Hippeastrum, a highly diverse genus in the Amaryllidaceae family, is a valuable ornamental bulbous flowering plant. Somatic embryogenesis(SE) is an efficient method for mass production of Hippeastrum plantlets. Previo...Hippeastrum, a highly diverse genus in the Amaryllidaceae family, is a valuable ornamental bulbous flowering plant. Somatic embryogenesis(SE) is an efficient method for mass production of Hippeastrum plantlets. Previous studies have been devoted to the in vitro propagation of Hippeastrum, but the SE and its regulatory networks are rarely reported. In this study, we established a direct SE method of Hippeastrum Bangkok Rose' using leaf bases as explants. MS supplemented with 1.00 mg·L^(-1)NAA +1.00 mg·L^(-1)KT + 0.25 mg·L^(-1)TDZ was the optimal medium for SE. Histological observations showed that the bipolar somatic embryo originated from the epidermal cell layer and underwent initiation,globular, scutellar and coleoptile stages. During SE, endogenous hormones of IAA, CTK, ABA, and SA were highly accumulated. Transcriptomic analysis revealed the genes encoding auxin biosynthesis/metabolic enzymes and efflux carriers were induced, while the auxin receptor of TIR1 and ARF transcriptional repressor of Aux/IAA were down-regulated and up-regulated, respectively, leading to suppression of auxin signaling. In contrast, cytokine signaling was promoted at the early stage of SE, as biosynthesis, transport, and signaling components were up-regulated.Various stress-related genes were up-regulated at the early or late stages of SE. Chromatin remodeling could also be dynamically regulated via distinct expression enzymes that control histone methylation and acetylation during SE. Moreover, key SE regulators, including WOXs and SERKs were highly expressed along with SE. Overall, the present study provides insights into the SE regulatory mechanisms of the Hippeastrum.展开更多
The main aim of this study was to investigate the prevalence of intramammary infection (IMI) in early-lactation of primiparous cows using milk recording cow composite somatic cell count (CSCC) categories (combining th...The main aim of this study was to investigate the prevalence of intramammary infection (IMI) in early-lactation of primiparous cows using milk recording cow composite somatic cell count (CSCC) categories (combining the first 2 milk recording results after calving). Another aim was to evaluate the milk urea (MU) content as a potential supplementary indicator to SCC or CSCC for the identification of IMI in primiparous cows after calving. This retrospective observational study was conducted on records of test-day of primiparous cows over a period of 6 years (January 2016 to December 2021. The SCC data for 158 Holstein Friesian primiparous cows, with their first milk recording 5 to 35 days after calving and their second milk recording 28 to 56 days in milk (DIM), were identified. Each primiparous cow was assigned a CSCC category (low-low, low-high, high-low or high-high) based on the CSCC at the first 2 milking recordings using the following cut-offs: ≤150,000 cells/ml (low), >150,000 cells/ml (high). The association between CSCC categories and MV content was analyzed using correlation models. At the first milk recording, a proportion of 63.29% was in the low SCC category, and the rest (36.71%) was in the high SCC category. At the second milk recording, a proportion of primiparous cows in CSCC categories was 59.49%, 3.80%, 27.85% and 8.86% in low-low, low-high, high-low and high-high, respectively. At the second milk recording, a proportion of 12.66% of primiparous cows was in the high CSCC category and a proportion of 87.34% of primiparous cows was in the low CSCC category, indicating a poor and a good udder health, respectively. The association of SCC with MU content in low and in high SCC categories at the first milk recording was positive and moderate (+0.49) and negative and strong (-0.97), respectively. The association of CSCC categories with MU contents at the second milk recording was inconclusive. We concluded that CSCC categories may be a useful tool for identifying success and problems regarding the udder health of primiparous cows in early lactation.展开更多
Background Subclinical intramammary infection(IMI)represents a significant problem in maintaining dairy cows’health.Disease severity and extent depend on the interaction between the causative agent,environment,and ho...Background Subclinical intramammary infection(IMI)represents a significant problem in maintaining dairy cows’health.Disease severity and extent depend on the interaction between the causative agent,environment,and host.To investigate the molecular mechanisms behind the host immune response,we used RNA-Seq for the milk somatic cells(SC)transcriptome profiling in healthy cows(n=9),and cows naturally affected by subclinical IMI from Proto-theca spp.(n=11)and Streptococcus agalactiae(S.agalactiae;n=11).Data Integration Analysis for Biomarker discov-ery using Latent Components(DIABLO)was used to integrate transcriptomic data and host phenotypic traits related to milk composition,SC composition,and udder health to identify hub variables for subclinical IMI detection.Results A total of 1,682 and 2,427 differentially expressed genes(DEGs)were identified when comparing Prototheca spp.and S.agalactiae to healthy animals,respectively.Pathogen-specific pathway analyses evidenced that Proto-theca’s infection upregulated antigen processing and lymphocyte proliferation pathways while S.agalactiae induced a reduction of energy-related pathways like the tricarboxylic acid cycle,and carbohydrate and lipid metabolism.The integrative analysis of commonly shared DEGs between the two pathogens(n=681)referred to the core-mastitis response genes,and phenotypic data evidenced a strong covariation between those genes and the flow cytometry immune cells(r2=0.72),followed by the udder health(r2=0.64)and milk quality parameters(r2=0.64).Variables with r≥0.90 were used to build a network in which the top 20 hub variables were identified with the Cytoscape cyto-hubba plug-in.The genes in common between DIABLO and cytohubba(n=10)were submitted to a ROC analysis which showed they had excellent predictive performances in terms of discriminating healthy and mastitis-affected animals(sensitivity>0.89,specificity>0.81,accuracy>0.87,and precision>0.69).Among these genes,CIITA could play a key role in regulating the animals’response to subclinical IMI.Conclusions Despite some differences in the enriched pathways,the two mastitis-causing pathogens seemed to induce a shared host immune-transcriptomic response.The hub variables identified with the integrative approach might be included in screening and diagnostic tools for subclinical IMI detection.展开更多
Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other...Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods.This indicates the potential epigenetic variations between them.DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences.In this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using SCNT.To investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these animals.Results:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly correlated.DMR(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,respectively.Functional annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.Conclusion:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT animals.The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT.These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.展开更多
Energy metabolism reprogramming was recently identified as one of the cancer hallmarks.One of the underlying mechanisms of energy metabolism reprogramming is mitochondrial dysfunction caused by mutations in nuclear ge...Energy metabolism reprogramming was recently identified as one of the cancer hallmarks.One of the underlying mechanisms of energy metabolism reprogramming is mitochondrial dysfunction caused by mutations in nuclear genes or mitochondrial DNA(mtDNA).In the past decades,several types of somatic mtDNA alterations have been identified in gastric cancer.However,the role of these mtDNA alterations in gastric cancer progression remains unclear.In this review,we summarize recently identified somatic mtDNA alterations in gastric cancers as well as the relationship between these alterations and the clinicopathological features of gastric cancer.The causative factors and potential roles of the somatic mtDNA alterations in cancer progression are also discussed.We suggest that point mutations and mtDNA copy number decreases are the two most common mtDNA alterations that result in mitochondrial dysfunction in gastric cancers.The two primary mutation types(transition mutations and mononucleotide or dinucleotide repeat instability)imply potential causative factors.Mitochondrial dysfunction-generated reactive oxygen species may be involved in the malignant changes of gastric cancer.The search for strategies to prevent mtDNA alterations and inhibit the mitochondrial retrograde signaling will benefit the development of novel treatments for gastric cancer and other malignancies.展开更多
