scholarly journals Cryopreservation of shoot tips of endangered Hayachine-usuyukiso ( Leontopodium hayachinense (Takeda) Hara et Kitam.) using a vitrification protocol

2008 ◽  
Vol 6 (02) ◽  
pp. 164-166 ◽  
Author(s):  
Daisuke Tanaka ◽  
Takao Niino ◽  
Yoshiko Tsuchiya ◽  
Kazuto Shirata ◽  
Matsuo Uemura
Keyword(s):  
Survival Rate ◽  
Liquid Nitrogen ◽  
Shoot Tips ◽  
Endangered Plant ◽  
Alpine Plant ◽  
Promising Technique ◽  
Skoog Medium ◽  
Murashige Skoog ◽  
Iwate Prefecture

Hayachine-usuyukiso (Leontopodium hayachinense) is an alpine plant native to Mt Hayachine. This unique chrysanthemum is listed as an endangered plant by the Department of Conservation, Iwate Prefecture, and as a threatened plant by the Ministry of the Environment, Japan. We successfully cryopreserved the shoot tips fromin vitro-grownL. hayachinenseshoots using a vitrification protocol. Cold-hardened shoot tips were excised and pre-cultured on a solidified Murashige–Skoog medium containing 0.3 M sucrose for 1 d at 5°C. The shoot tips were then treated with loading solution for 20 min at 25°C, dehydrated in plant vitrification solution 2 for 120 min at 25°C and immersed in liquid nitrogen. The survival rate of the vitrified shoot tips was 63.3% after 30 d of regrowth. This protocol appears to be a promising technique for the cryopreservation ofin vitro-grown shoots of this endangered plant.

Cryopreservation of in vitro-grown shoot tips of strawberry by the vitrification method using aluminium cryo-plates

2011 ◽  
Vol 10 (1) ◽  
pp. 14-19 ◽  
Author(s):  
Shin-ichi Yamamoto ◽  
Kuniaki Fukui ◽  
Tariq Rafique ◽  
Nayyar Iqbal Khan ◽  
Carlos Roman Castillo Martinez ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Sucrose Solution ◽  
Shoot Tips ◽  
New Method ◽  
Fragaria Ananassa ◽  
Alginate Gel ◽  
Skoog Medium ◽  
Murashige And Skoog Medium ◽  
Strawberry Cultivars

Cryopreservation using an aluminium cryo-plate was successfully applied to in vitro-grown strawberry (Fragaria × ananassa Duch.) shoot tips. The shoots were cold-hardened at 5°C for 3 weeks with an 8-h photoperiod. The shoot tips (1.5–2.0 mm × 0.5–1.0 mm) were dissected from the shoot and pre-cultured at 5°C for 2 d on Murashige and Skoog medium containing 2 M glycerol and 0.3 M sucrose. The pre-cultured shoot tips were placed on the aluminium cryo-plate containing ten wells embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates in a loading solution (2 M glycerol and 0.8 M sucrose) for 30 min at 25°C. Dehydration was performed by immersing the cryo-plates in plant vitrification solution 2 for 50 min at 25°C. Then, the cryo-plate with shoot tips was transferred into an uncapped cryotube that was held on a cryo-cane and directly immersed into liquid nitrogen (LN). After storage in LN, shoot tips attached to the cryo-plate were directly immersed into 2 ml of a 1 M sucrose solution for regeneration. Using this procedure, the average regrowth level of vitrified shoot tips of 15 strawberry cultivars reached 81%. This new method has many advantages and will facilitate the cryostorage of strawberry germplasm.

scholarly journals Critical vitrification steps towards survival of Garcinia hombroniana (Clusiaceae) shoot tips after cryopreservation

2018 ◽  
Vol 66 (3) ◽  
pp. 1314
Author(s):  
Norafarain Sulong ◽  
Nurul Farhana Shahabudin ◽  
Normah Mohd Noor
Keyword(s):  
Water Content ◽  
Liquid Nitrogen ◽  
Shoot Tips ◽  
The Other ◽  
Skoog Medium ◽  
Murashige And Skoog Medium ◽  
Vitrification Solution ◽  
Cryopreservation Protocol ◽  
Recovery Medium

A cryopreservation protocol was developed for in vitro shoot tips of Garcinia hombroniana using the vitrification technique. Four critical steps in the technique were investigated, namely preculture, loading, dehydration with Plant Vitrification Solution 2 (PVS2), and unloading. Shoot tips precultured for 48 hr gave significantly higher survival (75 %) compared to 24 hr preculture (50 %) after cryopreservation. Treatment with 1 M glycerol plus 0.4 M sucrose as a loading solution gave higher survival (45.83 %) compared to the other treatments (0.4 M sucrose + 2 M glycerol; 0.4 M sucrose). Shoot tips dehydrated with PVS2 for 25 min gave the highest survival after immersion in liquid nitrogen. Stepwise PVS2 treatment for 15 min with 50 % PVS2 followed by 10 min with 100 % PVS2 solution improved survival of the shoot tips after cryopreservation (41.67 %). Murashige and Skoog medium with 0.4 M sucrose gave significantly higher survival (66.67 %) than MS with 1.2 M sucrose (25 %) as an unloading solution. Water content was shown to decrease throughout the whole vitrification steps from 6.83 ± 1.66 g g-1 dw for fresh shoot tips down to 2.93 ± 0.28 g g-1 dw after PVS2 treatment. Further study on each step including recovery medium is required to improve the survival. Nevertheless, the present study showed the potential of using the vitrification technique for cryopreservation of G. hombroniana.

