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 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 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.  

scholarly journals Effect of the Duration of Liquid Nitrogen Storage on the Regrowth of Blackberry Cryopreserved by Droplet Vitrification

2017 ◽  
Vol 66 (1-2) ◽  
pp. 44-50
Author(s):  
Tatjana Vujović ◽  
Đurđina Ružić ◽  
Radosav Cerović
Keyword(s):  
Liquid Nitrogen ◽  
Room Temperature ◽  
Shoot Tips ◽  
Lower Survival Rate ◽  
Droplet Vitrification ◽  
Vitrification Solution ◽  
Long Term Preservation ◽  
Direct Immersion

SummaryIn vitro shoot tips of the blackberry cultivar ‘Čačanska Bestrna’ were cryopreserved using the droplet vitrification technique. Upon loading (30 min) in a solution of 1.9 M glycerol and 0.5 M sucrose, the explants were dehydrated for 40 min on ice with the PVS A3 vitrification solution (glycerol 37.5%, dimethyl sulfoxide 15%, ethylene glycol 15% and sucrose 22.5%) and for 40 min at room temperature with the PVS3 solution (glycerol 50% and sucrose 50%). They were subsequently frozen in individual microdroplets of vitrification solution, by direct immersion in liquid nitrogen (LN), and kept therein for 2, 4, 8 and 24 h. The explant rewarming was performed in an unloading solution (0.8 M sucrose) for 30 min at room temperature. The duration of LN exposure did not exert significant effects on the survival and regrowth of explants in both types of vitrification solutions. The survival and regrowth of cryopreserved shoot tips dehydrated with PVS3 solution ranged between 90–95% and 80–90%, respectively. However, dehydration with PVS A3 resulted in a lower survival rate (80–90%) and a considerably lower regrowth rate (55–65%) of explants. Monitoring the shoots regenerated in the in vitro culture revealed their normal capacity for multiplication and rooting in comparison with the controls, which fully confirms the purpose of cryopreservation in the long-term preservation of plant material.

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 Cryopreservation of in vitro-grown shoot tips of chrysanthemum by encapsulation-dehydration

2013 ◽  
Vol 25 (2) ◽  
pp. 133-140 ◽  
Author(s):  
Małgorzata Zalewska ◽  
Dariusz Kulus
Keyword(s):  
Survival Rate ◽  
Sucrose Concentration ◽  
Multiple Shoots ◽  
Shoot Tips ◽  
Long Term Storage ◽  
The Media ◽  
And Storage ◽  
Cryopreservation Protocol

ABSTRACT Chrysanthemums are amongst the most economically important flowers in the world. The protection and storage of these valuable genetic resources is of great importance. Today, cryopreservation, or the storage of biological material at the temperature of liquid nitrogen (-196°C), is believed to be the most promising long-term storage method. To optimise the cryopreservation protocol, the shoot tips of Chrysanthemum × grandiflorum /Ramat./ Kitam. ‘Lady Orange’ and ‘Lady Salmon’ mutants were cryopreserved using the encapsulation-dehydration technique. During the experiment, the influence of sucrose concentration (2, 3 and 6%) during preculture and the concentration of kinetin (0.25, 0.5, 0.75 and 1.0 mg dm-3) in the regrowth medium were tested. A higher survival rate was observed for ‘Lady Salmon’. In general, the media with higher sucrose levels provided the best survival and recovery rates (35-40%). Kinetin had no influence on the survival rate; however, it influenced the morphogenesis of the plants. The lowest number of explants forming multiple shoots was observed on the medium with the lowest sucrose (during preculture) and kinetin (in the recovery medium) concentration. On the other hand, the best rhizogenesis efficiency was observed when 0.25 mg dm-3 kinetin was added. In conclusion, the composition of both preculture and recovery media need to be adjusted to single cultivars. The use of 3% sucrose (preculture) and 0.25 mg dm-3 kinetin (recovery) seems reasonable, since it guarantees a satisfying recovery rate of the explants and at the same time prevents the formation of callus and multiple shoots, stimulating the rooting instead.

