Plant Cell, Tissue and Organ Culture

, Volume 94, Issue 3, pp 319–328 | Cite as

Stress-induced changes important for effective androgenic induction in isolated microspore culture of triticale (×Triticosecale Wittm.)

  • Iwona ŻurEmail author
  • Ewa Dubas
  • Elżbieta Golemiec
  • Magdalena Szechyńska-Hebda
  • Franciszek Janowiak
  • Maria Wędzony
Original Paper


The accumulation of abscisic acid (ABA) and the activities of antioxidative enzymes along with cell metabolic activity were monitored during androgenesis induction in triticale (×Triticosecale Wittm.). Tested cultivars ‘Mieszko’ and ‘Wanad’ were selected due to their significantly different responses to androgenic induction. Significant variation was observed in respect of superoxide dismutase activity and endogenous ABA content in anthers isolated from freshly cut tillers. For both cultivars, tillers pretreatment with low temperature decreased peroxidase activity by 36%, highly accelerated respiration rate and reduced heat production. At the same time, the level of ABA in ‘Mieszko’ was increased to the level measured in ‘Wanad’. This effect was associated with higher microspore culture viability and increased stress tolerance in ‘Mieszko’. Low temperature and metabolic starvation during 4-day anther preculture did not influence activities of antioxidative enzymes, while it resulted in slight decrease in respiration rate and heat emission. The importance of these changes for effective androgenesis induction is discussed.


Abscisic acid Antioxidative enzymes Metabolic activity Oxidative stress Androgenesis 



Abscisic acid




Anthers isolated from cold pretreated tillers


Doubled haploids


Dry weight


Embryo-like structures


Anthers isolated from freshly cut tillers


Fluorescein diacetate


α-Naphthaleneacetic acid


Precultured anthers




Reactive oxygen species


Superoxide dismutase



The research was supported by the project KBN23/E189/SPB/COST/P06/Dz585/2002-2005.


