Advertisement

Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 132, Issue 1, pp 137–155 | Cite as

High gellan gum concentration and secondary somatic embryogenesis: two key factors to improve somatic embryo development in Pseudotsuga menziesii [Mirb.]

  • Marie-Anne Lelu-WalterEmail author
  • Florian Gautier
  • Kateřina Eliášová
  • Leopoldo Sanchez
  • Caroline Teyssier
  • Anne-Marie Lomenech
  • Claire Le Metté
  • Cathy Hargreaves
  • Jean-François Trontin
  • Cathie Reeves
Original Article

Abstract

Douglas-fir is a conifer species of major economic importance worldwide, including Western Europe and New Zealand. Herein we describe some characterization and significant refinement of somatic embryogenesis in Douglas-fir, with focus on maturation. The most typical structures observed in the embryonal masses were large polyembryogenic centres (up to 800–1500 µm) with a broad meristem, creating a compact cell “package” with suspensor cells. Singulated somatic embryos composed of both a embryonal head (300–400 µm) and long, tightly arranged suspensor were also frequent. Embryo development was enhanced following embryonal mass dispersion on filter paper discs at low density (50–100 mg fresh mass). Moreover, increasing gellan gum concentration in maturation medium (up to 10 g L−1) improved both the quantity and quality of cotyledonary somatic embryos (SEs), which were subsequently able to germinate and develop into plantlets at high frequency. Embryogenic yield was highly variable among the seven embryogenic lines tested (27–1544 SE g−1 fresh mass). Interestingly secondary somatic embryogenesis could be induced from cotyledonary SEs of both low- and highly-productive lines with some useful practical outcomes: secondary lines from low-performance lines (30–478 SE g−1 fresh mass) displayed significantly higher embryogenic yield (148–1343 SE g−1 fresh mass). In our best conditions, the total protein content in cotyledonary SEs increased significantly with maturation duration (up to 150 µg mg−1 fresh mass after 7 weeks) but remained below that of mature zygotic embryos (300 µg mg−1). The protein pattern was similar in both somatic and zygotic embryos, with major storage proteins identified as 7S-vicilin- and legumin-like proteins.

Keywords

Cell density Cleavage polyembryony Douglas-fir Embryogenic potential Protein pattern Vegetative propagation 

Notes

Acknowledgements

This research was partially funded by Future Forests Research Limited and a grant from the French Ministry of Foreign Affairs and the French Ministry of Higher Education and Research, and Technology Support Programme and Core funding provided by The Ministry of Business, Innovation and Employment in New Zealand through the France/New Zealand Science Cooperation Programme Dumont d’Urville (No. 25815PH). We would like to acknowledge the support of the University of Limoges, and The Chair of Excellence Forest Resources and Wood Uses, for the grant to Florian Gautier.

Author contributions

MALW designed and coordinated the study, carried out somatic embryogenesis and drafted the manuscript. FG participated in somatic embryogenesis and helped to draft the manuscript. KE performed histological and microscopic analyses and drafted the manuscript. LS performed the statistical analyses and drafted the manuscript. CT performed protein analysis and drafted the manuscript. AML carried out mass spectrometric analysis and helped to draft the manuscript. CLM carried out somatic embryogenesis and collected the material. CH participated in the design of the study and helped to draft the manuscript. JFT participated in the design of the study and drafted the manuscript. CR performed English editing and drafted the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11240_2017_1318_MOESM1_ESM.docx (45 kb)
Supplementary material 1 (DOCX 44 KB)
11240_2017_1318_MOESM2_ESM.docx (2.2 mb)
Supplementary material 2 (DOCX 2299 KB)

