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1.
Lee YG  Popov E  Cui HY  Kim HH  Park SU  Bae CH  Lee SC  Engelmann F 《Cryo letters》2011,32(6):487-497
A droplet-vitrification protocol has been established for cryopreserving Chrysanthemum morifolium cv. Peak using axillary shoot tips and apical shoots of in vitro plants. In the optimized procedure, explants were submitted to a step-wise preculture in liquid sucrose-enriched medium (0.3, 0.5 and 0.7 M for 31,17 and 7 h, respectively). Precultured explants were treated for 40 min with C4 loading solution comprising (w/v) 17.5 percent glycerol + 17.5 percent sucrose, then dehydrated with PVS3 vitrification solution (w/v, 50 percent glycerol + 50 percent sucrose) for 60 min (axillary shoot tips) or 90 min (apical shoots). Explants were cryopreserved by direct immersion in liquid nitrogen in minute drops of PVS3 attached to aluminum foil strips. The optimal age of donor plants was 4-5.5 weeks for apical shoots and 7 weeks for axillary shoot tips, producing post-cryopreservation regeneration percentages of 81.9 percent and 84.9 percernt, respectively. Plants regenerated from cryopreserved samples showed no phenotypical abnormalities and similar profiles of relative DNA content were recorded for control and cryopreserved plants. Our results suggest that the modified droplet-vitrification protocol described in this paper is highly effective and may prove user-friendlier than the cryopreservation protocols already published for chrysanthemum.  相似文献   

2.
An efficient protocol for the cryopreservation of madder (Rubia akane Nakai) hairy root cultures was developed using droplet-vitrification and alternative loading and vitrification solutions formulated previously in our laboratory. Among eight preculture treatments tested, the highest post-cryopreservation regeneration was obtained for explants incubated in liquid half-strength MS medium with progressively increased sucrose concentration (0.3 M for 54 h, then 0.5 M for 16 h). Loading of precultured explants improved their post-cryopreservation regeneration by 50-75% compared with non-loaded control. Combining loading solution C4 (35% PVS3) and vitrification solution B5 (80% PVS3) was the most effective, while applying six PVS2-based solutions at room temperature resulted in low post-cryo regeneration. Treatment duration was optimized to 30 min for loading and to 10-20 min for vitrification solution. Apices of primary and secondary hairy roots showed similar post-cryo regeneration (88 and 95%, respectively), which was significantly higher than regeneration of root sections without apices (65%). Droplet-vitrification produced higher post-cryo regeneration than 'classical' vitrification in cryovials. Our results suggest that droplet-vitrification using alternative loading and vitrification solutions is an efficient method for cryopreservation of R. akane hairy root cultures.  相似文献   

3.
In this paper, we compared three vitrification-based cryopreservation techniques, viz. vitrification, encapsulation-vitrification and droplet-vitrification for cryopreserving sugarcane somatic embryos. Viability of somatic embryos was evaluated by measuring electrolyte leakage and by regrowth on recovery medium. Droplet-vitrification was the most efficient technique. Optimal conditions included loading with a solution containing 1.5 M glycerol and 0.3 M sucrose for 30 min at 25 degree C, treatment with the PVS2 solution for 20-40 min at 0 degree C followed by rapid immersion in liquid nitrogen of clumps of somatic embryos placed in microdroplets of cryoprotectant solution. Under such conditions, viability of cryopreserved somatic embryos reached 55 percent.  相似文献   

4.
The droplet-vitrification method was adapted to Pelargonium apices by optimizing the duration of the loading solution (LS) as well as the plant vitrification solution 2 (PVS2). The excised apices were dehydrated in two steps (20 min in LS and 15 min in PVS2) and then immersed directly in liquid nitrogen (LN). After thawing and unloading in the recovery solution at room temperature for 15 min, apices were plated onto semi-solid Murashige and Skoog medium. This simple protocol without any pretreatment was successfully applied to eight cultivars with a survival level ranging between 55.6 - 96.2 percent and a regrowth level between 9.1 and 70.6 percent. These results prove the feasibility of the long-term storage of Pelargonium germplasm through cryopreservation.  相似文献   

