BACKGROUND: Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and...BACKGROUND: Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons. OBJECTIVE: To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral cortical neurons cultured in vitro. DESIGN: A comparative experiment. SETTING: Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine. MATERIALS: The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005. Totally 218 Wistar rats (male or female) of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College (certification number was 021-97-03). METHODS: ① Preparation of cerebral cortical neurons of rats: The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral cortical neurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27 Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons: After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages (1-20 g/L) was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③ Grouping: After hypoxia/reoxygenation, the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum polysaccharide group. In the normal control group, the neurons were cultured at 37 ℃ in CO2 with the volume fraction of 0.05 under saturated humidity for 6 days. In the apoptotic positive group, the neurons were cultured with hypoxia for 12 hours after 4-day culture, and followed by reoxygenation for 48 hours. In the polygonatum polysaccharide group, polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to some neurons at 10 hours before the hypoxia culture, and then the neurons were cultured with hypoxia for 12 hours, followed by reoxygenation for 48 hours; polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to the other neurons at 12 hours after hypoxia followed by reoxygenation for 48 hours. ④ The Hoechst33342 fluorescence staining, Annexin V/PI flow cytometer, appearance of DNA agarose gel electrophoresis gradient strap and immunohistochemical staining were used to observe the expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax, and observe the effect of polygonatum polysaccharide against the hypoxic apoptosis of cerebral cortical neurons of neonatal rats. MAIN OUTCOME MEASURES: ① Toxicity and non-toxic dosage of polygonatum polysaccharide on neurons; ② Apoptotic rate of neurons detected with Hoechst33342 fluorescence staining; ③ Early apoptotic rate and necrotic rate of neurons detected with Annexin V/PI flow cytometer; ④ DNA agarose gel electrophoresis ladder-like strap appeared or not; ⑤ Expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax. RESULTS: ① Polygonatum polysaccharide within 6 g/L had no cytotoxicity on the normal cultured cerebral cortical neurons (P > 0.05). ② The apoptotic rates of neurons detected with Hoechst33342 fluorescence staining had significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 10 hours before the hypoxia culture [(13.00±4.52)%,(12.72±2.15)%,(11.80±1.18)%,(38.03±1.05)%, P < 0.01], and had no significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 12 hours after the hypoxia culture (36.77±1.45)%, (36.60±1.61)%, (36.37±2.02)%, (38.03±1.05)%, P > 0.05].③ Annexin V/PI flow cytometer detected that the anti-necrotic effect was enhanced with the increased concentration of polygonatum polysaccharide within 0.5-1.5 g/L (P < 0.01). Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia could significantly decrease the apoptotic rate of neurons induced by hypoxia/reoxygenation (P < 0.01). ④ No DNA agarose gel electrophoresis ladder-like strap appeared in the groups with polygonatum polysaccharide of 0.5-1.5 g/L added at 10 hours before hypoxia; ⑤ After Polygonatum polysaccharide of 0.5-1.5 g/L was added before hypoxia, the expression of Bcl-2 protein of hypoxic neurons was increased (P < 0.01), and those of Bax protein and Caspase-3 protein were reduced (P < 0.01), and the ratio of Bcl-2/Bax was increased (P < 0.01). CONCLUSION: Polygonatum polysaccharide within 6 g/L has no cytotoxicity on the normal cultured cerebral cortical neurons. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia plays a role agains necrosis of neurons induced by hypoxia. Polygonatum polysaccharide of 0.5-1.5 g/L can significantly reduce the apoptosis of neurons induced by hypoxia through up-regulating the expression of Bcl-2 protein, down-regulating the expressions of Bax protein and Caspase-3 protein, and increasing the ratio of Bcl-2/Bax.展开更多
Objective To establish a method for quantitative detection of the sulfate glycosaminoglycans ( GAG) content in extracellular matrix of in vitro cultured chondrocytes so as to evaluate the biological characteristics of...Objective To establish a method for quantitative detection of the sulfate glycosaminoglycans ( GAG) content in extracellular matrix of in vitro cultured chondrocytes so as to evaluate the biological characteristics of epiphyseal, articular and rib chondrocytes. Methods Sulfate GAG content in extracellular matrix of three chondrocytes was measured by the modified dimethylmethylene blue (DMB) method. The changes of the toluidine blue (TB) stain of chondrocytes were observed by light microscope. Results Primary chondrocytes had the highest content of sulfate GAG in the extracellular matrix, ie, epiphyseal chondrocytes reached ( 70. 12 ± 7. 72 )μg/cm2, articular chondrocytes (92.00 ± 10.15) μg/cm2 and rib chondrocytes (80.61 ± 11. 40) μg/cm2, respectively. On the third pasage chondrocytes, epiphyceal chondrocytes decreased to (53.27 ± 9. 50 ) μg/cm2, articular chondrocytes to (63.88 ± 11.92) μg/cm2 and rib chondrocytes to (58.94 ±8.21) μg/cm2, respectively. The change of TB in every passage展开更多
[Objectives] This study was conducted to investigate the effects of lamb age and in vitro culture system of oocytes on the results of juvenile in vitro embryo transfer( JIVET). [Methods]Ten Dorper × small-tailed ...[Objectives] This study was conducted to investigate the effects of lamb age and in vitro culture system of oocytes on the results of juvenile in vitro embryo transfer( JIVET). [Methods]Ten Dorper × small-tailed Han lambs aged 5 to 10 weeks were induced to superovulate via i. p. injection of pregnant mare's serum gonadotropin( PMSG). The oocytes were matured in basal maturation solution or modified maturation solution,which was prepared by adding 200 μmol/L cysteine to the basal maturation solution. Then,the oocytes were fertilized in fertilization medium I containing 2% estrus sheep serum( ESS) or fertilization medium II containing 3 mg/ml bull serum albumin( BSA). Finally,the number of oocytes,oocyte maturation rate and cleavage rate of the lambs of different ages were determined. [Results]The average number of oocytes recovered per lamb was( 111. 00 ± 16. 97),( 139. 50 ± 28. 99),( 108. 50 ± 17. 68) and( 42. 00 ± 11. 31) for5-,7-,8-and 10-week-old Dorper × small-tailed Han lambs,respectively. The number of oocytes obtained from 5-,7-and 8-week-old lambs was significantly higher than that from 10-week-old lambs( P < 0. 05),but there was no significant difference among 5-,7-and 8-week-old lambs( P > 0. 05). The maturation rate of oocytes cultured in modified maturation solution was 3. 64% higher than that in basal maturation solution. The cleavage rate of oocytes in fertilization medium I was very significantly higher than that in fertilization medium II( P < 0. 01). [Conclusions] The results of JIVET can be improved by harvesting oocytes from lambs aged 5-8 weeks,adding a certain amount of cysteine into oocyte maturation solution,and a certain amount of ESS into fertilization medium.展开更多
