Zinc oxide and silver effects on the growth, pigment content and genetic stability of chrysanthemums propagated by the node culture method

Tymoszuk, Alicja; Szałaj, Urszula; Wojnarowicz, Jacek; Kowalska, Jolanta; Antkowiak, Małgorzata; Kulus, Dariusz

doi:10.2478/fhort-2024-0003

Figures & Tables

Sample pictures of Chrysanthemum × morifolium ‘UBG’ (A) and ‘UPG’ (B) shoot and root systems (C, D) developed from node explants cultured in vitro for 10 weeks on the modified MS medium, depending on the ZnO SMPs/ZnO NPs/ZnO + Ag NPs treatment; bar = 1 cm. MS, Murashige and Skoog; NPs, nanoparticles; SMPs, submicron particles; UBG, ‘UTP Burgundy Gold’; UPG, ‘UTP Pinky Gold’.

Leaf architecture parameters of Chrysanthemum × morifolium ‘UBG’ plantlets developed from node explants cultured in vitro on the modified MS medium, depending on the ZnO SMPs/ZnO NPs/ZnO + Ag NPs treatment. Means ± SD on graphs for each parameter analysed followed by the same letter do not differ significantly at p ≤ 0.05 (Fisher’s test). MS, Murashige and Skoog; NPs, nanoparticles; SD, standard deviation; SMPs, submicron particles; UBG, ‘UTP Burgundy Gold’.

Leaf architecture parameters of Chrysanthemum × morifolium ‘UPG’ plantlets developed from node explants cultured in vitro on the modified MS medium, depending on the ZnO SMPs/ZnO NPs/ZnO + Ag NPs treatment. Means ± SD on graphs for each parameter analysed followed by the same letter do not differ significantly at p ≤ 0.05 (Fisher’s test). MS, Murashige and Skoog; NPs, nanoparticles; SD, standard deviation; SMPs, submicron particles; UPG, ‘UTP Pinky Gold’.

Root system architecture of Chrysanthemum × morifolium ‘UBG’ plantlets developed from node explants cultured in vitro on the modified MS medium, depending on the ZnO SMPs/ZnO NPs/ZnO + Ag NPs treatment. Means ± SD on graphs for each parameter analysed followed by the same letter do not differ significantly at p ≤ 0.05 (Fisher’s test). MS, Murashige and Skoog; NPs, nanoparticles; SD, standard deviation; SMPs, submicron particles; UBG, ‘UTP Burgundy Gold’.

Root system architecture of Chrysanthemum × morifolium ‘UPG’ plantlets developed from node explants cultured in vitro on the modified MS medium, depending on the ZnO SMPs/ZnO NPs/ZnO + Ag NPs treatment. Means ± SD on graphs for each parameter analysed followed by the same letter do not differ significantly at p ≤ 0.05 (Fisher’s test). MS, Murashige and Skoog; NPs, nanoparticles; SD, standard deviation; SMPs, submicron particles; UPG, ‘UTP Pinky Gold’.

Example of RAPD (A) and SCoT (B) band profiles of the control and 400 mg · L−1 ZnO SMPs/ZnO NPs/ZnO + Ag NPs-treated plantlets of Chrysanthemum × morifolium ‘UBG’ and ‘UPG’ received as a result of electrophoresis of the DNA amplification products obtained with the R2 (RAPD) and S4 (SCoT) primers. Outermost lanes (wm) are DNA bp weight markers. NPs, nanoparticles; RAPD, randomly amplified polymorphic DNA; SCoT, start codon targeted polymorphism; SMPs, submicron particles; UBG, ‘UTP Burgundy Gold’; UPG, ‘UTP Pinky Gold’.

SEM images of samples: (A, B) ZnO SMPs; (C, D) ZnO NPs (1.5% H2O); (E, F) ZnO NPs (6% H2O); (G, H) ZnO + 0.1% Ag NPs (1.5% H2O); (I, J) ZnO + 0.1% Ag NPs (6% H2O); (K, L) ZnO + 1% Ag NPs (1.5% H2O); (M, N) ZnO + 1% Ag NPs (6% H2O) images taken with the immersion lens detector. NPs, nanoparticles; SMPs, submicron particles.

Chrysanthemum × morifolium ‘UBG’ and ‘UPG’ shoot and root systems developed from node explants cultured in vitro for 10 weeks on the modified MS medium, depending on the ZnO SMPs/ZnO NPs/ZnO + Ag NPs treatment; bar = 1 cm. MS, Murashige and Skoog; NPs, nanoparticles; SMPs, submicron particles; UBG, ‘UTP Burgundy Gold’; UPG, ‘UTP Pinky Gold’.