  1. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126PubMedCrossRefGoogle Scholar
  2. Asada K, Takahashi M (1987) Production and scavenging of active oxygen in photosynthesis. In: Kyle DJ, Osmond CB, Arntzen CJ (eds) Photoinhibition. Elsevier, Amsterdam, pp 227–287Google Scholar
  3. Blokhina O, Virolainen E, Fagerstedt KV (2003) Antioxidants, oxidative damage and oxygen deprivation stress: a review. Ann Bot 91:179–194PubMedCrossRefGoogle Scholar
  4. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  5. Dat J, Vandenabeele S, Vranová E, Van Montagu M, Inzé D, Van Breusegem F (2000) Dual action of the active oxygen species during plant stress responses. Cell Mol Life Sci 57:779–795PubMedCrossRefGoogle Scholar
  6. Davies WJ, Jones HG (eds) (1991) In: Abscisic acid physiology and biochemistry. Bio’s Scientific, Oxford UK, pp 63–77Google Scholar
  7. Devaux P, Hou L, Ullrich SE, Huang Z, Kleinhofs A (1993) Factors affecting anther culturability of recalcitrant barley genotypes. Plant Cell Rep 13:32–36CrossRefGoogle Scholar
  8. Dunwell JM, Francis RJ, Powell W (1987) Anther culture of Hordeum vulgare L.: a genetic study of microspore callus production and differentiation. Theor Appl Genet 74:60–64CrossRefGoogle Scholar
  9. Forster BP, Thomas WTB (2003) Doubled haploids in genetic mapping and genomics. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants. A manual. Kluwer Acad. Publ., Dordrecht/Boston/London, pp 367–390Google Scholar
  10. Foyer CH (2002) The contribution of photosynthetic oxygen metabolism to oxidative stress in plants. In: Inzé D, Van Montagu M (eds) Oxidative stress in plants. Taylor and Francis, pp 33–68Google Scholar
  11. Gorbunova VJ, Kruglova NN (1996) Genetical determination of cereal androgenesis in vitro: hormonal aspects. In: Abstracts of the 14th international congress of sexual plant reproduction, Lorne, 18–23 February 1996, p 146Google Scholar
  12. Gorbunova VJ, Kruglova NN, Abramov SN (2001) The induction of androgenesis in vitro in spring soft wheat. Balance of exogenus and endogenous phytohormones. Biol Bull 28(1):25–30CrossRefGoogle Scholar
  13. Gu HH, Hagberg P, Zhou WJ (2004) Cold pretreatment enhances microspore embryogenesis in oilseed rape (Brassica napus L.). Plant Growth Reg 42:137–143CrossRefGoogle Scholar
  14. Guzman M, Arias FJZ (2000) Increasing anther culture efficiency in rice (Oryza sativa L.) using anthers from ratooned plants. Plant Sci 151:107–114PubMedCrossRefGoogle Scholar
  15. Hou L, Ullrich SE, Kleinhofs A, Stiff CM (1993) Improvement of anther culture methods for doubled haploid production in barley breeding. Plant Cell Rep 12:334–338CrossRefGoogle Scholar
  16. Hu TC, Ziauddin A, Simion E, Kasha KJ (1995) Isolated microspore culture of wheat (Triticum aestivum L.) in a defined media: I. Effects of pretreatment, isolation methods and hormones. In Vitro Cell Dev Biol 31:79–83CrossRefGoogle Scholar
  17. Imamura J, Harada H (1980) Effects of abscisic acid and water stress on the embryo and plantlet production in anther culture of Nicotiana tabacum cv. Samsun Z Pflanzenphysiol 100:285–289Google Scholar
  18. Immonen S, Anttila H (1999) Cold pretreatment to enhance green plant regeneration from rye anther culture. Plant Cell Tiss Org Cult 57:121–127CrossRefGoogle Scholar
  19. Immonen S, Robinson J (2000) Stress treatments and ficoll for improving green plant regeneration in triticale anther culture. Plant Sci 150:77–84CrossRefGoogle Scholar
  20. Jähne A, Lörz H (1995) Cereal microspore culture. Plant Sci 109:1–12CrossRefGoogle Scholar
  21. Kyo M, Harada H (1986) Control of the developmental pathway of tobacco pollen in vitro. Planta 168:427–432CrossRefGoogle Scholar
  22. Leipner J, Fracheboud Y, Stamp P (1999) Effect of growing season on the photosynthetic apparatus and leaf antioxidative defences in two maize genotypes of different chilling tolerance. Environ Exp Bot 42:129–139CrossRefGoogle Scholar
  23. Lück H (1962) Katalase, Peroxydase, Reduktasen, Saccharase, Xanthinoxydase. In: Bergmeyer HU (ed) Methoden der enzymatischen Analyse. Verlag Chemie, Weinheim/Bergstraße, pp 895–897Google Scholar
  24. Ma R, Guo Y-D, Pulli S (2004) Comparison of anther and microspore culture in the embryogenesis and regeneration of rye (Secale cereale). Plant Cell Tiss Org Cult 76:147–157CrossRefGoogle Scholar
  25. McCord JM, Fridovich I (1969) Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244:6049–6055PubMedGoogle Scholar
  26. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7(9):405–410PubMedCrossRefGoogle Scholar
  27. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9(10):490–498PubMedCrossRefGoogle Scholar
  28. Pauk J, Puolimatka M, Tóth KL, Monostori T (2000) In vitro androgenesis of triticale in isolated microspore culture. Plant Cell Tiss Org Cult 61:221–229CrossRefGoogle Scholar
  29. Płażek A (2002) Physiological aspects of cold or ozone induced cross tolerance of barley (Hordeum vulgare L.), meadow fescue (Festuca pratensis Huds.) and rapeseed (Brassica napus L.) to biotic stress. Habilitation thesis, Agriculture University, Kraków, pp 68–82Google Scholar