Research on in vitro culture and gene editing of domestic spermatogonial stem cells (SSCs) is of considerable interest but remains a challenging issue in animal science. In recent years, some progress on the isolation...Research on in vitro culture and gene editing of domestic spermatogonial stem cells (SSCs) is of considerable interest but remains a challenging issue in animal science. In recent years, some progress on the isolation, purification, and genetic manipulation of porcine SSCs has been reported. Here, we summarize the characteristics of porcine SSCs as well current advances in their in vitro culture, potential usage, and genetic manipulation. Furthermore, we discuss the current application of gene editing in pig cloning technology. Collectively, this commentary aims to summarize the progress made and obstacles encountered in porcine SSC research to better serve animal husbandry, improve livestock fecundity, and enhance potential clinical use.展开更多
The effects of the microspore developmental stage,hormones and culture condition on anther in vitro culture of lily(Lilium spp.) were discussed.The results showed that when the flower buds were about 23-26 mm long,the...The effects of the microspore developmental stage,hormones and culture condition on anther in vitro culture of lily(Lilium spp.) were discussed.The results showed that when the flower buds were about 23-26 mm long,the microspores were at the uninucleate stage which was suitable for culture and the culture under the darkness would promote the callus induction of anther.The induction frequency could reach 42.5% in the optimized medium which was MS+[6-BA(0.5)+KT(2.0)+2,4-D(1.0)] mg·L-1.The rate of callus differentiation could reach 31.57% in the optimized medium which was MS+ NAA(1.5,2.0) mg·L-1.展开更多
Hybrid combinations of Eucalyptus have increased due to expansion of plantations into unconventional areas and to the search for higher quality timber.However,most of these species have difficulties surviving in vitro...Hybrid combinations of Eucalyptus have increased due to expansion of plantations into unconventional areas and to the search for higher quality timber.However,most of these species have difficulties surviving in vitro cultivation.Active chlorine and sealing systems are often used to reduce contamination and increase gas exchange.The aim of the present study is to evaluate the establishment,multiplication,elongation and adventitious rooting of E.grandis × E.urophylla.Two clones(C1 and C2) and four active chlorine concentrations(0.000%,0.001%,0.003%,and 0.005%) were tested in the establishment and multiplication phases.Three sealing forms(W/M,1/M and 3/M) and the same four active chlorine concentrations were applied to the elongation phase.Two luminosities(dark and light)and three sealings(W/M,1/M and 3/M) were tested during adventitious rooting.Active chlorine concentration of0.005% led to the lowest fungal contamination rate and to the highest in vitro establishment.Active chlorine concentration of 0.003% resulted in the greatest length and highest number of shoots per explant in the multiplication phase.There were no phytotoxicity problems and the quality of plants grown in an environment with active chlorine was maintained in comparison with those grown in an autoclave.The increase in gas exchange in ventilation systems had a positive impact on the in vitro growth and development of plants.展开更多
This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus (NP) cells in vitro in order to provide seed cells for intervertebral disc (IVD) tissue engineering. A total of 8 ad...This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus (NP) cells in vitro in order to provide seed cells for intervertebral disc (IVD) tissue engineering. A total of 8 adult New Zealand white rabbits underwent annulus puncture to establish models of intervertebral disc degeneration (IDD). Four weeks later, normal and degenerated NP cells were obtained. Cell morphology was observed by light and electron microscopy. Cell viability was measured by MTT assay. Cell cycle and expression of extracellular matrix (ECM)-related genes (aggrecan and type Ⅱ collagen) were determined by using flow cytometry and RT-PCR respectively. The growth curve of normal NP cells showed that the cells at passage 4 tended to slowly grow on the fifth day of culture. The density of normal NP cells at passages 5 to 7 was significantly less than that of the first-passage cells 2 or 3 days after seeding (P<0.05). The degenerated NP cells at passage 3 showed slow growth at 4th day. After 5 passages, the degenerated NP cells assumed stagnant growth and the growth seemed to stop at passage 7. The MTT assay revealed that for both normal and degenerated NP cells, the absorbance (A) value at passages 4-7 was obviously decreased as compared with that at passage 1 (P<0.05). Cell cycle analysis showed that the proportion of normal NP cells at G l phase was 65.4%±3.5%, significantly lower than that of degenerated NP cells at the same cell cycle phase with the value being 77.6%±4.8%. The degenerated NP cells were predominantly arrested at G 1 phase and failed to enter S phase. The expression of type Ⅱ collagen and aggrecan was significantly decreased with passaging. It was concluded that normal NP cells possessed good viability and proliferative capacity by the third passage, and they could secrete large amounts of ECM within this period. The normal NP cells may serve as seed cells for IVD tissue engineering.展开更多
For the mass production of Koelreuteria bipinnata var. integrifoliola with selected, hybrid or genetically engineered genotypes, one potentially desirable propagation strategy is based on embryo culture. The immature ...For the mass production of Koelreuteria bipinnata var. integrifoliola with selected, hybrid or genetically engineered genotypes, one potentially desirable propagation strategy is based on embryo culture. The immature embryo development in vitro from K. bipinnata var. integrifoliola was studied under different conditions of embryo age, basic culture media and plant growth regulators. The results show that: 1) germination rate of grade 3 embryos in immature seeds with 0.6–0.8 cm diameter was 98.9%. The germination rate of grade 2 embryos in immature seeds with 0.4–0.6 cm diameter was 77.8% and the germination rate of grade 1 embryos in immature seeds with 0.4 cm diameter was 15.6%. 2) The amounts of macroelements in MS medium had no clear effect on the germination rate of immature grade 3 embryos and had a modest effect on plantlet growth, where the best medium was MS or 1/2 MS. The rates were all greater than 90%. 3) The germination rate of grade 3 embryos was greater than 87% when the medium contained a low concentration of NAA or no plant growth regulators at all and decreased markedly when BAP alone or BAP and NAA together were added to the media. We suggest that in vitro culture of immature embryos from K. bipinnata var. integrifoliola can be enhanced when a small amount of plant growth regulators is added. The addition of BAP has an adverse reaction to the germination and development of immature embryos.展开更多
