scholarly journals Droplet vitrification technique for cryopreservation of a large diversity of blackcurrant (Ribes nigrum L.) cultivars

2020 ◽  
Author(s):  
Saija Rantala ◽  
Janne Kaseva ◽  
Anna Nukari ◽  
Jaana Laamanen ◽  
Saara Tuohimetsä ◽  
...  
Keyword(s):  
Cultural Values ◽  
Core Collection ◽  
Gene Bank ◽  
Ribes Nigrum ◽  
Ex Vitro ◽  
Droplet Vitrification ◽  
Successful Transfer ◽  
Future Utilization

AbstractThe aim of plant gene banks is to preserve genetic resources selected based on their phenotypic, agronomic, historical or other cultural values for future utilization. In the present study the modified PVS2 droplet vitrification technique was tested and optimized for cryopreservation of a large diversity of blackcurrant (R. nigrum L.) accessions propagated in vitro and selected into a national gene bank core collection. Out of four accessions tested to optimize the method, three recovered and regenerated by 89–97% on average, but one recalcitrant in vitro line only by 25%. The tested post-cryopreservation recovery media with different macronutrient and growth regulator levels showed no generalized effect on regenerated shoots, but the effect of recovery media was different between cultivars. When the whole regeneration chain from cryopreservation via micropropagation to greenhouse conditions was tested, shoots at least 1 cm in length were found necessary for successful transfer ex vitro. The long-term cryopreservation of 22 blackcurrant accessions was finally conducted, with practices slightly modified from the tested protocol. The estimated recovery of shoot tips after 9 weeks in vitro was 17–94% with at least 75% recovery in seven accessions and at least 40% recovery in 19 out of 22 accessions. Only one accession had no cryopreservation success. The results demonstrated that the modified droplet vitrification technique is appropriate for a large diversity of blackcurrant accessions. However, cultivar-related differences and recovery procedures are to be considered for success in regeneration and ex vitro adaptation.

Long-term in vitro Storage Technique of Valuable Birch Genotypes and Plant Production on its Basis

2019 ◽  
pp. 57-67
Author(s):  
T.M. Tabatskaya ◽  
N.I. Vnukova
Keyword(s):  
Plant Production ◽  
High Survival ◽  
Ex Vitro ◽  
Regenerative Capacity ◽  
In Vitro Storage ◽  
Interspecific Differences ◽  
Regenerated Plants ◽  
Growing Conditions

A technique for the long-term (up to 27 years) in vitro storage of valuable birch genotypes under normal (25 °C, 2.0 klx, 16-h day and 8-h night) and low temperature (4 °C, 0.5 klx, 6-h day and 18-h night) growing conditions on hormone-free media has been described. The study explored for the first time the influence of different strategies to store the clones of Betula pubescens and B. pendula var. сarelica (6 genotypes) on the regenerative capacity of collection samples, adaptive potential of regenerated plants and plant production by the in vitro and ex vitro techniques. It was established that both storage strategies provided a persistently high survival rate (82-100%) and regenerative capacity of in vitro shoots (the multiplication coefficient of 4.2-6.3 and rhizogenic activity of 90-100%). The clones retained their characteristics of height growth under the in vitro and ex vitro conditions, and demonstrated intraclonal homogeneity and lack of signs of somaclonal variability. The plants showed substantial interspecific differences at the stage of multiplication and transfer to the greenhouse. The highest percentage of acclimated plants (75-98% depending on the clone genotype) was obtained after planting of micro plants straight in the greenhouse, which simplified the technology and made plant production less costly. long-term in vitro storage, birch, species, genotype, micropropagation, ex vitro adaptation, plant material

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 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 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 Gametophyte and sporophyte of tree ferns in vitro culture

2011 ◽  
Vol 76 (3) ◽  
pp. 193-199 ◽  
Author(s):  
Katarzyna Goller ◽  
Jan J. Rybczyński
Keyword(s):  
Development Time ◽  
Culture Conditions ◽  
In Vitro Cultures ◽  
Ex Vitro ◽  
Stage Of Development ◽  
Tree Ferns ◽  
Time Required ◽  
New Generation

