Protoplast-Based Method for Genome Editing in Tetraploid Potato.

Area of Science:

• Plant genetics
• Genome editing technologies
• Agricultural biotechnology

Background:

• Cultivated potato is tetraploid with four gene loci, often genetically heterogeneous.
• Clonal propagation maintains desirable genetic traits but complicates homozygous edits.
• Achieving edits in all four loci is essential to avoid inbreeding depression and facilitate genetic improvement.

Purpose of the Study:

• To establish an efficient method for genome editing in potato protoplasts.
• To enable simultaneous mutation introduction across all four gene loci in tetraploid potato.
• To avoid stable DNA integration and reduce the risk of inbreeding depression.

Main Methods:

• Transient transfection of potato protoplasts.
• Delivery of genome editing tools via DNA vectors encoding TALEN, Cas9, or Cas9 deaminase and sgRNA.
• Delivery of genome editing tools via in vitro-produced ribonucleoprotein (RNP) complexes.

Main Results:

• The developed protocol is effective for introducing mutations using both DNA vectors and RNP complexes.
• Transient transfection of protoplasts allows for efficient genome editing in potato.
• The method facilitates the introduction of edits in all four loci of target genes.

Conclusions:

• Protoplast-based transient transfection is a highly efficient strategy for genome editing in tetraploid potato.
• This approach enables rapid generation of homozygous edited lines, accelerating potato breeding.
• The method avoids foreign DNA insertion, making it attractive for genetic improvement.