Pluripotent stem cells, which are capable of differentiating in various species of cells, are hoped to be donor cells in transplantation in regenerative medicine. Embryonic stem(ES) cells and induced pluripotent stem ...Pluripotent stem cells, which are capable of differentiating in various species of cells, are hoped to be donor cells in transplantation in regenerative medicine. Embryonic stem(ES) cells and induced pluripotent stem cells have the potential to differentiate in approximately all species of cells. However, the proliferating ability of these cells is high and the cancer formation ability is also recognized. In addition, ethical problems exist in using ES cells. Somatic stem cells with the ability to differentiate in various species of cells have been used as donor cells for neuronal diseases, such as amyotrophic lateral sclerosis, spinal cord injury, Alzheimer disease,cerebral infarction and congenital neuronal diseases.Human mesenchymal stem cells derived from bone marrow, adipose tissue, dermal tissue, umbilical cord blood and placenta are usually used for intractable neuronal diseases as somatic stem cells, while neural progenitor/stem cells and retinal progenitor/stem cells are used for a few congenital neuronal diseases and retinal degenerative disease, respectively. However,non-treated somatic stem cells seldom differentiate to neural cells in recipient neural tissue. Therefore, the contribution to neuronal regeneration using non-treated somatic stem cells has been poor and various differential trials, such as the addition of neurotrophic factors,gene transfer, peptide transfer for neuronal differentiation of somatic stem cells, have been performed. Here,the recent progress of regenerative therapies using various somatic stem cells is described.展开更多
Somatic embryogenesis(SE)is an effective approach of in vitro regeneration that depends on plant cell totipotency.However,largely unknown of molecular mechanisms of SE in woody plants such as Chinese chestnut(Castanea...Somatic embryogenesis(SE)is an effective approach of in vitro regeneration that depends on plant cell totipotency.However,largely unknown of molecular mechanisms of SE in woody plants such as Chinese chestnut(Castanea mollissima Blume),limits the development of the woody plant industry.Here,we report the MADS-box transcription factor Cm AGL11 in Chinese chestnut.Cm AGL11 transcripts specifically accumulated in the globular embryo.Overexpression of Cm AGL11 in chestnut callus enhanced its SE capacity,and the development of somatic embryos occurred significantly faster than in the control.RNA-seq results showed that Cm AGL11 affects the expression of several genes related to the gibberellin,auxin,and ethylene pathways.Moreover,the analysis of DNA methylation status indicated that the promoter methylation plays a role in regulation of Cm AGL11 expression during SE.Our results demonstrated that Cm AGL11 plays an important role in the SE process in Chinese chestnut,possibly by regulating gibberellin,auxin,and ethylene pathways.It will help establish an efficient platform to accelerate genetic improvement and germplasm innovation in Chinese chestnut.展开更多
With embryogenic callus of Larix olgensisis, we investigated the effects of inositol, glutamine, casein hydrolysate, carbohydrate, abscisic acid and silver nitrate concentration on the maturation of the somatic embryo...With embryogenic callus of Larix olgensisis, we investigated the effects of inositol, glutamine, casein hydrolysate, carbohydrate, abscisic acid and silver nitrate concentration on the maturation of the somatic embryo.Three dominant factors emerged, and we developed a response surface model based on the Box-Behnken design.We defined the optimal conditions for the maturation of somatic embryos. The contents of abscisic acid, silver nitrate, sucrose and casein hydrolysis significantly affected the amount of maturing embryos, but inositol, maltose and glutamine had no effect. By establishing a response surface model with multiple factors, we predicted that the optimal number of L. olgensis somatic embryos was 204 ± 4 g^(-1) on basal medium, containing 18.28 mg L^(-1) abscisic acid,5.46 mg L^(-1) silver nitrate and 82.67 g L^(-1) sucrose. In the verification experiments, the addition of 20 mg L^(-1) abscisic acid, 5 mg L^(-1) silver nitrate and 80 g L^(-1) sucrose to BM yielded an average of 202.06 somatic embryos per gram. These results should guide large-scale breeding of L. olgensis.展开更多
Somatic embryogenesis of Fraxinus mandshurica has the problems of low somatic embryo(SE) yield,unsynchronized SE development,and a high percentage of deformed SEs.We aimed to improve F.mandshurica SE production by syn...Somatic embryogenesis of Fraxinus mandshurica has the problems of low somatic embryo(SE) yield,unsynchronized SE development,and a high percentage of deformed SEs.We aimed to improve F.mandshurica SE production by synchronizing SE development,improving SE quality,and inducing root formation to obtain complete regenerated plants.Cotyledons of immature zygotic embryos of F.mandshurica were induced to form callus and then SEs.The SE induction percentage from explants differed among 32 mother trees,and the one with the highest SE induction percentage(29.8%) was used for further experiments.The highest callus induction percentage was94.2% on 1/2-strength Murashige and Skoog medium(MS1/2)supplemented with 0.15 mg·L^(-1) naphthalene acetic acid.The highest callus proliferation coefficient(240.5) was obtained on McCown's Woody Plant Medium containing 0.1 mg·L^(-1)6-benzyl adenine and 0.15 mg·L^(-1) 2,4-dichlorophenoxyacetic acid.The highest number of SEs(1020.5 g~(-1) fresh weight) was obtained on MS1/2 medium supplemented with1 mg·L^(-1) 6-benzyladenine.The highest number of cotyledon embryos(397/g fresh weight) was obtained by incubating materials on medium containing 1 mg·L^(-1) abscisic acid and then applying a drying treatment.The cotyledon embryos were milky white,uniformly sized(average length 4.7 mm),and 80% of them were normal.The SE rooting percentage on 1/2 MS medium containing 0.01 mg·L^(-1) NAA was 37.5%.Overall,the germination percentage of SEs was 26.4%,and complete regenerated plants were obtained after transplanting and acclimation.These results provide more possibilities for the preservation and breeding of F.mandshurica.展开更多
The induction and proliferation of embryogenic callus are key steps for large-scale propagation of somatic embryogenesis pathway and long-term preservation of coniferous germplasm.Callus can be induced from immature e...The induction and proliferation of embryogenic callus are key steps for large-scale propagation of somatic embryogenesis pathway and long-term preservation of coniferous germplasm.Callus can be induced from immature embryos of Korean pine(Pinus koraiensis Sieb.et Zucc.;Pinaceae)as explants,but there are problems,such as low proliferation efficiency,loss of embryogenicity,poor vigor;thus,best conditions for proliferation and culture of immature embryos of Korean pine are not yet clear.To solve the problems with somatic embryogenesis of Korean pine and determine the best culture conditions for callus induction and proliferation,we varied hormone concentration,subculture cycle of proliferation and other plant growth regulators combinations in media to induce callus formation by megagametophytes of three Korean pine families at different developmental stages,then analyzed the effects on embryogenic callus retention and cell proliferation using a quadratic regression orthogonal rotation design.The results showed that the family origin and collection date of explants significantly affected callus induction(induction rate reached 1.67%).Embryogenic maintenance and callus proliferation were best on DCR medium supplemented with 0.25 mg L^(−1)6-benzyl adenine,1 mg L^(−1)naphthaleneacetic acid,30 g L^(−1)sucrose,500 mg L^(−1),L-glutamine,500 mg L^(−1)casein hydrolysis and 6.5 g L^(−1)agar.In addition,the combination of 2,4-dichlorophenoxyacetic acid+6-benzyl adenine also had a better proliferative effect on callus.The effects of different combinations of growth regulators on callus proliferation efficiency were significantly different.Transfer to new medium every 13–15 days not only maintained robust callus vigor,but also yielded a larger proliferation coefficient.The techniques and conditions for embryogenic callus induction and proliferation of Korean determined here will serve as a foundation for establishing a large-scale system for somatic embryogenesis and propagation of Korean pine.展开更多
