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Improvement and Innovation of Cryopreservation and In Vitro Methods in Plant Resources Protection

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A special issue of Biology (ISSN 2079-7737). This special issue belongs to the section "Conservation Biology and Biodiversity".

Deadline for manuscript submissions: closed (30 April 2023) | Viewed by 28367

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Special Issue Editors

Dr. Haenghoon Kim

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Guest Editor

Department of Agricultural Life Science, Sunchon National University, Suncheon 57922, Korea
Interests: biodiversity conservation; cryopreservation; future crops; in vitro culture
Special Issues, Collections and Topics in MDPI journals

Dr. Elena Popova

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Guest Editor

K.A. Timiryazev Institute of Plant Physiology of Russian Academy of Sciences, 127276 Moscow, Russia
Interests: biotechnology; in vitro cell culture; cell biology; tissue culture

Special Issue Information

Dear Colleagues,

Plant genetic resources (PGR) are perhaps the most precious gift of nature to humanity: they provide food, shelter, medicines, and a large variety of goods of high economic value, not to mention their key importance for healthy ecosystems and inspirational beauty. Nonetheless, the depletion of plant biodiversity is a problem that have been escalated to a threatening level in recent decades. There is an urgent call for the scientific community to search and put into practice modern methods for PGR conservation through principal studies and practical applications of cryobiotechnology.

This Special Issue is focused specifically on using all aspects of in vitro culture and cryopreservation technology to approach the problem of PGR conservation, including crops, ornamental, medicinal and model plants as well as wild species. Studies offering new insights into conservation methods and physiological responses for species and genotypes that cannot be conserved as seeds in conventional genebanks are particularly welcomed. The submission categories include original research papers, as well as critical reviews (upon prior consultation with the editors) and case studies.

Dr. Haenghoon Kim
Dr. Elena Popova
Guest Editors

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Keywords

conservation
cryobiotechnology
in vitro culture
plant genetic resources
vitrification

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Published Papers (9 papers)

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Editorial

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4 pages, 197 KiB

Open AccessEditorial

Improvement and Innovation of Cryopreservation and In Vitro Methods in Plant Resource Protection

by Haeng-Hoon Kim and Elena Popova

Biology 2024, 13(9), 741; https://doi.org/10.3390/biology13090741 - 21 Sep 2024

Viewed by 857

Abstract

Plant genetic resources (PGRs) are perhaps the most precious gift of nature to humanity: they provide food, shelter, medicines, and many goods of high economic value, not to mention their key importance for healthy ecosystems and their aesthetic value [...] Full article

(This article belongs to the Special Issue Improvement and Innovation of Cryopreservation and In Vitro Methods in Plant Resources Protection)

Research

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11 pages, 811 KiB

Open AccessArticle

Conservation of the Bird Cherry (Padus Mill.) Germplasm by Cold Storage and Cryopreservation of Winter Cuttings

by Vladimir Verzhuk, Sergey Murashev, Liubov Novikova, Stepan Kiru and Svetlana Orlova

Biology 2023, 12(8), 1071; https://doi.org/10.3390/biology12081071 - 30 Jul 2023

Cited by 1 | Viewed by 1403

Abstract

Conservation at cryogenic temperatures, usually in liquid nitrogen (LN) or in its vapor, is the only reliable method for the long-term ex situ conservation of fruit and berry crops with vegetative reproduction. In this study, five bird cherry (Padus Mill.) varieties of different genetic origin from the bird cherry genebank at the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR, Russia) were tested for their response to cryopreservation in LN vapor (−183–−185 °C). The response included viability under laboratory and field conditions, morphological assessment of the developed plants and biochemical analysis of fruits produced during three consecutive years by plants developed from cryopreserved cuttings. All parameters were compared to those recorded after cold storage of cuttings (−5 °C), a routine mid-term conservation method currently used at the VIR genebank. The initial viability of winter cuttings varied from 86.7% to 93.3%. Six-month cold storage and cryopreservation reduced viability to 53.3–86.7% and 43.3–60.0%, respectively, which was above the 40% viability threshold in all varieties tested. Cuttings after cold storage showed better viability when recovered in the laboratory (80% mean viability) than in the field (58% mean viability); viability of cryopreserved cuttings was not affected by recovery conditions. The results of a two-way analysis of covariance suggested that storage and recovery conditions had the most significant effect on viability (p < 0.0001), while the effects of genotype (p = 0.062) and factor interactions (p = 0.921) were minor. Cryopreservation had little or no influence on morphological parameters of the plants recovered in the field, including plant height, number of shoots, internodes and roots, and root length. Similarly, no effect of cryopreservation was recorded on dry matter content, total sugar content and ascorbic acid concentration in fruits produced by plants developed from the cryopreserved cuttings. These results suggest that cryopreservation in LN vapor is a reliable method for conservation of the bird cherry genetic collection and is worth testing with a broader variety of genotypes. Full article

