Plant tissue culture provides a rapid and reliable system for the production of a large number of genetically uniform and disease-free plantlets in controlled laboratory conditions, in a small space, in a short time and regardless of seasonality (Regni et al., 2022). Due to the high popularity and demand for chrysanthemum, it has become one of the first commercial targets for micropropagation. Chrysanthemum is propagated
Nanotechnology is concerned with the design, synthesis, manipulation and application of atomic or molecular aggregates with a dimension between 1 nm and 100 nm. The engineering methodology and processing that produce nanoparticles (NPs) alter their physicochemical properties, as well as biological reactivity, due to nanometric size and high surface-to-volume ratio. Nanotechnology has been applied to modern agriculture and horticulture practices as innovative pesticides, fertilisers or growth stimulators (García-López et al., 2018). In recent years, the use of NPs has successfully led to the reduction of microbial contaminations in plant tissue cultures and demonstrated the positive role of NPs in callus formation, organogenesis, somatic embryogenesis, secondary metabolite production (Kim et al., 2017) and variability induction in plant breeding (Tymoszuk and Kulus, 2022).
Zinc is an indispensable plant micronutrient that controls the activity of numerous enzymes and hormones; regulates the metabolism of macromolecules, stabilising proteins, DNA and RNA structures; and controls antioxidant metabolism and gene expression. It contributes to cell proliferation and differentiation, and chloroplast development and functioning and participates in plant growth regulation affecting root and shoot development. Zinc deficiency in plants causes abnormal growth, reduced enzymatic activity and, finally, a disturbed metabolism. To fulfil the Zn requirements of plants, the smartest delivery tool for Zn may be NPs (Awan et al., 2021). Zinc in the form of zinc oxide NPs (ZnO NPs) may be more effectively absorbed by plants and increase nutrient uptake, pigment content, photosynthesis efficiency and biomass accumulation (da Cruz et al., 2019; Salachna et al., 2021; Regni et al., 2022). ZnO NPs belong to the most produced NPs worldwide and are commonly used in several industrial products such as components of solar cells, sunscreens, wall paints, ceramics, catalysis and biomedicine. ZnO NPs are also the most used NPs in agricultural applications due to easy availability, low chemical price, stability at high temperature and neutral pH (Elshoky et al., 2021; Rani et al., 2022).
ZnO NPs had a greater and more responsive impact on tobacco (
Due to unique physicochemical properties, i.e. great chemical stability, conductivity, catalytic activity and antimicrobial potential, silver NPs are the most commonly used NPs in numerous applications (Tariq et al., 2022). In plant production, they are used as plant growth stimulators, components of fertilisers and plant protection products. Ag NPs have also been used in plant tissue culture to improve seed germination and plant growth, stimulate the biosynthesis of bioactive compounds and enable genetic transformation (Mahendran et al., 2019). However, silver NPs may also show phytotoxicity, manifested by limited germination and seedling growth, decreased biomass of leaves and shoots and inhibition of photosynthesis. Therefore, further studies are needed to clarify these contradictory observations (Salachna et al., 2019; Parzymies, 2021).
Micropropagation protocols aiming to produce efficiently true-to-type plants should guarantee the genetic fidelity of propagated plants. Since the application of NPs may result in the induction of variability, the genetic analysis of
This study aimed to test, for the first time, the effects of ZnO NPs alone or combined with silver NPs (ZnO + Ag NPs), applied at the concentration of 100 mg · L−1, 200 mg · L−1 or 400 mg · L−1, on the growth and chlorophyll and carotenoid content, as well as genetic stability of chrysanthemums ‘UTP Burgundy Gold (UBG)’ and ‘UTP Pinky Gold (UPG)’ plantlets developed
The synthesis of nanostructured ZnO NPs and ZnO + x% Ag NPs included the use of several materials such as zinc acetate dihydrate (Zn(CH3COO)2 · 2H2O, Avantor Performance Materials Poland S.A., Gliwice, Poland), silver acetate anhydrous (Ag(CH3COO), Chempur, Piekary Śląskie, Poland), ethylene glycol (C2H4(OH)2, Chempur, Piekary Śląskie, Poland) and deionised water (H2O) (specific conductance below 0.1 μS · cm−1). All the chemical substances were analytically pure and used without further purification. SMPs of pharmaceutically pure zinc oxide (ZnO SMPs) were purchased from ZM SILESIA SA, Huta Oława, Oława, Poland.
ZnO NPs and ZnO + x% Ag NPs samples were obtained by microwave solvothermal synthesis (Wojnarowicz et al., 2020) using the authors’ own procedure described in previous papers (Pokrowiecki et al., 2019; Tymoszuk et al., 2022). Briefly, to approximate the synthesis, zinc acetate dihydrate was dissolved in ethylene glycol at 70°C using a magnetic stirrer. The obtained solution was tightly sealed in a bottle. When the solution reached room temperature, the water content was analysed, and a calculated amount of water was added to reach a final water concentration of 1.5% or 6% by weight in the precursor solution. The MSS2 (Microwave Solvothermal Synthesis model 2) microwave reactor was used to synthesise the nanopowders (270 mL, 12 min, 4 bar, 3 kW, 2.45 GHz, IHPP PAN (Warsaw, Poland), ITeE-PIB (Radom, Poland) and ERTEC (Wrocław, Poland) (Majcher et al., 2013). After the synthesis, the obtained suspension was centrifuged, then the liquid from above the precipitate was decanted. The sediment was washed with distilled water and centrifuged (washing and centrifugation processes were repeated four times). The resulting paste was frozen using liquid nitrogen and dried by freeze-drying. The synthesis procedure was repeated five times, and a total of six powder samples were obtained, which were ZnO NPs (1.5% H2O), ZnO NPs (6% H2O), ZnO + 0.1% Ag NPs (1.5% H2O), ZnO + 0.1% Ag NPs (6% H2O), ZnO + 1% Ag NPs (1.5% H2O) and ZnO + 1% Ag NPs (6% H2O). The compositions of the precursor solutions can be found in Supplementary Table S1. Commercial submicron zinc oxide (ZnO SMPs) was used as a reference material.
The testing of the samples was carried out at the Laboratory of Nanostructures (IHPP PAN, Warsaw, Poland), which is accredited with accreditation no. AB 1503. A description of the research procedures used can be found in Wojnarowicz et al. (2018). X-ray powder diffraction (XRD) patterns were tested with an X’Pert PRO X-ray diffractometer (CuKα, Panalytical, Almelo, The Netherlands). Morphology was tested using a scanning electron microscope (ULTRA PLUS, ZEISS, Oberkochen, Germany). Skeletal density was examined using a helium pycnometer (AccuPyc II 1340, FoamPyc V1.06, Micromeritics®, Norcross, GA, USA). The specific surface area (SSA) was measured by using the Brunauer–Emmett–Teller (BET) method (Gemini 2360, V 2.01, Micromerit-ics®, Norcross, GA, USA). The zinc and the silver content were determined by energy dispersive spectrometry (Quantax 400, Bruker, Billerica, MA, USA). The water content (wt%) of the glycol solution samples was measured using the Karl Fischer method (Cou-Lo AquaMAX KF, GR Scientific, Bedford, UK).
