Cytology and Genetics

, Volume 52, Issue 2, pp 117–126 | Cite as

Effects of Exogenous Phytohormones on Spore Germination and Morphogenesis of Polystichum aculeatum (L.) Roth Gametophyte in vitro Culture

  • L. M. Babenko
  • K. O. Romanenko
  • M. M. Shcherbatiuk
  • O. V. Vasheka
  • P. O. Romanenko
  • V. A. Negretsky
  • I. V. Kosakivska


The effects of exogenous phytohormones–gibberellic acid (GA3) and benzyl-aminopurine (BAP)–on the spore germination and morphogenesis of Polystichum aculeatum (L.) Roth gametophyte in vitro culture were studied. In the control, four stages of gametophyte morphogenesis were determined and their periods were established. Spore germination and protonema formation of P. aculeatum occurred according to the Vittaria-type and prothalium development according to the Aspidium-type. The spore germination percentage depended on the storage time duration. It was found that 80–95% of freshly collected spores germinated. Spore viability was within the range of 68–95% after 4–6-month storage under lab conditions and did not exceed 20% after 1.5 year of the storage period. High concentrations of exogenous GA3 (10–5 and 10–6 M) and BAP (10–5 M) significantly inhibited spore germination, whereas low concentrations (GA3 10–7–10–8 M) had an insignificant stimulating effect or did not affect germination at all (BAP 10–6, 10–7, 10–8 M). All concentrations of exogenous BAP were demonstrated to inhibit the development of P. aculeatum gametophyte at the protonema stage, which might be due to the removal of apical dominance. The inhibiting effect directly depended on BAP concentrations. The formation of abnormal thalli of the P. aculeatum gametophyte in the response to exogenous GA3 treatments occurred as a result of impairment of cell growth by elongation. A direct interrelationship between GA3 concentrations and level of morphological abnormalities and grade of thalli underdevelopment was demonstrated.


Polystichum aculeatum gametophyte protonema prothallium thallus benzylaminopurine gibberellic acid 