(This article belongs to the Special Issue Improvement and Innovation of Cryopreservation and In Vitro Methods in Plant Resources Protection)

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14 pages, 2168 KiB

Open AccessArticle

Cryopreservation of Malus and Pyrus Wild Species in the ‘Fruit Genebank’ in Dresden-Pillnitz, Germany

by Monika Höfer and Henryk Flachowsky

Biology 2023, 12(2), 200; https://doi.org/10.3390/biology12020200 - 28 Jan 2023

Cited by 9 | Viewed by 2034

Abstract

A unique and valuable diversity of the Malus and Pyrus wild species germplasm is maintained ex situ in field collections in the ‘Fruit Genebank’ in Dresden-Pillnitz, Germany. (1) Background: The establishment of a duplicate collection is necessary to preserve this material safely from abiotic and biotic stress factors. (2) Methods: Two different techniques, cryopreservation using dormant buds and PVS2 vitrification using in vitro shoot tips, were applied and compared. (3) Results: In Malus altogether 180 accessions belonging to 32 species were processed with an average recovery rate of 39% within ten years using the dormant bud method. Accessions, 116 in number, achieved the criterion of 40% recovery which was 64.44% of all accessions tested. In the case of Pyrus germplasm a total of 35 accessions of 21 species and both techniques for cryopreservation were tested. In the results of these experiments the PVS2 method led to better results compared to the dormant bud method. (4) Conclusions: In Malus the dormant bud technique will be the method of choice for the future to build up the duplicate collection. Further experiments using both techniques are planned in the future to make a final decision for Pyrus. Full article

(This article belongs to the Special Issue Improvement and Innovation of Cryopreservation and In Vitro Methods in Plant Resources Protection)

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14 pages, 2353 KiB

Open AccessArticle

Genetic Stability Assessment of Six Cryopreserved Strawberry (Fragaria × ananassa Duch.) Accessions by Phenotypic and Molecular Studies

by Jinjoo Bae, Yunseo Choi, Jae-Young Song, Jung-Ro Lee, Munsup Yoon and Young-Yi Lee

Biology 2022, 11(12), 1746; https://doi.org/10.3390/biology11121746 - 30 Nov 2022

Cited by 3 | Viewed by 1844

Abstract

For the long-term preservation of genetic resources, cryopreservation techniques have been developed for strawberry germplasm, mainly using in vitro-grown shoot tips. In this study, genetic stability was tested under greenhouse conditions for six strawberry accessions (IT232511, PHS0132, IT245810, IT245830, IT245852, and IT245860) derived from the following procedures: (1) conventional propagation (GH: greenhouse maintained); (2) in vitro propagation (TC: tissue culture); (3) pretreatment before cryopreservation (−LN: non-liquid nitrogen exposure); and (4) cryopreservation (+LN: liquid nitrogen exposure). To test the performance of phenotypic traits, we measured six vegetative and five fruit traits. There were no distinct differences in most of the characteristics, but a few traits, such as sugar content and pH of fruits in three accessions, showed higher values in +LN compared to GH. However, the differences disappeared in the first runner generation. To test genetic variations, a total of 102 bands were generated by twelve inter simple sequence repeat (ISSR) primers. A few polymorphic bands were found only in plants derived from TC of IT245860, which was not cryopreserved. The sequencing analysis of four polymorphic bands produced by ISSR_15 showed that none of these sequences matched the characterized genes in NCBI. Phenotypic abnormality was not observed across all plants. This study indicates that cryopreserved plants of the six strawberry accessions are phenotypically and genetically stable. Therefore, the results of this study can help to implement cryobanking of strawberry germplasm. Full article

(This article belongs to the Special Issue Improvement and Innovation of Cryopreservation and In Vitro Methods in Plant Resources Protection)

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18 pages, 4332 KiB

Open AccessArticle

Optimization of Propagation of the Polish Strain of Aldrovanda vesiculosa in Tissue Culture

by Marzena Parzymies, Magdalena Pogorzelec and Alicja Świstowska

Biology 2022, 11(10), 1389; https://doi.org/10.3390/biology11101389 - 23 Sep 2022