The average crystallite size (diameter) was obtained using the Scherrer equation. The average particle size (diameter) was calculated from the skeleton density results and SSA results. The results of sample characterisation can be found in the supplementary materials (Supplementary Table S2, Supplementary Table S3 and Supplementary Figure S1). The nanopowder samples obtained by using the microwave method were characterised by a uniform size with a homogeneous spherical shape, which was confirmed by Scanning Electron Microscopy (SEM) results (Supplementary Figure S2). For samples ZnO SMPs, ZnO NPs (1.5% H2O), ZnO NPs (6% H2O), ZnO + 0.1% Ag NPs (1.5% H2O), ZnO + 0.1% Ag NPs (6% H2O), ZnO + 1% Ag NPs (1.5% H2O) and ZnO + 1% Ag NPs (6% H2O), particle size was 240 nm, 25 nm, 65 nm, 29 nm, 79 nm, 27 nm and 53 nm, respectively.
For micropropagation, the modified Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) was used, with the content of calcium and iron increased by half. The medium was supplemented with 30 g · L−1 sucrose and contained 8 g · L−1 Plant Propagation LABAGAR™ (BIOCORP, Warsaw, Poland). No plant growth regulators were used. After adding all of the nutrients, the medium pH was adjusted to 5.8. Afterwards, 40 mL of the medium was poured into 350-mL glass jars sealed with plastic caps and autoclaved (105 kPa, 121° C, 20 min).
Two
To evaluate the effect of the tested SMPs and NPs on the growth and development of plantlets, the following biometric data were collected: number of leaves, micropropagation coefficient (number of descendant single node explants that could be isolated from the developed plantlet for further subculture), shoot length (cm), shoot fresh/dry weight (FW/DW) (mg) and root system fresh/dry weight (FW/DW) (mg). For the determination of shoot and root DW, the plant material was pre-dried at room temperature and then desiccated at 105° C for 180 min in a laboratory drier (SML 42/AM, ZALMED, Warsaw, Poland) to obtain a constant dry matter.
Excised leaves and root systems were scanned with an Epson Perfection V800 scanner (Suwa, Japan). The obtained pictures were analysed to measure the leaf area (cm2), leaf perimeter (cm), maximal leaf vertical length (cm) and maximal leaf horizontal width (cm) using the imaging software WinFOLIA™ (Reagen Instruments, Quebec, Canada), as well as the total length of the root system (cm), root system area (cm2), root system volume (mm3), number of root tips and number of root forks with the imaging software WinRHIZO™ (Reagen Instruments, Quebec, Canada).
The whole leaves were used as fresh tissue samples for the biochemical assay. Chlorophylls and carotenoids were extracted using 100 mg samples and 100% acetone (Chemia, Bydgoszcz, Poland) according to Lichtenthaler’s (1987) procedure. The spectrophotometric analyses were performed using a NanoPhotometer® NP80 (Implen, München, Germany) at specific wavelengths (λmax): for chlorophylls
The genetic fidelity of SMPs/NPs-treated plantlets was assessed using RAPD (Williams et al., 1990) and SCoT (Collard and Mackill, 2009) marker systems. A total of 32 ‘UBG’/’UPG’ plantlets were included in the analysis (four from each SMPs/NPs treatment at the highest tested concentration of 400 mg · L−1 and four controls).
Total genomic DNA was extracted from fresh leaf tissue (100 mg) samples. The Genomic Mini AX Plant SPIN Kit (A&A Biotechnology, Gdańsk, Poland) reagents and materials were used for DNA isolation. The DNA concentration was measured using a NanoPhotometer® NP80 (Implen, München, Germany). The DNA was stored at 4°C in Tris-EDTA (TE) buffer for a few days before the PCR.
The DNA samples were used as a template for the PCR analysis with a total of 10 primers (5 RAPD and 5 SCoT; Genomed S.A., Warsaw, Poland). PCR was performed using a BioRad C1000 Touch thermal cycler with a heated cover (Bio-Rad, Hercules, CA, USA) in the 25-μL reaction solution. Each reaction contained 2 mM MgCl2 in the reaction buffer, 1 mM dNTP solution mix, 0.05 U · μL−1 Taq DNA polymerase (PCR Master MixPlus, A&A Biotechnology, Gdańsk, Poland), 1 μM single primer, 0.8 ng · μL−1 template DNA (20 ng) and molecular water to volume. For the RAPD analysis, the following profile was applied: one cycle of 4 min at 94°C for initial DNA denaturation; 40 cycles of 1 min at 94°C for denaturation, 40 s at 42°C for annealing and 2 min at 72°C for DNA extension. The last cycle was followed by a final extension step of 4 min at 72°C. SCoT amplification was programmed as follows: one cycle of 4 min at 94°C for initial DNA denaturation; 35 cycles of 1 min at 94°C for denaturation, 50 s at 44°C for annealing and 2 min at 72°C for DNA extension. The last cycle was followed by a final extension step of 8 min at 72°C.
The PCR products were visualised on a ultraviolet (UV) light transilluminator (GelDoc XR + Gel Photodocumentation System with Image Lab 4.1 software, Bio-Rad, Hercules, CA, USA) after staining with ethidium bromide. The Gene Ruler™ Express DNA Ladder (Thermo Fisher Scientific, Waltham, MA, USA), 100–5000 bp DNA marker, was used as a size reference.
The banding patterns were scored with GelAnalyzer 23.1 software and then checked manually. For every 10 primers tested, the banding patterns were recorded as binary matrices, where ‘1/0’ indicates the presence/ absence, respectively, of a given fragment. The numbers of monomorphic (mono), polymorphic (poly) (present in the electrophoretic profile of more than one individual) and specific (spec) (unique; present in the electrophoretic profile of a single individual)
The experiment was set up in a completely randomised design. The obtained data were presented as mean ± standard deviation (SD) and subjected to one-way analysis of variance (ANOVA) and
The obtained plantlets were of high quality, with a fully developed stem, leaves and root system. No growth or physiological disorders were observed. The used material samples significantly stimulated the growth and development of plantlets in the two tested chrysanthemum cultivars. Control explants produced plantlets that were characterised by the lowest biometric parameters such as the number of leaves, micropropagation coefficient, shoot length and shoot and root system FW/DW as compared to the explants treated with all tested material samples at the whole range of applied concentrations (Figure 1, Supplementary Figure S3 and Table 1 and Table 2).
