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Plant Cryobiotechnology: Progress and Prospects
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Plant Cryobiotechnology: Progress and Prospects

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

Deadline for manuscript submissions: closed (28 February 2022) | Viewed by 17951

Special Issue Editor

Dr. Daniel Ballesteros

E-Mail Website
Guest Editor
1. Department of Comparative Plant and Fungal Biology, Royal Botanic Gardens Kew, Wakehurst Place, West Sussex RH17 6TN, UK
2. Department of Botany and Geology. University of Valencia, 46100 Burjassot, Valencia, Spain
Interests: plant desiccation and freezing tolerance; longevity of plant propagules; cryobiology; structural biology

Special Issue Information

Dear Colleagues,

We live in a changing world that is testimony to an unprecedented loss of living species. For example, 40% of plants are threatened with extinction. This has a profound impact on biodiversity but also affects all agricultural industries and, hence, the future of global economy and human feeding. The preservation of plant genetic resources is now more urgent than ever. We must provide efficient solutions for the long-term conservation of a vast number of species, meaning that we must be able to preserve a wide variety of germplasm types and phenotypes: from cells of recalcitrant algae through desiccation and freezing tolerant spores of ferns to desiccation and freezing sensitive seeds of tropical trees. Conventional seed storage at dry and sub-zero conditions (while still the most efficient tool) is not sufficient to accomplish this tremendous task, and cryopreservation is indicated for the long-term preservation of all plant species. However, there is not a single cryopreservation method that can be applied universally, and many challenges in the development of protocols arise due to the large phylogenetical variation and degree of complexity of the germplasm to be cryopreserved. The use of multidisciplinary and modern research is vital to understand the fundamental basis that allows successful cryopreservation and recovery, and it is encompassed in the term “cryobiotechnology”.

This Special Issue invites worldwide experts in plant cryobiotechnology to publish their recent discoveries. Contributions from different plant disciplines are welcome, such as genomics, proteomics, metabolomics, biophysics, structural physiology, physiology, biotechnology, etc. We intend to merge in the same issue the most novel evidence to understand processes such as desiccation and low-temperature stress tolerance, longevity at sub-zero (cryogenic) temperatures, or in vitro plant regeneration in a variety of germplasm types and phenotypes across the whole plant phylogeny. The final aim is to facilitate knowledge that will lead to the development of new cryopreservation protocols for difficult-to-preserve species.

Dr. Daniel Ballesteros
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.

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. Plants is an international peer-reviewed open access semimonthly 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 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

  • cryobiotechnology
  • cryopreservation
  • in vitro culture
  • micropropagation
  • seeds
  • embryos
  • spores
  • pollen
  • shoot tips
  • dormant buds
  • genomics
  • proteomics
  • metabolomics
  • biophysics
  • structural physiology
  • physiology
  • biotechnology
  • longevity

Published Papers (7 papers)

