Plants

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5 pages, 227 KiB

Open AccessEditorial

Plant Cryopreservation: A Look at the Present and the Future

by Carla Benelli

Plants 2021, 10(12), 2744; https://doi.org/10.3390/plants10122744 - 13 Dec 2021

Cited by 17 | Viewed by 3313

Abstract

Cryopreservation is known as an applied aspect of cryobiology or the study of life at low temperatures [...] Full article

(This article belongs to the Special Issue Plant Cryopreservation)

Research

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8 pages, 861 KiB

Open AccessCommunication

The Survival of Pear Dormant Buds at Ultra-Low Temperatures

by Alois Bilavcik, Milos Faltus and Jiri Zamecnik

Plants 2021, 10(11), 2502; https://doi.org/10.3390/plants10112502 - 18 Nov 2021

Cited by 7 | Viewed by 1608

Abstract

Currently, there is a varietal diversity decline in pear orchards of the Czech Republic. Thus, the safe storage of their gene pool collections is becoming increasingly important. Therefore, the ultra-low temperature survival after two-step cryopreservation treatment of dormant buds was tested for a [...] Read more.

Currently, there is a varietal diversity decline in pear orchards of the Czech Republic. Thus, the safe storage of their gene pool collections is becoming increasingly important. Therefore, the ultra-low temperature survival after two-step cryopreservation treatment of dormant buds was tested for a safe and rapid way to conserve pear germplasm in a broader range of varieties. The following varieties crucial for cultivation in the Czech Republic were tested; ‘Amfora’, ‘Beurré Hardy’, ‘Bosc’, ‘Clapp’s Favourite’, ‘Conference’, ‘Dicolor’, ‘Erika’, ‘Lucas’, ‘Williams’ and ‘Williams Red’. In 2011 and 2012, dormant pear buds were dehydrated to 40.1% and 36.0% water content, respectively, before cryopreservation. The average regeneration of the dormant pear buds after cryopreservation by the two-step cryoprotocol in 2011 and 2012 was 54.3% and 16.1%, respectively. The mentioned cryopreservation procedure is suitable for the safe storage of dormant buds in most tested pear varieties. Full article

(This article belongs to the Special Issue Plant Cryopreservation)

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19 pages, 2871 KiB

Open AccessArticle

Vitrification Ability of Combined and Single Cryoprotective Agents

by Milos Faltus, Alois Bilavcik and Jiri Zamecnik

Plants 2021, 10(11), 2392; https://doi.org/10.3390/plants10112392 - 06 Nov 2021

Cited by 8 | Viewed by 2020

Abstract

Cryoprotective agents (CPA) are an important part of many current vitrification methods. The vitrification ability of CPAs influences the probability of the glass transition and water crystallization occurrence. Thermal characteristics and the vitrification ability of two combined CPAs (PVS2 and PVS3), common plant [...] Read more.

Cryoprotective agents (CPA) are an important part of many current vitrification methods. The vitrification ability of CPAs influences the probability of the glass transition and water crystallization occurrence. Thermal characteristics and the vitrification ability of two combined CPAs (PVS2 and PVS3), common plant vitrification solutions, and four single CPAs (ethylene glycol, DMSO, glycerol, and sucrose), the components of the mentioned PVSs, were evaluated utilizing a differential scanning calorimetry (DSC) during standard cooling/warming rates of 10 °C min⁻¹. The effect of solute concentration on their vitrification ability was shown in the CPAs tested. Four typical concentration regions at which the glassy state and/or crystallization occurred were defined. We suggest the solute concentration of 0.7 g g⁻¹ as the universal vitrification concentration, characterized by an actual Tg of CPA solution and limited water crystallization. Knowledge of the thermal properties of CPAs allows the design of new combined CPAs with the required vitrification ability respecting the cryopreservation method used and the characteristics of the cryopreserved sample. Full article

(This article belongs to the Special Issue Plant Cryopreservation)

