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Tissue Culture and Micropropagation Techniques of Horticultural Crops

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

Deadline for manuscript submissions: 25 June 2025 | Viewed by 11921

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Special Issue Editors

Dr. Tolga Izgü

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National Research Council of Italy (CNR) IBE/Institute of BioEconomy Polo Scientifico, via Madonna del Piano n. 10, Sesto Fiorentino, 50019 Firenze, Italy
Interests: plant tissue culture; micropropagation; somatic embryogenesis; temporary immersion system; cryopreservation

Dr. Özhan Şimşek

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Horticulture Department, Agriculture Faculty, Erciyes University, Kayseri 38030, Türkiye
Interests: plant tissue culture; micropropagation; temporary immersion system; molecular markers; gene expression

Prof. Dr. Maria Angeles Pedreño

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Department of Plant Biology, Faculty of Biology, University of Murcia, Campus de Espinardo, 30100 Murcia, Spain
Interests: plant biotechnology; antioxidant secondary metabolites; plant bioactive molecules
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Dr. Haiying Liang

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Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA
Interests: improving economically important woody plant species using genomics and biotechnology plant; plant hormones

Special Issue Information

Dear Colleagues,

As the demands for enhanced efficiency, productivity, and sustainability in horticultural crop production continue to grow, we are delighted to announce our Special Issue focused on "Tissue Culture and Micropropagation Techniques of Horticultural Crops".

This dedicated issue aims to showcase cutting-edge research and advancements in various areas such as plant tissue culture, micropropagation, organogenesis, gynogenesis, androgenesis, double haploid production, somatic embryogenesis, meristem culture, embryo culture, callus culture, protoplast culture, the application of temporary immersion system (TIS), etc., within the realm of horticultural crops.

We invite contributions from researchers and experts in the field, encouraging the submission of innovative studies, methodologies, and breakthroughs that explore the transformative potential of these techniques in horticultural crop production.

Additionally, we are interested in practical and applied research, spanning from new insights into the in vitro propagation process to the conservation of horticultural crops through methods such as Slow Growth Storage (SGS) and Cryopreservation.

By bringing together a diverse range of perspectives, this Special Issue aims to offer a comprehensive overview of the latest developments in tissue culture and micropropagation. We anticipate that this collaborative effort will facilitate knowledge exchange and foster a sense of community within the horticultural research community.

We eagerly await your valuable contributions to propel the frontiers of horticultural science.

Dr. Tolga Izgü
Dr. Özhan Şimşek
Prof. Dr. Maria Angeles Pedreño
Dr. Haiying Liang
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

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 regeneration
shoot culture
plant tissue culture
micropropagation
cryopreservation

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Published Papers (8 papers)

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Research

22 pages, 5876 KiB

Open AccessArticle

Bioreactor-Based Liquid Culture and Production of Konjac Micro-Corm

by Yuqi Sun, Xian Sun, Yufan Pan, Changbin Liu, Lingye Su and Zongshen Zhang

Horticulturae 2025, 11(3), 235; https://doi.org/10.3390/horticulturae11030235 - 22 Feb 2025

Viewed by 448

Abstract

Konjac (Amorphophallus konjac K. Koch) has numerous health benefits, but traditional propagation is hindered by long growth periods and soil-borne diseases. This study developed a novel cell liquid culture system to directly produce micro-corms of konjac for large-scale production. The results demonstrated significant improvements in bud induction and rooting compared to solid culture. Under MS + 1.0 mg/L 6-BA + 0.5 mg/L NAA, the induced buds per culture vessel and final fresh weight were 24.87 ± 0.06 and 6.64 ± 0.12 g, respectively, 1.95 and 1.67 times higher than those in solid culture. Rooting experiments showed that 1/2 MS + 0.5 mg/L IBA + 1.0 mg/L NAA resulted in a root length of 25.23 ± 0.04 cm and 18.12 ± 0.01 roots per vessel. Using a 5 L bioreactor for micro-corm induction led to a 2.51-fold increase in fresh weight (52.67 ± 0.01 g) after 31 days, with glucomannan production reaching 0.48 g/g _{(fresh weight)}. The optimized culture system also significantly reduced the propagation time and increased the yield of healthy micro-corms. Bioreactor-based cultivation effectively enhances konjac induction efficiency and shortens breeding time, making it a promising approach for commercial production of konjac micro-corms and potentially improving the economic viability of konjac farming. Full article