Biometric parameters of
Treatment | Number of leaves | Micropropagation coefficient | Shoot length (cm) | Shoot FW (mg) | Shoot DW (mg) | Root system FW (mg) | Root system DW (mg) |
---|---|---|---|---|---|---|---|
Control | 14.75 ± 0.50 h | 12.00 ± 0.82 i | 5.20 ± 0.70 g | 318.52 ± 128.04 d | 34.10 ± 12.39 e | 14.85 ± 9.12 c | 1.12 ± 0.48 g |
100 mg · L−1 ZnO SMPs | 24.25 ± 1.89 b–e | 21.50 ± 1.73 b–f | 9.60 ± 1.70 ab | 916.72 ± 390.89 a–c | 122.80 ± 53.97 a | 94.80 ± 58.17 ab | 7.48 ± 3.97 a–d |
200 mg · L−1 ZnO SMPs | 22.50 ± 1.00 b–f | 19.75 ± 0.96 c–h | 9.00 ± 1.93 a–c | 868.42 ± 338.87 a–c | 91.75 ± 44.85 a–e | 94.12 ± 44.37 ab | 7.00 ± 3.13 a–e |
400 mg · L−1 ZnO SMPs | 25.25 ± 2.87 a–d | 22.25 ± 2.87 b–e | 10.25 ± 1.95 a | 1,002.95 ± 389.36 a | 120.58 ± 52.61 ab | 107.10 ± 74.91 ab | 8.00 ± 2.98 a–c |
100 mg · L−1 ZnO NPs (1.5% H2O) | 22.50 ± 1.73 b–f | 20.00 ± 1.41 c–h | 8.40 ± 1.77 a–d | 531.70 ± 149.45 a–d | 61.52 ± 21.16 b–e | 44.08 ± 27.16 ab | 2.32 ± 1.38 fg |
200 mg · L−1 ZnO NPs (1.5% H2O) | 18.50 ± 3.79 gh | 16.50 ± 3.11 h | 6.80 ± 1.35 c–g | 598.95 ± 136.25 a–d | 57.32 ± 12.32 c–e | 90.28 ± 49.57 ab | 4.62 ± 2.33 b–g |
400 mg · L−1 ZnO NPs (1.5% H2O) | 19.25 ± 0.96 fg | 16.50 ± 1.29 h | 5.32 ± 0.64 fg | 467.12 ± 222.54 cd | 47.28 ± 17.01 de | 52.85 ± 21.40 ab | 2.98 ± 1.61 e–g |
100 mg · L−1 ZnO NPs (6% H2O) | 28.75 ± 2.75 a | 26.00 ± 2.58 a | 8.62 ± 1.68 a–d | 885.00 ± 378.42 a–c | 104.15 ± 61.28 a–d | 54.20 ± 21.37 ab | 4.18 ± 2.82 b–g |
200 mg · L−1 ZnO NPs (6% H2O) | 22.00 ± 0.82 c–g | 20.00 ± 0.82 c–h | 6.40 ± 1.75 d–g | 594.45 ± 214.63 a–d | 59.82 ± 22.95 c–e | 66.62 ± 27.71 ab | 3.38 ± 1.23 d–g |
400 mg · L−1 ZnO NPs (6% H2O) | 25.75 ± 4.57 a–c | 23.25 ± 4.03 a–c | 7.48 ± 2.09 b–f | 886.85 ± 525.24 a–c | 99.62 ± 56.87 a–d | 133.65 ± 79.22 a | 7.25 ± 3.57 a–e |
100 mg · L−1 ZnO + 0.1% Ag NPs (1.5% H2O) | 26.00 ± 3.92 ab | 24.00 ± 3.92 ab | 8.98 ± 2.35 a–c | 848.98 ± 418.51 a–c | 107.55 ± 60.71 a–c | 71.10 ± 29.22 ab | 6.35 ± 2.15 a–f |
200 mg · L−1 ZnO + 0.1% Ag NPs (1.5% H2O) | 24.75 ± 3.30 b–e | 22.50 ± 3.11 a–e | 8.00 ± 1.06 a–d | 699.28 ± 299.83 a–d | 88.62 ± 39.66 a–e | 61.50 ± 35.19 ab | 4.88 ± 2.64 a–g |
400 mg · L−1 ZnO + 0.1% Ag NPs (1.5% H2O) | 19.50 ± 2.38 fg | 17.00 ± 2.58 gh | 5.25 ± 1.44 e–g | 503.45 ± 253.84 b–d | 60.68 ± 24.29 c–e | 98.58 ± 52.69 ab | 4.70 ± 2.21 b–g |
100 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O) | 21.50 ± 3.70 d–g | 19.50 ± 3.70 d–h | 8.28 ± 2.21 a–d | 822.58 ± 629.57 a–c | 94.30 ± 61.92 a–d | 48.22 ± 24.81 ab | 2.55 ± 1.37 fg |
200 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O) | 22.75 ± 2.22 b–f | 20.25 ± 2.50 c–g | 9.40 ± 1.15 ab | 699.20 ± 213.92 a–d | 73.52 ± 13.02 a–e | 78.00 ± 31.07 ab | 4.52 ± 2.71 b–g |
400 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O) | 22.75 ± 2.36 b–f | 20.25 ± 2.63 c–g | 8.40 ± 1.87 a–d | 798.75 ± 459.66 a–d | 86.70 ± 35.42 a–e | 146.67 ± 131.02 a | 8.32 ± 2.11 ab |
100 mg · L−1 ZnO + 1% Ag NPs (1.5% H2O) | 21.25 ± 2.22 e–g | 18.75 ± 1.71 e–h | 9.38 ± 0.95 ab | 849.22 ± 322.33 a–c | 96.32 ± 35.65 a–d | 67.18 ± 33.93 ab | 3.78 ± 1.60 c–g |
200 mg · L−1 ZnO + 1% Ag NPs (1.5% H2O) | 21.50 ± 2.08 d–g | 18.75 ± 2.50 e–h | 7.88 ± 0.83 b–d | 643.65 ± 229.90 a–d | 68.72 ± 19.61 a–e | 129.20 ± 53.27 a | 8.15 ± 2.53 ab |
400 mg · L−1 ZnO + 1% Ag NPs (1.5% H2O) | 20.00 ± 1.83 fg | 18.00 ± 1.83 f–h | 8.00 ± 1.01 a–d | 788.32 ± 228.25 a–d | 81.00 ± 26.56 a–e | 105.08 ± 42.26 ab | 7.10 ± 3.03 a–e |
100 mg · L−1 ZnO + 1% Ag NPs (6% H2O) | 19.00 ± 2.94 fg | 16.50 ± 2.65 h | 6.58 ± 0.91 d–g | 528.92 ± 268.54 a–d | 49.15 ± 22.13 c–e | 97.10 ± 40.77 ab | 5.62 ± 1.70 a–f |
200 mg · L−1 ZnO + 1% Ag NPs (6% H2O) | 21.50 ± 4.36 d–g | 19.00 ± 4.08 d–h | 9.58 ± 2.00 ab | 987.92 ± 370.32 ab | 99.85 ± 38.98 a–d | 143.60 ± 45.49 a | 9.20 ± 3.23 a |
400 mg · L−1 ZnO + 1% Ag NPs (6% H2O) | 19.50 ± 2.65 fg | 17.25 ± 2.22 gh | 7.60 ± 1.88 b–e | 724.85 ± 535.35 a–d | 71.80 ± 45.90 a–e | 62.32 ± 31.34 ab | 4.33 ± 3.57 b–g |
Means ± SD in columns followed by the same letter do not differ significantly at
DW, dry weight; FW, fresh weight; MS, Murashige and Skoog; NPs, nanoparticles; SD, standard deviation; SMPs, submicron particles; ‘UBG, UTP Burgundy Gold’.
