, Volume 223, Issue 4, pp 637–645 | Cite as

Abscisic acid and stress treatment are essential for the acquisition of embryogenic competence by carrot somatic cells

  • Akira Kikuchi
  • Nobuya Sanuki
  • Katsumi Higashi
  • Tomokazu Koshiba
  • Hiroshi Kamada
Original Article


Studies of carrot embryogenesis have suggested that abscisic acid (ABA) is involved in somatic embryogenesis. A relationship between endogenous ABA and the induction of somatic embryogenesis was demonstrated using stress-induced system of somatic embryos. The embryonic-specific genes C-ABI3 and embryogenic cell proteins (ECPs) were expressed during stress treatment prior to the formation of somatic embryos. The stress-induction system for embryogenesis was clearly distinguished by two phases: the acquisition of embryogenic competence and the formation of a somatic embryo. Somatic embryo formation was inhibited by the application of fluridone (especially at 10−4 M), a potent inhibitor of ABA biosynthesis, during stress treatment. The inhibitory effect of fluridone was nullified by the simultaneous application of fluridone and ABA. The level of endogenous ABA increased transiently during stress. However, somatic embryogenesis was not significantly induced by the application of only ABA to the endogenous level, in the absence of stress. These results suggest that the induction of somatic embryogenesis, in particular the acquisition of embryogenic competence, is caused not only by the presence of ABA but also by physiological responses that are directly controlled by stresses.


Somatic embryogenesis Carrot (Daucus carotaStress Abscisic acid 



Abscisic acid


2,4-dichlorophenoxyacetic acid


Embryogenic cells


Embryogenic cell protein


Gas chromatography-selected ion monitoring-mass spectrometry


Indole acetic acid


Non-embryogenic cells



This work was supported, in part, by grant-in-aids from the Research for the Future Program of the Japan Society for the Promotion of Science (JSPS-RFTF00L01601) and the Ministry of Education, Sports, Science, and Technology, Japan.


