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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

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Abstract

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 (30 μM, 24 h) and the spindle-toxin amiprophos-methyl (32 μM, 24 h). 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 1.8–10.2 μm 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.

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Abbreviations

APH:

Aphidicolin

APM:

Amiprophos-methyl

CA:

Cremart

CB:

Cytochalasin B

DAPI:

4,6-Diamidino-2-phenylindole

CLSM:

Confocal laser scanning microscopy

FCM:

Flow cytometry

HU:

Hydroxy urea

MI:

Mitotic index

MMCT:

Microcell-mediated chromosome transfer

MNI:

Micro-nuclei index

MPP:

Micro-protoplast

Micro-nucleated cell:

MN-cell

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Acknowledgements

The present work was partially supported by the EEC-Bridge programme (no BIOT 900038). Our thanks are due to Emeritus Prof. M. Jacobs, department of Plant Genetics, Vrije Universiteit Brussel, for useful suggestions and support and J. Blaas for technical assistance in CLSM.

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Authors and Affiliations

  1. Laboratory of Plant Genetics, Institute for Molecular Biology, Vrije Universiteit Brussel, Pleinlaan 2, 1050, Brussels, Belgium

    Ivan Famelaer

  2. Plant Research International, P.O. Box 16, 6700 AA, Wageningen, The Netherlands

    Ivan Famelaer, Harrie A. Verhoeven, Paul Dijkhuis & Kamisetti S. Ramulu

Authors
  1. Ivan Famelaer
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  2. Harrie A. Verhoeven
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  3. Paul Dijkhuis
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  4. Kamisetti S. Ramulu
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Correspondence to Ivan Famelaer.

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Famelaer, I., Verhoeven, H.A., Dijkhuis, P. et al. 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. Plant Cell Tiss Organ Cult 90, 169–179 (2007). https://doi.org/10.1007/s11240-007-9225-4

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  • DOI: https://doi.org/10.1007/s11240-007-9225-4

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