(This article belongs to the Special Issue Tissue Culture and Micropropagation Techniques of Horticultural Crops)

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20 pages, 4958 KiB

Open AccessArticle

In Vitro Production of Plantlets and Microrhizomes, Genetic Fidelity Assessment, and Metabolic Profiling of Boesenbergia rotunda (L.) Mansf.

by Kankamon Thepthong and Supanath Kanjanawattanawong

Horticulturae 2025, 11(2), 186; https://doi.org/10.3390/horticulturae11020186 - 8 Feb 2025

Viewed by 821

Abstract

Fingerroot (Boesenbergia rotunda (L.) Mansf.) is valued for its therapeutic benefits, both in Thailand and internationally. This study optimized in vitro propagation and induced microrhizomes (MRZ) to produce cleaned plantlets to support organic farming using disease-free plantlets, which is crucial for preventing and eradicating diseased plantlets, reducing the use of chemicals, and alternative approaches to enhancing phytochemical diversity. Shoots cultured on ½-strength MS medium with 1 mg L⁻¹ of 6-benzylaminopurine (BAP) showed the highest shoot formation (69%) and shoot multiplication (3.45 ± 0.29 shoots per explant). Plantlets acclimatized in peat moss or a peat moss–coconut coir (1:1) mixture achieved a 100% survival rate. Genetic fidelity was confirmed using SSR markers, showing genetic consistency with the mother plant. The MRZ formation was the highest (98.33%) under white LED light with 30 g L⁻¹ of sucrose. Nuclear magnetic resonance (NMR) analysis in MRZ revealed aspartate, a precursor to pinocembrin and pinostrobin. Additionally, nine unique metabolites not previously identified in fingerroot were detected in the MRZ, suggesting some potential in novel therapeutic applications. These findings support the development of efficient micropropagation methods and highlight MRZ as a source of diverse bioactive compounds, contributing to the medicinal value of B. rotunda in sustainable and large-scale production. Full article

(This article belongs to the Special Issue Tissue Culture and Micropropagation Techniques of Horticultural Crops)

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18 pages, 817 KiB

Open AccessArticle

Optimizing Microclonal Propagation of Red Currant Cultivars: The Role of Nutrient Media, Sterilizers, and LED Lighting in Plant Adaptation

by Olga Panfilova, Nelli Ryago, Gabrijel Ondrasek, Inna V. Knyazeva, Ibrahim Kahramanoğlu, Oksana Vershinina, Mikhail Tsoy, Andrey Yu Izmailov and Alexey S. Dorokhov

Horticulturae 2025, 11(2), 149; https://doi.org/10.3390/horticulturae11020149 - 1 Feb 2025

