Chromosome Instability in 'Carrizo' Citrange plus Citrus macrophylla Somatic Hybrids

Somatic hybridization by protoplast fusion is an effective technique for citrus rootstock breeding. It theoretically allows the accumulation of all genes of the parents irrespective of their heterozygosity level and therefore the addition of complementary dominant traits. In the present work, protoplasts were isolated from callus of C. macrophylla and from leaves of 'Carrizo' citrange (C. sinensis x P. trifoliata). Chemical, electro-chemical and electric fusions were performed and ploidy level of the regenerated plants was evaluated by flow-cytometry. Five tetraploid plants, one pentaploid plant, one mixoploid (3x-6x) plant and one heptaploid plant were recovered. All these plants were analyzed with SSR markers, distributed in the nine chromosomes of citrus. Cytoplasmic genomes were also characterized using chloroplastic and mitochondrial markers. Results showed that mitochondrial genome was inherited from C. macrophylla in all plants while segregation was observed for chloroplastic genome. Nuclear genome analysis revealed the loss of parental alleles in most of the regenerated plants. In tetraploids, it affected mainly C. macrophylla alleles, while the alleles lost in pentaploid and heptaploid plants were mostly from P. trifoliata. Results suggest chromosome instability in this complex intergeneric combination with apparent non-random loss of chromosome fragments. Two allotetraploid somatic hybrids were selected for evaluation as rootstocks.