Biometric parameters of
Treatment | Number of leaves | Micropropagation coefficient | Shoot length (cm) | Shoot FW (mg) | Shoot DW (mg) | Root system FW (mg) | Root system DW (mg) |
---|---|---|---|---|---|---|---|
Control | 14.75 ± 1.50 e | 11.75 ± 1.50 f | 3.37 ± 0.97 e | 183.88 ± 101.51 c | 30.15 ± 9.71 b | 14.22 ± 3.42 c | 1.82 ± 1.44 d |
100 mg · L−1 ZnO SMPs | 18.50 ± 1.00 b–d | 15.50 ± 1.00 c–e | 8.42 ± 1.69 a–d | 621.52 ± 246.24 a–c | 76.82 ± 21.71 ab | 97.58 ± 43.42 b | 6.90 ± 4.00 bd |
200 mg · L−1 ZnO SMPs | 17.75 ± 0.96 cd | 14.75 ± 0.96 e | 6.55 ± 2.38 cd | 525.30 ± 281.34 a–c | 59.50 ± 25.42 ab | 130.28 ± 45.46 b | 5.28 ± 2.97 cd |
400 mg · L−1 ZnO SMPs | 19.75 ± 1.50 a–d | 16.75 ± 1.50 b–e | 6.85 ± 0.47 cd | 610.65 ± 54.16 a–c | 68.82 ± 9.47 ab | 219.28 ± 82.43 b | 9.15 ± 4.19 a–c |
100 mg · L−1 ZnO NPs (1.5% H2O) | 18.75 ± 0.96 b–d | 15.75 ± 0.96 c–e | 6.50 ± 2.43 cd | 479.82 ± 182.32 bc | 58.68 ± 21.99 ab | 102.65 ± 66.78 b | 5.08 ± 1.42 cd |
200 mg · L−1 ZnO NPs (1.5% H2O) | 19.00 ± 0.82 b–d | 17.75 ± 2.36 a–c | 7.12 ± 1.66 b–d | 508.12 ± 195.29 a–c | 67.40 ± 25.50 ab | 122.02 ± 50.14 b | 6.72 ± 2.26 cd |
400 mg · L−1 ZnO NPs (1.5% H2O) | 20.25 ± 2.50 a–c | 17.25 ± 2.50 a–e | 7.58 ± 2.88 a–d | 665.65 ± 271.19 ab | 82.82 ± 29.18 ab | 91.95 ± 53.37 b | 6.08 ± 3.47 cd |
100 mg · L−1 ZnO NPs (6% H2O) | 19.00 ± 1.41 b–d | 16.00 ± 1.41 c–e | 6.30 ± 1.89 de | 543.68 ± 247.53 a–c | 60.38 ± 26.87 ab | 161.10 ± 77.52 b | 7.35 ± 5.83 a–d |
200 mg · L−1 ZnO NPs (6% H2O) | 19.75 ± 0.96 a–d | 16.75 ± 0.96 b–e | 7.85 ± 0.79 a–d | 665.58 ± 207.59 ab | 84.38 ± 32.54 ab | 168.35 ± 46.40 b | 7.92 ± 2.66 a–c |
400 mg · L−1 ZnO NPs (6% H2O) | 18.50 ± 1.29 b–d | 15.50 ± 1.29 c–e | 6.42 ± 1.50 cd | 511.45 ± 164.74 a–c | 51.02 ± 14.96 ab | 184.92 ± 81.48 b | 6.95 ± 4.82 bd |
100 mg · L−1 ZnO + 0.1% Ag NPs (1.5% H2O) | 20.50 ± 0.58 ab | 17.75 ± 0.96 a–c | 7.28 ± 1.22 a–d | 753.18 ± 305.43 ab | 74.85 ± 36.24 ab | 101.58 ± 50.87 b | 5.98 ± 2.69 cd |
200 mg · L−1 ZnO + 0.1% Ag NPs (1.5% H2O) | 18.75 ± 0.50 b–d | 15.75 ± 0.50 c–e | 6.82 ± 1.25 cd | 545.70 ± 131.47 a–c | 61.08 ± 15.74 ab | 229.28 ± 86.24 b | 9.90 ± 4.08 a–c |
400 mg · L−1 ZnO + 0.1% Ag NPs (1.5% H2O) | 19.75 ± 0.96 a–d | 16.75 ± 0.96 b–e | 7.08 ± 1.28 bd | 678.05 ± 265.25 ab | 77.95 ± 26.21 ab | 186.42 ± 75.81 b | 9.85 ± 4.45 a–c |
100 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O) | 20.50 ± 1.00 ab | 17.50 ± 1.00 a–d | 8.75 ± 1.13 a–d | 755.55 ± 350.31 ab | 85.68 ± 32.51 ab | 140.40 ± 49.83 b | 7.80 ± 3.77 a–c |
200 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O) | 22.25 ± 1.89 a | 19.50 ± 1.73 a | 8.82 ± 1.08 a–c | 858.52 ± 283.60 ab | 99.28 ± 38.17 a | 210.38 ± 64.28 b | 12.98 ± 1.17 a |
400 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O) | 20.75 ± 2.63 ab | 17.75 ± 2.63 a–c | 8.82 ± 1.11 a–c | 746.30 ± 342.36 ab | 83.68 ± 35.37 ab | 482.35 ± 145.86 a | 9.35 ± 4.15 a–c |
100 mg · L−1 ZnO + 1% Ag NPs (1.5% H2O) | 19.50 ± 3.11 b–d | 17.25 ± 3.20 a–e | 8.75 ± 1.37 a–d | 618.72 ± 238.85 a–c | 86.40 ± 39.95 ab | 135.98 ± 69.05 b | 8.22 ± 2.35 a–c |
200 mg · L−1 ZnO + 1% Ag NPs (1.5% H2O) | 18.50 ± 1.29 b–d | 15.75 ± 0.96 c–e | 8.80 ± 1.70 a–c | 691.82 ± 299.58 ab | 77.88 ± 37.48 ab | 150.38 ± 58.97 b | 8.92 ± 1.29 a–c |
400 mg · L−1 ZnO + 1% Ag NPs (1.5% H2O) | 19.00 ± 1.41 b–d | 16.50 ± 1.00 b–e | 9.48 ± 2.30 ab | 764.60 ± 356.38 ab | 78.62 ± 34.05 ab | 137.82 ± 37.44 b | 7.92 ± 1.30 a–c |
100 mg · L−1 ZnO + 1% Ag NPs (6% H2O) | 19.25 ± 3.77 b–d | 17.25 ± 3.77 a–e | 8.68 ± 3.04 a–d | 687.50 ± 265.24 ab | 81.50 ± 36.31 ab | 104.40 ± 51.14 b | 5.95 ± 5.35 cd |
200 mg · L−1 ZnO + 1% Ag NPs (6% H2O) | 20.50 ± 1.91 ab | 18.75 ± 1.50 ab | 9.72 ± 1.32 a | 946.78 ± 134.07 a | 406.50 ± 21.20 a | 231.98 ± 133.63 b | 12.62 ± 3.99 ab |
400 mg · L−1 ZnO + 1% Ag NPs (6% H2O) | 17.25 ± 2.99 de | 15.00 ± 2.58 de | 7.05 ± 2.36 b–d | 733.92 ± 454.71 ab | 74.20 ± 36.72 ab | 146.40 ± 62.51 b | 9.75 ± 4.49 a–c |
Means ± SD in columns followed by the same letter do not differ significantly at
DW, dry weight; FW, fresh weight; MS, Murashige and Skoog; NPs, nanoparticles; SD, standard deviation; SMPs, submicron particles; UPG, ‘UTP Pinky Gold’.
As for the ‘UBG’ cultivar, the highest number of formed leaves (28.75) and the highest micropropagation coefficient (26) were found for 100 mg · L−1 ZnO NPs (6% H2O), whereas the values of these traits for the control amounted to 14.75 and 12, respectively. The highest values of shoot length (10.25 cm) and shoot FW (1,002.95 mg) were reported for 400 mg · L−1 ZnO SMPs treatment. Shoots produced on the medium with 100 mg · L−1 ZnO SMPs had the highest DW (122.80 mg). Material samples with Ag NPs caused a significant increase in the root system FW (129.20–146.67 mg) and DW (8.15–9.20 mg), especially treatments with 400 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O), 200 mg · L−1 ZnO + 1% Ag NPs (1.5% H2O) and 200 mg · L−1 ZnO + 1% Ag NPs (6% H2O). Contrarily, the FW/DW of the control root system amounted to 14.85/1.12 mg, respectively (Table 1).
