Gold nanoparticles affect the cryopreservation efficiency of in vitro-derived shoot tips of bleeding heart

The popularity of nanoparticles (NPs) is continuously increasing. To date, however, there has been little research on the application of NPs in plant cryopreservation, i.e. storage of tissues in liquid nitrogen (LN). The aim of this study is to analyze the effect and evaluate the usefulness of gold nanoparticles (AuNPs) in regard to cryobiology studies. In vitro-derived shoot tips of Lamprocapnos spectabilis ‘Valentine’ were cryopreserved with the encapsulation-vitrification protocol. Gold nanoparticles (at 10–30 ppm concentration; 13 nm in size) were added either into the preculture medium; to the protective bead matrix during encapsulation; or to the recovery medium after rewarming of samples. The control plants were produced from cryopreserved explants non-treated with nanoparticles or treated with colloid dispersion medium without NPs. A non-LN-treated standard was also considered. The influence of AuNPs on the cryopreservation efficiency was determined by evaluating the recovery rate of explants and their morphogenic response; the membrane stability index (MSI); the concentration of pigments in shoots; and the antioxidant enzymes activity. The genetic stability of the plant material was evaluated using Start Codon Targeted Polymorphism (SCoT) markers. It was found that 10 ppm of AuNPs added into the alginate bead matrix improved the recovery level of LN-derived shoot tips (70.0%) compared to the non-NPs-treated cryopreserved control (50.5%). On the other hand, the presence of nanoparticles in the recovery medium had a deleterious effect on the survival of explants. AuNPs usually had no impact on the MSI (73.9–85.9%), except for those added into the recovery medium at the concentration of 30 ppm (decline to 55.8%). All LN-derived shoots were shorter and contained less chlorophyll and carotenoids than the untreated standard. Moreover, the application of AuNPs affected the enzymatic activity in L. spectabilis. Minor genetic variation was found in 8.6% of plants if AuNPs were added either into the preculture medium (at 10 and 20 ppm) or to the alginate matrix (at 30 ppm). In conclusion, AuNPs added at a lower concentration (10 ppm) into the protective bead matrix can significantly improve the cryopreservation efficiency in L. spectabilis with no alternation in the DNA sequence.

ADDITIONS TO THE VASCULAR PLANT FLORA OF THE REPUBLIC OF MORDOVIA (RUSSIA): CONTRIBUTION OF THE iNATURALIST PLATFORM

"The (re)check of the iNaturalist data related to the Republic of Mordovia (European Russia) allowed us to find 16 vascular plant species, considered to be new taxa for the vascular plant flora of this region, in comparison to Silaeva et al. (2010). These species are Nonea rossica, Erigeron strigosus, Medicago × varia, Dianthus chinensis, Symphytum × uplandicum, Lamprocapnos spectabilis, Petunia × atkinsiana, Oenothera villosa, Parthenocissus inserta, Reynoutria × bohemica, Eschscholzia californica, Quercus rubra, Digitaria sanguinalis, Lysimachia punctata, Acer ginnala and Stachys byzantina. In addition, we present new data about the distribution of the five species (Silene wolgensis, Amaranthus cruentus, Cruciata laevipes, Euphorbia cyparissias and Achillea leptophylla) found in the Republic of Mordovia since publication of Silaeva et al. (2010). A majority of the species under discussion are alien plants arrived in Mordovia accidentally or considered as cultivated plants escaping in the wild. Taking into account the newly revealed species, the vascular plant flora of the Republic of Mordovia currently includes 1464 species. In this study, we highlight again the relevance and feasibility of the use of the iNaturalist platform, as an effective tool for the study and monitoring of regional and national biodiversity."

Induction of Callogenesis, Organogenesis, and Embryogenesis in Non-Meristematic Explants of Bleeding Heart and Evaluation of Chemical Diversity of Key Metabolites from Callus