References

  1. Aberlenc-Bertossi F, Chabrillange N, Duval Y, Tregear J (2008) Contrasting globulin and cysteine proteinase gene expression patterns reveal fundamental developmental differences between zygotic and somatic embryos of oil palm. Tree Physiol 28:1157–1167CrossRefPubMedGoogle Scholar
  2. Abrahamsson M, Valladares S, Larsson E, Clapham D, von Arnold S (2012) Patterning during somatic embryogenesis in Scots pine in relation to polar auxin transport and programmed cell death. Plant Cell Tissue Organ Cult 109:391–400CrossRefGoogle Scholar
  3. Abrahamsson M, Valladares S, Merino I, Larsson E, von Arnold S (2017) Degeneration pattern in somatic embryos of Pinus sylvestris L. In Vitro Cell Dev Biol-Plant 53:88–96CrossRefGoogle Scholar
  4. Beneš K, Kamínek M (1973) The use of aluminium lake of nuclear fast red in plant material successively with alcian blue. Biol Plant 15:294–297CrossRefGoogle Scholar
  5. Bonga JM (2015) A comparative evaluation of the application of somatic embryogenesis, rooting of cuttings, and organogenesis of conifers. Can J For Res 45:1–5CrossRefGoogle Scholar
  6. Cvikrová M, Vondrakova Z, Eliášová K, Pešek B, Trávníčková A, Vágner M (2016) The impact of UV-B irradiation applied at different phases of somatic embryo development in Norway spruce on polyamine metabolism. Trees 30:113–124CrossRefGoogle Scholar
  7. Dean CA, Welty DE, Herold GE (2009) Performance and genetic parameters of somatic and zygotic progenies of coastal Douglas-fir at 71/2-years across Washington and Oregon, USA. Silvae Genetica 58:212–219Google Scholar
  8. Dungey HS, Low CB, Lee J, Miller MA, Fleet K, Yanchuk AD (2012) Developing breeding and deployment options for Douglas-fir in New Zealand: breeding for future forest conditions. Silvae Genetica 61:104–115CrossRefGoogle Scholar
  9. Durzan DJ, Gupta PK (1987) Somatic embryogenesis and polyembryogenesis in Douglas-fir cell suspension cultures. Plant Sci 52:229–235CrossRefGoogle Scholar
  10. Eastman PAK, Webster FB, Pitel JA, Roberts DR (1991) Evaluation of somaclonal variation during somatic embryogenesis of interior spruce (Picea glauca engelmannii complex) using culture morphology and isozyme analysis. Plant Cell Rep 10:425–430CrossRefPubMedGoogle Scholar
  11. Forward BS, Tranbarger TJ, Misra S (2001) Characterization of proteinase activity in stratified Douglas-fir seeds. Tree Physiol 21:625–629CrossRefPubMedGoogle Scholar
  12. Green MJ, Mc Leod JK, Misra S (1991) Characterization of Douglas fir protein body composition by SDS-PAGE and electron microscopy. Plant Physiol Biochem 29:49–55Google Scholar
  13. Gupta PK (1996) Method for reproducing conifers by somatic embryogenesis using a maltose enriched maintenance medium: US Patent No. 5,563,061. U.S. Patent and Trademark Office, Washington, DCGoogle Scholar
  14. Gupta PK, Pullman GS (1996) Method for reproducing Douglas-fir by somatic embryogenesis. U.S. Patent No. 5,482,857. U.S. Patent and Trademark Office, Washington, DCGoogle Scholar
  15. Gupta PK, Timmis R (2005) Mass propagation of conifer trees in liquid cultures-progress towards commercialization. Plant Cell Tissue Organ Cult 81:339–346CrossRefGoogle Scholar
  16. Gupta PK, Timmis R, Timmis KA, Carlson WC, Welty EDE (1995) Somatic embryogenesis in Douglas-fir (Pseudotsuga menziesii). In: Jain S, Gupta P, Newton R (eds) Somatic embryogenesis in woody plants, vol 3. Kluwer Academic Publishers, Dordrecht, pp 303–313CrossRefGoogle Scholar