Objective:To assess the effect of L-carnitine supplementation during in vitro oocyte maturation and in vitro culture process of bovine oocytes.Methods:L-carnitine(3.8 mM)was added to maturation medium and the effect w...Objective:To assess the effect of L-carnitine supplementation during in vitro oocyte maturation and in vitro culture process of bovine oocytes.Methods:L-carnitine(3.8 mM)was added to maturation medium and the effect was assessed in the quality(Experiment 1)and in the cleavage and 4-cells stage(Experiment 2).Besides,the effect of L-carnitine addition on maturation medium(3.8 mM)and culture medium(1.5 mM)on embryo rate production was assessed.In Experiment 1,bovine oocytes from abattoir were randomly separated into two groups(the control group and L-carnitine group)forin vitro maturation.Matured oocytes were examined for cumulus cells expansion as an indicator of maturation,and the content of the mitochondrial activity,the presence of lipid droplets,the reduced glutathione,and the reactive oxygen species were measured by using specific fluorochromes.In Experiment 2,oocytes were matured as performed in Experiment 1,afterward fertilized and cultured until day 3,and cleavage rate and 4-cells stage rate were determinated.In Experiment 3,in vitro maturation and fertilization were done as performed in Experiment 2,but at day 3 of culture,each group of embryos was separated into two new groups,and L-carnitine(1.5 mM)was added in culture media until day 8.The cleavage and embryo development rate were determined on the basis with the oocytes put on maturation.Hatching rate was calculated from cleaved embryos.Results:The cumulus expansion rate at gradeⅢand mitochondrial activity were significantly higher in the L-carnitine group in comparison with the control group(P0.05).In addition,cleavage and the proportion of embryo development and hatching rate were similar for all groups(P>0.05).Conclusions:L-carnitine as a supplement in culture media improves the cumulus expansion and increases the mitochondrial activity during in vitro maturation process but has no apparent effect on the cleavage and development of bovine embryos.Further investigations of L-carnitine addition on in vitroculture are needed to test their effect on embryo quality.展开更多
In recent years,bonamiosis has frequently occurred in European areas,which has caused the death of oyster in a wide range and brought enormous economic losses to the breeding industry of oyster. Nowadays,the study on ...In recent years,bonamiosis has frequently occurred in European areas,which has caused the death of oyster in a wide range and brought enormous economic losses to the breeding industry of oyster. Nowadays,the study on Bonamia sp. is still in the elementary phase. The technology of pathogen's culture in vitro is the basis for further study on the pathogenesis of Bonamia sp.,its interaction with hosts and the prevention and control of the related disease. In this study,total tissues of oyster identified by PCR were used as culture media to in vitro culture. After one month,they were identified by the method of in situ hybridization. It was found that the results of in situ hybridization were accordant with PCR results. And Bonamia ostreae were detected by in situ hybridization after B. ostreae were cultured for one month. We successfully established a simple and feasible method for in vitro culturing B. ostreae.展开更多
BACKGROUND:Midbrain-derived neural stem cells (mNSCs) can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson’s disease. OBJECTIVE: To is...BACKGROUND:Midbrain-derived neural stem cells (mNSCs) can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson’s disease. OBJECTIVE: To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN,TIME AND SETTING: An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine,China-Japan Friendship Hospital (China) from March to November 2007. MATERIALS: Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody,β-Ⅲ tubulin antibody,glial fibrillary acidic protein (GFAP) antibody and cyclic nucleotide 3’-phosphohydrolase (CNPase) antibody were provided by Abcam; DMEM/F12 medium and N2 supplement were provided by In-vitrogen; epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2) were provided by R&D Sys-tems. METHODS: The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin di-gestion and mechanical separation,the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/F12,1% N2 supplement,20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10 μg/mL polylysine and induced to differenti-ate in the DMEM/F12 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES: The neural spheres of the third passage were identified by nestin im-munofluorescence; at the same time,the cells were induced to differentiate,and the types of differentiated cell were identified by immunofluorescence for βⅢ tubulin,GFAP and CNPase. RESULTS: Seven days after primary culture,a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive,and after differentia-tion,the cells expressed GFAP,CNPase and β-Ⅲ-tubulin. CONCLUSION: Embryonic day 14 rat mNSCs can differentiate into neuron-like cells and glial cells fol-lowing induction by EGF,FGF2 and N2 additive.展开更多
Brassica carinata, a natural alloploid formed between B. oleracea and B. nigra, is a potential oil crop for the Mediterranean area in which genetic transformation could help to breeding. In vitro culture and shoot reg...Brassica carinata, a natural alloploid formed between B. oleracea and B. nigra, is a potential oil crop for the Mediterranean area in which genetic transformation could help to breeding. In vitro culture and shoot regeneration are key factors in developing an efficient transformation method in the genus Brassica. However, the studies for in vitro culture and shoot regeneration in B. carinata are limited to only a few genotypes. The aim of this study was to evaluate the in vitro culture response and shoot regeneration in a collection of B. carinata accessions to identify promising genotypes with high shoot regeneration for genetic transformation experiments. Cotyledonary explants from 51 genotypes were cultured in vitro and callus formation and swelling as well as the mode of shoot regeneration evaluated. A highly positive response to in vitro culture, i.e. callus formation or swelling, was observed in all the genotypes tested. Tissue blackening occurred only in eleven genotypes. Parameters like callus formation and swelling, and number of shoots per explant were highly variable among genotypes. Fourteen genotypes regenerated only via callus formation, whereas only one regenerated only via swelling. Most genotypes showed a higher percentage of callus formation than swelling. The average number of shoots regenerating per explant among genotypes was the most variable factor measured. Six genotypes regenerated more than 6 shoots per explant via callus phase. These genotypes have been identified as having a high regeneration potential and can be used in genetic transformation via Agrobacterium.展开更多
The genetic variability is considered as the major principle of plant breeding for durum wheat. This variability can be induced in vitro by selection pressure exerted by stress factors such as salinity in order to reg...The genetic variability is considered as the major principle of plant breeding for durum wheat. This variability can be induced in vitro by selection pressure exerted by stress factors such as salinity in order to regenerate the vitro plantlets tolerant. This study aims in the first step in the regeneration of plantlets tolerant to salinity from mature embryos culture derived from two Tunisian durum wheat varieties: improved (Razzek) and landrace (Jenah Khotifa (JK)) varieties. The tolerance evaluation to salt stress was applied in vitro (100 mmol·l-1 NaCl) and was based on various parameters. Our results showed that JK variety was distinguished by a stable response for all parameters tested: average weight of callus (368.1 mg for control and 307 mg under salt stress), callus regenerated percentage (36.6% for control and 35.7% under salt stress) and green shoots number/callus (17 for control and 17 under salt stress). This stability of response translates the adaptability of this variety to salinity. In order to fix regenerated JK plantlets in single generation and obtain HDs homozygous stable lines, in vitro gynogenesis technical is tested for this genotype. The Evaluation of gynogenetic capacity focused on about 1200 unfertilized ovaries of JK and was based on its ability to induction, differentiation, development of green shoots, and haploid plantlets regeneration. JK showed good tolerance to salinity and a relatively good response to gynogenesis.展开更多