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 The Dynamics of Cell Division in Some Local Potato Varieties Cultivated in Vitro on Micropropagation Medium

2012 ◽  
Vol 45 (3) ◽  
pp. 71-78
Author(s):  
Iustina Brînduşa Ciobanu ◽  
Dana Constantinovici ◽  
L. Creţu
Keyword(s):  
Solanum Tuberosum ◽  
Cell Division ◽  
Mitotic Index ◽  
Accumulation Rate ◽  
Solanum Tuberosum L ◽  
In Vitro Cultures ◽  
Local Varieties ◽  
Skoog Medium ◽  
Murashige Skoog

Abstract This study was performed to reveal the changes in cell division, as a result of the prolonged period of subculture on micropropagation medium, of five local varieties of Solanum tuberosum L. maintained on in vitro collection at Suceava Genebank, Romania. For this purpose it was used the Murashige-Skoog medium (MS- 1962) with addition of 40 g/l sucrose, and 6 mg/l daminozide. The effect of prolonged period of subculture up to two and 12 months was expressed as mitotic index and frequency of cells with abnormal division. Mitotic index ranged from 20.1 to 22.1% after 12 days, between 15.5 - 17.7% after two months and between 17.7 - 19.2% after 12 months of subculture. The results obtained showed that the frequency of aberrant cells increased with the preservation time on the in vitro cultures and their accumulation rate depended on the genotype. Were identified interphases with micronuclei, metaphases with retarded chromosomes, ana-telophases with chromosomal bridges, retarded chromosomes and chromosomal fragments, but their percentage was low in all the genotypes.

114 CRYOTOP VITRIFICATION FOR IN VITRO-PRODUCED BUFFALO (BUBALUS BUBALIS) EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 174 ◽  
Author(s):  
A. De Rosa ◽  
L. Attanasio ◽  
L. Boccia ◽  
G. Pellerano ◽  
G. Campanile ◽  
...  
Keyword(s):  
Survival Rate ◽  
Liquid Nitrogen ◽  
Sucrose Solution ◽  
Bubalus Bubalis ◽  
Thin Strip ◽  
Embryo Survival ◽  
Vitro Fertilization ◽  
Group A ◽  
Group B

The aim of this study was to compare the efficiency of different combinations of cryoprotectants for vitrification of IVP buffalo (Bubalus bubalis) embryos by the cryotop method (Kuwayama et al. 2005 RBM Online 11, 300–308). In group A, we evaluated the vitrification and warming solutions previously used to vitrify buffalo embryos in French straws (Gasparrini et al. 2001 Theriogenology 55, 307). Embryos were equilibrated in 1.4 M glycerol for 5 min and in 1.4 M glycerol and 3.6 M ethylene glycol (EG) for an additional 5 min. After being transferred into 3.4 M glycerol and 4.6 M EG for 25 s, individual embryos were picked up in an extremely small volume (<0.1 �L) of vitrification solution and placed on the top of a very fine polypropylene strip (0.4 mm wide � 20 mm long � 0.1 mm thick) attached to a hard plastic handle, kindly provided by M. Kuwayama. Each embryo was placed onto the thin strip of the Cryotop and immediately submerged into liquid nitrogen. For warming, the strip of the Cryotop was immersed directly into a 0.5 M sucrose solution; embryos were retrieved and transferred into 0.25 M sucrose for 5 min before culture in SOF medium. In group B, we examined the vitrification and warming solutions previously used for OPS vitrification of buffalo embryos (De Rosa et al. 2006 Reprod. Fertil. Dev. 18, 153). Embryos were equilibrated in 7.5% EG + 7.5% dimethyl sulfoxide (DMSO) for 3 min before transfer into 16.5% EG + 16.5% DMSO + 0.5 M sucrose. After 25 s, they were placed on the cryotop, as previously described, and submerged into liquid nitrogen. For warming, embryos were recovered into a 0.25 M sucrose solution for 1 min, transferred into 0.15 M sucrose for 5 min, and cultured in SOF. IVP buffalo embryos of excellent quality that, by Day 7 of culture (Day 0 = in vitro fertilization), had reached the blastocyst stage (n = 44 and 53 for groups A and B, respectively), over 6 replicates, were vitrified. Embryo survival rate was determined as the percentage of vitrified-warmed embryos undergoing further development during a 24-h in vitro culture period. Differences between methods were analyzed by chi-square test. A significantly higher embryo survival rate was recorded in Group B compared to Group A (67.9 vs. 43.2% respectively; P < 0.05). In conclusion, it was demonstrated that cryotop vitrification, with the combination of cryoprotectants used in group B, is a valid tool to cryopreserve IVP buffalo blastocysts.