  17. Hakman I, Hallberg H, Palovaara J (2009) The polar auxin transport inhibitor NPA impairs embryo morphology and increases the expression of an auxin efflux facilitator protein PIN during Picea abies somatic embryo development. Tree Physiol 29:483–496CrossRefPubMedGoogle Scholar
  18. Hargreaves CL, Reeves CB, Find JI, Gough K, Josekutty P, Skudder DB, Van der Maas SA, Sigley MR, Menzies MI, Low CB, Mullin TJ (2009) Improving initiation, genotype capture, and family representation in somatic embryogenesis of Pinus radiata by a combination of zygotic embryo maturity, media, and explant preparation. Can J For Res 39:1566–1574CrossRefGoogle Scholar
  19. Hargreaves CL, Reeves CB, Find JI, Gough K, Menzies MI, Low CB, Mullin TJ (2011) Overcoming the challenges of family and genotype representation and early cell line proliferation in somatic embryogenesis from control-pollinated seeds of Pinus radiata. NZ J Forest Sci 41:97–114Google Scholar
  20. Harvengt L, Trontin JF, Reymond I, Canlet F, Pâques M (2001) Molecular evidence of true-to-type propagation of a 3-year-old Norway spruce through somatic embryogenesis. Planta 213:828–832CrossRefPubMedGoogle Scholar
  21. Hermann RK, Lavender DP (1999) Douglas-fir planted forests. New Forest 17:53–70CrossRefGoogle Scholar
  22. Hong L, Boulay M, Gupta PK, Durzan DJ (1992) Variations in somatic polyembryogenesis: induction of adventitious embryonal-suspensor masses on developing Douglas-fir embryos. In: Ahuja MR (ed) Woody plant biotechnology, vol 210. Plenum Press, New York, pp 105–121Google Scholar
  23. Isah T (2016) Induction of somatic embryogenesis in woody plants. Acta Physiol Plant 38:118–139CrossRefGoogle Scholar
  24. Käll L, Canterbury JD, Weston J, Noble WS, MacCoss MJ (2007) Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat Methods 4:923–925CrossRefPubMedGoogle Scholar
  25. Klimaszewska K, Smith D (1997) Maturation of somatic embryos of Pinus strobus is promoted by a high concentration of gellan gum. Physiol Plant 100:949–957CrossRefGoogle Scholar
  26. Klimaszewska K, Bernier-Cardou M, Cyr DR, Sutton BCS (2000) Influence of gelling agents on culture medium gel strength, water availability, tissue water potential, and maturation response in embryogenic cultures of Pinus strobus L. Vitro Cell Dev Biol-Plant 36:279–286CrossRefGoogle Scholar
  27. Klimaszewska K, Noceda C, Pelletier G, Label P, Rodriguez R, Lelu-Walter M-A (2009) Biological characterization of young and aged embryogenic cultures of Pinus pinaster (Ait.). In Vitro Cell Dev Biol-Plant 45:20–33CrossRefGoogle Scholar
  28. Klimaszewska K, Overton C, Stewart D, Rutledge RG (2010) Initiation of somatic embryos and regeneration of plants from primordial shoots of 10-year-old somatic white spruce and expression profile of 11 genes followed during tissue culture process. Planta 233:635–647CrossRefPubMedGoogle Scholar
  29. Klimaszewska K, Hargreaves CL, Lelu-Walter M-A, Trontin J-F (2016) Advances in conifer somatic embryogenesis since year 2000. In: Germanà MA, Lambardi M (eds) In vitro embryogenesis in higher plants, chap. 7, methods in molecular biology. Springer, New York, pp 131–162. doi: 10.1007/978-1-4939-3061-6_8 CrossRefGoogle Scholar
  30. Kong L, von Aderkas P (2011) A novel method of cryopreservation without a cryoprotectant for immature somatic embryos of conifer. Plant Cell Tissue Organ Cult 106:115–125CrossRefGoogle Scholar
