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

Plants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2093
Author(s):  
Akansha Saxena ◽  
Wen-Lu Bi ◽  
Mukund R. Shukla ◽  
Syd Cannings ◽  
Bruce Bennett ◽  
...  
Keyword(s):  
Natural Habitat ◽  
Germplasm Conservation ◽  
Yukon Territory ◽  
Shoot Tips ◽  
Indole Butyric Acid ◽  
Special Concern ◽  
Basal Salts ◽  
Direct Immersion

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.

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 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.

scholarly journals In Vitro Propagation via Organogenesis and Formation of Globular Bodies of Salvia Plebeia: A Valuable Medicinal Plant

2020 ◽  
Author(s):  
Qinggui Wu ◽  
Honglin Yang ◽  
Yuxi Sun ◽  
Jinyao Hu ◽  
Lijuan Zou
Keyword(s):  
Medicinal Plant ◽  
Large Scale ◽  
Natural Habitat ◽  
Shoot Tips ◽  
Direct Organogenesis ◽  
Ms Medium ◽  
Hypocotyl Explants ◽  
Cotyledonary Nodes ◽  
Over Exploitation

Abstract Background: As a highly valued medicinal plant, Salvia plebeia R. Brown belongs to the Lamiaceae family that has been subjected to over exploitation in its natural habitat for phytochemical and pharmacological studies. Alternative collection methods need to be developed for the large-scale propagation of Salvia plebeian. Results: Here, efficient and simple, direct organogenesis (from shoot tips and cotyledonary nodes explants) and Globular bodies (GBs) induction (from hypocotyl explants) systems were developed for the in vitro propagation of Salvia plebeia. The highest and number of regenerated shoots (7.0±0.82) per shoot tips was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 0.1 mg L-1 indole-3-acetic acid (IAA) and 1.0 mg L-1 6-benzyladenine (6-BA), the proliferation of shoots and shoots rooted were carried out on the same medium treatments almost synchronously. Similarly, MS medium supplemented with 0.1 mg L-1 IAA and 1.0 mg L-1 thidiazuron (TDZ) yielded the maximum number of shoots (37.5±1.34) with 100% shoot sprouting frequency. Simultaneously, a protocol was developed for GBs induction from hypocotyl explants, and it produced 17.4 GBs per explant with 82.7% response on MS medium supplemented with TDZ (1.0 mg L-1) and IAA (0.1 mg L-1), and produced GBs that were morphologically similar to globular embryos and successfully germinated on hormone-free MS medium. The acclimatized plantlets with well-developed root systems were successfully shifted to the natural soils with a 100% survival rate. Conclusions: Taken together, this protocol can be efficiently used for mass propagation, germplasm preservation and likely also for gene transfer of Salvia plebeia.

scholarly journals Long-term in Vitro Regeneration of Hamelia patens

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 873G-874
Author(s):  
D. Sankhla ◽  
T.D. Davis ◽  
N. Sankhla ◽  
A. Upadhyaya
Keyword(s):  
Culture Medium ◽  
In Vitro Regeneration ◽  
Shoot Formation ◽  
Shoot Tips ◽  
Shoot Cultures ◽  
Ms Medium ◽  
Prolific Shoot ◽  
Ornamental Shrub

This report describes an efficient in vitro regeneration protocol for H. patens (firebush), a heat-tolerant ornamental shrub native to tropical and subtropical America. Shoot cultures were initially established using shoot tips placed on MS-revised medium containing 2.3 μM 2,4-D, 2.3 μM kinetin, and 0.25% polyvinylpyrrolidone. Other types of explants (nodal and internodal segments, leaf pieces, floral buds) did not regenerate shoots when placed on this medium. Two-month-old plantlets derived from the shoot tips were subcultured on MS medium supplemented with 0.5 μM thidiazuron (TDZ), and within 3 to 4 weeks, some callus was produced at the root–shoot junction. When this callus, with a small portion of the root and shoots, was placed on MS medium with 0.05 μM TDZ and 0.01 μM ABA, prolific shoot formation occurred within 3 to 4 weeks followed by root formation. By regular subculturing every 5 to 6 weeks, hundreds of plantlets have been obtained over the past 3 years with no apparent decline in regeneration potential. Addition of activated charcoal (0.5%) to the culture medium has greatly improved growth of the plantlets.