RAPD and SCoT band profiles of the control and 400 mg · L−1 ZnO SMPs/ZnO NPs/ ZnO + Ag NPs-treated plantlets of Chrysanthemum × morifolium ‘UBG’ and ‘UPG’. Outermost lanes (wm) are DNA bp weight markers. NPs, nanoparticles; RAPD, randomly amplified polymorphic DNA; SCoT, start codon targeted polymorphism; SMPs, submicron particles; UBG, ‘UTP Burgundy Gold’; UPG, ‘UTP Pinky Gold’.

Molecular products obtained from Chrysanthemum × morifolium ‘UBG’ and ‘UPG’ control and 400 mg · L−1 ZnO SMPs/ZnO NPs/ZnO + Ag NPs-treated plantlets analysed with RAPD (R 1–5) and SCoT (S 1–5) marker systems

Primer code	Primer sequence 5’ → 3’	Reference	Cultivar	No. of bands	Band sizes (bp)	No. of loci				No. of genotypes
Primer code	Primer sequence 5’ → 3’	Reference	Cultivar	No. of bands	Band sizes (bp)	Total	mono	poly	spec	No. of genotypes
RAPD
R1	GGG AAT TCG G	Lema-Rumińska et al. (2004)	UBG	192	367–1,478	6	6	0	0	1
R1	GGG AAT TCG G	Lema-Rumińska et al. (2004)	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
R2	GAC CGC TTG T		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
R3	GCT GCC TCA GG	Shibata et al. (1998)	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
R4	TAC CCA GGA GCG	Wolf (1996)	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
R5	CAA TCG CCG T		UPG	160	579–1,430	5	5	0	0	1
Σ			UBG	864		27	27	0	0	1
Σ			UPG	1,024		32	32	0	0	1
Mean from a single primer			UBG	172.8		5.4	5.4	0	0	-
Mean from a single primer			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
S1	CAA TGG CTA CCA CCT	Collard and Mackill (2009)	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
S2	CAA TGG CTA CCA CGT		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
S3	ACG ACA TGG CGA CCA ACG		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
S4	ACG ACA TGG CGA CCA TCG		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
S5	ACC ATG GCT ACC GTC		UPG	256	329–1,824	8	8	0	0	1
Σ			UBG	1,696		53	53	0	0	1
Σ			UPG	1,632		51	51	0	0	1
Mean from a single primer			UBG	339.2		10.6	10.6	0	0	-
Mean from a single primer			UPG	326.4		10.2	10.2	0	0	-

Composition of the precursor solution used for synthesis

Sample name	Ethylene glycol volume (mL)	Weight of zinc acetate dihydrate (g)	Weight of silver acetate anhydrous (g)	Final water content of the precursor solution (weight %)
ZnO NPs (1.5% H₂O)			0	1.5
ZnO NPs (6% H₂O)			0	6
ZnO + 0.1% Ag NPs (1.5% H₂O)	525	35 g	0.0266	1.5
ZnO + 0.1% Ag NPs (6% H₂O)	525	35 g	0.0266	6
ZnO + 1% Ag NPs (1.5% H₂O)			0.2689	1.5
ZnO + 1% Ag NPs (6% H₂O)			0.2689	6

Content and ratios of chlorophylls and carotenoids in Chrysanthemum × morifolium ‘UPG’ plantlets developed from node explants cultured in vitro on the modified MS medium, depending on the ZnO SMPs/ZnO NPs/ZnO + Ag NPs treatment_

Treatment	Chlorophyll a content (mg · g⁻¹ FW)	Chlorophyll b content (mg · g⁻¹ FW)	Chlorophyll a/b ratio	Chlorophyll (a ± b) content (mg · g⁻¹ FW)	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

Biometric parameters of Chrysanthemum × morifolium ‘UBG’ plantlets developed from node explants cultured in vitro on the modified MS medium, depending on the ZnO SMPs/ZnO NPs/ZnO + Ag NPs treatment_

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

Content and ratios of chlorophylls and carotenoids in Chrysanthemum × morifolium ‘UBG’ plantlets developed from node explants cultured in vitro on the modified MS medium, depending on the ZnO SMPs/ZnO NPs/ZnO + Ag NPs treatment_