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  1. 1.
    Babenko, L.M., Sheyko, O.A., Kosakivska, I.V., Vedenichova, N.P., Nehretsky, V.A., and Vasheka, V.O., Structural and functional characteristics of Pteridophytes (Polypodiophyta), Bull. Charkovsky Natl. Agr. Univ., Ser. Biol., 2015, vol. 1, no. 34, pp. 80–103.Google Scholar
  2. 2.
    Ross, M., Mapping the world’s pteridophyte diversity, systematics and floras, in Pteridology in Perspective, Camus, J.M. and Johns, R.J., Eds, Kew: Rojal Bot. Gardens, 1996, pp. 29–42.Google Scholar
  3. 3.
    Haufler, C.H., Pryer, K.M., Schuettpelz, E., Sessa, E.B., Farrar, D.R., Moran, R., Schneller, J.J., Watkins, J.E., Jr., and Windham, M.D., Sex and the single gametophyte: revising the homosporous vascular plant life cycle in light of contemporary research, BioScience, 2016, vol. 66, no. 11, pp. 928–937.CrossRefGoogle Scholar
  4. 4.
    Atallah, N.M. and Banks, J.A., Reproduction and the pheromonal regulation of sex type in fern gametophytes, Front. Plant Sci., 2015, vol. 6, pp. 100–107.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Anterola, A., Shanle, E., Mansouri, K., Schuette, S., and Renzaglia, K., Gibberellin precursor is involved in spore germination in the moss Physcomitrella patens, Planta, 2009, vol. 229, no. 4, pp. 1003–1007.CrossRefPubMedGoogle Scholar
  6. 6.
    Guillon, J.M. and Raquin, C., Environmental sex determination in the genus equisetum: sugars induce male sex expression in cultured gametophytes, Int. J. Plant Sci., 2002, vol. 163, no. 5, pp. 825–830.CrossRefGoogle Scholar
  7. 7.
    Tanaka, J., Yano, K., Aya, K., Hirano, K., Takehara, S., Koketsu, E., Ordonio, R.L., Park, S.-H., Nakajima, M., Ueguchi-Tanaka, M., and Matsuoka, M., Antheridiogen determines sex in ferns via a spatiotemporally split gibberellin synthesis pathway, Science, 2014, vol. 346, no. 6208, pp. 469–473.CrossRefPubMedGoogle Scholar
  8. 8.
    Tanurdzic, M. and Banks, J.A., Sex determining mechanisms in land plants, Plant Cell, 2004, vol. 16, suppl., pp. 61–71.CrossRefGoogle Scholar
  9. 9.
    Vasjuk, V.A. and Kosakivska, I.V., Gibberellins in ferns: participation in regulation of physiological processes, Ukr. Bot. J., 2015, vol. 72, no. 1, pp. 65–73.CrossRefGoogle Scholar
  10. 10.
    Gerashchenkov, G.A. and Rozhnova, N.A., The involvement of phytohormones in the plant sex regulation, Russ. J. Plant Physiol., 2013, vol. 60, no. 5, pp. 597–610.CrossRefGoogle Scholar
  11. 11.
    Gantait, S., Sinniah, U.R., Ali, N., and Sahu, N.C., Gibberellins-a multifaceted hormone in plant growth regulatory network, Curr. Protein Pept. Sci., 2015, vol. 16, no. 5, pp. 406–412.CrossRefPubMedGoogle Scholar
  12. 12.
    Gupta, R. and Chakraborty, S., Gibberellic acid in plant, Plant. Signal. Behav., 2013, vol. 8, no. 9, p. e25504.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kosakivska, I.V., Babenko, L.M., Shcherbatiuk, M.M., Vedenicheva, N.P., Voytenko, L.V., and Vasyuk, V.A., Phytohormones during growth and development of Polypodiophyta, Adv. Biol. Earth Sci., 2016, vol. 1, no. 1, pp. 26–44.Google Scholar
  14. 14.
    Nakajima, M., Shimada, A., Takashi, Y., Kim, Y.C., Park, S.H., Ueguchi-Tanaka, M., Suzuki, H., Katoh, E., Iuchi, S., Kobayashi, M., Maeda, T., Matsuoka, M., and Yamaguchi, I., Identification and characterization of Arabidopsis gibberellin receptors, Plant J., 2006, vol. 46, no. 5, pp. 880–889.CrossRefPubMedGoogle Scholar
  15. 15.
    Furber, M., Mander, L.N., Nester, J.E., Takahashi, N., and Yamane, H., Structure of an antheridiogen from the fern Anemia mexicana, Phytochemistry, 1989, vol. 28, no. 1, pp. 63–66.CrossRefGoogle Scholar
  16. 16.
    Menéndez, V., Revilla, M.A., Bernard, P., Gotor, V., and Fernández, H., Gibberellins and antheridiogen on sex in Blechnum spicant L., Plant Cell Rep., 2006, vol. 25, no. 10, pp. 1104–1110.CrossRefPubMedGoogle Scholar
  17. 17.
    Hollingsworth, N.S., Andres, E.A., and Greery, G.K., Pheromonal interactions among gametophytes of Osmundastrum cinnamomeum L. and the origins of antheridiogen systems in leptosporangiate ferns, Int. J. Plant Sci., 2012, vol. 173, no. 4, pp. 382–390.CrossRefGoogle Scholar
  18. 18.
    Kazmierczak, A., Induction of cell division and cell expansion at the beginning of gibberellin A3-induced precocious antheridia formation in Anemia phyllitidis gametophytes, Plant Sci., 2003, vol. 165, no. 5, pp. 933–939.CrossRefGoogle Scholar
