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13 pages, 1326 KiB

Open AccessArticle

The Development of a Procedure for the Cryopreservation of the Callus of Anthurium andraeanum by Vitrification

by Yiying Zhang, Shan Deng, Huifeng Lin, Yunxia Chu, Jingyan Huang, Shouguo Li, Fazhuang Lin, Sumei Zhang, Weilan Jiang, Li Ren and Hairong Chen

Plants 2024, 13(21), 3106; https://doi.org/10.3390/plants13213106 - 4 Nov 2024

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Abstract

The cryopreservation of Anthurium andraeanum germplasm resources is extremely important for the production and selection of new varieties. At present, the cryopreservation procedure for the callus of A. andraeanum has not been established. In this study, the leaves of A. andraeanum were used [...] Read more.

The cryopreservation of Anthurium andraeanum germplasm resources is extremely important for the production and selection of new varieties. At present, the cryopreservation procedure for the callus of A. andraeanum has not been established. In this study, the leaves of A. andraeanum were used as explants to culture the callus. The cryopreservation procedure of the callus by vitrification was initially established by using the orthogonal experimental method of four factors and three levels in the preculture, loading, and dehydration steps. Furthermore, the vitrification-based cryopreservation was optimized by changing the preculture temperature and loading solution and adding exogenous substances to the plant vitrification solution (PVS2). In this procedure, the callus was precultured at 25 °C for 2 d, and loaded in 50% PVS2 at 25 °C for 60 min. The callus was dehydrated with PVS2 containing 0.08 mM reduced glutathione (GSH) at 0 °C for 60 min. After rapid-cooling in liquid nitrogen for 1 h, it was rapid-warming in a water bath at 40 °C for 90 s and unloaded for 30 min. After 1 d of recovery, the cell relative survival rate of the cryopreserved callus was 64.60%. The results provide a valuable basic and effective method for the long-term conservation of A. andraeanum germplasm resources. Full article

(This article belongs to the Special Issue In Vitro Propagation and Cryopreservation of Plants)

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15 pages, 5833 KiB

Open AccessArticle

Comparative Studies for Cryopreservation of Agave Shoot Tips by Droplet-Vitrification

by Lourdes Delgado-Aceves, Santiago Corona, Ubaldo Richard Marin-Castro, Martha Paola Rascón-Díaz, Liberato Portillo, Antonia Gutiérrez-Mora and María Teresa González-Arnao

Plants 2024, 13(18), 2609; https://doi.org/10.3390/plants13182609 - 18 Sep 2024

Viewed by 693

Abstract

The objective of this work was to assess the suitability of the Droplet-vitrification protocol previously developed with Agave peacockii shoot tips for the cryopreservation of six Agave species. Shoot tips were precultured for 1 day on a medium with 0.3 M sucrose in [...] Read more.

The objective of this work was to assess the suitability of the Droplet-vitrification protocol previously developed with Agave peacockii shoot tips for the cryopreservation of six Agave species. Shoot tips were precultured for 1 day on a medium with 0.3 M sucrose in the dark, loaded in a solution with 1.6 M glycerol and 0.4 M sucrose for 20 min, and dehydrated by exposure to Plant Vitrification Solution 2 (PVS2) at 0 °C for 20 min. Complementary studies using histological analysis, Differential scanning calorimetry (DSC), and evaluation of morphological characteristics in cryo-derived plants were performed. Survival rates ranged from 84% to 100% and from 76% to 97% before and after cryopreservation regardless of the Agave species belonging to two taxonomic subgenera. Thermal analysis of shoot tips subjected to the successive steps of the Droplet-vitrification protocol identified ice crystal formation after loading treatment and glass transition after osmotic dehydration with PVS2. The average glass transition temperature (Tg) was −55.44 °C based on the results of four Agave species. The histological studies showed the anatomical differences that could be found in the meristematic structures depending on the loss of apical dominance. This is the most advanced research on cryopreservation of Agave shoot tips. Full article

(This article belongs to the Special Issue In Vitro Propagation and Cryopreservation of Plants)

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19 pages, 2882 KiB

Open AccessArticle

Liquid Overlay-Induced Donor Plant Vigor and Initial Ammonium-Free Regrowth Medium Are Critical to the Cryopreservation of Scrophularia kakudensis

by Hyoeun Lee, Hana Park, Sang-Un Park and Haenghoon Kim

Plants 2024, 13(17), 2408; https://doi.org/10.3390/plants13172408 - 28 Aug 2024

Viewed by 782

Abstract

Cryopreservation, storing biological material in liquid nitrogen (LN, −196 °C), offers a valuable option for the long-term conservation of non-orthodox seeds and vegetatively propagated species in the sector of agrobiodiversity and wild flora. Although the large-scale cryobanking of germplasm collections has been increasing [...] Read more.

Cryopreservation, storing biological material in liquid nitrogen (LN, −196 °C), offers a valuable option for the long-term conservation of non-orthodox seeds and vegetatively propagated species in the sector of agrobiodiversity and wild flora. Although the large-scale cryobanking of germplasm collections has been increasing worldwide, the wide application of cryopreservation protocols in wild flora is hampered by difficulties in vitro propagation and a lack of universal cryopreservation protocols, among others. This study established a systematic approach to developing an in vitro culture and droplet-vitrification cryopreservation procedure for shoot tips of Scrophularia kakudensis. The standard procedure includes a two-step preculture with 10% sucrose for 31 h and with 17.5% sucrose for 16 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 30 min, cryoprotection with A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose, w/v) at 0 °C for 60 min, and cooling and rewarming using aluminum foil strips. After unloading, a three-step regrowth procedure starting with an ammonium-free medium with growth regulators was essential for developing normal plantlets from cryopreserved shoot tips. Liquid overlay on the gelled medium two weeks after inoculation resulted in vigorous growth during subcultures. Moreover, liquid overlay increased LN regeneration by up to 80%, i.e., 23% higher than no liquid overlay. Full article

(This article belongs to the Special Issue In Vitro Propagation and Cryopreservation of Plants)

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14 pages, 2916 KiB

Open AccessArticle

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 974

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

Open AccessArticle

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

Cited by 1 | Viewed by 813

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

Open AccessArticle

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

Cited by 2 | Viewed by 980

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

Open AccessArticle

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 1281

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

Open AccessReview

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

Cited by 1 | Viewed by 1418

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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