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Biological characterization of young and aged embryogenic cultures of Pinus pinaster (Ait.)

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Abstract

Pinus pinaster (Ait.) somatic embryogenesis (SE) has been developed during the last decade, and its application in tree improvement programs is underway. Nevertheless, a few more or less important problems still exist, which have an impact on the efficiency of specific SE stages. One phenomenon, which had been observed in embryogenic tissue (embryonal mass, EM) initiated from immature seed, has been the loss of the ability to produce mature somatic embryos after the tissue had been cultured for several months. In an attempt to get insight into the differences between young cultures of EM (3-mo-old since the first subculture) of P. pinaster that produced mature somatic embryos and the same lines of significantly increased age (18-mo-old, aged EM) that stopped producing mature somatic embryos, we analyzed in both types of materials the levels of endogenous hormones, polyamines, the global DNA methylation, and associated methylation patterns. In addition, we included in the analysis secondary EM induced from mature somatic embryos. The analysis showed that the two tested genotypes displayed inconsistent hormonal and polyamine profiles in EM cultures of a similar phenotype and that it might be difficult to attribute one specific profile to a specific culture phenotype among genotypes. Experiments were also undertaken to determine if the global DNA methylation and/or the resulting methylation pattern could be manipulated by treatment of the cultures with a hypomethylating drug 5-azacytidine (5-azaC). An aged EM was exposed to different concentrations and durations of 5-azaC, and its response in culture was established by fresh mass increases and somatic embryo maturation potential. All of the analyses are new in maritime pine, and thus, they provide the first data on the biochemistry of EM in this species related to embryogenic potential.

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Acknowledgments

M. Bernier-Cardou (CFS-LFC, Canada) is gratefully acknowledged for her expert statistical analysis of the hormone, PA, and DNA global methylation data. KK and MALW thank le STUDIUM, France for their financial contribution to this project, P. Alazard (FCBA, France) and A. Raffin, P. Patuszka (INRA Bordeaux) for providing seed cones.

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Authors and Affiliations

  1. Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du P.E.P.S., PO Box 10380, Stn. Sainte-Foy, Québec, QC, G1V 4C7, Canada

    K. Klimaszewska & G. Pelletier

  2. Laboratorio Fisiología Vegetal, Departamento Biologia de Organismos y Sistemas, Univesidad de Oviedo, C/Catedrático Rodrigo Uría, s/n, 33071, Oviedo, Asturias, Spain

    C. Noceda & R. Rodriguez

  3. I.N.R.A., UR 588 Unité Amélioration, Génétique et Physiologie Forestières, 2163 Avenue de la Pomme de pin CS 4001 Ardon, 45 075, Orléans Cedex 2, France

    P. Label & M. A. Lelu-Walter

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  1. K. Klimaszewska
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  2. C. Noceda
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  3. G. Pelletier
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  4. P. Label
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  5. R. Rodriguez
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  6. M. A. Lelu-Walter
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Correspondence to K. Klimaszewska.

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Editor: Gregory C. Phillips

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Klimaszewska, K., Noceda, C., Pelletier, G. et al. Biological characterization of young and aged embryogenic cultures of Pinus pinaster (Ait.). In Vitro Cell.Dev.Biol.-Plant 45, 20–33 (2009). https://doi.org/10.1007/s11627-008-9158-6

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