In the ‘UPG’ cultivar, the development of plantlets was significantly stimulated by the treatment with 200 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O), which yielded the highest number of leaves (22.25), the highest micropropagation coefficient (19.50), a high shoot DW (99.28 mg) and the highest root system DW (12.98 mg). Distinctive values of biometric parameters were also found for 200 mg · L−1 ZnO + 1% Ag NPs (6% H2O) application (the highest shoot length – 9.72 cm, shoot FW – 946.78 mg and shoot DW – 406.50 mg) (Table 2).
The detailed analysis of the leaf architecture in the two studied chrysanthemum cultivars showed that the control plantlets developed leaves with the lowest area, perimeter and width; however, no differences were found for the leaf length depending on the experimental treatments (Figure 2 and Figure 3). The application of ZnO + 0.1% Ag NPs (6% H2O) sample at the concentration of 100 mg · L−1 caused a significant increase in the leaf architecture parameters in chrysanthemum ‘UBG’. High values of the leaf area, perimeter and width were also reported in both cultivars for the treatment with 400 mg · L−1 ZnO + 1% Ag NPs (6% H2O). However, plantlets from this experimental object did not develop as many leaves as the plantlets from the most efficient treatments in terms of the number of leaves (Table 1 and Table 2).
Supplementation with ZnO SMPs/ZnO NPs/ ZnO + Ag NPs significantly improved the growth and development of the root system in ‘UBG’ (Figure 4). The most efficient treatment in terms of the analysed parameters of the root system architecture was 200 mg · L−1 ZnO + 1% Ag NPs (6% H2O). High values of the root system total length, area, volume and number of root tips and forks were also reported for other samples, i.e. 400 mg · L−1 ZnO + 0.1% Ag NPs (1.5% H2O), 200 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O), 100 mg · L−1 ZnO + 1% Ag NPs (1.5% H2O) and 400 mg · L−1 ZnO + 1% Ag NPs (6% H2O). Generally, the ZnO SMPs and ZnO NPs samples less effectively stimulated the development of chrysanthemum root systems; nevertheless, the least developed roots were found in control plantlets.
On the contrary, in ‘UPG’, different effects of the tested material samples on the root system architecture were reported. Moreover, the obtained results were more uniform between each experimental object (Figure 5). Explants treated, both with ZnO + 1% Ag NPs (1.5% H2O) and ZnO + 1% Ag NPs (6% H2O) at the concentration of 200 mg · L−1, formed plantlets with root systems that were characterised by the highest total length and area. Intermediate values of these traits were found in the control root system, whereas the lowest value was in the 400 mg · L−1 ZnO + 0.1% Ag NPs (1.5% H2O) treatment. No significant differences between the tested experimental objects were found for the root system volume, with values ranging from 75.75 mm3 to 122.62 mm3. Interestingly, the highest number of root tips (46 and 45.71) was reported for the control object and 200 mg · L−1 ZnO + 1% Ag NPs (6% H2O), respectively. The control object also formed the highest number of root forks (174).
In ‘UBG’, the highest contents of chlorophyll
Content and ratios of chlorophylls and carotenoids in
Treatment | Chlorophyll |
Chlorophyll |
Chlorophyll |
Chlorophyll ( |
Carotenoid content (mg · g−1 FW) | Chlorophyll/carotenoid ratio |
---|---|---|---|---|---|---|
Control | 1.13 ± 0.16 ab | 0.43 ± 0.07 a–d | 2.63 ± 0.05 bc | 1.56 ± 0.23 ab | 0.25 ± 0.03 ab | 6.24 ± 0.18 c–f |
100 mg · L−1 ZnO SMPs | 1.08 ± 0.25 a–c | 0.44 ± 0.09 a–c | 2.45 ± 0.09 e–i | 1.52 ± 0.34 a–c | 0.22 ± 0.06 b–e | 6.91 ± 0.41 b |
200 mg · L−1 ZnO SMPs | 0.95 ± 0.15 c–g | 0.39 ± 0.03 c–f | 2.44 ± 0.23 f–i | 1.34 ± 0.18 c–f | 0.21 ± 0.02 c–f | 6.38 ± 0.27 c–f |
400 mg · L−1 ZnO SMPs | 0.83 ± 0.08 g–i | 0.30 ± 0.03 j | 2.77 ± 0.08 a | 1.13 ± 0.11 g–i | 0.17 ± 0.02 gh | 6.65 ± 0.25 bc |
100 mg · L−1 ZnO NPs (1.5% H2O) | 0.83 ± 0.10 g–i | 0.33 ± 0.04 g–j | 2.51 ± 0.04 d–h | 1.16 ± 0.14 f–i | 0.17 ± 0.02 gh | 6.82 ± 0.26 b |
200 mg · L−1 ZnO NPs (1.5% H2O) | 0.63 ± 0.03 j | 0.25 ± 0.02 k | 2.52 ± 0.07 c–h | 0.88 ± 0.05 j | 0.12 ± 0.01 i | 7.33 ± 0.07 a |
400 mg · L−1 ZnO NPs (1.5% H2O) | 1.20 ± 0.06 a | 0.46 ± 0.03 a | 2.61 ± 0.05 b–e | 1.66 ± 0.09 a | 0.26 ± 0.02 a | 6.38 ± 0.26 c–f |
100 mg · L−1 ZnO NPs (6% H2O) | 0.91 ± 0.06 e–h | 0.35 ± 0.02 e–i | 2.60 ± 0.06 b–f | 1.26 ± 0.07 d–h | 0.18 ± 0.01 f–h | 7.00 ± 0.06 ab |
