Plant Methods 16: 130

PMC: PMC7507619

PMID: 32973916

Single-wall carbon nanotubes improve cell survival rate and reduce oxidative injury in cryopreservation of <em>Agapanthus praecox</em> embryogenic callus

Abstract

Background

Cryopreservation 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.

Results

This 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.

Conclusions

The 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.

Keywords: Agapanthus praecox, Cryopreservation, Oxidative stress, Single-wall carbon nanotubes

Background

The long-term preservation of plants in vitro cultures of cell cultures, embryogenic materials, and endangered germplasm is a vital requirement around the world [1]. Cryopreservation, the storage of living cells, tissues, organs or whole plants in extra low temperature, such as in liquid nitrogen (LN), is thought to be the ideal method to realize the safe and cost-efficient medium- and long-term preservation of almost all economically important crops [2–4]. Vitrification-based cryopreservation has been successfully applied to long-term preservation by achieving the glassy state in cryopreservation of many plant species for its rapid and convenient procedure [5]. Plant vitrification solution (PVS), a commonly used cryoprotectant in vitrification-based cryopreservation, can replace cellular water, alter the freezing behavior, and prevent ultra-water loss [6].

Vitrification-based cryopreservation achieves the storage by enhancing cell viscosity, and accompanies many stresses leading to the accumulation of reactive oxygen species (ROS) [7]. ROS-induced oxidative stress is the fundamental trigger of cell death during cryopreservation [8]. Ren et al. [7, 9] utilized comparative transcriptomics to obtain genes responses to cryoinjury and revealed that peroxidation was a key element affecting viability, and genes related to oxidative stress played important roles in Arabidopsis thaliana cryopreservation. Zhang et al. [10] revealed that the oxidative stress and apoptosis were the major factors that injure embryogenic callus (EC) during Agapanthus praecox cryopreservation.

Plants have complex antioxidant systems including antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and ascorbate peroxidase (APX), and non-enzymatic antioxidants, like glutathione (GSH) and ascorbic acid (AsA) [11]. The high antioxidant level is related to the tolerance to cryopreservation [12]. In antioxidant enzymes, SOD removes O₂^{to H}₂O₂, and CAT catalyzes H₂O₂ to O₂ and H₂O [13, 14]. In non-enzymatic antioxidants, AsA and GSH helps keep a normal REDOX state to counteract oxidative damages [15].

Many researchers have been focusing on the improvement of the cryopreservation procedure by adding exogenous compounds to the cryoprotectant to reduce damages. Nanomaterials have a good effect to improve the cryoprotectant, due to its small particle size and large specific surface [16]. After adding nanomaterials to the cryoprotectant can enhance the thermal conductivity and relative viscosity, promote vitrification, improve the stability of solution during the rewarming process, and suppress the occurrence of devitrification [17–19]. Han et al. [20] added 0.2% Nano-diamond to the ethylene glycol cryoprotectant, and found it doubled the freezing rate and decreased the glass transition temperature significantly. In addition, adding 0.05% hydroxyapatite to the cryoprotectant can improve the pig oocytes developmental rate from 14.7% to 30.4% after cryopreservation [19, 21]. After adding Nanomaterials Rhodiola Sachanensis Polysaccharide (NRSP), the motility, acrosome integrity and membrane integrity of boar sperm were improved significantly compared to the control groups after freezing and thawing [22]. With the addition of NRSP in the cryoprotectant, the malondialdehyde (MDA) content decreased and the activity of SOD increased [21]. Furthermore, lecithin nanoparticles can enhance the cryosurvival of caprine sperm [23], and albumin coated copper-cysteine nanozyme and Au–Ag-AFT nanozyme both can improve survival of human sperm after cryopreservation by reducing oxidative stress [24, 25]. Enrichment of semen extender with selenium nanoparticles improved fertility rate through decreasing peroxidation and injury during cryopreservation of Holstein bulls [26].

However, some nanomaterials have been reported to induce cell death [17, 18], and carbon nanomaterials (CNMs) have better biosafety and biocompatibility [27, 28]. 60-h seedlings of Arabidopsis thaliana are often used as an experimental model to evaluate the optimal effect of exogenous additives on cryopreservation of plants [7]. In our previous studies, CNMs including graphene, single-walled carbon nanotubes (SWCNTs) and graphene quantum dots have been applied in this cryopreservation model to identify their effects on cryopreservation, and 0.1 g/L SWCNTs in PVS2 was the most effective one (unpublished results). Then we applied it to the callus or protocorm cryopreservation of lily, Cymbidium and Anoectochilu, and it also played a positive role in each process. Chen et al. [28] investigated the efficiency of SWCNTs, Graphene, Graphene quantum dots and fullerene (C₆₀) on the cryopreservation of Agapanthus praecox callus by adding to PVS2. 0.3 g/L fullerene and 0.1 g/L SWCNTs were the best two CNMs. The effect on vitrification behaviors was detected, and the glass transition temperatures showed no significant difference on Nano-PVS2. Raman spectroscopy and transmission electron microscopy (TEM) analyses demonstrated that the SWCNTs and fullerene were able to enter the callus cells and protect the cell structure [28].

Agapanthus praecox is a perennial herbaceous plant from Agapanthaceae family and is endemic to southern Africa [29, 30]. A. praecox is known for its ornamental and medicinal values. It is popular as the potted plant, cut flower, and landscape plant due to its multiflorous character, long flowering duration, and wide spectrum of colors [30, 31]. It contains various bioactive compounds such as saponins and chalconoids, which possess anti-inflammatory, antitussive, immunoregulatory, and antibacterial properties [32]. Some rapid micropropagation protocols for clonal propagation and conservation purposes were reported including the induction of EC [33–35]. The EC is an important germplasm resource, but somatic embryogenic ability is often lost during long-term subculture. Cryopreservation is an effective way to solve this problem. Generally, seeds, with characteristic dense cytoplasm and low water content, are easily cryopreserved by vitrification. However, callus cultures, suspension cultures, shoot tips, and recalcitrant seeds are more difficult to cryopreserve due to their higher water content, active metabolic activities, complex biological processes, and sensitive stress responses [7, 10]. A. praecox EC is representatively cryoinjury-susceptible materials for its high moisture content and low-temperature sensitivity [30]. In previous, we have initially cryopreserved the EC of A. praecox. In this study, SWCNTs adding into PVS2 evaluated the survival rate of EC after cryopreservation. It aims to point the positive effects to oxidative stress in cryopreservation, and provides a new application of CNMs using as protectants during cryopreservation. This study is the first time to systematically investigate the effects of nanomaterial to regulate cell responses to oxidative stress in plant cryopreservation applications.

