scholarly journals Cryopreservation of Shoot Tips of “Brazilian Ginseng” (Pfaffia glomerata (Spreng.) Pedersen) by Vitrification

2019 ◽  
Vol 11 (11) ◽  
pp. 146
Author(s):  
Daniela Vasconcelos de Oliveira ◽  
Izulmé Rita Imaculada Santos ◽  
Ildeu Soares Martins ◽  
Antonieta Nassif Salomão
Keyword(s):  
Plant Genetic Resources ◽  
Folk Medicine ◽  
Shoot Tips ◽  
Ex Situ ◽  
Medicinal Species ◽  
Pfaffia Glomerata ◽  
Long Term Storage ◽  
Brazilian Ginseng ◽  
Two Temperatures

Pfaffia glomerata (Amaranthaceae), “Brazilian Ginseng”, is a medicinal plant used in folk medicine. Roots are used as a tonic to restore and enhance wellbeing and for treatment of arthritis, gastritis and rheumatism. Conservation of P. glomerata germplasm is a priority and cryopreservation is the most promising technique for long-term storage of plant genetic resources. Hence, the objective of this work was to develop a cryopreservation protocol for shoot tips of P. glomerata using vitrification techniques. For cryopreservation, shoot tips (ST) from in vitro grown plants were pre-cultured for 19 hr on MS medium containing 0.3 M sucrose, treated with loading and vitrification solutions prior to rapid freezing by direct plunge in liquid nitrogen, rapid thawing on a water bath at 38±2 °C and treatment with a dilution solution. Three vitrification solutions (PVS2, PVS3 and PVS4), three exposure times (20 min., 40 min. and 60 min.) and two temperatures (25 °C and 0 °C) were tested. After cryopreservation, rewarmed shoot tips were inoculated on MS growth medium and the best regeneration percentages were 63%, 42% and 65% for shoot tips treated with PVS2, PVS3 and PVS4, respectively, for 60 min., at 25 °C. The results obtained show that vitrification with PVS2 and PVS4, at 25 °C, for 60 min were the best treatments for successful cryopreservation of shoot tips of in vitro grown plantlets of P. glomerata and that cryopreservation is suitable for ex situ conservation of the germplasm of this medicinal species.

scholarly journals A modified droplet vitrification method for cryopreservation of shoot tips from in vitro potato plants

2019 ◽  
Vol 23 (4) ◽  
pp. 422-429 ◽  
Author(s):  
T. A. Gavrilenko ◽  
N. A. Shvachko ◽  
N. N. Volkova ◽  
Yu. V. Ukhatova
Keyword(s):  
Plant Genetic Resources ◽  
Original Method ◽  
Shoot Tips ◽  
Droplet Vitrification ◽  
Modified Method ◽  
Long Term Storage ◽  
Common Potato ◽  
The Impact

Collections of common potato maintained in the field genebanks suffer significant losses due to the impact of extreme environmental factors, diseases and pests. The solution of the problem of safe long-term preservation of common potato accessions is to create doublet in vitro and cryo-collections. Cryogenic collections are stored at ultra-low temperatures in cryobanks. Several methods of potato cryoconservation are known, of which the droplet vitrification method developed by B. Panis with colleagues in 2005 is the most widely used in genebanks. This paper provides a detailed description of the modified method of droplet vitrification, which is used for cryopreservation of apexes (shoot tips) of potato in vitro plants at the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR). The method modified at VIR includes the main steps of the original droplet-vitrification method developed by B. Panis and colleagues: 1) preparation of plant material, 2) isolation of shoot tips, 3) treatment of explants with cryoprotector solutions, 4) freezing/immersion in liquid nitrogen, 5) thawing, 6) post-cryogenic recovery and evaluation of viability and regeneration capacity. The modifications of stages 1, 2 and 6 proposed at VIR lead to a significant reduction in the duration of cryopreservation experiments in comparison with the original method of B. Panis. This paper presents the results of cryopreservation of modern potato cultivars and South American landraces which were obtained using the method of droplet vitrification as modified at VIR. The majority (76.7 %) of the studied accessions of cultivated potato were characterized by high rates of postcryogenic recovery (40–95 %) and 23.3 % of the samples had the values of postcryogenic regeneration from 20 to 39 %, which corresponds to the minimal permissible values for long-term storage in a cryobank. Currently the modified droplet-vitrification method is used for further expanding of the VIR potato cryocollection.

