Plant Cell, Tissue and Organ Culture

, Volume 94, Issue 3, pp 313–318 | Cite as

Effect of UV-C irradiation on mesophyll protoplasts of Cucumis sativus

  • Božena Navrátilová
  • Lenka Luhová
  • Marek Petřivalský
Original Paper


In the present work we studied the effect of UV-C irradiation on short-term protoplast physiology, with the aim of identifying and assessing parameters which can provide valuable information for asymmetric fusion experiments. Protoplast viability, cell wall regeneration, density of cell suspension and intensity of DAPI signal were followed by using microscopy and by the detection of specific fluorescent or spectroscopic signals in a microplate reader. The control and irradiated mesophyll protoplasts of Cucumis sativus were used for this experiment. In contrast to control cells, viability of irradiated cells significantly decreased. Intensive cell wall regeneration was observed only in control cells, which also showed significantly higher DAPI fluorescence signal. Microscopy for determination of viability by FDA and cell wall regeneration by Calcofluor White were modified for microplate reader instrumentation. These methods are simple, fast and suitable for detection of the effectiveness of UV-C irradiation of cells intended to be used in asymmetric fusion experiments.


Asymmetric fusion Calcofluor White Cell wall regeneration Cucumis sativus DAPI FDA Mesophyll protoplast 





Calcofluor White




Fluorescein diacetate


Indole-3-butyric acid


Liquid medium


2-(N-morpholino)ethanesulfonic acid


Murashige and Skoog


α-Naphthalene acetic acid


Protoplast suspension


Relative fluorescence unit


Ultraviolet light


2,4-Dichlorophenoxyacetic acid



This work was supported by the Grant MSM 6198959215 from the Ministry of Education, Youth and Sports of the Czech Republic and by the project NAZV QF 4108. We would like to thank to Research Institute of Crop Production (Prague, Czech Republic) and to Department of Gene Bank (Olomouc, Czech Republic) for providing of seed material.


  1. Atanassov A, Dimantov D, Atanassov I, Dragoeva A, Vassileva Z, Vitanov V, Jankulova M, Djilianov D (1991) Transfer of resistance to tomato spotted wilt virus from wild Nicotiana species to N. tabacum via somatic hybridization. Physiol Plant 82:A23, poster session 8, No. 130Google Scholar
  2. Coohill TP (1989) Ultraviolet action spectra (280 nm to 380 nm) and solar effectiveness spectra for higher plants. Photochem Photobiol 50:451–457. doi: 10.1111/j.1751-1097.1989.tb05549.x CrossRefGoogle Scholar
  3. Debeaujon I, Branchard M (1992) Induction of somatic embryogenesis and callogenesis from cotyledons and leaf protoplasts-derived colonies of melon (Cucumis melo L.). Plant Cell Rep 12:37–40. doi: 10.1007/BF00232420 CrossRefGoogle Scholar
  4. Forsberg J, Dixelius CH, Lagerkrantz U, Glimelius K (1998) UV dose-dependent DNA elimination in asymmetric somatic hybrids between Brassica napus and Arabidopsis thaliana. Plant Sci 131:65–76. doi: 10.1016/S0168-9452(97)00242-2 CrossRefGoogle Scholar
  5. Gajdová J, Lebeda A, Navrátilová B (2004) Protoplast cultures of Cucumis and Cucurbita spp. In: Lebeda A, Paris HS (eds) Progress in cucurbit genetics and breeding research. Proceedings of Cucurbitaceae 2004, 8th EUCARPIA meeting on cucurbit genetics and breeding, Palacký University in Olomouc, Olomouc, Czech Republic, 2004, pp 441–454Google Scholar
  6. Gajdová J, Navrátilová B, Smolná J, Lebeda A (2007) Factors affecting protoplast isolation and cultivation of Cucumis spp. J Appl Bot Food Qual 81:1–6Google Scholar
  7. Hall RD, Krens FA, Rounwendall GA (1992) DNA radiation damage and asymmetric somatic hybridization: is UV a potential substitute or supplement to ionising radiation in fusion experiments? Physiol Plant 85:319–324. doi: 10.1111/j.1399-3054.1992.tb04741.x CrossRefGoogle Scholar
  8. Hughes J, McCully ME (1975) The use of an optical brightener in the study of plant structure. Stain Technol 50:319–329PubMedGoogle Scholar
  9. Larkin PJ (1976) Purification and viability determinations of plant protoplasts. Planta 128:213–216. doi: 10.1007/BF00393231 CrossRefGoogle Scholar
  10. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x CrossRefGoogle Scholar
  11. Rotman BR, Papermaster RD (1966) Membrane properties of living cells as studied by enzymatic hydrolysis of fluorogenic esters. Proc Natl Acad Sci USA 55:134–141. doi: 10.1073/pnas.55.1.134 PubMedCrossRefGoogle Scholar
  12. Seemüller E (1976) Investigation to demonstrate mycoplasma-like organism in diseased plants by fluorescence microscopy. Acta Hortic 67:109–112Google Scholar
  13. Wang ZP, Sonntag K, Rudloff E (2003) Development of rapeseed with high erucic acid content by asymmetric somatic hybridization between Brassica napus and Crambe abyssinica. Theor Appl Genet 106:1147–1155PubMedGoogle Scholar
  14. Xia G, Xiang F, Zhou A, Wang H, Chen H (2003) Asymmetric somatic hybridization between wheat (Triticum aestivum L.) and Agropyron elongatum (Host) Nevishi. Theor Appl Genet 107:299–305. doi: 10.1007/s00122-003-1247-7 PubMedCrossRefGoogle Scholar
  15. Xu XY, Hu ZY, Li JF, Liu JH, Deng XX (2007) Asymmetric somatic hybridization between UV-irradiated Citrus unshiu and C. sinensis: regeneration and characterization of hybrid shoots. Plant Cell Rep 26:1263–1273. doi: 10.1007/s00299-007-0350-7 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Božena Navrátilová
    • 1
  • Lenka Luhová
    • 2
  • Marek Petřivalský
    • 2
  1. 1.Department of Botany, Faculty of SciencePalacký University in OlomoucOlomouc-HoliceCzech Republic
  2. 2.Department of Biochemistry, Faculty of SciencePalacký University in OlomoucOlomouc-HoliceCzech Republic

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