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Horticulturae
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Innovative Micropropagation of Horticultural and Medicinal Plants
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Innovative Micropropagation of Horticultural and Medicinal Plants

A special issue of Horticulturae (ISSN 2311-7524). This special issue belongs to the section "Propagation and Seeds".

Deadline for manuscript submissions: 31 July 2025 | Viewed by 12949

Special Issue Editors

Dr. Biljana K. Filipović

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Guest Editor
Department of Plant Physiology, Institute for Biological Research “Siniša Stanković”—National Institute of the Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia
Interests: plant biotechnology; in vitro propagation of plants; morphogenesis in vitro; arabinogalactan proteins; specialized metabolism and metabolic engineering; plant stress responses
Dr. Milana Trifunović Momčilov

E-Mail Website
Guest Editor
Department of Plant Physiology, Institute for Biological Research “Siniša Stanković”—National Institute of the Republic of Serbia, University of Belgrade, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia
Interests: abiotic stress; endogenous phytohormone; morphogenesis in vitro; secondary metabolites; biotechnology; plant cell; tissue; organ culture

Special Issue Information

Dear Colleagues,

Micropropagation is a plant tissue culture technique used for clonal mass propagation under aseptic and controlled conditions. The main advantage of micropropagation is the rapid multiplication of high quality, disease-free plants throughout the year, starting from a small amount of initial plant tissue. Clonally propagated plants are genetically uniform, which enables desirable trait preservation of the chosen elite germplasm, and the cultivation of high-yielding chemotypes. Tissue culture technology has been applied to the large-scale production of many economically important plants, such as horticultural, silvicultural, and medicinal plants. However, the multiplication rate can be affected by various factors and limitations. This can be overcome by the development and application of new approaches to increase the production of biomass and specialized metabolites, and to reduce the overall costs, such as via novel lighting and low-cost systems, e.g., LEDs, bioreactor-based culture systems, and different elicitors (plasma treatment, etc.). Therefore, innovative tissue culture technologies can enable the development of efficient, reliable, and sustainable micropropagation protocols optimized for specific plant species. Micropropagation technology is a valuable tool for the fundamental study of basic plant developmental processes, for the screening of abiotic and biotic stresses, for the in vitro conservation of rare and endangered plant species, and for the production of specialized metabolites.

This Special Issue of Horticulturae aims to highlight innovative techniques and practices for improving the micropropagation systems of horticultural, silvicultural, and medicinal plants. Original research articles and reviews that address diverse applications of micropropagation are welcome.

Dr. Biljana K. Filipović
Dr. Milana Trifunović Momčilov
Guest Editors

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Horticulturae is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2200 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • in vitro propagation
  • somatic embryogenesis
  • organogenesis
  • plant hormones
  • secondary metabolites
  • elicitors
  • cryopreservation
  • abiotic and biotic stresses

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Published Papers (9 papers)