Results

Effects of SWCNTs on the cell viability after cryopreservation

Based on the previous studies, 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 (unpublished results). In this study, we applied it to the EC cryopreservation of A. praecox, and 0.1 g/L SWCNTs have improved the relative survival rate from 53.42% to 84.57% (Fig. 1). Comparing it to the effects of other compounds adding to PVS2 in previous studies including 0.08 mM GSH, 1 μM abscisic acid (ABA), 0.1 μM melatonin, 1 mM AsA, 10 mM betaine, 1 mM CaCl₂, 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 EC survival increasing by 58.31% higher than others (Fig. 1).

Fig. 1

Localizations of SWCNTs in EC during cryopreservation

Transmission electron microscopy imaging provided accurate results, which displayed the cellular status of EC and localizations of SWCNTs inside the EC. Many mitochondria, Golgi apparatus, and endoplasmic reticulum distributed in the EC with complete and compact cell structure before cryopreservation (Fig. 2a, b). After pre-culture, mitochondria, and cell membrane exhibited slight contraction in EC (Fig. 2c, d). The SWCNTs entered EC at the step of dehydration, and mainly located around the cell wall and in the vesicles (Fig. 2e–h). It is demonstrated that plasmolysis, protoplast concentrated, organelles damage and more intracellular vesicles were observed after dehydration (Fig. 2h). The dilution treatment caused most of SWCNTs to move out of EC, and the retained SWCNTs were mostly tube-like fragmented, mainly distributed in the cytoplasm and vesicles (Fig. 2i–l).

Fig. 2

Cell ultrastructure observation of Agapanthus praecox during the SWCNTs-added cryopreservation. a, b the EC before cryopreservation, c, d the EC after pre-culture, e–h the EC after dehydration, i–l the EC after dilution. Cell wall (CW), endoplasmic reticulum (ER), golgi apparatus (G), lipid body (LB), mitochondrion (M), starch grains (SG), vesicle (VES)

Effects of SWCNTs on the ROS and MDA contents during cryopreservation

In the control procedure, H₂O₂ contents highly increased in dehydration and reached the peak after rapid cooling-warming, which was more than twofold compared with CK (Fig. 3a). Adding SWCNTs could inhibit H₂O₂ content during the dehydration step, and H₂O₂ content kept at a low level in rapid cooling-warming and decreased significantly in dilution in the improved cryopreservation. O₂^{inhibitation activities were higher in all steps of the control group, and OH· generation activities were higher after rapid cooling-warming in the SWCNTs-added cryopreservation (Fig.}3b, c). The MDA content trend was similar to that of H₂O₂. The MDA accumulation increased apparently during the control cryopreservation, and the EC treated with SWCNTs had less MDA than in control through the whole procedure (Fig. 3d).

Fig. 3

Physiological indices detection of SWCNTs added in cryopreservation system. Physiological indices were determined with at least three repeats. Bars represent means and standard deviation over triplicate detection. Values with different lowercase letters are significantly different among samples in the control group at 0.05 level. Values with different uppercase letters are significantly different among samples in the SWCNTs group at 0.05 level. * are significantly different between different groups in the same stage at 0.05 level, and ** are highly significantly different between different groups in the same stage at 0.01 level. The CK group is the cryopreservation without adding SWCNTs, and the CNT group is the SWCNTs-added cryopreservation. CK, untreated; PC, after pre-culture; DH, after dehydration; RW, after rapid cooling-warming; DL, after dilution

Effects of SWCNTs on the antioxidant system during cryopreservation

Adding SWCNTs significantly increased the activity of the antioxidant system during cryopreservation. With the addition of SWCNTs to the PVS2, the activities of enzymatic antioxidants highly increased, especially POD and CAT activities (Fig. 3f, g). The changes of SOD activity were not significant (Fig. 3e). The increases of POD activity treated by SWCNTs were much greater than those in the control process (Fig. 3f). Furthermore, the non-enzymatic antioxidant GSH in the SWCNTs group increased nearly twofold compared to the control group during dehydration (Fig. 3i).

Correlation analysis of oxidative physiological indices

In the control cryopreservation (Table 1), MDA contents showed a significant positive correlation with H₂O₂ contents, which revealed that peroxidation was mainly caused by H₂O₂ in EC during cryopreservation. At the same time, OH· generation activities had a significant positive correlation with H₂O₂ and MDA contents. SOD activities had a significant positive correlation with CAT activities and AsA contents. In the SWCNTs cryopreservation (Table 2), O₂^{inhibitation activities had a significant negative correlation with POD activities, MDA and AsA contents, and OH· generation activities had a significant positive correlation with SOD activities and AsA contents. MDA contents had a significant positive correlation with SOD activities, and SOD activities had a significant positive correlation with AsA contents like in the control cryopreservation. In addition, there was a significant positive correlation between POD and CAT activities indicating that the two enzymes may work together to decrease the ROS level during cryopreservation.}

Table 1

The correlation analysis of physiological indices in the control cryopreservation

Indices	H₂O₂	O₂^{inhibitation activity}	OH· generation activity	MDA	SOD	POD	CAT	AsA	GSH
H₂O₂	1	0.263	0.918*	0.974**	0.630	− 0.141	0.510	0.702	0.830
O₂^{inhibitation activity}		1	− 0.076	0.146	− 0.477	0.381	− 0.678	− 0.319	0.190
OH· generation activity			1	0.888*	0.764	− 0.447	0.709	0.747	0.718
MDA				1	0.707	− 0.046	0.621	0.794	0.844
SOD					1	− 0.107	0.935*	0.973**	0.764
POD						1	− 0.277	0.075	0.269
CAT							1	0.878	0.527
AsA								1	0.859
GSH									1

All the data were correlation coefficients, and significant levels are indicated at * P < 0.05 or ** P < 0.01

Table 2

The correlation analysis of physiological indices in the SWCNTs-added cryopreservation

Indices	H₂O₂	O₂^{inhibitation activity}	OH· generation activity	MDA	SOD	POD	CAT	AsA	GSH
H₂O₂	1	− 0.388	0.042	0.440	0.195	0.451	0.490	0.166	0.529
O₂^{inhibitation activity}		1	− 0.793	− 0.904*	− 0.864	− 0.894*	− 0.867	− 0.884*	− 0.437
OH· generation activity			1	0.851	0.982**	0.568	0.550	0.979**	0.124
MDA				1	0.934*	0.868	0.875	0.877	0.147
SOD					1	0.693	0.683	0.980**	0.152
POD						1	0.996**	0.647	0.175
CAT							1	0.621	0.134
AsA								1	0.304
GSH									1

All the data were correlation coefficients, and significant levels are indicated at * P < 0.05 or ** P < 0.01

Quantitative expression analysis of genes related to oxidative stress response

Some genes related to oxidative stress response were chosen to study the molecular protection of SWCNTs during cryopreservation (Figs. 4, ,5).5). As a ROS signal transduction related gene, oxidative signal-inducible 1 (OXI1) was significantly upregulated at rapid cooling-warming stage in the control group, but maintained a low expression level in the SWCNTs group. MAPK3/6 is located downstream of OXI1, and their expression patterns were very similar to that of OXI1. As a ROS signal amplification related gene, NADPH oxidase (RbohA) maintained a low expression level in the control group, and was significantly upregulated after dehydration in the SWCNTs group.