BACKGROUND: The ability to maintain isolated human islet preparation in tissue culture has recently been adopted by most islet transplant centers to improve the safety and practicality of islet transplantation. Howeve...BACKGROUND: The ability to maintain isolated human islet preparation in tissue culture has recently been adopted by most islet transplant centers to improve the safety and practicality of islet transplantation. However, maintaining islet viability and recovery remains a challenge in clinical setting. Extracellular matrix (ECM) is one of the most important components of islet microenvironment. The reconstruction of the cell-matrix relationship seems to be effective in improving the loss of differentiated islet structure and function. Small intestinal submucosa ( SIS ) , a naturally occurring ECM, has been investigated to be able to promote wound healing, tissue remodeling, and cell growth. The purpose of this study was to evaluate the recovery and function of isolated rat pancreatic islets after in vitro culture with SIS. METHODS: Pancreatic islets were isolated from Wistar rats by using standard surgical procurement followed by intra-ductal collagenase distension, mechanical dissociation, and EuroFicoll purification. Groups of purified islets were cultured in plates which were coated with multilayer SIS (SIS-treated group) or without ( standard cultured group) for 7 days and 14 days in standard islet culture conditions of RP-MI 1640 tissue culture media in humidified atmosphere containing 95% air and 5% CO2 at 37 ℃. The mean recovery of islets after the culture period was determined by sizing duplicate counts of a known volume and their viability was assessed by static incubation with low glucose (2.7 mmol) , high glucose (16.7 mmol) and high glucose solution supplemented with 50 μm 3-isobutyl-1-methylxanthine (IB-MX) solution. RESULTS: After 7 days and 14 days of in vitro tissue culture, the SIS-treated group showed a significantly higher recovery compared with those cultured under standard conditions. The recovery in the SIS-treated group was about two times of the control group cultured in standard conditions after 14 days culture. In the SIS-treated group, there was no statistically difference between the short and long periods of culture ( 95. 8 ± 1.0% vs. 90. 8±1. 5% , P 】 0.05). During incubation in high glucose (16.7 mmol) solution, there was a 2-3 fold increase in insulin secretion from both groups, but the SIS-treated group showed a higher increase than the standard cultured group after 14-day culture (20.7 ±1.1 mU/L vs. 11. 8 ±1.1 mU/L, P 【 0. 05). When islets were placed in the high glucose solution supplemented with IBMX, the stimulated insulin response in the SIS-treated group was higher than that in the standard cultured group in spite of the duration of the culture. The stimulation index of the SIS-treated group was about 2-3 times of the standard cultured group. In addition , after a long period of culture, the stimulation index of the SIS-treated group was statistically equivalent with that of the short period of culture (9.5 ±0.2 vs. 10.2 ±1.2, P】0.05). CONCLUSIONS: The co-culture of isolated rat islets with native sheet-like SIS can provide an excellent extracellular matrix, possible biotrophic and growth factors that promote the recovery and subsequent function of islets after in vitro tissue culture. In view of results of this study and rapid degradation of SIS in vitro, future studies will investigate the extended duration of culture and the effect of SIS on islets in vitro.展开更多
The purpose of our study was to establish a regeneration system for micropropagation of Populus euphratica Olivier. On the basis of an analysis of plant leaf mineral nutrients, a special medium was proposed, called MP...The purpose of our study was to establish a regeneration system for micropropagation of Populus euphratica Olivier. On the basis of an analysis of plant leaf mineral nutrients, a special medium was proposed, called MP2. In optimizing media for in vitro plant cultures including MS, B5 and MP2 media we employed hormones, auxin IAA, cytokine benzyladenine (BAP) and gibberellic acid (GA) in our factorial experiments on media. Adventitious shoots were derived from cuttings of adult plants taken from Xingjiang, west China, on selected media with MP2+0.5 mg·L-1 BA+ 0.1 mg·L-1 NAA. The shoots were elongated on a medium with 0.25 mg·L-1 BAP, 0.1 mg·L-1 NAA and 2 mg·L-1 GA and were then rooted on a medium with 0.2-0.5 mg·L-1 IBA. All the media were incorporated with 30 g·L-1 sucrose and an adjusted pH at 6.3.展开更多
The present investigation was undertaken to study the effect of gamma irradiation (dose from 10 to 100 Gy) and in vitro selection with fungus filtrate as selecting agent (concentration from 20% to 100%) on the suscept...The present investigation was undertaken to study the effect of gamma irradiation (dose from 10 to 100 Gy) and in vitro selection with fungus filtrate as selecting agent (concentration from 20% to 100%) on the susceptibility of the common bean to Rhizoctonia solani. The best results were found with a dose of 20 Gy or a concentration of 20% of fungus filtrate applied separately. These conditions were used to evaluate the combined effect of both approaches in a second experiment. The combined effect of irradiation and then selection adversely affected growth (height and roots) and survival of the in vitro plants. It may not be necessary to combine the variation generated by irradiation with the selection technique. For future assays we propose the application of: 1) gamma radiation, thereby inducing not only mutants with pathogen resistance, but also with other agronomic traits of interest. Later in the subculture MV4 potential fungus-resistant mutants will be evaluated in the field;or 2) selection pressure using fungus filtrate during three subcultures, which may be sufficient to induce the variation necessary to obtain in vitro plants resistant to fungus.展开更多
Fertilized rabbit eggs injected with SMTPGH gene were cultured in vitro and retention of theinjected gene was studied using PCR technique and nonradioactive labelling methed. In a mediumof TC199 + 10% FCS, three quart...Fertilized rabbit eggs injected with SMTPGH gene were cultured in vitro and retention of theinjected gene was studied using PCR technique and nonradioactive labelling methed. In a mediumof TC199 + 10% FCS, three quarters of the fertilized eggs developed to the blastocyst stage. Noapparent change of the injected gene was found before the 8-cell stage, after which it was eitherintegrated into the chromosome of the host or lost gradually. But finally, the retention rate of theinjected gene should be equal to its integration rate.展开更多
