In Vitro Shoot Multiplication and Rooting of 'Kashan' and 'Hervy Azerbaijan' Damask Rose (Rosa damascena Mill.) Genotypes for Cosmetic and Ornamental Applications.

The damask rose (Rosa damascena Mill.) is an ornamental-medicinal plant from the Rosaceae family, and its aromatic compounds and essential oils are applied globally in the food, cosmetic, and pharmaceutical industries. Due to its economic value, this research aimed to establish a protocol for an efficient, rapid, and cost-effective method for in vitro shoot multiplication and rooting of the R. damascena 'Kashan' and 'Hervy Azerbaijan' genotypes. Nodal segments (as primary explants) were cultured on the Murashige and Skoog (MS) medium with combinations of various plant growth regulators (PGRs) such as gibberellic acid (GA3), 6-benzylaminopurine (BAP), and indole-3-butyric acid (IBA), as well as a PGR-like substance, phloroglucinol (PG), vitamins such as ascorbic acid (AA), and activated carbon in the form of active charcoal (AC). For the establishment stage, 0.1 mg·L-1 PG, 0.2 mg·L-1 GA3, and 1 mg·L-1 BAP were added to the media. Secondary explants (nodal segments containing axillary buds produced from primary explants) were obtained after 30 days of in vitro culture and transferred to the proliferation media supplemented with different concentrations of BAP (0, 0.5, 1, 1.5, 2, and 2.5 mg·L-1) and GA3 (0, 0.1, 0.2, 0.4, 0.8, and 1 mg·L-1) together with 0.1 mg·L-1 PG and 20 mg·L-1 of AA. The rooting media were augmented with different concentrations of BAP and GA3 with 0.1 mg·L-1 of IBA, PG and 20 mg·L-1 of AA and AC. The results showed that the highest regeneration coefficient (4.29 and 4.28) and the largest number of leaves (23.33-24.33) were obtained in the explants grown on the medium supplemented with 2 mg·L-1 BAP and 0.4 mg·L-1 GA3 for the 'Kashan' and 'Hervy Azerbaijan' genotypes, respectively. Likewise, this PGR combination provided the shortest time until bud break (approximately 6.5 days) and root emergence (approximately 10 days) in both genotypes. The highest number of shoots (4.78 per explant) and roots (3.96) was achieved in this medium in the 'Kashan' rose. Stem and root lengths, as well as stem and root fresh and dry weights, were also analyzed. In most measured traits, the lowest values were found in the PGRs-free control medium. Rooted plantlets were transferred to pots filled with perlite and peat moss in a 2:1 proportion and were acclimatized to ambient greenhouse conditions with a mean 90.12% survival rate. This research contributes significantly to our understanding of Damask rose propagation and has practical implications for the cosmetic and ornamental plant industries. By offering insights into the manipulation of regeneration processes, our study opens up new possibilities for the effective production of high-quality plant material.

Nanoparticle-mediated enhancement of plant cryopreservation: Cultivar-specific insights into morphogenesis and biochemical responses in Lamprocapnos spectabilis (L.) Fukuhara 'Gold Heart' and 'Valentine'.

The integration of nanoparticles (NPs) holds promising potential to bring substantial advancements to plant cryopreservation, a crucial technique in biodiversity conservation. To date, little attention has been focused on using nanoparticles in cryobiology research. This study aimed to assess the effectiveness of NPs in enhancing the efficiency of plant cryopreservation. In-vitro-derived shoot tips of bleeding heart (Lamprocapnos spectabilis (L.) Fukuhara) 'Gold Heart' and 'Valentine' were used as the plant material. The encapsulation-vitrification cryopreservation protocol included preculture, encapsulation, dehydration, storage in liquid nitrogen, rewarming, and recovery steps. Gold (AuNPs), silver (AgNPs), or zinc oxide (ZnONPs) nanoparticles were added at various concentrations either into the preculture medium or the protective bead matrix during encapsulation. The explant survival and further morphogenic and biochemical events were studied. Results showed that the impact of NPs on cryopreservation outcomes was cultivar-specific. In the 'Valentine' cultivar, incorporating 5 ppm AgNPs within the alginate bead matrix significantly improved cryopreservation efficiency by up to 12%. On the other hand, the 'Gold Heart' cultivar benefited from alginate supplementation with 5 ppm AgNPs and 5-15 ppm ZnONPs, leading to an over 28% increase in the survival rate of shoot tips. Interestingly, adding NPs to the preculture medium was less effective and sometimes counterproductive, despite promoting greater shoot proliferation and elongation in 'Valentine' explants compared to the control. Moreover, nanoparticles often induced oxidative stress (and enhanced the activity of APX, GPOX, and SOD enzymes), which in turn affected the biosynthesis of plant primary and secondary metabolites. It was found that supplementation of preculture medium with higher concentration (15 ppm) of gold, silver and zinc oxide nanoparticles stimulated the production of plant pigments, but in a cultivar-dependent matter. Our study confirmed the beneficial action of nanoparticles during cryopreservation of plant tissues.