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

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 277
Author(s):  
Carla Benelli ◽  
Lara S. O. Carvalho ◽  
Soumaya EL merzougui ◽  
Raffaella Petruccelli
Keyword(s):  
Plant Genetic Resources ◽  
Stevia Rebaudiana ◽  
Preliminary Test ◽  
Shoot Tips ◽  
Axillary Shoot ◽  
Suitable Material ◽  
Long Term Storage ◽  
Stevia Rebaudiana Bertoni

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.

scholarly journals Cryopreservation of Woody Crops: The Avocado Case

Plants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 934
Author(s):  
Chris O’Brien ◽  
Jayeni Hiti-Bandaralage ◽  
Raquel Folgado ◽  
Alice Hayward ◽  
Sean Lahmeyer ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Ex Situ Conservation ◽  
Plant Genetic Resources ◽  
Ex Situ ◽  
Conservation Strategies ◽  
Recalcitrant Seed ◽  
In Vitro Storage ◽  
Germplasm Collections ◽  
Seed Crops

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.

scholarly journals Improved Conservation of Coffee (Coffea arabica L.) Germplasm via Micropropagation and Cryopreservation

Agronomy ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1861
Author(s):  
Yanelis Castilla Valdés ◽  
Mukund R. Shukla ◽  
María Esther González Vega ◽  
Praveen K. Saxena
Keyword(s):  
Coffea Arabica ◽  
Plant Genetic Resources ◽  
Ex Situ ◽  
Zygotic Embryos ◽  
Agricultural Crop ◽  
Temporary Immersion ◽  
Droplet Vitrification ◽  
Coffea Arabica L

Coffee (Coffea spp.) is an important tropical agricultural crop that has significant economic and social importance in the world. The ex situ conservation of plant genetic resources through seeds is not feasible due to the sensitivity of coffee seed to desiccation and low temperatures. The cryopreservation of zygotic embryos may allow for an efficient and long-term storage of coffee germplasm. This study describes the cryopreservation methods for conserving zygotic embryos of Coffea arabica L. for the long-term conservation of currently available germplasm. Zygotic embryos were successfully cryopreserved in liquid nitrogen at −196 °C under controlled environmental conditions with either droplet-vitrification or encapsulation–vitrification protocols without dehydration. Zygotic embryos had the highest regrowth (100%) following droplet-vitrification cryopreservation using the Plant Vitrification Solution 3 (PVS3) for 40 min at 23 °C. In the case of encapsulation–vitrification using PVS3 for 40 min at 23 °C, the embryo regeneration response was 78%. Plantlets were recovered following shoot multiplication using a temporary immersion system (TIS) and in vitro rooting. The prolific rooting of shoots was observed after 4 weeks of culture in the liquid medium with plugs made of the inert substrate Oasis® In vitro Express (IVE) compared to the semi-solid medium. The successful cryopreservation of coffee zygotic embryos using droplet vitrification and encapsulation–vitrification followed by micropropagation in temporary immersion culture system has not been reported earlier and together these technologies are anticipated to further facilitate the initiatives for the conservation and distribution of coffee germplasm.

scholarly journals Safeguarding and using global banana diversity: a holistic approach

2020 ◽  
Vol 1 (1) ◽  
Author(s):  
Ines Van den houwe ◽  
Rachel Chase ◽  
Julie Sardos ◽  
Max Ruas ◽  
Els Kempenaers ◽  
...  
Keyword(s):  
Genetic Resources ◽  
Agronomic Traits ◽  
Plant Genetic Resources ◽  
Holistic Approach ◽  
Global Strategy ◽  
Ex Situ ◽  
Phenotypic Traits ◽  
Research Activities ◽  
Genebank Management

AbstractThe CGIAR genebank International Musa Germplasm Transit Centre (ITC) currently holds 1617 banana accessions from 38 countries as an in vitro collection, backed-up by a cryopreserved collection to safeguard global Musa diversity in perpetuity. The ITC also serves as a vital safety backup and transit centre for national banana genebanks and ensures that germplasm is clean of pests and diseases and freely available under the International Treaty on Plant Genetic Resources for Food and Agriculture. In more than 35 years of activity, the ITC has distributed over 18,000 banana accession samples to researchers and farmers in 113 countries. Ex situ conservation of vegetatively-propagated crops such as banana poses very particular challenges. Maintaining the ITC genebank is labor intense and costly. Efficiencies are sought through research and development of techniques on detecting viruses, the genetic integrity of accessions, and on innovative means of safeguarding banana diversity, such as conserving populations of wild species by seed banking. Although the conservation of global banana diversity is the main objective of the ITC, significant value comes from its holistic approach to better understand and promote its germplasm through numerous research activities and resources. Techniques for morphological and molecular characterization serve to identify and describe the collection, while also determining what gaps should be filled by collecting missions with national partners. The evaluation of desirable agronomic traits inherent in Musa spp. are investigated by a high-throughput phenotyping platform, which helps breeding programs to select cultivars resistant or tolerant to biotic and abiotic stresses. Genomic and bioinformatic studies of several banana wild relatives greatly enhance our understanding of Musa genetic diversity, links to important phenotypic traits and bring new methods for management of the collection. Collectively, these research activities produce enormous amounts of data that require curation and dissemination to the public. The two information systems at the ITC, Musa Genebank Management System and the Musa Germplasm Information System, serve to manage the genebank activities and to make public germplasm-related data for over 30 banana collections worldwide, respectively. By implementing the 10-year workplan set out in the Global Strategy for the Conservation and Use of Musa Genetic Resources, the network MusaNet supports Musa researchers and stakeholders, including the ITC, and most importantly, links to the world’s banana-producing countries via three regional banana networks.

