TY - JOUR
T1 - Determining DNA strand breakage from embryogenic cell cultures of a conifer species using the singlecell gel electrophoresis assay
AU - Costa, Pedro M.
AU - Milhinhos, Ana
AU - Horta Simões, Marta Andreia
AU - Marum, Liliana Maria
AU - Costa, Maria Helena Ferrão Ribeiro da
AU - Miguel, C.
PY - 2012/1/1
Y1 - 2012/1/1
N2 - Although a routine procedure to detect mutagen- esis by DNA strand breakage in animal cells, the single-cell gel electrophoresis (“comet”) assay is difficult to apply in plant material due to constraints in obtaining suitable nucleoids (formed by DNA trapped in the agarose matrix after the cell lysis process) in either quality or quantity. A suitable protocol is described for the first time to perform the comet assay in conifer somatic embryogenic cultures by determining total DNA strand breakage in protoplasts, after having failed to acquire nuclei by standard mechanical techniques. The results show that protoplasts obtained from embryogenic cultures of the Norway spruce (Picea abies) are suitable to be lysed and surveyed for DNA damage through the standard alkaline version of the comet assay. Several common comet metrics were compared and all were found suitable for analysis, with the percentage of DNA in the comets' tail (constituted by DNA fragments that migrated during electrophoresis), given by the propor- tion between tail fluorescence intensity and total nucleoid intensity, being simplest and the most sensitive to compare between control and hydrogen peroxide-treated cells. The established procedures may be useful, for instance, for a comparative evaluation of somatic embryogenesis protocols and selection of less damaging treatments for clonal propagation or for mutagenesis-related studies with conifer cell cultures.
AB - Although a routine procedure to detect mutagen- esis by DNA strand breakage in animal cells, the single-cell gel electrophoresis (“comet”) assay is difficult to apply in plant material due to constraints in obtaining suitable nucleoids (formed by DNA trapped in the agarose matrix after the cell lysis process) in either quality or quantity. A suitable protocol is described for the first time to perform the comet assay in conifer somatic embryogenic cultures by determining total DNA strand breakage in protoplasts, after having failed to acquire nuclei by standard mechanical techniques. The results show that protoplasts obtained from embryogenic cultures of the Norway spruce (Picea abies) are suitable to be lysed and surveyed for DNA damage through the standard alkaline version of the comet assay. Several common comet metrics were compared and all were found suitable for analysis, with the percentage of DNA in the comets' tail (constituted by DNA fragments that migrated during electrophoresis), given by the propor- tion between tail fluorescence intensity and total nucleoid intensity, being simplest and the most sensitive to compare between control and hydrogen peroxide-treated cells. The established procedures may be useful, for instance, for a comparative evaluation of somatic embryogenesis protocols and selection of less damaging treatments for clonal propagation or for mutagenesis-related studies with conifer cell cultures.
U2 - 10.1007/s11295-011-0434-y
DO - 10.1007/s11295-011-0434-y
M3 - Article
SN - 1614-2942
VL - 8
SP - 425
EP - 430
JO - Tree Genetics & Genomes
JF - Tree Genetics & Genomes
IS - NA
ER -