ArticleMolecular biology reports2022
Exploring the Agrobacterium-mediated transformation with CRISPR/Cas9 in cucumber (Cucumis sativus L.).
Article in Molecular biology reports, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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5 citing papers in PubMed, 18 citations in OpenAlex.
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- TALE gene family: identification, evolutionary and expression analysis under various exogenous hormones and waterlogging stress in Cucumis sativus L.BMC plant biology · 2024Article
- ImprovingGenes · 2023Article
- A highly efficient genetic transformation system for broccoli and subcellular localization.Frontiers in plant science · 2023Article
- The Phosphoserine Phosphatase Alters the Free Amino Acid Compositions and Fecundity inInternational journal of molecular sciences · 2022Article
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Authors and funding
9 authors at 6 institutions in 1 country.
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Abstract
backgroundsThe narrow genetic basis of cucumber makes breeding of this species difficult. CRISPR/Cas9 system is characteristic of simple design, low cost and high efficiency, which has opened a new path for cucumber functional genetics and the development of cucumber mocular breeding. However, the immature genetic transformation system is the main limiting factor for applying this technology in cucumber. METHODS AND
resultsIn this study, a Histochemical β-glucuronidase (GUS) assay was used to analyze the effect of various parameters, including slight scratch of explants, pre-culture time, acetosyringone (AS) concentration, infection time in Agrobacterium solution, and co-culture period on the transformation efficiency. The results showed that the explants slightly scratched after cutting, pre-cultured for 1 day, Agrobacterium bacterial solution containing AS, and 20 min length of infection could significantly increase the GUS staining rate of explants. On this basis, two sequences with high specificity (sgRNA-1 and sgRNA-2) targeted different loci of gene CsGCN5 were designed. The corresponding vectors Cas9-sgRNA-1 and Cas9-sgRNA-2 were constructed and transformed using the above-optimized cucumber genetic transformation system, and three and two PCR positive lines were obtained from 210 and 207 explants, respectively. No sequence mutation at target loci of CsGCN5 was detected in the Cas9-sgRNA-1 transformed three PCR positive lines. However, one mutant line with targeted homozygous change was recognized from the Cas9-sgRNA-2 transformed two PCR positive lines.
conclusionIn this study, 2.4‰ of total explants had directed mutation in the CsGCN5 gene. The results in the present study would be beneficial to further optimize and improve the efficiency of the genetic transformation of cucumber.
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