The impact of Ceftriaxone on Relative Copy Numbers of Mitochondrial and Chloroplast DNA in Grapevine Leaves

Abstract

The cultivation of micropropagated grapevine plants in vitro is currently undergoing improvements in existing methods and the development of new cultivation techniques due to the problem of bacterial contamination caused by multidrug-resistant microorganisms. Explants (Vitis vinifera L., ‘Chardonnay’ variety) were cultured on Murashige-Skoog basal medium supplemented with different concentrations of ceftriaxone: 0 mg/L, 250 mg/L, and 1000 mg/L. After 30 days, morphometric characteristics of the micropropagated plants and relative copy numbers of mitochondrial and chloroplast DNA were evaluated. Leaf samples (5-10 mg) were randomly selected from each plant group for subsequent total DNA extraction. Quantitative RT-PCR was performed using LightCycler 480 SYBR Green I Master Mix (LifeScience, Roche) and analyzed with a LightCycler 96 automated analyzer (Roche Life Science). The relative copy numbers of NAD1 (mitochondrial DNA) and rps16 (chloroplast DNA) genes were determined using the GAPDH gene (chromosomal DNA) as the reference. The 2^-DCt and 2^-DDCt algorithms were used for quantitative assessment. Ceftriaxone at concentrations of 250 and 1000 mg/l reduces the relative number of copies of chloroplast and mitochondrial DNA, which indicates the suppression of photosynthesis and oxidative phosphorylation in grape microplants. The experimental scheme developed by us can be successfully used as a test system for assessing the degree of influence of various biogenic and abiogenic factors on plant objects in order to optimize their cultivation.