Effects of Cytokinins on In Vitro Seed Germination and Early Seedling Morphogenesis in Lotus corniculatus L.

  • Radomirka Nikolić
  • Nevena Mitić
  • Rade Miletić
  • Mirjana Nešković


We determined the effects of zeatin (ZEA), isopentenyl adenine (2iP), kinetin (KIN), benzyladenine (BA), and thidiazuron (TDZ) on seed germination, elongation of seedling shoots and roots, frequency of regeneration, and the number of regenerants per seedling in Lotus corniculatus L. Sterilized seeds were cultured in vitro on Murashige and Skoog (1962) medium containing 3% sucrose, 0.7% agar, and various cytokinins (0, 0.08, 0.22, 0.35, 0.80, 2.20, and 3.50 μM). After 30 days, seedlings were transferred to cytokinin-free medium for another 60 days. All cytokinins stimulated the rate and percentage of seed germination at least twofold in optimum concentrations; TDZ and ZEA were the most active, followed closely by BA, whereas KIN and 2iP stimulated germination in higher concentrations only. Elongation of shoots and roots was strongly inhibited at the lowest TDZ and BA concentrations, whereas ZEA, KIN, and 2iP exerted moderate, dose-dependent inhibition. The frequency of regenerant-producing seeds was highest on ZEA and BA, whereas the greatest number of regenerants per seedling was found on TDZ. It is concluded that the culture of seeds on cytokinin-containing media, followed by transfer to cytokinin-free medium, is a suitable procedure for rapid production of a large number of uniform regenerants. The presumed role of particular cytokinins is discussed.


Cytokinins In vitro cultures Seed germination Shoot elongation Root elongation Shoot regeneration 



The present work was supported by the Ministry of Science and Environment Protection of the Republic of Serbia (grant 3026).


