2024-03-28T08:41:33Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/530592022-02-02T10:31:42Zcom_10261_86com_10261_1col_10261_339
González-Melendi, Pablo
Ramírez, Carmen
Testillano, P. S.
Kumlehn, Jochen
Risueño, María Carmen
2012-07-10T09:19:26Z
2012-07-10T09:19:26Z
2005-09
Planta,222(1):47-57(2005)
0032-0935
http://hdl.handle.net/10261/53059
10.1007/s00425-005-1515-7
1432-2048
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
eng
closedAccess
Androgenesis
Electron microscopy
Genome doubling
Hordeum
Nuclear fusion
3D-confocal imaging
Three dimensional confocal and electron microscopy imaging define the dynamics and mechanisms of diploidisation at early stages of barley microspore-derived embryogenesis
artículo