Fig. 4

Real-time PCR quantitative analysis of ROS signal transduction and antioxidant system related genes between the control and the SWCNTs-added cryopreservation. Bars show means of gene expression level and standard deviation over triplicate detections. Values with different lowercase letters are significantly different among samples in the control group at 0.05 level. Values with different uppercase letters are significantly different among samples in the SWCNTs group at 0.05 level. * are significantly different between different groups in the same stage at 0.05 level, and ** are highly significantly different between different groups in the same stage at 0.01 level. The CK group is the cryopreservation without adding SWCNTs, and the CNT group is the SWCNTs-added cryopreservation. CK, untreated; PC, after pre-culture; DH, after dehydration; RW, after rapid cooling-warming; DL, after dilution

Fig. 5

The heat-map of ROS signal transduction and antioxidant system related genes in the control and the SWCNTs-added cryopreservation. The up-regulated genes are indicated in red, and the down-regulated genes are indicated in green. The intensity of the colors increases as the expression differences increase, as shown in the bar at the bottom. CK, untreated; PC, after pre-culture; DH, after dehydration; RW, after rapid cooling-warming; DL, after dilution

In the ROS-scavenging network, SOD and CAT catalyzed O₂^{by forming H}₂O₂ and H₂O, respectively. The expression levels of Cu/Zn and Fe SOD were higher in the control group than SWCNTs group, but those of CAT were quite the opposite which shown that CAT was upregulated in SWCNTs group. The glutathione peroxidase (GPX) and AsA-GSH cycle were also essential to scavenge H₂O₂. Glutathione reductase (GR) and GPX drive the GPX cycle, and GR, monodehydroascorbate reductase (MDHAR), and ascorbate peroxidase (APX) regulate the AsA-GSH cycle. The expression levels of GR and APX were higher in the control group than SWCNTs group. The expression patterns of GPX1 and MDHAR had the similar trend. They were only significantly upregulated in the rapid cooling-warming step of the control cryopreservation, and the dilution step of the SWCNTs cryopreservation. POD only significantly upregulated in the dilution step of the SWCNTs cryopreservation.

Effects of SWCNTs on the antioxidant system during cryopreservation

Correlation analysis of oxidative physiological indices

Table 1

The correlation analysis of physiological indices in the control cryopreservation

Indices	H₂O₂	O₂^{inhibitation activity}	OH· generation activity	MDA	SOD	POD	CAT	AsA	GSH
H₂O₂	1	0.263	0.918*	0.974**	0.630	− 0.141	0.510	0.702	0.830
O₂^{inhibitation activity}		1	− 0.076	0.146	− 0.477	0.381	− 0.678	− 0.319	0.190
OH· generation activity			1	0.888*	0.764	− 0.447	0.709	0.747	0.718
MDA				1	0.707	− 0.046	0.621	0.794	0.844
SOD					1	− 0.107	0.935*	0.973**	0.764
POD						1	− 0.277	0.075	0.269
CAT							1	0.878	0.527
AsA								1	0.859
GSH									1

All the data were correlation coefficients, and significant levels are indicated at * P < 0.05 or ** P < 0.01

Table 2

The correlation analysis of physiological indices in the SWCNTs-added cryopreservation

Indices	H₂O₂	O₂^{inhibitation activity}	OH· generation activity	MDA	SOD	POD	CAT	AsA	GSH
H₂O₂	1	− 0.388	0.042	0.440	0.195	0.451	0.490	0.166	0.529
O₂^{inhibitation activity}		1	− 0.793	− 0.904*	− 0.864	− 0.894*	− 0.867	− 0.884*	− 0.437
OH· generation activity			1	0.851	0.982**	0.568	0.550	0.979**	0.124
MDA				1	0.934*	0.868	0.875	0.877	0.147
SOD					1	0.693	0.683	0.980**	0.152
POD						1	0.996**	0.647	0.175
CAT							1	0.621	0.134
AsA								1	0.304
GSH									1

All the data were correlation coefficients, and significant levels are indicated at * P < 0.05 or ** P < 0.01

Quantitative expression analysis of genes related to oxidative stress response

Fig. 4

Fig. 5

Discussion

SWCNTs contribute to the better efficiency of cryoprotectant

The field of plant cryobiology seeks to modify existing techniques that allow for the more efficient storage by reducing multiple stresses. Adding some exogenous compounds can improve the survival of cryopreserved cells in many species, like antioxidants [30, 36–39], anti-stress compounds [40, 41], metabolism related compounds [42, 43] and ice inhibitors [44, 45]. Previously we found that CNMs especially SWCNTs adding to PVS2 can improve the survival rate of Arabidopsis seedlings, callus or protocorm of lily, Cymbidium and Anoectochilu (unpublished results) and A. praecox callus [28] 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 the 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 slightly decreased [28]. 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 [28]. Since the glass-transition temperature is not significantly changed, do SWCNTs regulate the physiological response of plant cells?

Effective antioxidant response during the dehydration step improved EC survival after cryopreservation

ROS-induced oxidative stress is a major reason of low survival in samples after cryopreservation [10, 46–53]. In many species cryopreservation, H₂O₂ is the major component of ROS leading to oxidative stress [8, 50]. 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 [54], and MDA acting as a breakdown product of lipid peroxidation increased in cryopreserved Oryza sativa [52, 55], Azadirachta indica [56], Arabidopsis thaliana [8], Agapanthus praecox [10], Hancornia speciose [57], and Passiflora suberosa [53]. With the addition of NRSP in the cryoprotectant, the MDA content decreased and the SOD activity increased leading to more intact membrane and better quality of boar sperm [21]. The MDA content significantly increased in the control process, and led to the low survival rate of EC after cryopreservation.

The antioxidant system works to prevent plant cells from oxidative injury through cleaning ROS [11]. 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 the 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 [58], and high tolerance was related to high CAT activity in Haematococcus pluvialis cryopreservation [59]. In the study of Liu [60], 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 [61]. 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 [62]. Adding SWCNTs increased GSH contents. Expression levels of GPX and GR in SWCNTs-added 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, and 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 [63–65]. In this pathway, plants sense ROS by three ways: (a) unknown ROS receptors; (b) redox-sensitive transcription factors; (c) direct inhibition of phosphatases [66]. The ROS signal is detected by unknown ROS receptors leading to the accumulation of Ca^{signal [}67–69], and the signal is transmitted to oxidative signal-inducible 1 (serine/threonine protein kinase, OXI1) which was significantly upregulated 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 upregulated in many H₂O₂-generating stimulus [70]. MAPK3/6 following OXI1 [71, 72] were mainly upregulated during 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 [73, 74], which were upregulated 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 [66]. The interaction between the producing and scavenging pathway determines the intensity of ROS signals [66]. In summary, EC in the control group suffered excessive ROS after dehydration, which broke the ROS metabolism balance. By contrast, SWCNTs both enhanced the producing and scavenging pathway smoothly, and maintained ROS signals balanced and stable in EC during cryopreservation.