  31. Kong L, Denchev P, Radley R, Lobatcheva II, Attree SM (2012) Method of culturing conifer somatic embryos using S (+)- abscisic acid. U.S. Patent No. US 8,124,412 B2. U.S. Patent and Trademark Office, Washington, DCGoogle Scholar
  32. Lelu M-A, Klimaszewska K, Charest P (1994) Somatic embryogenesis from immature and mature zygotic embryos and from cotyledons and needles of somatic plantlets of Larix. Can J For Res 24:100–106CrossRefGoogle Scholar
  33. Lelu M-A, Bastien C, Drugeault A, Gouez ML, Klimaszewska K (1999) Somatic embryogenesis and plantet development in Pinus sylvestris and Pinus pinaster on medium with and without growth regulators. Physiol Plant 105:719–728CrossRefGoogle Scholar
  34. Lelu-Walter M-A, Pâques LE (2009) Simplified and improved somatic embryogenesis of hybrid larches (Larix × eurolepis and Larix × marschlinsii). Perspectives for breeding. Ann For Sci 66:104CrossRefGoogle Scholar
  35. Lelu-Walter M-A, Bernier-Cardou M, Klimaszewska K (2008) Clonal plant production from self- and cross-pollinated seed families of Pinus sylvestris (L.) through somatic embryogenesis. Plant Cell Tissue Organ Cult 92:31–45CrossRefGoogle Scholar
  36. Lelu-Walter M-A, Thompson D, Harvengt L, Sanchez L, Toribio M, Pâques LE (2013) Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction. Tree Gene Genomes 9:883–899CrossRefGoogle Scholar
  37. Lelu-Walter M-A, Klimaszewska K, Miguel C, Aronen T, Hargreaves C, Teyssier C, Trontin J-F (2016) Somatic embryogenesis for more effective breeding and deployment of improved varieties in Pinus spp.: bottlenecks and recent advances. In: Loyola-Vargas VM, Ochoa-Alejo N (eds) Somatic embryogenesis—fundamental aspects and applications, chapter 19. Springer, Switzerland, pp 319–365. doi: 10.1007/978-3-319-33705-0_19 CrossRefGoogle Scholar
  38. Litvay JD, Verma DC, Johnson MA (1985) Influence of a loblolly pine (Pinus taeda L.). Culture medium and its components on growth and somatic embryogenesis of the wild carrot (Daucus carota L.). Plant Cell Rep 4:325–328CrossRefPubMedGoogle Scholar
  39. Merkle S, Cunningham M (2011) Southern hardwood varietal forestry: a new approach to short-rotation woody crops for biomass energy. J Forest 109:7–14Google Scholar
  40. Miguel CM, Rupps A, Raschke J, Rodrigues AS, Trontin J-F (2016) Impact of molecular studies on somatic embryogenesis development for implementation in conifer multi-varietal forestry. In: Park Y-S, Bonga JM, Moon H-K (eds) Vegetative propagation of forest trees. Korea Forest Research Institute, Seoul, pp 373–421. ISBN 978-89-8176-064-9Google Scholar
  41. Miller JT, Knowles FB (1994) Introduced forest trees in New Zealand: recognition, role and seed source. Forest Research Bulletin No. 124. No. 14. Douglas-fir Pseudotsuga menziesii (Mirbel) FrancoGoogle Scholar
  42. Ministry of Agriculture and Forestry (2011) A national exotic forest description as at April 15, 2010. Retrieved 27 Nov 2011 from http://www.maf.govt.nz/newsresources/publications
  43. Morel A, Teyssier C, Trontin JF, Eliášová K, Pešek B, Beaufour M, Morabito D, Boizot N, Le Metté C, Belal-Bessai L, Reymond I, Harvengt L, Cadene M, Corbineau F, Vágner M, Label P, Lelu-Walter MA (2014a) Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability: transcriptomic and proteomic analyses. Physiol Plant 152:184–201CrossRefPubMedGoogle Scholar
