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Micropropagation and Cryopreservation of Plants

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A special issue of Plants (ISSN 2223-7747). This special issue belongs to the section "Plant Physiology and Metabolism".

Deadline for manuscript submissions: closed (30 June 2023) | Viewed by 19575

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Special Issue Editor

Dr. Abel Piqueras Castilo

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Guest Editor

Department of Fruit Breeding, CEBAS (CSIC), Campus Universitario de Espinardo, 30100 Murcia, Spain
Interests: micropropagation; cryopreservation; somatic embryogenesis; oxidative stress; antioxidant activity

Special Issue Information

Dear Colleagues,

Micropropagation is, at the present moment, the most efficient and productive biotechnological application of plant tissue culture. Micropropagation has enlarged its field of application to many sectors related to plant production, conservation of selected genotypes, preservation of plant biodiversity, and the production of transgenic plants. Although micropropagation can be considered a well-established technology, there are several aspects that could be improved and will require more research for the quality control during the different stages of the process and the genetic stability of the plant material. This Special Issue will explore the most recent advances in micropropagation and cryopreservation as the required innovative tools to achieve new opportunities in crop improvement, productivity, and food security.

Dr. Abel Piqueras Castilo
Guest Editor

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.

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Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 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

micropropagation
cryopreservation
somatic embryogenesis
clonal multiplication
long term-conservation
post-thawing regrowth
ex situ conservation
longevity

Published Papers (6 papers)

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Research

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25 pages, 3402 KiB

Open AccessArticle

Cryopreservation of Duckweed Genetic Diversity as Model for Long-Term Preservation of Aquatic Flowering Plants

by Anton Peterson, Olena Kishchenko, Markus Kuhlmann, Henning Tschiersch, Joerg Fuchs, Natalia Tikhenko, Ingo Schubert and Manuela Nagel

Plants 2023, 12(18), 3302; https://0-doi-org.brum.beds.ac.uk/10.3390/plants12183302 - 18 Sep 2023

Cited by 1 | Viewed by 1032

Abstract

Vegetatively propagating aquatic angiosperms, the Lemnaceae family (duckweeds) represents valuable genetic resources for circular bioeconomics and other sustainable applications. Due to extremely fast growth and laborious cultivation of in vitro collections, duckweeds are an urgent subject for cryopreservation. We developed a robust and fast DMSO-free protocol for duckweed cryopreservation by vitrification. A single-use device was designed for sampling of duckweed fronds from donor culture, further spin-drying, and subsequent transferring to cryo-tubes with plant vitrification solution 3 (PVS3). Following cultivation in darkness and applying elevated temperatures during early regrowth stage, a specific pulsed illumination instead of a diurnal regime enabled successful regrowth after the cryopreservation of 21 accessions of Spirodela, Landoltia, Lemna, and Wolffia genera, including interspecific hybrids, auto- and allopolyploids. Genome size measurements revealed no quantitative genomic changes potentially caused by cryopreservation. The expression of CBF/DREB1 genes, considered as key factors in the development of freezing tolerance, was studied prior to cooling but was not linked with duckweed regrowth after rewarming. Despite preserving chlorophyll fluorescence after rewarming, the rewarmed fronds demonstrated nearly zero photosynthetic activity, which did not recover. The novel protocol provides the basis for future routine application of cryostorage to duckweed germplasm collections, saving labor for in vitro cultivation and maintaining characterized reference and mutant samples. Full article

(This article belongs to the Special Issue Micropropagation and Cryopreservation of Plants)

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15 pages, 3068 KiB

Open AccessArticle

Tracking Permeation of Dimethyl Sulfoxide (DMSO) in Mentha × piperita Shoot Tips Using Coherent Raman Microscopy

by Heidi D. Kreckel, Fionna M. D. Samuels, Remi Bonnart, Gayle M. Volk, Dominik G. Stich and Nancy E. Levinger

Plants 2023, 12(12), 2247; https://0-doi-org.brum.beds.ac.uk/10.3390/plants12122247 - 8 Jun 2023

Cited by 1 | Viewed by 1769

Abstract

Cryopreservation has emerged as a low-maintenance, cost-effective solution for the long-term preservation of vegetatively propagated crops. Shoot tip cryopreservation often makes use of vitrification methods that employ highly concentrated mixtures of cryoprotecting agents; however, little is understood as to how these cryoprotecting agents protect cells and tissues from freezing. In this study, we use coherent anti-Stokes Raman scattering microscopy to directly visualize where dimethyl sulfoxide (DMSO) localizes within Mentha × piperita shoot tips. We find that DMSO fully penetrates the shoot tip tissue within 10 min of exposure. Variations in signal intensities across images suggest that DMSO may interact with cellular components, leading to its accumulation in specific regions. Full article