  1. Arcioni S, Mariotti D, Damiani F, Pezzotti M. 1988. Bird’s foot trefoil (Lotus corniculatus L), crownvetch (Coronilla varia L) and sainfoin (Onobrychis viciifolia Scop). In YPS Bajaj (ed), Biotechnology in Agriculture and Forestry, Vol. 6, Crops II, Springer-Verlag, Berlin Heidelberg, p 548–572Google Scholar
  2. Atici Ö, Ağar G, Battal P. 2005. Changes in phytohormone contents in chickpea seeds germinating under lead or zink stress. Biol Plant 49:215–222CrossRefGoogle Scholar
  3. Auer CA, Motyka V, Březinová A, Kaminek M. 1999. Endogenous cytokinin accumulation and cytokinin oxidase activity during shoot organogenesis of Petunia hybrida. Physiol Plant 105:141–147CrossRefGoogle Scholar
  4. Badzian T, Rybczyński JJ. 1994. Cytokinin control of shoot regeneration in root segment culture of Lotus corniculatus seedling. Acta Physiol Plant 16:61–67Google Scholar
  5. Bilyeu KD, Laskey JG, Morris RO. 2003. Dynamics of expression and distribution of cytokinin oxidase/dehydrogenase in developing maize kernels. Plant Growth Regul 39:195–203CrossRefGoogle Scholar
  6. Chaitanya KSK, Naithani SC. 1998. Kinetin-mediated prolongation of viability in recalcitrant sal (Shorea robusta Gaertn f.) seeds at low temperature: role of kinetin in delaying membrane deterioration during desiccation-induced injury. J Plant Growth Regul 17:63–69CrossRefGoogle Scholar
  7. Chiwocha SDS, Cutler AJ, Abrams SR, Ambrose SJ, Yang J, et al. 2005. The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and germination. Plant J 42:35–48PubMedCrossRefGoogle Scholar
  8. Gadallah MAA, El-Enany AE. 1999. Role of kinetin in alleviation of copper and zinc toxicity in Lupinus termis plants. Plant Growth Regul 29:151–160CrossRefGoogle Scholar
  9. Galuszka P, Frébort I, Šebela M, Peč P. 2000. Degradation of cytokinins by cytokinin oxidases in plants. Plant Growth Regul 32:315–327CrossRefGoogle Scholar
  10. Galuszka P, Frébort I, Šebela M, Sauer P, Jacobsen S, et al. 2001. Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals. Eur J Biochem 268:450–461PubMedCrossRefGoogle Scholar
  11. Gaudinová A, Dobrev PI, Šolcová B, Novák O, Strnad M, et al. 2005. The involvement of cytokinin oxidase/dehydrogenase and zeatin reductase in regulation of cytokinin levels in pea (Pisum sativum L.) leaves. J Plant Growth Regul 24:188–200CrossRefGoogle Scholar
  12. Gerhäuser D, Bopp M. 1990. Cytokinin oxidases in mosses. 2. Metabolism of kinetin and benzyladenine in vitro. J Plant Physiol 135:714–718Google Scholar
  13. Gulzar S, Khan MA. 2002. Alleviation of salinity-induced dormancy in perennial grasses. Biol Plant 45:617–619CrossRefGoogle Scholar
  14. Hare PD, van Staden J. 1994. Cytokinin oxidase: Biochemical features and physiological significance. Physiol Plant 91:128–136CrossRefGoogle Scholar
  15. Khan MA, Ungar IA. 1997. Alleviation of seed dormancy in the desert forb Zygophyllum simplex L. from Pakistan. Ann Bot 80:395–400CrossRefGoogle Scholar
  16. Khan MA, Gul B, Weber DJ. 2004. Action of plant growth regulators and salinity on seed germination of Ceratoides lanata. Can J Bot 82:37–42CrossRefGoogle Scholar
  17. Malik KA, Saxena PK. 1992a. Somatic embryogenesis and shoot regeneration from intact seedlings of Phaseolus acutifolius A., P. aureus (L.) Wilczek, P. coccineus L., and P. wrightii L. Plant Cell Reports 11:163–168CrossRefGoogle Scholar
  18. Malik KA, Saxena PK. 1992b. Thidiazuron induces high-frequency shoot regeneration in intact seedlings of pea (Pisum sativum), chickpea (Cicer arietinum) and lentil (Lens culinaris). Aust J Plant Physiol 19:731–740CrossRefGoogle Scholar
  19. Malik KA, Saxena PK. 1992c. Regeneration in Phaseolus vulgaris L.: high-frequency induction of direct shoot formation in intact seedlings by N6-benzylaminopurine and thidiazuron. Planta 186:384–389CrossRefGoogle Scholar
  20. Martin RC, Mok MC, Shaw G., Mok DWS. 1989. An enzyme mediating the conversion of zeatin to dihydrozeatin in Phaseolus embryos. Plant Physiol 90:1630–1635PubMedCrossRefGoogle Scholar
  21. McKersie BD, Stinson RH. 1980. Effect of dehydration on leakage and membrane structure in Lotus corniculatus L. seeds. Plant Physiol 66:316–320PubMedGoogle Scholar
  22. Mijatović M, Milijić S, Spasić M, Petrović R, Mitrović S. 1986. Morphology, biology and productivity in new cultivars of bird’s-foot trefoil Zora and Bokor. Arhiv za Poljoprivredne Nauke 47:149–155 (in Serbian)Google Scholar
  23. Motyka V, Faiss M, Strnad M, Kaminek M, Schmülling T. 1996. Changes in cytokinin content and cytokinin oxidase activity in response to derepression of ipt gene transcription in transgenic tobacco calli and plants. Plant Physiol 112:1035–1043PubMedGoogle Scholar
  24. Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–479CrossRefGoogle Scholar
  25. Nikolić R, Mitić N, Nešković M. 1997. Evaluation of agronomic traits in tissue culture-derived progeny of bird’s-foot trefoil. Plant Cell Tiss Org Cult 48:67–69CrossRefGoogle Scholar
  26. Nikolić R, Mitić N, Ninković S, Miljuš-Djukić J, Nešković M. 2003/4. Efficient genetic transformation of Lotus corniculatus L. and growth of transformed plants in field. Biol Plant 47:137–140CrossRefGoogle Scholar
  27. Rybczyński JJ, Badzian T. 1987. High regeneration potential of root segments of Lotus corniculatus L. seedlings on hormone free media. Plant Sci 51:239–244CrossRefGoogle Scholar
  28. Spíchal L, Rakova NYu, Riefler M, Mizuno T, Romanov GA, et al. 2004. Two cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, differ in their ligand specificity in a bacterial assay. Plant Cell Physiol 45:1299–1305PubMedCrossRefGoogle Scholar
  29. Stirk WA, Gold JD, Novák O, Strnad M, van Staden J. 2005. Changes in endogenous cytokinins during germination and seedling establishment of Tagetes minuta L. 2005. Plant Growth Regul 47:1–7CrossRefGoogle Scholar
  30. Strnad M. 1997. The aromatic cytokinins. Physiol Plant 101:674–688CrossRefGoogle Scholar
  31. Victor JMR, Murch SJ, KrishnaRaj S, Saxena PK. 1999. Somatic embryogenesis and organogenesis in peanut: the role of thidiazuron and N6-benzylaminopurine in the induction of plant morphogenesis. Plant Growth Regul 28:9–15CrossRefGoogle Scholar
  32. Webb KJ, Watson EJ. 1991. Lotus corniculatus L. Morphological and cytological analysis of regenerants from three sources of tissue and selected progeny. Plant Cell Tiss Org Cult 25:27–33CrossRefGoogle Scholar
  33. Werner T, Motyka V, Strnad M, Schmülling T. 2001. Regulation of plant growth by cytokinin. Proc Natl Acad Sci USA 98:10487–10492PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Radomirka Nikolić
    • 1
    • 3
  • Nevena Mitić
    • 1
    • 3
  • Rade Miletić
    • 1
  • Mirjana Nešković
    • 2
  1. 1.Agricultural Research Institute “Serbia”Center for Agricultural and Technological Research
  2. 2.Institute of Botany, Faculty of Biology and Institute for Biological Research “S. Stanković”Belgrade UniversityBelgradeSerbia
  3. 3.Institute for Biological Research “S. Stanković”Belgrade UniversityBelgradeSerbia

Personalised recommendations