A marker-free cisgenesis/genome editing system, a new tool to produce fortified citrus fruits

In the last few years, citrus varietal innovation has focused on improved fruit colour, determined by carotenoids or their derived molecules, or by anthocyanins. Both carotenoids and anthocyanins are nutraceuticals, and antioxidant molecules known to prevent human diseases. Lycopene- (a carotenoid) and anthocyanin-rich fruits are widely present among cultivars and in the citrus germplasm collections. However, conventional breeding still struggles to get an accession containing both antioxidant compounds in the pulp due to several biological limitations characterising the Citrus species. New plant breeding techniques (cisgenesis and genome editing) might overcome some limitations of traditional breeding approaches. The causative event for anthocyanin accumulation is represented by Ruby (MYB-like) and its Long-terminal Repeats upstream insertion. The aim of our work was the generation of cisgenic plants transferring Ruby from an anthocyanin-pigmented cultivar to a lycopene-pigmented one. In our experimental plan, we developed two constructs, one consisting of a cisgenic vector for the Ruby gene, and the other consisting of a Cas9 system triggered for TFL1 gene editing to shorten the juvenility phase. Both vectors contained an FLP/FRT system to excise the cassette after a heat shock treatment and obtain marker-free cisgenic plants. The lycopene-rich accessions chosen for transformation were ‘Vaniglia Sanguigno’ (acidless sweet orange) and ‘Star Ruby’ (grapefruit). The regenerated plantlets were mini-grafted onto ‘Carrizo’ rootstock. The PCR screening identified four ‘Star Ruby’ positive lines obtained from epicotyl transformation, and 32 ‘Vaniglia Sanguigno’ positive lines (six obtained by the transformation of cotyledons and 26 of epicotyls). These results showed the potential of cisgenesis as a precise and fast approach to improving the existing cultivar, while keeping the quality of a typical fruit and overcoming the obstacles of pyramiding traits by crossing.