Maturation and Conversion of Somatic Embryos Derived from Seeds of Olive (Olea europaea L.) cv. Dahbia: Occurrence of Secondary Embryogenesis and Adventitious Bud Formation

Maturation and conversion of somatic embryos are two crucial steps that hamper the development of efficient somatic embryogenesis systems in olive. Herein, a simple and efficient protocol for the maturation and conversion of olive somatic embryos is reported. Globular somatic embryos derived from seeds of cv. Dahbia were cultured on either half-strength olive (OM) or olive cyclic embryogenesis (ECO) media, with and without plant growth regulators (PGRs). The embryos reached the cotyledonary stage in 9 weeks, but those cultured on ECO medium containing 0.1 mg·L−1 6-(dimethylallylamino)purine (2iP), 0.1 mg·L−1 6-benzyladenine (BA) and 0.05 mg·L−1 indole-3-butyric acid (IBA) exhibited the largest sizes, with an average of 4.7 mm. Somatic embryo conversion into plantlets was evaluated using different culture media (half-strength OM or one-third strength Murashige and Skoog (MS)), light conditions (light or dark) and desiccation pretreatments. The highest rate of somatic embryo conversion (45%) was observed under a 16 h photoperiod on half strength OM medium containing 0.1 mg·L−1 gibberellic acid (GA3) and 0.1 mg·L−1 1-naphthalene acetic acid (NAA). The embryos that failed to germinate showed either necrosis, cotyledon greening with no further conversion, adventitious bud formation or secondary embryogenesis. The findings of this study will be beneficial for biotechnological applications in olive.

Manifestation of embryogenic potential in culture of zygotic embryos of Quercus robur L.

For the initiation of somatic embryogenesis early cotyledonary stage of zygotic embryo explants (from 15th July until late August) was suitable. The highest frequency of differentiation of somatic embryos was obtained on cotyledons of zygotic embryos cultured on basal modified medium MS (with 1/2 concentration macronutrients) or WPM medium containing 500 mg•l^-1 glutamine, proline and casein hydrolysate and supplemented with 2,4-D (1,0-2,0 mg•l^-1) and BAP (0,5-1,0 mg•l^-1). The development of somatic embryos was direct and indirect and the process was continuous over a long period. Primary somatic embryos were able to produce secondary embryos. Repetitive somatic embryogenesis led to the proliferation of a large number of new somatic embryos on their cotyledons, hypocotyl or radicula. The process of embryo differentation is asynchronous - various stages of somatic embryos could be observed in embryogenic culture. A somatic embryo conversion was rare on tested media. Embryo germination occured on medium containing BAP (0,1 mg•l^-1) or on medium with ABA and GA₃ (each 0,2 mg•l^-1) after a previous culture on WPM medium without plant growth regulators supplemented with sorbitol (6%). The embryo germination occurred also on WPM medium with 0.2 mg•l^-1 BAP when cultures were mantained at 2^oC for 4 weeks. Only 8 somatic embryos developed into plantlets. Their transplantation to <em>in vivo</em> conditions was unsuccessful.

Anatomy, histochemistry and ultrastructure of seed and somatic embryo of Acrocomia aculeata (Arecaceae)

Macaw palm (Acrocomia aculeata (Jacq.) Lodd. ex Mart.) is a tropical species with multiple uses, including oil supply for biodiesel production. However, structural and physiological studies of the seed are still scarce, as well as in vitro propagation techniques. The aim of this study was to characterize the anatomy, histochemistry and ultrastructure of the seed and the in vitro somatic embryo of A. aculeata, gaining insight into relationships between the post-harvest seed behavior and the somatic embryo conversion to plant. The zygotic embryo and the endosperm show high quantities of protein and lipids, stored in protein and lipid bodies, respectively. The cell wall storage polysaccharides occur in the thickened cell wall of the endosperm. The absence of vacuoles and the few organelles, except for the storage ones, indicate low metabolic state of the zygotic embryo, which suggests a orthodox behavior of the seed at maturity. The somatic embryo shows a shoot meristem with few leaf primordia, vacuolated cells and, occasionally, amyloplasts , but not lipid or protein reserves. These characteristics, common in somatic embryogenesis, could be associated with the maturation phase, and, consequently, with the low conversion of these embryos into plants.

Carbon sources and polyethylene glycol on soybean somatic embryo conversion

Somatic embryogenesis is an efficient method for the production of target cells for soybean genetic transformation. However, this method still offers low percentages of plant regeneration, and perhaps is related to the maturation process and high morphological abnormalities of the matured embryos. This study aimed to identify a maturation medium that could contribute to the outcome of more efficient plant regeneration results. Embryogenic clusters, derived from cotyledons of immature seeds of the soybean cultivars Bragg and IAS5, were used as starting material for embryos development. Different maturation media were tested by using 6% maltose, 3% sucrose or 6% sucrose, combined with or without 25 g L-1 of the osmotic regulator polyethylene glycol (PEG-8000). The histodifferentiated embryos were quantified and classified in morphological types. Percentages of converted embryos were analyzed. Cultivar Bragg resulted in higher matured embryo quantities, but lower percentages were obtained for the conversion in comparison to cultivar IAS5. While the addition of PEG did not affect the number of embryos converted into plants, 6% sucrose enhanced the conversion percent significantly.

GENE EXPRESSION PATTERNS OF PECAN ZYGOTIC AND SOMATIC EMBRYOS DURING MATURATION AND GERMINATION

The coordinate expression of mRNA classes in pecan (Carya illinoensis) zygotic and somatic embryos has been studied. MRNA was isolated from zygotic embryos at early and late maturation stages (12 to 22 weeks post-pollination) and during germination. Additionally, mRNA was isolated from somatic embryos derived from a repetitive embryogenic system prior and after cold (6 weeks at 4°C) and desiccation treatments (5 days). These treatments have been determined to enhance somatic embryo conversion. The abundance of embryogenic mRNA classes was determined using various cloned cotton mRNA probes (Hughes and Galau, 1989). This study is a part of our efforts to elucidate the developmental and physiological differences between zygotic and somatic embryo systems in pecan.

DEVELOPMENTAL AND REGULATORY MECHANISMS INVOLVED IN PECAN SOMATIC EMBRYO MATURATION

Although somatic embryogenesis in vitro has been carried out successfully in a number of plants, a limiting factor in many somatic embryogenic systems is that plantlet regeneration is not obtainable or restricted to low frequencies. We have developed a repetitive, high frequency somatic embryogenic system in pecan (Carya illinoensis) and have identified effective treatments for improved somatic embryo conversion. A 6 to 10 week cold treatment followed by a 5 day desiccation, promoted enhanced root germination and extension, and epicotyl elongation. Light and transmission electron microscopic evaluations of somatic embryo cotyledon development will be presented and related to conversion enhancing treatments and their possible roles in embryo maturation.

DEVELOPMENTAL AND REGULATORY MECHANISMS INVOLVED IN PECAN SOMATIC EMBRYO MATURATION

Although somatic embryogenesis in vitro has been carried out successfully in a number of plants, a limiting factor in many somatic embryogenic systems is that plantlet regeneration is not obtainable or restricted to low frequencies. We have developed a repetitive, high frequency somatic embryogenic system in pecan (Carya illinoensis) and have identified effective treatments for improved somatic embryo conversion. A 6 to 10 week cold treatment followed by a 5 day desiccation, promoted enhanced root germination and extension, and epicotyl elongation. Light and transmission electron microscopic evaluations of somatic embryo cotyledon development will be presented and related to conversion enhancing treatments and their possible roles in embryo maturation.