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In Vitro Propagation and Cryopreservation of Plants
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In Vitro Propagation and Cryopreservation of Plants

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

Deadline for manuscript submissions: closed (31 July 2024) | Viewed by 4355

Special Issue Editor

Prof. Haeng-hoon Kim

E-Mail Website
Guest Editor
Department of Agricultural Life Science, Sunchon National University, Suncheon 57922, Republic of Korea
Interests: biodiversity conservation; cryopreservation; plant biotechnology
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Call upon the importance of plant biodiversity for humankind and their threatening level. Cryopreservation, the storing of biological samples in liquid nitrogen (LN), can offer valuable options for non-orthodox seeds, vegetatively propagated species, and cell cultures. In vitro propagation is also helpful for preparing plant materials for cryopreservation, especially threatened wild species. Moreover, as is common sense among cryobiologists, the success of cryopreservation depends on the vigor of plant materials provided by the in vitro culture and the regrowth protocol. In the era of cryobanking germplasm collections of food and agriculture, we still need to develop cryo-biotechnology through principle studies, systematic approaches, and practical applications. Since cryopreservation is a multidisciplinary process, approaches for tuning the whole process or focusing on specific stages, i.e., plant material preparation, pre-LN, cooling/rewarming and unloading, post-LN regrowth, etc., are welcome. This Special Issue of Plants will highlight all aspects of in vitro propagation and cryopreservation technologies to solve plant conservation problems.

Prof. Dr. Haeng-hoon Kim
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

  • Keywords: Breeding; cryoconservation; Humulus lupulus; pollination; Solanum tuberosum; variety

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Published Papers (5 papers)

