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In Vitro Conservation of Endangered and Value-Added Plant Species

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

Deadline for manuscript submissions: closed (31 January 2022) | Viewed by 37424

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Dr. Praveen K. Saxena

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Gosling Research Institute for Plant Preservation (GRIPP), Department of Plant Agriculture, University of Guelph, Guelph, ON N1G 2W1, Canada
Interests: plant morphogenesis; in vitro conservation; plant production systems; neurotransmitters; stress adaptations
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Special Issue Information

Dear Colleagues,

Plant biodiversity is crucial for sustaining human life on our planet. There are approximately half a million plants on earth and of these around 50,000 species are used globally for food, feed, fiber, medicine and horticulture. It is estimated that at least 21% of all known vascular plants are either threatened, endangered or at the risk of extinction due to habitat loss, overexploitation, and the rapidly changing climate. In vitro technologies can play an important role in the species recovery projects and enhance plant populations in natural habitats. Micropropagation, an advanced plant tissue culture technique, is a tool that can be used to maintain living germplasm and produce large quantities of plants for replenishment, conservation and global distribution of endangered species and crops of economic importance. Cryopreservation allows the storage of genetic material at an ultra-low temperature (−196 °C), and the tissues can be maintained for decades with minimal loss of viability and genetic uniformity. Over 200 plant species including staple food crops, endangered species and plants of horticultural importance have been cryopreserved with varying degree of success. Thus, integrated plant systems utilizing micropropagation and cryopreservation technologies hold great potential in mitigating the impact of current ecological crisis while complementing global conservation strategies.

This Special Issue is dedicated to highlight the application of in vitro technologies for plant biodiversity conservation. Contributions are welcome on all aspects of in vitro technologies in relation to species recovery, conservation, cryopreservation, and industrial applications of cryo-biotechnologies for producing plant-derived bioactive compounds with potential application in pharmaceutical, cosmetic, and natural health product industries. The submission categories include original research papers, critical reviews (prior consultation with the editor recommended), and research reports or case studies describing a single noteworthy accomplishment.

Dr. Praveen K. Saxena
Guest Editor

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Keywords

plant conservation
endangered species
food security
horticultural crops
medicinal plants
micropropagation
cryopreservation
cryobanking

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Published Papers (9 papers)

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Research

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21 pages, 3276 KiB

Open AccessEditor’s ChoiceArticle

In Vitro Technologies for American Chestnut (Castanea dentata (Marshall) Borkh) Conservation

by Zhuoya Liu, Wen-Lu Bi, Mukund R. Shukla and Praveen K. Saxena

Plants 2022, 11(3), 464; https://doi.org/10.3390/plants11030464 - 8 Feb 2022

Cited by 6 | Viewed by 3769

Abstract

American chestnut (Castanea dentata), a native species of eastern North America, is an economically important deciduous hardwood tree that has been designated as endangered in Canada. The population of American chestnut trees has dwindled significantly across Southern Ontario due to chestnut blight and many of the surviving trees continue to show blight disease symptoms. American chestnut requires efficient strategies for propagation and preservation for species recovery. The objective of this study was to develop a long-term plant conservation program using micropropagation and cryopreservation protocols. An in vitro technology using a liquid-based temporary immersion system (TIS) was developed for micropropagation of American chestnut. The highest rate of shoot multiplication was observed in cultures grown in the DKW (Driver and Kuniyuki 1984) basal medium supplemented with 2.2 µM 6-benzylaminopurine and 1.0 µM gibberellic acid. More than 95% of proliferated microshoots, about 40–50 mm in size, developed roots after 30 days of culture within bioreactor vessels containing DKW basal medium supplemented with 15 µM 3-Indolebutyric acid. Rooted plantlets transplanted to the greenhouse had a survival efficiency of 82% after one month of growth. The cryopreservation protocol for germplasm preservation was developed through droplet vitrification of shoots. Optimal regeneration of shoot tips occurred from explants precultured on stepwise concentrations of sucrose and subsequent dehydration in PVS3 for 30 min. Cryopreserved shoot tips were regenerated to whole plants using pre-optimized conditions of micropropagation. This study confirms the potential of TIS for micropropagation in ex situ conservation and reintroduction of endangered American chestnuts and possibly other woody plant species. Full article

(This article belongs to the Special Issue In Vitro Conservation of Endangered and Value-Added Plant Species)

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15 pages, 3490 KiB

Open AccessArticle

Seed Cryopreservation and Germination of Rhus glabra and the Critically Endangered Species Rhus michauxii

by Gerald S. Pullman, Kylie Bucalo, Ron O. Determann and Jennifer M. Cruse-Sanders