We examined how reactive oxygen species, in the form of hydrogen peroxide (H2O2), affect osmotic stress–induced programmed cell death during somatic embryogenesis from cotyledon explants of Manchurian ash (Fraxinus m...We examined how reactive oxygen species, in the form of hydrogen peroxide (H2O2), affect osmotic stress–induced programmed cell death during somatic embryogenesis from cotyledon explants of Manchurian ash (Fraxinus mandshurica Rupr.). We found that substantial osmotic stress was essential for Manchurian ash somatic cells to obtain embryogenic competence. The explant cells displayed hallmarks of programmed cell death, chromatin condensation, and DNA fragmentation to oligonucleotides during somatic embryogenesis. Increasing concentrations of plant growth regulators and sucrose in the medium increased osmotic stress thereby inducing H2O2 accumulation in the explant cells. We found that H2O2 concentration was significantly decreased in explant cells when the induction medium was modified, i.e., when reducing the concentration of sucrose, which reduces the osmotic pressure of the medium, or by withdrawing plant growth regulators at mid-culture. These treatments also decreased the proportion of explant cells undergoing programmed cell death. Accordingly, a decreased rate of somatic embryo induction was observed. These results show that PCD occurred during tissue browning and death of some explant cells during somatic embryogenesis in F. mandshurica. The ROS contributed to PCD in abiotic stress stimulated F. mandshurica cells.展开更多
I studied the influence of various combinations of auxin and cytokinin concentrations, and the increased content of zinc and enzymatic casein hydrolizate in SH medium on initiation and proliferation of embryogenic cal...I studied the influence of various combinations of auxin and cytokinin concentrations, and the increased content of zinc and enzymatic casein hydrolizate in SH medium on initiation and proliferation of embryogenic callus of Abies nordmanniana(Steven) Spach. Additionally, the effect of ABA, PEG-4000 and different wavelengths on the maturation of somatic embryos was tested.The use of optimum composition of modified SH medium with BA, KIN and 2.4-D while simultaneously ensuring appropriate external conditions resulted in 15.5 % embryogenesis. Finally, satisfactory results of micropropagation of A. nordmanniana by somatic embryogenesis were obtained providing seven lines of embryogenic callus with high proliferation capacity. Those lines gave properly developed seedlings in white LED light with a wavelength of 400-700 nm, preceded by eight-week vernalization treatment of the callus. This paper may provide a protocol by which all stages of somatic embryogenesis of A. nordmanniana can be carried out, including the preceding 24-h seed disinfection with Na OCl and PVP, which resulted in 100 % frequency of uninfected zygotic embryos that were capable of starting embryogenesis.展开更多
Somatic embryogenesis in upland cotton isstrongly genotype-dependent,which is a troublein cotton genetic engineering.Cloning genesrelated to somatic embryogenesis and thenintroducing the gene into mainly cultivatedvar...Somatic embryogenesis in upland cotton isstrongly genotype-dependent,which is a troublein cotton genetic engineering.Cloning genesrelated to somatic embryogenesis and thenintroducing the gene into mainly cultivatedvarieties would be greatly helpful for cottonimprovement by gene transfer.To study展开更多
PiggyBac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggyBac transposon marked ...PiggyBac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggyBac transposon marked with enhanced green fluorescent protein (eGFP) and showed efficient transposition in porcine somatic cells and cloned embryos. Our results demonstrated that piggyBac transposase could efficiently catalyze transposition in porcine fetal fibroblast cells, as well as in embryos. PiggyBac transposition generated 18-fold more eGFP-positive cell colonies compared to pEGFP-C1 random insertion mutagenesis, but excessive transposase might affect the transfection rate. Also piggyBac mediated 4-fold more eGFP expression than random insertion in cells and 17-fold in cloned embryos at mRNA level. When the mutagenized cells were used for somatic cell nuclear transfer (SCNT), the cleavage rate and blastocyst rate of constructed embryos harboring piggyBac transposition had no difference with random insertion group. This study provides key information on the piggyBac transposon system as a tool for creating transgenic pigs.展开更多
Somatic embryogenesis (SE) is a prominent mode of regeneration in plants. The acquisition of SE is predominantly invoked by the oxidative stress which plays an important role in signal transduction and cellular redox....Somatic embryogenesis (SE) is a prominent mode of regeneration in plants. The acquisition of SE is predominantly invoked by the oxidative stress which plays an important role in signal transduction and cellular redox. Since balanced generation of oxidants is important to cellular differentiation, modulation in cell redox could be responsive to genotypic refinement for SE. To study the dynamics of cellular redox during SE, we conducted comparative expression analyses of cotton (Gossypium hirsutum), using two independently purified near-isogenic lines for the trait of SE. We interrogated expression changes in cell-signaling factor Somatic Embryogenesis Receptor Kinase (SERK) and activity of antioxidant Glutathione in different developmental stages including cotyledonary leaf, calli from different stages of regeneration of fully-regenerating (FR) and non-regenerating (NR) lines of Coker310 cultivar. At evolutionary scale, the cotton SERKs showed high sequence similarity in receptor kinase domain with diverse systems. Exclusively, SERK1 responsible for potential signaling processes during SE revealed significant expression up-regulation in the embryogenic calli of FR line. Similarly, activity of antioxidant glutathione was substantially up-regulated in embryogenic calli of FR line in comparison to its counterpart form. In contrast, calli from early-stages of regeneration of both FR and NR lines had no significant influences on the regulation of SERK and glutathione levels prior to the acquisition of embryogenesis. These results highlight that in vitro purification of FR line in cotton for enhanced regeneration potential (through SE) resulted in signaling and metabolic transformations of the manner in which cellular redox levels have become modulated.展开更多
Pinus patula is a species commonly used for reforestation in Mexico.However,efficient methods for the mass production seedlings are required.Micropropagation particularly by somatic embryogenesis provides an option fo...Pinus patula is a species commonly used for reforestation in Mexico.However,efficient methods for the mass production seedlings are required.Micropropagation particularly by somatic embryogenesis provides an option for the rapid multiplication of high-quality,genetically improved material.This study induces somatic embryogenesis in this species using the thin cell layer(TCL)technique.Two sources of explants(complete immature embryos;lTCL segments from immature embryos)were evaluated.The efficiency of TCL from longitudinal sections[lTCL]and transverse[tTCL]was evaluated.The results show using thin cell layers from immature embryos cultivated in 16 light/8 dark hours achieves induction of somatic embryos.A higher percentage of embryogenic callus was obtained when tTCL segments were used as an explant source.These results produced somatic embryos from tTCL segments of an immature embryo without germinating the seed,making the process more time efficient.In addition,this technique can be used to generate somatic embryogenesis in forest species that have low germination rates.展开更多
BACKGROUND: Scorpion venom from Buthus martensii Karsch is a kind of protein toxin secreted by scorpion tail. According to records of Pen-ts’ao Kan-mu, the dried scorpion’s body and tail are used as medicine, which ...BACKGROUND: Scorpion venom from Buthus martensii Karsch is a kind of protein toxin secreted by scorpion tail. According to records of Pen-ts’ao Kan-mu, the dried scorpion’s body and tail are used as medicine, which mainly treating rheumatism numbness, pain and convulsion. Some researches consider that scorpion toxin is the main pharmacological active component of the dried scorpion, and it has 4 times stronger analgesic effect on somatic pain than morphine. OBJECTIVE: To observe the interventional effect of scorpion venom of Buthus martensii Karsch analgesic active peptide on somatic pain. DESIGN: A randomized and controlled trial. SETTING: Department of Physiology, Shenyang Medical College. MATERIALS: Totally 80 Wistar rats (provided by Animal Center of Shenyang Medical College), male and female in half, aged 3 to 4 months, weighed 250 to 350 g, were used in this trial. They were randomly divided into 4 groups: experimental group, control group, morphine group and naloxone group, with 20 in each group. Analgesia active peptide from buthus martensii karsch venom (provided by Biochemical Department of Shenyang Pharmaceutical University, 1 mL/dosage), morphine (produced by Shanghai First Pharmaceutical Manufacturing Plant), naloxone (opium acceptor inhibitor, American Sigma Corporation), ATAC-350 data-processing machine (Nihon Kohden Corporation), X-Y recording instrument (Nihon Kohden Corporation). METHODS: The experiment was conducted in the Department of Physiology, Shenyang Medical College from July 2003 to July 2005. ① After all the animals were anesthetized, common peroneal nerve is dissected and ligated at middle part. Posterior nucleus group of thalamus (PO) and the tail nuclear were oriented according to G. Paxino rat brain three-dimensional orientation atlas. Glass micro electrode was inserted into PO as guiding electrode, connected with ATAC-350 data-processing machine and X-Y recording instrument, to record the unit evoked potential of PO, taking the evoked potential discharge of the common peroneal nerve of PO as the somatic pain index.