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- Title
Green fluorescent protein gene as a tool to examine the efficacy of Agrobacterium-delivered CRISPR/Cas9 reagents to generate targeted mutations in the potato genome.
- Authors
Toinga-Villafuerte, Stephany; Janga, Madhusudhana R.; Isabel Vales, M.; Rathore, Keerti S.
- Abstract
CRISPR/Cas9 has emerged as a simple, yet efficient gene editing tool to generate targeted mutations in desired genes in crops plants. Agrobacterium tumefaciens, a reliable and inexpensive DNA-delivery mechanism into plant cells, has been used for the generation of CRISPR/Cas9-mediated mutations in crop plants, including potato. However, little information is available as to the progression of gene knockout during various stages of culture following the introduction of CRISPR components in this species. In the current study, the green fluorescent protein (gfp) transgene was first introduced in the genome of a potato variety, Yukon Gold. Two GFP-expressing lines, one with a single gfp copy integrated and another with four gfp copies integrated, were subjected to CRISPR/Cas9-mediated mutations in the transgene(s) using three different gRNAs. Disappearance of GFP fluorescence was monitored during the entire culture/regeneration process. Although all three gRNAs successfully knocked out the transgene(s), their efficiencies differed greatly and did not completely match the predicted scores by some guide RNA prediction tools. The nature of mutations in various knockout events was analyzed. Several lines containing four gfp-copies showed four different types of mutations. These findings suggest that it is possible to target all four alleles of a desired native gene in the tetraploid potato. Key message: Potato lines, with a single or four copies of the gfp transgene integrated, were subjected to CRISPR/Cas9-mediated mutations. The results indicated that it should be possible to target and knock out multiple copies of a native gene in the tetraploid genotypes of this crop.
- Subjects
GREEN fluorescent protein; PLANT mutation; CRISPRS; GENOMES; CROPS; POTATO diseases &; pests; POTATOES
- Publication
Plant Cell, Tissue & Organ Culture, 2022, Vol 150, Issue 3, p587
- ISSN
0167-6857
- Publication type
Article
- DOI
10.1007/s11240-022-02310-8