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.
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Acknowledgements
We thank members of the Park Lab, especially, Young-suk La, You-sun Chae for plant generation and Sujeevan Rajendran for plant care. We also thank NIBR for providing all S. nigrum seeds.
Funding
This research was supported by a grant from ‘the Next-Generation BioGreen 21 Program (SSAC, PJ0134212019; PMBC, PJ0133032019) funded by Rural Development Administration’ to S.J.P., ‘the National Research Foundation of Korea (NRF, 2017R1A4A1015594) funded by the Korean Ministry of Science, ICT and Future Planning’ to D.H.K., and ‘National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE, NIBR201921101)’ to W.Y.B.
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11816_2020_616_MOESM1_ESM.jpg
Supplementary Figure 1. Time period for generating S. nigrum transgenic plants on the medium. Representative regeneration stages of S. nigrum cotyledon explants with time scales from seed germination, according to the transgenic processing steps. Notably, 97% of 100 explants grew on selection media (kanamycine 100 mg/L) and all of the explants regenerated shoots. PCM, Scale bar, 1 cm. PCM, Preculture and Co-cultivation Medium; SM, Shooting Medium; RM, Rooting Medium (JPG 1910 kb)
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Park, S., Lee, E., Heo, J. et al. Rapid generation of transgenic and gene-edited Solanum nigrum plants using Agrobacterium-mediated transformation. Plant Biotechnol Rep 14, 497–504 (2020). https://doi.org/10.1007/s11816-020-00616-7
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DOI: https://doi.org/10.1007/s11816-020-00616-7