Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 111, Issue 2, pp 255–258 | Cite as

In vitro pollination in poplar of section Populus

  • Dietrich Ewald
  • Kristina Ulrich
Research Note


Two methods of in vitro pollination of aspen were tested and compared, first with moist pollen by transferring swollen pollen grains from a wet agar surface to the stigma and, second, using dry pollen grains. In vitro pollination with dry pollen grains appeared to be more efficient and this method was applied in three subsequent years. Additionally, pollination was performed with selected pollen grains in order to obtain triploid plants. Diploid pollen was induced by heat-treatment of pollen mother cells, but the enrichment of unreduced pollen from natural pollen mixtures was also successful. The selection of diploid pollen, which is larger in diameter than haploid pollen, was performed by sieving using micro sieves. Five triploid plants from different poplar combinations were obtained from 1,227 zygotic embryos cultured after in vitro pollination of 2,676 ovaries with dry pollen over 3 years. Embryo rescue was used to facilitate the development of immature zygotic embryos. The results of this study demonstrate the possibility to effect pollination using selected single pollen grains with the help of in vitro techniques.


Populus Diploid pollen Triploids Embryo rescue Triploid breeding Heat-treatment 



In vitro pollination


Stereo-zoom microscope



This work was supported by the Agency for Renewable Resources e.V. (FNR) within the framework of the project FastWOOD FKZ: 22011107. We are grateful to Hannelore Enkisch for her excellent technical assistance. We also thank Gisela Naujoks for supporting the flow cytometry work.


  1. Brewbaker JL, Kwack BH (1963) The essential role of calcium ion in pollen germination and pollen tube growth. Am J Bot 50:859–865CrossRefGoogle Scholar
  2. Dusi DMA, Alves ER, Willemse MTM, Falcão R, Do Valle CB, Carneiro VTC (2010) Toward in vitro fertilization in Brachiaria spp. Sex Plant Reprod 23:187–197PubMedCrossRefGoogle Scholar
  3. Ewald D, Ulrich K, Naujoks G, Schröder MB (2009) Induction of tetraploid poplar and black locust plants using colchicine: chloroplast number as an early marker for selecting polyploids in vitro. Plant Cell Tissue Organ Cult 99:353–357CrossRefGoogle Scholar
  4. Huang QJ, Zhang ZY, Kang XY (2002) 2n pollen of P. tomentosa × P. bolleana induced by four antimicrotubule agents. J Beijing For Univ 24:12–15Google Scholar
  5. Illies ZM (1974) Induction of haploid parthenogenesis in aspen by postpollination treatment with toluidine-blue. Silvae Genet 23:221–226Google Scholar
  6. Johnsson H, Eklundh C (1940) Colchicine treatment as a method in breeding hardwood species. Svensk Papp Tidn 43:373–377Google Scholar
  7. Kang XY, Zhu ZT (1997) A study on the 2n pollen vitality and germinant characteristics of white poplar. Acta Bot Yunnanica 19:402–406Google Scholar
  8. Kang XY, Zhu ZT, Lin HB (2000) Radiosensitivity of different ploidy pollen in poplar and its application. Acta Genet Sinica 27:78–82Google Scholar
  9. Liesebach H, Naujoks G, Ewald D (2010) Successful hybridisation of hybrid aspen (Populus tremula × P. tremuloides) and eastern cottonwood (P. deltoides). Sex Plant Reprod 24:189–198PubMedCrossRefGoogle Scholar
  10. Mashkina OS, Burdaeva IM, Belozerova MM, V’Yunova LN (1989) A method of inducing diploid pollen in woody species. Lesovedenie 1:19–25Google Scholar
  11. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  12. Ronald WG (1982) Intersectional hybridization of Populus sections Leuce-Aigeiros and Leuce-Tacamahaca. Silvae Genet 31:94–99Google Scholar
  13. Savka MA, Dawson JO, Jokela JJ, Skirvin RM (1987) A liquid culture method for rescuing immature embryos of eastern cottonwood. Plant Cell Tissue Organ Cult 10:221–226CrossRefGoogle Scholar
  14. Schenk RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocolyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199–204CrossRefGoogle Scholar
  15. Skàlovà D, Navràtilovà B, Ondrej V, Lebeda A (2010) Optimizing culture for in vitro pollination and fertilization in Cucumis sativus and C. melo. Acta Biol Crac Ser Bot 52:111–115CrossRefGoogle Scholar
  16. Van Tuyl JM, Van Diën MP, Van Creij MGM, Van Kleinwee TCM, Franken J, Bino RJ (1991) Application of in vitro pollination, ovary culture, ovule culture and embryo rescue for overcoming incongruity barriers in interspecific Lilium crosses. Plant Sci 74:115–126CrossRefGoogle Scholar
  17. Zhang Z, Kang X (2010) Cytological characteristics of numerically unreduced pollen production in Populus tomentosa Carr. Euphytica 173:151–159CrossRefGoogle Scholar
  18. Zhang Z, Kang X, Zhang P, Li Y, Wang J (2007) Incidence and molecular markers of 2n pollen in Populus tomentosa. Euphytica 154:145–152CrossRefGoogle Scholar
  19. Zhu Z, Kang X, Zhang Z (1997) Advances in the triploid breeding program of Populus tomentosa in China. J Beijing For Univ 6:1–8Google Scholar
  20. Zhu ZT, Kang XY, Zhang ZY (1998) Studies on selection of natural triploid of Populus tomentosa. Sci Silvae Sin 34:22–31Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  1. 1.Johann Heinrich von Thünen-Institute (vTI), Institute of Forest GeneticsWaldsieversdorfGermany

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