Viewed by 643

Abstract

This study focuses on refining in vitro propagation protocols for red currant cultivars of the Ribes genus and evaluating the role of LED lighting in the adaptation of microplants. The cultivars ‘Red Lake’, ‘Englische Grosse Weisse’, ‘Marmeladnitsa’, and ‘Podarok Leta’ were successfully introduced into in vitro culture during their dormancy phase using 0.1% HgCl₂ as a sterilizing agent. The period of spring introduction is not very efficient in connection with the intensive development of saprophytic microflora and weak morphogenesis microplants. Using 0.01% C₉H₉HgNaO₂S sterilizer resulted in a decrease in the necrosis percentage, but an increase in mold proportion. The preparation of the plants with 12% H₂O₂ was considered environmentally not effective enough to obtain a large number of healthy microplants. The use of 12% H₂O₂ resulted in increased necrosis rates by 24.76% compared to 0.01% C₉H₉HgNaO₂S and 0.1% HgCl₂ sterilizers. The variety specificity of Ribesia plants in terms of the content of MS and LF nutrient media components was determined by the survival rate of explants, the formation of additional micro-shoots, and the morphological development. The MS medium with the addition of 1 mg∙L⁻¹ BAP decreased the percentage of mold and necrosis infection and provided a high percentage of viable plants with optimal growth and reproduction rate. In contrast, the LF medium with the same concentration of 6-BAP resulted in poor explant quality and leaf chlorosis at later stages. The study also investigated the effects of different LED light spectra on morphological and physiological traits. For ‘Red Lake’ and ‘Englische Grosse Weisse’, RWUV-A (625–740 nm) lighting enhanced biomass and chlorophyll (Chl a and Chl a + b) accumulation, while the White (W) spectrum benefited ‘Podarok Leta’. Conversely, the RW spectrum with minimal green and no ultraviolet light restricted growth and photosynthetic pigment accumulation across all cultivars, promoting compact plant structures. The RWUV-A lighting condition resulted in the highest NDVI values across all cultivars, indicating an improved physiological status and biomass accumulation. These findings underscore the importance of refining the microclonal reproduction protocols for Ribesia subgenus representatives, emphasizing the genotype-specific light modulation during the proliferation stage. The study highlights the utility of the MS medium and tailored light conditions in enhancing the effectiveness of propagation techniques for producing high-quality planting material. Full article

(This article belongs to the Special Issue Tissue Culture and Micropropagation Techniques of Horticultural Crops)

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15 pages, 13154 KiB

Open AccessArticle

The First Protocol for In Vitro Propagation of Kalanchoe beharensis Through Adventitious Shoots, a Preliminary Study

by Ignacio Moreno-García, Victoria Rodríguez, Begoña García-Sogo, Cristhian Ventura, Vicente Moreno and Benito Pineda

Horticulturae 2024, 10(12), 1379; https://doi.org/10.3390/horticulturae10121379 - 23 Dec 2024

Viewed by 921

Abstract

Kalanchoe beharensis, a vulnerable species according to the International Union for Conservation of Nature, is highly prized for its ornamental value and medicinal properties. Therefore, an efficient methodology to propagate this ecologically significant species would be of particular interest. The propagation of K. beharensis has traditionally been achieved by seed or cuttings, but these methods are limited in efficiency. Micropropagation provides a more efficient and controlled alternative by enabling the in vitro production of numerous plants in a small space and in a short period of time. Despite its advantages, no micropropagation protocol for K. beharensis has been reported in the literature. In this study, we report an efficient in vitro regeneration protocol for K. beharensis. In order to implement this, we evaluated the morphogenetic response of leaf and root explants in media supplemented with auxins, cytokinins, or a combination of both growth regulators. Surprisingly, the best results were observed in indole-3-acetic acid-supplemented media. Adventitious shoots were rooted in either hormone-free or auxin-supplemented media, with indole-3-acetic acid yielding the best results. Rooted plants were acclimatized in the greenhouse, achieving over 80% survival during acclimatization. This protocol improves multiplication rate, space utilization, and uniformity, providing a viable alternative to conventional propagation methods. Full article

(This article belongs to the Special Issue Tissue Culture and Micropropagation Techniques of Horticultural Crops)

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21 pages, 8822 KiB

Open AccessArticle

Are Cytological and Morphological Analyses Sufficient in Ploidy Determination of Watermelon Haploid Plants?

by Ecem Kara, Hatıra Taşkın, Şenay Karabıyık, İlknur Solmaz, Nebahat Sarı, Tolga Karaköy and Gökhan Baktemur

Horticulturae 2024, 10(8), 818; https://doi.org/10.3390/horticulturae10080818 - 2 Aug 2024