(This article belongs to the Special Issue Micropropagation and Cryopreservation of Plants)

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15 pages, 2562 KiB

Open AccessEditor’s ChoiceArticle

Micropropagation of Endemic Endangered Taxa of the Italian Flora: Adenostyles alpina subsp. macrocephala (Asteraceae), as a Case Study

by Valeria Gianguzzi, Giulio Barone, Emilio Di Gristina, Francesco Sottile and Gianniantonio Domina

Plants 2023, 12(7), 1530; https://0-doi-org.brum.beds.ac.uk/10.3390/plants12071530 - 1 Apr 2023

Cited by 4 | Viewed by 1595

Abstract

The conservation of endangered, rare, and endemic plant species is based on in situ and ex situ conservation strategies. When in situ conservation alone is not sufficient to guarantee the survival of the species, ex situ techniques are adopted in support. This study aimed to develop an efficient micropropagation protocol for Adenostyles by evaluating the effect of different plant growth regulators on leaf explants. Adenostyles alpina subsp. macrocephala (Asterace) is a perennial herbaceous plant endemic to Calabria (Southern Italy). The genus Adenostyles includes three species confined to the mountains of the Mediterranean and southern Europe. For callus induction, media supplemented with different concentrations of Benzylaminopurine (BAP) (0.5, 1, 2, and 3 mg L⁻¹), Naphthaleneacetic Acid (NAA) (1 mg L⁻¹), and 2,4-Dichlorophenoxyacetic Acid (2,4-D) (1 mg L⁻¹) were tested. Shoot regeneration and proliferation were obtained in media supplemented with BAP (1, 2, and 3 mg L⁻¹) and NAA (1 mg L⁻¹). Root induction was obtained in media supplemented with IBA (0.25, 0.50, and 1 mg L⁻¹) and NAA (0.25, 0.50, and 1 mg L⁻¹). Statistically significant differences in callus induction and shoot regeneration were observed between the various media tested. The medium containing Murashige and Skoog (MS) supplemented with 3 mg L⁻¹ of BAP and 1 mg L⁻¹ of NAA showed the highest percentage of callus induction and increased shoot regeneration. The regenerated shoots showed more effective root induction in the hormone-free MS medium and in the presence of Indole-3-Butyric Acid (IBA) at concentrations of 0.25, 0.50, and 1 mg L⁻¹. These results can be used as a basis for the preparation of a micropropagation protocol for different taxa of Adenostyles, as well as other species of Asteraceae specialized to the Mediterranean mountain habitat. Full article

(This article belongs to the Special Issue Micropropagation and Cryopreservation of Plants)

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19 pages, 4346 KiB

Open AccessArticle

Transcriptome Profiling during Sequential Stages of Cryopreservation in Banana (Musa AAA cv Borjahaji) Shoot Meristem

by Chaw Su Su Htwe, Subramani Rajkumar, Pooja Pathania and Anuradha Agrawal

Plants 2023, 12(5), 1165; https://0-doi-org.brum.beds.ac.uk/10.3390/plants12051165 - 3 Mar 2023

Cited by 2 | Viewed by 2163

Abstract

Cryopreservation approaches have been implemented in gene banks as a strategy to back up plant genetic resource collections that are vegetatively propagated. Different strategies have been employed to effectively cryopreserve plant tissue. There is little information on the cellular processes and molecular adjustments that confer resilience to the multiple stresses imposed during a cryoprotocol. In the present work, the cryobionomics of banana (Musa sp.), a non-model species, was investigated through the transcriptomic approach using RNA-Seq. Proliferating meristems of in vitro explants (Musa AAA cv ‘Borjahaji’) were cryopreserved using the droplet-vitrification technique. Transcriptome profiling analysis of eight cDNA libraries including the bio-replicates for T0 (stock cultures (control tissue), T1 (high sucrose pre-cultured), T2 (vitrification solution-treated) and T3 (liquid nitrogen-treated) meristem tissues was carried out. The raw reads obtained were mapped with a Musa acuminata reference genome sequence. A total of 70 differentially expressed genes (DEGs) comprising 34 upregulated and 36 downregulated were identified in all three phases as compared to control (T0). Among the significant DEGs (>log FC 2.0), during sequential steps, 79 in T1, 3 in T2 and the 4 in T3 were upregulated and 122 in T1, 5 in T2 and 9 in T3 were downregulated. Gene ontology (GO) enrichment analysis showed that these significant DEGs were involved in the upregulation of biological process (BP-170), cellular component (CC-10) and molecular function (MF-94) and downregulation of biological process (BP-61), cellular component (CC-3) and molecular function (MF-56). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEGs were involved in the biosynthesis of secondary metabolites, glycolysis/gluconeogenesis, MAPK signaling, EIN 3-lke 1 protein, 3-ketoacy-CoA synthase 6-like, and fatty acid elongation during cryopreservation. For the first time, a comprehensive transcript profiling during four stages of cryopreservation in banana were carried out, which will pave the way for devising an effective cryopreservation protocol. Full article

