SEM and Histological Analysis of Somatic Embryogenesis Performed on Cactus Pear (Opuntia ficus-indica (L.) Mill.) Ovules Explants

Somatic embryogenesis can be induced in vitro in many plant species and is interesting for its potential applications in clonal propagation, genetic improvement and embryo development studies. In cactus pear, embryogenesis has been previously tried on vegetative explants such as shoot apices and in vitro plantlets discs. In this work, embroyogenic process was induced from generative explants consisting in ovule, extracted from flowers, 10 days after anthesis. These explants were cultivated in MS medium containing 87 mM fructose and 3 mu M GA(3). Cultures were held in darkness in growth chamber at 25 +/- 2 degrees C. Light Microscopy, Scanning Electron Microscopy (SEM) and histology were used in this study to describe the main morpho-anatomical events during the whole embryogenic process. Indirect somatic embryogenesis was induced on ovule explants through callus formation near the micropylar region. SEM observation of a regenerated callus revealed that it is covered by proeminent structures which were small and tightly packed. These structures progressed asynchronously giving rise to somatic embryos at different developmental stages ranging from globular to cotyledonary. Moreover, different embryo phenotypes including normal embryos with two distinct cotyledons and abnormal monocotylated or polycotylated ones have been observed. Secondary somatic embryos were also observed during induction of embryogenic process resulting in interruption of primary embryo development. Histological observations showed that callus cells involved in somatic embryo formation contained a large nucleus with conspicuous nucleolus and dense cytoplasm. Longitudinal section of a well developed embryo showed that it constituted with two cotyledons, shoot apex and leaf primordia. Concerning secondary embryos, their regeneration took place at the expense of the primary embryo protoderm. Conversion of the normal embryos to plantlets was easily realized in 0.3 mu M GA(3) containing medium.