5.
Kim HH  Yoon JW  P YE  Cho EG  Sohn JK  Kim TK  Engelmann F 《Cryo letters》2006,27(4):223-234
The applicability of cryopreservation protocols to a broad range of genotypes is a key issue for genebanks. We tried to identify the critical factors causing differences in survival of cryopreserved shoot tips using potato varieties coming from cultivated and wild species. The droplet-vitrification method, a combination of droplet-freezing and solution-based vitrification, was selected from several protocols. High survival after freezing was observed after dehydration with PVS2 for 20 min, cooling shoot tips placed in a droplet of PVS2 solution on aluminum foil strips by immersing the foil strips in liquid nitrogen, warming them by plunging the foil strips into a 0.8 M sucrose solution (at 40 degrees C) for 30 s and unloading in 0.8 M sucrose for 30 min. This optimized protocol was successfully applied to 12 accessions with survival ranging between 64.0 and 94.4%.  相似文献   

6.
7.
Palm cryobanking     
We describe the development of an efficient cryopreservation protocol for proembryogenic masses (PEMs) of date palm variety 'Barhee'. Proembryos were induced by inoculating small pieces of juvenile leaves on MS medium supplemented with 0.3 mg per liter 2,4-D. Application of these in vitro conditions led to true-to-type plants as observed after plant fructification. When compared to the standard vitrification protocol, the ultra-rapid droplet-vitrification technique proved to be superior. Sucrose preculture considerably increased post-cryopreservation recovery. The highest regeneration after cryogenic exposure reached 63.3 percent when PEMs were treated with PVS2 for 30 min at 0 degree C and 56.7 percent when PVS2 treatment lasted for 15 min at 25 degree C. The first signs of regrowth of cryopreserved PEMs were observed after 2 to 3 weeks. Cryopreservation did not affect the morphogenetic capacities of the plant material. Moreover, highly proliferating suspension cultures could be established from the cryopreserved material. The overall production of somatic embryos from 500 mg cryopreserved PEMs reached 1030 +/- 50 units after 1 month. The morphological study of date palms regenerated from cryopreserved material confirmed the stability of clonal material following cryopreservation.  相似文献   

8.
This paper reviews a 10-year experience in establishing a cryopreserved Allium germplasm collection at the genebank of the National Agrobiodiversity Center, Republic of Korea. A systematic approach to Allium cryopreservation included: 1. revealing the most critical factors that affected regeneration after cryostorage; 2. understanding the mechanisms of cryoprotection by analyzing the thermal behavior of explants and cryoprotectant solutions using DSC and influx/efflux of cryoprotectants using HPLC; 3. assessing genetic stability of regenerants; and 4. revealing the efficiency of cryotherapy. Bulbil primordia, i.e. asexual bulbs formed on unripe inflorescences, proved to be the most suitable material for conservation of bolting varieties due to high post-cryopreservation regrowth and lower microbial infection level, followed by apical shoot apices from single bulbs and cloves. A total of 1,158 accessions of garlic as well as some Allium species have been cryopreserved during 2005-2010 using the droplet-vitrification technique with a mean regeneration percentage of 65.9 percent after cryostorage. These results open the door for large-scale implementation of cryostorage and for simplifying international exchange for clonal Allium germplasm.  相似文献   