② Single square-wave stimulation of intensity 17-19 volt, wave wide 0.2 ms, time delay 20 ms was exerted on common peroneal nerve. The time interval was 5 minutes. X-Y recording instrument was used to draw the graph. 0.002 mg scorpion venom analgesic active peptide was injected into the rats of the experimental group; 0.002 mg normal saline was injected into the rats of the control group through caudate nucleus; 0.002 mg morphine was injected into the rats of morphine group through caudate nucleus; 1.0 mg/kg naloxone was intraperitoneally into the rats of naloxone group, then 0.002 mg scorpion venom was injected into the rats of control group through caudate nucleus.Changes of evoked potential of PO of 3 groups were observed. ③After experiment, 1 μA direct current was given to the guiding electrode and micro electrode , and it lasted for 5 minutes. Pontamine sky blue was used to label the peak of electrode by electrophoresis. Brain tissue was soaked in formalin for 1 week then sliced into 1.0 to 1.5 mm sections. Electrode was orientated according to blue spots. Compared with G . Parino rat brain three-dimensional orientation atlas, we confirmed if the electrode orientation is consistent with PO orientation of G. Parino rat brain three-dimensional orientation atlas. MAIN OUTCOME MEASURES: Changes of the evoked potential of PO in each group. RESULTS: Totally 80 Wistar rats were enrolled in this experiment. Four rats in the experimental group and two in the morphine group died of overdose of anesthesia, and finally 74 rats entered the stage of result analysis. The inhibitory action time of evoked potential of PO has no statistical difference in between experiment group and morphine group (P > 0.05). The whole inhibitory action time, timeof initiate recovery and time of complete recovery of PO of experiment group were longer than those in the morphine group [(45±0.7),(50±9.2),(65±8.1);(35±7.8),(40±8.9),(50±7.6) min,P < 0.05].The change of evoked potential of PO was not obviously in the control group and naloxone group (P > 0.05), and the above-mentioned 4 indexes were nearly 0. CONCLUSION: Scorpion venom possesses obviously inhibitory effect on somatic pain, and its inhibitory effect is stronger than that of the same dosage and concentration of morphine. Scorpion venom exerts analgesic effect on somatic pain through opium acceptor.展开更多
Leptadenia pyrotechnica is an important multipurpose endangered plant in the Kingdom of Bahrain with restricted distribution. Nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with different...Leptadenia pyrotechnica is an important multipurpose endangered plant in the Kingdom of Bahrain with restricted distribution. Nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of indole acetic acid (IAA) and 6-benzylaminopurine (BAP). Initially, 80% and 60% explants responded in direct shoot and callus initiation response respectively in presence of 8.88 μM BAP with 5.71 μM IAA in modified MS media after two weeks of culture. The highest frequency of plant regeneration was observed in presence of 8.88 μM BAP with 1.14 μM IAA following organogenic pathway of differentiation. Hundred percent callus proliferation was observed while initial callus developed in presence of 4.44 μM BAP with 2.85 μM IAA and was transferred in media containing 4.44 μM, 6.66 μM BAP with 2.85 μM IAA and 13.32 μM BAP with 5.71 μM IAA. The callus derived plants were regenerated following the pathway of indirect somatic embryogenesis. The induction of somatic embryogenesis and plant regeneration from callus was also observed in modified MS media supplemented with 4.44 μM BAP and 2.85 μM IAA. The plant regeneration protocol we developed for Leptadenia pyrotechnica will be very beneficial for biodiversity conservation and environment protection of Bahrain. Moreover, the present paper reports for the first time specifically the somatic embryogenesis in this multipurpose desert plant Leptadenia pyrotechnica.展开更多
To enhance the understanding of artemisinin biosynthesis, we have successfully bred self-pollination Artemisia annua plants. Here, we report efficient somatic embryogenesis and organogenesis of self-pollination plants...To enhance the understanding of artemisinin biosynthesis, we have successfully bred self-pollination Artemisia annua plants. Here, we report efficient somatic embryogenesis and organogenesis of self-pollination plants and artemisinin formation in regenerated plants. The first through sixth nodal leaves of seedlings are used as explants. On agar-solidified MS basal medium supplemented with TDZ (0.6 mg/l) and IBA (0.1 mg/l), all explants after inoculation of less than 3 weeks start to form embryogenic calli, which further produce globular, torpedo, heart and early cotyledon embryos. In all six positional leaves, explants from the sixth leaf show the rapidest responses to induction of embryogenic calli and somatic embryos. On this medium, somatic embryos continuously develop into adventitious buds, which can form adventitious roots on a rooting medium containing NAA (0.5 mg/l). Meanwhile, on agar-solidified MS basal medium supplemented with BAP (1 mg/l) and NAA (0.05 mg/l), approximately 100% of explants from leaves #3-6 form calli in less than 3 weeks of inoculation and adventitious buds via organogenesis in 3-4 weeks. In all six positional leaves, explants from the sixth leaf exhibit the rapidest response to induction of calli and adventitious buds. Nearly 100% adventitious buds can form adventitious roots on the rooting medium. Regenerated plants from both somatic embryogenesis and organogenesis complete self-pollination to produce seeds in 80-90 days of growth in growth chamber. LC-ESI-MS analysis demonstrates that regenerated plants biosynthesize artemisinin. These results show the highly efficient regeneration capacity of self-pollination A. annua plants that can form a new platform to enhance the understanding of artemisinin biosynthesis and metabolic engineering.展开更多
基金funded by Guangdong Basic and Applied Basic Research Foundation (Grant No.2023A1515010237)the 2021 Dongguan Provincial Rural Revitalization Program (Grant No.20211800400022)+2 种基金the Guangdong Key Technology Research and Development Program (Grant Nos.2020B020220005,2022B1111040003)the Guangdong Modern Agricultural Industry Technology System Program (Grant No.2023KJ121)the South China Botanical Garden,the Chinese Academy of Sciences (Grant No.QNXM-02)。
文摘Hippeastrum, a highly diverse genus in the Amaryllidaceae family, is a valuable ornamental bulbous flowering plant. Somatic embryogenesis(SE) is an efficient method for mass production of Hippeastrum plantlets. Previous studies have been devoted to the in vitro propagation of Hippeastrum, but the SE and its regulatory networks are rarely reported. In this study, we established a direct SE method of Hippeastrum Bangkok Rose' using leaf bases as explants. MS supplemented with 1.00 mg·L^(-1)NAA +1.00 mg·L^(-1)KT + 0.25 mg·L^(-1)TDZ was the optimal medium for SE. Histological observations showed that the bipolar somatic embryo originated from the epidermal cell layer and underwent initiation,globular, scutellar and coleoptile stages. During SE, endogenous hormones of IAA, CTK, ABA, and SA were highly accumulated. Transcriptomic analysis revealed the genes encoding auxin biosynthesis/metabolic enzymes and efflux carriers were induced, while the auxin receptor of TIR1 and ARF transcriptional repressor of Aux/IAA were down-regulated and up-regulated, respectively, leading to suppression of auxin signaling. In contrast, cytokine signaling was promoted at the early stage of SE, as biosynthesis, transport, and signaling components were up-regulated.Various stress-related genes were up-regulated at the early or late stages of SE. Chromatin remodeling could also be dynamically regulated via distinct expression enzymes that control histone methylation and acetylation during SE. Moreover, key SE regulators, including WOXs and SERKs were highly expressed along with SE. Overall, the present study provides insights into the SE regulatory mechanisms of the Hippeastrum.