Regulation of the physiological response by 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 the 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 [75]. Giraldo et al. [76] pointed out that SWCNTs localized in the lipid envelope of Arabidopsis chloroplasts, promoted 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 [77]. In this study, SWCNTs contributed to the better efficiency of the 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 the similar results. The MWCNTs enhanced the tobacco cell growth, and upregulated genes related to water transport and cell division [78]. 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 [79]. For instance, MAPK was upregulated in leaves exposed to MWCNTs, and played a positive role in promoting plant development and stress response of carbon nanotubes [79]. In this study, MAPK3/6 were higher in the control rapid cooling-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 [80]. On the contrary, experiment on suspension rice cells with MWCNTs showed that it induced the accumulation of ROS leading to cell death [81]. It also decreases the dry weight and the SOD activity in Arabidopsis suspension cells [82]. 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 [83, 84]. However, the mechanism of how the nanotubes can regulate the physiological response especially regulate the gene expression is still an unresolved issue and deserved further studies.

SWCNTs contribute to the better efficiency of cryoprotectant

Effective antioxidant response during the dehydration step improved EC survival after cryopreservation

Effects of SWCNTs on ROS signal transduction during cryopreservation

Regulation of the physiological response by carbon nanotubes in plants

Conclusions

This study found that the relative survival rate was highly improved by adding SWCNTs to PVS2 in cryopreservation of A. praecox EC, and analyzed the oxidative response of EC at some steps in the control and SWCNTs-added cryopreservation. 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. The SWCNTs affected the ROS signal transduction pathway and antioxidant system response through the physiological and gene expression results. The EC treated with SWCNTs exhibited higher antioxidant levels, including POD, CAT and GSH than the EC in the control group. The EC mainly depended on the GPX and AsA-GSH cycle to scavenge H₂O₂ in the control cryopreservation, but depended on CAT in the SWCNTs-added cryopreservation which led to low levels of H₂O₂ and MDA. The elevated antioxidant level in dehydration by adding SWCNTs improved cells tolerance to injury from the cryopreservation procedure. The ROS signals of EC were balanced and stable in the SWCNTs-added cryopreservation. Overall, SWCNTs regulated the oxidative stress response of EC in cryopreservation and controlled cell oxidative injury by keeping ROS homeostasis to achieve a high survival rate after cryopreservation.

Methods

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. After 14 days of subculture, 0.2 g of EC was used as the replication for subsequent experiments.

Experimental design

0.2 g of EC was cryopreserved as described by Chen et al. [30]. The cryopreservation procedure included pre-culture (PC), osmoprotection (OP), dehydration with PVS2 (DH), rapid cooling-warming (RW), dilution (DL), and recovery (RC) (Fig. 6). The SWCNTs aqueous solution (5.0 g/L, particle diameter 1 nm, length 1 μm) was kindly provided by Prof. Yafei Zhang (Shanghai Jiao Tong University, Shanghai, China). In the SWCNTs-added cryopreservation procedure, we added SWCNTs at 0.1 g/L in PVS2 at the dehydration step. The EC as samples for the physiological experiments and qRT-PCR analysis were taken after some key steps including CK, PC, DH, RW, and DL in the control and SWCNTs-added cryopreservation (Fig. 6). Experiments were performed three times individually.

Fig. 6

The samples in the study of SWCNTs to improve cryopreservation. The EC during the control and SWCNTs-added cryopreservation was taken as samples, including CK, after pre-culture, dehydration, rapid cooling-warming, and dilution

Viability detection

In order to detect the survival of cryopreserved EC, the 2,3,5-triphenyltetrazolium chloride (TTC) protocol was used in this study [30]. The EC (0.05 g) after 24 h-recovery was put into 2 mL TTC and kept in the dark for 20 h. After rinsing 3 times with sterile water, EC was immersed in 95% ethanol and 85 °C water bathed for 1 h. The EC was centrifuged at 3000g for 5 min, and the optical density value of supernatant was tested at 485 nm. The relative survival rate was the ratio of cryopreserved and non-cryopreserved value. Each sample procedure was repeated 3 times.

Transmission electron microscopy

The EC (0.05 g) was treated as described by Chen et al. [28], and observed by 120 kV biology transmission electron microscope (Tecnai G2 spirit Biotwin).

Detection of physiological indices

The EC (0.2 g) was used to detect the physiological indices. 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].

qRT-PCR analysis

The qRT-PCR was performed as described by Chen et al. [30]. The EC (0.1 g) was collected for total RNA extraction using MiniBEST plant RNA extraction kit (TaKaRa, Otsu, Shiga, Japan) according to the manufacturer’s instructions. The amplifications were repeated for 3 times. The relative quantitative expression was calculated using the 2^{method. 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.

Statistical analysis

The one-way ANOVA was used to analyze differences followed by the least significant difference multiple range test using Statistics Analysis System 9.1.3 software (SAS Institute, Inc., Cary, NC, USA). Correlation analysis was calculated by Statistics Analysis System 9.1.3 software, and P < 0.05 was considered as significant.

Plant material

Experimental design

Fig. 6

Viability detection

Transmission electron microscopy

The EC (0.05 g) was treated as described by Chen et al. [28], and observed by 120 kV biology transmission electron microscope (Tecnai G2 spirit Biotwin).

Detection of physiological indices

qRT-PCR analysis

Statistical analysis

Additional file 1: Table S1. Primers sequences of qRT-PCR.^{(15K, docx)}

^{Institute for Agri-food Standards and Testing Technology, Shanghai Academy of Agricultural Sciences, NO. 888, Rd. Yezhuang, Shanghai, 201403 China}

^{Department of Landscape Science and Engineering, School of Design, Shanghai Jiao Tong University, NO. 800, Rd. Dong Chuan, Shanghai, 200240 China}

Hairong Chen, Email: moc.361@sudsaas.

Contributor Information.

^{Corresponding author.}

^{Contributed equally.}

Received 2020 May 10; Accepted 2020 Sep 15.

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Background

Results

Conclusions

Effects of different exogenous substances on the survival rate of Agapanthus praecox EC cryopreservation. Concentration of exogenous substances added are: 0.1 g/L SWCNTs, 0.08 mM GSH, 1 μM ABA, 0.1 μM melatonin, 1 mM AsA, 10 mM betaine, 1 mM CaCl₂, 6 mM LA, 6 mM PVA, 3% PVP. Values with different small letters are significantly different between different treatment at 0.05 levelCell ultrastructure observation of Agapanthus praecox during the SWCNTs-added cryopreservation. a, b the EC before cryopreservation, c, d the EC after pre-culture, e–h the EC after dehydration, i–l the EC after dilution. Cell wall (CW), endoplasmic reticulum (ER), golgi apparatus (G), lipid body (LB), mitochondrion (M), starch grains (SG), vesicle (VES)

The correlation analysis of physiological indices in the control cryopreservation

All the data were correlation coefficients, and significant levels are indicated at * P < 0.05 or ** P < 0.01

The correlation analysis of physiological indices in the SWCNTs-added cryopreservation

All the data were correlation coefficients, and significant levels are indicated at * P < 0.05 or ** P < 0.01

Supplementary information

Additional file 1: Table S1. Primers sequences of qRT-PCR.^{(15K, docx)}

Supplementary information

Additional file 1: Table S1. Primers sequences of qRT-PCR.^{(15K, docx)}

Acknowledgements

The authors thank Prof. Yafei Zhang (Shanghai Jiao Tong University, Shanghai, China) for providing the SWCNTs aqueous solution.