Synthesis, Characteristics, and Effect of Zinc Oxide and Silver Nanoparticles on the In Vitro Regeneration and Biochemical Profile of Chrysanthemum Adventitious Shoots.

Studies on nanoparticles' effects on plants are relevant for horticulture. This study aimed to test the influence of zinc oxide submicron particles (ZnO SMPs), zinc oxide nanoparticles (ZnO NPs), and zinc oxide nanoparticles combined with silver nanoparticles (ZnO+1%Ag NPs) applied at 100 and 500 mg·L-1 on the regeneration and biochemical activity of adventitious shoots in Chrysanthemum × morifolium (Ramat.) Hemsl. 'UTP Burgundy Gold' and 'UTP Pinky Gold'. The original microwave solvothermal synthesis and characteristics of the ZnO samples were described. Internodes were cultured on the MS medium with 0.6 mg∙L-1 6-benzylaminopurine (BAP) and 2 mg∙L-1 indole-3-acetic acid (IAA). In 'UTP Burgundy Gold', the highest shoot regeneration efficiency was obtained for 100 mg·L-1 ZnO SMPs and 500 mg·L-1 ZnO NPs treatments (6.50 and 10.33 shoots per explant, respectively). These shoots had high or moderate chlorophyll and carotenoid contents. In 'UTP Pinky Gold', the highest shoot number was produced in the control (12.92), for 500 mg·L-1 ZnO SMPs (12.08) and 500 mg·L-1 ZnO NPs (10.42). These shoots had increased chlorophyll (a+b)-to-carotenoid ratios. In 'UTP Pinky Gold', the ZnO SMPs and ZnO NPs affected the anthocyanins biosynthesis, whereas ZnO + 1%Ag NPs decreased the phenolics accumulation. These results are important for the improvement of chrysanthemum micropropagation.

Endophytic Fungi as Potential Biocontrol Agents against Sclerotium rolfsii Sacc.-The Causal Agent of Peanut White Stem Rot Disease.

Peanut stem white rot caused by Sclerotium rolfsii Sacc. is a soil-borne disease that is widely prevailing across peanut farms, leading to serious economic losses. Screening for biocontrol agents against this pathogen is urgent. In this research, 166 fungal isolates including 136 isolates of S. rolfsii and 30 isolates of antagonistic endophytic fungi were obtained from a total of 220 samples collected from peanut farms in Guilan province, Iran. After morphological and molecular identification, six superior endophytic isolates were finally selected for the in vitro and greenhouse trials, including four isolates from Trichoderma viride, Trichoderma virens, Penicillium decaturense, and Aspergillus flavus and two isolates from Penicillium rubens. Four methods of biocontrol were used during the in vitro phase, i.e., dual culture, volatile metabolites assay, non-volatile metabolites assay (culture extract), and slide culture. It was found that T. virens had the highest capability of suppressing the mycelial growth of S. rolfsii in the dual culture method (90.98%). As for the volatile metabolites assay, the most effective isolates in inhibiting the pathogen's mycelial growth were P. rubens (MN395854.1) and A. flavus (84.30% and 73.50% inhibition, respectively). In the non-volatile metabolites method, the isolates that performed the best in suppressing the mycelial growth of S. rolfsii were T. viride and P. rubens (MN395854.1) with 91.80% and 90.20% inhibitory effects, respectively. On the other hand, in the slide culture method, all isolates, except for T. virens and T. viride, successfully controlled the development of S. rolfsii hyphae. The greenhouse trials also supported the effectiveness of endophytic fungi in controlling the pathogen on the host plants. According to the results, T. viride, A. flavus, and P. rubens (MN395854.1) were 44%, 42%, and 38% effective in alleviating the disease incidence and severity. Moreover, the application of these antagonistic fungi in the greenhouse conditions increased the height, fresh weight, and dry weight of the Arachis hypogaea plants infected with the disease causal agent compared to the plants treated only with the pathogen. The results of the in vitro and greenhouse experiments revealed that the endophytic fungi occurring in the natural microbiota of peanut are capable of bio-controlling S. rolfsii, the causal agent of peanut stem white rot disease. These findings shed new insights into the possible resistance induction in A. hypogaea plants through biological protection.

Endophytic Fungi as Potential Biocontrol Agents against Rhizoctonia solani J.G. Kühn, the Causal Agent of Rice Sheath Blight Disease.