  1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1987) Phenol/SDS Method for plant RNA preparation. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current protocols in molecular biology. Greene Publishing Associates and Wiley-Interscience, New York, pp 4.3.1–4.3.4Google Scholar
  2. Bartels PG, Watson CW (1978) Inhibition of carotenoid synthesis by fluridone and norflurazon. Weed Sci 26:198–203Google Scholar
  3. Bentley B, Morgan CB, Morgan DG, Sand FA (1975) Plant growth substances and effects of phytoperiod on flower and flower bud development in Phaseolus vulgaris. Nature 256:121–122CrossRefGoogle Scholar
  4. Czarncka E, Edelman L, Schoffl F, Key JL (1984) Comparative analysis of physical stress responses in soybean seedlings using cloned heat shock cDNAs. Plant Mol Biol 3:45–58CrossRefGoogle Scholar
  5. Fedina IS, Tsonev TD, Guleva EI (1994) ABA as modulator of the response of Pisum sativum to salt stress. J Plant Physiol 143:245–249Google Scholar
  6. Galli MG, Levi M (1982) Increased drought resistance induced by pretreatment with abscisic acid in germinating embryos of Haplopappus gracilis. Physiol Plant 54:425–430CrossRefGoogle Scholar
  7. Harada H, Kiyosue T, Kamada H, Kobayashi K (1990) Stress-induced carrot somatic embryogenesis and their application to synthetic seeds. In: Sangwan RS, Sangwan-Norreel BS (eds) The impact of biotechnology in agriculture. Kluwer Academic Publishers, Dordrecht, pp 129–157Google Scholar
  8. Imamura J, Okabe E, Kyo M, Harada H (1982) Embryogenesis and plantlet formation through direct pollen culture of isolated pollen of Nicotiana tabacum cv. Samsun and Nicotiana rustica cv. Rustica. Plant Cell Physiol 23:713–716Google Scholar
  9. Kamada H, Harada H (1979) Studies on organogenesis in carrot tissue culture. I. Effects of growth regulation on somatic embryogenesis and root formation. Z Pflanzenphysiol 91:225–266Google Scholar
  10. Kamada H, Kobayashi K, Kiyosue T, Harada H (1989) Stress-induced somatic embryogenesis in carrot and its application to synthetic seed production. In Vitro Cell Dev Biol 25:1163–1166CrossRefGoogle Scholar
  11. Kamada H, Ishikawa K, Saga H, Harada H (1993) Induction of somatic embryogenesis in carrot by osmotic stress. Plant Tissue Cult Lett 10:38–44Google Scholar
  12. Kamada H, Tachikawa Y, Saitou T, Harada H (1994) Heat stress induction of carrot somatic embryogenesis. Plant Tissue Cult Lett 11:229–232Google Scholar
  13. Keller WA, Armstrong KC (1978) High-frequency production of microspore-derived plants from Brassica napus anther cultures. Z Pflanzenphysiol 80:100–108Google Scholar
  14. Kiyosue T, Kamada H, Harada H (1989a) Induction of somatic embryogenesis from carrot seeds by hypochlorite treatment. Plant Tissue Cult Lett 6:138–143Google Scholar
  15. Kiyosue T, Kamada H, Harada H (1989b) Induction of somatic embryogenesis by salt stress in carrot. Plant Tissue Cult Lett 6:162–164Google Scholar
  16. Kiyosue T, Takano K, Kamada H, Harada H (1990) Induction of somatic embryogenesis in carrot by heavy metal ions. Can J Bot 68:2021–2033CrossRefGoogle Scholar
  17. Kiyosue T, Satoh S, Kamada H, Harada H (1992a) Purification and immunohistochemical detection of an embryogenic cell protein in carrot. Plant Physiol 95:1077–1083Google Scholar
  18. Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K, Higashi K, Satoh S, Kamada H, Harada H (1992b) Partial amino-acid sequence of ECP31, a carrot embryogenic-cell protein, and enhancement of its accumulation by abscisic acid in somatic embryos. Planta 186:337–342CrossRefGoogle Scholar
  19. Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K, Higashi K, Satoh S, Kamada H, Harada H (1992c) Isolation and characterization of a cDNA that encodes ECP31, an embryogenic-cell protein from carrot. Plant Mol Biol 19:239–249PubMedCrossRefGoogle Scholar
  20. Kiyosue T, Nakajima M, Yamaguchi I, Satoh S, Kamada H, Harada H (1992) Endogeneous levels of abscisic acid in embryogenic cells, non-embryogenic cells and somatic embryos of carrot (Daucus carota L.). Biochem Physiol Pflanzen 188:343–347Google Scholar
  21. Kiyosue T, Satoh S, Kamada H, Harada H (1993a) Somatic embryogenesis in higher plants. J Plant Res Special Issue 3:75–82Google Scholar
  22. Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K, Kamada H, Harada H (1993b) cDNA cloning of ECP40, an embryogenic-cell protein in carrot, and its expression during somatic and zygotic embryogenesis. Plant Mol Biol 21:1053–1068PubMedCrossRefGoogle Scholar