Viewed by 1499

Abstract

Watermelon (Citrullus lanatus L. Thunb. Matsum. and Nakai) is a vegetable species with high water content that is rich in lycopene and refreshing; therefore, breeding studies are carried out intensively to develop new varieties. The breeding period in these plants can be shortened with the use of the haploidization technique, and determining the ploidy levels of plants in haploidization studies is very important. In this study, it was examined whether morphological and cytological analyses would be sufficient for ploidy determination of haploid plants obtained by ovary culture in watermelon. With this aim, the stomatal, morphological and cytological characteristics were determined, taken from 15 haploid and 19 double haploid plants. The ploidy level of the plants was detected by flow cytometry before these analyses. In this study, “Principal Component Analysis” was performed based on average values to analyze the structure of the relationship between the parameters examined. It is thought that stomatal features can be used as morphological markers in determining the ploidy levels of plants. The differences obtained from the study results may help to develop effective strategies in determining the ploidy levels of plants. Full article

(This article belongs to the Special Issue Tissue Culture and Micropropagation Techniques of Horticultural Crops)

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21 pages, 6701 KiB

Open AccessArticle

Preserving Nature’s Treasure: A Journey into the In Vitro Conservation and Micropropagation of the Endangered Medicinal Marvel—Podophyllum hexandrum Royle

by Zahoor Khan, Bushra Khan, Syed Tanveer Shah, Javaid Iqbal, Abdul Basit, Muhammad Suleman Khan, Waleed Iqbal, Mohamed Farouk Elsadek, Aftab Jamal, Mohammad Ajmal Ali and Domenico Prisa

Horticulturae 2024, 10(8), 809; https://doi.org/10.3390/horticulturae10080809 - 30 Jul 2024

Viewed by 2403

Abstract

Podophyllum hexandrum Royle, also known as Podophyllum emodi Wall, holds significant ecological, ornamental, and medicinal values. However, it has become endangered due to overexploitation, prolonged seed dormancy, slow natural regeneration, and climate change. This study developed an efficient in vitro protocol for callogenesis and micropropagation of P. hexandrum to conserve germplasm in in vitro conditions. Callus formation from various plant parts, including the leaf, stem, rhizome, radicle, and cotyledon, was induced using Murashige and Skoog (MS) medium supplemented with different plant growth regulators. The combination of benzyladenine at 1 mg L⁻¹ and 4-dichlorophenoxy acetic acid at 3 mg L⁻¹ was optimal for biomass production, yielding 215.88 ± 0.31 mg, with growth per gram at 8.32 ± 0.32 and a growth rate of 13.62 ± 0.25 mg/day on MS medium. For shoot proliferation, benzyladenine (3.5 mg L⁻¹) and naphthalene acetic acid (0.5 mg L⁻¹) combined with activated charcoal showed the highest shoot induction percentage per explant. For shoot regeneration from calluses, 6-benzylaminopurine (0.5 mg L⁻¹) and thidiazuron (2 mg L⁻¹) were most effective, producing superior shoot length, number of regenerations, and regeneration percentage. Root induction was successful with α-naphthalene acetic acid supplementation (0.5 to 1.5 mg L⁻¹) in MS medium, resulting in the highest number per explant (4.08 ± 0.08), length (5.45 ± 0.15 cm), and rooting rate (87.00 ± 1.66%) of roots in plantlets. Subculturing for callus culture was performed every 28 days for up to four subcultures to prevent nutrient depletion and toxic metabolite accumulation, ensuring tissue health and viability. Continuous subculturing of callus on MS medium maintained healthy P. hexandrum germplasm in vitro. Overall, this micropropagation protocol provides a rapid system for conserving P. hexandrum germplasm. Full article

(This article belongs to the Special Issue Tissue Culture and Micropropagation Techniques of Horticultural Crops)

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11 pages, 1928 KiB

Open AccessArticle

In Vitro Micropropagation of the Vulnerable Chilean Endemic Alstroemeria pelegrina L.

by Francesca Guerra, Ricardo Cautín and Mónica Castro

Horticulturae 2024, 10(7), 674; https://doi.org/10.3390/horticulturae10070674 - 25 Jun 2024

