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Long-term subculture randomly affects morphology and subsequent maturation of early somatic embryos in maritime pine

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Abstract

Somatic embryogenesis is affected by highly variable maturation yields in Pinus pinaster. Origins of this variability were investigated by testing effects of spatial and temporal division of initial embryogenic tissue into independently cultivated pieces. One embryogenic cell-line was proliferated to obtain six embryonal-suspensor masses (ESM) treated as six sub-lines within a single dish. After proliferation to reach 2, 4 and 8 dishes (12, 24, 48 ESM), the 48 ESM (six sub-lines) were maintained by weekly subculture (34 weeks). ESM observations and maturation tests were regularly performed during the amplification and maintenance periods. Relations between maturation yields as well as cotylfedonary embryo length and immature embryo morphology were analyzed. ESM yielded variable results, independently from culture spatial (dish) and temporal (sub-line) subdivision. Maturation yields and length of regenerated embryos globally decreased as a function of subculture number. Concomitantly, morphological degradation of immature embryos occurred, indicating a global loss of embryogenic ability. Maturation variability probably results from immature embryos heterogeneity, which could be increased by manipulations during subculture. This is the first time that this evolution along time in culture is precisely described in conifers somatic embryos.

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Abbreviations

ESM:

embryonal-suspensor masses

f.wt:

fresh weight

CE:

cotyledonary somatic embryos

AE:

abnormally developed embryos

PC:

precotyledonary and early cotyledonary embryos

References

  • Bajaj S, Rajam MV (1996) Polyamine accumulation and near loss of morphogenesis in long-term callus cultures of rice (restoration of plant regeneration by manipulation of cellular polyamine levels). Plant Physiol 112(3):1343–1348

    PubMed  CAS  Google Scholar 

  • Bercetche J, Pâques M (1995) Somatic embryogenesis in maritime pine (Pinus pinaster). In: Jain SM, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, Vol. 3—gymnosperms. Kluwer Academic Publishers, Dordrecht, pp 221–242

    Google Scholar 

  • Bercetche J, Galerne M, Dereuddre J (1990) Enhancement regeneration from embryogenic callus of Picea abies L. karst after freezing in liquid nitrogen. Bull Soc Bot Fr Actual Bot 137:136–137

    Google Scholar 

  • Berlyn GP, Beck RC, Renfroe MH (1986) Tissue culture and the propagation and genetic improvement of conifers: problems and possibilities. Tree Physiol 1(2):227–240

    PubMed  CAS  Google Scholar 

  • Breton D, Harvengt L, Trontin JF, Bouvet A, Favre JM (2005) High subculture frequency, maltose-based and hormone-free medium sustained early development of somatic embryos in maritime pine. In Vitro Cell Dev Biol 41:494–504

    Article  CAS  Google Scholar 

  • Brisibe EA, Miyake H, Taniguchi T, Maeda E (1994) Regulation of somatic embryogenesis in long-term callus cultures of sugarcane (Saccharum officinarum L.). New Phytol 126:301–307

    Article  CAS  Google Scholar 

  • Cairney J, Xu N, MacKay J, Pullman J (2000) Transcript profiling: a tool to assess the development of conifer embryos. In Vitro Cell Dev Biol Plant 36:155–162

    CAS  Google Scholar 

  • Ciavatta VT, Morillon R, Pullman GS, Chrispeels MJ, Cairney J (2001) An aquaglyceroporin is abundantly expressed early in the development of the suspensor and the embryo proper of Loblolly pine. Plant Physiol 127:1556–1567

    Article  PubMed  CAS  Google Scholar 

  • Dunstan D, Bethune TD (1996) Variability in maturation and germination from white spruce somatic embryos, as affected by age and use of solid or liquid culture. In Vitro Cell Dev Biol Plant 32:165–170

    Google Scholar 

  • Find J, Grace L, Krogstrup P (2002) Effect of anti-auxins on maturation of embryogenic tissue cultures of Nordmanns fir (Abies nordmanniana). Physiol Plant 116(2):231–237

    Article  PubMed  CAS  Google Scholar 

  • Fourré JL, Berger P, Niquet L, André P (1997) Somatic embryogenesis and somaclonal variation in Norway spruce: morphogenetic, cytogenetic and molecular approaches. Theor Appl Genet 94(2):159–169

    Article  Google Scholar 

  • Gupta PK, Durzan DJ (1985) Shoot multiplication from mature trees of Douglas fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana). Plant Cell Rep 4:177–179

    Article  CAS  Google Scholar 

  • Harvengt L (2005) Somatic embryogenesis in Maritime pine (Pinus pinaster Ait.). In: Jain SM, Gupta PK (eds) Protocol of somatic embryogenesis in woody plants, 77, Springer Verlag, Berlin, pp 107–120

