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Citrus are subject to many biotic and abiotic stresses and markets are continuously requiring fruits of higher quality. These pose important problems in most citrus growing areas that can only be solved with the establishment of citrus improvement programs to recover new and healthy genotypes to be used as rootstocks and varieties. Success of traditional citrus breeding strategies is limited by the peculiar genetic and reproductive characteristics of citrus. Biotechnology offers different approaches that can overcome many of these limitations. In this paper the following technologies used in Spain are reviewed: a) Shoot tip grafting in vitro. It is used worldwide to recover plants free of all known citrus pathogens, and already hundreds of millions of trees originated in this technique have been planted in the field; b) Embryo rescue. It is used in large programs to recover triploid seedless hybrid varieties from aborted seeds from 2n x 4n and 2n x 2n crosses; c) Protoplast fusion. It allows the production of allotetraploid hybrids among sexually compatible and incompatible parents to be used as rootstocks or as parentals for interploid breeding. Fusions between diploid and haploid cell lines are also used to produce triploid hybrids. d) Genetic transformation. Efficient protocols for transformation of several genotypes have been developed and introduction in citrus of genes of potential agronomical interest has been accomplished.

Polyploidy has long been recognized as an important force in the evolution of flowering plants and often results in novel phenotypes having advantages in adaptation and major interest for agriculture. Many crops, including citrus, are bred to a higher level of ploidy in search of desirable traits. In previous works of our group synthetic clementines with different levels of ploidy, specifically haploid, dihaploid and autotetraploid, were generated. This plant material represents a valuable tool for studying the effects of polyploidization at the molecular level since important traits are expressed in different manners. By using a microarray approach, we have accomplished a comprehensive analysis of transcriptome divergence among the newly created clementine lineages with different genome dosage. Differences in expression in the bark tissue of the four lineages were found significant for 89 (haploid-dihaploid comparison) or 93 genes (diploid-tetrapolid comparison). These genes were classified on the basis of their structure in different families.

A proteomic study was used to know how the primary metabolism in Citrus leaves is affected by the fruit load. To this end, we researched the differential expression of proteins related to these processes between on-crop and off-crop ‘Moncada’ mandarin leaves. Samples were collected in November and proteins were extracted. From 2D DIGE gel of these extracts, 33 spots related to primary metabolism were isolated: 26 spots being associated with photosynthesis and carbohydrate metabolism, 4 spots related to Krebs cycle, 1 spot related to pentose phosphate pathway, and 2 spots related to nutrient reservoir activity. These spots were identified by MALDI-MS or LC-MS-MS. Between the proteins related to photosynthesis and carbohydrate metabolism, 16 were up-expressed in off-crop leaves, such as NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, RuBisCO large subunit, ADP-glucose pyrophosphorylase, cinnamoyl-CoA reductase, and carbonic anhydrase; while 10 proteins were up-expressed in on-crop leaves such as NADP-dependent malic enzyme, and 3,4-dihydroxy-2-butanone kinase. The isolated proteins related to pentose phosphate pathway (6-phosphogluconate dehydrogenase) and to nutrient reservoir activity (Granule-bound starch synthase Ib precursor) were up-expressed in off-crop leaves. Regarding the proteins related to Krebs cycle, only malate dehydrogenase was up-expressed in off-crop leaves, while the other proteins were up-expressed in on-crop leaves (citrate synthase and NADP-isocitrate dehydrogenase). According to these results, the primary metabolism seems to be more active in off-crop leaves, suggesting that fruit load inhibits the primary metabolism in ‘Moncada’ mandarins.

Alternaria brown spot (ABS) in citrus is a serious disease caused by the tangerine pathotype of the fungus Alternaria alternata, which induces necrotic lesions on fruit and young leaves, defoliation and fruit drop in susceptible genotypes. It is a strong concern for triploid breeding programs aiming to produce seedless mandarin cultivars. In the present work, we have taken advantage of the particular genetic structures of unreduced megagametophytes resulting from second division restitution (SDR) (leading to triploid hybrid recovery) to map a genome region linked to ABS resistance in citrus. The monolocus dominant inheritance of susceptibility, proposed on the basis of diploid population studies, was corroborated in triploid progeny. Bulk segregant analysis coupled with genome scan using a large set of genetically mapped SNP markers and targeted genetic mapping by half tetrad analysis, using SSR and SNP markers, allowed locating a region linked to ABS resistance near the centromere of chromosome III. SSR and SNP markers were developed for efficient early marker-assisted selection (MAS) of ABS resistant hybrids.

Alternaria brown spot (ABS) of mandarins, caused by a pathotype of the fungus Alternaria alternata, is a serious disease both in humid and semi-arid citrus-growing regions of the world. The pathogen affects leaves and fruit of susceptible mandarin cultivars such as 'Fortune', 'Nova', 'Minneola', and 'Murcott'. The epidemiology of ABS was studied mainly in humid areas in Florida but, due to climatic differences, this information cannot be extrapolated to semi-arid regions like Spain. Field studies were conducted in 2011 in the experimental orchards at IVIA, Valencia. Dynamics of the airborne Alternaria conidia were monitored weekly by a spore trap, and the percentage of pathogenic isolates was determined periodically using the selective medium, ARSA, and pathogenicity tests. The presence of inoculum on affected leaves, shoots, leaf litter, and weeds was also determined. Infection periods were monitored weekly by exposing trap or indicator plants of 'Fortune' and 'Nova' mandarins. Environmental variables were recorded by an automated meteorological station. Although Alternaria conidia were detected through the experimental period, only 5% of all the isolates were pathogenic. As affected leaves and shoots were determined to be the main source of inoculum, the survival of the fungus in the leaf litter was higher than previously reported in Florida. Pathogenic isolates were detected also in weeds, but only at very low levels. Infections in trap plants occurred mainly in May-June and September-October. A significant positive correlation was detected between disease incidence and rainfall.