Somatic embryogenesis from different tissues of Spanish populations of maritime pine
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The somatic embryogenesis (SE) capacity of megagametophytes belonging to Continental and Mediterranean Spanish provenances of maritime pine (Pinus pinaster Aiton) was studied, noting factors (megagametophyte developmental stage and culture medium) that enhanced the induction and establishment of SE lines. In both provenances, initiation and establishment of embryogenic calli was higher on megagametophytes in which the dominant zygotic embryo had begun to develop. In the Mediterranean provenance, however, SE lines were also established from megagametophytes enclosing zygotic embryos with well-developed cotyledons. A modified Litvay medium (mLV) containing 9.9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM 6-benzyladenine (BA) was superior to DCR medium containing 13.6 μM 2,4-D and 4.4 μM BA for SE induction, but there were no differences between media in terms of the number of SE lines established after 4 months in culture (153 vs. 155 established SE lines, for mLV and DCR media respectively). Of the 26 embryogenic lines tested for maturation, 15 (58 %) produced cotyledonary somatic embryos and 75 % of these gave rise to plants on germination medium. SE-like cultures from adult maritime pine trees were also initiated, but embryogenic lines could not be established. This is the first report on the production of SE in maritime pine of Continental and Mediterranean origin. The micropropagation protocols presented here provide an important tool for the vegetative multiplication of selected families and breeding programs for maritime pines from Spain.
KeywordsEmbryo development Histology Pinus pinaster Somatic embryogenesis
This work was supported by The Spanish Ministry of Science and Innovation; FEDER founds (AGL2005-07585-02FOR, AGL2007-66345-C02-02/FOR, grant for M. B., PSE310000-2009-4), and the Generalitat Valenciana (PROMETEO2009/075). We acknowledge the critical review of the manuscript to Prof. S. A. Merkle (University of Georgia, USA). Technical assistance by M. A. Morcillo, J. Muñoz and plant material supply by the Generalitat Valenciana (Centro para la Investigación y Experimentación Forestal) and by the Dirección General de Biodiversidad, are also acknowledged.
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