Skip to main content
SpringerLink
Log in

Selection of haploid cell lines from megagametophyte cultures of maritime pine as a DNA source for massive sequencing of the species

  • Original Paper
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

The potential of haploid tissues for genetic studies in conifers is hampered by the lack of abundant and homogeneous plant material suitable for DNA isolation. In this work we have determined factors promoting haploid callus induction and proliferation from megagametophytes of Oria 6, a genotype of Pinus pinaster Aiton (maritime pine) from the natural population Sierra de Oria (Almería, Spain), selected based on its response to extreme drought conditions. The generation of haploid callus was restricted to megagametophytes isolated from light-brown cones with no dehydrated seeds collected in September. Culture medium composition did not significantly affect callus induction, but a modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine favored further multiplication. The ploidy status of the callus lines was determined by flow cytometry and seven polymorphic microsatellites. A total of sixteen haploid callus lines were established and one of these is being used as a source of DNA for massive sequencing of maritime pine genome.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Abacus Concepts (1989) SuperAnova 1.01. Abacus Concepts, Berkeley

    Google Scholar 

  • Aleza P, Juárez J, Hernández M, Pina JA, Ollitrault P, Navarro L (2009) Recovery and characterization of a Citrus clementina Hort. ex Tan. ‘Clemenules’ haploid plant selected to establish the reference whole Citrus genome sequence. BMC Plant Biol 9:110–127

    Article  PubMed Central  PubMed  Google Scholar 

  • Andersen SB (2005) Haploids in the improvement of woody species. In: Palmer CE, Keller WA, Kasha KJ (eds) Haploids in crop improvement II. Biotechnology in agriculture and forestry, vol 56. Springer, Berlin, pp 243–257

    Google Scholar 

  • Aranda I, Alía R, Ortega U, Dantas AK, Majada J (2010) Intra-specific variability in biomass partitioning and carbon isotopic discrimination under moderate drought stress in seedlings from four Pinus pinaster populations. Tree Genet Genomes 6:169–178

    Article  Google Scholar 

  • Azevedo H, Dias A, Tavares RM (2008) Establishment and characterization of Pinus pinaster suspension cell cultures. Plant Cell Tiss Org Cult 93:115–121

    Article  CAS  Google Scholar 

  • Baldursson S, Ahuja MR (1996) Haploidy in forest tress. In: Jain MS, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants, vol 1. Kluwer, Dordrecht, pp 49–66

    Chapter  Google Scholar 

  • Baldursson S, Nørgaard J, Krogstrup P (1993) Factors influencing Haploid callus initiation and proliferation in megagametophyte cultures of Sitka spruce (Picea sitchensis). Silvae Genet 42:79–86

    Google Scholar 

  • Birol I, Raymond A, Jackman SD, Pleasance S, Coope R, Taylor GA, Yuen MM, Keeling CI, Brand D, Vandervalk BP, Kirk H, Pandoh P, Moore RA, Zhao Y, Mungall AJ, Jaquish B, Yanchuk A, Ritland C, Boyle B, Bousquet J, Ritland K, Mackay J, Bohlmann J, Jones SJ (2013) Assembling the 20 Gb white spruce (Picea glauca) genome from whole-genome shotgun sequencing data. Bioinformatics 29:1492–1497

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Blasco M, Barra A, Brisa C, Corredoira E, Segura J, Toribio M, Arrillaga I (2013) Somatic embryogenesis in holm oak male catkins. Plant Growth Regul 71:261–270

    Article  CAS  Google Scholar 

  • Bonga JM (1974) In vitro culture of microsporophylls and megagametophyte tissue of Pinus. In Vitro 9:270–278

    Article  CAS  PubMed  Google Scholar 

  • Cao H, Biswas MK, Lü Y, Amar MH, Tong Z, Xu Q, Xu J, Guo W, Deng X (2011) Doubled haploid callus lines of Valencia sweet orange recovered from anther culture. Plant Cell Tiss Organ Cult 104:415–423

    Article  Google Scholar 

  • de Lucas AI, Robledo-Arnuncio JJ, Hidalgo E, González-Martínez SC (2008) Mating system and pollen gene flow in Mediterranean maritime pine. Heredity 100:390–399

    Article  Google Scholar 

  • Dunwell JM (2010) Haploids in flowering plants: origins and exploitation. Plant Biotechnol J 8:377–424

    Article  CAS  PubMed  Google Scholar 

  • Forster BP, Heberle-Bors E, Kasha KJ, Touraev A (2007) The resurgence of haploids in higher plants. Trends Plant Sci 12:368–375

