The development of an efficient system of somatic embryogenesis from nucellar callus (Ollitrault et al., 1992, 1994; Cabasson et al., 1995, 1997; Engelmann et al., 1994) opened up the way for somatic hybridization by protoplast fusion (Ollitrault et al., 1994, 2000a, 2007; Grosser et al., 2000). Molecular analyses revealed the regeneration of frequent diploid cybrids in addition to tetraploid hybrids. In combinations between leaf protoplasts from one of the parents and callus from the other parent, mitochondria are systematically inherited from the callus parent (Cabasson et al., 2001), while chloroplasts are passed on in a random uniparent manner (Lotfy et al., 2003; Ollitrault et al., 2007).

Somatic hybridization has concrete uses in breeding for:

• Creating allotetraploid rootstocks by adding the nuclear genomes of complementary diploid parents (Dambier et al., 2011). The combinations created mainly associate accessions of the Citrus genus providing tolerance of abiotic stress, and rootstocks of the Poncirus genus, or Poncirus hybrids that are resistant to Phytophthora sp. and the Tristeza virus.
• Creating triploid varieties through the diversification of tetraploid parents (Ollitrault et al., 2000a;  Grosser et al., 2000) or the direct synthesis of triploid hybrids by diploid and haploid protoplast fusion
•  Diversifying nucleo-cytoplasmic associations at diploid level (Dambier et al., 2015) to manipulate the male sterility system and test the potential role of cytoplasmic organite diversity in that of the response to oxidative stress.

The tetraploid somatic hybrids and cybrids created have also formed the basis for studying the heredity of phenotypic traits (Gancel et al., 2003, 2005a, 2005b; Fanciullino et al., 2005; Bassene et al., 2008, 2009a, 2009b), of the meiotic functioning of allotetraploids (Kamiri et al., 2011), of proteome expression (Gancel et al., 2006) and of genome expression (Bassene et al., 2009, 2010, 2011, Gmitter et al., 2012).

