Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato

• Published in: Planta Vol. 256; no. 1; p. 14
• Main Authors: Rather, Gulzar A., Ayzenshtat, Dana, Teper-Bamnolker, Paula, Kumar, Manoj, Forotan, Zohar, Eshel, Dani, Bocobza, Samuel
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-07-2022; Springer Nature B.V
• Subjects: Agriculture; Biomedical and Life Sciences; CRISPR; Deoxyribonucleic acid; DNA; Ecology; Forestry; Genetic modification; Genome editing; Genomes; Kanamycin; Life Sciences; Linearization; Neomycin; Original Article; Plant breeding; Plant Sciences; Plant species; Polyploidy; Potatoes; Promoters; Protoplasts; Regeneration; Transcription factors; Transfection; Transgenic plants; Vegetables; Potato; Regeneration; Genome editing; promoter; Transfection; Protoplast
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/s00425-022-03933-z

Main conclusion
An efficient method of DNA-free gene-editing in potato protoplasts was developed using linearized DNA fragments,
UBIQUITIN10
promoters of several plant species, kanamycin selection, and transient overexpression of the
BABYBOOM
transcription factor.
Plant protoplasts represent a reliable experimental system for the genetic manipulation of desired traits using gene editing. Nevertheless, the selection and regeneration of mutated protoplasts are challenging and subsequent recovery of successfully edited plants is a significant bottleneck in advanced plant breeding technologies. In an effort to alleviate the obstacles related to protoplasts’ transgene expression and protoplasts’ regeneration, a new method was developed. In so doing, it was shown that linearized DNA could efficiently transfect potato protoplasts and that
UBIQUITIN10
promoters from various plants could direct transgene expression in an effective manner. Also, the inhibitory concentration of kanamycin was standardized for transfected protoplasts, and the
NEOMYCIN PHOSPHOTRANSFERASE2
(
NPT2
) gene could be used as a potent selection marker for the enrichment of transfected protoplasts. Furthermore, transient expression of the
BABYBOOM
(
BBM
) transcription factor promoted the regeneration of protoplast-derived calli. Together, these methods significantly increased the selection for protoplasts that displayed high transgene expression, and thereby significantly increased the rate of gene editing events in protoplast-derived calli to 95%. The method developed in this study facilitated gene-editing in tetraploid potato plants and opened the way to sophisticated genetic manipulation in polyploid organisms.