Somatic embryogenesis in holm oak male catkins

Somatic embryogenesis (SE) of tree species is the most promising method for the implementation of multivarietal forestry and for biotechnological approaches. To date, however, the application of this technology to mature trees is restricted to a few species. This is the first report on the induction of SE from male catkins of 100-year-old holm oaks (Quercus ilex L.). Embryogenic competence was mainly dependent on genotype and restricted to the most advanced catkin developmental stage with distinguishable closed flowers along the axis. Following a three-stage treatment procedure, embryogenic response (frequencies up 3.3 %) was obtained in three [Remedio, Villar del Arzobispo (VA) and Hunde (HU)] out of the five genotypes evaluated. In the culture conditions tested, the preferred protocol to induce SE in holm oak catkins should include: induction on MS medium with 6-benzyladenine and naphthaleneacetic acid, subculture onto medium with a reduced concentration of both plant growth regulators and a final transference to medium without growth regulators. Under these conditions, cotyledonary-stage somatic embryos developed from brown calli with or without nodular structures. Secondary SE, favored by the addition of sorbitol to the manifestation medium, allowed the establishment of 14 embryogenic lines belonging to VA and HU genotypes. Histological observations of the proliferating cultures revealed the presence of globular, torpedo and cotyledonary somatic embryos. Somatic embryos were diploid as verified by flow cytometry analysis, suggesting that they originated from the perianthic tissue of the male flower.