文摘The main aim of this study was to investigate the prevalence of intramammary infection (IMI) in early-lactation of primiparous cows using milk recording cow composite somatic cell count (CSCC) categories (combining the first 2 milk recording results after calving). Another aim was to evaluate the milk urea (MU) content as a potential supplementary indicator to SCC or CSCC for the identification of IMI in primiparous cows after calving. This retrospective observational study was conducted on records of test-day of primiparous cows over a period of 6 years (January 2016 to December 2021. The SCC data for 158 Holstein Friesian primiparous cows, with their first milk recording 5 to 35 days after calving and their second milk recording 28 to 56 days in milk (DIM), were identified. Each primiparous cow was assigned a CSCC category (low-low, low-high, high-low or high-high) based on the CSCC at the first 2 milking recordings using the following cut-offs: ≤150,000 cells/ml (low), >150,000 cells/ml (high). The association between CSCC categories and MV content was analyzed using correlation models. At the first milk recording, a proportion of 63.29% was in the low SCC category, and the rest (36.71%) was in the high SCC category. At the second milk recording, a proportion of primiparous cows in CSCC categories was 59.49%, 3.80%, 27.85% and 8.86% in low-low, low-high, high-low and high-high, respectively. At the second milk recording, a proportion of 12.66% of primiparous cows was in the high CSCC category and a proportion of 87.34% of primiparous cows was in the low CSCC category, indicating a poor and a good udder health, respectively. The association of SCC with MU content in low and in high SCC categories at the first milk recording was positive and moderate (+0.49) and negative and strong (-0.97), respectively. The association of CSCC categories with MU contents at the second milk recording was inconclusive. We concluded that CSCC categories may be a useful tool for identifying success and problems regarding the udder health of primiparous cows in early lactation.
基金the Ministero delle politiche agricole alimentari,forestali e del turismo(MIPAAF),Rome,Italy.Moreover,the study was conducted within the Agritech National Research Center and received funding from the European Union Next-GenerationEU(PIANO NAZIONALE DI RIPRESA E RESILIENZA(PNRR)-MISSIONE 4 COMPONENTE 2,INVESTIMENTO 1.4-D.D.103217/06/2022,CN00000022).
文摘Background Subclinical intramammary infection(IMI)represents a significant problem in maintaining dairy cows’health.Disease severity and extent depend on the interaction between the causative agent,environment,and host.To investigate the molecular mechanisms behind the host immune response,we used RNA-Seq for the milk somatic cells(SC)transcriptome profiling in healthy cows(n=9),and cows naturally affected by subclinical IMI from Proto-theca spp.(n=11)and Streptococcus agalactiae(S.agalactiae;n=11).Data Integration Analysis for Biomarker discov-ery using Latent Components(DIABLO)was used to integrate transcriptomic data and host phenotypic traits related to milk composition,SC composition,and udder health to identify hub variables for subclinical IMI detection.Results A total of 1,682 and 2,427 differentially expressed genes(DEGs)were identified when comparing Prototheca spp.and S.agalactiae to healthy animals,respectively.Pathogen-specific pathway analyses evidenced that Proto-theca’s infection upregulated antigen processing and lymphocyte proliferation pathways while S.agalactiae induced a reduction of energy-related pathways like the tricarboxylic acid cycle,and carbohydrate and lipid metabolism.The integrative analysis of commonly shared DEGs between the two pathogens(n=681)referred to the core-mastitis response genes,and phenotypic data evidenced a strong covariation between those genes and the flow cytometry immune cells(r2=0.72),followed by the udder health(r2=0.64)and milk quality parameters(r2=0.64).Variables with r≥0.90 were used to build a network in which the top 20 hub variables were identified with the Cytoscape cyto-hubba plug-in.The genes in common between DIABLO and cytohubba(n=10)were submitted to a ROC analysis which showed they had excellent predictive performances in terms of discriminating healthy and mastitis-affected animals(sensitivity>0.89,specificity>0.81,accuracy>0.87,and precision>0.69).Among these genes,CIITA could play a key role in regulating the animals’response to subclinical IMI.Conclusions Despite some differences in the enriched pathways,the two mastitis-causing pathogens seemed to induce a shared host immune-transcriptomic response.The hub variables identified with the integrative approach might be included in screening and diagnostic tools for subclinical IMI detection.
基金Key Research and Development Project of Hainan Province(ZDYF2021XDNY174)Science and Technology Major Project of Inner Mongolia(2021ZD0023–1)National Transgenic Key Project of the Ministry of Agriculture of China(2018ZX0800801B)。
文摘Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods.This indicates the potential epigenetic variations between them.DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences.In this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using SCNT.To investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these animals.Results:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly correlated.DMR(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,respectively.Functional annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.Conclusion:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT animals.The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT.These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.