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Research

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14 pages, 2916 KiB  
Article
In Vitro Propagation and Conservation of Lavandula stoechas subsp. luisieri and Pterospartum tridentatum, Two Important Medicinal and Aromatic Species from Portugal
by Joana Domingues, Anabela Eira, Isa Ramalho, Inês Barrocas and José Carlos Gonçalves
Plants 2024, 13(15), 2124; https://doi.org/10.3390/plants13152124 - 1 Aug 2024
Viewed by 513
Abstract
Lavandula stoechas subsp. luisieri and Pterospartum tridentatum are two valuable aromatic and medicinal plants. Their biometric and morphological parameters, such as the number of new shoots, length of the longest shoot, multiplication rate, and fresh weight, were evaluated using the multiplication MS medium [...] Read more.
Lavandula stoechas subsp. luisieri and Pterospartum tridentatum are two valuable aromatic and medicinal plants. Their biometric and morphological parameters, such as the number of new shoots, length of the longest shoot, multiplication rate, and fresh weight, were evaluated using the multiplication MS medium protocol. The rooting protocols involved immersing the explants in IBA (1 g L−1) and a commercial IBA (3.3 g L−1) preparation (Clonex®). Slow-growth conservation assays were carried out using two different sucrose concentrations (15 g L−1 and 30 g L−1), and a control, with the cultures kept at 4 °C for 12 months. The multiplication rate for L. stoechas subsp. luisieri was 6.8, and that of P. tridentatum was 13.3, achieved using the MS medium supplemented with 0.2 mg L−1 BAP, 1 mg L−1 BAP, and 0.5 mg L−1 IBA. The application of Clonex® showed the best ex vitro rooting results in L. stoechas subsp. luisieri (77%) and P. tridentatum (90%). In the slow-growth conservation assays, at 4 °C, in darkness for 12 months, an excellent survival rate was achieved in L. stoechas subsp. luisieri (>80%) and P. tridentatum (>90%), even at the reduced sucrose concentration. This study demonstrates the effectiveness of in vitro multiplication and ex vitro rooting protocols for two valuable aromatic and medicinal plants. These findings are significant for the ex situ conservation of these species, as they provide effective long-term preservation and utilization strategies. Full article
(This article belongs to the Special Issue In Vitro Propagation and Cryopreservation of Plants)
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21 pages, 2802 KiB  
Article
Conservation Potential Trough In Vitro Regeneration of Two Threatened Medicinal Plants Ungernia sewertzowii and U. victoris
by Feruza Usmanovna Mustafina, Hanifabonu Kobul kizi Juraeva, Dilafruz Nematilla kizi Jamalova, Abbos Tulkin ogli Hazratov, Ayimxan Jalgasbaevna Janabaeva, Hoe Jin Kim, Chae Sun Na, Min Sung Lee, Yu Jin Oh, Komiljon Sharobiddinovich Tojibaev and Sodikjon Kholiknazarovich Abdinazarov
Plants 2024, 13(14), 1966; https://doi.org/10.3390/plants13141966 - 18 Jul 2024
Viewed by 387
Abstract
Ungernia sewertzowii (US) and U. victoris (UV) are medicinal plants and sources of biologically active compounds for pharmaceutical needs. The leaves of US contain 0.29–0.81% sum of alkaloids with a predominance of lycorine, which is 0.04–0.46% in leaves and 0.15–0.38% in bulbs. Lycorine [...] Read more.
Ungernia sewertzowii (US) and U. victoris (UV) are medicinal plants and sources of biologically active compounds for pharmaceutical needs. The leaves of US contain 0.29–0.81% sum of alkaloids with a predominance of lycorine, which is 0.04–0.46% in leaves and 0.15–0.38% in bulbs. Lycorine is used to treat acute and chronic bronchitis. The leaves of UV contain 0.27–0.71% sum of alkaloids with a predominance of galanthamine—0.13–1.15%. Galanthamine is used to treat mild-to-moderate dementia (Alzheimer’s disease). The natural populations of US and UV are in danger as sources of income for local people. To resolve this problem, two protocols for microclonal propagation were developed to replace natural raw materials with in vitro regenerated plants. Callusogenesis of US and UV was induced on Murashige and Skoog (MS) nutrient media with 2.4D (0.5 mg/L) in combination with BAP (0.5 mg/L), Kin (0.5 mg/L), or Zea (0.5 mg/L). Direct (for US) and indirect (for US and UN) organogenesis were observed on MS with BAP (0.5 mg/L) or Kin (0.5 mg/L) in combination with IAA (0.5 mg/L) or NAA (0.5 mg/L). Direct organogenesis resulted in 3–5 bulbs of US on one explant; indirect organogenesis resulted in up to 100–150 bulbs of US and UV on one explant within 6 months, or five to six subcultures after transferring the callus to the nutrient medium. The tissue cultures of US and UV were characterized by very low data on antioxidant activity based on IC50 values for DPPH and ABTS radical scavenging activities, whereas in vitro regenerated plants (leaves and bulbs) had higher data. We concluded that in vitro regenerated plants are valuable sources of lycorine and galanthamine, which allow the protection of the natural populations of these two species from extinction. Full article
(This article belongs to the Special Issue In Vitro Propagation and Cryopreservation of Plants)
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17 pages, 6863 KiB  
Article
Analysis of Thermal Characteristics of Potato and Hop Pollen for Their Cryopreservation and Cross-Breeding
by Milos Faltus, Jaroslava Domkářová, Petr Svoboda, Vendulka Horáčková, Vladimír Nesvadba, Vladislav Klička, Jiří Ptáček, Alois Bilavcik and Jiri Zamecnik
Plants 2024, 13(11), 1578; https://doi.org/10.3390/plants13111578 - 6 Jun 2024
Viewed by 596
Abstract
This study investigated the thermal properties of potato and hop pollen for cryopreservation and subsequent cross-breeding. Phase transitions and frozen water content in selected pollen samples were measured using a differential scanning calorimeter (DSC). Unlike hop pollen, potato pollen showed high variability in [...] Read more.
This study investigated the thermal properties of potato and hop pollen for cryopreservation and subsequent cross-breeding. Phase transitions and frozen water content in selected pollen samples were measured using a differential scanning calorimeter (DSC). Unlike hop pollen, potato pollen showed high variability in thermal properties and water content. Three specific types of pollen samples based on their thermal characteristics and water content were distinguished by DSC in potato: (1) ‘glassy’, with a water content lower than 0.21 g water per g dry matter; (2) ‘transient’, with a water content between 0.27 and 0.34 g of water per g of dry matter; (3) ‘frozen’, with a water content higher than 0.34 g of water per g of dry matter. Only the ‘glassy’ pollen samples with a low water content showed suitable properties for its long-term storage using cryopreservation in potato and hops. Cryopreservation of pollen did not significantly reduce its viability, and cryopreserved pollen was successfully used to produce both potato and hop hybrids. The results indicate that cryopreservation is a feasible technique for the preservation and utilization of pollen of these crops in the breeding process. Full article
(This article belongs to the Special Issue In Vitro Propagation and Cryopreservation of Plants)
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21 pages, 5353 KiB  
Article
Cryopreservation of Indigenous Plums and Monitoring of Multiplication and Rooting Capacity of Shoots Obtained from Cryopreserved Specimens
by Tatjana Vujović, Tatjana Anđelić, Bojana Vasilijević, Darko Jevremović and Florent Engelmann
Plants 2023, 12(17), 3108; https://doi.org/10.3390/plants12173108 - 30 Aug 2023
Cited by 2 | Viewed by 1084
Abstract
The objective of this study is to assess the suitability of vitrification cryo-plate (V cryo-plate) and dehydration cryo-plate (D cryo-plate) methods for the long-term conservation of eight autochthonous Prunus domestica L. genotypes originating from the Balkan Peninsula region. In vitro shoot tips were [...] Read more.
The objective of this study is to assess the suitability of vitrification cryo-plate (V cryo-plate) and dehydration cryo-plate (D cryo-plate) methods for the long-term conservation of eight autochthonous Prunus domestica L. genotypes originating from the Balkan Peninsula region. In vitro shoot tips were briefly pre-cultured for 1 day at 23 °C in the dark on a medium containing 0.3 M sucrose and then embedded in calcium alginate gel within the wells of the aluminum cryo-plates. In the V cryo-plate protocol, dehydration was carried out at room temperature using the following vitrification solutions: original plant vitrification solution 2 (PVS2) and 90% PVS2 solution (for 20 and 40 min) and plant vitrification solution 3 (PVS3) (for 60 and 80 min). In the D cryo-plate protocol, desiccation was performed for 2, 2.5, or 3 h over silica gel at 23 °C. The effect of different treatments was evaluated by monitoring the regrowth of both non-frozen and cryo-preserved explants. After cryo-preservation, five genotypes achieved regrowth rates over 40% in at least one of the applied protocols, while two genotypes showed regrowth rates of around 10%. A significant improvement in regrowth success for all genotypes using both cryo-plate methods was achieved by pre-culturing shoot tips for 7 days on a medium containing 0.5 M sucrose in complete darkness at 4 °C. Shoots regenerated from cryo-preserved explants were further monitored in vitro. By the third subculture, they had not only regained but had even exceeded the multiplication capacity (index of multiplication, length of axial, and lateral shoots) of shoots regenerated from dissection controls. Following multiplication, the cryo-preserved shoots were successfully rooted and rooting ability was assessed by monitoring the percentage of rooting, number and length of roots, and height of rooted plantlets. Full article
(This article belongs to the Special Issue In Vitro Propagation and Cryopreservation of Plants)
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Review

