Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. 'W. Murcott' mandarin (a hybrid of 'Murcott' and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. Transformation of 'W. Murcott' mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of 'W. Murcott' mandarin. Following citrus protoplast culture and regeneration, transformed micro calli were microscopically designated via GFP expression, physically isolated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryos were recovered and from them, ten transgenic lines were regenerated and cultured on rooting medium for shoot elongation. Transgenic shoots were micrografted and established in the greenhouse with 3-5 replicates per line. The insertion of Xa21 and GFP was confirmed by PCR and southern blot analysis. GFP expression was verified by fluorescence microscopy and western blot analysis revealed expression of Xa21 although it was variable among transgenic lines, as shown by RT-qPCR. Transgenic plants challenged with the citrus canker pathogen by syringe inoculation showed a reduction in lesion number and bacterial populations within lesions compared to non-transgenic control plants. Transgenic 'W. Murcott' mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance.

Host disease resistance is the most desirable strategy for control of citrus canker, a disease caused by a gram-negative bacterium Xanthomonas citri subsp. citri. However, no resistant commercial citrus cultivar has been identified. Cybridization, a somatic hybridization approach that combines the organelle and nuclear genomes from different species, was used to create cybrids between citrus canker resistant ‘Meiwa’ kumquat (Fortunella crassifolia Swingle snym. Citrus japonica Thunb.) and susceptible grapefruit (Citrus paradisi Macfad) cultivars. From these fusions, cybrids with grapefruit nucleus, kumquat mitochondria and kumquat chloroplasts and cybrids with grapefruit nucleus, kumquat mitochondria and grapefruit chloroplasts were generated. These cybrids showed a range of citrus canker response, but all cybrids with kumquat chloroplasts had a significantly lower number of lesions and lower Xanthomonas citri subsp. citri populations than the grapefruit controls. Cybrids with grapefruit chloroplasts had a significantly higher number of lesions than those with kumquat chloroplasts. To understand the role of chloroplasts in the cybrid disease defense, quantitative PCR was performed on both cybrid types and their parents to examine changes in gene expression during Xanthomonas citri subsp. citri infection. The results revealed chloroplast influences on nuclear gene expression, since isonuclear cybrids and ‘Marsh’ grapefruit had different gene expression profiles. In addition, only genotypes with kumquat chloroplasts showed an early up-regulation of reactive oxygen species genes upon Xanthomonas citri subsp. citri infection. These cybrids have the potential to enhance citrus canker resistance in commercial grapefruit orchards. They also serve as models for understanding the contribution of chloroplasts to plant disease response and raise the question of whether other alien chloroplast genotypes would condition similar results.

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