  19. 19.
    Takeno, K.M. and Furuya, M., Inhibitory effect of gibberellins on archegonial differentiation in Lygodium japonicum, Physiol. Plant., 1977, vol. 39, no. 2, pp. 135–138.CrossRefGoogle Scholar
  20. 20.
    Guo, Q.X., Shen, Y.X., Song, X.H., and Zhao, H.T., The effects of spores germination and planting rate of Athyrium multidentatum, Chin. Agric. Sci. Bull., 2007, vol. 23, no. 2, pp. 343–345.Google Scholar
  21. 21.
    Li, J., Yuan, Y.B., and Cao, Z.X., View of the cytology and biochemistry of sexual reproduction of algae and pteridophyta, Chin. Bull. Bot., 1995, vol. 12, no. 2, pp. 1–8.Google Scholar
  22. 22.
    Naf, U., On dark-germination and antheridium formation in Anemia phyllitidis, Physiol. Plant., 1966, vol. 19, no. 4, pp. 1079–1088.CrossRefGoogle Scholar
  23. 23.
    Nester, J.E. and Coolbaugh, R.C., Factors influencing spore germination and early gametophyte development in Anemia mexicana and Anemia phyllitidis, Plant Physiol., 1986, vol. 82, no. 1, pp. 230–235.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Suo, J., Chen, S., Zhao, Q., Shi, L., and Dai, Sh., Fern spore germination in response to environmental factors, Front. Biol., 2015, vol. 10, no. 4, pp. 358–376.CrossRefGoogle Scholar
  25. 25.
    Weinberg, E.S. and Voeller, B.R., Induction of fern spore germination, Proc. Natl. Acad. Sci. U. S. A., 1969, vol. 64, no. 2, pp. 835–842.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Zhang, Z. and Dai, S., Effect of environmental factors on fern spore germination, Acta Ecol. Sin., 2010, vol. 30, no. 7, pp. 1882–1893.Google Scholar
  27. 27.
    Zhang, J.W. and Niu, J.Y., The effects of culture ground substances, GA3 and B, on spores germination and planting rate of Pteridium aquilinum, Acta Pratac. Sin., 1999, vol. 8, no. 1, pp. 62–68.Google Scholar
  28. 28.
    Vasyuk, V.A., Vedenicheva, N.P., and Musatenko, L.I., Matteucia struthiopteris (L.) Tod. endogenous phytohormones, in Mat-ly Vseukr. nauk. konf. “Botanika ta mikolohiia: problemy i perspektyvy na 2011–2020 roky” (Proc. All-Ukr. Sci. Conf. “Botany and Mycology: Problems and Prospects for 2011–2020”), Kyiv, 2011, pp. 257–258.Google Scholar
  29. 29.
    Guiragossian, H.A. and Koning, R.E., Induction of spore germination in Schizaea pusilla (Schizaeaceae), Am. J. Bot., 1986, vol. 73, no. 11, pp. 1588–1594.CrossRefGoogle Scholar
  30. 30.
    Ren, B.R., Xia, B., Li, W.L., Wu, J.L., and Zhao, Y.Y., Investigation on spore germination of Sphenomeris chinensis (Lindsaeaceae), Acta Bot. Yun., 2008, vol. 30, no. 6, pp. 713–717.Google Scholar
  31. 31.
    Zhai, G.Y., Bian, K., Jia, K.G., and Zhu, L.X., Effect of GA3 and MS medium ratio treatments on spore germination of wild brake, China Veget., 2007, vol. 8, pp. 21–23.Google Scholar
  32. 32.
    Huang, S., Cerny, R.E., Qi, Y., Bhat, D., Aydt, C.M., Hanson, D.D., Malloy, K.P., and Ness, L.A., Transgenic studies on the involvement of cytokinin and gibberellin in male development, Plant Physiol., 2003, vol. 131, no. 3, pp. 1270–1282.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Vedenicheva, N.P. and Kosakivska, I.V., Modern aspects of cytokinins studies: evolution and crosstalk with other phytohormones, Plant Physiol. Genet., 2016, vol. 4, no. 1, pp. 3–15.Google Scholar
  34. 34.
    Kieber, J.J. and Schaller, G.E., Cytokinins, Arabidopsis Book, 2014, vol. 12, p. e0168.CrossRefPubMedGoogle Scholar
  35. 35.
    Vankova, R., Cytokinin regulation of plant growth and stress responses, phytohormones: a window to metabolism, in Signaling and Biotechnological Applications, Tran, L.-S. and Pal, S., Eds., New York: Springer, 2014, pp. 55–79.Google Scholar
  36. 36.
    Vedenicheva, N.P., Cytokinins as regulators of plant organs growth under different conditions, Bull. Kharkiv Nat. Agrar. Univ., Ser. Biol., 2016, vol. 1, no. 37, pp. 6–26.Google Scholar
  37. 37.
    Chia, S.G.E. and Raghavan, V., Abscisic acid effect on spore germination and protonemal growth in the fern, Mohria caffrorum, New Phytol., 1982, vol. 92, no. 1, pp. 31–37.CrossRefGoogle Scholar
  38. 38.
    Chen, S.Y. and Read, P.E., Micropropagation of leatherleaf fern (Rumohra adiantiformis), Proc. Fla. State Hort. Soc., 1983, vol. 96, pp. 266–269.Google Scholar
