In this study, genetic engineering methods, ranging from gene transformation to gene editing, were efficiently conducted on cotyledon explants in Solanum nigrum. Organogenic calli were observed on the explants following 10 days of growth on medium supplemented with 2 mg/L zeatin and 0.2 mg/L indole acetic acid (IAA), which we suggest to be an efficient shoot initiation medium (SM1). In vitro shoot production was enhanced in explants grown on media supplemented with 1 mg/L zeatin and 0.1 mg/L IAA, which we used as a proper shoot differentiation medium (SM2) in S. nigrum shoot regeneration. Direct infection of explants with Agrobacterium harboring CRISPR/Cas9 constructs can simplify the method of Agrobacterium-mediated T-DNA transformation by skipping the preincubation step. The method was applied to deliver transgenes for genome editing, such as CRISPR/Cas9 DNA. SnLazy1 locus, considered to be orthologs of tomato Lazy1, was edited using the CRISPR/Cas9 system in S. nigrum. Two independent deletion snlazy1-cr alleles were successively inherited and showed stem growth in a relatively downward direction. Our method offers rapid and simple procedure for gene transformation and genome editing that could be applicable for the enhancement of beneficial traits for precision plant breeding and engineering quantitative trait loci in S. nigrum.

中文翻译：

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利用农杆菌介导的转化快速产生转基因和基因编辑的龙葵

在这项研究中，有效地对茄茄子叶外植体进行了从基因转化到基因编辑的基因工程方法。在补充了2 mg / L玉米素和0.2 mg / L吲哚乙酸（IAA）的培养基上生长10天后，在外植体上观察到器官发生的愈伤组织，我们建议这是一种有效的芽萌生培养基（SM1）。在补充了1 mg / L玉米素和0.1 mg / L IAA的培养基上生长的外植体中，外植体的芽生长得以增强，我们将其用作黑链霉菌芽再生的合适芽分化培养基（SM2）。用带有CRISPR / Cas9构建体的农杆菌直接感染外植体可以简化农杆菌的方法-通过跳过预温育步骤介导的T-DNA转化。该方法已应用于传递用于基因组编辑的转基因，例如CRISPR / Cas9 DNA。SnLazy1轨迹，认为是直向同源物番茄Lazy1，使用CRISPR / Cas9系统中被编辑龙葵。两个独立的删除snlazy1-cr等位基因被连续遗传，并显示茎生长在相对向下的方向。我们的方法提供了快速，简单的基因转化和基因组编辑程序，可用于增强黑链霉菌的精确育种和工程数量性状位点的有益性状。