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

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 mM, 24 h) and the spindle-toxin amiprophos-methyl (32 mM, 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 microprotoplast 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 subdiploid 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 mm at a frequency of 34.2-34.5%. The DNA measurements of micro-nuclei and micro-protoplasts, confirmed the hypothesis that the process of micronucleation 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.

Asymmetric Somatic Plant Hybridization: Status and Applications

“…These can be further filtered through sequential filters of smaller pore width. Some recent examples of suspension cell derived MPPs are Citrus unshiu [33] and Beta vulgaris [34].…”

“…Oryzaline is the most widely used mitosis inhibitor, but its efficiency varies based on the plant species [38,39]. Famelaer et al quantified genome fragmentation in Beta microprotoplasts through flow cytometry and confocal microscopy [34].…”