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15 pages, 3780 KiB

Open AccessArticle

Effect of Cryopreservation on Proteins from the Ubiquitous Marine Dinoflagellate Breviolum sp. (Family Symbiodiniaceae)

by Hsing-Hui Li, Jia-Lin Lu, Hui-Esther Lo, Sujune Tsai and Chiahsin Lin

Plants 2021, 10(8), 1731; https://doi.org/10.3390/plants10081731 - 21 Aug 2021

Cited by 5 | Viewed by 2142

Abstract

Coral reefs around the world are exposed to thermal stress from climate change, disrupting the delicate symbiosis between the coral host and its symbionts. Cryopreservation is an indispensable tool for the preservation of species, as well as the establishment of a gene bank. [...] Read more.

Coral reefs around the world are exposed to thermal stress from climate change, disrupting the delicate symbiosis between the coral host and its symbionts. Cryopreservation is an indispensable tool for the preservation of species, as well as the establishment of a gene bank. However, the development of cryopreservation techniques for application to symbiotic algae is limited, in addition to the scarceness of related studies on the molecular level impacts post-thawing. Hence, it is essential to set up a suitable freezing protocol for coral symbionts, as well as to analyze its cryo-injury at the molecular level. The objective of this study was to develop a suitable protocol for the coral symbiont Breviolum subjected to two-step freezing. The thawed Breviolum were then cultured for 3, 7, 14, and 28 days before they were analyzed by Western blot for protein expression, light-harvesting protein (LHP), and red fluorescent protein (RFP) and tested by adenosine triphosphate bioassay for cell viability. The results showed the highest cell viability for thawed Breviolum that was treated with 2 M propylene glycol (PG) and 2 M methanol (MeOH) and equilibrated with both cryoprotectants for 30 min and 20 min. Both treatment groups demonstrated a significant increase in cell population after 28 days of culture post-thawing, especially for the MeOH treatment group, whose growth rate was twice of the PG treatment group. Regarding protein expression, the total amounts of each type of protein were significantly affected by cryopreservation. After 28 days of culture, the protein expression for the MeOH treatment group showed no significant difference to that of the control group, whereas the protein expression for the PG treatment group showed a significant difference. Breviolum that were frozen with MeOH recovered faster upon thawing than those frozen with PG. LHP was positively and RFP was negatively correlated with Symbiodiniaceae viability and so could serve as health-informing biomarkers. This work represents the first time to document it in Symbiodiniaceae, and this study established a suitable protocol for the cryopreservation of Breviolum and further refined the current understanding of the impact of low temperature on its protein expression. By gaining further understanding of the use of cryopreservation as a way to conserve Symbiodiniaceae, we hope to make an effort in the remediation and conservation of the coral reef ecosystem and provide additional methods to rescue coral reefs. Full article

(This article belongs to the Special Issue Plant Cryopreservation)

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22 pages, 3772 KiB

Open AccessArticle

Seed Cryopreservation, Germination, and Micropropagation of Eastern Turkeybeard, Xerophyllum asphodeloides (L.) Nutt.: A Threatened Species from the Southeastern United States

by Michelle Issac, Princy Kuriakose, Stacie Leung, Alex B. Costa, Shannon Johnson, Kylie Bucalo, Jonathan M. Stober, Ron O. Determann, Will L. Rogers, Jenifer M. Cruse-Sanders and Gerald S. Pullman

Plants 2021, 10(7), 1462; https://doi.org/10.3390/plants10071462 - 16 Jul 2021

Cited by 5 | Viewed by 2498

Abstract

Xerophyllum asphodeloides (Xerophyllaceae), known as eastern turkeybeard, is an herbaceous perennial found in eastern North America. Due to decline and destruction of its habitat, several states rank X. asphodeloides as “Imperiled” to “Critically Imperiled”. Protocols for seed cryopreservation, in vitro germination, sustainable shoot [...] Read more.