References cited

• Bassene J.B., Berti L., Carcouet E., Dhuique-Mayer C., Fanciullino A.L., Bouffin J., Ollitrault P., Froelicher Y. (2008) Influence of mitochondria origin on fruit quality in a citrus hybrid. Journal of Agricultural and Food Chemistry, 56 (18) : p. 8635-8640.
• Bassene J.B., Froelicher Y., Dhuique-Mayer C., Mouhaya W., Ferrer R.M., Ancillo G., Morillon R., Navarro L., Ollitrault P. (2009a) Non-additive phenotypic and transcriptomic inheritance in a citrus allotetraploid somatic hybrid between C. reticulata and C. limon: The case of pulp carotenoid biosynthesis pathway. Plant Cell Reports, 28 (11) : p. 1689-1697.
• Bassene J.B., Berti L., Costantino G., Carcouet E., Kamiri M., Tomi F., Dambier D., Ollitrault P., Froelicher Y. (2009b) Inheritance of characters involved in fruit quality in a citrus interspecific allotetraploid somatic hybrid. Journal of Agricultural and Food Chemistry, 57 (11) : p. 5065-5070.
• Bassene J.B., Froelicher Y., Dubois C., Ferrer R.M., Navarro L., Ollitrault P., Ancillo G. (2010) Non-additive gene regulation in a citrus allotetraploid somatic hybrid between C. reticulata Blanco and C. limon (L.) Burm. Heredity, 105 (3) : p. 299-308.
• Bassene J.B., Froelicher Y., Navarro L., Ollitrault P., Ancillo G. (2011) Influence of mitochondria on gene expression in a citrus cybrid. Plant Cell Reports, 30 (6) : p. 1077-1085.
• Cabasson C., Ollitrault P., Côte F.X., Michaux-Ferrière N., Dambier D., Dalnic R., Teisson C. (1995) Characteristics of Citrus cell cultures during undifferentiated growth on sucrose and somatic embryogenesis on galactose. Physiologia Plantarum, 93 (3) : p. 464-470.
• Cabasson C., Alvard D., Dambier D., Ollitrault P., Teisson C. (1997) Improvement of Citrus somatic embryo development by temporary immersion. Plant Cell, Tissue and Organ Culture, 50 (1) : p. 33-37.
• Cabasson C.M., Luro F., Ollitrault P., Grosser J.W. (2001) Non-random inheritance of mitochondrial genomes in Citrus hybrids produced by protoplast fusion. Plant Cell Reports, 20 (7) : p. 604-609.
• Dambier D, Benyahia H, Pensabene-Bellavia G, Aka Kacar Y, Froelicher Y, Belfalah Z, Lhou B, Handaji N, Printz B, Morillon R, Yesiloglu T, Navarro L, Ollitrault P (2011) Somatic hybridization for citrus rootstock breeding: an effective tool to solve some important issues of the Mediterranean citrus industry. Plant Cell Rep 30:883-900.
• Dambier D., Petit F., Barantin P., Ollitrault P. (2015) New cybrids resulting from asexual pathway: a promise of the cybridization for creating new citrus rootstocks and cultivars. In : Navarro Luis (ed.), Sabater-Muñoz Beatriz (ed.), Moreno Pedro (ed.), Peña Leandro (ed.). Proceedings of the XIIth International Citrus Congress: International Society of Citriculture. Leuven : ISHS, p. 501-510. (Acta Horticulturae, 1065). International Citrus Congress: Citrus and Health. 12, 2012-11-18/2012-11-23, Valence (Espagne).
• Engelmann F., Dambier D. and Ollitrault P. (1994) Cryopreservation of cell suspensions and embryogenic calluses of Citrus using a simplified freezing process. Cryo-Letters 15, 53-58.
• Fanciullino A.L., Gancel A.L., Froelicher Y., Luro F., Ollitrault P., Brillouet J.M. (2005) Effects of nucleo-cytoplasmic interactions on leaf volatile compounds from citrus somatic diploid hybrids. Journal of Agricultural and Food Chemistry, 53 (11) : p. 4517-4523.
• Gancel A.L., Ollitrault P., Froelicher Y., Tomi F., Jacquemond C., Luro F., Brillouet J.M. (2003) Leaf volatile compounds of seven citrus somatic tetraploid hybrids sharing willow leaf mandarin (Citrus deliciosa Ten.) as their common parent. Journal of Agricultural and Food Chemistry, 51 (20) : p. 6006-6013.
• Gancel A.L., Ollitrault P., Froelicher Y., Tomi F., Jacquemond C., Luro F., Brillouet J.M. (2005a) Leaf volatile compounds of six citrus somatic allotetraploid hybrids originating from various combinations of lime, lemon, citron, sweet orange, and grapefruit. Journal of Agricultural and Food Chemistry, 53 (6) : p. 2224-2230.
• Gancel A.L., Ollitrault P., Froelicher Y., Tomi F., Jacquemond C., Luro F., Brillouet J.M. (2005b) Citrus somatic allotetraploid hybrids exhibit a differential reduction of leaf sesquiterpenoid biosynthesis compared with their parents. Flavour and Fragrance Journal, 20 (6) : p. 626-632.
• Gancel A.L., Grimplet J., Sauvage F.X., Ollitrault P., Brillouet J.M. (2006) Predominant expression of diploid mandarin leaf proteome in two citrus mandarin-derived somatic allotetraploid hybrids. Journal of Agricultural and Food Chemistry, 54 (17) : p. 6212-6218.
• Gmitter F.G.J., Chen C., Machado M.A., De Souza A.A., Ollitrault P., Froelicher Y., Shimizu T. (2012) Citrus genomics. Tree Genetics and Genomes, 8 (3) : p. 611-626.
• Grosser J.W., Ollitrault P., Olivares-Fuster O. (2000) Somatic hybridization in Citrus : An effective tool to facilitate variety improvement. In Vitro Cellular and Developmental Biology. Plant, 36 : p. 434-449.
• Kamiri M., Stift M., Srairi I., Costantino G., El Moussadik A., Hmyene A., Bakry F., Ollitrault P., Froelicher Y. (2011) Evidence for non-disomic inheritance in a Citrus interspecific tetraploid somatic hybrid between C. reticulata and C. limon using SSR markers and cytogenetic analysis. Plant Cell Reports, 30 (8) : p. 1415-1425.
• Lotfy S., Luro F., Carreel F., Froelicher Y., Rist D., Ollitrault P. (2003) Application of cleaved amplified polymorphic sequence method for analysis of cytoplasmic genome among Aurantioideae intergeneric somatic hybrids. Journal of the American Society for Horticultural Science, 128 (2) : p. 225-230.
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• Ollitrault P., Dambier D., Froelicher Y., Carreel F., D'Hont A., Luro F., Bruyère S., Cabasson C., Lotfy S., Joumaa A., Vanel F., Maddi F., Treanton K., Grisoni M. (2000a) Ressources génétiques : apport de l'hybridation somatique pour l'exploitation des ressources génétiques des agrumes. Cahiers Agricultures, 9 (3) : p. 223-236.
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