(This article belongs to the Special Issue Micropropagation and Cryopreservation of Plants)

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14 pages, 10358 KiB

Open AccessArticle

IBA Delivery Technique and Media Salts Affected In Vitro Rooting and Acclimatization of Eight Prunus Genotypes

by John D. Lawson, William C. Bridges and Jeffrey W. Adelberg

Plants 2023, 12(2), 289; https://0-doi-org.brum.beds.ac.uk/10.3390/plants12020289 - 7 Jan 2023

Cited by 3 | Viewed by 2132

Abstract

Difficult-to-root plants often perform poorly during acclimatization and in vitro rooting can increase the survival and quality of plants. The influence of auxin application and mineral nutrition on in vitro rooting and subsequent effects on plant quality in eight Prunus genotypes were investigated. Microshoots were rooted in vitro on Murashige and Skoog (MS), ½ MS, Driver and Kuniyuki (DKW), or New Prunus Medium (NPM) media formulations in combination with 15 µM indole-3-butyric acid (IBA), 4-day 15 µM IBA pulse, 1 mM 30 s quick-dip, or IBA-free treatments. Shoots were observed pre- and post-acclimatization to determine rooting methods to maximize quality and minimize labor. A genotype-specific response to auxin application was observed with seven of eight genotypes achieving 100% survival when paired with the recommended IBA treatment. Peaches performed best when treated with 4-day IBA pulse or 30 s quick-dip. Rooting of P. cerasifera, it’s hybrid to P. persica, and P. munsoniana all benefitted from IBA application. Shoots rooted with 15 µM IBA were smaller and lower quality in most genotypes. DKW maximized size and quality in six genotypes. Better shoots and larger root systems during in vitro rooting produced better plants in the greenhouse with no detrimental effect of callus growth. Rooting techniques to maximize plant quality while reducing labor are specified. Full article

(This article belongs to the Special Issue Micropropagation and Cryopreservation of Plants)

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Review

Jump to: Research

34 pages, 646 KiB

Open AccessFeature PaperReview

Application of In Vitro Plant Tissue Culture Techniques to Halophyte Species: A Review

by Luísa Custódio, Gilbert Charles, Christian Magné, Gregorio Barba-Espín, Abel Piqueras, José A. Hernández, Karim Ben Hamed, Viana Castañeda-Loaiza, Eliana Fernandes and Maria João Rodrigues

Plants 2023, 12(1), 126; https://0-doi-org.brum.beds.ac.uk/10.3390/plants12010126 - 27 Dec 2022

Cited by 10 | Viewed by 9569

Abstract

Halophytes are plants able to thrive in environments characterized by severe abiotic conditions, including high salinity and high light intensity, drought/flooding, and temperature fluctuations. Several species have ethnomedicinal uses, and some are currently explored as sources of food and cosmetic ingredients. Halophytes are considered important alternative cash crops to be used in sustainable saline production systems, due to their ability to grow in saline conditions where conventional glycophyte crops cannot, such as salt-affected soils and saline irrigation water. In vitro plant tissue culture (PTC) techniques have greatly contributed to industry and agriculture in the last century by exploiting the economic potential of several commercial crop plants. The application of PTC to selected halophyte species can thus contribute for developing innovative production systems and obtaining halophyte-based bioactive products. This work aimed to put together and review for the first time the most relevant information on the application of PTC to halophytes. Several protocols were established for the micropropagation of different species. Various explant types have been used as starting materials (e.g., basal shoots and nodes, cotyledons, epicotyls, inflorescence, internodal segments, leaves, roots, rhizomes, stems, shoot tips, or zygotic embryos), involving different micropropagation techniques (e.g., node culture, direct or indirect shoot neoformation, caulogenesis, somatic embryogenesis, rooting, acclimatization, germplasm conservation and cryopreservation, and callogenesis and cell suspension cultures). In vitro systems were also used to study physiological, biochemical, and molecular processes in halophytes, such as functional and salt-tolerance studies. Thus, the application of PTC to halophytes may be used to improve their controlled multiplication and the selection of desired traits for the in vitro production of plants enriched in nutritional and functional components, as well as for the study of their resistance to salt stress. Full article

(This article belongs to the Special Issue Micropropagation and Cryopreservation of Plants)

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