    Article  CAS  PubMed  Google Scholar 

  • Guevara MA, Chagné D, Almeida H, Byrnes M, Collada C, Favre JM, Harvengt L, Jeandroz S, Orazio C, Plomion C, Ramboer A, Rocheta M, Sebastiani F, Soto A, Vendramin GG, Cervera MT (2005) Isolation and characterization of nuclear microsatellite loci in Pinus pinaster Ait. Mol Ecol Notes 5:57–59

    Article  CAS  Google Scholar 

  • Humánez A, Blasco M, Brisa C, Segura J, Arrillaga I (2012) Somatic embryogenesis from different tissues of Spanish populations of maritime pine. Plant Cell Tiss Organ Cult 11:373–383

    Article  Google Scholar 

  • Lelu-Walter MA, Bernier-Cardou M, Klimaszewska K (2006) Simplied and improved somatic embriogenesis for clonal propagation or Pinus pinaster (Ait.). Plant Cell Rep 25:767–776

    Article  CAS  PubMed  Google Scholar 

  • Mackay J, Dean JFD, Plomion C, Peterson DG, Cánovas FM, Pavy N, Ingvarsson PK, Savolainen O, Guevara MA, Fluch S, Vinceti B, Abarca D, Díaz-Sala C, Cervera MT (2012) Towards decoding the conifer giga-genome. Plant Mol Biol 80:555–569

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Neale DB, Kremer A (2011) Forest tree genomics: growing resources and applications. Nat Rev Genet 12:111–122

    Article  CAS  PubMed  Google Scholar 

  • Nystedt B, Street NR, Wetterbom A, Zuccolo A, Lin YC, Scofield DG, Vezzi F, Delhomme N, Giacomello S, Alexeyenko A, Vicedomini R, Sahlin K, Sherwood E, Elfstrand M, Gramzow L, Holmberg K, Hällman J, Keech O, Klasson L, Koriabine M, Kucukoglu M, Käller M, Luthman J, Lysholm F, Niittylä T, Olson A, Rilakovic N, Ritland C, Rosselló JA, Sena J, Svensson T, Talavera-López C, Theißen G, Tuominen H, Vanneste K, Wu ZQ, Zhang B, Zerbe P, Arvestad L, Bhalerao R, Bohlmann J, Bousquet J, Gil RG, Hvidsten TR, de Jong P, MacKay J, Morgante M, Ritland K, Sundberg B, Thompson SL, Van de Peer Y, Andersson B, Nilsson O, Ingvarsson PK, Lundeberg J, Jansson S (2013) The Norway spruce genome sequence and conifer genome evolution. Nature 497:579–584

    Article  CAS  PubMed  Google Scholar 

  • Ochatt SJ, Patat-Ochatt EM, Moessner A (2011) Ploidy level determination within the context of in vitro breeding. Plant Cell Tiss Org Cult 104:329–341

    Article  Google Scholar 

  • Owens JN, Kittirat T, Mahalovich MF (2008) Whitebark pine (Pinus albicaulis Engelm.) seed production in natural stands. For Ecol Manag 255:803–809

    Article  Google Scholar 

  • Pattanavibool R, von Aderkas P, Hanhijärvi A, Simola LK, Bonga JM (1995) Diploidization in megagametophyte-derived cultures of the gymnosperm Larix decidua. Theor Appl Genet 90:671–674

    Article  CAS  PubMed  Google Scholar 

  • Pichot C, El Maataoui M (1997) Flow cytometric evidence for multiple ploidy levels in the endosperm of some gymnosperm species. Theor Appl Genet 94:865–870

    Article  Google Scholar 

  • Ravi M, Chan SWL (2010) Haploid plants produced by centromere-mediated genome elimination. Nature 464:615–619

    Article  CAS  PubMed  Google Scholar 

  • Sánchez-Gómez D, Majada J, Alía R, Feito I, Aranda I (2010) Intraspecific variation in growth and allocation patterns in seedlings of Pinus pinaster Ait. Ann For Sci 67:505p1–505p8 (submitted to contrasting watering regimes: can water availability explain regional variation?)