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Research

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13 pages, 1847 KiB  
Article
Assessment of Vegetative Growth and Genetic Integrity of Vanilla planifolia Regenerants after Cryopreservation
by María Teresa González-Arnao, Carlos A. Cruz-Cruz, Fabiola Hernández-Ramírez, Jorge A. Alejandre-Rosas and Ana Constanza Hernández-Romero
Plants 2022, 11(13), 1630; https://doi.org/10.3390/plants11131630 - 21 Jun 2022
Cited by 1 | Viewed by 1490
Abstract
Vanilla planifolia Jacks. ex Andrews is the vanilla species with the most commercial and greatest economic importance. It has been used as a case study in different cryopreservation studies that involve three vitrification-based approaches: droplet-vitrification (D-V), V-cryoplate (V-Cp) and D-cryoplate (D-Cp). The aim [...] Read more.
Vanilla planifolia Jacks. ex Andrews is the vanilla species with the most commercial and greatest economic importance. It has been used as a case study in different cryopreservation studies that involve three vitrification-based approaches: droplet-vitrification (D-V), V-cryoplate (V-Cp) and D-cryoplate (D-Cp). The aim of this study was to compare the impact of these cryogenic techniques on vegetative growth (survival, stem length and leaf number) between cryo-derived plants and in vitro-derived controls during 12 months of greenhouse growth. Genetic stability was also assessed using the inter-simple sequence repeat (ISSR) markers. There were no significant differences found in the survival and stem lengths of the in vitro-derived regenerants and cryo-derived plants. A significant increase in the number of leaves was only detected in cryo-derived plants when using the V-Cp method. The electrophoretic profiles, based on seven ISSR primers, detected low variability: 81 total bands and 27% polymorphism. This is the first report on the assessment of vegetative growth and genetic integrity in cryo-derived V. planifolia plants recovered under greenhouse conditions. Of the three cryogenic approaches, D-Cp appears to yield V. planifolia regenerants plants with more vigorous vegetative growth and a lower level of polymorphism. Future research should focus on the reproductive growth of vanilla regenerants. Full article
(This article belongs to the Special Issue Plant Cryobiotechnology: Progress and Prospects)
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15 pages, 1197 KiB  
Article
The Tip of the Iceberg: Cryopreservation Needs for Meeting the Challenge of Exceptional Plant Conservation
by Valerie C. Pence and Emily Beckman Bruns
Plants 2022, 11(12), 1528; https://doi.org/10.3390/plants11121528 - 7 Jun 2022
Cited by 9 | Viewed by 2124
Abstract
Cryopreservation is increasingly important as a conservation tool, particularly for threatened exceptional species. The goal of this study was to investigate the current knowledge of plant cryopreservation through a search of the literature in Web of Science and align that with the 775 [...] Read more.
Cryopreservation is increasingly important as a conservation tool, particularly for threatened exceptional species. The goal of this study was to investigate the current knowledge of plant cryopreservation through a search of the literature in Web of Science and align that with the 775 species currently identified on the Working List of Exceptional Plants. While there is a good foundation in plant cryopreservation research, particularly with economically important species, there are significant gaps in research on families that contain the largest numbers of currently known exceptional species, including the Dipterocarpaceae, Rhizophoraceae, and Pittosporaceae. Even families well represented in both in the literature and on the List of Exceptional Plants had much less overlap at the level of genus. Tropical trees, a significant portion of exceptional species, were not as well represented in the literature as herbaceous species. Over 70% of all articles dealt with in vitro cryopreservation, with much less emphasis on other methods (seed, embryo, dormant bud, and pollen) that will be more cost-effective for species where they can be applied. While the research on plant cryopreservation to date provides a strong foundation and is being utilized effectively for conserving the diversity of a number of economically important species, this study revealed significant gaps that can help prioritize future research to more effectively conserve the diversity of threatened exceptional species. Full article
(This article belongs to the Special Issue Plant Cryobiotechnology: Progress and Prospects)
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16 pages, 1208 KiB  
Article
Cryopreservation of Holm Oak Embryogenic Cultures for Long-Term Conservation and Assessment of Polyploid Stability
by Maria Teresa Martínez, Sonia Suárez, Paloma Moncaleán and Elena Corredoira
Plants 2022, 11(9), 1266; https://doi.org/10.3390/plants11091266 - 8 May 2022
Cited by 6 | Viewed by 2006
Abstract
Holm oak populations are severely affected by oak decline syndrome, and reliable methods of conserving the plant material are required. A vitrification-based cryopreservation method was used for the first time for the long-term conservation of holm oak embryogenic cultures. Successful cryopreservation was achieved [...] Read more.
Holm oak populations are severely affected by oak decline syndrome, and reliable methods of conserving the plant material are required. A vitrification-based cryopreservation method was used for the first time for the long-term conservation of holm oak embryogenic cultures. Successful cryopreservation was achieved after determining the best developmental stage of the somatic embryos used and the optimal incubation period in plant vitrification solution 2 (PVS2). Embryos were recovered from individual nodular embryogenic structures (NES) derived from four embryogenic lines after preculture on a medium containing 0.3 M sucrose, incubation in PVS2 vitrification solution for 15 min at 25 °C and direct immersion in liquid nitrogen (LN). Embryo recovery rates of 16.7–63.3% were obtained after cryostorage for four years in LN. In addition to the embryo developmental stage and the PVS2 treatment time, the genotype can also significantly affect embryo recovery after LN storage. There were no significant differences in plant regeneration or polyploid stability between somatic embryos and plants derived from control embryos (not cryopreserved) and cryopreserved embryos. The findings indicate that embryo proliferation, plant conversion and polyploid stability are maintained in material recovered from the vitrification solution and subsequently cryopreserved. Full article
(This article belongs to the Special Issue Plant Cryobiotechnology: Progress and Prospects)
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20 pages, 1816 KiB  
Article