scholarly journals Integration of Cryopreservation and Tissue Culture for Germplasm Conservation and Propagation of Rosa pomifera ‘Karpatia’

2017 ◽  
Vol 45 (1) ◽  
pp. 208-214 ◽  
Author(s):  
Ewelina KWAŚNIEWSKA ◽  
Ewa DZIEDZIC ◽  
Bożena PAWŁOWSKA
Keyword(s):  
Treatment Time ◽  
Germplasm Conservation ◽  
Shoot Multiplication ◽  
Shoot Tips ◽  
Ms Medium ◽  
Droplet Vitrification ◽  
Multiplication Cycle ◽  
New Research

Cryopreservation is an useful technique for long-term conservation that requires minimal space and maintenance. Germplasm protection of Rosa is important to preserve genetic diversity, to store material for breeding and to expand new research. This study was conducted to develop a droplet vitrification cryopreservation and micropropagation of Rosa pomifera cv. ‘Karpatia’, whose large hypanthia are characterized by remarkable pro-health properties. Culture in vitro was stabilized and shoot tips collected from dormant buds served as initial explants. The multiplication of shoots was carried out on MS medium containing benzyladenine. For the droplet vitrification cryopreservation, shoot tips from in vitro cultures were used: small with exposed meristem, and large with a meristem covered with leaves, as well as shoot tips from in situ plants, which were collected in winter. Treatment time with plant vitrification solution (PVS2) was also tested (10-30 minutes). From in vitro culture, 32-41% small explants with exposed meristem survived, but they regenerated at a very low level. The best cryostorage results were obtained for shoot tips from dormant buds and a 20-minute PVS2 treatment: the survival was 84% and regeneration 72%. During the post-freezing regeneration multiplication index was 2.4 shoots per one multiplication cycle, after cryopreservation and in the control. On half MS medium without growth regulators, 97-99% of shoots rooted, and all rooted plants have adapted to ex vitro conditions and were planted into the soil. Biometric analyses during shoot multiplication, rooting and acclimatization stages did not reveal any changes compared to the non-cryopreserved samples.

scholarly journals Application of vitrification-derived cryotechniques for long-term storage of poplar and aspen (Populus spp.) germplasm

2008 ◽  
Vol 18 (2) ◽  
pp. 160 ◽  
Author(s):  
I. TSVETKOV ◽  
C. BENELLI ◽  
M. CAPUANA
Keyword(s):  
Woody Species ◽  
Growth Conditions ◽  
Shoot Tips ◽  
Hybrid Aspen ◽  
White Poplar ◽  
Long Term Storage ◽  
Apical Buds ◽  
High Level

The application of three different vitrification-based freezing strategies for the cryostorage of white poplar (Populus alba L.) and hybrid aspen (P. tremula L. × P. tremuloides Michx.) have been assessed. The PVS2 vitrification protocol was successfully applied to two white poplar in vitro clones stored for more than 6 months in slow-growth conditions (4 °C, in darkness) and showing clear signs of explant etiolation and decay. After 60 min of PVS2 treatment, P. alba L. (cv. Villafranca) explants isolated from axillary buds demonstrated significantly better potential for post-freeze regrowth (64%) compared to those obtained from apical buds (17%). Similarly, a high level of survival (78%) of the frozen hybrid aspen shoot tips was recorded following the application of the same technique. Using the ‘encapsulation-vitrification’ procedure, no toxic effects of the PVS2 treatment were noticed after 120 min exposure, however none of the cryopreserved (poplar and aspen) explants survived after 3 weeks. In contrast, the ‘droplet-vitrification’ technique appeared to be very efficient in the cryopreservation of white poplar shoot tips, which increases the opportunities for wider application of this method in other woody species.;

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