Biodiversity protection is important for systematics research, species evolution understanding, and breeding programs. Cryopreservation, i.e. maintenance of biological material at a cryogenic temperature of liquid nitrogen (-196oC), is considered the most effective long-term storage method. Nonetheless, this technology is still developing and requires more research. Application of nanoparticles (NPs) may greatly improve the effectiveness of cryopreservation protocols and ought to be carefully explored.
The popularity of nanoparticles (i.e. particles of a maximum size = 100 nm) is continuously increasing. They are commonly applied in medicine, pharmacy, cosmetology, and plant protection. To date, however, there has been no research on the application of NPs in plant cryopreservation. Moreover, there is a lack of comprehensive research demonstrating the influence of nanoparticles on living organisms.
The aim of the project was to broaden the scope of knowledge about the effect of various NPs on the in vitro and ex vitro development of plants, particularly concerning cryobiology studies.
The scientific hypothesis assumed that silver (AgNPs), gold (AuNPs), and zinc oxide (ZnONPs) nanoparticles can penetrate plant tissues, enhance the pace of cooling and rewarming during a cryopreservation procedure, and consequently, minimize the lethal ice-formation effects. Moreover, the addition of NPs into the protective bead matrix may positively affect the regrowth of explants and the biosynthesis of primary and/or secondary metabolites after rewarming.
Two cultivars of Lamprocapnos spectabilis – a vegetatively propagated species of great ornamental, medicinal, and economic value were used in the experiments.
Shoot tips of in vitro-derived plants were cryopreserved with the previously optimised encapsulation-vitrification protocol. Silver or gold nanoparticles at various concentrations were added either into the preculture medium (1st step of the cryopreservation procedure) or to the protective bead matrix during encapsulation (2nd step of the protocol). The control included plants recovered from cryopreserved explants 1) non-treated with nanoparticles and 2) treated with colloid dispersion medium without NPs.
After storage and rewarming, the influence of NPs on the cryopreservation efficiency was determined. This included evaluation of the recovery rate of explants and their morphogenetic response; i.e. number of shoots, leaves, and roots produced. Transmission electron microscopy (TEM) allowed to detect the presence of NPs in the cells, as well as to assess their structural integrity. Based on the spectrophotometric analyses of phenolic compounds concentration and antioxidant enzymes activity in shoots, the level of defence reaction against reactive oxygen species, resulting from NPs application, was evaluated. During the in vitro cultivation period, it was demonstrated, whether NPs cause alternations in the DNA sequence with the use of RAPD and SCoT molecular markers. The cytogenetic stability of the LN-derived plant material was evaluated using flow cytometry.
After acclimatisation to ex vitro glasshouse conditions, the biochemical stability of plants was assessed. The influence of NPs on the content of photosynthetic pigments and secondary metabolites (carotenoids and flavonoids) in the NP-treated and untreated control leaves and stems was monitored. HPLC chromatography was used to study the phenolic profile of plants. The fluorescence parameters of chlorophyll gave insights into stress reactions. The detailed phenotype analysis of plants at this stage was included. By those means, it was possible to evaluate the widely understood stability of plants at various developmental stages.
The results of the study suggest that different types and concentrations of nanoparticles, such as gold, silver and zinc oxide, have different effects on cryopreservation efficiency and further growth and physiological activity of plants, which highlights their specific effects in the context of different plant cultivars. The proper use of nanoparticles in cryopreservation protocols can significantly improve explant survival rates and their regenerative potential, which is crucial for the protection of genetic resources, as well as enable the modulation of plant secondary metabolism to achieve the desired production effects. However, the use of NPs in suboptimal conditions may have an adverse effect on the plants.
(2024-10-27)