文摘BACKGROUND: Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons. OBJECTIVE: To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral cortical neurons cultured in vitro. DESIGN: A comparative experiment. SETTING: Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine. MATERIALS: The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005. Totally 218 Wistar rats (male or female) of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College (certification number was 021-97-03). METHODS: ① Preparation of cerebral cortical neurons of rats: The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral cortical neurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27 Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons: After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages (1-20 g/L) was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③ Grouping: After hypoxia/reoxygenation, the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum polysaccharide group. In the normal control group, the neurons were cultured at 37 ℃ in CO2 with the volume fraction of 0.05 under saturated humidity for 6 days. In the apoptotic positive group, the neurons were cultured with hypoxia for 12 hours after 4-day culture, and followed by reoxygenation for 48 hours. In the polygonatum polysaccharide group, polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to some neurons at 10 hours before the hypoxia culture, and then the neurons were cultured with hypoxia for 12 hours, followed by reoxygenation for 48 hours; polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to the other neurons at 12 hours after hypoxia followed by reoxygenation for 48 hours. ④ The Hoechst33342 fluorescence staining, Annexin V/PI flow cytometer, appearance of DNA agarose gel electrophoresis gradient strap and immunohistochemical staining were used to observe the expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax, and observe the effect of polygonatum polysaccharide against the hypoxic apoptosis of cerebral cortical neurons of neonatal rats. MAIN OUTCOME MEASURES: ① Toxicity and non-toxic dosage of polygonatum polysaccharide on neurons; ② Apoptotic rate of neurons detected with Hoechst33342 fluorescence staining; ③ Early apoptotic rate and necrotic rate of neurons detected with Annexin V/PI flow cytometer; ④ DNA agarose gel electrophoresis ladder-like strap appeared or not; ⑤ Expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax. RESULTS: ① Polygonatum polysaccharide within 6 g/L had no cytotoxicity on the normal cultured cerebral cortical neurons (P > 0.05). ② The apoptotic rates of neurons detected with Hoechst33342 fluorescence staining had significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 10 hours before the hypoxia culture [(13.00±4.52)%,(12.72±2.15)%,(11.80±1.18)%,(38.03±1.05)%, P < 0.01], and had no significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 12 hours after the hypoxia culture (36.77±1.45)%, (36.60±1.61)%, (36.37±2.02)%, (38.03±1.05)%, P > 0.05].③ Annexin V/PI flow cytometer detected that the anti-necrotic effect was enhanced with the increased concentration of polygonatum polysaccharide within 0.5-1.5 g/L (P < 0.01). Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia could significantly decrease the apoptotic rate of neurons induced by hypoxia/reoxygenation (P < 0.01). ④ No DNA agarose gel electrophoresis ladder-like strap appeared in the groups with polygonatum polysaccharide of 0.5-1.5 g/L added at 10 hours before hypoxia; ⑤ After Polygonatum polysaccharide of 0.5-1.5 g/L was added before hypoxia, the expression of Bcl-2 protein of hypoxic neurons was increased (P < 0.01), and those of Bax protein and Caspase-3 protein were reduced (P < 0.01), and the ratio of Bcl-2/Bax was increased (P < 0.01). CONCLUSION: Polygonatum polysaccharide within 6 g/L has no cytotoxicity on the normal cultured cerebral cortical neurons. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia plays a role agains necrosis of neurons induced by hypoxia. Polygonatum polysaccharide of 0.5-1.5 g/L can significantly reduce the apoptosis of neurons induced by hypoxia through up-regulating the expression of Bcl-2 protein, down-regulating the expressions of Bax protein and Caspase-3 protein, and increasing the ratio of Bcl-2/Bax.
文摘Objective To establish a method for quantitative detection of the sulfate glycosaminoglycans ( GAG) content in extracellular matrix of in vitro cultured chondrocytes so as to evaluate the biological characteristics of epiphyseal, articular and rib chondrocytes. Methods Sulfate GAG content in extracellular matrix of three chondrocytes was measured by the modified dimethylmethylene blue (DMB) method. The changes of the toluidine blue (TB) stain of chondrocytes were observed by light microscope. Results Primary chondrocytes had the highest content of sulfate GAG in the extracellular matrix, ie, epiphyseal chondrocytes reached ( 70. 12 ± 7. 72 )μg/cm2, articular chondrocytes (92.00 ± 10.15) μg/cm2 and rib chondrocytes (80.61 ± 11. 40) μg/cm2, respectively. On the third pasage chondrocytes, epiphyceal chondrocytes decreased to (53.27 ± 9. 50 ) μg/cm2, articular chondrocytes to (63.88 ± 11.92) μg/cm2 and rib chondrocytes to (58.94 ±8.21) μg/cm2, respectively. The change of TB in every passage
基金Supported by Special Fund for National Hair Sheep Industrial Technology System(CARS-39-24)Science and Technology Development Program of Shanxi Province(20120311024-1)+2 种基金Science and Technology Innovation Team Project of Shanxi Province(201705D131028-20)Financial Support of Agriculture of Shanxi Province(NYGX2015-03)Talent Project for Science and Technology Development in Outlaying Poor Areas,Frontier Ethnic Minority Areas and Old Revolutionary Base Areas of Shanxi Province,China(2017Sy128)
文摘[Objectives] This study was conducted to investigate the effects of lamb age and in vitro culture system of oocytes on the results of juvenile in vitro embryo transfer( JIVET). [Methods]Ten Dorper × small-tailed Han lambs aged 5 to 10 weeks were induced to superovulate via i. p. injection of pregnant mare's serum gonadotropin( PMSG). The oocytes were matured in basal maturation solution or modified maturation solution,which was prepared by adding 200 μmol/L cysteine to the basal maturation solution. Then,the oocytes were fertilized in fertilization medium I containing 2% estrus sheep serum( ESS) or fertilization medium II containing 3 mg/ml bull serum albumin( BSA). Finally,the number of oocytes,oocyte maturation rate and cleavage rate of the lambs of different ages were determined. [Results]The average number of oocytes recovered per lamb was( 111. 00 ± 16. 97),( 139. 50 ± 28. 99),( 108. 50 ± 17. 68) and( 42. 00 ± 11. 31) for5-,7-,8-and 10-week-old Dorper × small-tailed Han lambs,respectively. The number of oocytes obtained from 5-,7-and 8-week-old lambs was significantly higher than that from 10-week-old lambs( P < 0. 05),but there was no significant difference among 5-,7-and 8-week-old lambs( P > 0. 05). The maturation rate of oocytes cultured in modified maturation solution was 3. 64% higher than that in basal maturation solution. The cleavage rate of oocytes in fertilization medium I was very significantly higher than that in fertilization medium II( P < 0. 01). [Conclusions] The results of JIVET can be improved by harvesting oocytes from lambs aged 5-8 weeks,adding a certain amount of cysteine into oocyte maturation solution,and a certain amount of ESS into fertilization medium.