Plants 2021, 10(11), 2277; https://doi.org/10.3390/plants10112277 - 24 Oct 2021

Cited by 2 | Viewed by 2421

Abstract

Rhus michauxii is a perennial rhizomatous shrub native to the southeastern United States that is found mainly in sunny, dry, open rocky or sandy woodlands. Moreover, it is found on ridges or river bluffs in the inner coastal plane and lower piedmont of Virginia, Georgia, and the Carolinas. Habitat conversion to agriculture, suppression of fires, and low reproduction have caused R. michauxii to become rare and it is now federally listed as threatened. Methods are needed to multiply and conserve R. michauxii. Protocols were developed for seed cryopreservation, in vitro germination, and micropropagation for R. glabra and R. michauxii. Seed scarification in concentrated sulfuric acid for 6 h and germination on ½ MS medium resulted in germination up to 96% for control and cryopreserved seeds of R. glabra and 70 and 40% for control and cryopreserved seeds of R. michauxii. Shortly after germination in vitro, young seedlings were established in a greenhouse potting mix providing new plants from the endemic Georgia R. michauxii populations. Several of the findings meet goals within the R. michauxii recovery plan by providing methods for sexual and asexual multiplication and long-term seed storage under cryogenic conditions. The protocols developed will assist in the safeguarding and conservation of dwindling natural R. michauxii populations. Full article

(This article belongs to the Special Issue In Vitro Conservation of Endangered and Value-Added Plant Species)

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19 pages, 2782 KiB

Open AccessArticle

Micropropagation and Cryopreservation of Yukon Draba (Draba yukonensis), a Special Concern Plant Species Endemic to Yukon Territory, Canada

by Akansha Saxena, Wen-Lu Bi, Mukund R. Shukla, Syd Cannings, Bruce Bennett and Praveen K. Saxena

Plants 2021, 10(10), 2093; https://doi.org/10.3390/plants10102093 - 3 Oct 2021

Cited by 6 | Viewed by 3547

Abstract

Yukon Draba (Draba yukonensis) is a small, short-lived perennial mustard species that is endemic to southwestern Yukon in Canada. This plant has been categorized as a species of Special Concern. It faces the threat of habitat loss due to natural and man-made causes and a population that is unevenly distributed to a few large and several small subpopulations in the area. It will therefore be judicious to undertake investigations on the conservation of this species to save it from further deterioration which may lead to its extinction. In this study, a protocol was developed for in vitro propagation and cryopreservation of Yukon Draba. The micropropagation protocol was optimized using shoot tips which enabled clonal propagation and in vitro storage of the species. Shoots grew best in the medium containing MS basal salts and had the highest multiplication with the addition of 2 µM 6-benzylaminopurine or 5 µM Kinetin with 3% sucrose. The addition of 10 µM Indole Butyric Acid (IBA) produced the highest number of adventitious roots on the shoots and the longest root length was observed at 2 µM IBA. The rooted plantlets were transferred to greenhouse and the highest survival (87.5%) was observed for the plantlets treated with a lower concentration of IBA (2 µM). Cryopreservation protocol was developed using the droplet-vitrification method for in vitro shoot tips. Two-week-old shoots had the highest survival and regrowth following exposure to plant vitrification solution 3 (PVS3) for 30 min, prior to direct immersion of the droplets into the liquid nitrogen. The optimized protocols for the micropropagation and cryopreservation may be useful for the long-term germplasm conservation and reintroduction of this species in its natural habitat. Full article

(This article belongs to the Special Issue In Vitro Conservation of Endangered and Value-Added Plant Species)

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17 pages, 1922 KiB

Open AccessArticle

Optimization of a Cryopreservation Method for the Endangered Korean Species Pogostemon yatabeanus Using a Systematic Approach: The Key Role of Ammonium and Growth Regulators

by Hyo-Eun Lee, Elena Popova, Ha-Na Park, Sang-Un Park and Haeng-Hoon Kim

Plants 2021, 10(10), 2018; https://doi.org/10.3390/plants10102018 - 26 Sep 2021

Cited by 9 | Viewed by 2100

Abstract

Cryopreservation provides a secure long-term conservation option for rare and endangered plant species with non-orthodox or limitedly available seeds. Wide application of cryopreservation to biobank wild flora is hampered by the need to re-optimize nearly all protocol steps for every new species. We applied a systematic approach to simplify optimization of a multi-stage droplet-vitrification method for the endangered wetland Korean species, Pogostemon yatabeanus. This approach consisted of a standard procedure pre-selected based on material type and size, which was complemented with 11 additional treatments to reveal the most impactful conditions. Effect of ammonium nitrate at various protocol steps was also tested. The highest shoot tip survival (92%) and plant regeneration (90%) after cryopreservation were achieved using preculture with 10% sucrose followed by 40 min osmoprotection and 60 min treatment with vitrification solution A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose) on ice. A three-step regrowth procedure starting with ammonium-free medium with 1 mg/L GA₃ and 1 mg/L BA followed by ammonium-containing medium with and without growth regulators was essential for the development of healthy plants from cryopreserved shoot tips. This approach enables fast optimization of the cryopreservation procedure for new osmotic stress-sensitive plant species. Full article

