培养到芽诱导培养基（SIM）中，S8含有3.0 mg L ^{- 1} BAP、0.05 mg L ^-1 2,4-D和0.3 mg L ^-1 GA ₃ 4至6周成功地再生芽。叶外植体未能对再生测试的任何处理组合做出反应。使用本研究中优化的基于下胚轴外植体的再生方案，使用uidA和hpt基因作为概念证明进行农杆菌介导的转化。虽然转基因植物没有再生，但转化愈伤组织的分子表征证实了转基因基因的存在，例如uidA和hpt。这是第一份关于水麻下胚轴愈伤组织的组织培养再生和农杆菌介导转化的报告，这是探索杂草科学基因组编辑研究可能性的一步。