  44. Morel A, Trontin JF, Corbineau F, Lomenech A-M, Beaufour M, Reymond I, Le Metté C, Ader K, Harvengt L, Cadene M, Label P, Teyssier C, Lelu-Walter M-A (2014b) Cotyledonary somatic embryos of Pinus pinaster Ait. most closely resemble fresh, maturing cotyledonary zygotic embryos: biological, carbohydrate and proteomic analyses. Planta 240:1075–1095CrossRefPubMedGoogle Scholar
  45. Muñoz F, Sanchez L (2015) breedR: statistical methods for forest genetic resources analysts. R package version 0.11. https://github.com/famuvie/breedR
  46. Noah AM, Niemenak N, Sunderhaus S, Haase C, Omokolo DN, Winkelmann T, Braun H-P (2013) Comparative proteomic analysis of early somatic and zygotic embryogenesis in Theobroma cacao L. J Proteomics 78:123–133CrossRefPubMedGoogle Scholar
  47. Pullman GS, Zhang Y, Phan BH (2003) Brassinolide improves embryogenic tissue initiation in conifers and rice. Plant Cell Rep 22:96–104CrossRefPubMedGoogle Scholar
  48. Pullman GS, Mein J, Johnson S, Zhang Y (2005) Gibberellin inhibitors improve embryogenic tissue initiation in conifers. Plant Cell Rep 23:596–605CrossRefPubMedGoogle Scholar
  49. R Core Team (2016) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. http://www.R-project.org/
  50. Reeves C, Hargreaves C, Trontin J-F, Lelu-Walter M-A (2017) Simple and efficient protocols for the initiation and proliferation of embryogenic tissue of Douglas-fir. Trees (in press)Google Scholar
  51. Ruaud JN (1993) Maturation and conversion into plantlets of somatic embryos derived from needles and cotyledons of 7, 56-day-old Picea abies. Plant Sci 92:213–220CrossRefGoogle Scholar
  52. Ruaud JN, Bercetche J, Pâques M (1992) First evidence of somatic embryogenesis from needles of 1-year-old Picea abies plants. Plant Cell Rep 11:563–566CrossRefPubMedGoogle Scholar
  53. Saly S, Joseph C, Corbineau F, Lelu M-A, Côme D (2002) Induction of secondary somatic embryogenesis in hybrid larch (Larix × leptoeuropaea) as related to ethylene. Plant Growth Regul 37:287–294CrossRefGoogle Scholar
  54. Shelbourne CJA, Low CB, Gea LD, Knowles RL (2007) Achievements in forest tree genetic improvement in Australia and New Zealand 5: genetic improvement of Douglas-fir in New Zealand. Aust Forest 70:28–32CrossRefGoogle Scholar
  55. Sterk P, de Vries S (1993) Molecular markers for plant embryos. In: Redenbaugh K (ed) Synseeds: applications of synthetic seeds to crop improvement. CRC Press, Boca Raton, pp 115–132Google Scholar
  56. Taber RP, Zhang C, Hu WS (1998) Kinetics of Douglas-fir (Pseudotsuga menziesii) somatic embryo development. Can J Bot 76:863–871Google Scholar
  57. Terskikh VV, Feurtado JA, Borchardt S, Giblin M, Abrams SR, Kermode AR (2005) In vivo 13C NMR metabolite profiling: potential for understanding and assessing conifer seed quality. J Exp Bot 56:2253–2265CrossRefPubMedGoogle Scholar
  58. Teyssier C, Grondin C, Bonhomme L, Lomenech A-M, Vallance M, Morabito D, Label P, Lelu-Walter MA (2011) Increased gelling agent concentration promotes somatic embryo maturation in hybrid larch (Larix × eurolepis): a 2-DE proteomic analysis. Physiol Plant 141:152–165CrossRefPubMedGoogle Scholar
  59. Teyssier C, Maury S, Beaufour M, Grondin C, Delaunay A, Le Metté C, Ader K, Cadene M, Label P, Lelu-Walter MA (2014) In search of markers for somatic embryo maturation in hybrid larch (Larix × eurolepis): global DNA methylation and proteomic analyses. Physiol Plant 150:271–291CrossRefPubMedGoogle Scholar