(This article belongs to the Special Issue In Vitro Conservation of Endangered and Value-Added Plant Species)

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22 pages, 2724 KiB

Open AccessArticle

Cryopreservation of 13 Commercial Cannabis sativa Genotypes Using In Vitro Nodal Explants

by Cassandra D. Downey, Gregory Golenia, Ekaterina A. Boudko and Andrew Maxwell P. Jones

Plants 2021, 10(9), 1794; https://doi.org/10.3390/plants10091794 - 28 Aug 2021

Cited by 8 | Viewed by 5839

Abstract

Cannabis has developed into a multi-billion-dollar industry that relies on clonal propagation of elite genetics with desirable agronomic and chemical phenotypes. While the goal of clonal propagation is to produce genetically uniform plants, somatic mutations can accumulate during growth and compromise long-term genetic fidelity. Cryopreservation is a process in which tissues are stored at cryogenic temperatures, halting cell division and metabolic processes to facilitate high fidelity germplasm preservation. In this study, a series of experiments were conducted to optimize various stages of cryopreservation and develop a protocol for long-term germplasm storage of Cannabis sativa. The resulting protocol uses a standard vitrification procedure to cryopreserve nodal explants from in vitro shoots as follows: nodes were cultured for 17 h in a pre-culture solution (PCS), followed by a 20-min treatment in a loading solution (LS), and a 60 min incubation in plant vitrification solution 2 (PVS2). The nodes were then flash frozen in liquid nitrogen, re-warmed in an unloading solution at 40 °C, and cultured on basal MS culture medium in the dark for 5 days followed by transfer to standard culture conditions. This protocol was tested across 13 genotypes to assess the genotypic variability. The protocol was successful across all 13 genotypes, but significant variation was observed in tissue survival (43.3–80%) and regrowth of shoots (26.7–66.7%). Plants grown from cryopreserved samples were morphologically and chemically similar to control plants for most major traits, but some differences were observed in the minor cannabinoid and terpene profiles. While further improvements are likely possible, this study provides a functional cryopreservation system that works across multiple commercial genotypes for long-term germplasm preservation. Full article

(This article belongs to the Special Issue In Vitro Conservation of Endangered and Value-Added Plant Species)

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12 pages, 1536 KiB

Open AccessArticle

Somatic Embryogenesis and Plant Regeneration in Viola canescens Wall. Ex. Roxb.: An Endangered Himalayan Herb

by Arun Kumar Khajuria, Christophe Hano and Narendra Singh Bisht

Plants 2021, 10(4), 761; https://doi.org/10.3390/plants10040761 - 13 Apr 2021

Cited by 11 | Viewed by 2974

Abstract

Viola canescens Wall. ex. Roxb. is an important but threatened medicinal herb found at 1500–2400 m above mean sea level in the Himalayas. Overexploitation and habitat preference have put the plant under serious threat. Thus, the present study was undertaken to develop an efficient protocol for in vitro propagation via somatic embryogenesis. The results revealed that plant can be regenerated successfully through somatic embryogenesis using leaf derived calli. Regular subculturing of calli on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D)/indole-3-butyric acid (IBA)/kinetin (Kn) and varying combinations of 2,4-D+Kn induced somatic embryogenesis. The maximum average number of somatic embryos (SE) (19.15 ± 2.66) was induced on the medium with 0.15 + 0.05 mg L⁻¹ of 2,4-D and Kn, respectively, and this medium was used as a control. To enhance somatic embryo induction, the control MS medium was supplemented with l-glutamine (200–400 mg L⁻¹) and casein hydrolysate (1–4%). The maximum average number of SE (27.66 ± 2.67) and average mature SE (13.16 ± 3.48) were recorded on the medium having 2 % l-glutamine and 50 mg L⁻¹ casein hydrolysate. The induced SE were asynchronous, so, to foster their maturation, the culture medium (free from growth regulators) was supplemented with abscisic acid (ABA) and silver nitrate (AgNO₃). The maximum average number (35.96 ± 3.68) of mature SE was noticed on MS medium supplemented with 1.5 mg L⁻¹ ABA. Mature embryos had two well-developed cotyledons and an elongated hypocotyl root axis. The development of SE into plantlets was significant for embryos matured on the medium with AgNO₃ and ABA, with 86.67% and 83.33% conversion on the medium with 0.20 mg L⁻¹ 6-benzylaminopurine (BAP). The plantlets thus produced acclimatized in a growth chamber before being transferred to the field, which showed 89.89% survival. The plants were morphologically similar to the mother plant with successful flowering. Full article