Bryophyte Spores Tolerate High Desiccation Levels and Exposure to Cryogenic Temperatures but Contain Storage Lipids and Chlorophyll: Understanding the Essential Traits Needed for the Creation of Bryophyte Spore Banks
by Giuseppe Tiloca, Giuseppe Brundu and Daniel Ballesteros
Plants 2022, 11(9), 1262; https://doi.org/10.3390/plants11091262 - 7 May 2022
Cited by 1 | Viewed by 2366
Abstract
Understanding the desiccation and freezing tolerance of bryophyte spores is vital to explain how plants conquered land and current species distribution patterns and help to develop efficient ex situ conservation methods. However, knowledge of these traits is scarce. We investigated tolerance to drying [...] Read more.
Understanding the desiccation and freezing tolerance of bryophyte spores is vital to explain how plants conquered land and current species distribution patterns and help to develop efficient ex situ conservation methods. However, knowledge of these traits is scarce. We investigated tolerance to drying (at 15% relative humidity [RH] for two weeks) and freezing (1 h exposure to liquid nitrogen) on the spores of 12 bryophyte species (23 accessions) from the UK. The presence of storage lipids and their thermal fingerprint, and the levels of unfrozen water content, were determined by differential scanning calorimetry (DSC). The presence of chlorophyll in dry spores was detected by fluorescence microscopy. All species and accessions tested tolerated the drying and freezing levels studied. DSC suggested that 4.1–29.3% of the dry mass is storage lipids, with crystallization and melting temperatures peaking at around −30 °C. Unfrozen water content was determined <0.147 g H2O g−1 dry weight (DW). Most of the spores investigated showed the presence of chlorophyll in the cytoplasm by red autofluorescence. Bryophyte spores can be stored dry at low temperatures, such as orthodox seeds, supporting the creation of bryophyte spore banks. However, the presence of storage lipids and chlorophyll in the cytoplasm may reduce spore longevity during conventional storage at −20 °C. Alternatively, cryogenic spore storage is possible. Full article
(This article belongs to the Special Issue Plant Cryobiotechnology: Progress and Prospects)
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17 pages, 3144 KiB  
Article
Impacts of Rapid Desiccation on Oxidative Status, Ultrastructure and Physiological Functions of Syzygium maire (Myrtaceae) Zygotic Embryos in Preparation for Cryopreservation
by Karin van der Walt, David J. Burritt and Jayanthi Nadarajan
Plants 2022, 11(8), 1056; https://doi.org/10.3390/plants11081056 - 13 Apr 2022
Cited by 8 | Viewed by 1898
Abstract
Syzygium maire is a highly threatened Myrtaceae tree species endemic to New Zealand. Due to its recalcitrant seed storage behaviour, cryopreservation is the only viable long-term ex situ conservation option for this species. This study investigated viability, oxidative stress, thermal properties, and ultrastructure [...] Read more.
Syzygium maire is a highly threatened Myrtaceae tree species endemic to New Zealand. Due to its recalcitrant seed storage behaviour, cryopreservation is the only viable long-term ex situ conservation option for this species. This study investigated viability, oxidative stress, thermal properties, and ultrastructure of zygotic embryo axes (EAs) desiccated to various moisture contents (MC). Fresh EAs had a MC of c. 1.9 g/g with 100% viability but rapid desiccation to MC < 0.3 g/g significantly reduced viability and decreased the activities of the enzymatic antioxidants superoxide dismutase, catalase and glutathione peroxidase, with a sevenfold increase in the production of protein carbonyls and lipid peroxides. Differential Scanning Calorimetry analysis showed no thermal events in EAs desiccated to a MC of <0.2 g/g, indicating that all freezable water had been removed, but this was lethal to both EAs and enzymatic antioxidants. The ultrastructure of desiccated EAs showed signs of plasmolysis, while fully hydrated EAs exposed to cryogenic temperature had ultrastructural disintegration and membrane damage. The decline in enzymatic antioxidant activities and the increase in lipid peroxidation suggest that S. maire EA viability loss is due to oxidative stress rather than structural impacts. Full article
(This article belongs to the Special Issue Plant Cryobiotechnology: Progress and Prospects)
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18 pages, 1359 KiB  
Article
Thermotherapy Followed by Shoot Tip Cryotherapy Eradicates Latent Viruses and Apple Hammerhead Viroid from In Vitro Apple Rootstocks
by Jean Carlos Bettoni, Gennaro Fazio, Larissa Carvalho Costa, Oscar P. Hurtado-Gonzales, Maher Al Rwahnih, Abby Nedrow and Gayle M. Volk
Plants 2022, 11(5), 582; https://doi.org/10.3390/plants11050582 - 22 Feb 2022
Cited by 15 | Viewed by 3881
Abstract
Virus and viroid-free apple rootstocks are necessary for large-scale nursery propagation of apple (Malus domestica) trees. Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) are among the most serious apple viruses that are prevalent in most apple [...] Read more.
Virus and viroid-free apple rootstocks are necessary for large-scale nursery propagation of apple (Malus domestica) trees. Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) are among the most serious apple viruses that are prevalent in most apple growing regions. In addition to these viruses, a new infectious agent named Apple hammerhead viroid (AHVd) has been identified. We investigated whether thermotherapy or cryotherapy alone or a combination of both could effectively eradicate ACLSV, ASGV, and AHVd from in vitro cultures of four apple rootstocks developed in the Cornell-Geneva apple rootstock breeding program (CG 2034, CG 4213, CG 5257, and CG 6006). For thermotherapy treatments, in vitro plants were treated for four weeks at 36 °C (day) and 32 °C (night). Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture in 2 M glycerol + 0.8 M sucrose for one day followed by exposure to PVS2 for 60 or 75 min at 22 °C, either without or with liquid nitrogen (LN, cryotherapy) exposure. Combinations of thermotherapy and PVS2/cryotherapy treatments were also performed. Following treatments, shoot tips were warmed, recovered on growth medium, transferred to the greenhouse, grown, placed in dormancy inducing conditions, and then grown again prior to sampling leaves for the presence of viruses and viroids. Overall, thermotherapy combined with cryotherapy treatment resulted in the highest percentage of virus- and viroid-free plants, suggesting great potential for producing virus- and viroid-free planting materials for the apple industry. Furthermore, it could also be a valuable tool to support the global exchange of apple germplasm. Full article
(This article belongs to the Special Issue Plant Cryobiotechnology: Progress and Prospects)
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Review