基金Supported by A grant from the Center of Excellence for Cancer Research at Taipei Veterans General,the Ministry of Health and Welfare,No.DOH102-TDC-111-007,Executive Yuana grant from the Ministry of Education,Aim for the Top University Planand grant from the National Science Council,No.NSC101-2320-B-010-068-MY3,Taiwan
文摘Energy metabolism reprogramming was recently identified as one of the cancer hallmarks.One of the underlying mechanisms of energy metabolism reprogramming is mitochondrial dysfunction caused by mutations in nuclear genes or mitochondrial DNA(mtDNA).In the past decades,several types of somatic mtDNA alterations have been identified in gastric cancer.However,the role of these mtDNA alterations in gastric cancer progression remains unclear.In this review,we summarize recently identified somatic mtDNA alterations in gastric cancers as well as the relationship between these alterations and the clinicopathological features of gastric cancer.The causative factors and potential roles of the somatic mtDNA alterations in cancer progression are also discussed.We suggest that point mutations and mtDNA copy number decreases are the two most common mtDNA alterations that result in mitochondrial dysfunction in gastric cancers.The two primary mutation types(transition mutations and mononucleotide or dinucleotide repeat instability)imply potential causative factors.Mitochondrial dysfunction-generated reactive oxygen species may be involved in the malignant changes of gastric cancer.The search for strategies to prevent mtDNA alterations and inhibit the mitochondrial retrograde signaling will benefit the development of novel treatments for gastric cancer and other malignancies.
文摘Pluripotent stem cells, which are capable of differentiating in various species of cells, are hoped to be donor cells in transplantation in regenerative medicine. Embryonic stem(ES) cells and induced pluripotent stem cells have the potential to differentiate in approximately all species of cells. However, the proliferating ability of these cells is high and the cancer formation ability is also recognized. In addition, ethical problems exist in using ES cells. Somatic stem cells with the ability to differentiate in various species of cells have been used as donor cells for neuronal diseases, such as amyotrophic lateral sclerosis, spinal cord injury, Alzheimer disease,cerebral infarction and congenital neuronal diseases.Human mesenchymal stem cells derived from bone marrow, adipose tissue, dermal tissue, umbilical cord blood and placenta are usually used for intractable neuronal diseases as somatic stem cells, while neural progenitor/stem cells and retinal progenitor/stem cells are used for a few congenital neuronal diseases and retinal degenerative disease, respectively. However,non-treated somatic stem cells seldom differentiate to neural cells in recipient neural tissue. Therefore, the contribution to neuronal regeneration using non-treated somatic stem cells has been poor and various differential trials, such as the addition of neurotrophic factors,gene transfer, peptide transfer for neuronal differentiation of somatic stem cells, have been performed. Here,the recent progress of regenerative therapies using various somatic stem cells is described.
基金supported by the National Key Research&Development Program of China(2018YFD1000605)the National Natural Science Foundation of China(31870671 and 31672135)+1 种基金the Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipality,China(IDHT20180509)the Supporting Plan for Cultivating High Level Teachers in Colleges and Universities in Beijing,China(CIT&TCD20180317)。
文摘Somatic embryogenesis(SE)is an effective approach of in vitro regeneration that depends on plant cell totipotency.However,largely unknown of molecular mechanisms of SE in woody plants such as Chinese chestnut(Castanea mollissima Blume),limits the development of the woody plant industry.Here,we report the MADS-box transcription factor Cm AGL11 in Chinese chestnut.Cm AGL11 transcripts specifically accumulated in the globular embryo.Overexpression of Cm AGL11 in chestnut callus enhanced its SE capacity,and the development of somatic embryos occurred significantly faster than in the control.RNA-seq results showed that Cm AGL11 affects the expression of several genes related to the gibberellin,auxin,and ethylene pathways.Moreover,the analysis of DNA methylation status indicated that the promoter methylation plays a role in regulation of Cm AGL11 expression during SE.Our results demonstrated that Cm AGL11 plays an important role in the SE process in Chinese chestnut,possibly by regulating gibberellin,auxin,and ethylene pathways.It will help establish an efficient platform to accelerate genetic improvement and germplasm innovation in Chinese chestnut.
基金supported by the National High-tech R&D Program(863 Program)of China(2013AA102704)
文摘With embryogenic callus of Larix olgensisis, we investigated the effects of inositol, glutamine, casein hydrolysate, carbohydrate, abscisic acid and silver nitrate concentration on the maturation of the somatic embryo.Three dominant factors emerged, and we developed a response surface model based on the Box-Behnken design.We defined the optimal conditions for the maturation of somatic embryos. The contents of abscisic acid, silver nitrate, sucrose and casein hydrolysis significantly affected the amount of maturing embryos, but inositol, maltose and glutamine had no effect. By establishing a response surface model with multiple factors, we predicted that the optimal number of L. olgensis somatic embryos was 204 ± 4 g^(-1) on basal medium, containing 18.28 mg L^(-1) abscisic acid,5.46 mg L^(-1) silver nitrate and 82.67 g L^(-1) sucrose. In the verification experiments, the addition of 20 mg L^(-1) abscisic acid, 5 mg L^(-1) silver nitrate and 80 g L^(-1) sucrose to BM yielded an average of 202.06 somatic embryos per gram. These results should guide large-scale breeding of L. olgensis.
基金supported by the Fundamental Research Funds for the Central Universities of China(2572018BW02)the National Natural Science Foundation of China (31400535 and 31570596)+1 种基金the Innovation Project of State Key Laboratory of Tree Genetics and Breeding (2016C01)the National Key R&D Program of China (2017YFD0600600)。
文摘Somatic embryogenesis of Fraxinus mandshurica has the problems of low somatic embryo(SE) yield,unsynchronized SE development,and a high percentage of deformed SEs.We aimed to improve F.mandshurica SE production by synchronizing SE development,improving SE quality,and inducing root formation to obtain complete regenerated plants.Cotyledons of immature zygotic embryos of F.mandshurica were induced to form callus and then SEs.The SE induction percentage from explants differed among 32 mother trees,and the one with the highest SE induction percentage(29.8%) was used for further experiments.The highest callus induction percentage was94.2% on 1/2-strength Murashige and Skoog medium(MS1/2)supplemented with 0.15 mg·L^(-1) naphthalene acetic acid.The highest callus proliferation coefficient(240.5) was obtained on McCown's Woody Plant Medium containing 0.1 mg·L^(-1)6-benzyl adenine and 0.15 mg·L^(-1) 2,4-dichlorophenoxyacetic acid.The highest number of SEs(1020.5 g~(-1) fresh weight) was obtained on MS1/2 medium supplemented with1 mg·L^(-1) 6-benzyladenine.The highest number of cotyledon embryos(397/g fresh weight) was obtained by incubating materials on medium containing 1 mg·L^(-1) abscisic acid and then applying a drying treatment.The cotyledon embryos were milky white,uniformly sized(average length 4.7 mm),and 80% of them were normal.The SE rooting percentage on 1/2 MS medium containing 0.01 mg·L^(-1) NAA was 37.5%.Overall,the germination percentage of SEs was 26.4%,and complete regenerated plants were obtained after transplanting and acclimation.These results provide more possibilities for the preservation and breeding of F.mandshurica.
基金supported by the National Key R&D Program of China(2017YFD0600600).