Treatment	Chlorophyll a content (mg · g⁻¹ FW)	Chlorophyll b content (mg · g⁻¹ FW)	Chlorophyll a/b ratio	Chlorophyll (a + b) content (mg · g⁻¹ FW)	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

Characteristics of samples

Sample name	Skeleton density, ρ_s ± σ (g · cm⁻³)	SSA, a_s (m² · g⁻¹)	Average particle size from SSA BET, d ± σ (nm)	Average crystallite size, Scherrer equation, d ± σ (nm)
ZnO SMPs	5.59 ± 0.03	4.5	240 ± 30	124 ± 11
ZnO NPs (1.5% H₂O)	5.09 ± 0.06	48.4	25 ± 2	31 ± 8
ZnO NPs (6% H₂O)	5.38 ± 0.05	17.2	65 ± 6	40 ± 10
ZnO + 0.1% Ag NPs (1.5% H₂O)	5.16 ± 0.07	40.0	29 ± 2	17 ± 5 (ZnO)
				31 ± 10 (Ag)
ZnO + 0.1% Ag NPs (6% H₂O)	5.37 ± 0.06	14.2	79 ± 2	39 ± 12 (ZnO)
				31 ± 10 (Ag)
ZnO + 1% Ag NPs (1.5% H₂O)	5.05 ± 0.05	44.4	27 ± 2	22 ± 3 (ZnO)
				45 ± 20 (Ag)
ZnO + 1% Ag NPs (6% H₂O)	5.31 ± 0.09	21.4	53 ± 2	35 ± 5 (ZnO)
				31 ± 15 (Ag)

Biometric parameters of Chrysanthemum × morifolium ‘UPG’ plantlets developed from node explants cultured in vitro on the modified MS medium, depending on the ZnO SMPs/ZnO NPs/ZnO + Ag NPs treatment_

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

Results of the analysis of the chemical composition of the sample

Sample name	Actual dopant content (mol%)		Nominal composition (mol%)		Weight content of ZnO and Ag in 100 mg of sample ⁽mg)
Sample name	Zinc	Silver	Zinc	Silver	ZnO	Ag
ZnO + 0.1% Ag NPs (1.5% H₂O)	99.91 ± 0.04	0.09 ± 0.04	99.9	0.1	99.881	0.119
ZnO + 0.1% Ag NPs (6% H₂O)	99.85 ± 0.06	0.15 ± 0.06	99.9	0.1	99.801	0.199
ZnO + 1% Ag NPs (1.5% H₂O)	99.05 ± 0.20	0.95 ± 0.20	99.00	1.00	98.745	1.255
ZnO + 1% Ag NPs (6% H₂O)	98.95 ± 0.20	1.05 ± 0.20	99.00	1.00	98.613	1.387

Zinc oxide and silver effects on the growth, pigment content and genetic stability of chrysanthemums propagated by the node culture method

Figures & Tables

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Supplementary Figure S1.

Supplementary Figure S2.

Supplementary Figure S3.

Supplementary Figure S4.

Molecular products obtained from Chrysanthemum × morifolium ‘UBG’ and ‘UPG’ control and 400 mg · L−1 ZnO SMPs/ZnO NPs/ZnO + Ag NPs-treated plantlets analysed with RAPD (R 1–5) and SCoT (S 1–5) marker systems

Composition of the precursor solution used for synthesis

Content and ratios of chlorophylls and carotenoids in Chrysanthemum × morifolium ‘UPG’ plantlets developed from node explants cultured in vitro on the modified MS medium, depending on the ZnO SMPs/ZnO NPs/ZnO + Ag NPs treatment_

Biometric parameters of Chrysanthemum × morifolium ‘UBG’ plantlets developed from node explants cultured in vitro on the modified MS medium, depending on the ZnO SMPs/ZnO NPs/ZnO + Ag NPs treatment_

Content and ratios of chlorophylls and carotenoids in Chrysanthemum × morifolium ‘UBG’ plantlets developed from node explants cultured in vitro on the modified MS medium, depending on the ZnO SMPs/ZnO NPs/ZnO + Ag NPs treatment_

Characteristics of samples

Biometric parameters of Chrysanthemum × morifolium ‘UPG’ plantlets developed from node explants cultured in vitro on the modified MS medium, depending on the ZnO SMPs/ZnO NPs/ZnO + Ag NPs treatment_

Results of the analysis of the chemical composition of the sample

Paradigm

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