Xerophyllum asphodeloides (Xerophyllaceae), known as eastern turkeybeard, is an herbaceous perennial found in eastern North America. Due to decline and destruction of its habitat, several states rank X. asphodeloides as “Imperiled” to “Critically Imperiled”. Protocols for seed cryopreservation, in vitro germination, sustainable shoot micropropagation, shoot establishment in soil, and seed germination are presented. Seeds from two tested sources were viable after 20 months of cryopreservation. Germination of isolated embryos in vitro was necessary to overcome strong seed dormancy. Shoot multiplication and elongation occurred on ½ MS medium without PGRs. Shoots rooted in vitro without PGRs or with 0.5 mg/L NAA or after NAA rooting powder treatment and placement in potting mix. When planted in wet, peaty soil mixes, shoots grew for two months and then declined. When planted in a drier planting mix containing aged bark, most plants continued growth. In the field, plant survival was 73% after three growing seasons. Safeguarding this species both ex situ and in situ is possible and offers a successful approach to conservation. Whole seeds germinated after double dormancy was overcome by incubation under warm moist conditions for 12 weeks followed by 12 weeks cold at 4 °C and then warm. Full article

(This article belongs to the Special Issue Plant Cryopreservation)

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17 pages, 2508 KiB

Open AccessArticle

Successful Cryopreservation of Dormant Buds of Blackcurrant (Ribes nigrum L.) by Using Greenhouse-Grown Plants and In Vitro Recovery

by Saija Rantala, Janne Kaseva, Anna Nukari, Jaana Laamanen, Merja Veteläinen, Hely Häggman and Saila Karhu

Plants 2021, 10(7), 1414; https://doi.org/10.3390/plants10071414 - 10 Jul 2021

Cited by 3 | Viewed by 2546

Abstract

The cryopreservation of dormant buds can be a feasible method for preserving germplasm of cold-tolerant woody plants. In the present study, we evaluated the effects of pre-desiccation, thawing method, and the rehydration of bud sections on the post-cryopreservation recovery of dormant blackcurrant buds [...] Read more.

The cryopreservation of dormant buds can be a feasible method for preserving germplasm of cold-tolerant woody plants. In the present study, we evaluated the effects of pre-desiccation, thawing method, and the rehydration of bud sections on the post-cryopreservation recovery of dormant blackcurrant buds in vitro. The estimated recovery of small- and medium-sized buds was 80.1 and 62.7% respectively for desiccated buds and 67.8 and 72.3% respectively for non-desiccated buds. The pre-desiccation of bud sections enhanced the number of the shoots regenerated from vegetative buds (2.3 vs. 4.7). The estimated recovery of fast-thawed buds was better after 14-day than after 7-day rehydration (85 vs. 59%). In slowly thawed buds the difference between 14-day and 7-day rehydration was not significant (73 vs. 62%). The estimated recovery of vegetative and flower buds was 77.7 and 41.1% respectively after 7-day rehydration, and 95.2 and 43.6% respectively after a 14-day rehydration period. The rehydration of bud sections was not necessary for the in vitro recovery of non-desiccated, fast-thawed buds. Of the 23 blackcurrant cultivars cryopreserved using non-desiccated dormant buds collected from a greenhouse, the estimated recovery of 22 cultivars ranged between 42 and 90%. Full article

(This article belongs to the Special Issue Plant Cryopreservation)

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10 pages, 1348 KiB

Open AccessCommunication

Non-Uniform Distribution of Cryoprotecting Agents in Rice Culture Cells Measured by CARS Microscopy

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

Plants 2021, 10(3), 589; https://doi.org/10.3390/plants10030589 - 21 Mar 2021

Cited by 4 | Viewed by 3299

Abstract

Cryoprotectants allow cells to be frozen in liquid nitrogen and cryopreserved for years by minimizing the damage that occurs in cooling and warming processes. Unfortunately, how the specific cryoprotectants keep the cells viable through the cryopreservation process is not entirely evident. This contributes [...] Read more.

