Cold hardening and slow cooling: tools for successful cryopreservation and recovery of in vitro shoot tips of silver birch

1996 ◽  
Vol 26 (11) ◽  
pp. 2015-2022 ◽  
Author(s):  
Leena Ryynänen
Keyword(s):  
Dimethyl Sulfoxide ◽  
Liquid Nitrogen ◽  
Callus Formation ◽  
Shoot Tips ◽  
Silver Birch ◽  
Mature Trees ◽  
Slow Cooling ◽  
Freezing Procedure ◽  
Light:Dark Cycle

Shoot tips of silver birch (Betulapendula Roth) derived from in vitro cultures originating from mature trees were used as explant material. Several pretreatments prior to cryopreservation were studied in order to enhance shoot tip recovery after storage in liquid nitrogen. Cold acclimation and the classical slow freezing procedure proved to be essential for successful survival, while both nonhardening and vitrification resulted in minimal survival. The optimum procedure was the following: Shoots were cold hardened (5 °C on a 8 h: 16 h (light:dark) cycle) for 3 weeks, and dissected shoot tips were then precultivated on woody plant medium with 5% dimethyl sulfoxide for 72 h. The material was transferred to cryotubes, and a mixture of 10% polyethylene glycol, 10% glucose, and 10% dimethyl sulfoxide was used as cryoprotectant. The cryotubes were slowly cooled to −38 °C before immersion in liquid nitrogen for 8 d. After fast thawing, axillary buds formed shoots without callus formation, but the growth of the new shoots was delayed from a few days to several weeks. Variability in regrowth was considerable as a result of genotype differences.

Effect of early spring birch bud type on post-thaw regrowth after prolonged cryostorage

1999 ◽  
Vol 29 (1) ◽  
pp. 47-52 ◽  
Author(s):  
Leena Ryynänen
Keyword(s):  
Liquid Nitrogen ◽  
Betula Pendula ◽  
Early Spring ◽  
Silver Birch ◽  
Slow Cooling ◽  
Betula Pendula Roth ◽  
Vegetative Buds ◽  
Female Catkin

Early spring buds of silver birch (Betula pendula Roth), collected with and without a female catkin in the middle of April, were cryopreserved using slow cooling followed by immersion in liquid nitrogen at -196°C for 8 days, 6 months, 1 year, 3 years, or 5 years. After fast thawing the buds were cultured in vitro according to the published protocol. The regrowth ability of the two types of buds was different. The regrowth of vegetative buds without a female catkin was good after all the cryopreservation times. There was a significant decrease in the regrowth ability of buds growing in the axil of a female catkin compared with the corresponding unfrozen controls after 1 year in cryostorage. In addition to the effect of the presence of a catkin on the regrowth ability, the late collecting time of the buds also probably decreased the regrowth and regrowth rates of both types of bud. The regrowth rates of buds without a catkin were 66, 67 and 24% after 1, 3, and 5 years of cryostorage, respectively, while those of buds with a catkin were 13.5, 32, and 2.6%, respectively.

scholarly journals In vitro Conservation and Cryopreservation of Nandina domestica, an Outdoor Ornamental Shrub

2013 ◽  
Vol 41 (2) ◽  
pp. 638 ◽  
Author(s):  
Aylin OZUDOGRU ◽  
Diogo Pedrosa Corrêa Da SILVA ◽  
Ergun KAYA ◽  
Giuliano DRADI ◽  
Renato PAIVA ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Storage Temperature ◽  
Sucrose Concentration ◽  
Aluminum Foil ◽  
Shoot Tips ◽  
In Vitro Conservation ◽  
Shoot Cultures ◽  
Storage Medium ◽  
Droplet Vitrification

The study focused on an economically-important ornamental outdoor shrub, Nandina domestica, with the aims to (i) optimize an effective in vitro conservation method, and (ii) develop a cryopreservation protocol for shoot tips by the PVS2 vitrification and droplet-vitrification techniques. For in vitro conservation of shoot cultures, the tested parameters were sucrose content in the storage medium (30, 45, 60 g/L) and storage temperature (4 °C or 8 °C). Cryopreservation was performed by applying the PVS2 vitrification solution, in 2-ml cryovials or in drops over aluminum foil strips, for 15, 30, 60 or 90 min at 0 °C, followed by the direct immersion in liquid nitrogen of shoot tips. Results show that N. domestica shoots can be conserved successfully for 6 months at both the temperatures tested, especially when 60 g/L sucrose is used in the storage medium. However, conservation at 4 °C showed to be more appropriate, as hyperhydricity was observed in post-conservation of shoots coming from storage at 8 °C. As for cryopreservation, a daily gradual increase of sucrose concentration (from 0.25 to 1.0 M) produced better protection to the samples that were stored in liquid nitrogen. Indeed, with this sucrose treatment method, a 30-min PVS2 incubation time was enough to produce, 60 days after thawing, the best recovery (47% and 50%) of shoot tips, cryopreserved with PVS2 vitrification and droplet-vitrification, respectively.