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Research

14 pages, 4406 KiB  
Article
Establishment of an In Vitro Micropropagation System for Cannabis sativa ‘Cheungsam’
by Sang-Cheol Baek, Sang-Yoon Jeon, Yoon-Jung Choi, Bo-Hyun Byun, Da-Hoon Kim, Ga-Ram Yu, Hyuck Kim and Dong-Woo Lim
Horticulturae 2024, 10(10), 1060; https://doi.org/10.3390/horticulturae10101060 - 3 Oct 2024
Viewed by 1205
Abstract
Cannabis has been cultivated for thousands of years for a variety of purposes, including fiber, seeds, oil, and medicinal compounds. The cannabis industry is growing rapidly because several countries have recently legalized the use of cannabis. In these countries, the industry related to [...] Read more.
Cannabis has been cultivated for thousands of years for a variety of purposes, including fiber, seeds, oil, and medicinal compounds. The cannabis industry is growing rapidly because several countries have recently legalized the use of cannabis. In these countries, the industry related to cannabinoid ingredients such as cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) is steadily increasing every year. High concentrations of cannabinoids are mainly produced in unfertilized female flowers. Maintaining plants with high cannabinoid content is essential for producing uniform substances in large quantities. This study established an in vitro micropropagation protocol that can maintain the mother plant characteristics of Cannabis sativa ‘Cheungsam’. As a result of this experiment, the shoot tips and nodes of Cannabis sativa ‘Cheungsam’ at various concentrations (0, 0.25, 0.5, 1.0 mg/L) of 2iP, BA, and mT plant growth regulators (PGRs), and all concentrations of 2iP showed better results compared to two other hormones. However, the cut surfaces turned black, and excessive hyperhydricity occurred. Based on these symptoms, activated charcoal was added to the medium with the assumption that necrosis and hyperhydricity occur due to the accumulation of reactive oxygen species (ROS). When treated with 0.5 g/L charcoal, hyperhydricity was not overcome, and there was no difference compared to the control. As a new alternative, we divided the experiments into MS (Murashige and Skoog) and DKW (Driver and Kuniyuki Walnut) medium, which were commercially available. As a result, the rate of hyperhydricity was reduced, the cut surface did not turn black, and the growth conditions were also improved. Subsequently, ½ MS medium and ½ DKW medium were treated with various concentrations of IBA alone and with combinations of IBA and NAA for rooting. As a result, ½ DKW with IBA 0.5 mg/L showed the highest rooting rate and the best root condition for Cheungsam. After 4 weeks, when considering rooted plants with a height above 5 cm that were acclimatized, the acclimatization rate reached 100%. In conclusion, the Cannabis sativa ‘Cheungsam’ plants used in this study produced healthy shoots on DKW medium containing 1.0 mg/L 2iP and 0.5 mg/L of IBA in ½ DKW medium showed the best rooting rate. Full article
(This article belongs to the Special Issue Innovative Micropropagation of Horticultural and Medicinal Plants)
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17 pages, 10831 KiB  
Article
An Efficient In Vitro Shoot Organogenesis and Comparative GC-MS Metabolite Profiling of Gaillardia pulchella Foug
by Mahima Bansal, A. Mujib, Yashika Bansal, Yaser Hassan Dewir and Nóra Mendler-Drienyovszki
Horticulturae 2024, 10(7), 728; https://doi.org/10.3390/horticulturae10070728 - 11 Jul 2024
Cited by 4 | Viewed by 918
Abstract
Gaillardia pulchella Foug. is a widely studied plant because of its high pharmacological and ornamental value. The leaves of G. pulchella were used for inducing callus and subsequent plant regeneration as it is the primary source of phytocompounds. The purpose of the present [...] Read more.
Gaillardia pulchella Foug. is a widely studied plant because of its high pharmacological and ornamental value. The leaves of G. pulchella were used for inducing callus and subsequent plant regeneration as it is the primary source of phytocompounds. The purpose of the present investigation was to formulate an in vitro propagation method for Gaillardia by using leaf explants in MS (Murashige and Skoog) medium. The best callus induction was observed on high (2.0 mg/L) α-naphthalene acetic acid (NAA) and a low (0.5 mg/L) 6-benzylaminopurine (BAP) with callus induction frequency of 91.66%. The leaf callus also demonstrated high caulogenesis ability (95.83%), with an average 5.2 shoots/callus mass at 0.5 mg/L BAP and 2.0 mg/L NAA. Indole Acetic acid (IAA) at 1.0 mg/L had the maximum rooting percentage (79.17%) with 12.4 roots per shoot. Rooted plantlets were later transferred to greenhouse conditions, showing a survivability rate of 75–80%. The physiological parameters, i.e., phenolic compounds and the flavonoids’ level, in the DPPH assay were higher in leaves obtained in vitro compared to callus formed from leaves and field-obtained (mother) leaves. Gas chromatography–mass spectrometry (GC–MS) analysis of methanol extracts of leaves (in vivo and in vitro) and leaf callus presented a wide array of compounds. In callus extract, some 34 phytocompounds were identified. Some of them were 3-hydroxy-2,3-dihydromaltol (25.39%), isoamyl acetate (11.63%), palmitic acid (11.55%), 4-methyloxazole (7.54%), and 5-methoxypyrrolidin-2-one (7.49%). Leaves derived in vivo and in vitro had 45 and 28 phytocompounds, respectively, belonging to different classes like lignans, phenols, terpenoids, alkaloids and fatty acids, etc. Those findings demonstrated that the leaf derived callus and the leaves are the potential stable source of several compounds with medicinal importance. The developed protocol may provide an alternative source of compounds without affecting wild flora. Full article
(This article belongs to the Special Issue Innovative Micropropagation of Horticultural and Medicinal Plants)
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10 pages, 1405 KiB  
Article
Metabolism of Fluorinated Topolin Cytokinins in Micropropagated Phalaenopsis amabilis
by Nino Murvanidze, Karel Doležal, Lenka Plačková and Stefaan P. O. Werbrouck
Horticulturae 2024, 10(7), 727; https://doi.org/10.3390/horticulturae10070727 - 10 Jul 2024
Viewed by 524
Abstract
Fluorinated cytokinins have emerged as promising alternatives to traditional cytokinins in Phalaenopsis plant tissue culture, offering enhanced stability and bioactivity. However, their metabolic fate and impact on endogenous cytokinin profiles remain largely unexplored. This study builds upon previous research to investigate the comparative [...] Read more.
Fluorinated cytokinins have emerged as promising alternatives to traditional cytokinins in Phalaenopsis plant tissue culture, offering enhanced stability and bioactivity. However, their metabolic fate and impact on endogenous cytokinin profiles remain largely unexplored. This study builds upon previous research to investigate the comparative metabolism of the traditional cytokinin 6-Benzylaminopurine (BA) with the successful alternatives 6-(3-fluorobenzylamino)purine (FmT) and 6-(3-fluorobenzylamino)purine 9-riboside (FmTR). Additionally, this study examines the impact of another crucial factor, the use of ventilated versus closed containers, on metabolic processes. The results revealed the distinct metabolic profiles associated with each treatment, highlighting the complex interplay between exogenous and endogenous cytokinin levels. This study is the first to investigate the effects of these stable, synthetic, and exogenous cytokinins on the naturally occurring cytokinin levels and their metabolites in micropropagated Phalaenopsis. Additionally, we proposed an alternative inactivation pathway involving the conversion of FmTR and BA to pT and pTR. These findings provide valuable insights into the intricate relationship between cytokinin metabolism and plant growth under in vitro conditions. Full article
(This article belongs to the Special Issue Innovative Micropropagation of Horticultural and Medicinal Plants)
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16 pages, 8224 KiB  
Article
Saffron In Vitro Propagation: An Innovative Method by Temporary Immersion System (TIS), Integrated with Machine Learning Analysis
by Waed Tarraf, Tolga İzgü, Özhan Şimşek, Nunzia Cicco and Carla Benelli
Horticulturae 2024, 10(5), 454; https://doi.org/10.3390/horticulturae10050454 - 30 Apr 2024
Cited by 3 | Viewed by 2196
Abstract
The propagation of Crocus sativus L. relies exclusively on corm multiplication. As underground storage organs, corms are susceptible to a wide range of pathogens, environmental stresses, and diseases, making traditional propagation methods often ineffective with the loss of valuable material. In vitro propagation [...] Read more.