MICROPROPAGATION OF Helianthus maximiliani (Schrader) BY SHOOT APEX CULTURE / MICROPROPAGACION DE Helianthus maximiliani (Schrader) POR EL CULTIVO DE VAINAS DE LOS VASTAGOS / MICROPROPAGATION DE L’Helianthus maximiliani (Schrader) AU MOYEN DE LA CULTURE DE L’APEX DE LA POUSSE

Helia ◽  
2001 ◽  
Vol 24 (34) ◽  
pp. 63-68 ◽  
Author(s):  
Vasić Dragana ◽  
Škorić Dragan ◽  
Alibert Gilbert ◽  
Miklič Vladimir
Keyword(s):  
Silver Nitrate ◽  
Shoot Apex ◽  
Casein Hydrolysate ◽  
Shoot Tips ◽  
Nodal Segments ◽  
Shoot Apices ◽  
Skoog Medium ◽  
Leaf Mesophyll ◽  
Murashige And Skoog Medium

SUMMARYH.maximiliani was micropropagated using culture of shoot apices on modified Murashige and Skoog medium (DV). Further propagation of in vitro grown plants was done by culture of their nodal segments and shoot tips on the same medium supplemented with phloridzin, silver nitrate and casein hydrolysate (DV'). Rooting was induced by dipping the explants into IBA solution prior culture. Viable protoplasts (90%) were isolated from leaf mesophyll. These protoplasts divided (18%) in culture in agarose droplets.

scholarly journals The elimination of Plum pox virus in plum cv. Bluefree and apricot cv. Hanita by chemotherapy of in vitro cultures

2011 ◽  
Vol 38 (No. 2) ◽  
pp. 49-53 ◽  
Author(s):  
A. Hauptmanová ◽  
J. Polák
Keyword(s):  
In Vitro Cultures ◽  
Plum Pox Virus ◽  
Rt Pcr ◽  
Virus Elimination ◽  
Skoog Medium ◽  
Negative Results ◽  
Murashige Skoog ◽  
One Year

In vitro cultures of plum cv. Bluefree and apricot cv. Hanita infected with Plum pox virus (PPV) were used for the virus elimination by chemotherapy. Low ribavirin concentrations of 5 and 10 mg/l in Murashige-Skoog medium were applied in the treatment. Plum pox virus was completely eliminated by 5 mg/l of ribavirin in plum cv. Bluefree within twenty weeks and in apricot cv. Hanita in twelve weeks of the application. Plum pox virus was completely eliminated by 10 mg/l of ribavirin both in plum cv. Bluefree and apricot cv. Hanita within twelve weeks. The presence of PPV was not proved by RT-PCR. Clones of plum cv. Bluefree and apricot cv. Hanita were re-tested by RT-PCR one year after the termination of the ribavirin treatment and negative results confirmed the elimination of Plum pox virus.

scholarly journals In Vitro Regeneration of The Endangered Cactus Turbincarpus Mombergeri Riha, A Hybrid of T. Laui x T. Pseudopectinatus

2021 ◽  
Author(s):  
Maria del Socorro Santos-Diaz ◽  
Ma. Lourdes Santos-Díaz ◽  
Juana Alvarado-Rodríguez
Keyword(s):  
Survival Rate ◽  
Indoleacetic Acid ◽  
In Vitro Regeneration ◽  
Salt Medium ◽  
Natural Conditions ◽  
Normal Morphology ◽  
Culture Techniques ◽  
Seed Disinfection ◽  
Murashige Skoog

Abstract Turbinicarpus mombergeri is a cacti species formed by a hybridization process between Turbinicarpus laui and Turbinicarpus pseudopectinatus. Under natural conditions, it is very difficult for two species be genetically compatible for hybridization, and to produce flowers at the same time. Thus, T. mombergeri is a very interesting and a rare species. Unfortunately, the current populations are decreasing and now it is considered critically endangered. The aim of this research was to develop a successful protocol for propagating T. mombergeri using the in vitro culture techniques. Seed disinfection was performed with Plant Preservative Mixture, and 80% of germination occurred at day 45 in Murashige-Skoog medium. The shoots were cut longitudinally, and the segments were transferred to media containing 2.22 or 4.44 µM benzyladenine to induce shooting. The generated shoots were highly hydrated, and presented abundant callus. The hyperhydricity was controlled by reducing salt medium concentration, by increasing calcium levels and by using polyethylenglycol. The reduction of callus was attained by adding tri-iodo benzoic acid. Vigorous and thick shoots were generated in medium containing urea, and rooting improved in the presence of 0.5 µM indoleacetic acid. Plantlets with normal morphology were obtained, and the survival rate of the plants in soil was 80%. The methodology developed represents an alternative for propagation of T. mombergeri under controlled conditions for commercial or conservation purposes.

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