基金supported by the Fundamental Research Funds for the Central Universities in China(KYDS201807)Ministry of Science and Technology,China(2016YFE0128500)
文摘Research on in vitro culture and gene editing of domestic spermatogonial stem cells (SSCs) is of considerable interest but remains a challenging issue in animal science. In recent years, some progress on the isolation, purification, and genetic manipulation of porcine SSCs has been reported. Here, we summarize the characteristics of porcine SSCs as well current advances in their in vitro culture, potential usage, and genetic manipulation. Furthermore, we discuss the current application of gene editing in pig cloning technology. Collectively, this commentary aims to summarize the progress made and obstacles encountered in porcine SSC research to better serve animal husbandry, improve livestock fecundity, and enhance potential clinical use.
基金Supported by Program of Postdoctoral Funds Project of Heilongjiang Province (LBH-Z08259)Program for Innovative Research Team of Northeast Agricultural University (CXZ004)
文摘The effects of the microspore developmental stage,hormones and culture condition on anther in vitro culture of lily(Lilium spp.) were discussed.The results showed that when the flower buds were about 23-26 mm long,the microspores were at the uninucleate stage which was suitable for culture and the culture under the darkness would promote the callus induction of anther.The induction frequency could reach 42.5% in the optimized medium which was MS+[6-BA(0.5)+KT(2.0)+2,4-D(1.0)] mg·L-1.The rate of callus differentiation could reach 31.57% in the optimized medium which was MS+ NAA(1.5,2.0) mg·L-1.
基金the support of the Coordination for the Improvement of Higher Education Personnel-Brazil (CAPES)the National Council for Scientific and Technological Development (CNPq)the Research Support Foundation of the State of Minas (FAPEMIG)。
文摘Hybrid combinations of Eucalyptus have increased due to expansion of plantations into unconventional areas and to the search for higher quality timber.However,most of these species have difficulties surviving in vitro cultivation.Active chlorine and sealing systems are often used to reduce contamination and increase gas exchange.The aim of the present study is to evaluate the establishment,multiplication,elongation and adventitious rooting of E.grandis × E.urophylla.Two clones(C1 and C2) and four active chlorine concentrations(0.000%,0.001%,0.003%,and 0.005%) were tested in the establishment and multiplication phases.Three sealing forms(W/M,1/M and 3/M) and the same four active chlorine concentrations were applied to the elongation phase.Two luminosities(dark and light)and three sealings(W/M,1/M and 3/M) were tested during adventitious rooting.Active chlorine concentration of0.005% led to the lowest fungal contamination rate and to the highest in vitro establishment.Active chlorine concentration of 0.003% resulted in the greatest length and highest number of shoots per explant in the multiplication phase.There were no phytotoxicity problems and the quality of plants grown in an environment with active chlorine was maintained in comparison with those grown in an autoclave.The increase in gas exchange in ventilation systems had a positive impact on the in vitro growth and development of plants.
文摘This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus (NP) cells in vitro in order to provide seed cells for intervertebral disc (IVD) tissue engineering. A total of 8 adult New Zealand white rabbits underwent annulus puncture to establish models of intervertebral disc degeneration (IDD). Four weeks later, normal and degenerated NP cells were obtained. Cell morphology was observed by light and electron microscopy. Cell viability was measured by MTT assay. Cell cycle and expression of extracellular matrix (ECM)-related genes (aggrecan and type Ⅱ collagen) were determined by using flow cytometry and RT-PCR respectively. The growth curve of normal NP cells showed that the cells at passage 4 tended to slowly grow on the fifth day of culture. The density of normal NP cells at passages 5 to 7 was significantly less than that of the first-passage cells 2 or 3 days after seeding (P<0.05). The degenerated NP cells at passage 3 showed slow growth at 4th day. After 5 passages, the degenerated NP cells assumed stagnant growth and the growth seemed to stop at passage 7. The MTT assay revealed that for both normal and degenerated NP cells, the absorbance (A) value at passages 4-7 was obviously decreased as compared with that at passage 1 (P<0.05). Cell cycle analysis showed that the proportion of normal NP cells at G l phase was 65.4%±3.5%, significantly lower than that of degenerated NP cells at the same cell cycle phase with the value being 77.6%±4.8%. The degenerated NP cells were predominantly arrested at G 1 phase and failed to enter S phase. The expression of type Ⅱ collagen and aggrecan was significantly decreased with passaging. It was concluded that normal NP cells possessed good viability and proliferative capacity by the third passage, and they could secrete large amounts of ECM within this period. The normal NP cells may serve as seed cells for IVD tissue engineering.