scholarly journals CLONAL PROPAGATION OF NEEM (Azadirachta indica A. Juss.) VIA DIRECT AND INDIRECT IN VITRO REGENERATION

2015 ◽  
Vol 39 (3) ◽  
pp. 439-445 ◽  
Author(s):  
Laureen Michelle Houllou ◽  
Robson Antônio de Souza ◽  
Elizabete Cristina Pacheco dos Santos ◽  
José Jackson Pereira da Silva ◽  
Marta Ribeiro Barbosa ◽  
...  
Keyword(s):  
Azadirachta Indica ◽  
Callus Formation ◽  
In Vitro Regeneration ◽  
Regenerative Process ◽  
Shoot Tips ◽  
Shoot Tip ◽  
Stem Base ◽  
Different Characteristics

ABSTRACTThe study was conducted with shoot tip explants of neem (Azadirachta indica A. Juss) to identify a viable regenerative process. Shoot tips were obtained from neem embryos cultured alternatingly in DKW medium supplemented with BAP and medium without hormones. Initial shoot development was influenced by cotyledon presence. Basal callus, excised from in vitro stem base, also presented organogenic potential. In some cases, plant lines, obtained from each seed, presented different characteristics. The most common characteristic observed in vitro was callus formation at the stem base. However, the rarest characteristics were stem callus formation and leaf senescence. The regenerated shoot tips were further subculture and rooted on a medium supplemented with IBA so that complete plants could be obtained. The rooted plants were transplanted to a greenhouse and successfully acclimatized. No significant differences in in vivo development were observed between neem plants from callus and from shoot tip propagation.

scholarly journals Stimulation of Stem Callus and Regeneration of Ficus elastica "Decora" and from Apical Tips in vitro

2013 ◽  
Vol 7 (2) ◽  
pp. 5-12
Author(s):  
Ragad M. Abdullah ◽  
Mazahim K. AL-Mallah
Keyword(s):  
Callus Formation ◽  
Potassium Nitrate ◽  
Shoot Tips ◽  
Peat Moss ◽  
Ms Medium ◽  
Ability To Regenerate ◽  
Calli Cultures ◽  
Clear Increase ◽  
Stimulation Of

Calli cultures of stems and leaves explants excised from field -grown rubber, Ficus elastica Decora, plants were formed on agar-solidified Murashige and Skoog (MS) medium.The results proved that MS medium supplemented with 1.0 mg L-1 benzyl adenin (BA) and 0.8mg L-1 2,4-dichloro-phenoxy acetic acid (2,4-D) was suitable to stimulate stem’s callus at ratio 87.5% . Whereas supplementation of MS medium with 0.5 mg L-1 of both BA and Indole -3-butyric acid (IBA)encouraged callus formation to reach76.6%. Leaves showed responses for callus initiation up to 75% at the same media. Stem calli showed limited ability to regenerate shoots on both agar solidified MS medium containing 1.0 mg L-1 2,4-D and kinetin (kin) 0.5 mg L-1 and MS medium supplemented with 0.5 mg L-1 of both BA and IBA. Transferring of shoots to differentiation medium (MS+0.5 mg L-1 BA+ 0.1 mg L-1 IBA) stimulated the growth and elongation of these shoots. Shoots were transferred to agar-solidified MS medium free from growth regulators failed to form roots. Because of the less number of shoots, other rooting media were not tested. The data showed, that shoot tips succeeded to regenerate shoots when they were cultured in different MS. The results proved clear increase in chlorophyll and protein content of the rubber shoots as compared with content of field grown rubber plants. It was noticed that agar-solidified MS medium supplemented with 4.0 mg L-1 BA was considered the optimum medium for shoots regeneration. All plants regenerated from shoot tips were readily rooted in agar-solidified MS medium with increasing Potassium Nitrate KNO3 from 1900 mg L-1 to 2000 mg L-1, and at the same medium supplemented with 3.0 mg L-1 IBA and 1.0 mg L-1 BA. All these plants were successfully acclimated and transferred to peat moss.