scholarly journals Cryopreservation of Shoot Tips and Pollen of Potato

2017 ◽  
Vol 76 (1) ◽  
pp. 75-80
Author(s):  
Paulina Smyda-Dajmund
Keyword(s):  
Liquid Nitrogen ◽  
Shoot Tips ◽  
Popular Method ◽  
Long Term Storage ◽  
Term Storage ◽  
Direct Immersion

Abstract Cryopreservation is a frequently used method of long-term storage of potato meristems and pollen in liquid nitrogen (LN) in temperature of -196°C. This technique allows for theoretically unlimited storage of potato material. The most popular method of potato shoot tips preservation is cryopreservation by the solidification of liquids without crystallization (vitrification).The best method of pollen conservation is its direct immersion in LN. The successful regeneration after vitrification is genotype-dependent, which require optimization of protocol.

scholarly journals Initial stages of Gymnadenia conopsea (Orchidaceae) morphogenesis in in vitro culture

2020 ◽  
Vol 24 ◽  
pp. 00059
Author(s):  
Alexandra Nabieva ◽  
Elena Zhmud ◽  
Yulianna Zaytseva
Keyword(s):  
Environmental Changes ◽  
Germplasm Conservation ◽  
West Siberia ◽  
Habitat Destruction ◽  
Ex Situ ◽  
Naphthaleneacetic Acid ◽  
Gymnadenia Conopsea ◽  
Initial Stage

In the West Siberia there is very little data on the biology of cenopopulations of Gymnadenia conopsea. Habitat destruction and environmental changes, which potentially affected species long-term viability, led to G. conopsea cenopopulations fragmentation and isolation. A detailed study of the morphology variability of the Fragrant orchid representatives was carried out in the cenopopulation in Novosibirsk region. Our results indicate that asymbiotic germination of G. conopsea seeds is difficult to achieve and the species has serious conservation issues. It is the first report when the introduction of G. conopsea in tissue culture was undertaken as the initial stage of germplasm conservation of Siberian cenopopulation. Three modified nutrient media with different growth additives were tested to promote protocorm and seedling formation. The advanced G. conopsea seedlings establishment was obtained in 1/3 Murashige and Skoog (MS) medium, supplemented by 1.0 mgl−1 2-isopentenyladenine (2iP), 0.1 mgl−1 1-Naphthaleneacetic acid (NAA) and 10% coconut water. This study allowed establishing a reliable and reproducible system for the G. conopsea maintenance and conservation ex situ.

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 Survival of and In Situ Gene Expression by Vibrio vulnificus at Varying Salinities in Estuarine Environments

2007 ◽  
Vol 74 (1) ◽  
pp. 182-187 ◽  
Author(s):  
Melissa K. Jones ◽  
Elizabeth Warner ◽  
James D. Oliver
Keyword(s):  
Gene Expression ◽  
Vibrio Vulnificus ◽  
Natural Habitat ◽  
Term Survival ◽  
Long Term Survival ◽  
Wide Range ◽  
Opportunistic Human Pathogen

ABSTRACT The opportunistic human pathogen Vibrio vulnificus survives in a wide range of ecological environments, which demonstrates its ability to adapt to highly variable conditions. Survival and gene expression under various conditions have been extensively studied in vitro; however, little work has been done to evaluate this bacterium in its natural habitat. Therefore, this study monitored the long-term survival of V. vulnificus in situ and simultaneously evaluated the expression of stress (rpoS, relA, hfq, and groEL) and putative virulence (vvpE, smcR, viuB, and trkA) genes at estuarine sites of varying salinity. Additionally, the survival and gene expression of an rpoS and an oxyR mutant were examined under the same conditions. Differences between the sampling sites in the long-term survival of any strain were not seen. However, differences were seen in the expression of viuB, trkA, and relA but our findings differed from what has been previously shown in vitro. These results also routinely demonstrated that genes required for survival under in vitro stress or host conditions are not necessarily required for survival in the water column. Overall, this study highlights the need for further in situ evaluation of this bacterium in order to gain a true understanding of its ecology and how it relates to its natural habitat.

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