SWCNTs contribute to the better efficiency of cryoprotectant
The field of plant cryobiology seeks to modify existing techniques that allow for more efficient storage by reducing multiple stresses. Adding some exogenous compounds can improve the survival of cryopreserved cells in many species, like antioxidants [29-33], anti-stress compounds [34, 35], metabolism related compounds [36, 37] and ice inhibitors [38, 39]. Previously we found that CNMs especially SWCNTs adding to PVS2 can improve survival rate of Arabidopsis seedlings, callus or protocorm of lily, Cymbidium and Anoectochilu (unpublished results) and A. praecox callus  after cryopreservation. In this study, the relative survival rate was highly improved by adding SWCNTs to PVS2 in cryopreservation of A. praecox EC compared to above compounds (Fig. 1). Thus, SWCNTs are the potential and important exogenous additions of cryoprotectant.
In our previous study, the differential scanning calorimetry (DSC) analysis suggested that the glass-transition temperature of SWCNTs-PVS2 was slightly decreased . Adding 0.1 g/L SWCNTs to PVS2 reduced the glass-transition temperature from -112.67 to -114.18 °C still within the reported temperature range of PVS2 (-115 °C to -112 °C). Moreover, a melting peak around -38.87 °C and an endothermic peak at -50.12 °C were observed in the DSC curve of 0.1 g/L SWCNTs-PVS2. It indicated that SWCNTs-PVS2 may be more stable than PVS2, but no significant changes on glass transition parameters were detected . Since the glass-transition temperature is not changed, do SWCNTs regulate the physiological response of plant cells?
Effective antioxidant response during dehydration step improved EC survival after cryopreservation
ROS-induced oxidative stress is a major reason of low survival in samples after cryopreservation [40-48]. In many species cryopreservation, H2O2 is the major component of ROS leading to oxidative stress [8, 45]. Adding SWCNTs in the cryoprotectant might suppress H2O2 production and maintain the H2O2 content in the lower level than that in the control process, in which H2O2 content increased dramatically otherwise.
Membrane lipids are the primary target in oxidative damage , and MDA acting as a breakdown product of lipid peroxidation increased in cryopreserved Oryza sativa [47, 50], Azadirachta indica , Arabidopsis thaliana  and Agapanthus praecox , Hancornia speciose , Passiflora suberosa . With the addition of NRSP in the cryoprotectant, the MDA content decreased and the activity of SOD increased leading to more intact membrane and better quality of boar sperm . MDA content significantly increased in the control process, and led to low survival rate of EC after cryopreservation.
Antioxidant system works to prevent plant cells from oxidative injury through cleaning ROS . SWCNTs can activate related antioxidant enzymes, and improve survival after cryopreservation. In general, antioxidant enzyme activities increased after rapid cooling-warming (Fig. 3). In the SWCNTs cryopreservation, the enzyme activities rose up when cells were treated with SWCNTs-added PVS2, and maintained in a relative high level through the process. Like CAT was involved in scavenging the intracellular H2O2 in SWCNTs treated group, it is also found that cryopreserved of Dendrobium suffered serious oxidative stress because of decreasing CAT activity after cryopreservation leading to low survival , and high tolerance was related to high CAT activity in Haematococcus pluvialis cryopreservation . In the study of Liu , the graphene-treated rice produced oxidative stress response, and the activities of SOD, POD and CAT were all increased in seedlings treated with graphene.
AsA-GSH cycle is a key way to scavenge H2O2 . In this cycle, APX can scavenge H2O2 followed by a series of catalytic reactions involving GR, MDHAR and DHAR, in which GSH and AsA work as reducing substrates . Adding SWCNTs increased GSH contents. Expression levels of GPX and GR in SWCNTs cryopreservation were lower than those in the control group, and the difference between these two processes became very obvious after rapid cooling-warming. This study indicated that PVS2 with SWCNTs improved the survival of Agapanthus praecox. SWCNTs promoted dehydration protection. This improvement is due to the scavenging of ROS and improving of antioxidative system activity, especially POD and CAT.
Effects of SWCNTs on ROS signal transduction during cryopreservation
Because of their key signaling roles (at low levels) and toxic roles (at high levels), the levels of ROS are regulated by the complex pathway including many genes [58-60]. In this pathway, plants sense ROS by three ways: (a) unknown ROS receptors; (b) redox-sensitive transcription factors; (c) direct inhibition of phosphatases . The ROS signal is detected by unknown ROS receptors leading to the accumulation of Ca2+ signal [62-64], and the signal is transmitted to oxidative signal-inducible 1 (serine/threonine protein kinase, OXI1) which was significantly up-regulated in the control rapid cooling-warming with ROS level reaching the maximum which is much higher than that in the SWCNTs-added cryopreservation. OXI1 acts as a central factor in the ROS sensing, and is up-regulated in many H2O2-generating stimulus . MAPK3/6 following OXI1 [66, 67] were mainly up-regulated during the rapid cooling-warming of the control group.
The ROS signals further influenced following pathways including ROS producing and scavenging. The producing pathway has NADPH oxidases [68, 69], which were up-regulated in the SWCNTs treated steps. The producing pathway might be activated by ROS at low levels leading to the ROS production and amplification, and the scavenging pathway might be activated by ROS accumulation leading to the ROS suppression . The interaction between producing and scavenging pathway determines the intensity of ROS signals . In summary, EC in the control group suffered excessive ROS after dehydration, which broke the ROS metabolism balance. By contrast, SWCNTs both enhanced producing and scavenging pathway smoothly, and maintained ROS signals balanced and stable in EC during cryopreservation.
Regulation of physiological response of carbon nanotubes in plants
In the past decade, researchers have applied carbon nanotubes to plant studies and found that they have a certain regulatory effect on physiological response, especially on enzyme activity and gene expression. The effective impact of CNMs on plant development and growth has been studied by many research groups . Giraldo et al.  pointed out that SWCNTs localized in the lipid envelope of Arabidopsis chloroplasts, promote over three times higher photosynthetic activity than that of controls, and concentrations of ROS inside chloroplasts were significantly suppressed. Industrialized MWCNTs can stimulate the growth of Onobrychis arenaria and enhance peroxidase activity . In this study, SWCNTs contribute to the better efficiency of cryoprotectant, and also improved enzyme activities including POD and CAT. At the molecular level, SWCNTs regulated gene expression levels including NADPH oxidase, CAT and POD. Other studies have found similar results. MWCNTs enhanced the tobacco cell growth, and up-regulated genes related to water transport and cell division . A number of genes regulated by MWCNTs were related to plant stress-signal transduction in tomato. Important stress signal pathways could be regulated in response to the uptake of carbon nanotubes . For instance, MAPK was up-regulated in leaves exposed to MWCNTs, and played a positive role to promote plant development and stress response of carbon nanotubes . In this study, MAPK3/6 were higher in control rapid cooling and warming than those treated with SWCNTs. Adding MWCNTs to the seeds of soybean (Glycine max), corn (Zea mays) and barley (Hordeum vulgare) led to the improvement of germination, and activated expression levels of aquaporins . On the contrary, experiment on suspension rice cells with MWCNTs showed that it induced the accumulation of ROS leading to cell death . It also decreases the dry weight and the activity of SOD in Arabidopsis suspension cells . Whether carbon nanotubes play a positive or negative regulatory role, they can regulate a variety of biological processes in plants, like water transport, cell division, stress response, electron transfer, ROS generation and metabolism [78, 79]. However, the mechanism of how the nanotubes can regulate the physiological response especially regulate the gene expression is an unresolved issue and deserved further studies.