The propagation of Crocus sativus L. relies exclusively on corm multiplication. As underground storage organs, corms are susceptible to a wide range of pathogens, environmental stresses, and diseases, making traditional propagation methods often ineffective with the loss of valuable material. In vitro propagation offers an alternative for the saffron culture under controlled conditions. In particular, the innovative application of the Temporary Immersion System (TIS) represents a technological advancement for enhancing biomass production with a reduction in operational costs. The current study utilized the Plantform™ bioreactor to propagate in vitro saffron corms from the ‘Abruzzo’ region (Italy), integrating machine learning models to assess its performance. The evaluation of saffron explants after 30, 60, and 90 days of culture showed a marked improvement in growth and microcorm production compared to conventional in vitro culture on semisolid medium, supported by the machine learning analysis. Indeed, the Random Forest algorithm revealed a predictive accuracy with an R2 value of 0.81 for microcorm number, showcasing the capability of machine learning models to forecast propagation outcomes effectively. These results confirm that applying TIS in saffron culture could lead to economically viable, large biomass production within a controlled environment, irrespective of seasonality. This study represents the first endeavor to use TIS technology to enhance the in vitro propagation of saffron in conjunction with machine learning, suggesting an innovative approach for cultivating high-value crops like saffron. Full article
(This article belongs to the Special Issue Innovative Micropropagation of Horticultural and Medicinal Plants)
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15 pages, 2938 KiB  
Article
Exploring the Production of Secondary Metabolites from a Halophyte Tetragonia tetragonoides through Callus Culture
by Ka Youn Lee, Do-Hyeon Nam, Yongsam Jeon, Sang Un Park, Jongki Cho, Md Ashrafuzzaman Gulandaz, Sun-Ok Chung and Geung-Joo Lee
Horticulturae 2024, 10(3), 244; https://doi.org/10.3390/horticulturae10030244 - 3 Mar 2024
Viewed by 1390
Abstract
Considering Korea’s gradual shift toward an aging society, consumer interest in compounds with physiological benefits, including antioxidant and anticancer effects, has surged. This study explored the potential of Tetragonia tetragonoides (Pall.) Kunze, commonly known as New Zealand Spinach (NZS), a halophyte with reported [...] Read more.
Considering Korea’s gradual shift toward an aging society, consumer interest in compounds with physiological benefits, including antioxidant and anticancer effects, has surged. This study explored the potential of Tetragonia tetragonoides (Pall.) Kunze, commonly known as New Zealand Spinach (NZS), a halophyte with reported health benefits, including efficacy in treating gastrointestinal diseases, high blood pressure, diabetes, and obesity. This study also introduced a novel callus culture system for NZS, allowing for the rapid in vitro production of secondary metabolites. Optimal callus induction (100%) and biomass production (0.416 g) were achieved by adding 2.0 mg·L−1 6-BA (6-Benzylaminopurine) and 0.5 mg·L−1 2,4-D (2,4-Dichlorophenoxyacetic acid) among five auxin and cytokinin combinations. Two distinct callus types, TGC [TDZ (Thidiazuron)-supplemented Green Callus] and TNYC [TDZ + NAA (Naphthalene acetic acid)-supplemented Yellow Callus], were identified, each with unique characteristics. The calli showed total phenolic and flavonoid contents comparable to those of NZS leaves grown in the greenhouse. An expression analysis of six genes (CHS, CHI, F3H, F3′H, FLS, and DFR) involved in the kaempferol biosynthesis revealed an enhanced flavonoid biosynthesis-related gene expression in TGC, emphasizing its potential for compound production. GC-MS analysis identified distinct compound profiles in TGC and TNYC, with 2,3-butanediol and succinic acid being the predominant compounds among the nine and forty-four components, respectively. These calli offer a stable supply of functional compounds and present an environmentally sustainable solution. The derived callus culture system is anticipated to contribute to the development of healthy functional foods or pharmaceuticals from halophyte NZS. Full article
(This article belongs to the Special Issue Innovative Micropropagation of Horticultural and Medicinal Plants)
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14 pages, 12242 KiB  
Article
Unlocking the Potential of In Vitro Photoautotrophy for Eryngium foetidum: Biomass, Morphophysiology, and Acclimatization
by Darlyara Reis Silva, Marion Nayon Braga Soares, Maria Cristina Rocha Silva, Mariana Costa Lima, Vitória Karla de Oliveira Silva-Moraes, Givago Lopes Alves, Anyela Marcela Ríos-Ríos, Aldilene da Silva Lima, Marcos Vinícius Marques Pinheiro, Thais Roseli Corrêa, Diego Silva Batista, Fábio Afonso Mazzei Moura de Assis Figueiredo, Fabrício de Oliveira Reis, Tiago Massi Ferraz and Sérgio Heitor Sousa Felipe
