Abstract
BackgroundCryopreservation is the best way for long-term in vitro preservation of plant germplasm resources. The preliminary studies found that reactive oxygen species (ROS) induced oxidative stress and ice-induced membrane damage are the fundamental causes of cell death in cryopreserved samples. How to improve plant cryopreservation survival rate is an important scientific issue in the cryobiology field.ResultsThis study found that the survival rate was significantly improved by adding single-wall carbon nanotubes (SWCNTs) to plant vitrification solution (PVS) in cryopreservation of Agapanthus praecox embryogenic callus (EC), and analyzed the oxidative response of cells during the control and SWCNTs-added cryopreservation protocol. The SWCNTs entered EC at the step of dehydration and mainly located around the cell wall and in the vesicles, and most of SWCNTs moved out of EC during the dilution step. Combination with physiological index and gene quantitative expression results, SWCNTs affect the ROS signal transduction and antioxidant system response during plant cryopreservation. The EC treated by SWCNTs had higher antioxidant levels, like POD, CAT, and GSH than the control group EC. The EC mainly depended on the AsA-GSH and GPX cycle to scavenge H2O2 in the control cryopreservation, but depended on CAT in the SWCNTs-added cryopreservation which lead to low levels of H2O2 and MDA. The elevated antioxidant level in dehydration by adding SWCNTs enhanced cells resistance to injury during cryopreservation. The ROS signals of EC were balanced and stable in the SWCNTs-added cryopreservation.ConclusionsThe SWCNTs regulated oxidative stress responses of EC during the process and controlled oxidative damages by the maintenance of ROS homeostasis to achieve a high survival rate after cryopreservation. This study is the first to systematically describe the role of carbon nanomaterial in the regulation of plant oxidative stress response, and provided a novel insight into the application of nanomaterials in the field of cryobiology.
Highlights
Cryopreservation is the best way for long-term in vitro preservation of plant germplasm resources
Effects of single-wall carbon nanotubes (SWCNTs) on the cell viability after cryopreservation Based on the previous studies, carbon nanomaterials (CNMs) have been applied in the Arabidopsis cryopreservation model to identify their effects on cryopreservation, and 0.1 g/L SWCNTs in PVS2 was the most effective one
Betaine, 1 mM C aCl2, 6 mM lipoic acid (LA), 6 mM polyvinyl alcohol (PVA), or 3% polyvinyl pyrrolidone (PVP), 0.1 g/L SWCNTs had a significant effect on the embryogenic callus (EC) survival increasing by 58.31% higher than others (Fig. 1)
Summary
Plant material The EC was induced and cultured from pedicel tissue of A. praecox as described by Wang et al [34], and was maintained at 25 ± 2 °C in the dark by subculturing monthly onto the MS medium supplemented with 1.5 mg/L picloram. Experimental design 0.2 g of EC was cryopreserved as described by Chen et al [30]. H2O2 levels, O2− inhabitation and OH· generation activities, MDA content, SOD, CAT and POD activities, AsA and GSH contents were tested using the related biological assay kits (Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer’s instructions with some modifications according to Yang et al [85]. The actin was used as an internal control parameter for normalization, and all primer sequences were listed in Additional file 1: Table S1 including APX, CAT, Fe SOD, Cu/Zn SOD, POD, MDHAR, GPX1, GR, NADPH oxidase, OXI1, MAPK3, and MAPK6. The heat-map figures were made by GENESIS Software after the normalization of gene expression data
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