200 mg · L−1 ZnO NPs (6% H2O) | 0.89 ± 0.10 f–i | 0.34 ± 0.03 f–j | 2.62 ± 0.10 b–d | 1.23 ± 0.13 e–i | 0.22 ± 0.05 b–e | 5.60 ± 1.66 f |
400 mg · L−1 ZnO NPs (6% H2O) | 0.86 ± 0.07 f–i | 0.32 ± 0.04 h–j | 2.69 ± 0.10 a–c | 1.18 ± 0.11 e–i | 0.18 ± 0.02 f–h | 6.56 ± 0.11 b–d |
100 mg · L−1 ZnO + 0.1% Ag NPs (1.5% H2O) | 1.04 ± 0.03 b–d | 0.40 ± 0.01 b–e | 2.60 ± 0.10 b–f | 1.44 ± 0.03 b–d | 0.24 ± 0.02 a–c | 6.00 ± 0.34 d–f |
200 mg · L−1 ZnO + 0.1% Ag NPs (1.5% H2O) | 0.88 ± 0.17 f–i | 0.36 ± 0.05 e–i | 2.44 ± 0.28 f–i | 1.24 ± 0.21 e–h | 0.19 ± 0.03 e–h | 6.53 ± 0.11 b–d |
400 mg · L−1 ZnO + 0.1% Ag NPs (1.5% H2O) | 0.92 ± 0.11 d–h | 0.38 ± 0.07 d–g | 2.63 ± 0.14 bc | 1.30 ± 0.18 d–g | 0.22 ± 0.03 b–e | 5.91 ± 0.11 ef |
100 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O) | 1.03 ± 0.07 b–e | 0.40 ± 0.04 b–e | 2.58 ± 0.13 b–g | 1.43 ± 0.11 b–d | 0.23 ± 0.01 a–d | 6.22 ± 0.36 d–f |
200 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O) | 0.79 ± 0.12 h–i | 0.31 ± 0.05 ij | 2.55 ± 0.06 b–g | 1.10 ± 0.17 hi | 0.17 ± 0.03 gh | 6.47 ± 0.29 b–e |
400 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O) | 0.98 ± 0.03 c–g | 0.39 ± 0.02 c–f | 2.72 ± 0.04 ab | 1.37 ± 0.05 c–e | 0.22 ± 0.02 b–e | 6.23 ± 0.24 c–f |
100 mg · L−1 ZnO + 1% Ag NPs (1.5% H2O) | 0.99 ± 0.06 c–f | 0.38 ± 0.03 d–g | 2.61 ± 0.10 b–e | 1.37 ± 0.09 c–e | 0.22 ± 0.01 b–e | 6.23 ± 0.24 c–f |
200 mg · L−1 ZnO + 1% Ag NPs (1.5% H2O) | 1.07 ± 0.06 a–c | 0.45 ± 0.03 ab | 2.38 ± 0.04 hi | 1.52 ± 0.08 a–c | 0.22 ± 0.02 b–e | 6.91 ± 0.20 b |
400 mg · L−1 ZnO + 1% Ag NPs (1.5% H2O) | 0.76 ± 0.03 ij | 0.29 ± 0.02 jk | 2.62 ± 0.14 b–d | 1.05 ± 0.04 i | 0.16 ± 0.01 h | 6.56 ± 0.18 b–f |
100 mg · L−1 ZnO + 1% Ag NPs (6% H2O) | 0.92 ± 0.23 d–h | 0.36 ± 0.08 e–i | 2.56 ± 0.15 b–g | 1.28 ± 0.31 d–h | 0.21 ± 0.06 c–f | 6.10 ± 0.47 d–f |
200 mg · L−1 ZnO + 1% Ag NPs (6% H2O) | 0.92 ± 0.08 d–h | 0.38 ± 0.02 d–g | 2.42 ± 0.12 g–i | 1.30 ± 0.10 d–g | 0.20 ± 0.02 d–g | 6.50 ± 0.22 b–d |
400 mg · L−1 ZnO + 1% Ag NPs (6% H2O) | 0.85 ± 0.15 f–i | 0.37 ± 0.08 e–h | 2.30 ± 0.12 i | 1.22 ± 0.22 e–i | 0.20 ± 0.04 d–g | 6.10 ± 0.19 d–f |
Means ± SD in columns followed by the same letter do not differ significantly at
MS, Murashige and Skoog; NPs, nanoparticles; SD, standard deviation; SMPs, submicron particles; UBG, ‘UTP Burgundy Gold’.
Content and ratios of chlorophylls and carotenoids in
Treatment | Chlorophyll |
Chlorophyll |
Chlorophyll |
Chlorophyll ( |
Carotenoid content (mg · g−1 FW) | Chlorophyll/carotenoid ratio |
---|---|---|---|---|---|---|
Control | 1.46 ± 0.06 a | 0.51 ± 0.02 a–c | 2.86 ± 0.06 a | 1.97 ± 0.08 a | 0.29 ± 0.01 ab | 6.79 ± 0.07 ef |
100 mg · L−1 ZnO SMPs | 1.03 ± 0.04 fg | 0.42 ± 0.03 d–f | 2.45 ± 0.11 d–g | 1.45 ± 0.07 d–f | 0.23 ± 0.01 c–f | 6.30 ± 0.26 ik |
200 mg · L−1 ZnO SMPs | 1.08 ± 0.15 e–g | 0.45 ± 0.05 c–f | 2.40 ± 0.06 f–h | 1.53 ± 0.20 c–f | 0.21 ± 0.03 d–g | 7.29 ± 0.48 bc |
400 mg · L−1 ZnO SMPs | 1.03 ± 0.16 fg | 0.42 ± 0.08 d–f | 2.45 ± 0.08 d–g | 1.45 ± 0.24 d–f | 0.21 ± 0.04 d–g | 6.91 ± 0.12 d–f |
100 mg · L−1 ZnO NPs (1.5% H2O) | 1.16 ± 0.19 c–f | 0.47 ± 0.06 b–e | 2.47 ± 0.09 c–f | 1.63 ± 0.26 b–d | 0.24 ± 0.04 c–e | 6.79 ± 0.30 ef |
200 mg · L−1 ZnO NPs (1.5% H2O) | 0.97 ± 0.29 g | 0.40 ± 0.12 ef | 2.42 ± 0.02 e–g | 1.37 ± 0.42 ef | 0.18 ± 0.06 g | 7.61 ± 0.36 a |
400 mg · L−1 ZnO NPs (1.5% H2O) | 1.26 ± 0.03 b–d | 0.50 ± 0.03 a–c | 2.52 ± 0.11 b–d | 1.76 ± 0.07 a–c | 0.26 ± 0.01 bc | 6.77 ± 0.15 e–g |
100 mg · L−1 ZnO NPs (6% H2O) | 1.40 ± 0.13 ab | 0.53 ± 0.04 ab | 2.64 ± 0.07 b | 1.93 ± 0.17 a | 0.30 ± 0.03 a | 6.43 ± 0.29 hi |
200 mg · L−1 ZnO NPs (6% H2O) | 1.14 ± 0.32 c–g | 0.46 ± 0.09 c–e | 2.48 ± 0.23 c–e | 1.60 ± 0.42 bc | 0.25 ± 0.07 c–d | 6.40 ± 0.17 hj |
400 mg · L−1 ZnO NPs (6% H2O) | 1.17 ± 0.20 c–f | 0.45 ± 0.07 c–f | 2.60 ± 0.08 bc | 1.62 ± 0.26 b–d | 0.24 ± 0.04 c–e | 6.75 ± 0.06 e–g |
100 mg · L−1 ZnO + 0.1% Ag NPs (1.5% H2O) | 1.19 ± 0.09 c–f | 0.48 ± 0.03 a–d | 2.48 ± 0.08 c–e | 1.67 ± 0.12 b–d | 0.24 ± 0.01 c–e | 6.96 ± 0.34 de |
200 mg · L−1 ZnO + 0.1% Ag NPs (1.5% H2O) | 1.09 ± 0.05 d–g | 0.48 ± 0.01 a–d | 2.27 ± 0.06 i | 1.57 ± 0.06 c–f | 0.24 ± 0.02 c–e | 6.54 ± 0.23 e–h |
400 mg · L−1 ZnO + 0.1% Ag NPs (1.5% H2O) | 1.16 ± 0.09 c–f | 0.49 ± 0.03 a–c | 2.37 ± 0.08 g–i | 1.65 ± 0.11 b–d | 0.22 ± 0.02 d–g | 7.50 ± 0.19 ab |
100 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O) | 1.09 ± 0.18 d–g | 0.46 ± 0.08 c–f | 2.37 ± 0.06 g–i | 1.55 ± 0.26 c–f | 0.25 ± 0.04 c–d | 6.20 ± 0.08 jk |
