Genome Instability in Solanum tuberosum Plants Regenerated from Protoplast

Most examples of crop genome modification through transgene and genome editing involve the regeneration of whole plants from callus derived from explants or protoplasts. Interest in the latter has spiked because delivery of ribonucleoproteins (RNP) made of Cas9 and guide RNA can modify a genomic target without the use of transgenes. This can make regulatory approval less complicated. A drawback of this system, however, is somaclonal variation. Somaclonal variation is a syndrome entailing the appearance of novel and unexpected phenotypes in plants regenerated from callus in tissue culture. Historical cytological investigations of somaclonal variants reported frequent aneuploidy and possible chromosomal changes. Before applying the RNP editing technique in protoplasts, it is important to fully understand if and how genomic instability may result. We have established a protoplast regeneration platform in a tetraploid S. tuberosum variety, demonstrating strong genotype dependency in the efficiency of regeneration. To elucidate the impact of genome structural variation on this system, we analyzed 15 potato plants regenerated from protoplasts comparing them to 8 propagated cutting controls. Plant DNA was subject to Illumina sequencing and resulting mapped reads were counted by chromosomal bin providing dosage. Deviations from the expected four copies were evaluated by plotting the bin counts by chromosomes. All regenerated plants analyzed displayed some genome alteration regardless of whether their phenotype appeared normal or altered. In addition to aneuploidy, we also observed segmental deletion and duplications, chromothripsis-like changes and chimerism. All chromosomes could be affected but some displayed higher frequency of changes. This variation, observed by sequence-based chromosome dosage profiles, greatly exceeds what was originally anticipated, validating the importance of considering such analysis when generating plants from protoplasts, as can be done with gene editing via RNPs.