44 THE EFFECT OF SUCROSE CONCENTRATION FOR SINGLE-STEP DILUTION ON THE VIABILITY OF CRYOTOP-VITRIFIED IN VITRO-PRODUCED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 115
Author(s):  
S. Kondo ◽  
K. Imai ◽  
O. Dochi
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Liquid Nitrogen ◽  
Sucrose Concentration ◽  
Calf Serum ◽  
Single Step ◽  
Bovine Embryos ◽  
Vitrification Solution ◽  
Phosphate Buffered Saline

The aim of this study was to test sucrose concentrations for single-step dilution on the viability of vitrified in vitro-produced bovine embryos. Blastocysts (n = 173, 7 to 8 days after fertilization) were vitrified using the Cryotop (Kitazato, Tokyo, Japan) method placement by incubating the blastocysts in Dulbecco's phosphate buffered saline supplemented with 20% calf serum, 7.5% ethylene glycol, and 7.5% dimethyl sulfoxide for 3 min and then transferring into vitrification solution (Dulbecco's phosphate buffered saline supplemented with 20% calf serum, 16.5% ethylene glycol, 16.5% dimethyl sulfoxide, and 0.5 M sucrose). Each embryo was placed on a Cryotop with minimum volume of vitrification solution, and then the Cryotop was plunged into liquid nitrogen. Total time from placement in vitrification solution to plunging into liquid nitrogen was 1 min. The blastocysts were warmed by incubation in the single-step dilution medium for 5 min [0 M sucrose (n = 42), 0.25 M sucrose (n = 44), 0.5 M sucrose (n = 43), and 1.0 M sucrose (n = 44)] at 38.0°C. After dilution, the embryos were washed in TCM-199 supplemented with 20% calf serum and 0.1 mM β-mercaptoethanol and were cultured for 72 h in the same medium at 38.5°C in an atmosphere of 5% CO2. The rates of re-expanded blastocysts and hatched blastocysts were determined at 24 and 72 h after warming, respectively. Data were analysed using the chi-squared test. The percent of re-expanded blastocysts at 24 h after warming in dilution medium supplemented with any level of sucrose was significantly higher (P < 0.05) than in blastocysts warmed without sucrose (Table 1). The hatched blastocyst rate of embryos at 72 h after warming in dilution medium with 0.5 M sucrose was significant higher than that with no sucrose. There were no differences in hatched blastocyst rates between the sucrose concentrations supplemented to the dilution medium. These results suggest that embryos vitrified by the Cryotop method can be diluted in single-step dilution using 0.25, 0.5, or 1.0 M sucrose supplemented to the medium. Table 1.The effect of sucrose concentration for single-step dilution on the viability of Cryotop vitrified in vitro-produced bovine embryos

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 Micropropagation of Citrus indica Tanaka using Shoot tips from Mature Trees

2021 ◽  
Vol 31 (1) ◽  
pp. 13-23
Author(s):  
Kiran Chhetri ◽  
Binu Mathew ◽  
Lolly S Pereira
Keyword(s):  
Plant Tissue ◽  
Shoot Tips ◽  
Direct Regeneration ◽  
Root Induction ◽  
Ms Medium ◽  
Mature Trees ◽  
Shoot Initiation ◽  
Potting Medium ◽  
Shoot Tip Explants

A study was conducted to standardize a protocol for in-vitro direct regeneration and mass multiplication of Citrus indica Tanaka using shoot tip explants excised from mature trees. Shoot tips were inoculated in MS medium supplemented with varying concentrations and combinations of cytokinins and auxins. MS media when fortified with BAP 0.5 mg/l and 0.5 mg/l BAP + 1.0 mg/l Kn were found to be the best treatments for shoot initiation while MS supplemented with 1 mg/l IBA; 0.5 mg/l NAA + 0.5 mg/l IBA and 0.5 mg/l NAA + 0.5 mg/l IAA were the best treatments for root induction. Among the different media used for hardening, 100 % survivability was obtained when plantlets were hardened using vermicompost as the potting medium. Subsequently, these plantlets were transferred to larger pots and acclimatization was achieved gradually in outdoor conditions. Plant Tissue Cult. & Biotech. 31(1): 13-23, 2021 (June)

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