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22 pages, 760 KiB  
Review
Optimizing the Droplet-Vitrification Procedure by Balancing the Cryoprotection and Cytotoxicity of Alternative Plant Vitrification Solutions Based on the Nature of Donor Plant Vigor
by Haenghoon Kim
Plants 2023, 12(23), 4040; https://doi.org/10.3390/plants12234040 - 30 Nov 2023
Viewed by 1077
Abstract
Over 30 years of plant vitrification, droplet vitrification (DV) of in vitro propagules and slow freezing of dormant buds are typical methods of large-scale cryobanking worldwide. One-step sucrose preculture and Plant Vitrification Solution 2 (PVS2) cryoprotection in solution-based vitrification often face unacceptably low [...] Read more.
Over 30 years of plant vitrification, droplet vitrification (DV) of in vitro propagules and slow freezing of dormant buds are typical methods of large-scale cryobanking worldwide. One-step sucrose preculture and Plant Vitrification Solution 2 (PVS2) cryoprotection in solution-based vitrification often face unacceptably low regeneration, and the results are on a case-by-case basis depending on the plant species, like a blind test. The absence of a universal protocol applicable across all plant diversity is considered one of the limiting factors. For wild flora, limits of source material available and difficulties in in vitro propagation make it worse to re-optimize the protocol steps for new species. Since cryoprotectant toxicity is the most crucial barrier to the vitrification of organized explants, selecting alternative plant vitrification solutions (PVS) based on the cytotoxicity of cryoprotectants is vital. This review proposes the concept of donor plant vigor (DPV), which refers to the donor plant properties that determine the potential to regenerate normal plantlets under various cryopreservation procedures. DV is a multi-stage procedure with many factors from stage (1) material preparation to (2) pre-liquid nitrogen (pre-LN) (preculture, osmoprotection, cryoprotection), (3) LN (cooling), (4) warming conditions (rewarming, unloading), and (5) regrowth. Since the cytotoxicity of PVS is a primary limiting factor in DV approaches, DPV is crucial for coping with the toxicity of PVS. The DPV is innate and can be maximized with appropriate material preparations, i.e., vigorously growing in subcultures aided by a liquid overlay on top of the gelled medium, selecting proper explants, optimizing the two-step preculture conditions, and media supplements. Developing the DV protocol starts with testing the material with a tentative standard protocol, which includes a two-step preculture (10% sucrose for 31 h and 17.5% sucrose for 16 h), osmoprotection with C4-35%, cryoprotection with A3-80% (60 min at 0 °C), cooling, and rewarming using aluminum foil strips. Using a three-step regrowth initially with ammonium-free regrowth medium, regrowth of shoot tips in one plate following the successive stages of the tentative standard protocol for shoot tips, i.e., fresh, PC, OP, CP (LNC), and LN, is a valuable tool to characterize the sensitivity of the material and to standardize the procedure by tuning the cryoprotection and cytotoxicity of cryoprotectants. A-series PVS (A3-90%, A3-80%, A3-70%) and B-series PVS (PVS3, B5-85%) can be tested based on the DPV. These alternative PVSs have been applied in over 30 pieces of literature with an 8.5~67.3% increase in LN regeneration compared to PVS2 and Plant Vitrification Solution 3 (PVS3) treatments. Using this approach as an alternative to blind condition screening would be influential in broadening the cryopreservation of diverse wild species and problem materials. Full article
(This article belongs to the Special Issue In Vitro Propagation and Cryopreservation of Plants)
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