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1.
This study aimed to evaluate different vitrification methods using distinct cryoprotectants (CPAs) for the preservation of collared peccary ovarian preantral follicles (PFs). Ovarian pairs from six females were fragmented and three fragments (fresh control group) were immediately evaluated for morphology, viability, cell proliferation capacity (assessed by quantifying the number of argyrophilic nucleolus organizer regions – NORs), and apoptosis (by the identification of activated caspase-3 expression). The remaining 18 fragments were vitrified using the solid surface vitrification (SSV) method or the ovarian tissue cryosystem (OTC) with 3 M ethylene glycol (EG), 3 M dimethylsulfoxide (DMSO), or a combination of the two (1.5 M EG/1.5 M DMSO). After two weeks, samples were rewarmed and evaluated as described previously. The OTC with any of the CPAs provided a similar conservation of morphologically normal PFs as the fresh control group (75.6 ± 8.6%); however, the SSV was only efficient with DMSO alone (63.9 ± 7.6%). Regarding the viability or cell proliferation, all tested groups provided post rewarming values similar to those observed for the fresh control group, 84.0 ± 2.9% viable cells with 2.0 ± 0.2 NORs. Related to apoptosis analysis, only the OTC with EG (46.7%) and the SSV method with EG (43.4%) or the combination of EG and DMSO (33.4%) provided similar values to those found for the fresh control group (36.7%). Our findings indicate the utilization of a closed system, the OTC, with 3 M EG as the CPA for the vitrification of collared peccary ovarian tissue.  相似文献   

2.
Currently, it has been observed that a considerable segment of the jaguar population is declining mainly because of hunting, and destruction and fragmentation of habitat. Given this scenario, efforts of the scientific community have been concentrated on the development of conservation strategies, such as the formation and use of somatic sample banks. We aimed to assess the effects of cryopreservation techniques of the ear skin of jaguar [slow freezing (SF) or direct vitrification in cryovials (DVC) or solid-surface vitrification (SSV)] on the morphological analysis and cell ability during the culture. All cryopreserved fragments regardless of the technique used, showed a reduction in the dermis and total thickness of the skin. Although a collagen matrix similar to the control group (fresh) has been observed only for the fragments from SF and SSV groups, all cryopreserved techniques were able to maintain normal patterns of the fibroblasts. Moreover, DVC and SSV methods maintained the proliferative activity of the tissues even after warming. After the culture, SF and SSV techniques were efficient for the recovery of the somatic cells according to most of the evaluated parameters, especially with regard to the duration of culture and cell metabolic activity. In conclusion, SSV was found to be a more efficient technique for cryopreserving jaguar skin when compared to DVC and SF. These results are relevant for the formation of somatic resource banks of this species, directed at cryopreserving adequate samplings of different individuals and generations for future applications in regenerative medicine, and assisted reproductive technologies.  相似文献   

3.
Sweet sorghum is a climate smart crop with multiple uses. The crop is susceptible to attack by the spotted stemborer, Chilo partellus (Swinhoe). This causes deadheart formation, leading to lodging of plants and consequent high economic losses. Lack of stable sources of resistance make any genetic enhancement through breeding difficult. We report a study to build up host plant resistance using transgenic technology by introducing two different classes of Bt genes (cry1Aa and cry1B) into two elite sweet sorghum genotypes of India (SSV84 and RSSV9). We devised tissue culture methods to suit the genotypes of our interest, SSV84 and RSSV9, and employed two methods of genetic transformation: the particle bombardment and in planta method of Agrobacterium. Modification of in vitro culture methods involved subculture every 3 days in the initial stages of culture and the use of precultured embryos as target tissues. For the in planta method, a floral dip for 1 h in Agrobacterium suspension supplemented with l-cysteine and Tween-20 was used. Sixteen transgenic events were generated; inheritance, integration and stable expression of the transgenes till the T4 generation were confirmed. The amount of Bt Cry1Aa protein at 25–30 days of growth ranged from 24.8 to 72.8 ng/g of fresh leaf tissue. We recorded 78.4 % larval mortality, reduced leaf damage (3.0 out of 9.0) and reduced feeding (41.0 %) over the controls in insect feed assays. Stable inheritance and expression in the in planta-derived transgenics are presented.  相似文献   

4.
The objective was to compare the efficiency of various vitrification techniques and solutions for preserving morphology and viability of preantral caprine follicles enclosed in ovarian tissue. Fragments of ovarian cortex were cryopreserved by conventional vitrification (CV) in French straws, vitrification in macrotubes (MTV), or solid-surface vitrification (SSV). Six solutions containing 6 M ethylene glycol, with or without sucrose (SUC; 0.25 or 0.50 M) and/or 10% fetal calf serum (FCS) were tested (Experiment I). After 1 wk, samples were warmed and preantral follicles were examined histologically. To evaluate follicular viability (Experiment II), ovarian fragments were vitrified with the three techniques listed above, in a solution containing 0.25 M SUC and 10% FCS. After warming, follicles were assessed by the trypan blue dye exclusion test. In Experiment III, preantral follicles enclosed in ovarian tissue were vitrified using the protocol which yielded the highest percentage of viable preantral follicles (SSV with 0.25 M SUC and 10% SFB). After warming, the preantral follicles enclosed in ovarian tissue were cultured in vitro and then, were analyzed by histology and fluorescence microscopy (calcein-AM and ethidium homodimer-1). Every vitrification protocol significantly reduced the percentages of morphologically normal follicles relative to the control (88.0%); however, the addition of 0.25 M SUC and 10% FCS to the vitrification solution improved preservation of follicular morphology (67.4, 67.4, and 72.0% for CV, MTV, and SSV, respectively). Although follicular viability after SSV (80.7%) did not differ from that in fresh (non-vitrified) ovarian tissues (88.0%), after in vitro culture, percentages of viable follicles were significantly reduced (70.0%). Percentages of morphologically normal follicles after in vitro culture of vitrified ovarian tissue were similar (76.0%) to those in ovarian cortex fragments cultured without previous vitrification (83.2%). In conclusion, SSV using a solution containing 0.25 M SUC and 10% FCS, was the most efficient method for vitrifying caprine ovarian tissue.  相似文献   

5.
Petiveria alliacea L. is a medicinal plant originating from the Amazon region. This study describes an efficient cryopreservation protocol for somatic embryos (SEs) produced from roots of P. alliacea based on the comparison of vitrification, encapsulation-dehydration, and D cryo-plate techniques. With the vitrification technique, SEs treated with PVS2 solution (0.4 M sucrose, 3.3 M glycerol, 2.4 M ethylene glycol, and 1.9 M DMSO) for 30 min displayed high viability (85%) and intermediate proliferation recovery (about 12 adventitious SEs produced from original SEs [SEs/SE] after 90 d of culture). With the encapsulation-dehydration technique, lower viability (70%) and very low proliferation recovery (about two SEs/SE) were achieved with cryopreserved SEs dehydrated for 10 min in a laminar air flow cabinet. The D cryo-plate technique led to high viability (85%) and proliferation recovery (19 SEs/SE) of cryopreserved SEs after 90 min dehydration. In the experimental conditions tested, the D cryo-plate method was the most efficient technique for cryopreservation of P. alliacea SEs.  相似文献   

6.
An efficient cryopreservation protocol was developed for mature seeds of Oncidium flexuosum Sims. Seed morphology, protocorm formation, and early seedling development were also assessed. The effects of phloroglucinol and Supercool X-1000® as cryoprotectant additives in the vitrification solution were investigated. Dehydration using the plant vitrification solution 2 (PVS2) for 60 and 120 min prior to immersion in liquid nitrogen promoted the highest frequency of in vitro seed germination 6 weeks following culture on half-strength Murashige and Skoog (½ MS) medium. Mature seeds submitted to vitrification for 120 min in PVS2 and 1 % phloroglucinol at 0 °C enhanced germination by 68 %, whereas in PVS2 and 1 % Supercool X-1000® germination was just moderately enhanced (26 %). In vitro-germinating seedlings developed healthy shoots and roots without the use of plant growth regulators. After 6 months of growth, there were no differences between in vitro- and ex vitro-grown seedlings for various phenotypic characteristics, including shoot length, number of leaves, number and length of roots, and fresh and dry weight. Seedlings were transferred to greenhouse conditions and successfully acclimatized, further developing into normal plants with over 90 % survival. Comparative analysis of seedlings from control and vitrified seeds using flow cytometry indicated that no change in ploidy levels occurred as a result of cryopreservation, therefore maintaining seedlings genetic stability. In this study, vitrification with PVS2 for 120 min with the addition of 1 % phloroglucinol offers a simple, safe, and feasible protocol for cryopreservation of O. flexuosum mature seeds.  相似文献   

7.
Cryopreservation of pronuclear-stage embryos would be useful for transgenic technology and genome preservation purposes. We compared a novel vitrification technique (solid surface vitrification, SSV) with another vitrification method in straws for cryosurvival and to generate transgenic progeny from cryopreserved mouse zygotes following microinjection. The SSV solution consisted of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4 M trehalose in M2 supplemented with 4 mg/ml BSA; the in straw vitrification solution was 7 M EG in M2 plus BSA. In experiment I, we compared the effect of the vitrification solutions alone, without cooling. No reduction was detected in survival and cleavage rates. In experiment II, SSV yielded a significantly higher percentage of morphologically normal zygotes (96%) that also cleaved at significantly higher rates (80%) when compared to that following "in straw" vitrification (68 and 66%, respectively). Cleavage rate in the non-vitrified control group (93%) was significantly higher than that of both vitrified groups. Following embryo transfer, there was no difference in the rate of pups obtained from the SSV, "in straw" vitrified, and control groups (97/457, 21%; 15/75, 20% and 56/209, 27%, respectively). In experiment III, SSV vitrified and fresh embryos were used for pronuclear DNA injection. Survival rate of vitrified embryos after microinjection was reduced compared to nonvitrified ones (64 vs. 72%, respectively; P < 0.05); however, development to two-cell stage was not different (76 vs. 72%, respectively). Following embryo transfer of vitrified vs. fresh microinjected embryos, in both cases 10% live pups were generated, including transgenic pups. The results demonstrated that the efficiency of generating transgenic pups from SSV vitrified pronuclear zygotes is comparable to that from fresh embryos.  相似文献   

8.
Reports on the vitrification of somatic cells are scarce. Here, we show that Leydig cells (murine cell line TM3) could be successfully vitrified by both open vitrification [plastic straw (PS) and plastic vials (PV)] and closed ultravitrification [microdrop (MD) and solid surface vitrification (SSV)], after protocol optimization. However, open ultravitrification resulted in better post-warming viability than closed systems of vitrification with highest success obtained in modified SSV (84.8 ± 1.86%; p < 0.05). Leydig cells vitrified-warmed by modified SSV also showed superior (p < 0.05) cell growth, mitochondrial activity and cytoplasmic esterase enzyme activity, than MD, PS and PV, respectively. It was also observed that vitrified-warmed cells had higher level of ROS activity than non-vitrified control cells (41.6 ± 4.0 vs. 16.7 ± 1.0; p < 0.05). Treatment of cells with glutathione (GSH) or 2-mercaptoethanol (2-ME) (0, 10, 50, 100 μM) significantly (p < 0.05) reduced the ROS activity but had no significant (p > 0.05) effect on post-warm viability. Nevertheless, antioxidant-treated cells had improved mitochondrial activity, cytoplasmic esterase activity and cell growth during in vitro culture (p < 0.05). In conclusion, our results suggest that modified SSV offers a viable method for vitrifying single cell suspension of Leydig cells. To the best of our knowledge, this is the first report on cryopreservation of Leydig cells by vitrification.  相似文献   

9.
Defensive behavioral patterns in response to human-induced rapid environmental change can affect animals’ fitness and may play a role in species conservation status. To test this hypothesis, we compared the risk assessment and defensive behavioral responses of captive white-lipped peccary (WLP; Tayassu pecari) and collared peccary (CP; Pecari tajacu), which retain different conservation status; WLP are considered vulnerable and CP of least concern. We used an adapted paradigm of the mouse defense test battery (MDTB) comprising four consecutive tests. Two of these tests simulated a novel environment, while the other two stimulated the expression of defensive behavioral patterns. Besides differences in risk assessment and defensive threat/attack behavioral patterns between species, we compared flight initiation distance, flight speed, and plasma glucocorticoid concentrations. When facing a novel environment and risk challenges from humans’ predator-like cues, the white-lipped peccary showed more exploratory and defensive threat/attack behavioral patterns, shorter flight initiation distances, and lower flight speeds, whereas the collared peccaries showed more cautious and retreat patterns, longer flight initiation distances, and higher flight speeds. There were also correlations between physiological and behavioral parameters. We confirmed our hypothesis that the collared peccary’s cautiousness may help to prevent a decrease in its population, while the white-lipped peccary’s exploratory and confrontational behavioral patterns in overhunted areas, together with other simultaneous factors as forest fragmentation, might contribute to placing this species in the vulnerable category.  相似文献   

10.
In this study conservation of Castilleja levisecta Greenm., a globally endangered species was addressed through in vitro cryopreservation of shoot tips. In vitro cultures were successfully established using seedlings received from British Columbia, Canada. Shoot tips excised from in vitro propagated plants were cryopreserved using a droplet-vitrification method following optimization of individual protocol steps such as pre-culture, treatment with vitrification solutions, and unloading. The highest plant regrowth after cryopreservation (66%) was achieved when shoot tips were pre-cultured in 0.3 M sucrose for 17 h followed by 0.5 M sucrose for 4 h, incubated in an osmo-protectant solution (17.5% [v/v] glycerol and 17.5% [w/v] sucrose) for 20 min, exposed to vitrification solution A3 (37.5% [v/v] glycerol plus 15% [v/v] dimethylsulfoxide (DMSO) plus 15% [v/v] ethylene glycol (EG) plus 22.5% [w/v] sucrose) on ice for 40 min, and unloaded in 0.8 M sucrose solution for 30 min. Healthy plants were developed from cryopreserved shoot tips and propagated in vitro using nodal segments. Plants derived from in vitro culture and from cryopreserved tissues were successfully rooted and acclimated in a greenhouse with 100% survival rate. Acclimatized plants were reintroduced in a naturalized propagation area at the Conservation Nursery at Fort Rodd Hill, Canada. Twenty of 94 reintroduced plants (21%) survived the transit from lab to field and some had started to flower. This is the first report for cryopreservation of C. levisecta, an important step in conserving and re-introducing this critically imperiled species in nature.  相似文献   

11.
Animal cloning is a promising technology for biodiversity conservation, and its success depends on the recovery of nucleus donor cells. Specifically for collared peccaries, found sometimes in regions that are difficult to access, the storage at 4–6°C of skin tissues would be an alternative for the conservation of genetic material. Therefore, we aimed to evaluate different storage periods and the presence of a nutrient medium at 4–6°C on the recovery of somatic cells from the skin of collared peccaries. To analyze cell recovery rates, ear explants were distributed in non-refrigerated samples and samples refrigerated for 10, 30, and 50 d in the absence or presence of nutrient medium. All explants were analyzed by histologically and cultured. Only the fragments stored for 50 d without medium showed an increase in the total thickness of skin. Moreover, increased storage period, regardless of the presence of medium, increased the halo number and reduced the metabolic activity. After culture, only the fragments stored without medium for 50 d did not yield any somatic cells. Cells recovered from explants stored for 10 d showed similar characteristics to these recovered from non-refrigerated explants, regardless of the presence of medium, including the day at which explants achieved attachment and the total time to reach subconfluence. In conclusion, viable cells can be recovered from somatic tissues of collared peccaries stored for up to 50 d in the presence of medium, and tissues refrigerated for up to 10 d in the presence of medium yielded more viable cells.  相似文献   

12.
In this study, importance of Wnt-β-catenin pathway in the development of uterine cervical carcinoma was evaluated. For this purpose, the profiles (expression/methylation/deletion) of β-catenin, p-β-catenin (Y654), Wnt3a, and APC were studied in disease free normal cervical epithelium (n = 9), adjacent normal cervical epithelium of primary tumors (n = 70), CIN (n = 28), CACX (n = 102) samples, and two CACX cell lines (HeLa and SiHa). Immunohistochemical analysis revealed high/medium (74–95%) expression of β-catenin/p-β-catenin (Y654) and Wnt3a and low expression (23–26%) of APC in proliferating basal–parabasal layers contrary to differentiated spinous layer in normal cervix irrespective of HPV16 infection. The expression profile of the genes in the basal–parabasal layers did not change significantly during development of CACX. High (66%) promoter methylation of APC was seen in basal–parabasal layers and the cervical lesions (42–69%), unlike in spinous layers (25%). The promoter methylation status of APC was validated by in vitro demethylation experiments using 5-aza-dC in CACX cell lines. However, additional deletion of APC was significantly increased from CIN (12%) to stage I/II (40%) and became comparable in stage III/IV (48%) of the tumor. Patients with alterations (deletion/methylation) of APC and high/medium expression of Wnt3a/β-catenin/p-β-catenin (Y654) showed significantly poor survival. Thus our data indicate that cumulative effect of Wnt3a overexpression and APC inactivation are needed for overexpression of β-catenin during the development of CACX.  相似文献   

13.
14.
Developmental deficiency of somatic embryos and regeneration to plantlets, especially in the case of transformation, are major problems of somatic embryo regeneration in alfalfa. One of the ways to overcome these problems is the use of natural plant regulators and nutrients in the culture medium of somatic embryos. For investigating the influence of Cuscuta campestris extract on the efficiency of plant regeneration and transformation, chimeric tissue type plasminogen activator was transferred to explants using Agrobacterium tumefaciens, and transgenic plants were recovered using medium supplemented with different concentration of the extract. Transgenic plants were analyzed by PCR and RT-PCR. Somatic embryos of Medicago sativa L. developed into plantlets at high frequency level (52 %) in the maturation medium supplemented with 50 mg 1?1 C. campestris extract as compared to the medium without extract (26 %). Transformation efficiency was 29.3 and 15.2 % for medium supplemented with dodder extract and without the extract, respectively. HPLC and GC/MS analysis of the extract indicated high level of ABA and some compounds such as Phytol, which can affect the somatic embryo maturation. The antibacterial assay showed that the extract was effective against some strains of A. tumefaciens. These results have provided a scientific basis for using of C. campestris extract as a good natural source of antimicrobial agents and plant growth regulator as well, that can be used in tissue culture of transgenic plants.  相似文献   

15.
Arachis glabrata Benth (perennial peanut) is a rhizomatous legume with high forage value and great potential for soil conservation as well as it displays valuable plant genetic resources for the cultivated edible peanut improvement. In this study, we developed for the first time successful protocols for micropropagation and cryopreservation of A. glabrata. First fully expanded leaflets from greenhouse-growing plants were efficiently established in vitro (93%) and displayed high frequency of bud induction (58%) on MS medium with 6 mg L?1 1-fenil-3-(1,2,3-tiadiazol-5-il)urea [TDZ]. Whole plant regeneration was achieved via direct organogenesis by transferring the induced buds to MS media. Immature unexpanded leaves from micropropagated plants were effectively cryopreserved by using the droplet-vitrification technique. Maximum survival (~ 70%) and further regeneration (60–67%) were obtained by preconditioning immature leaves on semisolid MS with 0.3 M sucrose (1 d), exposing to loading solution consisting of 0.4 M sucrose plus 2 M glycerol (30 min) followed by glycerol-sucrose plant vitrification solution PVS3 (150 min in ice), and direct plunging into liquid nitrogen in droplets of PVS3 deposited on cryoplates. Tissues were rewarmed by plunging the aluminum foils directly in liquid MS enriched with 1.2 M sucrose (15 min) at room temperature. Growth recovery and plant regeneration were efficiently achieved via shoot organogenesis, and somatic embryogenesis by culturing cryostored explants on MS added with 6 mg L?1 TDZ. Genetic stability of plants derived from cryopreserved leaves was confirmed by random amplified polymorphic DNA markers. The protocols established in this study have great potential for rapid multiplication and conservation of selected A. glabrata genotypes.  相似文献   