  30. Ponitka A, Ślusarkiewicz-Jarzina A, Wędzony M, Marcińska I, Woźna J (1999) The influence of various in vitro culture conditions on androgenic embryo induction and plant regeneration from hexaploid triticale (×Triticosecale Wittm.) anther culture. J Appl Genet 40(3):165–170Google Scholar
  31. Powell W (1990) Environmental and genetical aspects of pollen embryogenesis. In: Bajaj VPS (ed) Haploids in crop improvement. I. Biotechnology in agriculture and forestry, vol 12. Springer-Verlag, Berlin, pp 45–65Google Scholar
  32. Prasad TK, Anderson MD, Martin BA, Steward RC (1994) Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide. Plant Cell 6:65–74PubMedCrossRefGoogle Scholar
  33. Ritala A, Mannonen L, Osman-Caldentey K-M (2001) Factors affecting the regeneration capacity of isolated barley microspore (Hordeum vulgare L.). Plant Cell Rep 20:403–407CrossRefGoogle Scholar
  34. Ryöppy PH (1997) Haploidy in triticale. In: Jain SM, Sopory SK, Veilleux RE (eds) In vitro haploid production in higher plants, vol 4. Springer, Berlin Heidelberg, pp 117–131Google Scholar
  35. Sato S, Katoh N, Iwai S, Hagimori M (2002) Effect of low temperature pretreatment of buds or inflorescence on isolated microspore culture in Brassica rapa (syn. B. campestris). Breed Sci 52:23–26CrossRefGoogle Scholar
  36. Schumann G (1990) In vitro production of haploids in triticale. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, wheat, vol 13. Springer-Verlag, Berlin Heidelberg, pp 383–402Google Scholar
  37. Shim YS, Kasha KJ (2003) Barley microspore transformation protocol by biolistic gun. In: Maluszynski M, Kasha KJ, Forster BP, Szarejko I (eds) Doubled haploid production in crop plants. A manual. Kluwer Acad. Publ., Dordrecht/Boston/London, pp 363–367Google Scholar
  38. Seppänen MM, Fagerstedt K (2000) The role of superoxide dismutase activity in response to cold acclimation in potato. Physiol Plant 108:279–285CrossRefGoogle Scholar
  39. Szarejko I, Forster BP (2007) Doubled haploidy and induced mutation. Euphytica 158:359–370CrossRefGoogle Scholar
  40. Tenhola-Roininen T, Tanhuanpää P, Immonen S (2005) The effect of cold and heat treatments on the anther culture response of diverse rye genotypes. Euphytica 145:1–9CrossRefGoogle Scholar
  41. Touraev A, Vicente O, Heberle-Bors E (1997) Initiation of microspore embryogenesis by stress. Trends Plant Sci 2(8):297–302CrossRefGoogle Scholar
  42. Touraev A, Tashpulatov A, Indrianto A, Barinova J, Katholnigg H, Akimcheva S, Ribarits A, Voronin V, Zhexsembekova M, Heberle-Bors E (2000) Fundamental aspects of microspore embryogenesis. Proc COST Action 824, “Biotechnological approaches for utilisation of gametic cells” Bled, Slovenia 1–5 July 2000, pp 205–214Google Scholar
  43. Tsang EW, Bowler C, Herouart D, Van Camp W, Inzé D (1991) Differential regulation of superoxide dismutases in plants exposed to environmental stress. Plant Cell 3:783–792PubMedCrossRefGoogle Scholar
  44. Van Bergen S, Kottenhagen MJ, Van Der Meulen RM, Wang M (1999) The role of abscisic acid in induction of androgenesis: a comparative study between Hordeum vulgare L. cvs. Igri and Digger. J Plant Growth Regul 18:135–143PubMedCrossRefGoogle Scholar
  45. Walker-Simmons MK, Abrams SR (1991) Use of ABA immunoassays. In: Davies WJ, Jones HG (eds) Abscisic acid, physiology and biochemistry. Bios Scientific Publishers, Oxford, pp 53–63Google Scholar
  46. Wang M, Oppedijk BJ, Lu X, Van Duijn B, Schilperoort RA (1996) Apoptosis in barley aleurone during germination and its inhibition by abscisic acid. Plant Mol Biol 32:1125–1134PubMedCrossRefGoogle Scholar
  47. Wang M, Hoekstra S, Van Bergen S, Lamers GEM, Oppedijk BJ, Van Der Heijden MW, De Priester W, Schilperoort RA (1999) Apoptosis in developing anthers and the role of ABA in this process during androgenesis in Hordeum vulgare L. Plant Mol Biol 39:489–501PubMedCrossRefGoogle Scholar
  48. Wang M, Van Bergen S, Van Duijn B (2000) Insights into a key developmental switch and its importance for efficient plant breeding. Plant Physiol 124:523–530PubMedCrossRefGoogle Scholar
  49. Zhuang JJ, Xu J (1983) Increasing differentiation frequencies in wheat pollen callus. In: Hu H, Vega MR (eds) Cell and tissue culture techniques for cereal crop improvement. Science Press, Beijing, p 431Google Scholar
  50. Zoriniants S, Tashpulatov AS, Heberle-Bors E, Touraev A (2005) The role of stress in the induction of haploid microspore embryogenesis. In: Palmer CE (ed) Haploids in crop improvements. II. Springer-Verlag, Berlin Heidelberg New York, pp 35–52CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Iwona Żur
    • 1
    Email author
  • Ewa Dubas
    • 1
  • Elżbieta Golemiec
    • 1
  • Magdalena Szechyńska-Hebda
    • 1
  • Franciszek Janowiak
    • 1
  • Maria Wędzony
    • 1
    • 2
  1. 1.Institute of Plant Physiology Polish Academy of SciencesKrakowPoland
  2. 2.Pedagogical University of KrakówKrakowPoland

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