200 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O) | 1.15 ± 0.15 c–f | 0.48 ± 0.05 a–d | 2.40 ± 0.07 f–h | 1.63 ± 0.20 b–d | 0.24 ± 0.03 c–e | 6.79 ± 0.04 ef |
400 mg · L−1 ZnO + 0.1% Ag NPs (6% H2O) | 1.30 ± 0.07 a–c | 0.54 ± 0.04 a | 2.41 ± 0.06 e–g | 1.84 ± 0.11 ab | 0.26 ± 0.01 bc | 7.08 ± 0.24 de |
100 mg · L−1 ZnO + 1% Ag NPs (1.5% H2O) | 1.02 ± 0.27 fg | 0.42 ± 0.12 d–f | 2.43 ± 0.08 e–g | 1.44 ± 0.39 d–f | 0.20 ± 0.05 e–g | 7.20 ± 0.32 cd |
200 mg · L−1 ZnO + 1% Ag NPs (1.5% H2O) | 1.25 ± 0.11 b–e | 0.51 ± 0.03 a–c | 2.45 ± 0.15 d–g | 1.76 ± 0.14 a–c | 0.27 ± 0.02 a–c | 6.52 ± 0.21 f–h |
400 mg · L−1 ZnO + 1% Ag NPs (1.5% H2O) | 1.02 ± 0.12 fg | 0.45 ± 0.04 c–f | 2.27 ± 0.09 i | 1.47 ± 0.16 d–f | 0.19 ± 0.03 f–g | 7.74 ± 0.27 a |
100 mg · L−1 ZnO + 1% Ag NPs (6% H2O) | 1.03 ± 0.07 fg | 0.45 ± 0.03 c–f | 2.29 ± 0.09 hi | 1.48 ± 0.10 d–f | 0.24 ± 0.02 c–e | 6.17 ± 0.10 k |
200 mg · L−1 ZnO + 1% Ag NPs (6% H2O) | 1.11 ± 0.11 d–g | 0.44 ± 0.03 c–f | 2.52 ± 0.06 b–d | 1.55 ± 0.14 c–f | 0.24 ± 0.02 c–e | 6.46 ± 0.03 gi |
400 mg · L−1 ZnO + 1% Ag NPs (6% H2O) | 0.96 ± 0.07 g | 0.39 ± 0.03 f | 2.46 ± 0.02 c–g | 1.35 ± 0.10 f | 0.18 ± 0.01 g | 7.50 ± 0.16 ab |
Means ± SD in columns followed by the same letter do not differ significantly at
MS, Murashige and Skoog; NPs, nanoparticles; SD, standard deviation; SMPs, submicron particles; UPG, ‘UTP Pinky Gold’.
A total of 5,216 scorable bands were detected by five RAPD (1,888) and five SCoT (3,328) primers in the tested ‘UBG’ and ‘UPG’ plantlets. As for the RAPD marker system, primer R2 generated the highest number of bands (160 and 320 in ‘UBG’ and ‘UPG’, respectively), whereas the lowest number of bands was reported for primers R4 and R5 (160 in each cultivar). The primers R1 and R3 R2/R4 generated 5
All tested RAPD and SCoT primers did not generate polymorphic products, confirming the genetic uniformity of the ZnO SMPs/ZnO NPs/ZnO + Ag NPs-treated plantlets as compared to the control within each tested cultivar. Simultaneously, different band profiles were generated for ‘UBG’ and ‘UPG’ chrysanthemums, indicating the genetic distinctiveness of these two cultivars (Table 5, Figures 6 and Supplementary Figure S4).
Molecular products obtained from
Primer code | Primer sequence 5’ → 3’ | Reference | Cultivar | No. of bands | Band sizes (bp) | No. of loci | No. of genotypes | |||
---|---|---|---|---|---|---|---|---|---|---|
Total | mono | poly | spec | |||||||
RAPD | ||||||||||
R1 | GGG AAT TCG G | Lema-Rumińska et al. (2004) | UBG | 192 | 367–1,478 | 6 | 6 | 0 | 0 | 1 |
UPG | 192 | 355–1,500 | 6 | 6 | 0 | 0 | 1 | |||
R2 | GAC CGC TTG T | UBG | 160 | 564–1,207 | 5 | 5 | 0 | 0 | 1 | |
UPG | 320 | 195–2,418 | 10 | 10 | 0 | 0 | 1 | |||
R3 | GCT GCC TCA GG | Shibata et al. (1998) | UBG | 192 | 432–2,094 | 6 | 6 | 0 | 0 | 1 |
UPG | 192 | 414–2,020 | 6 | 6 | 0 | 0 | 1 | |||
R4 | TAC CCA GGA GCG | Wolf (1996) | UBG | 160 | 500–1,807 | 5 | 5 | 0 | 0 | 1 |
UPG | 160 | 500–1,910 | 5 | 5 | 0 | 0 | 1 | |||
R5 | CAA TCG CCG T | UBG | 160 | 534–1,449 | 5 | 5 | 0 | 0 | 1 | |
UPG | 160 | 579–1,430 | 5 | 5 | 0 | 0 | 1 | |||
Σ | UBG | 864 | 27 | 27 | 0 | 0 | 1 | |||
UPG | 1,024 | 32 | 32 | 0 | 0 | 1 | ||||
Mean from a single primer | UBG | 172.8 | 5.4 | 5.4 | 0 | 0 | - | |||
UPG | 204.8 | 6.4 | 6.4 | 0 | 0 | - | ||||
SCoT | ||||||||||
S1 | CAA TGG CTA CCA CCT | Collard and Mackill (2009) | UBG | 352 | 425–1,762 | 11 | 11 | 0 | 0 | 1 |
UPG | 416 | 374–1,730 | 13 | 13 | 0 | 0 | 1 | |||
S2 | CAA TGG CTA CCA CGT | UBG | 256 | 531–1,757 | 8 | 8 | 0 | 0 | 1 | |
UPG | 256 | 531–1,758 | 8 | 8 | 0 | 0 | 1 | |||
S3 | ACG ACA TGG CGA CCA ACG | UBG | 352 | 509–1,888 | 11 | 11 | 0 | 0 | 1 | |
UPG | 288 | 400–1,989 | 9 | 9 | 0 | 0 | 1 | |||
S4 | ACG ACA TGG CGA CCA TCG | UBG | 416 | 409–2,036 | 13 | 13 | 0 | 0 | 1 | |
UPG | 416 | 409–2,037 | 13 | 13 | 0 | 0 | 1 | |||
S5 | ACC ATG GCT ACC GTC | UBG | 320 | 416–2,988 | 10 | 10 | 0 | 0 | 1 | |
UPG | 256 | 329–1,824 | 8 | 8 | 0 | 0 | 1 | |||
Σ | UBG | 1,696 | 53 | 53 | 0 | 0 | 1 | |||
UPG | 1,632 | 51 | 51 | 0 | 0 | 1 | ||||
Mean from a single primer | UBG | 339.2 | 10.6 | 10.6 | 0 | 0 | - | |||
UPG | 326.4 | 10.2 | 10.2 | 0 | 0 | - |
mono, monomorphic; NPs, nanoparticles; poly, polymorphic; RAPD, randomly amplified polymorphic DNA; SCoT, start codon targeted polymorphism; SMPs, submicron particles; spec, specific; UBG, ‘UTP Burgundy Gold’; UPG, ‘UTP Pinky Gold’.
