Adventitious organogenesis induced in sweet orange (Citrus sinensis L.) var. “half-blood” maltese: morphogenetic and histological study

Tunisian citrus crops are faced to several abiotic and biotic constraints among which virus and virus-like diseases are incurable. The production of virus-free plants systematically needs the use of in vitro techniques. In this context, somatic embryogenesis and further plantlet regeneration of the Tunisian “half-blood” Maltese orange were obtained using explants consisting in style/stigma collected from unopened flowers.Somatic embryos were induced on Murashige and Skoog medium containing 13.3 μM 6-benzylaminopurine and 500 mg.l-1 malt extract, but their germination was obtained on hormone free-medium. Somatic embryogenesis was induced indirectly from intermediate friable callus initiated at the basal part of the style. Somatic embryos exhibited central procambial cells and were surrounded by a protoderm isolating them from the callus. These embryos had bipolar structure confirmed by the presence of shoot and root apices at cotyledonary stage. The use of cotyledon excised from those embryos failed to regenerate somatic embryos, but gave rise to direct organogenesis in two forms, true buds and protuberances both evolved in shoots after transfer in hormone-free medium. According to histological observations, protuberances are induced from epidermal and subepidermal cells of the cotyledon explant and remain closely attached to their mother tissue even at the shoot stage.