9.
The aim of this study was to develop a cryopreservation protocol for Dioscorea rotundata with maintenance of genetic stability of regenerated plants after cryopreservation. In vitro shoot tips were cryopreserved using vitrification and encapsulation-dehydration to compare the efficacy of the two methods. Both methods produced high levels of plant regeneration from cryopreserved shoot tips. The regeneration level obtained using vitrification (71%) was not significantly different from that obtained using encapsulation-dehydration (67%). Genetic stability of plants derived from cryopreserved shoot tips was evaluated using RAPD markers. Analysis of 50 cryopreserved-derived and 20 in vitro- maintained (control) plantlets showed that 10 primers produced 77 clear, reproducible bands, with the amplification products being monomorphic for all the plantlets tested. A total of 5,390 bands obtained from this study exhibited no aberration in RAPD banding. Thus, the present study showed that both vitrification and encapsulation-dehydration methods are equally applicable to D. rotundata for cryopreservation. The in vitro plantlets derived from cryopreservation were genetically stable at the molecular level tested.  相似文献   

10.
Zhang T  Rawson DM 《Cryo letters》2002,23(3):191-196
The effect of cryopreservation on the survival of luc gene transfected bluegill sunfish fibroblast (BF-2) cells was investigated. Propane-1,2-diol was found to be the least toxic cryoprotectant when compared with DMSO and methanol. Both propane-1,2-diol and DMSO are effective in protecting cells from freezing damage. Whilst there were no significant differences in cell survival between cryoprotectant concentration (10 or 15%) and culture age used in this study, 7-day old culture appeared to be more resistant to freezing without cryoprotectant when compared with 3- and 14-day old culture. The highest cell survival values obtained with propane-1,2-diol (10%) and DMSO (10%) protection were 96.2 1.2% and 94.0 3.1% respectively. Initial subsequent cell growth after cryopreservation was slower than their non-frozen controls. The survival of transfected BF-2 cells (BF-2/luc1) after cryopreservation were very similar to those obtained with wild type cells being: 94.0 3.1% and 95.2 1.5% respectively with 10% DMSO protection. These results suggested that genetically modified fish cell lines may be equally amenable to cryopreservation as the wild type.  相似文献   

11.
The rust fungus Puccinia spegazzinii (Basidiomycotina: Uredinales) has been identified as a potential classical biological control agent for the invasive weed Mikania micrantha (Asteraceae). Long-term, live storage of this pathogen is required for reference. As biotrophs, almost all rusts species cannot be preserved by traditional cryopreservation protocols, which rely on in vitro culture techniques. In addition, the embedded teliospores and delicate basidiospores of this microcyclic rust are not amenable to direct plunge freezing. Continuous culture of the rust on living plants is both laborious and expensive, so a variety of approaches for cryopreservation and storage were tested. These methods included traditional approaches to fungal cryopreservation such as variation of cooling rate regime and alginate encapsulation techniques. However, an in situ cryopreservation technique was the only method identified as having any potential for the long-term cryopreservation of the 10 isolates tested. Material from either petiole or stem tissue remained viable after cryopreservation, determined by the ability of the material to produce basidiospores. However, despite great progress being made in developing an optimal cryopreservation method, infection of the host plant by basidiospores produced from previously cryopreserved teliospores, embedded in leaf petioles, was not achieved.  相似文献   

12.
Kim HH  Lee JK  Yoon JW  Ji JJ  Nam SS  Hwang HS  Cho EG  Engelmann F 《Cryo letters》2006,27(3):143-153
The droplet-vitrification protocol, a combination of droplet-freezing and solution-based vitrification was applied for cryopreserving garlic bulbil primordia. The highest survival and regeneration percentages of cryopreserved primordia (90.1 to 95.0 percent and 82.7 to 85.0 percent, respectively) were achieved after preculture for 2-4 days at 10 degree C on solid medium with 0.1 - 0.3 M sucrose, loading for 50 minutes in liquid medium with 2 M glycerol + 0.5 M sucrose, dehydration with PVS3 vitrification solution for 90-150 min, cooling primordia in 5 microl droplets of PVS3 vitrification solution placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into pre-heated (40 degree C) 0.8 M sucrose solution for 30 s and further incubation in the same solution for 30 minutes. The optimized droplet-vitrification protocol was successfully applied to bulbil primordia of five garlic varieties originating from various countries and to immature bulbils of two vegetatively propagated Allium species, with regeneration percentages ranging between 77.4 - 95.4 percent.  相似文献   