Cryoprotectants allow cells to be frozen in liquid nitrogen and cryopreserved for years by minimizing the damage that occurs in cooling and warming processes. Unfortunately, how the specific cryoprotectants keep the cells viable through the cryopreservation process is not entirely evident. This contributes to the arduous process of optimizing cryoprotectant formulations for each new cell line or species that is conserved. Coherent anti-Stokes Raman scattering microscopy facilitates the visualization of deuterated cryoprotectants within living cells. Using this technique, we directly imaged the location of fully deuterated dimethyl sulfoxide (d₆-DMSO), the deuterated form of a commonly used cryoprotectant, DMSO, within rice suspension cells. This work showed that d₆-DMSO does not uniformly distribute throughout the cells, rather it enters the cell and sequesters within organelles, changing our understanding of how DMSO concentration varies within the cellular compartments. Variations in cryoprotectant concentration within different cells and tissues will likely lead to differing protection from liquid nitrogen exposure. Expanding this work to include different cryoprotectants and mixtures of cryoprotectants is vital to create a robust understanding of how the distributions of these molecules change when different cryoprotectants are used. Full article

(This article belongs to the Special Issue Plant Cryopreservation)

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12 pages, 1078 KiB

Open AccessArticle

Two Advanced Cryogenic Procedures for Improving Stevia rebaudiana (Bertoni) Cryopreservation

by Carla Benelli, Lara S. O. Carvalho, Soumaya EL merzougui and Raffaella Petruccelli

Plants 2021, 10(2), 277; https://doi.org/10.3390/plants10020277 - 31 Jan 2021

Cited by 7 | Viewed by 2949

Abstract

Cryopreservation is a useful tool for the long-term storage of plant genetic resources, and different cryogenic procedures have recently been developed. The present study focused on the use of the Droplet-vitrification (DV) and V cryo-plate protocol for the cryopreservation of Stevia rebaudiana in [...] Read more.

Cryopreservation is a useful tool for the long-term storage of plant genetic resources, and different cryogenic procedures have recently been developed. The present study focused on the use of the Droplet-vitrification (DV) and V cryo-plate protocol for the cryopreservation of Stevia rebaudiana in vitro-derived apical shoot tips and axillary shoot tips. A preliminary test showed that 90 and 120 min PVS2 (Plant Vitrification Solution 2) treatment significantly reduced the regrowth of the explants before immersion in liquid nitrogen (LN). For both procedures tested, the best osmoprotective condition for obtaining a higher regrowth of cryopreserved explants occurred when explants were PVS2 treated for 60 min. After direct immersion in LN, thawing and plating, the highest regrowth recorded was 80% with DV and 93% with V cryo-plate. Moreover, shoot tips proved to be a more suitable material for Stevia cryopreservation. A satisfactory vegetative regrowth was observed in the subcultures following cryopreservation by DV and V cryo-plate cryogenic procedures. Full article

(This article belongs to the Special Issue Plant Cryopreservation)

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9 pages, 949 KiB

Open AccessArticle

Efficient Protocol for Improving the Development of Cryopreserved Embryonic Axes of Chestnut (Castanea sativa Mill.) by Encapsulation–Vitrification

by Mariam Gaidamashvili, Eka Khurtsidze, Tamari Kutchava, Maurizio Lambardi and Carla Benelli

Plants 2021, 10(2), 231; https://doi.org/10.3390/plants10020231 - 25 Jan 2021

Cited by 9 | Viewed by 2754

Abstract

An optimized cryopreservation protocol for embryonic axes (EAs) of chestnut (Castanea sativa Mill.) has been developed based on the encapsulation–vitrification procedure. EAs of mature seeds were aseptically dissected and encapsulated in alginate beads with or without 0.3% (w/v) [...] Read more.

