Enhancing Germination and Growth of Chrysanthemum Synthetic Seeds Through Iron Oxide Nanoparticles and Indole-3-Acetic Acid: Impact of Treatment Duration on Metabolic Activity and Genetic Stability

Abstract

Background: This study investigated the effects of pure iron oxide nanoparticles (Fe₃O₄ NPs), citrate-stabilized iron oxide nanoparticles (Fe₃O₄CA NPs), and indole-3-acetic acid (IAA), applied at various time regimes, on the germination, growth, and ex vitro development of chrysanthemum synthetic seeds. The genetic and metabolic stability of the plants was also assessed.

Methods: Nodal segments of Chrysanthemum × morifolium /Ramat./ Hemsl. 'Richmond', with a single axillary bud, were encapsulated in 3% calcium alginate with the addition of IAA (1 mg·L^-1) and/or NPs (7.7 mg·L^-1). The synthetic seeds were cultured in vitro for 30 or 60 days on a water-agar medium and then transplanted to the greenhouse for further analyses.

Results: Results indicated that IAA and Fe₃O₄CA NPs applied singularly significantly enhanced germination rates (83.33-92.18%) compared with the IAA- and NP-free control (56.67-64.18%), regardless of treatment time. The simultaneous use of IAA and Fe₃O₄CA NPs promoted longer shoot development after 30 days of treatment but showed negative effects after extended exposure. The same combination improved rooting efficiency compared to IAA alone. Supplementation with NPs improved acclimatization rates for younger plants but had variable effects on older plants. Leaf growth metrics were enhanced with Fe₃O₄CA NPs in plants after 30 days of treatment, yet no significant differences were observed in leaf dimensions after 60 days. The content of flavonoids, anthocyanins, and chlorophyll was affected by the exposure duration. Biochemical analyses revealed increased total polyphenol content and antioxidant capacity (FRAP, ABTS) in treated plants, particularly with IAA and Fe₃O₄CA NPs. Start codon targeted (SCoT) analyses showed no polymorphisms among treated plants, confirming their genetic stability.

Conclusion: The study found that the combination of IAA and Fe₃O₄CA NPs improved germination and shoot development in chrysanthemum synthetic seeds, while maintaining genetic stability, although prolonged exposure negatively affected plant growth metrics.

Keywords: Chrysanthemum × morifolium /Ramat./ Hemsl.; SCoT; antioxidant capacity; molecular markers; nanotechnology; polyphenols.

Figure 1

Effect of indole-3-acetic acid (IAA) and iron nanoparticles in pure (Fe₃O₄ NPs) or stabilized form (Fe₃O₄CA NPs), applied singularly or in combination, on the root number per shoot and length of the longest root in chrysanthemum ‘Richmond’ after 30 days of in vitro culture. Means ± standard errors (SE) marked with the same letter do not differ significantly according to Fisher’s test at p ≤ 0.05.

Figure 2

Effect of indole-3-acetic acid (IAA) and iron nanoparticles in pure (Fe₃O₄ NPs) or stabilized form (Fe₃O₄CA NPs), applied singularly or in combination, on the biometrical parameters of leaves in glasshouse-grownchrysanthemum’Richmond’, acclimatized after 30 days of in vitro culture. Means ± standard errors (SE) marked with the same letter do not differ significantly according to Fisher’s test at p ≤ 0.05.

Figure 3

Effect of indole-3-acetic acid (IAA) and iron nanoparticles in pure (Fe₃O₄ NPs) or stabilized form (Fe₃O₄CA NPs), applied singularly or in combination, on the biometrical parameters of leaves in glasshouse-grown chrysanthemum ‘Richmond’, acclimatized after 60 days of in vitro culture. Means ± standard errors (SE) marked with the same letter do not differ significantly according to Fisher’s test at p ≤ 0.05.

Figure 4

Effect of indole-3-acetic acid (IAA) and iron nanoparticles in pure (Fe₃O₄ NPs) or stabilized form (Fe₃O₄CA NPs), applied singularly or in combination, on the relative content of flavonoids, anthocyanins, chlorophyll (CCI) and SPAD value in the leaves of glasshouse-grownchrysanthemum’Richmond’, acclimatized after 30 and 60 days of in vitro culture. Means ± standard errors (SE) marked with the same letter do not differ significantly according to Fisher’s test at p ≤ 0.05.

Figure 5

Example SCoT band profiles of chrysanthemum ‘Richmond’ received with primer S2 as a result of auxin and/or nanoparticle treatments. The outermost lanes (wm) serve as DNA base pair (bp) weight markers (GeneRuler Express DNA Ladder from Thermo Scientific, Waltham, MA, USA), while inner lines represent control plants, plants treated with indole-3-acetic acid (IAA), iron nanoparticles in pure (Fe₃O₄ NPs) or stabilized form (Fe₃O₄CA NPs), applied singularly or in combination.