文摘The induction and proliferation of embryogenic callus are key steps for large-scale propagation of somatic embryogenesis pathway and long-term preservation of coniferous germplasm.Callus can be induced from immature embryos of Korean pine(Pinus koraiensis Sieb.et Zucc.;Pinaceae)as explants,but there are problems,such as low proliferation efficiency,loss of embryogenicity,poor vigor;thus,best conditions for proliferation and culture of immature embryos of Korean pine are not yet clear.To solve the problems with somatic embryogenesis of Korean pine and determine the best culture conditions for callus induction and proliferation,we varied hormone concentration,subculture cycle of proliferation and other plant growth regulators combinations in media to induce callus formation by megagametophytes of three Korean pine families at different developmental stages,then analyzed the effects on embryogenic callus retention and cell proliferation using a quadratic regression orthogonal rotation design.The results showed that the family origin and collection date of explants significantly affected callus induction(induction rate reached 1.67%).Embryogenic maintenance and callus proliferation were best on DCR medium supplemented with 0.25 mg L^(−1)6-benzyl adenine,1 mg L^(−1)naphthaleneacetic acid,30 g L^(−1)sucrose,500 mg L^(−1),L-glutamine,500 mg L^(−1)casein hydrolysis and 6.5 g L^(−1)agar.In addition,the combination of 2,4-dichlorophenoxyacetic acid+6-benzyl adenine also had a better proliferative effect on callus.The effects of different combinations of growth regulators on callus proliferation efficiency were significantly different.Transfer to new medium every 13–15 days not only maintained robust callus vigor,but also yielded a larger proliferation coefficient.The techniques and conditions for embryogenic callus induction and proliferation of Korean determined here will serve as a foundation for establishing a large-scale system for somatic embryogenesis and propagation of Korean pine.
基金supported by the National Key R&D Program of China(2017YFD0600600)the National Natural Science Foundation of China(Nos.31400535 and 31570596)+2 种基金the National Basic Science Personnel Training Fund of China(No.J1210053)the Innovation Project of State Key Laboratory of Tree Genetics and Breeding(Northeast Forestry University,No.2016C01)the Fundamental Research Funds for the Central Universities(Nos.2572014CA13 and 2572018BW02)
文摘We examined how reactive oxygen species, in the form of hydrogen peroxide (H2O2), affect osmotic stress–induced programmed cell death during somatic embryogenesis from cotyledon explants of Manchurian ash (Fraxinus mandshurica Rupr.). We found that substantial osmotic stress was essential for Manchurian ash somatic cells to obtain embryogenic competence. The explant cells displayed hallmarks of programmed cell death, chromatin condensation, and DNA fragmentation to oligonucleotides during somatic embryogenesis. Increasing concentrations of plant growth regulators and sucrose in the medium increased osmotic stress thereby inducing H2O2 accumulation in the explant cells. We found that H2O2 concentration was significantly decreased in explant cells when the induction medium was modified, i.e., when reducing the concentration of sucrose, which reduces the osmotic pressure of the medium, or by withdrawing plant growth regulators at mid-culture. These treatments also decreased the proportion of explant cells undergoing programmed cell death. Accordingly, a decreased rate of somatic embryo induction was observed. These results show that PCD occurred during tissue browning and death of some explant cells during somatic embryogenesis in F. mandshurica. The ROS contributed to PCD in abiotic stress stimulated F. mandshurica cells.
基金supported by research topic DS No.3414 of the Ministry of Science and Higher Education and funded from international corporation Vitroflora
文摘I studied the influence of various combinations of auxin and cytokinin concentrations, and the increased content of zinc and enzymatic casein hydrolizate in SH medium on initiation and proliferation of embryogenic callus of Abies nordmanniana(Steven) Spach. Additionally, the effect of ABA, PEG-4000 and different wavelengths on the maturation of somatic embryos was tested.The use of optimum composition of modified SH medium with BA, KIN and 2.4-D while simultaneously ensuring appropriate external conditions resulted in 15.5 % embryogenesis. Finally, satisfactory results of micropropagation of A. nordmanniana by somatic embryogenesis were obtained providing seven lines of embryogenic callus with high proliferation capacity. Those lines gave properly developed seedlings in white LED light with a wavelength of 400-700 nm, preceded by eight-week vernalization treatment of the callus. This paper may provide a protocol by which all stages of somatic embryogenesis of A. nordmanniana can be carried out, including the preceding 24-h seed disinfection with Na OCl and PVP, which resulted in 100 % frequency of uninfected zygotic embryos that were capable of starting embryogenesis.
文摘Somatic embryogenesis in upland cotton isstrongly genotype-dependent,which is a troublein cotton genetic engineering.Cloning genesrelated to somatic embryogenesis and thenintroducing the gene into mainly cultivatedvarieties would be greatly helpful for cottonimprovement by gene transfer.To study
基金Supported by the National Projects of Genetic Modified Organism Breeding Technology (2008ZX08006-002)the State Transgenic Research Programme of China (2008ZX08006-002)
文摘PiggyBac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggyBac transposon marked with enhanced green fluorescent protein (eGFP) and showed efficient transposition in porcine somatic cells and cloned embryos. Our results demonstrated that piggyBac transposase could efficiently catalyze transposition in porcine fetal fibroblast cells, as well as in embryos. PiggyBac transposition generated 18-fold more eGFP-positive cell colonies compared to pEGFP-C1 random insertion mutagenesis, but excessive transposase might affect the transfection rate. Also piggyBac mediated 4-fold more eGFP expression than random insertion in cells and 17-fold in cloned embryos at mRNA level. When the mutagenized cells were used for somatic cell nuclear transfer (SCNT), the cleavage rate and blastocyst rate of constructed embryos harboring piggyBac transposition had no difference with random insertion group. This study provides key information on the piggyBac transposon system as a tool for creating transgenic pigs.
文摘Somatic embryogenesis (SE) is a prominent mode of regeneration in plants. The acquisition of SE is predominantly invoked by the oxidative stress which plays an important role in signal transduction and cellular redox. Since balanced generation of oxidants is important to cellular differentiation, modulation in cell redox could be responsive to genotypic refinement for SE. To study the dynamics of cellular redox during SE, we conducted comparative expression analyses of cotton (Gossypium hirsutum), using two independently purified near-isogenic lines for the trait of SE. We interrogated expression changes in cell-signaling factor Somatic Embryogenesis Receptor Kinase (SERK) and activity of antioxidant Glutathione in different developmental stages including cotyledonary leaf, calli from different stages of regeneration of fully-regenerating (FR) and non-regenerating (NR) lines of Coker310 cultivar. At evolutionary scale, the cotton SERKs showed high sequence similarity in receptor kinase domain with diverse systems. Exclusively, SERK1 responsible for potential signaling processes during SE revealed significant expression up-regulation in the embryogenic calli of FR line. Similarly, activity of antioxidant glutathione was substantially up-regulated in embryogenic calli of FR line in comparison to its counterpart form. In contrast, calli from early-stages of regeneration of both FR and NR lines had no significant influences on the regulation of SERK and glutathione levels prior to the acquisition of embryogenesis. These results highlight that in vitro purification of FR line in cotton for enhanced regeneration potential (through SE) resulted in signaling and metabolic transformations of the manner in which cellular redox levels have become modulated.
基金supported by grant from the Mexican Forest Service(CONAFOR)(No.11385232709—“Promotion and Operation of the Forest Genetic Resources Subsystem,within CNRGINIFAP’’)
文摘Pinus patula is a species commonly used for reforestation in Mexico.However,efficient methods for the mass production seedlings are required.Micropropagation particularly by somatic embryogenesis provides an option for the rapid multiplication of high-quality,genetically improved material.This study induces somatic embryogenesis in this species using the thin cell layer(TCL)technique.Two sources of explants(complete immature embryos;lTCL segments from immature embryos)were evaluated.The efficiency of TCL from longitudinal sections[lTCL]and transverse[tTCL]was evaluated.The results show using thin cell layers from immature embryos cultivated in 16 light/8 dark hours achieves induction of somatic embryos.A higher percentage of embryogenic callus was obtained when tTCL segments were used as an explant source.These results produced somatic embryos from tTCL segments of an immature embryo without germinating the seed,making the process more time efficient.In addition,this technique can be used to generate somatic embryogenesis in forest species that have low germination rates.