An optimized cryopreservation protocol for embryonic axes (EAs) of chestnut (Castanea sativa Mill.) has been developed based on the encapsulation–vitrification procedure. EAs of mature seeds were aseptically dissected and encapsulated in alginate beads with or without 0.3% (w/v) activated charcoal (AC). Embedded EAs were dehydrated with Plant Vitrification Solution 2 for different treatment times up to 120 min, followed by direct immersion in liquid nitrogen. Cryopreserved embryonic axes encapsulated with AC showed higher survival (70%) compared to those encapsulated without AC (50%). Sixty-four percent of embryonic axes, from synthetic seeds with AC, subsequently developed as whole plants. Plantlet regrowth was faster in AC-encapsulated EAs and showed enhanced postcryopreservation shoot and root regrowth over 2 cm after five weeks from rewarming. Results indicate that encapsulation–vitrification with activated charcoal added to the beads is an effective method for the long-term preservation of Castaneasativa embryonic axes. Full article

(This article belongs to the Special Issue Plant Cryopreservation)

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Review

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17 pages, 380 KiB

Open AccessReview

Vitrification Solutions for Plant Cryopreservation: Modification and Properties

by Jiri Zamecnik, Milos Faltus and Alois Bilavcik

Plants 2021, 10(12), 2623; https://doi.org/10.3390/plants10122623 - 29 Nov 2021

Cited by 31 | Viewed by 4741

Abstract

Many plants cannot vitrify themselves because they lack glassy state-inducing substances and/or have high water content. Therefore, cryoprotectants are used to induce vitrification. A cryoprotectant must have at least the following primary abilities: high glass-forming property, dehydration strength on a colligative basis to [...] Read more.

Many plants cannot vitrify themselves because they lack glassy state-inducing substances and/or have high water content. Therefore, cryoprotectants are used to induce vitrification. A cryoprotectant must have at least the following primary abilities: high glass-forming property, dehydration strength on a colligative basis to dehydrate plant cells to induce the vitrification state, and must not be toxic for plants. This review introduces the compounds used for vitrification solutions (VSs), their properties indicating a modification of different plant vitrification solutions, their modifications in the compounds, and/or their concentration. An experimental comparison is listed based on the survival or regeneration rate of one particular species after using more than three different VSs or their modifications. A brief overview of various cryopreservation methods using the Plant Vitrification Solution (PVS) is also included. This review can help in alert researchers to newly introduced PVSs for plant vitrification cryoprotocols, their properties, and the choice of their modifications in the compounds and/or their concentration. Full article

(This article belongs to the Special Issue Plant Cryopreservation)

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23 pages, 2271 KiB

Open AccessEditor’s ChoiceReview

Cryopreservation of Woody Crops: The Avocado Case

by Chris O’Brien, Jayeni Hiti-Bandaralage, Raquel Folgado, Alice Hayward, Sean Lahmeyer, Jim Folsom and Neena Mitter

Plants 2021, 10(5), 934; https://doi.org/10.3390/plants10050934 - 07 May 2021

Cited by 13 | Viewed by 5797

Abstract

Recent development and implementation of crop cryopreservation protocols has increased the capacity to maintain recalcitrant seeded germplasm collections via cryopreserved in vitro material. To preserve the greatest possible plant genetic resources globally for future food security and breeding programs, it is essential to [...] Read more.

Recent development and implementation of crop cryopreservation protocols has increased the capacity to maintain recalcitrant seeded germplasm collections via cryopreserved in vitro material. To preserve the greatest possible plant genetic resources globally for future food security and breeding programs, it is essential to integrate in situ and ex situ conservation methods into a cohesive conservation plan. In vitro storage using tissue culture and cryopreservation techniques offers promising complementary tools that can be used to promote this approach. These techniques can be employed for crops difficult or impossible to maintain in seed banks for long-term conservation. This includes woody perennial plants, recalcitrant seed crops or crops with no seeds at all and vegetatively or clonally propagated crops where seeds are not true-to-type. Many of the world’s most important crops for food, nutrition and livelihoods, are vegetatively propagated or have recalcitrant seeds. This review will look at ex situ conservation, namely field repositories and in vitro storage for some of these economically important crops, focusing on conservation strategies for avocado. To date, cultivar-specific multiplication protocols have been established for maintaining multiple avocado cultivars in tissue culture. Cryopreservation of avocado somatic embryos and somatic embryogenesis have been successful. In addition, a shoot-tip cryopreservation protocol has been developed for cryo-storage and regeneration of true-to-type clonal avocado plants. Full article

(This article belongs to the Special Issue Plant Cryopreservation)

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