基金supported by the Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of Ministry of Education, Beijing Forestry University, China (05–04)
文摘For the mass production of Koelreuteria bipinnata var. integrifoliola with selected, hybrid or genetically engineered genotypes, one potentially desirable propagation strategy is based on embryo culture. The immature embryo development in vitro from K. bipinnata var. integrifoliola was studied under different conditions of embryo age, basic culture media and plant growth regulators. The results show that: 1) germination rate of grade 3 embryos in immature seeds with 0.6–0.8 cm diameter was 98.9%. The germination rate of grade 2 embryos in immature seeds with 0.4–0.6 cm diameter was 77.8% and the germination rate of grade 1 embryos in immature seeds with 0.4 cm diameter was 15.6%. 2) The amounts of macroelements in MS medium had no clear effect on the germination rate of immature grade 3 embryos and had a modest effect on plantlet growth, where the best medium was MS or 1/2 MS. The rates were all greater than 90%. 3) The germination rate of grade 3 embryos was greater than 87% when the medium contained a low concentration of NAA or no plant growth regulators at all and decreased markedly when BAP alone or BAP and NAA together were added to the media. We suggest that in vitro culture of immature embryos from K. bipinnata var. integrifoliola can be enhanced when a small amount of plant growth regulators is added. The addition of BAP has an adverse reaction to the germination and development of immature embryos.
基金This study was funded by the Administrative Department of Science Technology and Innovation(COLCIENCIAS)(Grant No.727,2015).
文摘Objective:To assess the effect of L-carnitine supplementation during in vitro oocyte maturation and in vitro culture process of bovine oocytes.Methods:L-carnitine(3.8 mM)was added to maturation medium and the effect was assessed in the quality(Experiment 1)and in the cleavage and 4-cells stage(Experiment 2).Besides,the effect of L-carnitine addition on maturation medium(3.8 mM)and culture medium(1.5 mM)on embryo rate production was assessed.In Experiment 1,bovine oocytes from abattoir were randomly separated into two groups(the control group and L-carnitine group)forin vitro maturation.Matured oocytes were examined for cumulus cells expansion as an indicator of maturation,and the content of the mitochondrial activity,the presence of lipid droplets,the reduced glutathione,and the reactive oxygen species were measured by using specific fluorochromes.In Experiment 2,oocytes were matured as performed in Experiment 1,afterward fertilized and cultured until day 3,and cleavage rate and 4-cells stage rate were determinated.In Experiment 3,in vitro maturation and fertilization were done as performed in Experiment 2,but at day 3 of culture,each group of embryos was separated into two new groups,and L-carnitine(1.5 mM)was added in culture media until day 8.The cleavage and embryo development rate were determined on the basis with the oocytes put on maturation.Hatching rate was calculated from cleaved embryos.Results:The cumulus expansion rate at gradeⅢand mitochondrial activity were significantly higher in the L-carnitine group in comparison with the control group(P0.05).In addition,cleavage and the proportion of embryo development and hatching rate were similar for all groups(P>0.05).Conclusions:L-carnitine as a supplement in culture media improves the cumulus expansion and increases the mitochondrial activity during in vitro maturation process but has no apparent effect on the cleavage and development of bovine embryos.Further investigations of L-carnitine addition on in vitroculture are needed to test their effect on embryo quality.
基金Supported by National Science and Technology Supporting Program(2012BAK11B04)
文摘In recent years,bonamiosis has frequently occurred in European areas,which has caused the death of oyster in a wide range and brought enormous economic losses to the breeding industry of oyster. Nowadays,the study on Bonamia sp. is still in the elementary phase. The technology of pathogen's culture in vitro is the basis for further study on the pathogenesis of Bonamia sp.,its interaction with hosts and the prevention and control of the related disease. In this study,total tissues of oyster identified by PCR were used as culture media to in vitro culture. After one month,they were identified by the method of in situ hybridization. It was found that the results of in situ hybridization were accordant with PCR results. And Bonamia ostreae were detected by in situ hybridization after B. ostreae were cultured for one month. We successfully established a simple and feasible method for in vitro culturing B. ostreae.
基金the National Natural Science Foundation of China,No.30672151
文摘BACKGROUND:Midbrain-derived neural stem cells (mNSCs) can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson’s disease. OBJECTIVE: To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN,TIME AND SETTING: An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine,China-Japan Friendship Hospital (China) from March to November 2007. MATERIALS: Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody,β-Ⅲ tubulin antibody,glial fibrillary acidic protein (GFAP) antibody and cyclic nucleotide 3’-phosphohydrolase (CNPase) antibody were provided by Abcam; DMEM/F12 medium and N2 supplement were provided by In-vitrogen; epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2) were provided by R&D Sys-tems. METHODS: The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin di-gestion and mechanical separation,the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/F12,1% N2 supplement,20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10 μg/mL polylysine and induced to differenti-ate in the DMEM/F12 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES: The neural spheres of the third passage were identified by nestin im-munofluorescence; at the same time,the cells were induced to differentiate,and the types of differentiated cell were identified by immunofluorescence for βⅢ tubulin,GFAP and CNPase. RESULTS: Seven days after primary culture,a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive,and after differentia-tion,the cells expressed GFAP,CNPase and β-Ⅲ-tubulin. CONCLUSION: Embryonic day 14 rat mNSCs can differentiate into neuron-like cells and glial cells fol-lowing induction by EGF,FGF2 and N2 additive.
文摘Brassica carinata, a natural alloploid formed between B. oleracea and B. nigra, is a potential oil crop for the Mediterranean area in which genetic transformation could help to breeding. In vitro culture and shoot regeneration are key factors in developing an efficient transformation method in the genus Brassica. However, the studies for in vitro culture and shoot regeneration in B. carinata are limited to only a few genotypes. The aim of this study was to evaluate the in vitro culture response and shoot regeneration in a collection of B. carinata accessions to identify promising genotypes with high shoot regeneration for genetic transformation experiments. Cotyledonary explants from 51 genotypes were cultured in vitro and callus formation and swelling as well as the mode of shoot regeneration evaluated. A highly positive response to in vitro culture, i.e. callus formation or swelling, was observed in all the genotypes tested. Tissue blackening occurred only in eleven genotypes. Parameters like callus formation and swelling, and number of shoots per explant were highly variable among genotypes. Fourteen genotypes regenerated only via callus formation, whereas only one regenerated only via swelling. Most genotypes showed a higher percentage of callus formation than swelling. The average number of shoots regenerating per explant among genotypes was the most variable factor measured. Six genotypes regenerated more than 6 shoots per explant via callus phase. These genotypes have been identified as having a high regeneration potential and can be used in genetic transformation via Agrobacterium.