  60. Timmis R, Grob JA, Gupta PK, Rayfield SD (2011) Methods for increasing germination vigor by early singulation of conifer somatic embryos. U.S. Patent No. 7,964,404. U.S. Patent and Trademark Office, Washington, DCGoogle Scholar
  61. Trontin J-F, Klimaszewska K, Morel A, Hargreaves C, Lelu-Walter M-A (2016a). Molecular aspects of conifer zygotic and somatic embryo development: a review of genome-wide approaches and recent insights. In: Germanà MA, Lambardi M (eds) In vitro embryogenesis in higher plants, methods in molecular biology, vol 1359, chapter 8. Springer, New York, pp 167–207. doi: 10.1007/978-1-4939-3061-6_8 CrossRefGoogle Scholar
  62. Trontin J-F, Aronen T, Hargreaves C, Montalbán IA, Moncaleán P, Reeves C, Quoniou S, Lelu-Walter M-A, Klimaszewska K (2016b) International effort to induce somatic embryogenesis in adult pine trees. In: Park Y-S, Bonga JM, Moon H-K (eds) Vegetative propagation of forest trees. Korea Forest Research Institute Seoul, Korea, pp 211–260. ISBN 978-89-8176-064-9Google Scholar
  63. Uddenberg D, Valladares S, Abrahamsson M, Sundström JF, Sundås-Larsson A, von Arnold S (2011) Embryogenic potential and expression of embryogenesis-related genes in conifers are affected by treatment with a histone deacetylase inhibitor. Planta 234:527–539CrossRefPubMedPubMedCentralGoogle Scholar
  64. von Aderkas P, Bonga JM (2000) Influencing micropropagation and somatic embryogenesis in mature trees by manipulation of phase change, stress and culture environment. Tree Physiol 20:921–928CrossRefGoogle Scholar
  65. von Aderkas P, Teyssier C, Charpentier JP, Gutmann M, Pâques L, Le Metté C, Ader K, Label P, Kong L, Lelu-Walter M-A (2015) Effect of light conditions on anatomical and biochemical aspects of somatic and zygotic embryos of hybrid larch (Larix × marschlinsii). Ann Bot 115:605–615CrossRefGoogle Scholar
  66. Vondráková Z, Cvikrová M, Eliášová K, Martincová O, Vágner M (2010) Cryotolerance in Norway spruce and its association with growth rates, anatomical features and polyamines of embryogenic cultures. Tree Physiol 30:1335–1348CrossRefPubMedGoogle Scholar
  67. Vondráková Z, Eliášová K, Fischerová L, Vágner M (2011) The role of auxins in somatic embryogenesis of Abies alba. Cent Eur J Biol 6:587–596Google Scholar
  68. Vondráková Z, Eliášová K, Vágner M, Martincová O, Cvikrová M (2015) Exogenous putrescine affects endogenous polyamine levels and the development of Picea abies somatic embryos. Plant Growth Regul 75:405–414CrossRefGoogle Scholar
  69. Vooková B, Kormuták A (2006) Comparison of induction frequency, maturation capacity and germination of Abies numidica during secondary somatic embryogenesis. Biol Plant 50:785–788CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  • Marie-Anne Lelu-Walter
    • 1
    Email author
  • Florian Gautier
    • 1
    • 2
  • Kateřina Eliášová
    • 3
  • Leopoldo Sanchez
    • 1
  • Caroline Teyssier
    • 1
  • Anne-Marie Lomenech
    • 4
  • Claire Le Metté
    • 1
  • Cathy Hargreaves
    • 5
  • Jean-François Trontin
    • 6
  • Cathie Reeves
    • 5
  1. 1.INRA, UR 0588, Amélioration, Génétique et Physiologie ForestièreOrléans Cedex 2France
  2. 2.Université de Limoges, Laboratoire de Chimie des Substances NaturellesLimogesFrance
  3. 3.Institute of Experimental Botany CASPraha 6-LysolajeCzech Republic
  4. 4.Université de Bordeaux, Centre de Génomique Fonctionnelle, Plateforme ProtéomeBordeauxFrance
  5. 5.ScionRotoruaNew Zealand
  6. 6.FCBA, Pôle Biotechnologie et Sylviculture AvancéeCestasFrance

Personalised recommendations