RESEARCH NOTE Somatic embryogenesis from ovaries of sweet orange cv. Tobias Jean C. Cardoso • Adriana P. Martinelli • Rodrigo R. Latado Received: 2 July 2011 / Accepted: 16 October 2011 / Published online: 29 October 2011 Ó Springer Science+Business Media B.V. 2011 Abstract Callogenesis, somatic embryogenesis, and regeneration were obtained from tissues of unfertilized ovaries of sweet orange (Citrus sinensis Osbeck.) cv. Tobias. The influence of two modified basal media, woody plant medium (WPM) and N6 medium, to induce callus formation from pistils was determined. Overall, high frequencies of callogenesis were observed when either medium was used. However, initial culture of explants in WPM medium fol- lowed by transfer of callus to N6 medium resulted in higher frequency of callus induction (of 2.30 callus per explant that were larger than 0.5 cm in size), and of subsequent devel- opment of embryogenic callus (10%). A total of 125 somatic embryos were obtained. After 6 months of culture, 72% of somatic embryos germinated into plantlets. These plantlets were subsequently micrografted in vitro, and then acclima- tized. Ploidy of these plants were determined using flow cytometry and TRAPS molecular markers were used to confirm their maternal origin. Keywords Citrus sinensis Á Pistil culture Á Callus Á Culture medium Á Plantlet regeneration Introduction Conventional genetic breeding in Citrus species, including sweet oranges, presents limitations associated with the reproductive biology of the genus, such as nucellar poly- embryony, a long juvenile period and a high level of het- erozygosity (Grosser and Gmitter 1990). The use of biotechnological tools has, hence, attracted important interest in this family (Germana ` et al. 2011; Grosser and Gmitter 2011), to overcome these limitations. Biotechnological methods can aid in the improvement of sweet oranges, as a method for regeneration of plants from somatic hybrids (Grosser and Gmitter 2011), haploid and doubled haploid plants (Germana ` et al. 2011; Cao et al. 2011) and for genetic transformation (Cai et al. 2006). The establishment of efficient protocols for callus and somatic embryo induction and plant regeneration from different genotypes and explants are important for a more effective use of somatic embryogenesis. Embryogenic callus has been obtained in different Citrus species and explants such as nucellus (Kayim and Koc 2006); ovule (Froelicher et al. 2007); style and stigma (Carimi et al. 1999); anther (Germana ` and Chiancone 2003); leaf, cotyledon and root segments (Gill et al. 1995). Although citrus somatic embryogenesis can be obtained using different culture conditions and explant types, best results have been shown on callus induction from ovular origin (ovules or nucellus) (Fiore et al. 2002), except in monoembryonic citrus varie- ties, which in some cases have proved recalcitrant to somatic embryo induction from in vitro cultured ovules (Kobayashi et al. 1981), and also seedless varieties, where ovules and nucellus are absent. In these cases the induction of somatic embryogenesis from female floral organ culture (stigma, style and ovary) can be an option (Carimi et al. 1999; Fiore et al. 2002). Carimi et al. (1999) obtained various responses in the induction of somatic embryogenesis from thin cell layers (TCL) from stigma, style and ovary in five Citrus species, but failed to regenerate embryos from ovary tissue cultures of sweet orange. In fact, there are no reports in the J. C. Cardoso (&) Á A. P. Martinelli Centro de Energia Nuclear na Agricultura, Universidade de Sa ˜o Paulo, Av. Centena ´rio, 303, Piracicaba, SP 13416-000, Brazil e-mail: jeancardosoctv@gmail.com R. R. Latado Centro APTA Citros Sylvio Moreira, IAC, Rod Anhanguera, km 158, CP 04, Cordeiro ´polis, SP CEP 13490-970, Brazil 123 Plant Cell Tiss Organ Cult (2012) 109:171–177 DOI 10.1007/s11240-011-0073-x