    Article  Google Scholar 

  • Sederoff R (2013) A spruce sequence. Nature 497:569–570

    Article  CAS  PubMed  Google Scholar 

  • Simola LK, Santanen A (1990) Improvement of nutrient medium for growth and embryogenesis of megagametophyte and embryo callus lines of Picea abies. Physiol Plant 80:27–35

    Article  CAS  Google Scholar 

  • Tukey JW (1953) Some selected quick and easy methods of statistical analysis. Trans NY Acad Sci 16:88–97

    Article  CAS  Google Scholar 

  • von Aderkas P, Bonga JM (1988) Formation of haploid embryoids of Larix decidua: early embryogenesis. Am J Bot 75:690–700

    Article  Google Scholar 

  • von Aderkas P, Bonga JM (1993) Plants from haploid tissue of Larix decidua. Theor Appl Genet 87:225–228

    Article  Google Scholar 

  • von Aderkas P, Klimaszewska K, Bonga JM (1990) Diploid and haploid embryogenesis in Plants from haploid tissue of Larix leptoletis, L. decidua, and their reciprocal hybrids. Can J For Res 20:9–14

    Article  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 75:27–34

    Article  Google Scholar 

  • Wegrzyn JL, Lin BY, Zieve JJ, Dougherty WM, Martínez-García PJ, Koriabine M, Holtz-Morris A, deJong P, Crepeau M, Langley CH, Puiu D, Salzberg SL, Neale DB, Stevens KA (2013) Insights into the loblolly pine genome: characterization of BAC and fosmid sequences. PLoS One 8:e72439

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Xu Q, Chen LL, Ruan X, Chen D, Zhu A, Chen C, Bertrand D, Jiao WB, Hao BH, Lyon MP, Chen J, Gao S, Xing F, Lan H, Chang JW, Ge X, Lei Y, Hu Q, Miao Y, Wang L, Xiao S, Biswas MK, Zeng W, Guo F, Cao H, Yang X, Xu XW, Cheng YJ, Xu J, Liu JH, Luo OJ, Tang Z, Guo WW, Kuang H, Zhang HY, Roose ML, Nagarajan N, Deng XX, Ruan Y (2013) The draft genome of sweet orange (Citrus sinensis). Nat Genet 45:59–66

    Article  CAS  PubMed  Google Scholar 

  • Yu J (2009) The potential of ultrahigh throughput genomic technologies in crop improvement. Plant Genome 2:2

    Article  Google Scholar 

Download references

Acknowledgments

Research funds were provided by Spanish National projects (BIO2007-29814 and BIO2010-12302-E) and Generalitat Valenciana (Prometeo/2009/075). The research leading to these results has also received funding from the European Union’s Seventh Framework Programme (FP7/2007–2013) under Grant Agreement No. 289841 (ProCoGen). We thank M. A. Morcillo for technical assistance, Dr. José Juarez (Instituto Valenciano de Investigaciones Agrarias, IVIA, Spain) and Dr. Vesselina Nikolova (Vegetable Crops Research Institute, Plovdiv, Bulgaria) for their support in the flow cytometry and karyological analyses, respectively.

Author information

Author notes

  1. J. Muñoz-Bertomeu

    Present address: Instituto de Biologia Molecular y Celular de Plantas, Universidad Politécnica de Valencia-Consejo Superior de Investigaciones Cientificas, 46022, Valencia, Spain

Authors and Affiliations

  1. Departamento de Biología Vegetal, Facultad de Farmacia, ERI Biotecmed, Universidad de Valencia, Avda. Vicent Andrés Estellés s/n, 46100, Burjassot, Valencia, Spain

    I. Arrillaga, J. Muñoz-Bertomeu, D. Lázaro-Gimeno, L. Torralba, I. Mendoza-Poudereux & J. Segura

  2. Departamento de Ecología y Genética Forestal, INIA-CIFOR, Carretera de la Coruña Km 7.5, 28040, Madrid, Spain

    M. A. Guevara, E. Sáez-Laguna, L. M. Díaz & M. T. Cervera

  3. Unidad Mixta de Genómica y Ecofisiología Forestal, INIA/UPM, Madrid, Spain

    M. A. Guevara, E. Sáez-Laguna, L. M. Díaz & M. T. Cervera

Authors
  1. I. Arrillaga
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. A. Guevara
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. Muñoz-Bertomeu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D. Lázaro-Gimeno
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. E. Sáez-Laguna
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. L. M. Díaz
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. L. Torralba
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. I. Mendoza-Poudereux
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. J. Segura
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. M. T. Cervera
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to I. Arrillaga.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11240_2014_470_MOESM1_ESM.jpg

Supplementary data legend: Profiles of SSR marker genetic analysis of lines generated from megagametophyte cultures. Oria 6, mother tree; L1-L12 and L14-L24 putative haploid lines; L13, L25 and L26 diploid controls. (JPEG 1055 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Arrillaga, I., Guevara, M.A., Muñoz-Bertomeu, J. et al. Selection of haploid cell lines from megagametophyte cultures of maritime pine as a DNA source for massive sequencing of the species. Plant Cell Tiss Organ Cult 118, 147–155 (2014). https://doi.org/10.1007/s11240-014-0470-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-014-0470-z

Keywords

Search

Navigation