(This article belongs to the Special Issue In Vitro Conservation of Endangered and Value-Added Plant Species)

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Review

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20 pages, 1031 KiB

Open AccessEditor’s ChoiceReview

In Vitro Technology in Plant Conservation: Relevance to Biocultural Diversity

by Verena Kulak, Sheri Longboat, Nicolas D. Brunet, Mukund Shukla and Praveen Saxena

Plants 2022, 11(4), 503; https://doi.org/10.3390/plants11040503 - 12 Feb 2022

Cited by 29 | Viewed by 6378

Abstract

Plant diversity is critical to the functioning of human societies, and evidence shows that plant conservation success is driven by integrative approaches that include social and biological factors. Plants have a unique capacity to reproduce asexually, and propagation practices can yield large numbers of plantlets. These plantlets can be used in several ways to fulfil conservation goals including the repopulation of regions with declining densities of threatened species that hold cultural meaning. However, the potential of in vitro technologies in the conservation of plants that hold cultural meaning is understudied. In this paper we focus upon the roles of in vitro technologies in the conservation of plants relevant to biocultural environments and provide an overview of potential knowledge gaps at the interface of in vitro and plants used traditionally, including those meaningful to Indigenous Peoples. We conclude that in vitro technologies can be powerful tools in biocultural conservation if they are deployed in a manner respectful of the socio-cultural context in which plants play a role, but that further research is needed in this regard. We suggest several epistemological points to facilitate future research. Full article

(This article belongs to the Special Issue In Vitro Conservation of Endangered and Value-Added Plant Species)

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19 pages, 850 KiB

Open AccessReview

Epigenetic and Genetic Integrity, Metabolic Stability, and Field Performance of Cryopreserved Plants

by Min-Rui Wang, Wenlu Bi, Mukund R. Shukla, Li Ren, Zhibo Hamborg, Dag-Ragnar Blystad, Praveen K. Saxena and Qiao-Chun Wang

Plants 2021, 10(9), 1889; https://doi.org/10.3390/plants10091889 - 13 Sep 2021

Cited by 37 | Viewed by 5000

Abstract

Cryopreservation is considered an ideal strategy for the long-term preservation of plant genetic resources. Significant progress was achieved over the past several decades, resulting in the successful cryopreservation of the genetic resources of diverse plant species. Cryopreservation procedures often employ in vitro culture techniques and require the precise control of several steps, such as the excision of explants, preculture, osmo- and cryoprotection, dehydration, freeze-thaw cycle, unloading, and post-culture for the recovery of plants. These processes create a stressful environment and cause reactive oxygen species (ROS)-induced oxidative stress, which is detrimental to the growth and regeneration of tissues and plants from cryopreserved tissues. ROS-induced oxidative stresses were documented to induce (epi)genetic and somatic variations. Therefore, the development of true-to-type regenerants of the source germplasm is of primary concern in the application of plant cryopreservation technology. The present article provides a comprehensive assessment of epigenetic and genetic integrity, metabolic stability, and field performance of cryopreserved plants developed in the past decade. Potential areas and the directions of future research in plant cryopreservation are also proposed. Full article

(This article belongs to the Special Issue In Vitro Conservation of Endangered and Value-Added Plant Species)

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Other

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7 pages, 1172 KiB

Open AccessTechnical Note

Short-Term Storability of Alginate-Encapsulated Persian Violet Microshoots for Germplasm Exchange

by Saowaros Phanomchai, Kitti Bodhipadma, Sompoch Noichinda and David W. M. Leung

Plants 2022, 11(2), 185; https://doi.org/10.3390/plants11020185 - 11 Jan 2022

Cited by 3 | Viewed by 1972

Abstract

Microshoots have been widely used for micropropagation. It may be necessary to store microshoots for a short period of time, for example in germplasm exchange needing transport to other research groups. Here, we investigated the short-term storability of alginate-encapsulated Persian violet (Exacum affine Balf. f. ex Regel) microshoots at 4 °C and 25 °C. After storage, the encapsulated microshoots were sown on basal Murashige and Skoog medium for germination and viability determination using tetrazolium chloride staining. The results showed that one or five microshoots encapsulated with a single alginate layer could be stored at 4 °C for up to 30 days, while the percentages of germination and viability of the microshoots encapsulated with two layers of alginate were greatly reduced upon storage. This is the first report on the storability of alginate-encapsulated multiple microshoots, which could be a more efficient way to encapsulate microshoots used for short-term cold storage. Full article

(This article belongs to the Special Issue In Vitro Conservation of Endangered and Value-Added Plant Species)

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