Abstract
BackgroundMost plants encounter water stress at one or more different stages of their life cycle. The maintenance of genetic stability is the integral component of desiccation tolerance that defines the storage ability and long-term survival of seeds. Embryonic axes of desiccation-sensitive recalcitrant seeds of Acer pseudoplatnus L. were used to investigate the genotoxic effect of desiccation. Alkaline single-cell gel electrophoresis (comet assay) methodology was optimized and used to provide unique insights into the onset and repair of DNA strand breaks and 8-oxo-7,8-dihydroguanine (8-oxoG) formation during progressive steps of desiccation and rehydration.ResultsThe loss of DNA integrity and impairment of damage repair were significant predictors of the viability of embryonic axes. In contrast to the comet assay, automated electrophoresis failed to detect changes in DNA integrity resulting from desiccation. Notably, no significant correlation was observed between hydroxyl radical (٠OH) production and 8-oxoG formation, although the former is regarded to play a major role in guanine oxidation.ConclusionsThe high-throughput comet assay represents a sensitive tool for monitoring discrete changes in DNA integrity and assessing the viability status in plant germplasm processed for long-term storage.
Highlights
Most plants encounter water stress at one or more different stages of their life cycle
Viability of explants: TTC staining assay, survival, and regrowth The impact of desiccation on embryonic axes was assessed as the survival of tissue and regrowth of seedlings in in vitro culture, as well as the level of metabolic activity of the tissues as measured with a 2,3,5-triphenyltetrazolium chloride (TTC) staining assay
Gradual desiccation resulted in a decrease in survival, which became statistically significant after 4 h of desiccation (45.2%)
Summary
Most plants encounter water stress at one or more different stages of their life cycle. Alkaline single-cell gel electrophoresis (comet assay) methodology was optimized and used to provide unique insights into the onset and repair of DNA strand breaks and 8-oxo-7,8-di‐ hydroguanine (8-oxoG) formation during progressive steps of desiccation and rehydration. Single-cell gel electrophoresis, known as the comet assay, is a technique that is widely used to evaluate the formation and repair of DNA strand breaks and nucleobase modifications. The method is sensitive and costefficient, with proven diagnostic value in several areas of research, including biomonitoring, genotoxicity, carcinogenesis, and aging. Interest in this method has attracted considerable attention from industry [1,2,3,4,5,6]. While both versions provide evidence of double- and single-strand breaks, when performed under alkaline conditions, the comet assay can detect apurinic or apyrimidinic sites
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