New plant breeding techniques (NPBTs) aim to overcome traditional breeding limits for fruit tree species, in order to obtain new varieties with improved organoleptic traits and resistance to biotic and abiotic stress, and to maintain fruit quality achieved over centuries by (clonal) selection. Knowledge on the gene(s) controlling a specific trait is essential for the use of NPBTs, such as genome editing and cisgenesis. In the framework of the international scientific community working on fruit tree species, including citrus, NPBTs have mainly been applied to address pathogen threats. Citrus could take advantage of NPBTs because of its complex species biology (seedlessness, apomixis, high heterozygosity, and long juvenility phase) and aptitude for in vitro manipulation. To our knowledge, genome editing in citrus via transgenesis has successful for induced resistance to Citrus bacterial canker in sweet orange and grapefruit using the resistance gene CsLOB1. In the future, NPBTs will also be used to improve fruit traits, making them healthier. The regeneration of plants following the application of NPBTs is a bottleneck, making it necessary to optimize the efficiency of current protocols. The strengths and weaknesses of using explants from young in vitro plantlets, and from mature plants, will be discussed. Other major issues addressed in this review are related to the requirement for marker-free systems and shortening the long juvenility phase. This review aims to summarize methods and approaches available in the literature that are suitable to citrus, focusing on the principles observed before the use of NPBTs.

Citrus fruit quality and scion productivity are influenced by the choice of rootstock. We aimed to evaluate the effect of rootstocks on yield and fruit quality of Mandared, a triploid pigmented mandarin. To do so, we established a rootstock field trial on a high pH soil (8.6) in which Mandared was grafted onto 11 rootstocks. These included some standard rootstocks, such as trifoliate orange ((Poncirus trifoliata (L.) Raf.), Troyer citrange (Citrus sinensis (L.) Osb. × P. trifoliata), Swingle citrumelo (Citrus paradisi Macf. × P. trifoliata), and C35 citrange (C. sinensis × P. trifoliata), as well as new releases from the Council for Agricultural Research and Economics (CREA, Acireale, Italy) and the University of California Riverside (UCR). The cumulative yield was measured over five consecutive years, while fruit quality was analyzed for two years. The trees on C35, C57 (Citrus sunki Hort. ex. Tan. × P. trifoliata), and C22 (C. sunki × P. trifoliata), started to set fruits one year earlier than the others. The trees on C57 provided some of the highest cumulative yields and canopy volumes. The production of Mandared grafted onto C57 was double that of Mandared grafted onto Troyer, while Mandared grafted onto C35 and C22 resulted in the best yield efficiency. The trees on Swingle and C57 significantly reduced the pre-harvest fruit drop, to which Mandared is particularly sensitive. However, grafting Mandared onto Swingle resulted in the highest variation among replicates, probably due to its high sensitivity to iron chlorosis. Most of the fruit quality parameters, such as fruit size, total soluble solids (TSS), and acidity were not significantly different among the rootstock treatments. However, fruits produced by Mandared grafted onto C22 had one of the highest rates of anthocyanin accumulation. The results indicate that C57, C35, and C22 were the most suitable rootstocks for Mandared in South-Eastern Sicily.

Alternaria alternata is a fungus that causes a serious disease in susceptible genotypes of citrus, particularly in mandarins. The Alternaria citri toxin (ACT) produced by the pathogen induces necrotic lesions on young leaves and fruits, defoliation and fruit drop. Here, we describe two methods of marker-assisted selection (MAS) that could be used for the early identification of Alternaria brown spot (ABS)-resistant mandarin hybrids. The first method is based on a nested PCR coupled to high resolution melting (HRM) analysis at the SNP08 locus, which is located at 0.4 cM from the ABS resistance locus, and was previously indicated as the most suitable for the selection of ABS-resistant hybrids. The method was validated on 41 mandarin hybrids of the CREA germplasm collection, and on 862 progenies generated from five crosses involving different susceptible parents. Four out of five populations showed Mendelian segregation at the analyzed locus, while a population involving Murcott tangor as male parent showed distorted segregation toward the susceptible hybrids. The second method is based on a cleaved amplified polymorphic sequences (CAPS) marker that was developed using the same primers as the nested PCR at the SNP08 locus, coupled with BccI restriction enzyme digestion. To verify the reliability of the two genotyping methods, in vitro leaf phenotyping was carried out by inoculating A. alternata spores onto young leaves of 101 hybrids, randomly chosen among the susceptible and resistant progenies. The phenotyping confirmed the SNP08 genotyping results, so the proposed method of selection based on HRM or CAPS genotyping could be routinely used as an alternative to KBioscience competitive allele specific polymerase chain reaction (KASPar) single nucleotide polymorphism (SNP) genotyping system to improve citrus breeding programs. While the study confirmed that the SNP08 marker is a reliable tool for MAS of new citrus hybrids with different genetic backgrounds, it also identified a small group of genotypes where the resistance mechanism requires further investigation.