Horticulturae 2024, 10(1), 107; https://doi.org/10.3390/horticulturae10010107 - 22 Jan 2024
Viewed by 1748
Abstract
Eryngium foetidum L., a biennial herb with diverse applications in food and traditional medicine, holds economic and pharmacological significance. Given its growing commercial interest, implementing biotechnological approaches like plant tissue culture is vital for sustainable propagation and metabolite production. In this study, we [...] Read more.
Eryngium foetidum L., a biennial herb with diverse applications in food and traditional medicine, holds economic and pharmacological significance. Given its growing commercial interest, implementing biotechnological approaches like plant tissue culture is vital for sustainable propagation and metabolite production. In this study, we investigated the in vitro photoautotrophic potential of Eryngium foetidum, examining growth, chlorophyll a fluorescence, photosynthetic pigments, and anatomical features under sucrose concentrations (0 and 30 g L−1) and gas exchange rate (14 and 25 μL L−1 s−1 CO2). Acclimatization and survival rates of plants after ex vitro transfer were also assessed. Eryngium foetidum exhibited robust growth in both photoautotrophic and photomixotrophic conditions, with natural ventilation significantly enhancing plant development. Chlorophyll a fluorescence and photosynthetic performance were influenced by sucrose and gas exchange, highlighting the importance of these factors in plant micropropagation. Moreover, the species demonstrated remarkable plasticity during acclimatization, with high survival rates and rapid inflorescence development. The research provides valuable insights into optimizing in vitro cultivation conditions for Eryngium foetidum, emphasizing the potential for large-scale clonal propagation and exploring secondary metabolites. The observed phenotypic plasticity underscores the adaptability of the species to diverse environments. These biotechnological strategies open avenues for future studies, including the application of elicitors for enhanced secondary metabolite production. Full article
(This article belongs to the Special Issue Innovative Micropropagation of Horticultural and Medicinal Plants)
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9 pages, 1997 KiB  
Communication
Optimization of In Vitro Regeneration of Pinus peuce (Gris.)
by Dragana Stojičić, Snežana Budimir, Vlado Čokeša and Branka Uzelac
Horticulturae 2024, 10(1), 97; https://doi.org/10.3390/horticulturae10010097 - 19 Jan 2024
Viewed by 1257
Abstract
Pinus peuce (Macedonian pine) is considered a valuable ornamental tree that is frequently planted in parks and gardens, especially in Western Europe. This endemic pine is one of the most valuable conifer species in its native range, which currently consists of only two [...] Read more.
Pinus peuce (Macedonian pine) is considered a valuable ornamental tree that is frequently planted in parks and gardens, especially in Western Europe. This endemic pine is one of the most valuable conifer species in its native range, which currently consists of only two disjunct populations restricted to small mountainous areas of the Balkans and is listed as a near-threatened species. The reproduction of Macedonian pine by seed is limited, so in vitro propagation methods have emerged as a promising tool for large-scale propagation. The objective of this study was to develop an improved system for the micropropagation of P. peuce from juvenile plant material using a short-term liquid cytokinin pulse. For that, explants derived from 4-week-old seedlings were pulse-treated with different concentrations of N6-benzyladenine (BA) for 1 or 2 h to stimulate the induction of axillary buds. The highest axillary shoot formation was achieved with 222 µM BA pulse treatment, with an average number of ~six shoots per explant. Elongated shoots (≥10 mm) were detached from the explants and pulse-treated with 0.27 or 1.08 mM α-naphthaleneacetic acid (NAA) or 0.25 or 0.98 mM indole-3-butyric acid (IBA) for 1 or 2 h. IBA was more effective than NAA and led to a maximum rooting percentage (up to 40%) and the highest number of acclimatized plants (15–20%). Rooted plants were successfully transferred to ex vitro conditions. Full article
(This article belongs to the Special Issue Innovative Micropropagation of Horticultural and Medicinal Plants)
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8 pages, 4071 KiB  
Communication
Histochemical Localization of Alkaloids in the Bulbs of In Vitro-Regenerated Snake’s Head Fritillary (Fritillaria meleagris L.): The Effect of a Temperature Regime
