Studies on Enhancing Yield of Embryos in Microspore Culture of Brassica napus L.

In ecological zone of Chengdu, Sichuan, microspore culture was carried out in Brassica napus L. to study the influencing factors on microspore culture. The results showed that the temperature on microspore formation stage, day and night temperature, disinfection solution of buds, cultivation concentration on microspore and strain-age were both important influencing factors on microspore culture. At a temperature below 5 ℃ or above 20 ℃, the material had a much lower embryo producing rate or even could not produce any embryo, but at the optimum temperature of 10 -15 ℃ the embryo yield was up to 300 pieces per bud; the best cultivation effect appeared when 0. 1% HgCl2 was used for disin- fection; the best density of microspore was 3 -4 buds per dish; In 2009, while strain-age was from 125 d to 150 d, the microspore embryo yield increased as strain-age increased. When stain-age was 150 days, the microspore embryo yield was up to the highest, but the yield declined after 150 days.

Establishment on Isolated Microspore Culture Optimized System for Restorer of New Cytoplasmic Male Sterile (NER) of Brassica napus L.

[Objective]The aim was to establish optimized system for NER isolated microspore culture of Brassica napus L.. [Method]Twenty varieties of NER of Brassica napus were grown under uncontrolled temperature and light conditions,and their isolated microspore from anthers were used as explants in vitro culture. The influencing factors of microspore culture were preliminarily studied. [Result]The difference of genotypes was important influencing factors to embryoid yield. The embryoid yield increased by supplementing with 6-BA and NAA,culturing in solid-liquid double layer medium with activated charcoal; The difference was not significant of embryoid yield between culturing in medium supplemented with colchicines and the CK. The rates of cotyledonous embryoids directly developed into normal plantlets increased through enriching with 0.1-0.2 mg/L NAA and being treated on slim illumination two days before being inducted into normal plantlets. [Conclusion]The technical system of microspore culture of restorer of new cytoplasmic male sterile （NER） was established,which and lays a foundation for accelerating genotype purification of NER introgressive line.

Isolated Microspore Culture and Plant Regeneration of Leaf Mustard(Brassica juncea(L.)Czern.et Coss.)

Isolated micmspore culture was carried out with Brassica juncea to study the effects of sterilization methods, culture period of embryos, hormone formula and the concentration of aetivatod carbon on embryogenesis, and the concentration of NAA on induction of roots. The results showed that the embryogenesis on medi- um subjected to filter sterilization is better than medium subjected to autoclaved sterilization. The embryos cultured for 15 d had the highest plantlat regenexafion rate （52%）. NLN-13 liquid medium including 0. 1mg/L 6-BA and 0.2 g/L activated carbon significantly improved the planflet regeneration rate. 1/2 MS inclu- ding 0.3 mg/L NAA had the highest plantlet regeneration rate （ 100% ）. Key words Brassica juncea ; Isolated micmspore culture ; Embryogenesis ; Planflet regeneration

Cell culture isolation can miss the laboratory diagnosis of HSV ocular infection

AIMWe compared polymerase chain reaction (PCR) to cell culture isolation for the laboratory diagnosis of ocular herpes simplex virus (HSV) disease.

Anther Culture of Chinese Radish(Raphanus sativus L. var. Longinnatus Bailey):Response of Different Genotypes to the Embryogenesis and the Traits of Regenerated Plantlets

[Objective] The aims were to ① conduct anther culture of Chinese radish varieties; ② observe the development of embryos from anther culture; ③ study the response of different genotypes to embryogenesis in anther culture; ④ observe the morphology of regenerated plantlets; ⑤ analyze the ploidy level of regenerated plantlets arising from the anther culture process. [Method]Anthers of 15 genotypes with diverse genetic backgrounds of Chinese radish have been cultured in vitro and induced to undergo embryogenesis and plant formation. [Result] Of 15 genotypes evaluated,four produced embryos. The genotype was the main factor to influence the embryogenesis. The morphology and the ploidy of the regenerated plantlets were observed,and the anther-derived plantlets included a mix of haploids,diploids and hexaploids. Of the plants that regenerated from anther embryos 60% were diploid. [Conclusion] The plantlets had the high ability to double spontaneously.

