An efficient regeneration system via somatic embryogenesis in olive

Olive is one of the most important oil crops in the Mediterranean area. Biotechnological improvement of this species is hampered by the recalcitrant nature of olive tissue regeneration in vitro. In this investigation, we have developed an efficient regeneration system for juvenile olive explants via somatic embryogenesis. Embryogenic cultures were obtained at a rate of 25% by culturing isolated radicles from mature seeds in a modified olive medium (OMc) containing 2.5 μM 6-(dimethylallylamino) purine (2iP) and 25 μM indole-3-butyric acid (IBA) over 3 weeks and later transferring to the same medium without 2iP and with a lower IBA concentration. Two different basal formulations, OMc and olive cyclic embryogenesis medium (ECO) [1/4 OM macroelements, 1/4 Murashige and Skoog (MS) microelements and 1/2 OM vitamins supplemented with 550 mg l⁻¹ glutamine], were tested for embryogenic callus proliferation and maturation. The growth rate of embryogenic calli was similar in both media. However, the regeneration of mature embryos, achieved by culturing embryogenic masses in the same medium without hormones and supplemented with 1 g l⁻¹ activated charcoal, was significantly higher when embryos were cultured in the ECO basal formulation. Pre-culturing embryogenic masses in liquid medium for up to 4 weeks did not affect subsequent callus proliferation in solid medium. The maturation rate of small globular somatic embryos, 1–3 mm size, obtained after filtering liquid cultures through a 3 × 3 mm mesh, was also similar to control embryos cultured in solid medium. To improve the maturation and germination rates, the effect of culturing globular somatic embryos on semi-permeable cellulose acetate membranes was also tested. Membrane treatments reduced the regeneration of mature embryos from 56.5% in the control treatment to 40.6% when the membrane was applied during the first half of the 8-week maturation phase and to 18% when the membrane was applied during last 4 weeks of the maturation period. However, membrane treatments significantly enhanced the conversion of mature embryos to plants, increasing the embryo conversion rate from 1.5% in the control to an average value of 37.8% in the membrane treatment. Cotyledonary embryos that were matured on the membranes showed lower values of water and solute potential than controls, indicating that this treatment exerted a controlled desiccation rate that enhanced the recovery of plants.