by Marija Marković, Milana Trifunović-Momčilov, Olga Radulović, Danijela M. Paunović, Dragana D. Antonić Reljin, Slađana Jevremović and Branka Uzelac
Horticulturae 2024, 10(1), 17; https://doi.org/10.3390/horticulturae10010017 - 22 Dec 2023
Viewed by 1175
Abstract
The genus Fritillaria is characterized by the production of alkaloids, primarily of the isosteroidal type. The aim of this preliminary study was to perform a histolocalization screening aimed at revealing the presence of alkaloids in the bulbs of F. meleagris, cultured in [...] Read more.
The genus Fritillaria is characterized by the production of alkaloids, primarily of the isosteroidal type. The aim of this preliminary study was to perform a histolocalization screening aimed at revealing the presence of alkaloids in the bulbs of F. meleagris, cultured in vitro under different growth regimes and at different developmental stages, as a powerful tool to identify preferable in vitro plant material for alkaloid isolation. Histochemical localization of alkaloids was performed using Wagner’s and Dragendorff’s reagents in fresh sections of bulbs cultured at 24 °C or 7 °C for 4 weeks, as well as those cultured at 24 °C following the 4-week chilling treatment, which were sampled at the beginning of sprouting. A positive reaction was observed with both reagents and was particularly intense in cold-treated bulbs. Alkaloids were mainly distributed in the bulbs and partly in the sprouts of the in vitro grown F. meleagris. The most intense staining, indicative of high alkaloid content, was observed in the bulb scales of pre-chilled bulbs that sprouted at 24 °C, rendering them preferable as in vitro plant material for alkaloid isolation. The results suggest that alkaloid production in the bulbs of F. meleagris can be improved by manipulating growth in the microenvironment of in vitro cultures, in order to meet the increasing industrial demand for medicinally or commercially important metabolites used as traditional medicines and herbal remedies. Full article
(This article belongs to the Special Issue Innovative Micropropagation of Horticultural and Medicinal Plants)
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15 pages, 6072 KiB  
Article
Polyethylene Glycol (PEG)-Induced Dehydration Alters Enzymatic and Non-Enzymatic Components of the Antioxidant Defense System in Nepeta nervosa Royle ex Bentham
by Jasmina Nestorović Živković, Neda Aničić, Dragana Matekalo, Marijana Skorić, Biljana Filipović, Tijana Marković and Slavica Dmitrović
Horticulturae 2023, 9(12), 1277; https://doi.org/10.3390/horticulturae9121277 - 28 Nov 2023
Cited by 1 | Viewed by 1436
Abstract
Plants have developed a variety of defense mechanisms that allow them to recognize and respond to specific stressors by triggering complex signaling networks that cause appropriate biochemical changes to overcome the stress. In this study, we subjected in vitro grown N. nervosa plants [...] Read more.
Plants have developed a variety of defense mechanisms that allow them to recognize and respond to specific stressors by triggering complex signaling networks that cause appropriate biochemical changes to overcome the stress. In this study, we subjected in vitro grown N. nervosa plants to PEG-induced dehydration stress for 1 day (1DPT), 3 days (3DPT) and 6 days (6DPT). Our study investigated antioxidant enzyme activities, including catalase (CAT), peroxidase (POX) and superoxide dismutase (SOD), unveiling dynamic responses to PEG-induced water stress. CAT levels increased initially (1DPT) but declined with prolonged treatment; while POX activity significantly increased at 3DPT and 6DPT; and SOD, particularly the Mn-SOD3 isoform, demonstrated a substantial increase, emphasizing its role in the enzymatic free-radical scavenging activity. Furthermore, examination of the phenolic acid content revealed that rosmarinic acid (RA) was the predominant phenolic compound, followed by chlorogenic acid (CHLA), while ferulic acid (FA) and caffeic acid (CAFFA) were present in lower concentrations. Notably, PEG-induced dehydration significantly boosted RA content in N. nervosa plants at 3DPT. This increase highlights the plant’s response to oxidative stress conditions and its role in non-enzymatic antioxidant defense mechanisms. These findings significantly contribute to our comprehension of N. nervosa’s adaptive strategies under PEG-induced dehydration stress, offering valuable insights into plant stress physiology within industrial and agricultural contexts. Full article
(This article belongs to the Special Issue Innovative Micropropagation of Horticultural and Medicinal Plants)
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