Haploids and doubled haploids in Citrus ssp.

Abstract

Citrus breeding is based either on conventional (hybridization, selection, mutation) or biotechnological methods, the latter employing in vitro tissue culture, regeneration from protoplasts, somatic hybridization, in vitro mutant selection, genetic transformation and haploid/doubled haploid production. All cultivated forms of Citrus spp. and related genera (Poncirus, Fortunella, etc.) are diploid with a monoploid number of chromosomes (n=x=9). Triploid and tetraploid forms of Citrus spp. also exist. Haploid plants, with a gametophytic set of chromosomes in the sporophyte, have potential use in mutation research, selection, genetic analysis and genetic transformation. The possibility of obtaining triploid somatic hybrids (important for the seedlessness of their fruits) by fusion between haploid and diploid protoplasts is an important application of haploidy in Citrus spp. breeding. Doubled haploids are also important in genome mapping and in exploring gametoclonal variation. Haploids can be induced in woody plants mainly through two approaches: gynogenesis, where they arise from the female gamete (in situ parthenogenesis induced by irradiated pollen and followed by in vitro culture of embryos, in situ or in vitro parthenogenesis induced by triploid pollen followed by in vitro culture of embryos), and androgenesis where they are regenerated from the male gametes (anther culture, isolated microspore culture). Although in our laboratory haploid plantlet regeneration through gynogenesis in C. clementina Hort. ex Tan., variety ‘Nules’, has been induced by in vitro pollination with the pollen of ‘Oroblanco’, a triploid grapefruit-type (triploidy of pollen, like irradiation, does not hinder pollen germination, but prevents pollen fertilization and stimulates the development of haploid embryoids from ovules (Germana and Chiancone, 2001), the experimental procedure in this Manual will be limited to the protocol of anther culture because of its proven efficiency. In particular, the protocol given is for anther culture of several varieties (Nules, ‘SRA 63’ and ‘Monreal’) of C. clementina Hort. ex Tan, which is currely the only Citrus species that responds well to androgenesis. In addition to genotypic effects, physiological conditions of donor plants and seasonal dependency are important factors for anther culture in Citrus and other woody plants (Germana et al., 2000a). Although a lot of research has been carried out on gametic embryogenesis in Citrus spp. and their relatives, not much of it has been successful. For further details regarding Citrus haploidy, see the review by Germanà (1997).