Plant materials and SWCNTs
EC of A. praecox was induced from pedicel tissue [80] and is under continuous subculture on MS medium supplemented with 1.5 mg L− 1 picloram at 25 °C.
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).
Cryopreservation procedure
EC were cryopreserved by vitrification as described by Chen et al. [33] with some modifications as detailed below:
(1) Pre-culture (PC): 0.2 g EC were cultured on MS medium with 0.5 M sucrose at 4 °C for 48 h.
(2) Osmoprotection (OP): EC were immersed in 2.0 mL loading solution (MS liquid medium + 2.0 M glycerol + 0.4 M sucrose + 10 mM KNO3) at 25 °C for 60 min.
(3) Dehydration (DH): the loading solution was replaced by PVS2 (30% w/v glycerol, 15% w/v ethylene glycol, and 15% w/v dimethyl sulfoxide in MS liquid medium with 0.4 M sucrose) at 0 °C for 40 min.
(4) Rapid cooling-warming (RW): EC after dehydration in cryovials were rapidly plunged into LN, held for 1 h, and rewarmed in 40 °C water bath for 90 s.
(5) Dilution (DL): PVS2 was replaced by the unloading solution (MS liquid medium + 1.2 M sucrose + 10 mM KNO3) for 30 min, and the solution was replaced with fresh unloading solution every 10 min.
(6) Recovery (RC): the EC were cultured on MS medium under the same conditions as for subculture.
In the SWCNTs-added cryopreservation procedure, we added SWCNTs at 0.1 g/L in PVS2 at the dehydration step. The samples for physiological assay and qRT-PCR were taken after key steps including pre-culture, dehydration, rapid cooling-warming and dilution. Experiments were performed three times individually.
Viability detection
In order to detect the survival of cryopreserved EC, the 2,3,5-triphenyltetrazolium chloride (TTC) method was used in this study [33]. EC (0.05 g) after 24 h-recovery was placed into 2 mL TTC buffer (0.8% w/v TTC in 0.05 M PBS) and kept in dark at 25 °C for 20 h. After 3 times sterile water rinse, EC were immersed in 95% ethanol and water bathed at 85 °C for 1 h. The EC were centrifuged at 3000 g for 5 min and the optical density value of supernatant was measured at 485 nm. The ratio of absorbance value of cryopreserved and non-cryopreserved samples was taken as the relative survival rate. Each sample procedure was repeated 3 times.
Transmission Electron Microscopy
EC were submerged in 2% glutaraldehyde fixing solution for over 6 h. Then EC were stained with 1% osmium tetroxide at 4 °C for 15 min, and rinsed by 0.1 M PBS for 3 times. After rinsing, EC were dehydrated in an ascending ethanol-acetone series (50% ethanol for 15 min, 70% ethanol for 15 min, 90% ethanol for 15 min, 90% ethanol, 90% acetone (1: 1) for 20 min, 90% acetone 20 min) at 4 °C. Dehydration was continued by washing EC in 100% acetone 3 times. After dehydration, samples were embedded in epoxy resin at room temperature (pure acetone + 812 epoxy resin (1: 1) overnight, pure acetone + 812 epoxy resin (1: 2) 8 h, 812 pure epoxy 4 h, 2 times). Then cured the resin (37 °C overnight, 45 °C 12 h,60 °C 48 h). Finally, the ultrathin sections with a thickness of 70 nm were cut from the embedded samples using an ultramicrotome (Leica UC6), and placed onto copper grids stained with TI Blue (10 min) and lead citrate (6 min). The sections were observed by 120 kV biology transmission electron microscope (Tecnai G2 spirit Biotwin).
Detection of physiological indices
H2O2 levels, O2− inhabitation and OH· generation activities, SOD, CAT and POD activities, AsA, GSH and MDA contents were detected using biological assay kits (Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer’s instructions according to Yang et al. [81].
Quantitative gene expression analysis
The method was as described by Chen et al. [33]. About 0.2 g EC were collected for total RNA extraction using RNAiso Plus (TaKaRa, Otsu, Shiga, Japan) according to the manufacturer’s instructions. The cDNA was synthesized using the PrimeScriptTM 1st strand cDNA synthesis Kit (TaKaRa) according to the manufacturer’s instructions. Real-Time quantitative PCR (qRT-PCR) was performed according to SYBR Premix Ex Taq II Kit (TaKaRa) on the Bio-Rad CHROMO4 Gradient cycler system as follows: one cycle of 1 min at 95 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at Tm, and 1 min at 72 °C. All the amplifications were repeated at least three times. The relative quantitative expression of each gene was calculated via the 2−△△CT method. The primer sequences used for quantitative gene expression analysis are listed in Additional file 1: Table S1.
Statistical analysis
All data were expressed as the mean values ± standard deviation (SD) of three replicates. Statistical analysis was performed with Statistics Analysis System 9.1.3 software (SAS Institute, Inc., Cary, NC, USA). For oxidative stress-related indices and qRT-PCR data, a one-way ANOVA was used followed by least significant difference (LSD) multiple range test when significance differences were detected (P < 0.05). Correlation analysis between oxidative stress-related indices was calculated, and P < 0.05 was considered to be significant.