scholarly journals Long-term conservation of potato genetic resources: Methods and status of conservation

2019 ◽  
pp. 717-725
Author(s):  
Jane Muthoni ◽  
Hussein Shimelis ◽  
Rob Melis
Keyword(s):  
Genetic Resources ◽  
Slow Growth ◽  
Plant Genetic Resources ◽  
Ex Situ ◽  
Growth Media ◽  
Medium Term ◽  
Natural Habitats

Plant genetic resources (PGRs) play an important role in agriculture, environment protection, cultural property and trade; they need to be conserved. There are two fundamental approaches for the conservation of PGRs: in situ and ex situ. In situ conservation is the conservation of ecosystems and natural habitats and the maintenance and recovery of viable populations of species in their natural surroundings. Ex situ preservation is the storage of seeds or plant materials under artificial conditions to maintain their long term viability and availability for use. Genebanks employ seed storage, field collections of living plants and in vitro storage (tissue culture or cryopreservation) for ex situ preservation of PGR. Storage of orthodox seeds, which are tolerant to low moisture content and low temperatures at appropriate temperature and humidity, is the most convenient ex situ conservation method. Plants that produce recalcitrant seeds or non-viable seeds are conserved in field genebanks as well as in-vitro in slow growth media for short-to-medium term and cryopreservation in liquid nitrogen at -1960C for long-term periods. Cryopreservation is very expensive and needs trained personnel; this could explain why this method is rarely used for conservation of plant genetic resources in most developing countries. Potato tubers are bulky and highly perishable; the crop is generally conserved as clones either in field genebanks (with annual replanting), in-vitro conservation in slow growth media for short-to-medium term and cryopreservation for long term. Field genebanks are expensive to maintain and the crop is exposed to many dangers; hence, cryopreservation is the only feasible method for long term conservation. However, given the high cost of cryopreservation, long-term conservation of potato genetic resources is poorly developed in most resource-poor countries leading to high rates of genetic erosion. This paper looks into the various methods that that can be applied to conserve potato genetic resources and the status of conservation of potatoes in major genebanks and some countries.

scholarly journals CO2-enriched atmosphere and supporting material impact the growth, morphophysiology and ultrastructure of in vitro Brazilian-ginseng [Pfaffia glomerata (Spreng.) Pedersen] plantlets

2014 ◽  
Vol 118 (1) ◽  
pp. 87-99 ◽  
Author(s):  
Cleber Witt Saldanha ◽  
Caio Gomide Otoni ◽  
Diego Ismael Rocha ◽  
Paulo Cézar Cavatte ◽  
Kelly da Silva Coutinho Detmann ◽  
...  
Keyword(s):  
Pfaffia Glomerata ◽  
Brazilian Ginseng ◽  
Supporting Material

scholarly journals Comparative Study of Three Vitrification Solutions for an Effective Droplet-vitrification Cryopreservation Procedure for Pfaffia Glomerata (Spreng.) Pedersen

2021 ◽  
pp. 30-38
Author(s):  
Daniela Vasconcelos de Oliveira ◽  
Antonieta Nassif Salomão ◽  
Ildeu Soares Martins ◽  
Izulmé Rita Imaculada Santos
Keyword(s):  
Aluminum Foil ◽  
Medicinal Species ◽  
Droplet Vitrification ◽  
Germplasm Collections ◽  
Randomized Design ◽  
Two Temperatures ◽  
Study Laboratory ◽  
Suitable Technique ◽  
Cryopreservation Protocol

