Successful protoplast transfection in Cichorium

Cichorium intybus var. sativum (chicory) and var. foliosum (witloof) are economically important crops with a high nutritional value. Many specialized metabolites have been isolated, such as polyphenols and terpenoids, with beneficial effects on human health. Cichorium plants are rich in sesquiterpene lactones (STL) which are responsible for a bitter taste, limiting the use of these crops for industrial purposes. Editing specific genes from the STL pathway in these Cichorium species would lead to changes in the STL metabolite pathway and thus result in altered bitterness.
In this study CRISPR/Cas9 was successfully used on witloof , using protoplast transfection with plasmid DNA of a CRISPR/Cas9 vector targeting the first exon in the phytoene desaturase gene (CiPDS). CiPDS mutation frequencies of up to 20% were observed in callus-stage and albino and dwarf phenotypes were obtained. Indel Detection by Amplicon Analysis (IDAA) and Sanger sequencing revealed that mostly frameshift mutations and fragment insertions up to 153bp occurred, leading to a knock-out of the PDS gene and albino plantlets. Our developed protocol for successful protoplast transfection and regeneration in Cichorium opens opportunities to alter the STL pathway genes to change the bitterness of the crops.