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15 pages, 356 KiB  
Review
Myrtaceae in Australia: Use of Cryobiotechnologies for the Conservation of a Significant Plant Family under Threat
by Lyndle K. Hardstaff, Karen D. Sommerville, Bryn Funnekotter, Eric Bunn, Catherine A. Offord and Ricardo L. Mancera
Plants 2022, 11(8), 1017; https://doi.org/10.3390/plants11081017 - 8 Apr 2022
Cited by 6 | Viewed by 3359
Abstract
The Myrtaceae is a very large and diverse family containing a number of economically and ecologically valuable species. In Australia, the family contains approximately 1700 species from 70 genera and is structurally and floristically dominant in many diverse ecosystems. In addition to threats [...] Read more.
The Myrtaceae is a very large and diverse family containing a number of economically and ecologically valuable species. In Australia, the family contains approximately 1700 species from 70 genera and is structurally and floristically dominant in many diverse ecosystems. In addition to threats from habitat fragmentation and increasing rates of natural disasters, infection by myrtle rust caused by Austropuccinia psidii is of significant concern to Australian Myrtaceae species. Repeated infections of new growth have caused host death and suppressed host populations by preventing seed set. Although most Myrtaceae species demonstrate orthodox seed storage behavior, exceptional species such as those with desiccation sensitive seed or from myrtle rust-suppressed populations require alternate conservation strategies such as those offered by cryobiotechnology. Targeting seven key Australian genera, we reviewed the available literature for examples of cryobiotechnology utilized for conservation of Myrtaceae. While there were only limited examples of successful cryopreservation for a few genera in this family, successful cryopreservation of both shoot tips and embryonic axes suggest that cryobiotechnology provides a viable alternative for the conservation of exceptional species and a potential safe storage method for the many Myrtaceae species under threat from A. psidii. Full article
(This article belongs to the Special Issue Plant Cryobiotechnology: Progress and Prospects)
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