Lamprocapnos spectabilis (L.) Fukuhara is a perennial plant species valued in the horticultural, cosmetic, and pharmaceutical markets. To date, however, there were no studies on tissue culture systems in this species when adjusted from non-meristematic explants. The aim of this study is to induce callogenesis, organogenesis, and somatic embryogenesis in non-meristematic explants of Lamprocapnos spectabilis ‘Alba’ cultured in various media and to analyze the chemical diversity of the produced callus. Leaf, petiole, and internode explants were cultured on the modified Murashige and Skoog (MS) medium fortified with various combinations and concentrations of 6-benzyladenine (BA), indole-3-acetic acid (IAA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorphenoxyacetic acid (2,4-D), and picloram (PIC). After 10 weeks of culturing, the morphogenetic response of explants was evaluated and the concentration of chlorophylls, carotenoids, anthocyanins, and polyphenols in callus was analyzed. There was no influence of explant type on the callogenesis efficiency (62.1–65.3%). The highest fresh weight of callus was produced on leaf explants in the presence of 2,4-D or PIC. In contrast, the highest share of dry weight was found in internode-derived calli and cultured on IAA-supplemented medium (up to 30.8%). Only 2.5% of all explants regenerated adventitious shoots, while rhizogenesis was reported in 4.5% of explants. Somatic embryos were produced indirectly by 0% to 100% of explants, depending on the culture medium and explant type. The highest mean number of embryos (11.4 per explant) was found on petioles cultured in the MS medium with 0.5 mg·L−1 BA and 1.0 mg·L−1 PIC. Calli cultured in media with NAA usually contained a higher content of primary and secondary metabolites. There was also a significant impact of explant type on the content of anthocyanins, polyphenols, and carotenoids in callus. Further studies should focus on the elicitation of metabolites production in callus culture systems of the bleeding heart.

Effect of Bead Composition, PVS Type, and Recovery Medium in Cryopreservation of Bleeding Heart ‘Valentine’—Preliminary Study

Bleeding heart (Lamprocapnos spectabilis (L.) Fukuhara) is a valuable ornamental and medicinal perennial. To date, there are few studies focused on cryopreservation of this species, although it could be useful in storage and breeding. This research is aimed at analyzing the effect of bead composition, type of plant vitrification solution (PVS), and the recovery medium of cryopreservation of bleeding heart. Shoot tips of L. spectabilis ‘Valentine’ were used in the study. The explants were precultured on modified Murashige and Skoog medium (MS; 1962), supplemented with 9% sucrose, 1.0-mg·L−1 kinetin (KIN), and 2.62-mg·L−1 abscisic acid. Next, in the first experiment, the shoot tips were embedded in 3% calcium alginate, based either on an MS medium or distilled sterile water. The produced synseeds were inoculated on the recovery medium with 3.0-mg·L−1 KIN, 0.5-mg·L−1 6-benzyladenine (BA), or cytokinin–free control. Based on the results of the first study, in the second experiment, precultured shoot tips were embedded in 3% calcium alginate based on MS medium and dehydrated with PVS2 or PVS3 for various durations. The pre-treated explants were plunged in liquid nitrogen and, after rewarming, inoculated on the recovery MS medium with 0.5-mg·L−1 BA. PVS3 was more effective in securing the shoot tips than PVS2. The highest recovery level (68.3%) was reported after a 150-min pretreatment with PVS3. Explants from this experimental combination also proliferated the highest number of shoots, as well as those with the greatest length. On the other hand, a higher share of dry weight was found in PVS2-derived shoots (13.5–18.2%) compared with plants produced after PVS3 treatment (10.6–11.4%). The obtained results here can serve as a good basis for further studies related to synthetic seeds and cryopreservation of bleeding heart.

Shoot Tip Cryopreservation of Lamprocapnos spectabilis (L.) Fukuhara Using Different Approaches and Evaluation of Stability on the Molecular, Biochemical, and Plant Architecture Levels

The aim of this study is to optimize and evaluate the effectiveness of vitrification, droplet-vitrification, and encapsulation-vitrification techniques in the cryopreservation of Lamprocapnos spectabilis (L.) Fukuhara ‘Gold Heart’, a popular medicinal and ornamental plant species. In vitro-derived shoot tips were used in the experiments. All three techniques were based on explant dehydration with plant vitrification solution 3 (PVS3; 50% glycerol and 50% sucrose) for 0, 30, 60, 90, 120, 150, or 180 min. The recovered microshoots were subjected to morphometric, biochemical, and molecular analyses (RAPD, ISSR, SCoT). The highest recovery level was reported with the encapsulation-vitrification protocol based on 150 min dehydration (73.1%), while the vitrification technique was the least effective (maximum 25.8% recovery). Explants cryopreserved with the encapsulation-vitrification technique produced the highest mean number of shoots (4.9); moreover, this technique was optimal in terms of rooting efficiency. The highest fresh weight of shoots, on the other hand, was found with the vitrification protocol based on a 30-min PVS3 treatment. The concentrations of chlorophyll a and b were lower in all cryopreservation-derived plants, compared to the untreated control. On the other hand, short dehydration and cryopreservation of non-encapsulated explants stimulated the synthesis of anthocyanins. A small genetic variation in 5% of all samples analyzed was detected by RAPD and ISSR marker systems. Only plants recovered from the encapsulation-vitrification protocol had no DNA sequence alternations.