The rice sheath blight disease, caused by Rhizoctonia solani J.G. Kühn fungus, is a major disease of Oryza sativa L. occurring all over the world. Therefore, efforts need to be undertaken to limit the spread of this pathogen, preferably by using environmentally friendly methods. In the present study, 57 fungal isolates were recovered by surface sterilization technique from 120 rice samples collected from paddy fields in Guilan province, Iran. Biological characterizations of the isolated taxa were performed in vitro, in the dual culture, volatile metabolites, and slide culture methods. Among the studied isolates, Trichoderma virens (J. H. Miller, Giddens and A. A. Foster) Arx was most effective in inhibiting the mycelial growth of R. solani in the dual culture (44.16% inhibition level), while Aspergillus fumigatus Fresen and T. virens had a 62.50-68.75% inhibition efficiency by volatile metabolites. In the slide culture, all of the isolates, except for T. harzianum Rifai, were effective in inhibiting the hyphae growth of R. solani. Under greenhouse conditions, rice plants inoculated with these potential antagonistic fungi showed a reduction in disease severity by even 41.4% as in the case of T. virens. Moreover, phenotypic properties of rice, such as plant height, fresh weight, and dry weight were increased in the plants inoculated with all antagonistic fungi tested, compared to the infected plants, except for the fresh weight of plants inoculated with Curnularia lunata (Wakker) Boedijn. The present in vivo and in vitro studies revealed that T. virens and A. fumigatus are the most effective antagonists in rice sheath blight disease control and could be applied in agricultural practice.

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

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.

Cryopreservation of Endangered Ornamental Plants and Fruit Crops from Tropical and Subtropical Regions.

Horticultural crops comprise various economic species extending from fruits, nuts, vegetables, spices and condiments, ornamentals, aromatic, and medicinal plants. Ornamental and fruit plants are produced mainly for their nutritional and aesthetic values, respectively. Unfortunately, many tropical and subtropical species are in danger of extinction because of climate change and (a)biotic stresses. It is imperative to preserve the germplasms of these species for the present and future genetic improvement programs. Cryopreservation, i.e., maintenance of tissues at the ultralow temperature of liquid nitrogen, is a promising long-term preservation technique, alternative to seed or in vitro banks, which can be applied for both vegetatively and generatively (through seeds) propagated crops, including those with recalcitrant seeds. It is a technology of choice not only for the preservation of plant biodiversity but also for virus elimination in the proficient administration of large-scale micropropagation. The main advantages of cryopreservation are the lowering of in vitro culture expenditures, needed space, contamination risk, and operator errors. However, tropical species are temperature delicate and one of the foremost challenging issues is preconditioning treatments that stimulate physiological reactions to sufficiently enhance tolerance to dehydration and cryogenic procedures. In recent years, several cryopreservation methods based on encapsulation-vitrification, droplet-vitrification, the use of aluminum cryo-plates, and cryo-mesh have been established. Combined cryo-techniques, gene/DNA conservation, as well as studies on perceiving bio-molecular events and exploring the multistage process from the beginning to end of cryopreservation are receiving more emphasis. The development of cryobiomics delivers a conceptual framework to assess the significance of cell signaling mechanisms on cellular functions, the influence of cryoinjury factors on sample viability, and the implications for genetic stability following cryo-storage. The aim of this mini-review article is to provide a succinct synthesis of the developed cryogenic procedures and their use for the storage and exchange of genetic resources of tropical and subtropical horticultural crops, particularly fruit crops and ornamental plants under the threat of extinction.

Cryopreservation of Agronomic Plant Germplasm Using Vitrification-Based Methods: An Overview of Selected Case Studies.

Numerous environmental and endogenous factors affect the level of genetic diversity in natural populations. Genetic variability is the cornerstone of evolution and adaptation of species. However, currently, more and more plant species and local varieties (landraces) are on the brink of extinction due to anthropopression and climate change. Their preservation is imperative for the sake of future breeding programs. Gene banks have been created worldwide to conserve different plant species of cultural and economic importance. Many of them apply cryopreservation, a conservation method in which ultra-low temperatures (-135 °C to -196 °C) are used for long-term storage of tissue samples, with little risk of variation occurrence. Cells can be successfully cryopreserved in liquid nitrogen (LN) when the adverse effect of ice crystal formation and growth is mitigated by the removal of water and the formation of the so-called biological glass (vitrification). This state can be achieved in several ways. The involvement of key cold-regulated genes and proteins in the acquisition of cold tolerance in plant tissues may additionally improve the survival of LN-stored explants. The present review explains the importance of cryostorage in agronomy and presents an overview of the recent works accomplished with this strategy. The most widely used cryopreservation techniques, classic and modern cryoprotective agents, and some protocols applied in crops are considered to understand which parameters provide the establishment of high quality and broadly applicable cryopreservation. Attention is also focused on the issues of genetic integrity and functional genomics in plant cryobiology.

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.

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.