文摘The genetic variability is considered as the major principle of plant breeding for durum wheat. This variability can be induced in vitro by selection pressure exerted by stress factors such as salinity in order to regenerate the vitro plantlets tolerant. This study aims in the first step in the regeneration of plantlets tolerant to salinity from mature embryos culture derived from two Tunisian durum wheat varieties: improved (Razzek) and landrace (Jenah Khotifa (JK)) varieties. The tolerance evaluation to salt stress was applied in vitro (100 mmol·l-1 NaCl) and was based on various parameters. Our results showed that JK variety was distinguished by a stable response for all parameters tested: average weight of callus (368.1 mg for control and 307 mg under salt stress), callus regenerated percentage (36.6% for control and 35.7% under salt stress) and green shoots number/callus (17 for control and 17 under salt stress). This stability of response translates the adaptability of this variety to salinity. In order to fix regenerated JK plantlets in single generation and obtain HDs homozygous stable lines, in vitro gynogenesis technical is tested for this genotype. The Evaluation of gynogenetic capacity focused on about 1200 unfertilized ovaries of JK and was based on its ability to induction, differentiation, development of green shoots, and haploid plantlets regeneration. JK showed good tolerance to salinity and a relatively good response to gynogenesis.
文摘BACKGROUND: The ability to maintain isolated human islet preparation in tissue culture has recently been adopted by most islet transplant centers to improve the safety and practicality of islet transplantation. However, maintaining islet viability and recovery remains a challenge in clinical setting. Extracellular matrix (ECM) is one of the most important components of islet microenvironment. The reconstruction of the cell-matrix relationship seems to be effective in improving the loss of differentiated islet structure and function. Small intestinal submucosa ( SIS ) , a naturally occurring ECM, has been investigated to be able to promote wound healing, tissue remodeling, and cell growth. The purpose of this study was to evaluate the recovery and function of isolated rat pancreatic islets after in vitro culture with SIS. METHODS: Pancreatic islets were isolated from Wistar rats by using standard surgical procurement followed by intra-ductal collagenase distension, mechanical dissociation, and EuroFicoll purification. Groups of purified islets were cultured in plates which were coated with multilayer SIS (SIS-treated group) or without ( standard cultured group) for 7 days and 14 days in standard islet culture conditions of RP-MI 1640 tissue culture media in humidified atmosphere containing 95% air and 5% CO2 at 37 ℃. The mean recovery of islets after the culture period was determined by sizing duplicate counts of a known volume and their viability was assessed by static incubation with low glucose (2.7 mmol) , high glucose (16.7 mmol) and high glucose solution supplemented with 50 μm 3-isobutyl-1-methylxanthine (IB-MX) solution. RESULTS: After 7 days and 14 days of in vitro tissue culture, the SIS-treated group showed a significantly higher recovery compared with those cultured under standard conditions. The recovery in the SIS-treated group was about two times of the control group cultured in standard conditions after 14 days culture. In the SIS-treated group, there was no statistically difference between the short and long periods of culture ( 95. 8 ± 1.0% vs. 90. 8±1. 5% , P 】 0.05). During incubation in high glucose (16.7 mmol) solution, there was a 2-3 fold increase in insulin secretion from both groups, but the SIS-treated group showed a higher increase than the standard cultured group after 14-day culture (20.7 ±1.1 mU/L vs. 11. 8 ±1.1 mU/L, P 【 0. 05). When islets were placed in the high glucose solution supplemented with IBMX, the stimulated insulin response in the SIS-treated group was higher than that in the standard cultured group in spite of the duration of the culture. The stimulation index of the SIS-treated group was about 2-3 times of the standard cultured group. In addition , after a long period of culture, the stimulation index of the SIS-treated group was statistically equivalent with that of the short period of culture (9.5 ±0.2 vs. 10.2 ±1.2, P】0.05). CONCLUSIONS: The co-culture of isolated rat islets with native sheet-like SIS can provide an excellent extracellular matrix, possible biotrophic and growth factors that promote the recovery and subsequent function of islets after in vitro tissue culture. In view of results of this study and rapid degradation of SIS in vitro, future studies will investigate the extended duration of culture and the effect of SIS on islets in vitro.
文摘The purpose of our study was to establish a regeneration system for micropropagation of Populus euphratica Olivier. On the basis of an analysis of plant leaf mineral nutrients, a special medium was proposed, called MP2. In optimizing media for in vitro plant cultures including MS, B5 and MP2 media we employed hormones, auxin IAA, cytokine benzyladenine (BAP) and gibberellic acid (GA) in our factorial experiments on media. Adventitious shoots were derived from cuttings of adult plants taken from Xingjiang, west China, on selected media with MP2+0.5 mg·L-1 BA+ 0.1 mg·L-1 NAA. The shoots were elongated on a medium with 0.25 mg·L-1 BAP, 0.1 mg·L-1 NAA and 2 mg·L-1 GA and were then rooted on a medium with 0.2-0.5 mg·L-1 IBA. All the media were incorporated with 30 g·L-1 sucrose and an adjusted pH at 6.3.
文摘The present investigation was undertaken to study the effect of gamma irradiation (dose from 10 to 100 Gy) and in vitro selection with fungus filtrate as selecting agent (concentration from 20% to 100%) on the susceptibility of the common bean to Rhizoctonia solani. The best results were found with a dose of 20 Gy or a concentration of 20% of fungus filtrate applied separately. These conditions were used to evaluate the combined effect of both approaches in a second experiment. The combined effect of irradiation and then selection adversely affected growth (height and roots) and survival of the in vitro plants. It may not be necessary to combine the variation generated by irradiation with the selection technique. For future assays we propose the application of: 1) gamma radiation, thereby inducing not only mutants with pathogen resistance, but also with other agronomic traits of interest. Later in the subculture MV4 potential fungus-resistant mutants will be evaluated in the field;or 2) selection pressure using fungus filtrate during three subcultures, which may be sufficient to induce the variation necessary to obtain in vitro plants resistant to fungus.
基金This work was supported by National High Technoloqy Grants 86310105.
文摘Fertilized rabbit eggs injected with SMTPGH gene were cultured in vitro and retention of theinjected gene was studied using PCR technique and nonradioactive labelling methed. In a mediumof TC199 + 10% FCS, three quarters of the fertilized eggs developed to the blastocyst stage. Noapparent change of the injected gene was found before the 8-cell stage, after which it was eitherintegrated into the chromosome of the host or lost gradually. But finally, the retention rate of theinjected gene should be equal to its integration rate.
