Colour change in potato (Solanum tuberosum L.) tubers by disruption of the anthocyanin pathway via ribonucleoprotein complex delivery of the CRISPR/Cas9 system

Potato is an important part of the traditional Norwegian diet, and the crop faces several challenges with respect to pests and diseases, as well as the increasingly challenging changes in climate. Genome editing may provide tools to improve the resilience of Norwegian potato cultivars to new climate challenges. We have altered the skin colour of two potato cultivars, ‘Desirée’ and ‘Nansen’ from red to yellow, as a proof-of-concept for the use of CRISPR/Cas9 in a Norwegian cultivar. Our method has involved the use of protoplasts and we have grown the regenerants for three successive clonal tuber generations to evaluate the stability of the edited plants over time and under varying temperature conditions in contained rooms in a greenhouse. We found that the protoplast method is well suited to achieving CRISPR/Cas9 applications. The results show that the yellow skin is consistent over the three generations of tuber propagation. We found some suspected somaclonal variation in the protoplast regenerants. Some of the variation which we observed under high temperatures (up to nearly 40ºC) during the second growth cycle, disappeared when cultivated under lower temperatures in the third cultivation cycle. Protoplast derived potato obtained RNA knock-outs by CRISPR of F3H in a ribonucleoprotein complex to interrupt with the anthocyanin biosynthesis. The analysis of phenotypes revealed knock-outs in 1–4 alleles. We followed three successive tuber generations, and revealed change of skin colour and how temperature influenced the tuber appearances, as well as some somaclonal variation. Without trials in three tuber generations, we would not have picked up on the influence of temperature on the tubers.