S01 - Session O1 - A marker-free cisgenesis/genome editing system, a new tool to produce fortified citrus fruits

Authors: Angelo Ciacciulli, Helena Domenica, Marco Caruso, Massimo Pindo, Stefano Piazza, Mickael Malnoy, Concetta Licciardello *

In the last few years, the varietal innovation in Citrus has been focused on fruit colour from carotenoids or their derived molecules to anthocyanins. Both carotenoids and anthocyanins are nutraceutical and antioxidant molecules, known to prevent and cure many human diseases, like cancer, obesity, cardiovascular and anti-inflammatory disorders. In particular, lycopene- (a carotenoid) and anthocyanins-rich fruits are present in the wide citrus germplasm; for example, 'Star Ruby' grapefruit and 'Moro' orange, respectively. Conventional breeding still struggles to get an accession highly enriched in both antioxidant compounds in the pulp due to several biological limits characterizing Citrus . Therefore, New Plant Breeding Techniques (cisgenesis and genome editing) intend to overcome some of the drawbacks in order to obtain these fortified fruits. The causative event for anthocyanin accumulation is represented by Ruby (MYB-like) and its Long Terminal Repeats upstream insertion. Therefore, our objective could be achieved by transferring Ruby from an anthocyanin-rich to a lycopene-rich accession. In our experimental plan, the FLP/FRT system was chosen to transfer Ruby and the cassette that contained the selection marker NptII, the Cas9 system triggered for TFL1 gene editing (to shorten the juvenility phase), and the FLP/FRT system (to excise the cassette after heat shock treatment as to produce marker-free cisgenic plants). The lycopene-rich accessions chosen to be transformed were 'Vaniglia Sanguigno' (acid-less red-orange) and 'Star Ruby' (pink grapefruit). The regenerated plantlets were mini-grafted on 'Carrizo' rootstock. PCR screening identified four positive lines of 'Star Ruby', obtained from epicotyls transformation, 32 lines of 'Vaniglia Sanguigno' (six obtained by the transformation of cotyledons and 26 of epicotyls ). These results show the potential of cisgenesis as a precise and fast approach to improve existing varieties while maintaining their characteristic quality traits, as well as bypassing the obstacles of combining characters by crossing.