13.
Hao YJ  You CX  Deng XX 《Cryo letters》2002,23(1):37-46
Shoot-tips of 10 strawberry genotypes were successfully cryopreserved using a modified encapsulation-dehydration method. All genotypes survived cryopreservation with high survival and regeneration rates. Eight Joho single-bud sibling lines were established as a model system for genetic analysis. Although cytological examination found chromosomal variation in both non-cryopreserved and cryopreserved samples, the ploidy constitution remained relatively stable after cryopreservation. DNA samples digested with MseI and PstI were used for amplified fragmentation length polymorphism (AFLP) assay. In 16 primer combinations, only one, namely, PCCA-MCAG, detected one site where band pattern changed after cryopreservation, which might be contributed to the change in DNA methylation status at PstI recognition site. Methylation sensitive amplified polymorphism (MSAP) assay was carried out for further investigation on the influence of cryopreservation on DNA methylation status. It was found that cryopreservation induced a significant change in DNA methylation status.  相似文献   

14.
Homolka's perlite protocol (HPP) for cryopreservation of fungal cultures was evaluated in 12 strains (7 species) of cryosensitive basidiomycete cultures maintained in NBRC culture collection by investigating viability, time to recover, and basic morphological study after freezing and storing at -80 degree C for 6 months. The viability of the fungal strains was 60 percent in Phallus hadriani and 100 percent in remaining 11 strains, indicating the efficacy of HPP method for cryopreservation of some cryosensitive basidiomycetes. The HPP method was modified by changing the addition of cryoprotectant (glycerol) from prior precultivation to post precultivation, limiting the cryoprotectant exposure time to 48 hours, and increasing the glycerol concentration from 5 percent to 12 percent. The viability of P. hadriani strain increased from 60 percent to 100 percent with the modified perlite protocol after storage at -80 degree C for 6 months.  相似文献   

15.
Hirai D 《Cryo letters》2011,32(4):287-296
The droplet vitrification method was improved for maneuverability by embedding shoot tips in gelled droplets before osmoprotection. This newly modified cryopreserving method -gelled droplet vitrification - was compared with other PVS2-based cryopreservation methods using potato shoot tips. Survival rates of each cryogenic procedure held at 25 degree C were about 40 percent by cryotube-vitrification procedures (vitrification and encapsulation vitrification methods) and about 70 percent by PVS2-droplet procedures (droplet vitrification and gelled droplet vitrification methods). Much higher cooling rates of PVS2-droplet procedures than cryotube- vitrification procedures increased their survival rates. The gelled droplet vitrification method was applied to shoot tips of 26 potato cultivars and six wild potatoes. After a little modifications of the conditions for preculture, osmoprotection and dehydration, all cultivars and wild potatoes produced high enough survival rates to be of value to genebanks and all surviving shoot tips developed normal shoots within 3 weeks.  相似文献   

16.
Morphological assessment of spermatozoa has a long history and it is generally accepted that specific morphologic structural deviations correlate with male sub- and infertility. Although many different and also new methods are used in semen analysis, light microscopy is still used for routine morphological evaluation. This paper gives an overview about the detailed structure of physiological mammalian spermatozoa as well as the most common morphological deviations in correlation to fertility. This should be the basis for explanation of problems resulting from semen cryopreservation. General aspects of semen cryopreservation should be regarded before to facilitate the understanding of methods and mechanisms.  相似文献   

