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. 2022 Jul 3;23(13):7406.
doi: 10.3390/ijms23137406.

Effect of Silver Nanoparticles on the In Vitro Regeneration, Biochemical, Genetic, and Phenotype Variation in Adventitious Shoots Produced from Leaf Explants in Chrysanthemum

Alicja Tymoszuk  1 , Dariusz Kulus  1
Affiliations

Effect of Silver Nanoparticles on the In Vitro Regeneration, Biochemical, Genetic, and Phenotype Variation in Adventitious Shoots Produced from Leaf Explants in Chrysanthemum

Alicja Tymoszuk et al. Int J Mol Sci. .

Abstract

Novel and unique properties of nanomaterials, which are not apparent in larger-size forms of the same material, encourage the undertaking of studies exploring the multifaced effects of nanomaterials on plants. The results of such studies are not only scientifically relevant but, additionally, can be implemented to plant production and/or breeding. This study aimed to verify the applicability of silver nanoparticles (AgNPs) as a mutagen in chrysanthemum breeding. Chrysanthemum × grandiflorum (Ramat.) Kitam. 'Lilac Wonder' and 'Richmond' leaf explants were cultured on the modified MS medium supplemented with 0.6 mg·L-1 6-benzylaminopurine (BAP) and 2 mg·L-1 indole-3-acetic acid (IAA) and treated with AgNPs (spherical; 20 nm in diameter size; 0, 50, and 100 mg·L-1). AgNPs strongly suppressed the capability of leaf explants to form adventitious shoots and the efficiency of shoot regeneration. The content of primary and secondary metabolites (chlorophyll a, chlorophyll b, total chlorophylls, carotenoids, anthocyanins, phenolic compounds) and the activity of enzymatic antioxidants (superoxide dismutase and guaiacol peroxide) in leaf explants varied depending on the AgNPs treatment and age of culture. Phenotype variations of ex vitro cultivated chrysanthemums, covering the color and pigment content in the inflorescence, were detected in one 50 mg·L-1 AgNPs-derived and five 100 mg·L-1 AgNPs-derived 'Lilac Wonder' plants and were manifested as the color change from pink to burgundy-gold. However, no changes in inflorescence color/shape were found among AgNPs-treated 'Richmond' chrysanthemums. On the other hand, the stem height, number of leaves, and chlorophyll content in leaves varied depending on the AgNPs treatment and the cultivar analyzed. A significant effect of AgNPs on the genetic variation occurrence was found. A nearly two-fold higher share of polymorphic products, in both cultivars studied, was generated by RAPD markers than by SCoTs. To conclude, protocols using leaf explant treatment with AgNPs can be used as a novel breeding technique in chrysanthemum. However, the individual cultivars may differ in biochemical response, the efficiency of in vitro regeneration, genetic variation, and frequency of induced mutations in flowering plants.

Keywords: Chrysanthemum × grandiflorum (Ramat.) Kitam.; induced mutagenesis; molecular markers; nanotechnology; oxidative stress; phenotype alternation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Dynamics of adventitious shoots regeneration on the inoculated Chrysanthemum × grandiflorum ‘Lilac Wonder’ and ‘Richmond’ leaf explants cultured on the modified MS medium with 0.6 mg·L−1 BAP and 2 mg·L−1 IAA, depending on the AgNPs treatment (0–100 mg·L−1).
Figure 2
Figure 2
Shoot induction rate of Chrysanthemum × grandiflorum ‘Lilac Wonder’ and ‘Richmond’ leaf explants after 10 weeks of culture on the modified MS medium with 0.6 mg·L−1 BAP and 2 mg·L−1 IAA, depending on the AgNPs treatment (0–100 mg·L−1). Means on graphs for each cultivar tested followed by the same letter do not differ significantly at p ≤ 0.05 (Tukey’s test).
Figure 3
Figure 3
Mean number of adventitious shoots regenerated per one inoculated leaf explant in Chrysanthemum × grandiflorum ‘Lilac Wonder’ and ‘Richmond’ after 10 weeks of culture on the modified MS medium with 0.6 mg·L−1 BAP and 2 mg·L−1 IAA, depending on the AgNPs treatment (0–100 mg·L−1). Means ± SD on graphs for each cultivar tested followed by the same letter do not differ significantly at p ≤ 0.05 (Tukey’s test).
Figure 4
Figure 4
Activity of superoxide dismutase in Chrysanthemum × grandiflorum ‘Lilac Wonder’ (graphs A–C) and ‘Richmond’ (graphs D–F) leaf explants cultured in vitro on the modified MS medium with 0.6 mg·L−1 BAP and 2 mg·L−1 IAA, depending on the AgNPs treatment (0–100 mg·L−1) and week of culture (first–third). Means and means ± SD on graphs for each cultivar tested followed by the same letter do not differ significantly at p ≤ 0.05 (Tukey’s test). Upper-case letters refer to the main effects (irrespectively) (graphs A,B,D,F), lower-case letters refer to the interaction between the two studied independent variables (graphs C,F).
Figure 5
Figure 5
Activity of guaiacol peroxidase in Chrysanthemum × grandiflorum ‘Lilac Wonder’ (graphs A–C) and ‘Richmond’ (graphs D–F) leaf explants cultured in vitro on the modified MS medium with 0.6 mg·L−1 BAP and 2 mg·L−1 IAA, depending on the AgNPs treatment (0–100 mg·L−1) and week of culture (first–third). Means and means ± SD on graphs for each cultivar tested followed by the same letter do not differ significantly at p ≤ 0.05 (Tukey’s test). Upper-case letters refer to the main effects (irrespectively) (graphs A,B,D,F), lower-case letters refer to the interaction between the two studied independent variables (graphs C,F).
Figure 6
Figure 6
Chrysanthemum× grandiflorum ‘Lilac Wonder’ and ‘Richmond’ and their mutants, created as a result of AgNPs treatment (0–100 mg·L−1); arrows indicate a chimeric structure of the ligulate floret; bar = 1 cm.
Figure 7
Figure 7
Dendrograms based on the estimation of genetic distance coefficient and UPGMA clustering present in the relationships between the populations of AgNPs-treated, control, and standard plants, revealed by the randomly amplified polymorphic DNA (RAPD) and start codon targeted polymorphism (SCoT) analyses.
Figure 7
Figure 7
Dendrograms based on the estimation of genetic distance coefficient and UPGMA clustering present in the relationships between the populations of AgNPs-treated, control, and standard plants, revealed by the randomly amplified polymorphic DNA (RAPD) and start codon targeted polymorphism (SCoT) analyses.
Figure 8
Figure 8
Graphs of principal coordinates analysis (PCoA) of Chrysanthemum × grandiflorum ‘Lilac Wonder’ and ‘Richmond’ plants obtained after 0 (control), 50, and 100 mg·L−1 AgNPs treatment and in standard plants, based on randomly amplified polymorphic DNA (RAPD) and start codon targeted polymorphism (SCoT) analyses. Plants representing the same band pattern as the predominant standard are collected within a single group named ‘monomorphic’.
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