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 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 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 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 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.;

Current situation with the core collection of spring triticale of the National Center of Plant Genetic Resources of Ukraine

2018 ◽  
pp. 75-85
Author(s):  
S.V. Chernobai ◽  
V.K. Riabchun ◽  
T.B. Kapustina ◽  
V.S. Melnyk ◽  
O.E. Shchechenko
Keyword(s):  
Genetic Resources ◽  
Plant Genetic Resources ◽  
Plant Production ◽  
Educational Institutions ◽  
Breeding Lines ◽  
Growing Season Length ◽  
Long Term Storage ◽  
Foreign Countries ◽  
Spring Triticale

Goal. To build up a spring triticale genetic bank to provide breeding, scientific and educational institutions with initial material and to preserve the existing diversity. To update the database of accessions with a set of valuable economic and morphological features. Results and discussion. The methodology and results of the collection formation and evaluation of spring triticale accessions in the National Center for Plant Genetic Resources of Ukraine of Plant Production Institute nd. a V. Ya. Yuriev are presented. The formed collection includes 1,935 accessions from 27 countries: 42 varieties and 1,478 breeding lines from Ukraine, 92 varieties and 248 lines from foreign countries and also 75 genetic lines. The collection was formed by major valuable economic features (plant height, growing season length, spike threshing, yield, 1000-grain weight, disease resistance, technological properties, etc.). Accessions with the majority of morpho-biological and valuable economic features were selected. All the accessions in the collection are certificated. 1,762 accessions were packed for storage into the National Depository; 1,507 of them were packed for long-term storage. Conclusions. The gene pool of spring triticale from the collection of the Gene Bank of Plants of Ukraine is widely used for breeding. This allows conducting hybridization of genetically and ecologically remote forms with various expressions of features and obtaining whole new breeding material. Involvement of collection accessions in breeding allows generating new genetic sources of valuable economic features.

scholarly journals Salinity Effects on Gene Expression, Morphological, and Physio-Biochemical Responses of Stevia rebaudiana Bertoni In Vitro

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 820
Author(s):  
Clara Azzam ◽  
Sudad Al-Taweel ◽  
Ranya Abdel-Aziz ◽  
Karim Rabea ◽  
Alaa Abou-Sreea ◽  
...  
Keyword(s):  
Salt Tolerance ◽  
Biochemical Parameters ◽  
Large Scale ◽  
Stevia Rebaudiana ◽  
Multiple Shoots ◽  
High Salt ◽  
Stress Factors ◽  
Stevia Rebaudiana Bertoni ◽  
Nacl Concentration

Stevia rebaudiana Bertoni is a little bush, which is cultivated on a large scale in many countries for medicinal purposes and used as a natural sweetener in food products. The present work aims to conduct a protocol for stevia propagation in vitro to produce and introduce Stevia rebaudiana plants as a new sweetener crop to Egyptian agriculture. To efficiently maximize its propagation, it is important to study the influence of stress factors on the growth and development of Stevia rebaudiana grown in vitro. Two stevia varieties were investigated (Sugar High A3 and Spanti) against salt stress. Leaves were used as the source of explants for callus initiation, regeneration, multiplication and rooting. Some stress-related traits, i.e., photosynthetic pigments, proline contents, and enzyme activity for peroxidase (POD), polyphenol oxidase (PPO), and malate dehydrogenase (MDH) were studied. Murashig and Skoog (MS) medium was supplemented with four NaCl concentrations: 500, 1000, 2000, and 3000 mgL−1, while a salt-free medium was used as the control. The data revealed that salinity negatively affected all studied characters: the number of surviving calli, regeneration%, shoot length, the number of multiple shoots, number of leaf plantlets−1, number of root plantlets−1, and root length. The data also revealed that Sugar High A3 is more tolerant than Spanti. The total chlorophyll content decreased gradually with increasing NaCl concentration. However, the opposite was true for proline content. Isozyme’s fractionation exhibited high levels of variability among the two varieties. Various biochemical parameters associated with salt tolerance were detected in POD. Namely, POD4, POD6, POD 9 at an Rf of 0.34, 0.57, and 0.91 in the Sugar High A3 variety under high salt concentration conditions, as well as POD 10 at an Rf of 0.98 in both varieties under high salt concentrations. In addition, the overexpression of POD 5 and POD 10 at Rf 0.52 and 0.83 was found in both varieties at high NaCl concentrations. Biochemical parameters associated with salt tolerance were detected in PPO (PPO1, PPO2 and PPO4 at an Rf of 0.38, 0.42 and 0.62 in the Sugar High A3 variety under high salt concentrations) and MDH (MDH 3 at an Rf of 0.40 in both varieties at high salt concentrations). Therefore, these could be considered as important biochemical markers associated with salt tolerance and could be applied in stevia breeding programs (marker-assisted selection). This investigation recommends stevia variety Sugar High A3 to be cultivated under salt conditions.

scholarly journals Efficient Cryopreservation of Populus tremula by In Vitro-Grown Axillary Buds and Genetic Stability of Recovered Plants

Plants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 77
Author(s):  
Elena O. Vidyagina ◽  
Nikolay N. Kharchenko ◽  
Konstantin A. Shestibratov
Keyword(s):  
Survival Rate ◽  
Axillary Buds ◽  
Long Term Storage ◽  
Average Survival ◽  
Transgenic Lines ◽  
Ssr Loci ◽  
One Step ◽  
The One

Axillary buds of in vitro microshoots were successfully frozen at –196 °C by the one-step freezing method using the protective vitrification solution 2 (PVS2). Microshoots were taken from 11 transgenic lines and three wild type lines. Influence of different explant pretreatments were analyzed from the point of their influence towards recovery after cryopreservation. It was found out that the use of axillary buds as explants after removal of the apical one increases recovery on average by 8%. The cultivation on growth medium of higher density insignificantly raises the regenerants survival rate. Pretreatment of the osmotic fluid (OF) shows the greatest influence on the survival rate. It leads to the increase in survival rate by 20%. The cryopreservation technology providing regenerants average survival rate of 83% was developed. It was based on the experimental results obtained with explant pretreatment. Incubation time in liquid nitrogen did not affect the explants survival rate after thawing. After six months cryostorage of samples their genetic variability was analyzed. Six variable simple sequence repeat (SSR) loci were used to analyze genotype variability after the freezing-thawing procedure. The microsatellite analysis showed the genetic status identity of plants after cryopreservation and of the original genotypes. The presence of the recombinant gene in the transgenic lines after cryostorage were confirmed so as the interclonal variation in the growth rate under greenhouse conditions. The developed technique is recommended for long-term storage of various breeding and genetically modified lines of aspen plants, as it provides a high percentage of explants survival with no changes in genotype.

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