Acknowledgements

The authors thank Prof. Yafei Zhang (Shanghai Jiao Tong University, Shanghai, China) for providing the SWCNTs aqueous solution.

Abbreviations

ABA	Abscisic acid
APX	Ascorbate peroxidase
AsA	Ascorbic acid
CAT	Catalase
CNMs	Carbon nanomaterials
CNTs	Carbon nanotubes
CW	Cell wall
DH	Dehydration
DHAR	Dehydroascorbate reductase
DL	Dilution
DSC	Differential scanning calorimetry
EC	Embryogenic callus
ER	Endoplasmic reticulum
G	Golgi apparatus
GPX	Glutathione peroxidase
GR	glutathione reductase
GSH	Glutathione
H₂O₂	Hydrogen peroxide
LA	Lipoic acid
LB	Lipid body
LN	Liquid nitrogen
LSD	Least significant difference
M	Mitochondrion
MAPK	Mitogen-activated protein kinase
MDA	Malonaldehyde
MDHAR	Monodehydroascorbate reductase
MS	Murashige and Skoog
NRSP	Nanomaterials Rhodiola Sachanensis Polysaccharide
O₂⁻	Superoxide anion
OH	Hydroxyl radicals
OP	Osmoprotection
OXI1	Oxidative signal-inducible 1
PC	Pre-culture
PVA	polyvinyl alcohol
PVP	Polyvinyl pyrrolidone
PVS	Plant vitrification solution
qRT-PCR	Quantitative reverse-transcription polymerase chain reactions
RC	Recovery
ROS	Reactive oxygen species
RW	Rapid cooling-warming
SG	Starch grains
SOD	Superoxide dismutase
SWCNTs	Single-wall carbon nanotubes
TEM	Transmission electron microscopy
TTC	Triphenyltetrazolium chloride
VES	Vesicle

Abbreviations

Authors’ contributions

HC and DZ conceived and designed research. LR and SD conducted experiments. YC, YZ and HZ analyzed the data. LR was a major contributor in writing the manuscript. All authors read and approved the final manuscript.

Authors’ contributions

Funding

This work was supported by the National Natural Science Foundation of China [Nos. 31870686, 31670693 and 31971705], and Academic Specialty Development Project of Shanghai Academy of Agricultural Sciences (2019-13).

Funding

Availability of data and materials

All data generated or analysed during this study are included in this published article and its Additional file 1: Table S1.

Availability of data and materials

All data generated or analysed during this study are included in this published article and its Additional file 1: Table S1.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Li Ren and Shan Deng contributed equally to this work

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Li Ren and Shan Deng contributed equally to this work

Contributor Information

Hairong Chen, Email: moc.361@sudsaas.

Di Zhang, Email: nc.ude.utjs@3102idgnahz.

Contributor Information

Supplementary information

Supplementary information accompanies this paper at 10.1186/s13007-020-00674-6.

Supplementary information

Supplementary information accompanies this paper at 10.1186/s13007-020-00674-6.

