caa a22 4500 46323619X CHVBK 20180405153257.0 cr unu---uuuuu 170326e20070801xx s 000 0 eng 10.1007/s11240-007-9225-4 doi (NATIONALLICENCE)springer-10.1007/s11240-007-9225-4 A study of the process of synchronisation and micronucleation in Beta vulgaris and the monitoring of an isolation procedure for micro-nuclei and micro-protoplasts by confocal laser scanning microscopy and flow cytometry [Elektronische Daten] [Ivan Famelaer, Harrie Verhoeven, Paul Dijkhuis, Kamisetti Ramulu] The process of synchronization and micro-nuclei induction in a suspension culture of Beta vulgaris, was induced by the sequential treatment with the DNA-synthesis inhibitor aphidicolin (30μM, 24h) and the spindle-toxin amiprophos-methyl (32μM, 24h). Mitotic arrest of divisions, spreading of G2-metaphase chromosomes, re-grouping of chromosomes, formation of a nuclear cell wall around single and re-grouped chromosomes and restitution of nuclei with a doubled DNA content was observed. The process of micro-nucleation was induced much less efficiently in Beta vulgaris than in Nicotiana plumbaginifolia. Cytological observations and monitoring of the process with flow cytometry and confocal laser scanning microscopy, was essential to follow up the course of events and to monitor the development of an efficient procedure for micro-protoplast isolation. Micro-nucleated protoplasts were fractioned by iso-osmotic Percoll gradient centrifugation to obtain heterogeneous micro-protoplast populations with cytoplasts and micro-protoplasts of different size. An enriched fraction with small sub-diploid micro-protoplasts was obtained with the equivalent DNA content of 1-4 chromosomes, as revealed by confocal laser scanning microscopy and flow cytometry. Sub-diploid micro-protoplasts with DNA amounts equivalent to 1-4 chromosomes were predominantly observed in the size classes of 1.8-10.2μm at a frequency of 34.2-34.5%. The DNA measurements of micro-nuclei and micro-protoplasts, confirmed the hypothesis that the process of micro-nucleation followed the same course of cellular events as observed in N. plumbaginifolia. The correlation between DNA content and size of micro-nuclei and micro-protoplasts was not linear and affected by the degree of DNA condensation, total amount of DNA, and the presence of cytoplasm. Springer Science+Business Media B.V., 2007 Sugar beet nationallicence Amiprophos-methyl nationallicence Synchronization nationallicence Micro-nucleated protoplast nationallicence Micro-protoplast nationallicence Micro-nuclei nationallicence Confocal laser scanning microscopy nationallicence Flow Cytometry nationallicence APH : Aphidicolin nationallicence APM : Amiprophos-methyl nationallicence CA : Cremart nationallicence CB : Cytochalasin B nationallicence DAPI : 4,6-Diamidino-2-phenylindole nationallicence CLSM : Confocal laser scanning microscopy nationallicence FCM : Flow cytometry nationallicence HU : Hydroxy urea nationallicence MI : Mitotic index nationallicence MMCT : Microcell-mediated chromosome transfer nationallicence MNI : Micro-nuclei index nationallicence MPP : Micro-protoplast nationallicence Micro-nucleated cell : MN-cell nationallicence Famelaer Ivan Laboratory of Plant Genetics, Institute for Molecular Biology, Vrije Universiteit Brussel, Pleinlaan 2, 1050, Brussels, Belgium aut Verhoeven Harrie Plant Research International, P.O. Box 16, 6700 AA, Wageningen, The Netherlands aut Dijkhuis Paul Plant Research International, P.O. Box 16, 6700 AA, Wageningen, The Netherlands aut Ramulu Kamisetti Plant Research International, P.O. Box 16, 6700 AA, Wageningen, The Netherlands aut Plant Cell, Tissue and Organ Culture Kluwer Academic Publishers 90/2(2007-08-01), 169-179 0167-6857 90:2<169 2007 90 11240 https://doi.org/10.1007/s11240-007-9225-4 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11240-007-9225-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Famelaer Ivan Laboratory of Plant Genetics, Institute for Molecular Biology, Vrije Universiteit Brussel, Pleinlaan 2, 1050, Brussels, Belgium aut NATIONALLICENCE 700 1- Verhoeven Harrie Plant Research International, P.O. Box 16, 6700 AA, Wageningen, The Netherlands aut NATIONALLICENCE 700 1- Dijkhuis Paul Plant Research International, P.O. Box 16, 6700 AA, Wageningen, The Netherlands aut NATIONALLICENCE 700 1- Ramulu Kamisetti Plant Research International, P.O. Box 16, 6700 AA, Wageningen, The Netherlands aut NATIONALLICENCE 773 0- Plant Cell, Tissue and Organ Culture Kluwer Academic Publishers 90/2(2007-08-01), 169-179 0167-6857 90:2<169 2007 90 11240 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer