scholarly journals In vitro cloning potential and phytochemical evaluations of aneuploid individuals produced from reciprocal crosses between diploid and triploid in Echinacea purpurea L.

2017 ◽  
Vol 86 (3) ◽  
Author(s):  
Qingling Li ◽  
Weizhen Jiang ◽  
Yi Ren ◽  
Rong Chen ◽  
Xinglian Li ◽  
...  
Keyword(s):  
Chlorogenic Acid ◽  
Acid Content ◽  
Survival Rates ◽  
Female Parent ◽  
Echinacea Purpurea ◽  
Adventitious Bud ◽  
High Survival ◽  
Cichoric Acid ◽  
Reciprocal Crosses

Aneuploidy often presents large variations in morphology, physiology, biochemistry, and genetics owing to karyotypic imbalance. This study aimed to evaluate the efficacy of aneuploid breeding in <em>Echinacea purpurea</em> L, an important medicinal plant. Reciprocal crosses between diploid and triploid plants were performed to generate aneuploid plants. Cross with triploid as female parent resulted in increased production of aneuploid individuals (19 of 23; 82.61%), while using diploid as female parent yielded much higher percentage of diploid progenies (130 of 133; 97.74%). Each aneuploid had particular karyotypic characteristics compared to the parents. The proportions of median, submedian, and subterminal centromere location chromosomes in gross chromosomes among aneuploids and two parents showed large variations. Although aneuploids had relatively lower adventitious bud regeneration rates than their parents, almost half of them looked morphologically normal, with high survival rates when transplanted to ex vitro conditions. Among the bioactive compounds assessed, cichoric acid and chlorogenic acid contents were extremely encouraging. Most aneuploids had higher cichoric acid and chlorogenic acid contents than their parents. For example, A2 had the highest cichoric acid content of 21.98 mg/g dry weight, more than twice the values of diploid and triploid. Meanwhile, A21 had the highest chlorogenic acid content of 1.84 mg/g, approximately five times more than the parental values. Eleven superior aneuploid lines were successfully screened as breeding candidates. The present findings indicated <em>E. purpurea</em> is highly tolerant of karyotypic imbalance and aneuploid plants could serve as prospective breeding resources in <em>E. purpurea</em>.

scholarly journals In Vitro Segregation of Tetraploid and Octoploid Plantlets from Colchicine-induced Ploidy Chimeras in Echinacea purpurea L.

HortScience ◽  
2016 ◽  
Vol 51 (5) ◽  
pp. 549-557 ◽  
Author(s):  
Qingling Li ◽  
Yuesheng Yang ◽  
Hong Wu
Keyword(s):  
Plant Species ◽  
Guard Cells ◽  
Echinacea Purpurea ◽  
Source Material ◽  
Cichoric Acid ◽  
Leaf Petiole ◽  
Stunted Growth ◽  
Ploidy Chimeras ◽  
Ploidy Manipulation

Echinacea purpurea L. is one of the important ornamental and medicinal plant species. Ploidy manipulation is a valuable tool for improving plant quality or production in E. purpurea as well as in many other plants. To study the segregation of pure ploidy plantlets from colchicine-induced ploidy chimeras in E. purpurea, we used a chimera plantlet that consisted of 1.93% diploid, 35.04% tetraploid, and 63.03% octoploid cells as the source material for experiments. The results showed that three factors significantly influenced the segregation, i.e., the component ratios of different ploidy cells in the chimera, the number of sequential passages, and the methods of segregation culture of the chimera plantlets. Other factors, such as explant types (i.e., leaf, petiole, or root) and 6-benzyladenine (BA) concentrations (i.e., 0.2, 0.4, 0.8, and 1.2 mg·L−1) occasionally influenced the segregation. Pure chromosome-doubled polyploids are not easily obtained in various plant species, so segregation culture of ploidy chimeras may potentially be more effective. The morphological characteristic and content of cichoric acid were compared among diploid, tetraploid, and octoploid plants. Results indicated that tetraploid and octoploid plants had more stunted growth, larger stomata, lower stomata frequency, more chloroplast number in guard cells, and higher cichoric acid content than original diploid lines.

scholarly journals 97 ASSESSMENT OF VIABILITY OF IN VITRO PRODUCED BOVINE EMBRYOS FOLLOWING VITRIFICATION BY CVM OR SLOW FREEZING WITH ETHYLENE GLYCOL AND TRIPLE TRANSFER

2005 ◽  
Vol 17 (2) ◽  
pp. 199 ◽  
Author(s):  
B. Peachey ◽  
K. Hartwich ◽  
K. Cockrem ◽  
A. Marsh ◽  
A. Pugh ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Slow Freezing ◽  
High Survival ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Frozen Embryos ◽  
Morula Stage ◽  
Fresh Embryos

Vitrification has become the method of choice for the preservation of in vitro derived embryos of a number of species, and several methods of vitrification have been developed. One such method, the cryoLogic vitrification method (CVM) yields high survival rates of warmed embryos (Lindemans W et al. 2004 Reprod. Fertil. Dev. 16, 174 abst). In this study, the post-warm viability of bovine IVP embryos following either vitrification using CVM or slow freezing using ethylene glycol (EG) was compared. In addition, the survival of embryos following triple transfer to synchronized recipients was measured and the embryo (“e”) and recipient (“r”) contributions to embryo survival was determined using the “er” model for embryo survival (McMillan WH et al. 1998 Theriogenology 50, 1053–1070). Bovine IVP methods were those of van Wagtendonk et al. 2004 Reprod. Fertil. Dev. 16, 214 (abst). On day 7 of culture (Day 0 = IVF), Grade 1 and 2 embryos that had reached at least the late morula stage were selected for vitrification (20% DMSO, 20% ethylene glycol) or freezing in 1.5 M ethylene glycol + 0.1 M sucrose (0.5°C/min to −35°C). Following storage in LN2 for at least 24 h the embryos were thawed, the cryoprotectant removed, and the embryos cultured for 72 h in mSOF medium under 5% CO2, 7% O2, 88% N2. The number of hatching embryos was recorded at 24-h intervals. In addition, blastocyst and expanded blastocyst embryos were thawed and immediately transferred nonsurgically to recipients (three embryos of the same grade to each recipient) on Day 7 of a synchronized cycle (Day 0 = heat). The recipients were ultrasound-scanned for the presence of, and number of, fetuses on Days 35 and 62, respectively. The invitro assessment of 148 CVM and 230 EG frozen embryos indicated that more vitrified than EG embryos hatched by 72 h (73% vs. 62%; CVM vs. EG, χ2 = 4.5, P < 0.05). Overall, more Grade 1 embryos hatched than Grade 2 (74% vs. 60%, χ2 = 7.2, P < 0.01). CVM embryos (105) were triple-transferred to 35 recipients, and EG embryos (30) were triple-transferred to 10 recipients. Recipient pregnancy rates at Day 62 were 80% and 70%, respectively. Overall embryo survival was 38.5% (41% for CVM and 30% for EG). The overall calculated “e” and “r” values were 0.39 and 1.0 (“e”: 0.42 and 1.0, and “r”: 0.31 and 1.0, respectively, CVM and EG groups). Survival rates of CVM embryos to Day 62 (41%) were slightly lower than that previously obtained for fresh embryos produced using an identical IVP procedure (44% – van Wagtendonk AM 2004).

scholarly journals High in vitro survival rate of sheep in vitro produced blastocysts vitrified with a new method and device

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Sergio Ledda ◽  
Jen M. Kelly ◽  
Stefano Nieddu ◽  
Daniela Bebbere ◽  
Federica Ariu ◽  
...  
Keyword(s):  
Survival Rate ◽  
Survival Rates ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
High Survival ◽  
Apoptotic Cells ◽  
Embryo Survival ◽  
New Device ◽  
In Vitro Survival

Abstract Background To advance the use of embryo vitrification in veterinary practice, we developed a system in which embryo vitrification, warming and dilution can be performed within a straw. Ovine in vitro produced embryos (IVEP) were vitrified at either early (EBs: n = 74) or fully expanded blastocyst stage (FEBs: n = 195), using a new device named “E.Vit”, composed by a 0.25-mL straw with a 50-μm pore polycarbonate grid at one end. Embryos at each stage (EBs and FEBs) were vitrified by either Two-step (TS) or Multi-step (MS; 6 different concentrations of vitrification solutions) protocol. Non-vitrified embryos (n = 102) were maintained in in vitro culture as a control. Warming consisted of placing the straws directly into 1.5 mL tubes containing a TCM-199 solution with three decreasing concentrations of sucrose. Blastocyst re-expansion, embryo survival and hatching rate were evaluated at 2, 24 and 48 h post warming. The number of apoptotic cells was determined by TUNEL assay. Results Blastocyst re-expansion (2 h) after warming was higher (P < 0.05) in FEBs group, vitrified with the MS and TS methods (77.90% and 71.25%, respectively) compared with the EBs group (MS: 59.38% and TS: 48.50%, respectively). Survival rates of vitrified FEBs after 24 h IVC were higher (P < 0.001) in both methods (MS and TS) than vitrified EBs (MS: 56.25%; TS: 42.42%) and was higher (P < 0.05) in the MS method (94.19%) compared with those in TS (83.75%). After 48 h of culture the hatching rate for FEBs vitrified in MS system (91.86%) was similar to control (91.89%), but higher than FEB TS (77.5%) and EBs vitrified in MS (37.5%) and TS (33.33%). Number of apoptotic cells were higher in EBs, irrespective of the system used, compared to FEBs. The number of apoptotic cells in FEBs vitrified with MS was comparable to the control. Conclusions A high survival rate of IVP embryos can be achieved by the new “E.Vit” device with hatching rates in vitro comparable with control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.

scholarly journals Generation of UCiPSCs-Derived Neurospheres for Cell Therapy and its Application in Cell Shipment at Ambient Temperature

2020 ◽  
Author(s):  
Shuai Li ◽  
Huifang Zhao ◽  
Xiaobo Han ◽  
Lang He ◽  
Omar Mukama ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Therapy ◽  
Neural Stem Cells ◽  
Ambient Temperature ◽  
Low Cost ◽  
Survival Rates ◽  
High Survival ◽  
Simple Method

Abstract BackgroundNeural stem cells（NSCs）therapy remains one of the most potential approaches for neurological disorders treatment. The discovery of human induced pluripotent stem cells (hiPSCs) and the establishment of hiPSC-derived human neural stem cells (hiNSCs) have revolutionized our technique to cell therapy. Meanwhile, it is often required that NSCs are stored and transported long distances for research or treatment. Although high survival rates could be maintained, conventional methods of cell transport (dry ice or liquid nitrogen) are inconvenient and expensive. Therefore，the establishment of a safe, affordable, and frequent obtained hiPSCs and hiNSCs, with characteristics that match fetal hNSCs and a simple, low-cost way to store and transport, are incredibly urgent. MethodsWe reprogrammed human urinary cells to iPSCs using a virus-free technique and differentiated the iPSCs toward iNSCs/neurospheres and neurons, under Good Manufacturing Practice (GMP)-compatible conditions. The pluripotency of iPSCs and iNSCs was characterized by a series of classical methods (surface markers, karyotype analysis and in vitro and in vivo differentiation capabilities, etc).ResultsHere, our results showed that we successfully generated hiNSCs/neurospheres from more available, non-invasive, and more acceptable urinary cells by a virus-free technique and their differentiation into neural networks. Moreover，hiNSCs survived longer as neurospheres at ambient temperature than those cultured in a monolayer. Approximately 7 days, the neural viability remained at > 80%, while hiNSCs cultured in a monolayer died almost immediately. Neurospheres exposed to ambient temperature that were placed under standard culture conditions (37 ℃, 5% CO2) recovered their typical morphology, and retained their ability to proliferate and differentiate. ConclusionsIn this study, we provided a simple method for the storage of NSCs as neurospheres at ambient temperature as an alternative to more costly and inconvenient traditional methods of cryopreservation. This will enable hiNSCs to be transported over long distances at ambient temperature and facilitate the therapeutic application of NSCs as neurospheres without any further treatment.

Clonal propagation of Pelargonium x hortorum through tissue culture: Effects of salt dilution and growth regulator concentration

1993 ◽  
Vol 73 (3) ◽  
pp. 871-878 ◽  
Author(s):  
Hélène Desilets ◽  
Yves Desjardins ◽  
Richard R. Bélanger
Keyword(s):  
Growth Regulators ◽  
Doubling Time ◽  
Culture Media ◽  
Survival Rates ◽  
Basal Medium ◽  
Multiple Shoots ◽  
Naphthaleneacetic Acid ◽  
High Survival ◽  
Half Strength

Different culture media were compared at the initiation and multiplication steps to develop a rapid production system for geranium (Pelargonium × hortorum) in vitro. Different salt dilutions of the Murashige and Skoog (MS) (1962) mineral medium were used in combination with different concentrations of 1-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) in order to optimize initiation of shoots of four geranium cultivars. The use of a MS basal medium with half-strength macrosalts supplemented with 0.11 μM NAA and 0.89 μM BA gave the best results in initiation. More than 40% of the apices initiated on this medium produced multiple shoots within a month. Subsequently, the effect of different concentrations of growth regulators was quantified by the mean of "shoot doubling time" evaluation. The shortest time recorded was 10.5 d for a theoretical production of 1 × 109 plantlets/apex/year. This is the first quantitative evaluation of geranium production in vitro. Geranium plantlets rooted easily on a half-strength MS medium without growth regulators. Acclimatization of geranium plantlets was characterized by high survival rates (94%) and the plants thus produced were phenotypically comparable to seed-derived plants. Key words: Geranium, micropropagation, shoot doubling time, in vitro

scholarly journals Generation of UCiPSCs-derived neurospheres for cell therapy and its application

2021 ◽  
Author(s):  
Shuai Li ◽  
Huifang Zhao ◽  
Xiaobo Han ◽  
Ni Bin ◽  
Lang He ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Therapy ◽  
Neural Stem Cells ◽  
Cortical Neurons ◽  
Survival Rates ◽  
High Survival ◽  
Long Distance ◽  
Simple Method

Abstract Background Neural stem cells（NSCs）therapy remains one of the most potential approaches for the treatment of neurological disorders. The discovery of human induced pluripotent stem cells (hiPSCs) and the establishment of hiPSC-derived human neural stem cells (hiNSCs) have revolutionized the technique of cell therapy. Meanwhile, it is often required that NSCs are stored and transported to a long distance for research or treatment purposes. Although high survival rates could be maintained, conventional methods for cell transportation (dry ice or liquid nitrogen) are inconvenient and expensive. Therefore，the establishment of a safe, affordable, and low-cost strategy to store and transport easily accessible hiPSCs and hiNSCs, with characteristics that match fetal hNSCs, is incredibly urgent.Methods We reprogrammed human urinary cells to iPSCs using a non-integrating, virus-free technique and differentiated the iPSCs toward iNSCs/neurospheres and neurons, under Good Manufacturing Practice (GMP)-compatible conditions. The pluripotency of iPSCs and iNSCs was characterized by a series of classical methods (surface markers, karyotype analysis and in vitro as well as in vivo differentiation capabilities, etc).Results Here, our results showed that we successfully generated hiNSCs/neurospheres from more available, non-invasive, and more acceptable urinary cells by a virus-free technique. Next, we demonstrated that the iNSCs differentiated into mature cerebral cortical neurons and neural networks. Interestingly, hiNSCs survived longer as neurospheres at ambient temperature (AT) than those cultured in a monolayer. Within 7 days approximately, the neural viability remained at > 80%, while hiNSCs cultured in a monolayer died almost immediately. Neurospheres exposed to AT that were placed under standard culture conditions (37 ℃, 5% CO2) recovered their typical morphology, and retained their proliferation and differentiation abilities. Conclusions In this study, we provided a simple method for the storage of NSCs as neurospheres at AT as an alternative method to more costly and inconvenient traditional methods of cryopreservation. This will enable hiNSCs to be transported over long distances at AT and facilitate the therapeutic application of NSCs as neurospheres without any further treatment.

scholarly journals Vitrification Effects on the Transcriptome of in vivo-Derived Porcine Morulae

2021 ◽  
Vol 8 ◽  
Author(s):  
Cristina Cuello ◽  
Cristina A. Martinez ◽  
Josep M. Cambra ◽  
Alejandro González-Plaza ◽  
Inmaculada Parrilla ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Pathways ◽  
Survival Rates ◽  
Gene Expression Pattern ◽  
Control Group ◽  
High Survival ◽  
Embryo Survival ◽  
Developmental Potential

Despite the reported promising farrowing rates after non-surgical and surgical transfers of vitrified porcine morulae and blastocysts produced in vivo (range: 70–75%), the pregnancy loss is 5–15 fold higher with vitrified than with fresh embryos. The present study aimed to investigate whether vitrification affects the transcriptome of porcine morulae, using microarrays and RT-qPCR validation. Morulae were obtained surgically from weaned sows (n = 13) on day 6 (day 0 = estrus onset). A total of 60 morulae were vitrified (treatment group). After 1 week of storage, the vitrified morulae were warmed. Vitrified-warmed and non-vitrified fresh morulae (control; n = 40) were cultured for 24 h to assess embryo survival by stereomicroscopy after. A total of 30 vitrified/warmed embryos that were deemed viable and 30 fresh control embryos (three pools of 10 for each experimental group) were selected for microarray analysis. Gene expression was assessed with a GeneChip® Porcine Genome Array (Affymetrix). An ANOVA analysis p-unadjusted &lt;0.05 and a fold change cut-off of ±1.5 were set to identify differentially expressed genes (DEGs). Data analysis and biological interpretation were performed using the Partek Genomic Suite 7.0 software. The survival rate of morulae after vitrification and warming (92.0 ± 8.3%) was similar to that of the control (100%). A total of 233 DEGs were identified in vitrified morulae (38 upregulated and 195 downregulated), compared to the control group. Nine pathways were significantly modified. Go-enrichment analysis revealed that DEGs were mainly related to the Biological Process functional group. Up-regulated DEGs were involved in glycosaminoglycan degradation, metabolic pathways and tryptophan metabolism KEGG pathways. The pathways related to the down-regulated DEGs were glycolysis/gluconeogenesis, protein export and fatty acid elongation. The disruption of metabolic pathways in morulae could be related to impaired embryo quality and developmental potential, despite the relatively high survival rates after warming observed in vitro. In conclusion, vitrification altered the gene expression pattern of porcine morulae produced in vivo, generating alterations in the transcriptome that may interfere with subsequent embryo development and pregnancy after embryo transfer.

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