16.
The objective of the present study was the cryopreservation of monotypic endemic Hladnikia pastinacifolia Rchb. shoot tips from an in vitro culture, via encapsulation-dehydration (ED) or encapsulation-vitrification (EV). For all tested genotypes, the highest rates of shoot regrowth and multiplication were obtained after overnight preculture in 0.4 M sucrose, encapsulation in Murashige and Skoog (MS) medium with 0.4 M sucrose and 1 M glycerol, followed by polymerization in 3% (w/v) Na-alginate in MS with 0.4 M sucrose. Optimal osmoprotection was achieved for ED with 0.4 M sucrose plus 1 M glycerol and for EV with 0.4 M sucrose plus 2 M glycerol. The best dehydration time for ED was 150 min in a desiccation chamber with silica gel, and the best vitrification time for EV was 85 min in plant vitrification solution 2 (PVS2). For ED, dehydration for 150 min resulted in explant water content of 22%. When the encapsulation method was combined with ED, 53% regrowth was achieved, and when it was combined with EV, 64% regrowth was achieved. Both methods could become applicable for the long-term cryopreservation of H. pastinacifolia germplasm, although EV was faster and resulted in better final regrowth success. Genetic stability analysis of cryopreserved plant samples was carried out for two genotypes, using random amplified polymorphic DNA (RAPD) markers to compare the two different cryopreservation protocols. Significant genetic differences between the genotypes were detected and a low level of genomic variation was observed.  相似文献   

17.
Vitrification is considered a viable method for cryopreservation of ovarian tissue and selection of methods that minimize follicular damage is important. The objective of the present study was to evaluate the effects of two vitrification methods on ovarian tissue morphology, preantral follicles survival rate during in vitro culture, and relative expression of genes associated with oocyte maturation and cumulus expansion. Ovaries from 12-day-old mice were vitrified in media containing ethylene glycol, dimethyl sulphoxide, and sucrose. Before plunging in liquid nitrogen, ovaries were first loaded into an acupuncture needle (needle immersion vitrification [NIV]) or placed on a cold steel surface for 10 to 20 seconds (solid surface vitrification [SSV]). The integrity of the ovarian tissue was well-preserved after vitrification and was similar controls. Follicle viability in the SSV group was lower (P < 0.05) than in the control group after 6 days of culture and the NIV group after 10 day of culture. Follicle viability after 12 day of culture was 92.8%, 82.1%, and 58.4% in control, NIV, and SSV groups, respectively. Bmp15, Gdf9, BmprII, Alk6, Alk5, Has2, and Ptgs2 gene expression patterns were similar among groups. However, the level of gene expression in the vitrification groups during Days 6 to 10 were higher compared with the control group. In conclusion, ovarian tissue morphologic integrity was well-preserved, regardless of the vitrification method. Vitrification using the needle immersion method resulted in greater follicular survival after 12 day of culture than the SSV method. Gene expression patterns during culture did not seem to explain the reduced survival rate observed in the solid surface group.  相似文献   

