Peroxidase Enzyme Fractions as Markers of Somatic Embryogenesis Capacities in Olive (Olea europaea L.).

As part of the search for biochemical markers of somatic embryogenesis in tissue cultures of olive (Olea europaea L.), peroxidases (POXs) in both the soluble and ionically wall-bound fractions were studied in two reputed olive cultivars (cvs.): "Picholine Marocaine" and "Dahbia". In order to carry out embryogenesis induction, proximal cotyledons were cultured in modified olive medium (OMc) supplemented with 25 µM indole-3-butylic acid (IBA) and 2.5 µM 2-isopentenyladenine (2iP), while distal leaf fragments (somatic explants) were cultured in OMc supplemented with 4.56 µM zeatin riboside (ZR) and 10.25 µM 1-naphthaleneacetic acid (NAA). Regarding embryogenic potentials, the zygotic explants (cv. Picholine Marocaine: 43.39%; cv. Dahbia: 53.41%) were more regenerative than the somatic explants (cv. Picholine Marocaine: 13.05%; cv. Dahbia: 19.51%). The enzyme assay showed a higher POX activity in embryogenic calluses (ECs) than in nonembryogenic calluses (NECs) for the zygotic explants in both studied cultivars. When expressed as units per milligram of proteins (U mg-1 proteins), the highest total POXs activities (soluble POXs + ionically wall-bound POXs) were found in the ECs derived from the zygotic explants; for cv. Dahbia, 65% of the enzyme activities came from the ionically wall-bound fractions. Polyacrylamide gel electrophoresis showed that the ECs of the highly active cv. Dahbia were characterized by highly active isoperoxidases that were revealed in four migration zones, particularly a doublet in the A4 zone (Rf 0.70-0.73) present in the ionically wall-bound POXs. The fast-moving anodic POXs of the ionically wall-bound fractions could be adopted as an early electrophoretic test to determine the embryogenesis capacities in olive tissue culture materials. As biochemical markers, the POX enzyme and its profile in fractions, i.e., as soluble POXs and ionically wall-bound POXs, can offer a valuable tool for improving the tissue culture of olive via somatic embryogenesis.

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.

A combined pathway of organogenesis and somatic embryogenesis for an efficient large-scale propagation in date palm (Phoenix dactylifera L.) cv. Mejhoul.

An efficient regeneration system via a combined pathway of organogenesis and somatic embryogenesis was developed for date palm (Phoenix dactylifera L.) cv. Mejhoul. Adventitious buds were obtained from shoot-tip explants with a frequency of 53.3% after 9 months of culture: 6 months on half-strength Murashige and Skoog (MS/2) medium containing 14.2 µM indole-3-acetic acid (IAA), 13.4 µM 1-naphthaleneacetic acid (NAA) and 0.5 µM 6-(dimethylallylamino) purine (2iP), and 3 months on MS/2 medium supplemented with 1.1 µM IAA, 1.1 µM NAA, 0.5 µM 2iP, 2.2 µM 6-benzyladenine (BA) and 0.4 µM kinetin. Adventitious bud segments were used as explants to induce somatic embryogenesis, and the effects of different concentrations (22.5, 45, 90, 225 or 450 µM) of 3,6-dichloro-o-anisic acid (dicamba) and 4-amino-3,5,6-trichloropicolinic acid (picloram) were evaluated. The optimal medium for somatic embryogenesis induction was MS medium supplemented with 45 µM picloram and 5 µM 2iP, in which the somatic embryogenesis rate was 70%. For somatic embryo maturation, the effects of sorbitol, mannitol, polyethylene glycol (PEG) and abscisic acid (ABA) were tested. The highest maturation rate (88.6 mature somatic embryos per 100 mg fresh weight callus) was observed on liquid MS medium supplemented with 20 g L-1 PEG. Subsequent somatic embryo germination was achieved with up to 52.0% in MS medium containing 2.5 µM NAA and 2.5 µM BA. The regenerated plantlets were transferred to the glasshouse where 76.0% of them survived.

Somatic embryogenesis from bud and leaf explants of date palm (Phoenix dactylifera L.) cv. Najda.

An efficient regeneration system through somatic embryogenesis was developed for date palm cv. Najda. Adventitious bud and proximal leaf segments cultured on Murashige and Skoog (MS) medium supplemented with various combinations of auxins and cytokinins induced embryogenesis after at least 6 months of culture. Somatic embryogenesis induction seemed correlated with the type of the explant, the induction period and the auxin used. The highest rate of somatic embryogenesis (86.0%) was obtained on bud explants cultured on MS medium supplemented with 45.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), and 4.5 µM kinetin or 4.5 µM 6-(dimethylallylamino) purine (2iP). Whereas, low levels of embryogenesis were obtained on media supplemented with 1-naphthalene acetic acid (NAA) or 2-naphthoxyacetic acid (NOA). Proximal leaf segments showed somatic embryogenesis only when cultured on media supplemented with 2,4-D or picloram. Statistical analysis revealed significant effects of explant type and plant growth regulators (PGRs) combination on somatic embryogenesis. Somatic embryos were germinated successfully on PGR-free MS medium with or without activated charcoal (50.0-60.0 and 26.6-36.6%, respectively), and 80.0% of plantlets survived after transferring to a glasshouse for 6 months. Our results will be useful for large-scale propagation of date palm cv. Najda, characterized by high fruit quality and bayoud disease resistance.