Somatic embryogenesis of a wild passion fruit species Passiflora cincinnata Masters: histocytological and histochemical evidences

Abstract

The characterization of cellular changes that occur during somatic embryogenesis is essential for understanding the factors involved in the transition of somatic cells into embryogenically competent cells and determination of cells and/or tissues involved. The present study describes the anatomical and ultrastructural events that lead to the formation of somatic embryos in the model system of the wild passion fruit (Passiflora cincinnata). Mature zygotic embryos were inoculated in Murashige and Skoog induction media supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Zygotic embryo explants at different development stages were collected and processed by conventional methods for studies using light, scanning, and transmission electron microscopy (TEM). Histochemical tests were used to examine the mobilization of reserves. The differentiation of the somatic embryos began in the abaxial side of the cotyledon region. Protuberances were formed from the meristematic proliferation of the epidermal and mesophyll cells. These cells had large nuclei, dense cytoplasm with a predominance of mitochondria, and a few reserve compounds. The protuberances extended throughout the abaxial surface of the cotyledons. The ongoing differentiation of peripheral cells of these structures led to the formation of proembryogenic zones, which, in turn, dedifferentiated into somatic embryos of multicellular origin. In the initial stages of embryogenesis, the epidermal and mesophyll cells showed starch grains and less lipids and protein reserves than the starting explant. These results provide detailed information on anatomical and ultrastructural changes involved in the acquisition of embryogenic competence and embryo differentiation that has been lacking so far in Passiflora.

Cellular Markers for Somatic Embryogenesis

“…The embryogenic ones are characterised by their small size, with rich cytoplasm and filled with starch. The similar character of embryogenic cells was described in Passiflora cincinnata (Rocha et al, 2011) and cork oak (Puigderrajols et al, 2001). Cells with the same characteristics were described for the embryogenic parts of the explants of Carya (Rodriguez & Wetzstein, 1998).…”

“…The unicellular origins of somatic embryos was described (among others) in the leaf explant of Coffea arabica (Quiroz-Figueroa et al, 2002), coconut (Verdeil et al, 2001) and Dactylis glomerata (Trigiano et al, 1989). In some species, only the multicellular origins of somatic embryos were described, as, for example, in Carya illinoinensis (Rodriguez & Wetzstein, 1998) and Passiflora cincinnata (Rocha et al, 2011).…”

“…In some species, zygotic embryos are the best source of somatic ones and the explant organs involved in SE are cotyledons or shoot apical meristem (e.g. Gaj, 2001;Kurczyńska et al, 2007;Raghavan, 2004;Rocha et al, 2011). Cultures of leafs, stems and roots parts are also efficient in SE induction (Mathews et al, 1993;Quiroz-Figueroa et al, 2002).…”

Thidiazuron Induces High Frequency Direct Somatic Embryogenesis Growth from Cotyledon Culture of Eurycoma longifolia

“…Thus, it possesses a strong cytokinin-like activity that exceeds the other commonly used adenine-type cytokinins including benzylaminopurine or kinetin (Li et al 1998). Study from Rocha et al (2012) also showed that the greatest embryogenic response of Leucopogon verticillatus was from the treatment of TDZ and zeatin combined with IAA with the first appearance of somatic embryo growth after 3 weeks of inoculation.…”

“…However, to the best of our knowledge, in vitro mass propagation from direct somatic embryogenesis has not been reported. In addition, the embryogenic pathway is a suitable methods to learn about the morphophysiological and molecular aspects involved in cellular differentiation (Rocha et al 2012), the biochemical pathways during the development of embryogenesis (Balzon et al 2013) and the propagation of non-seed producing plants or plants that do not respond to conventional propagation techniques such as cuttings (Anthony et al 2004). Generally, the application of auxin and cytokinin is a key factor in the determination of embryogenic response.…”