18.
ABSTRACT Determining presence or absence of collared peccaries (Pecari tajacu) from surveys of sign (tracks and feces) requires information on whether animals in sample units are detected. We estimated detection probabilities of collared peccary from sign surveys using occupancy models. Because it was unlikely that residence status of collared peccary in sampling units remained the same over a survey season, which is a primary assumption of occupancy models, we first determined the time interval for which to pool data. We then examined the influence of rainfall and peccary abundance on detection probabilities. We placed 90 sign stations (25-m-diam circular plots) throughout Chaparral Wildlife Management Area, south Texas, USA. We surveyed plots weekly for the presence or non-presence of collared peccary during 2 11-week sampling seasons in spring and fall 2003. We examined sign data weekly and we pooled the data in intervals from 2 weeks to 5 weeks. Estimates of detection probabilities increased from 1 week to 3 weeks of pooled data and leveled off thereafter. We needed a 3-week time interval to meet the assumption of unchanging residence status. Using sign data pooled in 3-week increments, detection probabilities were influenced by areas that differed in peccary abundance, but they were not influenced by rainfall. Estimates of detection probabilities ranged from 0.43 to 0.77 for 3-week time intervals. Sign surveys and occupancy modeling of data can be used to measure spatial patterns of collared peccary in south Texas as long as multiple 3-week time intervals are sampled.  相似文献   

19.
Use of non-viable somatic cells for hand-made cloning (HMC) can enable production of cloned animals from tissues obtained from elite or endangered dead animals. Buffalo skin fibroblast cells were rendered non-viable by heat treatment and used for HMC. Although fusion (93.6 ± 1.72 vs 67.1 ± 2.83%) and cleavage (90.3 ± 1.79 vs 65.8 ± 1.56%) rate was lower (P < 0.001) than that for controls, blastocysts could be successfully produced. However, blastocyst rate (34.1 ± 2.43 vs 6.9 ± 2.18%, P < 0.001) and total cell number of blastocysts (TCN, 221.3 ± 25.14 vs 151.1 ± 21.69, P < 0.05) were lower and apoptotic index (4.8 ± 1.06 vs 10.9 ± 1.21) was higher (P < 0.001) than that of controls. In another experiment, ear tissue of slaughterhouse buffaloes was preserved in mustard oil at room temperature for 48 h following which somatic cells were harvested by enzymatic digestion and used for HMC. Although fusion (96.8 ± 1.48 vs 84.2 ± 3.19%), cleavage (89.6 ± 3.59 vs 77.2 ± 3.99%), and blastocyst rate (36.9 ± 7.45 vs 13.1 ± 6.87%) were lower (P < 0.01), TCN (223.0 ± 27.89 vs 213.3 ± 28.21) and apoptotic index (3.97 ± 0.67 vs 5.22 ± 0.51) of blastocysts were similar to those of controls. In conclusion, HMC can be successfully used for production of blastocysts from non-viable cells and from cells obtained from freshly slaughtered buffaloes. This can pave the way for the restoration of farm or wild animals by HMC if somatic cells could be obtained within a few hours after their death.  相似文献   

20.
A critical factor in somatic embryogenesis protocols in papaya (Carica papaya L.) has been incomplete germination of somatic embryos due to formation of a basal callus, which prevents the emission of the radicle. This work aims to achieve complete germination of somatic embryos in liquid and semi-solid culture media. The effect of the culture conditions on germination of somatic embryos using the RITA® temporary immersion system were evaluated as well as the effect of phloroglucinol on germination of somatic embryos in semi-solid culture medium. The results of using the RITA® culture medium with a combination of 0.02 μM BAP and 2.90 μM gibberellic acid had a good response for total germination (100%) but somatic embryos had only partial germination with 400 mg fresh mass. However, the optimum inoculum density was 200 mg fresh mass of somatic embryos which produced 100% total germination and 95% somatic embryos with complete germination. Also, it was possible to achieve complete germination of somatic embryos with low callus formation (13%) using phloroglucinol at a concentration of 475.8 μM on semi-solid culture medium. This is the first report of two biotechnological strategies for complete germination of plants from somatic embryos in the papaya cultivar `Maradol Roja´.  相似文献   

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