  23. Koornneef M, Reuling G, Karessen CM (1984) The isolation and characterization of abscisic acid-insensitive mutants of Arabidopsis thaliana. Plant Physiol 61:377–383CrossRefGoogle Scholar
  24. Kuwabara A, Ikegami K, Koshiba T, Nagata T (2003) Effects of ethylene and abscisic acid upon heterophylly in Ludwigia arcuata (Onagraceae). Planta 217:880–887PubMedCrossRefGoogle Scholar
  25. Kyo M, Harada H (1985) Studies on conditions for cell division and embryogenesis in isolated pollen culture of Nicotiana rustica. Plant Physiol 79:90–94PubMedGoogle Scholar
  26. Li Y, Wu YH, Hagen G, Guilfoyle T (1999) Expression of the auxin-inducible GH3 promoter/GUS fusion gene as a useful molecular marker for auxin physiology. Plant Cell Physiol 40:675–682Google Scholar
  27. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  28. Nishiwaki M, Fujino K, Koda Y, Masuda K, Kikuta Y (2000) Somatic embryogenesis induced by the simple application of abscisic acid to carrot (Daucus carota L.) seedlings in culture. Planta 211:756–759PubMedCrossRefGoogle Scholar
  29. Ogata Y, Iizuka M, Nakayama D, Ikeda M, Kamada H, Koshiba T (2005) Possible involvement of abscisic acid in the induction of secondary somatic embryogenesis on seed coat-derived carrot somatic embryos. Planta 221:417–423PubMedCrossRefGoogle Scholar
  30. Parcy F, Valon C, Raynal M, Gaubier-Comella P, Delseny M, Giraudat J (1994) Regulation of gene expression programs during Arabidopsis seed development: roles of the ABI3 locus and of endogenous abscisic acid. Plant Cell 6:1567–1582PubMedCrossRefGoogle Scholar
  31. Reinert J (1959) Uber die Kontrolle der morphogenese und die induction von adventivembryonen an Gewebekulturen aus Carotten. Planta 53:318–333CrossRefGoogle Scholar
  32. Ribnicky DM, Ilic N, Cohen JD, Cooke TJ (1996) The effects of exogenous auxins on endogenous indole-3-acetic acid metabolism. The implications for carrot somatic embryogenesis. Plant Physiol 112:549–558PubMedGoogle Scholar
  33. Saab IN, Sharp RE, Pritchard J (1992) Effect of inhibition of abscisic acid accumulation on spatial distribution of elongation in the primary root and mesocotyl of maize at low water potentials. Plant Physiol 99:26–33PubMedGoogle Scholar
  34. Satoh S, Kamada H, Harada H, Fujii T (1986) Auxin-controlled glycoprotein release into the medium of embryogenic carrot cells. Plant Physiol 81:931–933PubMedCrossRefGoogle Scholar
  35. Sauser C, Kwiatkowski J, Jung J, Grossamann K (1992) Accumulation of abscisic acid in cell suspension cultures of oilseed rape treated with the growth retardant BAS111.W: effects on osmotic potential and potassium, water and sugar contents. J Plant Physiol 140:747–753Google Scholar
  36. Shinozaki-Yamaguchi K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6:251–264CrossRefGoogle Scholar
  37. Shiota H, Satoh R, Watabe K, Harada H, Kamada H (1998) C-ABI3, the carrot homologue of Arabidopsis ABI3, is expressed during both zygotic and somatic embryogenesis and functions in the regulation of embryo-specific ABA-inducible genes. Plant Cell Physiol 39:1184–1193PubMedGoogle Scholar
  38. Shiota H, Kamada H (2000) Acquisition of desiccation tolerance by cultured carrot cells upon ectopic expression of C-ABI3, a carrot homologue of ABI3. J Plant Physiol 156:510–515Google Scholar
  39. Skriver K, Mundy J (1990) Gene expression in response to abscisic acid and osmotic stress. Plant Cell 2:503–512PubMedCrossRefGoogle Scholar
  40. Steward FC, Mapes MO, Mears K (1958) Growth and organized development of cultured cells. I. Growth and division of freely suspended cells. Am J Bot 45:693–703CrossRefGoogle Scholar
  41. Sung ZR, Fienberg A, Chorneau R, Borkird C, Furner I, Smith J (1984) Developmental biology of embryogenesis from carrot culture. Plant Mol Biol Rep 2:3–14CrossRefGoogle Scholar
  42. Tachikawa Y, Saitou T, Kamada H, Harada H (1998) Changes in protein pattern during stress-induction of carrot (Daucus carota L.) somatic embryogenesis. Plant Biotechnol 15:17–22Google Scholar
  43. Walton DC (1980) Biochemistry and physiology of abscisic acid. Annu Rev Plant Physiol 31:453–489CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Akira Kikuchi
    • 1
  • Nobuya Sanuki
    • 2
  • Katsumi Higashi
    • 3
  • Tomokazu Koshiba
    • 2
  • Hiroshi Kamada
    • 1
  1. 1.Graduate School of Life and Environmental SciencesUniversity of TsukubaTsukubaJapan
  2. 2.Department of Biological SciencesTokyo Metropolitan UniversityHachiojiTokyoJapan
  3. 3.Department of Biosciences, Faculty of Science and EngineeringTeikyo University of Science and TechnologyUenoharaYamanashiJapan

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