17.
Cryopreservation is the best method of storing germplasm efficiently and safely, particularly for the maintenance of vegetatively propagated material. In IPK cryopreservation is used for potato, garlic, mint and yam. IPK collaborates with other cryobanks and research groups (ECPGR, COST, EURALLIVEG) and finds considerable differences in the adoption of cryopreservation between crops and their host institutes, depending on crop, local and historical circumstances. A better understanding of the long-term benefits of cryopreservation and its further integration into general genebank management is therefore needed. Recommended approaches include: comparative validation of methods between different laboratories, detailed comparisons of crop-based methods, economical analyses, efficient integration strategies of cryobanks by genebanks; including safe duplication of cryopreserved resources for the limitation of risk of loss Importantly, there has been recent progress in the development of quality management systems. Cryopreservation is, however, characterized by high expectations. Therefore, to ensure its sustainable and practicable use, basic knowledge of storage protocols must be combined with increased awareness of the rationales required to validate, implement and apply cryobanking technologies in working genebanks.  相似文献   

18.
Ex situ conservation of endangered plants is an important aim in order to preserve biodiversity of flora in threatened ecosystems. Among the biotechnological techniques which can be used, cryopreservation is emerging as a preferred option in many instances. This study describes a cryopreservation technique developed for shoot tips of the endangered species Centaurea ultreiae (Compositae) using a vitrification procedure. Basal medium (BM) for preculture and loading phases consisted of 1/2 MS basal salts with modified vitamins (3 microM thiamine). For preculturing shoot tips, BM with five osmotic treatments were investigated: 0.3 M sucrose +/- 20 microM ABA, 0.6 M glycerol +/- 20 microM ABA and 0.25 M sucrose + 0.25 M glycerol + 10 microM ABA. A loading solution treatment (BM with 2 M glycerol and 0.4 M sucrose) was applied prior to exposure of shoot tips to PVS2 and found to be indispensable to obtaining successful post-LN recovery. Highest (95.5%) regrowth of LN immersed shoot tips was obtained following incubation on BM + 0.3 M sucrose + 20 microM ABA or 0.25 M sucrose + 0.25 M glycerol + 10 microM ABA, with loading treatment and PVS2 exposure for 20 minutes at 0 degree C. Keywords: cryopreservation, encapsulation, endangered species, ex situ conservation, vitrification.  相似文献   

19.
Datar SP  Bhonde RR 《Cryo letters》2010,31(6):485-492
The avian endocrine pancreas shares some similarities with those of mammals. Previously we have reported a technique of isolation of B islets from chick pancreas and also demonstrated their possible use for hypoglycemic drug screening as efficiently as mammalian islets. Here we describe a novel cryopreservative medium for the cryopreservation of chick islets. Isolated chick islets were suspended in a cryo medium consisting of Dulbecco's modified Minimum Essential Medium: Ham's F12 (1:1), Fetal Bovine Serum (20%), Dimethylsulfoxide (10%) with different concentrations (50 microg/ml to 500 microg/ml) of riboflavin or nicotinamide. The viability of revived islets after three and half months was checked by trypan blue dye exclusion and functionality was assessed by insulin secretion in response to glucose challenge. The maximum recovery of viable islets and insulin secretion was obtained in response to glucose challenge at 250 microg/ml concentration of Riboflavin or Nicotinamide. This is a first report on cryopreservation of chick islets exhibiting cryoprotective role of water soluble vitamins without vitamin C.  相似文献   

20.
Zhang A  Cheng S  Gao D  Xu LX 《Cryo letters》2005,26(2):113-120
Two methods used in artery deep cryopreservation, "Cryopreservation in Medium" and "Cryopreservation in Air", were studied. For the former method, samples were frozen together with a certain amount of cryoprotectants (CPA) in the cryovial or cryobag, while for the other method the arteries were first exposed to CPA and then frozen without the CPA medium surrounding in the cryovial. Study of the cryopreserved arteries using these two methods found that "cryopreservation in air" could substantially reduce the fracture rate of the arteries. To explain the difference theoretically, a two-compartment model is presented to study the thermal stresses generated during the freezing and thawing processes. The properties were measured as inputs to the model. Numerical results showed that the thermal stresses occurring in the "cryopreservation in air" process were much smaller than in the other method. The maximum thermal stress during cryopreservation occurs in the thawing process. The theoretical results could well explain published experimental results.  相似文献   

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