Abstract
In order to determine the timing and mechanisms of the spontaneous diploidisation throughout microspore-derived embryogenesis in barley, we have estimated the ploidy level of individual nuclei within young pro-embryos, from the first androgenetic division up to multinuclear structures still surounded by the exine. Our methodological approach was based on the measure of the intensity of fluorescence after 4,6-Diamidino-2-phenylindole dihydrochloride staining, nuclear size and number of nucleoli in the confocal microscope. This method avoids the overlapping of the fluorescence signal in multinuclear pro-embryos, which cannot be studied using cytophotometer methods based on other types of fluorescence microscopes. The identification of haploid and diploid nuclei enabled us to determine the timing of diploidisation at early stages throughout androgenetic development. We found that diploidisation is an ongoing process that can start after the first embyogenic division and continues in multinuclear pro-embryos. Reconstruction of 3D-images of entire pro-embryos and the observation of cross and longitudinal sections across stacks of optical sections, together with correlative light and electron microscopy, provided evidences of nuclear fusion as the main mechanism of diploidisation.
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Abbreviations
- DAPI:
-
4,6-Diamidino-2-phenylindole dihydrochloride
- NOR:
-
Nucleolar organiser region
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Acknowledgements
Pablo González-Melendi is a Researcher at the C.S.I.C. funded by the programme “Ramón y Cajal” of the Spanish Ministry of Education and Science. We thank Diego Megias of the “Servicio Interdepartamental De Investigación” (SIDI) of the Universidad Autónoma de Madrid (UAM) for excellent technical assistance in the use of the confocal microscope and fruitful discussions and Francisco Urbano (SIDI/UAM) for the facilities in the use of the electron microscope. This work was supported by the following grants: CICYT BOS2002-03572, Comunidad de Madrid 07G/0026/2003, and Spanish-German “Acción Integrada” HA01-86. Finally we would like to specially thank María Ángeles Ollacarizqueta of the CCD and Confocal Microscopy Service of the CIB for all her help throughout the years she has run the Service. This paper is dedicated to her on her retirement.
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This paper is dedicated to María Ángeles Ollacarizqueta (CCD and Confocal Service of the CIB) on her retirement
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González-Melendi, P., Ramírez, C., Testillano, P.S. et al. Three dimensional confocal and electron microscopy imaging define the dynamics and mechanisms of diploidisation at early stages of barley microspore-derived embryogenesis. Planta 222, 47–57 (2005). https://doi.org/10.1007/s00425-005-1515-7
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DOI: https://doi.org/10.1007/s00425-005-1515-7