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Protocol for the in vitro multiplication of Fraxinus excelsior by somatic embryogenesis

A complete protocol for somatic embryogenesis in Fraxinus excelsior has been defined. The starting material consists of zygotic embryos harvested at an immature developmental stage corresponding to the early formation of cotyledons. For somatic embryogenesis induction, embryo axes are cultured on modified Murashige and Skoog (MS) medium enriched with acid 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyl-adenine (BA); cultures are incubated in darkness, at 23°C. The outgrowing embryogenic tissues are transferred to hormone-free MS medium and somatic embryos can spontaneously mature up to the cotyledonary stage. A faster maturation can be achieved by isolating the embryos; their successive germination and conversion is improved by a 3-week storage at 4°C, followed by transfer to Lloyd and McCown (WPM) medium. Acclimatization of plantlets is carried out in a 'misted' greenhouse in a period of 4-5 weeks.

These are the first achievements on somatic embryogenesis in common ash. The use of this technique opens new perspectives in the large-scale utilization of the species, particularly if we take into account the difficulties encountered in the vegetative propagation by rooted cuttings.

By somatic embryogenesis, in fact, millions of plants can be multiplied in a short time. It is worth-while to point out that in our case the genetic identity of the cloning material is not defined, being the original explant of zygotic origin; however, the technique is advantageously applied, for instance, on seeds obtained by controlled crossing and can successfully flank strategies of genetic improvement. A suitable scheme can include progeny tests for the plants growing from controlled crosses and long-term conservation of the corresponding embryogenic lines by slow growth or cryo-preservation. In clonal propagation it is always recommendable to stress the importance of the utilization of a large number of clones for any forest use.

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