Cited by 1 | Viewed by 1483

Abstract

The Chilean rhizomatous geophyte Alstroemeria pelegrina (A. pelegrina), a species endemic to the Coquimbo (31°45′ S) and Valparaíso (33°12′ S) regions, is currently classified as vulnerable and experiencing population decline due to loss of habitat. This heightened threat underscores the need to develop effective mass propagation techniques to support its conservation efforts. Consequently, the present study aimed to establish an optimized in vitro propagation protocol tailored specifically for A. pelegrina to facilitate its large-scale propagation and promote the preservation of this vulnerable geophyte. In our experiment, explants obtained from in vitro germination were used. These explants were then cultured on a Murashige and Skoog (MS) medium solidified with agar (6 g L⁻¹) and supplemented with 2.22 μM 6-Benzylaminopurine (BAP) and 30 g L⁻¹ sucrose. The results indicated an average of 4.6 new shoots produced per initial explant. The implementation of light-emitting diode (LED) illumination with a red:blue ratio of 3:1 and 2.22 μM BAP resulted in an average shoot length of 6.2 cm. For rhizome induction, the addition of either 5.37 μM 1-naphthaleneacetic acid (NAA) or 5.37 μM indole-3-butyric acid (IBA) resulted in average rhizome lengths of 1.9 cm and 1.7 cm, respectively, with fresh weights of the vitroplants ranging from 2.9 g to 3.1 g, and rooting percentages between 100% and 96%, respectively. Notably, the survival rate of rooted plants obtained through in vitro propagation was 90% after a 4-week acclimatization period in a cold greenhouse, indicating the efficacy of the developed protocol for mass propagation and conservation efforts of this vulnerable geophyte species. Full article

(This article belongs to the Special Issue Tissue Culture and Micropropagation Techniques of Horticultural Crops)

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16 pages, 4472 KiB

Open AccessArticle

Assessing Cadmium Stress Resilience in Myrtle Genotypes Using Machine Learning Predictive Models: A Comparative In Vitro Analysis

by Mehmet Tütüncü, Musab A. Isak, Tolga İzgü, Dicle Dönmez, Yıldız Aka Kaçar and Özhan Şimşek

Horticulturae 2024, 10(6), 542; https://doi.org/10.3390/horticulturae10060542 - 22 May 2024

Cited by 2 | Viewed by 1667

Abstract

This study investigated the effects of cadmium (Cd) stress on the micropropagation and rooting dynamics of two myrtle (Myrtus communis L.) genotypes with different fruit colors under controlled in vitro conditions. We evaluated the response of these genotypes to varying concentrations of Cd (0, 100, 200, 300, 400, and 500 µM) to determine dose-dependent effects on plantlet multiplication and root formation. Our results demonstrate that the white-fruited (WF) genotype exhibits greater resilience than the black-fruited (BF) genotype across all concentrations, maintaining higher multiplication rates and shoot heights. For instance, the multiplication rate at 100 µM Cd was highest for WF at 6.73, whereas BF showed the lowest rate of 1.94 at 500 µM. Similarly, increasing Cd levels significantly impaired root length and the number of roots for both genotypes, illustrating the detrimental impact of Cd on root system development. Additionally, this study incorporated machine learning (ML) models to predict growth outcomes. The multilayer perceptron (MLP) model, including random forest (RF) and XGBoost, was used to analyze the data. The MLP model performed notably well, demonstrating the potential of advanced computational tools in accurately predicting plant responses to environmental stress. For example, the MLP model accurately predicted shoot height with an R² value of 0.87 and root length with an R² of 0.99, indicating high predictive accuracy. Overall, our findings provide significant insights into the genotypic differences in Cd tolerance and the utility of ML models in plant science. These results underscore the importance of developing targeted strategies to enhance plant resilience in contaminated environments. Full article

(This article belongs to the Special Issue Tissue Culture and Micropropagation Techniques of Horticultural Crops)

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