Transitioning to Automated Microbiologic Era: Blood Culture Isolates in Children and Adults in Federal Teaching Hospital in Gombe, North East Nigeria 2016-2020

Introduction: Automated blood culture systems for incubation and growth monitoring have become the standard in high-income countries (HICs), but are still relatively expensive and not universally available for implementation in most low- and middle-income countries (LMIC). We aimed to report blood culture isolates using Automated technique in children and adults admitted into the Federal Teaching Hospital Gombe from 2016 to 2020. Materials and Methods: Blood Culture Isolates in children (0 - 18 years) and adults (>19 yrs) by Bactec 9050 Automated culture system from 2016-2020 were retrieved from the medical and laboratory register. Information analyzed included, age, sex, month, and year and culture growth and reported antibiotic sensitivity. A Bactec Blood culture tests is $20 in this facility. In Nigeria, the minimum monthly wage is $70 (Official currency exchange rate is N423/US Dollar). Results: Of the 1713 blood cultures performed, children 0 - 18 years were 1322 (77.2%) and adult (19 years above) (22.8%). Overall positivity was 733 (42.2%) with males 385 (52.5%). Of the 1322 Blood cultures (BC) in children 615 (46.5%) were positive for isolates and adults 118 (30.2)%. Blood culture positivity decreased with increasing age with newborns 251 (34.5%) and adults > 65 years 18 (2.5%). Staphylococcus aureus constituted 61.3% of all isolates and was the leading isolates in all age groups;Alkaligenes (9.1%);Citrobacter 8.1%, Klebsiella 6.7%;Pseudomonas 6.1%;E. coli 2.7%;Enterococcus 2%;Proteus 1%. Of the Antimicrobial resistance priority isolates E. coli susceptibility ranged from 71% to Gentamycin and 100% to Cefixime;Klebsiella from 25% sensitivity to Amikacin to 78% each to chloramphenicol and ciprofloxacin;Salmonella was 100% sensitive to chloramphenicol, ciprofloxacin and cefuroxime. Klebsiella was 100% sensitive to Cefoxitin;Proteus sensitivity ranged from 35% to ampicillin and 100% to ciprofloxacin and cefuroxime. Staph aureus sensitivity was 35% to cefoxitin, 70% to amoxicillin/clavulanate and 70% to cefuroxime. Conclusion: Blood culture yield by Automated method was high. Staph aureus was the predominant pathogen and bacterial yield reduced with increasing age. Antibiotic sensitivity was variably reduced against gram negative bacteria.

Trends in Bacterial Blood Culture Isolates and Resistance in Children in Two Microbiologic Eras from a Tertiary Health Facility in North East Nigeria

Introduction: Antimicrobial Resistance surveillance is predicated on blood culture as a priority clinical specimen in especially resource limited settings. Establishing trends in blood stream infections and resistance patterns can inform institutional and national policy on antimicrobial stewardship, surveillance, infection prevention and control. Methodology: Blood Culture isolates in children (0 - 18 years) by conventional method from 2008-2012 and Bactec Automated culture system from 2015-2020 were retrieved. Information analyzed included age, sex, month, and year and culture growth/identity of microorganisms and their sensitivity/resistance patterns. Clinical and Laboratory Standards Institute (CLSI) guideline for antibiotic susceptibility testing was used. Results: 20,540 children were admitted: 8964 (44.6%) and 11,630 (55.4%) in the Manual and Bactec blood culture era respectively. Blood cultures were done in 5271 in the manual culture era and 1077 in the Bactec culture era;of these cultures, 514 (9.7%) and 461 (42.8%) were positive for isolates in the respective era (p = 0.01). There were no statistically significant differences in trend between positive and negative blood cultures in males and females. Newborns, followed by children 1 - 5 years had more blood culture performed on them than other age categories. In general, there is no significant relationship in blood culture outcomes between the age categories and sex of the patients. The isolation of Staph aureus, Citrobacter and Alkaligenes increased two-fold with Bactec automated system. Resistance to the quinolones and the penicillin was high. Resistance trend to Genticin, an aminoglycoside was less than 40%. Resistance to Ceftazidime was high. Conclusion: Antimicrobial resistance surveillance is critical to reduce AMR related morbidity and mortality.