Cited by 4 | Viewed by 2301

Abstract

Aldrovanda vesiculosa is a rare and critically endangered carnivorous plant species. Its populations have declined worldwide, so there is a need to protect the species from extinction. The research was conducted to establish an effective method of in vitro propagation of the species in order to obtain plants for reintroduction in the wild. The procedures included disinfection, multiplication, and acclimatization of plants. Contamination-free in vitro cultures were established using shoots and turions, which were disinfected with 0.25% sodium hypochlorite. The shoots were first defoliated. The explants regenerated better in liquid 1/5 MS medium than in solidified one. The optimum medium for the multiplication phase contained MS macro- and microelements diluted to 1/10. Plants cultivated in that medium were of good quality, long, and branched. The advantageous effect of medium was also confirmed by the content of photosynthetic pigments in the plant material. The content of chlorophyll a was highest in plants cultivated in 1/5 or 1/10 MS medium. The plants obtained were acclimatized to ex vitro conditions and reintroduced in the wild. Full article

(This article belongs to the Special Issue Improvement and Innovation of Cryopreservation and In Vitro Methods in Plant Resources Protection)

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Review

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43 pages, 6158 KiB

Open AccessReview

Plants, Cells, Algae, and Cyanobacteria In Vitro and Cryobank Collections at the Institute of Plant Physiology, Russian Academy of Sciences—A Platform for Research and Production Center

by Natalya Yuorieva, Maria Sinetova, Ekaterina Messineva, Irina Kulichenko, Artem Fomenkov, Olga Vysotskaya, Ekaterina Osipova, Angela Baikalova, Olga Prudnikova, Maria Titova, Alexander V. Nosov and Elena Popova

Biology 2023, 12(6), 838; https://doi.org/10.3390/biology12060838 - 9 Jun 2023

Cited by 6 | Viewed by 3834

Abstract

Ex situ collections of algae, cyanobacteria, and plant materials (cell cultures, hairy and adventitious root cultures, shoots, etc.) maintained in vitro or in liquid nitrogen (−196 °C, LN) are valuable sources of strains with unique ecological and biotechnological traits. Such collections play a vital role in bioresource conservation, science, and industry development but are rarely covered in publications. Here, we provide an overview of five genetic collections maintained at the Institute of Plant Physiology of the Russian Academy of Sciences (IPPRAS) since the 1950–1970s using in vitro and cryopreservation approaches. These collections represent different levels of plant organization, from individual cells (cell culture collection) to organs (hairy and adventitious root cultures, shoot apices) to in vitro plants. The total collection holdings comprise more than 430 strains of algae and cyanobacteria, over 200 potato clones, 117 cell cultures, and 50 strains of hairy and adventitious root cultures of medicinal and model plant species. The IPPRAS plant cryobank preserves in LN over 1000 specimens of in vitro cultures and seeds of wild and cultivated plants belonging to 457 species and 74 families. Several algae and plant cell culture strains have been adapted for cultivation in bioreactors from laboratory (5–20-L) to pilot (75-L) to semi-industrial (150–630-L) scale for the production of biomass with high nutritive or pharmacological value. Some of the strains with proven biological activities are currently used to produce cosmetics and food supplements. Here, we provide an overview of the current collections’ composition and major activities, their use in research, biotechnology, and commercial application. We also highlight the most interesting studies performed with collection strains and discuss strategies for the collections’ future development and exploitation in view of current trends in biotechnology and genetic resources conservation. Full article

(This article belongs to the Special Issue Improvement and Innovation of Cryopreservation and In Vitro Methods in Plant Resources Protection)

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35 pages, 1419 KiB

Open AccessReview

Critical Role of Regrowth Conditions in Post-Cryopreservation of In Vitro Plant Germplasm

by Elena Popova, Irina Kulichenko and Haeng-Hoon Kim

Biology 2023, 12(4), 542; https://doi.org/10.3390/biology12040542 - 2 Apr 2023

Cited by 19 | Viewed by 4588

Abstract

Cryopreservation is an effective option for the long-term conservation of plant genetic resources, including vegetatively propagated crops and ornamental plants, elite tree genotypes, threatened plant species with non-orthodox seeds or limited seed availability, as well as cell and root cultures useful for biotechnology. With increasing success, an arsenal of cryopreservation methods has been developed and applied to many species and material types. However, severe damage to plant material accumulating during the multi-step cryopreservation procedure often causes reduced survival and low regrowth, even when the optimized protocol is applied. The conditions at the recovery stage play a vital role in supporting material regrowth after cryopreservation and, when optimized, may shift the life-and-death balance toward a positive outcome. In this contribution, we provide an overview of the five main strategies available at the recovery stage to improve post-cryopreservation survival of in vitro plant materials and their further proliferation and development. In particular, we discuss the modification of the recovery medium composition (iron- and ammonium-free), exogenous additives to cope with oxidative stress and absorb toxic chemicals, and the modulation of medium osmotic potential. Special attention is paid to plant growth regulators used at various steps of the recovery process to induce the desired morphological response in cryopreserved tissues. Given studies on electron transport and energy provision in rewarmed materials, we discuss the effects of light-and-dark conditions and light quality. We hope that this summary provides a helpful guideline and a set of references for choosing the recovery conditions for plant species that have not been cryopreserved. We also propose that step-wise recovery may be most effective for materials sensitive to cryopreservation-induced osmotic and chemical stresses. Full article

