Molecular Cell Biology, Department of Biology, Lund University, Sölvegatan 35, 223 62 Lund, Sweden.
Cells. 2022 Mar 22;11(7):1068. doi: 10.3390/cells11071068.
(Camelina) is an oilseed crop that in recent years has gained importance due to its closeness to the plant model organism (Arabidopsis), its low agronomical requirements, and the ability to grow under temperate conditions. To explore all the agronomical and biotechnological possibilities of this crop, it is important to evaluate the usability of the molecular procedures currently available for plants. One of the main tools for plant genetic modification and genetic studies is stable plant transformation. In the case of Arabidopsis, as well as Camelina, floral dipping is the easiest and most used method, which is followed by a selection for stable transformants. Commonly used selection methods for Camelina involve sp. red protein (DsRed) fluorescence screening. However, many widely used plant transformation vector systems, for example those used in Arabidopsis and grasses, rely on antibiotic resistance selection. In this study, we evaluated the usability of different antibiotics including kanamycin (Kan), hygromycin (Hyg) and BASTA, and propose optimised protocols for selecting T1 and subsequent generation Camelina transformants, as well as crossing of Camelina lines expressing different transgenes. Finally, we also showed that overexpression of genes encoding enzymes from the seco-iridoid pathway of using Hyg or BASTA-based expression constructs could be successfully achieved in Camelina, demonstrating the potential of these methods for metabolic engineering. Overall, in this study we show an efficient way to sterilize seeds, handle and perform selection of Camelina for use with transformation vectors designed for . We also demonstrate a successful method to cross and provide qRT-PCR results to prove its effectiveness.
荠蓝是一种油料作物，近年来由于其与植物模式生物拟南芥的接近性、低农业要求以及在温带条件下生长的能力而变得越来越重要。为了探索该作物的所有农业和生物技术可能性，评估当前可用于植物的分子程序的可用性非常重要。植物遗传修饰和遗传研究的主要工具之一是稳定的植物转化。在拟南芥和荠蓝的情况下，花浸是最简单和最常用的方法，随后是对稳定转化体的选择。荠蓝常用的选择方法涉及 sp 红色蛋白（DsRed）荧光筛选。然而，许多广泛使用的植物转化载体系统，例如在拟南芥和禾本科植物中使用的系统，依赖于抗生素抗性选择。在这项研究中，我们评估了不同抗生素的可用性，包括卡那霉素（Kan）、潮霉素（Hyg）和 BASTA，并提出了优化的 T1 和后续世代荠蓝转化体选择以及表达不同转基因的荠蓝系杂交的方案。最后，我们还表明，使用 Hyg 或 BASTA 为基础的表达构建体表达来自 secoiridoid 途径的基因编码酶可以在荠蓝中成功实现，这证明了这些方法在代谢工程中的潜力。总的来说，在这项研究中，我们展示了一种有效的荠蓝种子消毒、处理和转化载体选择方法，这些方法专为拟南芥设计。我们还成功地展示了一种交叉荠蓝的方法，并提供了 qRT-PCR 结果来证明其有效性。