References

1. Reed BMPlant cryopreservation: a continuing requirement for food and ecosystem security. Vitro Cell Dev. 2017;53:285–288. doi: 10.1007/s11627-017-9851-4.[PubMed][Google Scholar]
2. Sakai ASurvival of the twigs of woody plants at − 196 °C. Nature. 1960;185:392–394.[PubMed][Google Scholar]
3. Engelmann FCryopreservation: progress and prospects. Vitro Cell Dev. 2004;40:427–433. doi: 10.1079/IVP2004541.[PubMed][Google Scholar]
4. Wang MR, Yang W, Zhao L, Li JW, Liu K, Yu JW, Wu YF, Wang QCCryopreservation of virus: a novel biotechnology for long-term preservation of virus in shoot tips. Plant Methods. 2018;14:47. doi: 10.1186/s13007-018-0312-9.] [[Google Scholar]
5. Sakai A, Hirai D, Niino T. Development of PVS-based vitrification and encapsulation-vitrification protocols. In: Reed BM, editor. Plant cryopreservation: a practical guide. New York: Springer-Verlag; 2008. pp. 33–57. [PubMed]
6. Volk GM, Walters CPlant vitrification solution 2 lowers water content and alters freezing behaviour in shoot tips during cryoprotection. Cryobiology. 2006;52:48–61. doi: 10.1016/j.cryobiol.2005.09.004.] [[PubMed][Google Scholar]
7. Ren L, Zhang D, Jiang X, Gai Y, Wang W, Reed BM, Shen XPeroxidation due to cryoprotectant step is a vital factor for cell survival in Arabidopsis cryopreservation. Plant Sci. 2013;212:37–47. doi: 10.1016/j.plantsci.2013.07.011.] [[PubMed][Google Scholar]
8. Benson EE Free radical damage in stored plant germplasm. Italy: International board for plant genetic resources; 1990. [PubMed][Google Scholar]
9. Ren L, Zhang D, Chen G, Reed BM, Shen X, Chen HTranscriptomic profiling revealed the regulatory mechanism of Arabidopsis seedlings response to oxidative stress from cryopreservation. Plant Cell Rep. 2015;34:2161–2178. doi: 10.1007/s00299-015-1859-9.] [[PubMed][Google Scholar]
10. Zhang D, Ren L, Chen G, Zhang J, Reed BM, Shen XROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox. Plant Cell Rep. 2015;34:1499–1513. doi: 10.1007/s00299-015-1802-0.] [[PubMed][Google Scholar]
11. Gill SS, Tuteja NReactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Bioch. 2010;48:909–930. doi: 10.1016/j.plaphy.2010.08.016.] [[PubMed][Google Scholar]
12. Volk GMApplication of functional genomics and proteomics to plant cryopreservation. Curr Genomics. 2010;11:24–29. doi: 10.2174/138920210790217945.] [[Google Scholar]
13. Van Breusegem F, Vranová E, Dat JF, Inzé DThe role of active oxygen species in plant signal transduction. Plant Sci. 2001;161:405–414. doi: 10.1016/S0168-9452(01)00452-6.[PubMed][Google Scholar]
14. Scandalios JG. Molecular genetics of SOD in plants. In: Scandalios JG, editor. Oxidative stress and the molecular biology of antioxidant defense. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1997. pp. 527–568. [PubMed]
15. Meyer AJThe integration of glutathione homeostasis and redox signaling. J Plant Physiol. 2008;165:1390–1403. doi: 10.1016/j.jplph.2007.10.015.] [[PubMed][Google Scholar]
16. Husen A, Siddiqi KSCarbon and fullerene nanomaterials in plant system. J Nanobiotech. 2014;12:1–10. doi: 10.1186/1477-3155-12-1.[PubMed][Google Scholar]
17. Hao BTApplication of nanopaticles in cell cryopreservation. J Clin Rehabilita Tiss Eng Res. 2008;12:8140–8142.[PubMed][Google Scholar]
18. Derui DI, Zhizhu HE, Liu JAdvances in nano-cryopreservation of biological materials. Ciesc J. 2011;62:1781–1789.[PubMed][Google Scholar]
19. Li W, Zhou X, Lv F, Liu BPreliminary study on the mechanism of promoting the survival rate of vitrified oocytes with nano-cryoprotectants. J Refrigeration. 2014;35:114–118.[PubMed][Google Scholar]
20. Han X, Ma HB, Wilson C, Critser JKEffects of nanoparticles on the nucleation and devitrification temperatures of polyol cryoprotectant solutions. Microfluid Nanofluid. 2008;4:357–361. doi: 10.1007/s10404-007-0186-z.[PubMed][Google Scholar]
21. Li W, Zhou XL, Liu BL, Dai JJ, Lv FK, Zhang DF, Xu LThe effect of nanoparticle on vitrification of porcine GV-stage oocytes. Chin J Biomed Eng. 2013;32:601–605.[PubMed][Google Scholar]
22. Han X, Dai JJ, Zhang SS, Zhang TY, Wu CF, Gu XL, Li X, Xu L, Li FF, Zhang DFEffects of nonomaterials rhodiola sachalinensis polysaccaride on cryopreservation of boar semen. Chin J Vet Sci. 2013;33:1937–1942.[PubMed][Google Scholar]
23. Nadri T, Towhidi A, Zeinoaldini S, Martínez-Pastor F, Mousavi M, Noei R, Tar M, Mohammadi Sangcheshmeh ALecithin nanoparticles enhance the cryosurvival of caprine sperm. Theriogenology. 2019;133:38–44. doi: 10.1016/j.theriogenology.2019.04.024.] [[PubMed][Google Scholar]
24. Dashtestani F, Ghourchian H, Najafi AAlbumin coated copper-cysteine nanozyme for reducing oxidative stress induced during sperm cryopreservation. Bioorg Chem. 2018;80:621–630. doi: 10.1016/j.bioorg.2018.07.020.] [[PubMed][Google Scholar]
25. Dashtestani F, Ghourchian H, Najafi ASilver-gold-apoferritin nanozyme for suppressing oxidative stress during cryopreservation. Mat Sci Eng C Mater. 2019;94:831–840. doi: 10.1016/j.msec.2018.10.008.] [[PubMed][Google Scholar]
26. Khalil WA, El-Harairy MA, Zeidan AEB, Hassan MAEImpact of selenium nano-particles in semen extender on bull sperm quality after cryopreservation. Theriogenology. 2019;126:121–127. doi: 10.1016/j.theriogenology.2018.12.017.] [[PubMed][Google Scholar]
27. Zhang J, Yang K, Zhang H, Liang XApplication status and prospect of carbon-based nanomaterials in biomedical field. Prog Chem. 2013;25:397–408.[PubMed][Google Scholar]
28. Chen SM, Ren L, Zhang D, Zhang YF, Shen XHCarbon nanomaterials enhance survival of Agapanthus praecox callus after cryopreservation by vitrification. Cryo Lett. 2017;38:125–136.[PubMed][Google Scholar]
29. Zhang D, Ren L, Yue J, Wang L, Zhuo L, Shen XA comprehensive analysis of flowering transition in Agapanthus praecox ssp. orientalis (Leighton) Leighton by using transcriptomic and proteomic techniques. J Proteomics. 2013;80:1–25. doi: 10.1016/j.jprot.2012.12.028.] [[PubMed][Google Scholar]
30. Chen GQ, Ren L, Zhang D, Shen XHGlutathione improves survival of cryopreserved embryogenic calli of Agapanthus praecox subsp. orientalis. Acta Physiol Plant. 2016;38:250. doi: 10.1007/s11738-016-2271-y.[PubMed][Google Scholar]
31. Zhang D, Ren L, Shen XH, Zhuo LHFertilization and embryogenesis in Agapanthus praecox ssp. orientalis (Leighton) Leighton. Plant Syst Evol. 2011;293:25–30. doi: 10.1007/s00606-010-0378-y.[PubMed][Google Scholar]