Experiments had been carried out on gametophytes and very young fronds of sporophytes with application of Murashige and Skoog (1962) medium. The paper described the results of 15 years in vitro experiments on 16 species of tree ferns belonging to various genera: <em>Blechnum</em>, <em>Cibotium</em>, <em>Cyathea</em> and <em>Dicksonia</em>. Genus <em>Cyathea</em> was represented by: <em>C. australis</em> (R.Br.) Domin., <em>C. capensis</em> (L.f.) Sm., <em>C. cooperi</em> (F.Muell.) Domin, <em>C. brownii</em> Domin, <em>C. dealbata</em> (G.Forest) Sw., <em>C. dregei</em> Kunze, <em>C. leichhardtiana</em> (F.Muell.) Copel., <em>C. robertsiana</em> (F.Muell.) Domin., <em>C. schanschin</em> Mart., <em>C. smithii</em> Hook.f. and <em>Cyathea</em> sp. In case of genus <em>Dicksonia</em> only two species were introduced into our experiments: <em>D. fibrosa</em> Colenso and <em>D. sellowiana</em> Hook.. Taxa <em>Blechnum</em> was presented by <em>B. brasiliense</em> Desv. and <em>Cibotium</em> by <em>C. glaucum</em> (Sm.) Hook. and Arn. and <em>C. schiedei</em> Schltdl. and Cham.. The studied species presented various responses on culture conditions depending on the level of stage of development. Time required for spores germination differed between species and took from only a few to 16 weeks. Prothalium formations showed various types of growth presented by marginal meristems. For all investigated species long term gametophyte in vitro cultures was established. Mature gametophyte possessed functional antheridia and archegonia. Spontaneous fertilization helped to establish the culture of young sporophytes. For all species the ex vitro culture in greenhouse collection was established. Manipulation of sucrose content in the medium stimulated the multiplication of gametophytes, but its lack induced formation of gemmae. Apospory was observed when culture of very young fronds was extended for 6 months and new generation of gametophytes was developed. Finally, sporophytes of 12 species were obtained and they have been growing in our greenhouse.

scholarly journals Long-Term Subculture Affects Rooting Competence via Changes in the Hormones and Protein Profiles in Cedrela Fissilis Vell. (Meliaceae) Shoots

2021 ◽  
Author(s):  
Tadeu dos Reis Oliveira ◽  
Damián Balfagón ◽  
Kariane Rodrigues Sousa ◽  
Victor Paulo Mesquita Aragão ◽  
Leandro Francisco de Oliveira ◽  
...  
Keyword(s):  
Large Scale ◽  
Molecular Mechanisms ◽  
Monodehydroascorbate Reductase ◽  
Protein Profiles ◽  
Ex Vitro ◽  
Plantlet Production ◽  
Auxin Binding Protein ◽  
Indole 3 Acetic Acid

Abstract Long-term subculture plays an essential role in the large-scale multiplication and production of somatic plantlets. We investigated the effects of long-term subculture on in vitro shoot development and ex vitro rooting associated with changes in the hormones and protein profiles in C. fissilis. The number of subcultures of shoots induced a decrease in the ex vitro rooting response. The reduction in adventitious root (AR) formation was associated with decreases in the contents of indole-3-acetic acid (IAA), abscisic acid (ABA), 12-oxo phytodienoic acid (OPDA), putrescine (Put), and spermine and increases in jasmonic acid (JA), jasmonoyl-isoleucine, trans-cinnamic acid, and salicylic acid contents in shoots at the fourth subculture compared to the first. The ornithine decarboxylase enzyme preferentially functions in the Put biosynthesis pathway and was related to the highest AR formation in shoots at the first subculture. Down-accumulation of the auxin-binding protein ABP19a in shoots from the fourth subculture compared to the first subculture was related to a decrease in both IAA contents and AR formation. In addition, down-accumulation of glucose-6-phosphate isomerase, glutamine synthetase leaf isozyme chloroplastic, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, L-ascorbate peroxidase, cytosolic, monodehydroascorbate reductase, and 2-Cys peroxiredoxin BAS1-like, chloroplastic and up-accumulation of caffeoyl-CoA O-methyltransferase 1 and isoforms of peroxidase 4 proteins in shoots from the fourth relative to the first subculture were associated with a reduction in AR formation. These results showed that the understanding of hormonal and molecular mechanisms related to the potential of AR formation in shoots under successive subcultures is relevant to improving large-scale plantlet production in C. fissilis.

