TY - JOUR
T1 - 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
AU - Famelaer, I.
AU - Verhoeven, H.A.
AU - Dijkhuis, P.
AU - Ramulu, K.S.
N1 - 000248624100005
PY - 2007
Y1 - 2007
N2 - 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 mu M, 24 h) and the spindle-toxin amiprophos-methyl (32 mu M, 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 mu m 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.
AB - 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 mu M, 24 h) and the spindle-toxin amiprophos-methyl (32 mu M, 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 mu m 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.
KW - partial genome transfer
KW - cell-suspension cultures
KW - nicotiana-plumbaginifolia
KW - gametic microprotoplasts
KW - hybridization
KW - hybrids
KW - fusion
KW - plants
KW - regeneration
KW - chromosomes
U2 - 10.1007/s11240-007-9225-4
DO - 10.1007/s11240-007-9225-4
M3 - Article
SN - 0167-6857
VL - 90
SP - 169
EP - 179
JO - Plant Cell, Tissue and Organ Culture: an international journal on in vitro culture of higher plants
JF - Plant Cell, Tissue and Organ Culture: an international journal on in vitro culture of higher plants
IS - 2
ER -