文摘BACKGROUND: Scorpion venom from Buthus martensii Karsch is a kind of protein toxin secreted by scorpion tail. According to records of Pen-ts’ao Kan-mu, the dried scorpion’s body and tail are used as medicine, which mainly treating rheumatism numbness, pain and convulsion. Some researches consider that scorpion toxin is the main pharmacological active component of the dried scorpion, and it has 4 times stronger analgesic effect on somatic pain than morphine. OBJECTIVE: To observe the interventional effect of scorpion venom of Buthus martensii Karsch analgesic active peptide on somatic pain. DESIGN: A randomized and controlled trial. SETTING: Department of Physiology, Shenyang Medical College. MATERIALS: Totally 80 Wistar rats (provided by Animal Center of Shenyang Medical College), male and female in half, aged 3 to 4 months, weighed 250 to 350 g, were used in this trial. They were randomly divided into 4 groups: experimental group, control group, morphine group and naloxone group, with 20 in each group. Analgesia active peptide from buthus martensii karsch venom (provided by Biochemical Department of Shenyang Pharmaceutical University, 1 mL/dosage), morphine (produced by Shanghai First Pharmaceutical Manufacturing Plant), naloxone (opium acceptor inhibitor, American Sigma Corporation), ATAC-350 data-processing machine (Nihon Kohden Corporation), X-Y recording instrument (Nihon Kohden Corporation). METHODS: The experiment was conducted in the Department of Physiology, Shenyang Medical College from July 2003 to July 2005. ① After all the animals were anesthetized, common peroneal nerve is dissected and ligated at middle part. Posterior nucleus group of thalamus (PO) and the tail nuclear were oriented according to G. Paxino rat brain three-dimensional orientation atlas. Glass micro electrode was inserted into PO as guiding electrode, connected with ATAC-350 data-processing machine and X-Y recording instrument, to record the unit evoked potential of PO, taking the evoked potential discharge of the common peroneal nerve of PO as the somatic pain index.② Single square-wave stimulation of intensity 17-19 volt, wave wide 0.2 ms, time delay 20 ms was exerted on common peroneal nerve. The time interval was 5 minutes. X-Y recording instrument was used to draw the graph. 0.002 mg scorpion venom analgesic active peptide was injected into the rats of the experimental group; 0.002 mg normal saline was injected into the rats of the control group through caudate nucleus; 0.002 mg morphine was injected into the rats of morphine group through caudate nucleus; 1.0 mg/kg naloxone was intraperitoneally into the rats of naloxone group, then 0.002 mg scorpion venom was injected into the rats of control group through caudate nucleus.Changes of evoked potential of PO of 3 groups were observed. ③After experiment, 1 μA direct current was given to the guiding electrode and micro electrode , and it lasted for 5 minutes. Pontamine sky blue was used to label the peak of electrode by electrophoresis. Brain tissue was soaked in formalin for 1 week then sliced into 1.0 to 1.5 mm sections. Electrode was orientated according to blue spots. Compared with G . Parino rat brain three-dimensional orientation atlas, we confirmed if the electrode orientation is consistent with PO orientation of G. Parino rat brain three-dimensional orientation atlas. MAIN OUTCOME MEASURES: Changes of the evoked potential of PO in each group. RESULTS: Totally 80 Wistar rats were enrolled in this experiment. Four rats in the experimental group and two in the morphine group died of overdose of anesthesia, and finally 74 rats entered the stage of result analysis. The inhibitory action time of evoked potential of PO has no statistical difference in between experiment group and morphine group (P > 0.05). The whole inhibitory action time, timeof initiate recovery and time of complete recovery of PO of experiment group were longer than those in the morphine group [(45±0.7),(50±9.2),(65±8.1);(35±7.8),(40±8.9),(50±7.6) min,P < 0.05].The change of evoked potential of PO was not obviously in the control group and naloxone group (P > 0.05), and the above-mentioned 4 indexes were nearly 0. CONCLUSION: Scorpion venom possesses obviously inhibitory effect on somatic pain, and its inhibitory effect is stronger than that of the same dosage and concentration of morphine. Scorpion venom exerts analgesic effect on somatic pain through opium acceptor.
文摘Leptadenia pyrotechnica is an important multipurpose endangered plant in the Kingdom of Bahrain with restricted distribution. Nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of indole acetic acid (IAA) and 6-benzylaminopurine (BAP). Initially, 80% and 60% explants responded in direct shoot and callus initiation response respectively in presence of 8.88 μM BAP with 5.71 μM IAA in modified MS media after two weeks of culture. The highest frequency of plant regeneration was observed in presence of 8.88 μM BAP with 1.14 μM IAA following organogenic pathway of differentiation. Hundred percent callus proliferation was observed while initial callus developed in presence of 4.44 μM BAP with 2.85 μM IAA and was transferred in media containing 4.44 μM, 6.66 μM BAP with 2.85 μM IAA and 13.32 μM BAP with 5.71 μM IAA. The callus derived plants were regenerated following the pathway of indirect somatic embryogenesis. The induction of somatic embryogenesis and plant regeneration from callus was also observed in modified MS media supplemented with 4.44 μM BAP and 2.85 μM IAA. The plant regeneration protocol we developed for Leptadenia pyrotechnica will be very beneficial for biodiversity conservation and environment protection of Bahrain. Moreover, the present paper reports for the first time specifically the somatic embryogenesis in this multipurpose desert plant Leptadenia pyrotechnica.
文摘To enhance the understanding of artemisinin biosynthesis, we have successfully bred self-pollination Artemisia annua plants. Here, we report efficient somatic embryogenesis and organogenesis of self-pollination plants and artemisinin formation in regenerated plants. The first through sixth nodal leaves of seedlings are used as explants. On agar-solidified MS basal medium supplemented with TDZ (0.6 mg/l) and IBA (0.1 mg/l), all explants after inoculation of less than 3 weeks start to form embryogenic calli, which further produce globular, torpedo, heart and early cotyledon embryos. In all six positional leaves, explants from the sixth leaf show the rapidest responses to induction of embryogenic calli and somatic embryos. On this medium, somatic embryos continuously develop into adventitious buds, which can form adventitious roots on a rooting medium containing NAA (0.5 mg/l). Meanwhile, on agar-solidified MS basal medium supplemented with BAP (1 mg/l) and NAA (0.05 mg/l), approximately 100% of explants from leaves #3-6 form calli in less than 3 weeks of inoculation and adventitious buds via organogenesis in 3-4 weeks. In all six positional leaves, explants from the sixth leaf exhibit the rapidest response to induction of calli and adventitious buds. Nearly 100% adventitious buds can form adventitious roots on the rooting medium. Regenerated plants from both somatic embryogenesis and organogenesis complete self-pollination to produce seeds in 80-90 days of growth in growth chamber. LC-ESI-MS analysis demonstrates that regenerated plants biosynthesize artemisinin. These results show the highly efficient regeneration capacity of self-pollination A. annua plants that can form a new platform to enhance the understanding of artemisinin biosynthesis and metabolic engineering.