32. Baskaran P, Chukwujekwu JC, Amoo SO, Van Staden JAnticholinesterase and mutagenic evaluation of in vitro-regenerated Agapanthus praecox grown ex vitro. Vitro Cell Dev. 2014;50:271–275. doi: 10.1007/s11627-013-9567-z.[PubMed][Google Scholar]
33. Suzuki S, Oota M, Nakano MEmbryogenic callus induction from leaf explants of the Liliaceous ornamental plant, Agapanthus praecox ssp. orientalis (Leighton) Leighton Histological study and response to selective agents. Sci Hortic. 2002;95:123–132. doi: 10.1016/S0304-4238(02)00033-X.[PubMed][Google Scholar]
34. Wang Y, Fan X, Zhang D, Shen XRegeneration of Agapanthus praecox ssp. orientalis ‘big blue’ via somatic embryogenesis. Propag Ornam Plants. 2012;12:148–154.[PubMed][Google Scholar]
35. Baskaran P, Van Staden JRapid in vitro micropropagation of Agapanthus praecox. S Afr J Bot. 2013;86:46–50. doi: 10.1016/j.sajb.2013.01.008.[PubMed][Google Scholar]
36. Wang ZC, Deng XXCryopreservation of shoot-tips of citrus using vitrification: effect of reduced form of glutathione. Cryo Lett. 2004;25:43–50.[PubMed][Google Scholar]
37. Uchendu EE, Leonard SW, Traber MG, Reed BMVitamins C and E improve regrowth and reduce lipid peroxidation of blackberry shoot tips following cryopreservation. Plant Cell Rep. 2010;29:25–35. doi: 10.1007/s00299-009-0795-y.] [[PubMed][Google Scholar]
38. Uchendu EE, Muminova M, Gupta S, Reed BMAntioxidant and anti-stress compounds improve regrowth of cryopreserved Rubus shoot tips. Vitro Cell Dev Biol. 2010;46:386–393. doi: 10.1007/s11627-010-9292-9.[PubMed][Google Scholar]
39. Zhao Y, Qi LW, Wang WM, Saxena PK, Liu CZMelatonin improves the survival of cryopreserved callus of Rhodiola crenulata. J Pineal Res. 2011;50:8–88. doi: 10.1111/j.1600-079X.2010.00817.x.] [[PubMed][Google Scholar]
40. Hu MJCryopreservation of Arabidopsis thaliana suspension cells and the role of Abscisic Acid in stress signaling transduction pathways. Dissertation, Zhejiang University, 2003.
41. Lardet L, Martin F, Dessailly F, Carron MP, Montoro PEffect of exogenous calcium on post-thaw growth recovery and subsequent plant regeneration of cryopreserved embryogenic calli of Hevea brasiliensis (Müll. Arg.) Plant Cell Rep. 2007;26:559–569. doi: 10.1007/s00299-006-0278-3.] [[PubMed][Google Scholar]
42. Ryynanen L, Haggman HRecovery of cryopreserved silver birch shoot tips is affected by the pre-freezing age of the cultures and ammonium substitution. Plant Cell Rep. 2001;20:354–360. doi: 10.1007/s002990100336.[PubMed][Google Scholar]
43. Decruse SW, Seeni S, Nair GMPreparative procedures and culture medium affect the success of cryostorage of Holostemma annulare shoot tips. Plant Cell Tiss Org. 2004;76:179–182. doi: 10.1023/B:TICU.0000007283.28280.a3.[PubMed][Google Scholar]
44. Kami D, Kasuga J, Arakawa K, Fujikawa SImproved cryopreservation by diluted vitrification solution with supercooling-facilitating flavonol glycoside. Cryobiology. 2008;57:242–245. doi: 10.1016/j.cryobiol.2008.09.003.] [[PubMed][Google Scholar]
45. Wang HY, Inada T, Funakoshi K, Lu SSInhibition of nucleation and growth of ice by poly (vinyl alcohol) in vitrification solution. Cryobiology. 2009;59:83–89. doi: 10.1016/j.cryobiol.2009.04.013.] [[PubMed][Google Scholar]
46. Johnston JW, Harding K, Benson EEAntioxidant status and genotypic tolerance of Ribes in vitro cultures to cryopreservation. Plant Sci. 2007;172:524–534. doi: 10.1016/j.plantsci.2006.11.001.[PubMed][Google Scholar]
47. Fang JY, Wetten A, Johnston JHeadspace volatile markers for sensitivity of cocoa (Theobroma cacao L.) somatic embryos to cryopreservation. Plant Cell Rep. 2008;27:453–461. doi: 10.1007/s00299-007-0487-4.] [[PubMed][Google Scholar]
48. Whitaker C, Beckett RP, Minibayeva FV, Kranner IProduction of reactive oxygen species in excised, desiccated and cryopreserved explants of Trichilia dregeana Sond. S Afr J Bot. 2010;76:112–118. doi: 10.1016/j.sajb.2009.09.008.[PubMed][Google Scholar]
49. Skyba M, Petijová L, Košuth J, Koleva DP, Ganeva TG, Kapchina-Toteva VM, Cěllárová EOxidative stress and antioxidant response in Hypericum perforatum L. plants subjected to low temperature treatment. J Plant Physiol. 2012;169:955–964. doi: 10.1016/j.jplph.2012.02.017.] [[PubMed][Google Scholar]
50. Chen GQ, Ren L, Zhang J, Reed BM, Zhang D, Shen XHCryopreservation affects ROS-induced oxidative stress and antioxidant response in Arabidopsis seedlings. Cryobiology. 2015;70:38–47. doi: 10.1016/j.cryobiol.2014.11.004.] [[PubMed][Google Scholar]
51. Jia MX, Shi Y, Di W, Jiang XR, Xu J, Liu YROS-induced oxidative stress is closely related to pollen deterioration following cryopreservation. Vitro Cell Dev Biol-Pl. 2017;53:433–439. doi: 10.1007/s11627-017-9844-3.[PubMed][Google Scholar]
52. Huang B, Zhang JM, Chen XL, Xin X, Yin GK, He JJ, Lu XX, Zhou YCOxidative damage and antioxidative indicators in 48 h germinated rice embryos during the vitrification-cryopreservation procedure. Plant Cell Rep. 2018;37:1325–1342. doi: 10.1007/s00299-018-2315-4.] [[PubMed][Google Scholar]
53. Vianna MG, Garcia RO, Mansur E, Engelmann F, Pacheco GOxidative stress during the cryopreservation of Passiflora suberosa L. shoot tips using the V-Cryo-plate technique: determination of the critical stages of the protocol. Plant Cell Tiss Org. 2019;139:369–379. doi: 10.1007/s11240-019-01690-8.[PubMed][Google Scholar]
54. Benson EE, Bremner D. Oxidative stress in the frozen plant: a free radical point of view. In: Fuller BJ, Lane N, Benson EE, editors. Life in the frozen state. Boca Raton: CRC Press; 2004. pp. 205–241. [PubMed]
55. Benson EE, Lynch PT, Jones JThe detection of lipid peroxidation products in cryoprotected and frozen rice cells—consequences for post thaw survival. Plant Sci. 1992;85:107–114. doi: 10.1016/0168-9452(92)90099-8.[PubMed][Google Scholar]
56. Varghese B, Naithani SCOxidative metabolism-related changes in cryogenically stored neem (Azadirachta indica A. Juss) seeds. J Plant Physiol. 2008;165:755–765. doi: 10.1016/j.jplph.2007.03.009.] [[PubMed][Google Scholar]
57. Prudente DO, Paiva R, Nery FC, Paiva PDO, Alves JD, Máximo WPF, Silva LCCompatible solutes improve regrowth, ameliorate enzymatic antioxidant systems, and reduce lipid peroxidation of cryopreserved Hancornia speciosa Gomes lateral buds. Vitro Cell Dev Biol. 2017;53:352–362. doi: 10.1007/s11627-017-9830-9.[PubMed][Google Scholar]
