Memorias de investigación
Communications at congresses:
Genetic transformation of Pinus pinaster Ait. with genes involved in water stress response

Research Areas
  • Natural sciences and health sciences

Current climate change is specially affecting the Mediterranean region imposing strong restrictions in forest tree species. Some Mediterranean species are already suffering a severe decline, since their adaptation rate cannot keep pace with this fast change. Even pine species, considered as drought stress tolerant, are suffering important damages in the Iberian Peninsula. In such scenario it is of utmost importance to obtain further knowledge of the molecular mechanisms by which forest tree species face drought, as well as to consider potential applications of this knowledge in breeding programs to catalyse the production of individuals highly resistant to water stress. An exhaustive analysis using Pinus pinaster as model species has enabled us to identify an important number of reliable drought candidate genes for conifers. Three genes were selected due to their remarkable up-regulation during a prolonged drought stress in soil, and presumably they are involved in different steps of the water stress response. These genes putatively encode (1) a dehydrin (DH2) which would be involved in protecting cells from structural collapse suffered during dehydration; (2) a nodulin (NOD) presumably involved in sugar transport through membranes and (3) an AP2 transcription factor which may control the activation of new pathways during dehydration process. For all of them, the exon/intron structure was established and their promoter region was sequenced. An ubiquitin promoter-based binary vector (pMBb7Fm21GW-UBIL) was used to prepare four plasmid constructs for constitutive overexpression of these genes (NOD, AP2, DH2 and DH2-GFP as fusion protein). A selected embryogenic line of Pinus pinaster (PN519) provided by FCBA (France) was transformed by Agrobacterium with the four gene constructs and an empty vector as positive control. The transformation protocol was based on phosphinothricin (PPT) selection and on previously described procedures [1]. After 12 weeks, a high percentage of tissue clumps showed PPT resistant lines (from 37.8% for AP2 to 87.2% for NOD) corresponding to a high number of putative transformation events per gram of embryogenic tissue (from 34.5 for AP2 to 74.5 for NOD). All analysed events were PCR-positive and were able to proliferate both in selective medium as well as in proliferation medium. Transgene expression in the embryogenic tissues was highly variable among lines transformed with the same vector reaching, in 2 transgenic lines over-expressing NOD, expression levels approximately 100-times higher than in the control. Three selected lines from every construct were subjected to culture conditions for promoting embryo maturation. After three months in maturation medium high numbers of normal embryos were isolated (from 70 to 370 embryos per gram of mass) and transferred to germination medium to obtain complete plants. Rooting and acclimation were unsuccessful and only a few well developed plants were obtained. These plant regeneration experiments will be repeated using transgenic lines re-established from cryopreserved stocks in order to obtain enough transformed plants to analyse their performance under different conditions. [1] Trontin et al. (2013) Somatic embryogenesis as an effective regeneration support for reverse genetics in maritime pine: the Sustainpine collaborative project as a case study. In Park Y, Bonga J (eds) Proceeding of IUFRO Conference 25-28 Jun 2012 (Brno, Czech Republic). Pp 184-187 Acknowledgements: Sustainpine (Genomic tools in maritime PINE for enhanced biomass production and SUSTAINable forest managnement), 2010-2013, 2009 Plant KBBE initiative supported by transnational cooperation with funding from FCT (Portugal), MICINN (Spain), ANR (France), and BMBF (Germany); HIDROFOR (Estudio de la respuesta a sequía de P. pinaster Ait. utilizando aproximaciones genómicas y ecofisiológicas, AGL2006-03242/FOR; MEC, Spain).
3rd International Conference IUFRO Somatic embryogenesis and other vegetative propagation technologies.
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Proceedings Woody Plant Production Integrating Genetic and Vegetative Propagation Technologies

Research Group, Departaments and Institutes related
  • Creador: Grupo de Investigación: Genética, Fisiología e Historia Forestal
  • Departamento: Sistemas y Recursos Naturales