Rapid generation of transgenic and gene-edited Solanum nigrum plants using Agrobacterium-mediated transformation

Abstract

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.