The Italian experience on the use of new breeding technologies in citrus: advanced and marker-free approaches to improve fruit quality and resistance to plant diseases

Citrus improvement by conventional breeding is hampered by several factors, such as the high level of heterozygosity, polyembryony, partial or complete sterility, sexual (in)compatibility, and the long juvenility that imposes around 5 to 10 years to produce fruits. New breeding technologies (NBTs) might represent a complementary strategy for the development of improved new cultivars. However, there is still poor knowledge of genes controlling important horticultural traits and on the capacity to transform most of the cultivars of interest. The cisgenesis of Ruby (causative element for anthocyanin accumulation in citrus fruits) and the genome editing of β-LCY2 (converting lycopene in β-carotene) were successfully used at CREA (Italy) to improve citrus fruit quality, with the aim of enriching sweet orange and grapefruit cultivars in antioxidant compounds. The produced plants are marker-free thanks to the use of the FRT/Flp recombinase system and VvMYBA1 gene as phenotypic marker. Moreover, CRISPR/Cas9 was used to modify seed growth and development (to obtain seedless cultivars) and to speed up flowering. For most of the genotypes, the regeneration protocols were duly optimized considering different sources of material, either seeds, nucellar seedlings, and internodes of juvenile or mature tissues. The application of NBTs was possible thanks to the long history and experience in clonal selection and the large citrus germplasm available at CREA and Catania University, which offered a wide range of possibilities to select the best cultivar to improve. The expertise gained in the last 5 years and the new protocols developed for the NBTs applications on citrus are still employed for the improvement of new citrus quality traits and could now be used to generate resistant cultivars to diseases such as Huanglongbing and Alternaria brown spot.