  39. 39.
    Paull, R.E. and Chantrachit, T., Benzyladenine and the vase life of tropical ornamentals, Postharvest Biol. Technol., 2001, vol. 21, no. 3, pp. 303–310.CrossRefGoogle Scholar
  40. 40.
    Spiro, M.D., Torabi, B., and Cornell, C.N., Cytokinins induce photomorphogenic development in dark-grown gametophytes of Ceratopteris richardii, Plant Cell Physiol., 2004, vol. 45, no. 9, pp. 1252–1260.CrossRefPubMedGoogle Scholar
  41. 41.
    Menéndez, V., Abul, Y., Bohanec, B., Lafont, E., and Fernández, H., The effect of exogenous and endogenous phytohormones on the in vitro development of gametophyte and sporophyte in Asplenium nidus L., Acta Physiol. Plant., 2011, vol. 33, no. 6, pp. 2493–2500.CrossRefGoogle Scholar
  42. 42.
    Greer, G.K., Dietrich, M.A., DeVol, J.A., and Rebert, A., The effects of exogenous cytokinin on the morphology and gender expression of Osmunda regalis gametophytes, Am. Fern J., 2012, vol. 102, no. 1, pp. 32–46.CrossRefGoogle Scholar
  43. 43.
    Raghavan, V., Developmental Biology of Fern Gametophytes, Cambridge: Univ. Press, 1989.CrossRefGoogle Scholar
  44. 44.
    Vasheka, O.V. and Bezsmertna, I.O., Atlas of the Ferns Flora of Ukraine, Kyiv: Palyvoda, 2012.Google Scholar
  45. 45.
    Kotuhov, Yu.A., Method of phenological observations of ferns of the family Polypodiaceae, Byull. Gl. Bot. Sada, 1974, vol. 94, pp. 10–18.Google Scholar
  46. 46.
    Vasheka, O.V., Some biological characteristics of Dryopteris Adans introduced in open ground Fomin Botanical Garden, Bull. Nikit. Bot. Sad, 2004, vol. 89, pp. 12–15.Google Scholar
  47. 47.
    Grichuk, V.P. and Monoszon, M.H., The Determinant of Single-Beam Spores of the Ferns from the Family Polypodiaceae R. Br. Growing on the Territory of the USSR, Moscow: Nauka, 1971.Google Scholar
  48. 48.
    Arnautova, E.M., Gametophytes of Equisporous Ferns, St. Petersburg: Univ. Press, 2008.Google Scholar
  49. 49.
    Schneller, J.J., Untersuchungen an einheimischen Farnen, insbesondere der Dryopteris filix-mas-gruppe. 3. Teil. Okologiche Untersuchungen, Ber. Schweiz. Bot. Ges., 1975, vol. 85, no. 2, pp. 110–159.Google Scholar
  50. 50.
    Quintanilla, L.G., Amigo, J., Pangua, E., and Pajaron, S., Effect of storage method on spore viability in five globally threatened fern species, Ann. Bot., 2002, vol. 90, no. 4, pp. 461–467.CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Nayar, B.K. and Kaur, S., Gametophytes of homosporous ferns, Bot. Rev., 1971, vol. 37, pp. 295–396.CrossRefGoogle Scholar
  52. 52.
    Dyer, A.F., Fern gametophytes in culture—a simple system for studying plant development and reproduction, J. Biol. Educ., 1983, vol. 17, no. 1, pp. 23–39.CrossRefGoogle Scholar
  53. 53.
    Pittermann, J., Brodersen, C., and Watkins, J., The physiological resilience of fern sporophytes and gametophytes: advances in water relations offer new insights into an old lineage, Front. Plant Sci., 2013, vol. 4, art. 285, pp. 1–10.CrossRefGoogle Scholar
  54. 54.
    Menéndez, V., Villacorta, N.F., Revilla, M.A., Gotor, V., Bernard, P., and Fernández, H., Exogenous and endogenous growth regulators on apogamy in Dryopteris affinis (Lowe) Fraser-Jenkins, Plant Cell Rep., 2006, vol. 25, no. 2, pp. 85–91.CrossRefPubMedGoogle Scholar
  55. 55.
    Kazmierczak, A., Studies on morphology and metabolism of prothalli during GA3-induced formation of antheridia in Anemia phyllitidis, Acta Physiol. Plant., 1998, vol. 20, no. 3, pp. 277–283.CrossRefGoogle Scholar
  56. 56.
    Menéndez, V., Revilla, M.A., Fal, M.A., and Fernández, H., The effect of cytokinins on growth and sexual organ development in the gametophyte of Blechnum spicant L., Plant Cell Tiss. Organ Cult., 2009, vol. 96, no. 3, pp. 245–250.CrossRefGoogle Scholar

Copyright information

© Allerton Press, Inc. 2018

Authors and Affiliations

  • L. M. Babenko
    • 1
  • K. O. Romanenko
    • 1
  • M. M. Shcherbatiuk
    • 1
  • O. V. Vasheka
    • 2
  • P. O. Romanenko
    • 2
  • V. A. Negretsky
    • 1
  • I. V. Kosakivska
    • 1
  1. 1.Kholodny Institute of BotanyNational Academy of Sciences of UkraineKyivUkraine
  2. 2.Institute of Biology and MedicineTaras Shevchenko National University of KyivKyivUkraine

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