scholarly journals Valorisation of Ribes nigrum L. Pomace, an Agri-Food By-Product to Design a New Cosmetic Active

Cosmetics ◽  
2020 ◽  
Vol 7 (3) ◽  
pp. 56
Author(s):  
Hortense Plainfossé ◽  
Manon Trinel ◽  
Grégory Verger-Dubois ◽  
Stéphane Azoulay ◽  
Pauline Burger ◽  
...  
Keyword(s):  
Circular Economy ◽  
Raw Material ◽  
Waste Materials ◽  
Phytochemical Analysis ◽  
Ribes Nigrum ◽  
Cosmetic Ingredients ◽  
In Vitro Bioassays ◽  
By Products

The ethical and ecological concerns of today’s consumers looking for both sustainable and efficient ingredients in finished products, put a lot of pressure on the cosmetic market actors who are being driven to profoundly modify the strategies adopted to innovate in terms of actives while notably being urged to switch from petroleum- to plant-based ingredients. To produce such natural cosmetic ingredients, agri-food by-products are advocated as raw material due to their reduced carbon footprint as they actively contribute to the worldwide improvement of waste management. The process to transform plant waste materials into such powerful and objectified “green” cosmetic actives in compliance with circular economy principles is a long-term integrated process. Such a development is thoroughly exemplified in the present paper through the description of the design of liquid anti-age ingredients based on Ribes nigrum L. extract. This was obtained by maceration of blackcurrant pomace. and the embodiment of this extract following its phytochemical analysis notably by HPLC-DAD-ELSD and its bioguided fractionation using in vitro bioassays.

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 Cryopreservation of Shoot Tips of Elite Cultivars of Cannabis sativa L. by Droplet Vitrification

2019 ◽  
Vol 2 (1) ◽  
pp. 29-34 ◽  
Author(s):  
Esther Uchendu ◽  
Hemant Lata ◽  
Suman Chandra ◽  
Ikhlas A. Khan ◽  
Mahmoud A. ElSohly
Keyword(s):  
Exposure Duration ◽  
Cannabis Sativa ◽  
Germplasm Conservation ◽  
Shoot Tips ◽  
Fiber Production ◽  
Droplet Vitrification ◽  
Protocol Development ◽  
Significant Difference

Cannabis sativa L. (marijuana or hemp) is recognized worldwide for its psychoactive properties as well as for fiber production. This study focused on the evaluation of 3 droplet vitrification protocols for long-term conservation of shoot tips in liquid nitrogen (LN). Shoot tips (∼0.5 mm) were excised from 3- to 4-week-old in vitro-grown shoots of 3 cultivars (MX, VI-20, and B-5: high tetrahydrocannabinol [THC], high cannabidiol [CBD], and intermediate THC∼CBD, respectively) and pretreated on 5% dimethyl sulfoxide agar plates for 48 h. The shoot tips were then vitrified in LN using 3 separate cryoprotectant (plant vitrification solutions [PVS] #2, #3, and #4) droplets on an aluminum cryoplate. There was no significant difference between the regrowth of cryopreserved shoot tips exposed to PVS2 for 15 and 20 min, but regrowth of all 3 cultivars significantly declined after 20 min of exposure. Exposure duration of 15 min was adapted for subsequent experiments. Regrowth of cryopreserved MX was significantly higher with PVS2 (63%) than with PVS3 and PVS4 (≤5%). Regrowth of cryopreserved VI-20 was highest with PVS2 (57%) and significantly higher than with PVS3 and PVS4 (≤25%). The regrowth of cryopreserved shoot tips of B-5 was significantly different between all 3 protocols with PVS2 > PVS4 > PVS3. Both PVS2 and PVS4 produced regrowth above 55%, while regrowth with PVS3 was significantly lower (31%). These results indicate that 15–20 min of exposure to PVS2 are most suitable for cryopreservation of these varieties. This is the first report on protocol development for the cryopreservation of organized tissues of C. sativa L. for germplasm conservation.

Sign in / Sign up

Export Citation Format

Share Document