(This article belongs to the Special Issue Improvement and Innovation of Cryopreservation and In Vitro Methods in Plant Resources Protection)

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23 pages, 2470 KiB

Open AccessReview

Cryopreservation of Endangered Ornamental Plants and Fruit Crops from Tropical and Subtropical Regions

by Behzad Kaviani and Dariusz Kulus

Biology 2022, 11(6), 847; https://doi.org/10.3390/biology11060847 - 31 May 2022

Cited by 24 | Viewed by 4479

Abstract

Horticultural crops comprise various economic species extending from fruits, nuts, vegetables, spices and condiments, ornamentals, aromatic, and medicinal plants. Ornamental and fruit plants are produced mainly for their nutritional and aesthetic values, respectively. Unfortunately, many tropical and subtropical species are in danger of extinction because of climate change and (a)biotic stresses. It is imperative to preserve the germplasms of these species for the present and future genetic improvement programs. Cryopreservation, i.e., maintenance of tissues at the ultralow temperature of liquid nitrogen, is a promising long-term preservation technique, alternative to seed or in vitro banks, which can be applied for both vegetatively and generatively (through seeds) propagated crops, including those with recalcitrant seeds. It is a technology of choice not only for the preservation of plant biodiversity but also for virus elimination in the proficient administration of large-scale micropropagation. The main advantages of cryopreservation are the lowering of in vitro culture expenditures, needed space, contamination risk, and operator errors. However, tropical species are temperature delicate and one of the foremost challenging issues is preconditioning treatments that stimulate physiological reactions to sufficiently enhance tolerance to dehydration and cryogenic procedures. In recent years, several cryopreservation methods based on encapsulation-vitrification, droplet-vitrification, the use of aluminum cryo-plates, and cryo-mesh have been established. Combined cryo-techniques, gene/DNA conservation, as well as studies on perceiving bio-molecular events and exploring the multistage process from the beginning to end of cryopreservation are receiving more emphasis. The development of cryobiomics delivers a conceptual framework to assess the significance of cell signaling mechanisms on cellular functions, the influence of cryoinjury factors on sample viability, and the implications for genetic stability following cryo-storage. The aim of this mini-review article is to provide a succinct synthesis of the developed cryogenic procedures and their use for the storage and exchange of genetic resources of tropical and subtropical horticultural crops, particularly fruit crops and ornamental plants under the threat of extinction. Full article

(This article belongs to the Special Issue Improvement and Innovation of Cryopreservation and In Vitro Methods in Plant Resources Protection)

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Other

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13 pages, 1669 KiB

Open AccessPerspective

Effect of Explant Source on Phenotypic Changes of In Vitro Grown Cannabis Plantlets over Multiple Subcultures

by Mohsen Hesami, Kristian Adamek, Marco Pepe and Andrew Maxwell Phineas Jones

Biology 2023, 12(3), 443; https://doi.org/10.3390/biology12030443 - 13 Mar 2023

Cited by 17 | Viewed by 5522

Abstract

Drug-type cannabis is often multiplied using micropropagation methods to produce genetically uniform and disease/insect-free crops. However, micropropagated plantlets often exhibit phenotypic variation, leading to culture decline over time. In cannabis, the source of these changes remains unknown, though several factors (e.g., explant’s sources and prolonged in vitro culture) can result in such phenotypical variations. The study presented herein evaluates the effects of explant sources (i.e., nodal segments derived from the basal, near-basal, middle, and apical parts of the greenhouse-grown mother plant) over multiple subcultures (4 subcultures during 235 days) on multiplication parameters and leaf morphological traits of in vitro cannabis plantlets. While initial in vitro responses were similar among explants sourced from different regions of the plant, there were significant differences in performance over the course of multiple subcultures. Specifically, explant source and/or the number of subcultures significantly impacted plantlet height, number of nodes, and canopy surface area. The explants derived from the basal and near-basal parts of the plant resulted in the tallest shoots with the greatest number of nodes, while the explants derived from the middle and apical regions led to shorter shoots with fewer nodes. Moreover, the basal-derived explants produced cannabis plantlets with shorter but wider leaves which demonstrated the potential of such explants for in vitro rejuvenation practices with minimal culture decline. This study provides new evidence into the long-term impacts of explant source in cannabis micropropagation. Full article

(This article belongs to the Special Issue Improvement and Innovation of Cryopreservation and In Vitro Methods in Plant Resources Protection)

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