    Chapter  Google Scholar 

  • Lelu MA, Bastien C, Drugeault A, Gouez ML, Klimaszewska K (1999) Somatic embryogenesis and plantlet development in Pinus sylvestris and Pinus pinaster on medium with and without growth regulators. Physiol Planta 105(4):719–728

    Article  CAS  Google Scholar 

  • Liao YK, Amerson HV (1995) Slash pine (Pinus elliottii Engelm.) somatic embryogenesis II. Maturation of somatic embryos and plant regeneration. New Forest 10:165–182

    Google Scholar 

  • Miguel C, Gonçalves S, Tereso S, Marum L, Maroco J, Oliveira MM (2004) Somatic embryogenesis from 20 open-pollinated families of Portuguese plus trees of maritime pine. Plant Cell Tiss Org Cult 76(2):121–130

    Article  CAS  Google Scholar 

  • Mo LH, Egertsdotter U, von Arnold S (1996) Secretion of specific extracellular proteins by somatic embryos of Picea abies is dependent on embryo morphology. Ann Bot 77:143–152

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Planta 15:473–497

    Article  CAS  Google Scholar 

  • Ramarosandratana A, Harvengt L, Bouvet A, Calvayrac R, Pâques M (2001a) Effects of carbohydrate source, polyethylene glycol and gellan gum concentration on embryonal-suspensor mass (ESM) proliferation and maturation of maritime pine somatic embryos. In Vitro Cell Dev Biol 37:29–34

    Article  CAS  Google Scholar 

  • Ramarosandratana A, Harvengt L, Bouvet A, Calvayrac R, Pâques M (2001b) Influence of the embryonal-suspensor mass (ESM) sampling on development and proliferation of maritime pine somatic embryos. Plant Sci 160:473–479

    Article  CAS  Google Scholar 

  • Singh H (1978) Embryology of gymnosperms. Gebrüder Borntraeger, Berlin, Stuttgart

    Google Scholar 

  • Stasolla C, Belmonte MF, van Zyl L, Craig DL, Liu W, Yeung EC, Sederoff RR (2004) The effect of reduced glutathione on morphology and gene expression of white spruce (Picea glauca) somatic embryos. J Exp Bot 55(397):695–709

    Article  PubMed  CAS  Google Scholar 

  • Tremblay L, Levasseur C, Tremblay FM (1999) Frequency of somaclonal variation in plants of black spruce (Picea mariana, Pinaceae) and white spruce (Picea␣glauca, Pinaceae) derived from somatic embryogenesis and identification of some factors involved in genetic instability. Am J Bot 86(10):1373–1381

    Article  PubMed  Google Scholar 

  • van Zyl L, Bozhkov PV, Clapham DH, Sederoff RR, von Arnold S (2003) Up, down and up again is a signature of global gene expression pattern at the beginning of gymnosperm embryogenesis. Gene Expr Patterns 3:83–91

    Article  PubMed  Google Scholar 

  • von Aderkas P, Pattanavibool R, Hristoforoglu K, Ma Y (2003) Embryogenesis and genetic stability in long term megagametophyte-derived cultures of larch. Plant Cell Tiss Org Cult 75(1):27–34

    Article  Google Scholar 

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Acknowledgements

Participants of the European SEP project are acknowledged for productive exchanges during the last 4 years. Our thanks also go to the Biotechnology team at AFOCEL for the wonderful job done along years and to Yill-Sung Park for English reviewing. The SEP project, QLK5-CT−1999-00679—Propagation of Pines via Somatic Embryogenesis, was partly funded by EU but does not necessarily reflect the European commission’s views and in no way anticipates its future policy in this area.

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Authors and Affiliations

  1. Biotechnology Laboratory – Domaine de l’Etançon, AFOCEL, F-77370 , Nangis, France

    David Breton, Luc Harvengt & Jean-François Trontin

  2. Statistical Support – Domaine de l’Etançon, AFOCEL, F-77370, Nangis, France

    Alain Bouvet

  3. UMR-INRA IaM, Faculté des Sciences, Université Henri Poincaré, Nancy 1, BP 239, F-54506 , Vandoeuvre-lès-Nancy, France

    Jean-Michel Favre

Authors
  1. David Breton
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  2. Luc Harvengt
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  3. Jean-François Trontin
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  4. Alain Bouvet
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  5. Jean-Michel Favre
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Correspondence to Luc Harvengt.

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Breton, D., Harvengt, L., Trontin, JF. et al. Long-term subculture randomly affects morphology and subsequent maturation of early somatic embryos in maritime pine. Plant Cell Tiss Organ Cult 87, 95–108 (2006). https://doi.org/10.1007/s11240-006-9144-9

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  • DOI: https://doi.org/10.1007/s11240-006-9144-9

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