Recovery of citrus triploid hybrids by embryo rescue and flow cytometry from 2x x 2x sexual hybridisation and its application to extensive breeding programs

Seedlessness is one of the most important characteristics for mandarins for the fresh-fruit market and mandarin triploid hybrids have this trait. Citrus triploid plants can be recovered by 2x x 2x sexual hybridisations as a consequence of the formation of unreduced gametes at low frequency. Triploid embryos are found in small seeds that do not germinate under greenhouse conditions. Extensive breeding programs based on this type of hybridisation require very effective methodologies for embryo rescue and ploidy evaluation. In this work, we describe an effective methodology to recover triploid hybrids from 2x x 2x hybridisations based on in vitro embryo rescue and ploidy level determination by means of flow cytometry. The influence of parents and environmental conditions on obtaining triploid hybrids has been analysed. The strongest effect was associated with the genotype of the female parent while a strong interaction was found between the male parent genotype and environmental conditions. The effect of the female parent genotype on the length of the juvenile phase was also demonstrated by observing a large number of progenies over the last 10 years. The methodology described here has enabled us to obtain over 4,000 triploid hybrids so far, of which 13 have been protected in the European Union and two are being extensively planted by citrus growers to establish new commercial plots. These triploid hybrids have been analysed with simple sequence repeats markers to differentiate all the new triploid varieties and their parents, and thus molecular identification will help defend plant breeders' rights

Ploidy manipulation and citrus breeding, genetics and genomics

Polyploidy appears to have played a limited role in citrus germplasm evolution. However, today, ploidy manipulation is an important component of citrus breeding strategies. For varieties, the main objective is to develop triploid seedless varieties. For rootstock, the aim is to cumulate interesting traits in tetraploid hybrids and to improve adaptation to biotic and abiotic stresses. In this chapter we make a review of the recent knowledge acquired on the natural mechanisms of citrus polyploidization, and teraploid meiosis. Chromosome doubling of nucellar cells is frequent in apomictic citrus and results in tetraploid seedling production. Unreduced gametes are also frequently produced, mainly by second division restitution for ovules. First division restitution was described for pollen as well as alternative mechanisms for both ovules and pollen. Tetraploid plants display tetrasomic to disomic segregations in relation with their genome structure (autoteraploid versus allotetraploid) and the divergence of the parental species. The implications of the origin of diploid gametes, on the genetic diversity of polyploid progenies, are discussed. The biotechnological tools (haplo-methods, chromosome doubling by chemichal treatments, somatic hybridization and cytogenetic/molecular tools for polyploid genome studies) to optimize ploidy manipulation are presented. The interest of haploids and polyploid genotypes for basic genetic and genomic studies is discussed. The following research area are reviewed: haploids and doubled haploid for genome sequencing and haplotyping, centromere mapping from unreduced gametes, marker-trait association study in polyploids, phenome and gene expression in polyploids with a special focus on polyploidy and adaptation. Finally, we give an overview of the recent advances of concrete polyploid citrus breeding programs in China, Florida and the Mediterranean Basin