Zinc is an essential constituent of enzymes and cell membranes and acts as a binding domain in many proteins, i.e. structural and transcriptional regulatory proteins. This microelement plays an important role in the biosynthesis of phytohormones, chlorophyll, proteins and carbohydrates, thus modulating plant growth and development. Plants growing in zinc-deficient environments have reduced photosynthesis and nitrogen metabolism, short internodes, curly leaves and reduced flowering, fruit development and crop production. Considering the ability of plants to accumulate ZnO NPs, these NPs can be used as an effective nanofertiliser (Sohail et al., 2020; Sarkhosh et al., 2022).
Diverse effects on biometric and biochemical parameters of micropropagated plants, due to the medium supplementation with ZnO NPs, were observed in previous studies in different species.
Changes in the Zn status of the plant may modify its phytohormonal balance, significantly affecting the growth process (Oraghi Ardebili and Sharifi, 2018). Moreover, ZnO NPs stimulate the transfer of iron, potassium and phosphorus from roots to shoots, thus increasing the availability of these elements. This, in turn, causes an increase in carbohydrate biosynthesis in plants (Awan et al., 2021). Interestingly, the comparative transcriptomic analysis revealed that ZnO NPs can upregulate the expression of a set of genes encoding antioxidative enzymes, transporters and enzymes or regulators involved in nutrient element transport, carbon/nitrogen metabolism and secondary metabolism in plants (Sun et al., 2020). The mentioned multifaced interactions of Zn with physiological and growth processes in plants contributed most likely to the increases in biometric parameters of the tested ‘UBG’ and ‘UPG’ plantlets and allowed to improve the efficiency of micropropagation.
The ZnO NPs-induced increase in the chlorophyll content may result from the zinc involvement in chlorophyll formation by protochlorophyllide and chloroplast development when ZnO NPs are applied at low concentrations. In contrast, high ZnO NPs concentrations, by providing excessive zinc to the plant, can inhibit chlorophyll formation by interfering with the expression of genes associated with chlorophyll biosynthesis, reduce chlorophyll fluorescence parameters and photosynthetic efficiency and in turn, lead to a reduction in biomass accumulation (Wang et al., 2018; Del Buono et al., 2021). Similarly, specific, non-excessive concentrations of ZnO NPs can stimulate carotenoid biosynthesis; however, higher concentrations interfere with the biosynthesis of these pigments. Carotenoids are not only important light-harvesting pigments in the photosynthesis process, but they have also antioxidant activity and are involved in removing reactive oxygen species (ROS), protecting chloroplasts from NPs-induced oxidative stress through their ability to quench chlorophyll in a singlet or triplet form (Del Buono et al., 2021). In the present study, as compared to the control, the SMPs- and NPs-treated chrysanthemums were characterised by a similar, or most often, lower content of chlorophylls and carotenoids; however, no visible symptoms of zinc excess or deficiency were detected. These results, on the one hand, may reflect the use of high concentrations of ZnO SMPs/NPs and their inhibiting effect on plant pigment biosynthesis, and/or induction of oxidative stress. On the other hand, the SMPs/NPs-treated plantlets presented significantly higher biometric parameters than control plantlets, and we presume that most likely, intensive-growing young plant tissues might have accumulated less pigments.
Some of the tested ZnO NPs material samples contained 0.1% or 1% Ag NPs, and we observed positive effects of silver NPs on the analysed parameters of plantlets. The results obtained by Hegazi et al. (2021) indicate that the medium supplementation with 5 mg · L−1 Ag NPs increased bud sprouting, shoot length, number of shoots per explant and number of leaves per shoot in
In our study, the tested material samples highly improved the growth and development of root systems in the two studied cultivars. Similarly, significant increases in
Being an enzymatic constituent, Zn has an important function in the synthesis and accumulation of free amino acids. For example, tryptophan is a precursor of natural auxin IAA, which stimulates root formation and improves the root system architecture (Li et al., 2021; Sarkhosh et al., 2022). Pandey et al. (2010) proved that ZnO NPs gave a very positive response in root development in
Positive, negative or non-significant effects of various NPs on plants depend also on the NP’s size and shape, the method of NPs synthesis and the solvents used for synthesis (Thunugunta et al., 2018). The physicochemical properties of NPs differ significantly from the corresponding bulk material; thus, NPs can differently affect biological processes in living cells (Thunugunta et al., 2018). Higher efficiency of ZnO NPs for enhancing growth parameters than the macro size ZnSO4 salt was reported in
In the present study, for samples ZnO SMPs, ZnO NPs (1.5% H2O), ZnO NPs (6% H2O), ZnO + 0.1% Ag NPs (1.5% H2O), ZnO + 0.1% Ag NPs (6% H2O), ZnO + 1% Ag NPs (1.5% H2O) and ZnO + 1% Ag NPs (6% H2O), the particle size was 240 nm, 25 nm, 65 nm, 29 nm, 79 nm, 27 nm and 53 nm, respectively. The best developed plantlets were obtained after the use of samples containing a higher water content and larger particle size, especially ZnO NPs (6% H2O) (65 nm), ZnO + 0.1% Ag NPs (6% H2O) (79 nm) and ZnO + 1% Ag NPs (6% H2O) (53 nm), as compared to their counterparts with lower water content (1.5%) and smaller particle size (25–29 nm). It can, therefore, be concluded that smaller NPs are more toxic to plants, limiting their growth and development. Smaller NPs have a larger SSA and, thus, more available surface area to interact with cellular components such as nucleic acids, proteins, fatty acids and carbohydrates. The smaller size also likely makes it possible to enter the cell, causing cellular damage (Huang et al., 2017).
Each species responds differently to the application of ZnO NPs, either at a biometric or at a biochemical level. As was presented in the study performed by López-Reyes et al. (2022), some species are more sensitive (
Considering the data obtained in this study and results reported by other authors, it can be stated that highly differentiated properties of NPs, plant species specificity, different NPs treatments and variable experimental conditions are crucial factors determining multidirectional effects of NPs on plants and highlighting possible uses of NPs in plant production. The comprehensive understanding of nanoparticle
RAPD and SCoT markers are rapid and reliable tools for monitoring NPs-induced genetic effects in plants, being sensitive methods capable of detecting variations in genome profiles (Plaksenkova et al., 2020). As was demonstrated in the present study, based on the RAPD and SCoT analysis, the 400 mg · L−1 ZnO SMPs/ZnO NPs/ZnO + Ag NPs-treated chrysanthemums were genetically stable, presenting the same genomic profiles as the control plants. Interestingly, RAPD and SCoT markers were effective in polymorphism screening in adventitious shoots regenerated from leaf explants in 50 mg · L−1 and 100 mg · L−1 Ag NPs-treated ‘Lilac Wonder’ and ‘Richmond’ chrysanthemums (Tymoszuk and Kulus, 2022). Nevertheless, there is a fundamental difference between the regeneration of adventitious shoot from the
Excess zinc, typically >400 mg · kg−1 Zn in tissue dry weight, is toxic to plants. Zinc toxicity leads to several implications in many metabolic processes and can cause genetically related disorders since Zn is a constituent of proteins related to DNA and RNA stabilisation (da Cruz et al., 2019). ZnO NPs at the concentrations of 1 mg · L−1, 2 mg · L−1 and 4 mg · L−1 enhanced
Ag NPs are also known for their genotoxic properties in plants. As reported by Patlolla et al. (2012) in
Our study focused on the application of zinc oxide and silver NPs in chrysanthemum micropropagation