Aims: The objective of this research was to establish a cryopreservation protocol for shoot tips (ST) of in vitro P. glomerata using the droplet-vitrification technique. Study Design: The experimental design was a factorial, with four factors, arranged in a completely randomized design. Three vitrification solutions (PVS2, PVS3, PVS4), three times (20, 40, 60 min) and two temperatures (25 ± 2 °C and 0 °C) of treatment with the solutions, followed by freezing (LN+) or not (LN-) with liquid nitrogen (LN) were tested. All tests were performed using six replicates and the results analysed using Two-way ANOVA and Tukey’s tests and expressed as the mean ± the standard error of the means (SEM) deviation. Place and Duration of Study: Laboratory of Plant Cryobiology, Embrapa Genetic Resources and Biotechnology, over a two-year period. Methodology: ST excised from in vitro plantlets were pre-cultured overnight (19h), treated with a loading solution (LS) and three different vitrification solutions (PVS2, PVS3, PVS4) prior to freezing in LN. Treatment with the vitrification solutions was carried out at 0 or 25°C, for 20, 40 or 60 min. For freezing, drops of the vitrification solutions containing a single ST were dispensed on aluminum foil strips and the strips were submerged in LN (-196°C). For thawing, foil strips were submerged into unloading solution (US) at 40 ± 2°C, for three min. Thawed ST were transferred to regeneration medium and cultured in vitro. Results: Highest regeneration percentages after cryopreservation were 82% for ST treated with PVS3, at 0°C, for 60 min; 32% for ST treated with PVS4 at 25°C for 60 min or 0°C for 40 min and 22% for those treated with PVS2 at 0°C for 60 min. Conclusion: Droplet-vitrification is a suitable technique to ensure survival of P. glomerata ST after cryopreservation. This procedure can be applied to establish germplasm collections of this medicinal species in gene banks.

scholarly journals Preservation of In Vitro Grown Shoot Tips of Potato (Solanum tuberosum L.) by Different Methods of Cryopreservation

1970 ◽  
Vol 10 ◽  
pp. 15-20
Author(s):  
Shambhu P. Dhital ◽  
Hira K. Manandhar ◽  
Hak T. Lim
Keyword(s):  
Sucrose Concentration ◽  
Shoot Tips ◽  
Efficient Tool ◽  
Long Term Storage ◽  
In Vitro Plantlets ◽  
Tuber Morphology ◽  
Vegetatively Propagated Plants ◽  
Term Storage

Cryopreservation has been recognized as a practical and efficient tool for long-term storage of vegetatively propagated plants. This study was conducted to investigate the effects of sucrose concentration, hardening temperature and different cryopreservation methods on the survival rate of potato shoot tips after cryopreservation. Excised shoot tips of in vitro plantlets of potato cultivars, Atlantic and Superior were cryopreserved by vitrification, encapsulationvitrification and encapsulation-dehydration. Cryopreservation by vitrification method was used to determine the optimum concentration of sucrose and cold hardening temperature during sub-culturing period to the donor plantlets. Nine-percent sucrose gave 46.7% survival in Atlantic and 40% in Superior. The most optimum hardening temperature for 50% survival in Atlantic and 43.3% in Superior was 10°C. In the case of comparative study of three different cryopreservation methods, the highest survival (52%) as well as regeneration (46%) were observed when the shoot tips were cryopreserved by encapsulation-vitrification method, and the lowest survival (36%) and regeneration (28%) from the vitrification. Plant and tuber morphology of potato regenerated after cryopreservation were similar to those of the non-cryopreserved in vitro plantlets (control). Thus, this study demonstrated that encapsulation-vitrification method was the most effective one among other methods for higher survival as well as regeneration in in vitro shoot tips of potato.Key words: Cryopreservation; Dehydration; Encapsulation; Potato; Regeneration; VitrificationDOI: 10.3126/njst.v10i0.2804Nepal Journal of Science and Technology Volume 10, 2009 December Page: 15-20

scholarly journals Cryopreservation of cherry rootstock Gisela 5 using vitrification procedure

2014 ◽  
Vol 41 (No. 2) ◽  
pp. 55-63 ◽  
Author(s):  
Dj. Ružić ◽  
T. Vujović ◽  
R. Cerović
Keyword(s):  
Sucrose Concentration ◽  
Survival Rates ◽  
Shoot Tips ◽  
Prunus Cerasus ◽  
Ms Medium ◽  
Long Term Storage ◽  
Term Storage ◽  
Gisela 5

In vitro-grown shoot tips of Gisela 5 (Prunus cerasus × Prunus canescens) cherry rootstock were tested for regrowth after cryopreservation using vitrification technique. Explants were precultured in the dark at 23°C, in a liquid MS medium with a progressively increasing sucrose concentration (0.3 M for 15 h, then 0.7 M for 5 h), and subsequently loaded in a solution containing 2 M glycerol and 0.4 M sucrose for 20 minutes. Shoot tips were dehydrated at 0°C using either the original PVS2 or modified PVS2 solution (PVS A3 – 22.5% sucrose, 37.5% glycerol, 15% ethylene glycol and 15% DMSO) for 30, 40 and 50 minutes. The survival and regrowth of the cryopreserved shoot tips dehydrated with the original PVS2 solution ranged between 36–54% and 8–17%, respectively. However, the dehydration with the PVS A3 solution resulted in considerably higher survival rates (81–92%), as well as higher regrowth rates (39–56%) after cryopreservation. These results prove the feasibility of the PVS A3-based vitrification technique for a long-term storage of this genotype.  

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