58. Poobathy R, Sinniah UR, Xavier R, Subramaniam SCatalase and superoxide dismutase activities and the total protein content of protocorm-like bodies of Dendrobium Sonia-28 subjected to vitrification. Appl Biochem Biotechnol. 2013;170:1066–1079. doi: 10.1007/s12010-013-0241-z.] [[PubMed][Google Scholar]
59. Fleck RA, Benson EE, Bremner DH, Day JGA comparative study of antioxidant protection in cryopreserved unicellular algae Euglena gracilis and Haematococcus pluvialis. Cryo-lett. 2003;24:213–228.[PubMed][Google Scholar]
60. Liu SJ. The effects of graphene on the germination and seedling growth in rice. Yangtze University, 2013.
61. Noctor G, Foyer CHAscorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol. 1998;49:249–279. doi: 10.1146/annurev.arplant.49.1.249.] [[PubMed][Google Scholar]
62. Alscher RG, Donahue JL, Cramer CLReactive oxygen species and antioxidants: relationships in green cells. Physiol Plant. 1997;100:224–233. doi: 10.1111/j.1399-3054.1997.tb04778.x.[PubMed][Google Scholar]
63. Dat JF, Vandenabeele S, Vranová E, Van Montagu M, Inzé D, Van Breusegem FDual action of the active oxygen species during plant stress responses. Cell Mol Life Sci. 2000;57:779–795. doi: 10.1007/s000180050041.] [[PubMed][Google Scholar]
64. Mittler ROxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 2002;7:405–410. doi: 10.1016/S1360-1385(02)02312-9.] [[PubMed][Google Scholar]
65. Vranová E, Atichartpongkul S, Villarroel R, Van Montagu M, Inzé D, Van Camp WComprehensive analysis of gene expression in Nicotiana tabacum leaves acclimated to oxidative stress. Proc Natl Acad Sci USA. 2002;99:10870–10875. doi: 10.1073/pnas.152337999.] [[Google Scholar]
66. Mittler R, Vanderauwera S, Gollery M, Van Breusegem FReactive oxygen gene network of plants. Trends Plant Sci. 2004;9:490–498. doi: 10.1016/j.tplants.2004.08.009.] [[PubMed][Google Scholar]
67. Bowler C, Fluhr RThe role of calcium and activated oxygens as signals for controlling cross-tolerance. Trends Plant Sci. 2000;5:241–246. doi: 10.1016/S1360-1385(00)01628-9.] [[PubMed][Google Scholar]
68. Knight H, Knight MRAbiotic stress signalling pathways: specificity and cross-talk. Trends Plant Sci. 2001;6:262–267. doi: 10.1016/S1360-1385(01)01946-X.] [[PubMed][Google Scholar]
69. Coelho SM, Taylor AR, Ryan KP, Sousa-Pinto I, Brown MT, Brownlee CSpatiotemporal patterning of reactive oxygen production and Ca^{wave propagation in}Fucus rhizoid cells. Plant Cell. 2002;14:2369–2381. doi: 10.1105/tpc.003285.] [[Google Scholar]
70. Rentel MC, Lecourieux D, Ouaked F, Usher SL, Petersen L, Okamoto H, Knight H, Peck SC, Grierson CS, Hirt H, Knight MROXI1 kinase is necessary for oxidative burst-mediated signalling in Arabidopsis. Nature. 2004;427:858–861. doi: 10.1038/nature02353.] [[PubMed][Google Scholar]
71. Kovtun Y, Chiu WL, Tena G, Sheen JFunctional analysis of oxidative stress activated mitogen-activated protein kinase cascade in plants. Proc Natl Acad Sci USA. 2000;97:2940–2945. doi: 10.1073/pnas.97.6.2940.] [[Google Scholar]
72. Apel K, Hirt HReactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol. 2004;55:373–399. doi: 10.1146/annurev.arplant.55.031903.141701.] [[PubMed][Google Scholar]
73. Dat JF, Pellinen R, Beeckman T, Van De Cotte B, Langebartels C, Kangasjärvi J, Inzé D, Van Breusegem FChanges in hydrogen peroxide homeostasis trigger an active cell death process in tobacco. Plant J. 2003;33:621–632. doi: 10.1046/j.1365-313X.2003.01655.x.] [[PubMed][Google Scholar]
74. Rizhsky L, Davletova S, Liang HJ, Mittler RThe zinc finger protein Zat12 is required for cytosolic ascorbate peroxidase 1 expression during oxidative stress in Arabidopsis. J Biol Chem. 2004;279:11736–11743. doi: 10.1074/jbc.M313350200.] [[PubMed][Google Scholar]
75. Zaytseva O, Neumann GCarbon nanomaterials: production, impact on plant development, agricultural and environmental applications. Chem Biol Technol Agric. 2016;3:17. doi: 10.1186/s40538-016-0070-8.[PubMed][Google Scholar]
76. Giraldo JP, Landry MP, Faltermeier SM, McNicholas TP, Iverson NM, Boghossian AA, Reuel NF, Hilmer AJ, Sen F, Brew JA, Strano MSPlant nanobionics approach to augment photosynthesis and biochemical sensing. Nat Mater. 2014;13:400–408. doi: 10.1038/nmat3890.] [[PubMed][Google Scholar]
77. Elena S, Gusev A, Zaytseva O, Sheina O, Tkachev A, Kuznetsova E, Lazareva E, Onishchenko G, Feofanov A, Kirpichnikov MUptake and accumulation of multiwalled carbon nanotubes change the morphometric and biochemical characteristics of Onobrychis arenaria seedlings. Fron Chem Sci Eng. 2012;6:132–138. doi: 10.1007/s11705-012-1290-5.[PubMed][Google Scholar]
78. Khodakovskaya MV, De Silva K, Biris AS, Dervishi E, Villagarcia HCarbon nanotubes induce growth enhancement of tobacco cells. ACS Nano. 2012;6:2128–2135. doi: 10.1021/nn204643g.] [[PubMed][Google Scholar]
79. Khodakovskaya M, De Silva K, Nedosekin D, Dervishi E, Biris AS, Shashkov EV, Galanzha EI, Zharov VPComplex genetic, photothermal, and photoacoustic analysis of nanoparticle-plant interactions. Proc Natl Acad Sci USA. 2011;108:1028–1033. doi: 10.1073/pnas.1008856108.] [[Google Scholar]
80. Lahiani MH, Dervishi E, Chen J, Nima Z, Gaume A, Biris AS, Khodakovskaya MVImpact of carbon nanotube exposure to seeds of valuable crops. ACS Appl Mater Inter. 2013;5:7965–7973. doi: 10.1021/am402052x.] [[PubMed][Google Scholar]
81. Tan X, Lin C, Fugetsu BStudies on toxicity of multi-walled carbon nanotubes on suspension rice cells. Carbon. 2009;47:3479–3487. doi: 10.1016/j.carbon.2009.08.018.[PubMed][Google Scholar]
82. Lin C, Fugetsu B, Su Y, Watari FStudies on toxicity of multi-walled carbon nanotubes on Arabidopsis T87 suspension cells. J Hazard Mater. 2009;170:578–583. doi: 10.1016/j.jhazmat.2009.05.025.] [[PubMed][Google Scholar]
83. Aslani F, Bagheri S, Julkapli NM, Juraimi AS, Hashemi FSG Baghdadi A. Effects of engineered nanomaterials on plants growth: An overview. Sci World J; 2014. p. 641759. [Google Scholar]
84. Husen A, Siddiqi KSCarbon and fullerene nanomaterials in plant system. J Nanobiotechnol. 2014;12:16. doi: 10.1186/1477-3155-12-16.] [[Google Scholar]
85. Yang Z, Sheng J, Lv K, Ren L, Zhang DY₂SK₂ and SK₃ type dehydrins from Agapanthus praecox can improve plant stress tolerance and act as multifunctional protectants. Plant Sci. 2019;284:143–160. doi: 10.1016/j.plantsci.2019.03.012.] [[PubMed][Google Scholar]