Embryogenesis and Development of Isolated Microspore in Chinese Cabbage

[Objective] The aim was to observe embryogenesis and development of isolated microspores in Chinese cabbage. [Method] Chinese cabbage F 1 hybrids were used as the experimental materials, and optical microspore was employed to observe the embryogenesis and development of isolated microspores. [Result] Cells swelled after heat shock treatment, which was the critical factor of embryoid induction. Three pathways equal division, unequal division and germination of microspores were discovered to lead to the embryogenesis from isolated microspores after swelling. Microspore could grow directly to embryoid through germination path way. Equally divided microspores formed the original embryos after successive multiple equal divisions. Original embryos could develop into cotyledon-shaped embryos via globular, heart-shaped and torpedo-shaped embryos. The large one of the two cells from unequally divided microspores continued to divide and finally formed a polar embryoid. [Conclusion] The study will provide cytological basis for high induction frequency and embryoid of Chinese cabbage.

Isolation and differentiation of embryonic stem cells from BALB/c mouse

Objective To invest the efficient method which can culture and induce embryonic stem cells to neuroeyte in vitro. Methods Isolate the blastula o f 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass （inner cell mass, ICM） which were isolated by mechanical method on the mouse embryonic fibroblaste cell （MEF） feeder layer or 0.1% gelatin coated dishes. The stem ceils were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunoehemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 （ stage specific embryonic antigen 1 ）. Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated ceils presented the characters of ESCs. Then the isolated cells were able to differentiate into neuroeytes in vitro. Conclusion Mouse embryonic stem ceils isolation, culture and differentiation system has been established.

Stem cell properties and neural differentiation of sheep amniotic epithelial cells

This study was designed to verify the stem cell properties of sheep amniotic epithelial cells and their capacity for neural differentiation. Immunofluorescence microscopy and reverse transcription-PCR revealed that the sheep amniotic epithelial cells were positive for the embryonic stem cell marker proteins SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, and the totipotency-associated genes Oct-4, Sox-2 and Rex-1, but negative for Nanog. Amniotic epithelial cells expressed β-Ⅲ-tubulin, glial fibrillary acidic protein, nestin and microtubule-associated protein-2 at 28 days after induction with serum-free neurobasal-A medium containing B-27. Thus, sheep amniotic epithelial cells could differentiate into neurons expressing β-Ⅲ-tubulin and microtubule-associated protein-2, and glial-like cells expressing glial fibrillary acidic protein, under specific conditions.

On the Strategies Employed in Transmission of Cross-cultural Advertisements

Such an extremely difficult issue as cultural diversity is confronted by the existence and evolution of enterprises with the rapid development of global economy of the present. If the establishment is to boost competitiveness, it must have a quite good knowledge of target market so as to formulate some efficient strategies of broadcasting in its management, and the best way to resolve such problem is to eliminate cultural barriers with the adoption of the transmission of cross-cultural advertisements. This thesis shall research cultural diversities between eastern nations and western ones, and explore the effect the diversities have upon cross-cultural transmission, thus to propose an approach of eliminating cultural isolation with the adoption of effective techniques and manners of the transmission of cross-cultural advertisements.

Characterization, isolation, and culture of spermatogonial stem cells in Macaca fascicularis

Spermatogonial stem cells(SSCs)have great applications in both reproductive and regenerative medicine.Primates including monkeys are very similar to humans with regard to physiology and pathology.Nevertheless,little is known about the isolation,the characteristics,and the culture of primate SSCs.This study was designed to identify,isolate,and culture monkey SSCs.Immunocytochemistry was used to identify markers for monkey SSCs.Glial cell line-derived neurotrophic factor family receptor alpha-1(GFRAl)-enriched spermatogonia were isolated from monkeys,namely Macaca fascicularis(M.fascicularis),by two-step enzymatic digestion and magnetic-activated cell sorting,and they were cultured on precoated plates in the conditioned medium.Reverse transcription-polymerase chain reaction(RT-PCR),immunocytochemistry,and RNA sequencing were used to compare phenotype and transcriptomes in GFRAl-enriched spermatogonia between 0 day and 14 days of culture,and xenotransplantation was performed to evaluate the function of GFRAl-enriched spermatogonia.SSCs shared some phenotypes with rodent and human SSCs.GFRAl-enriched spermatogonia with high purity and viability were isolated from M.fascicularis testes.The freshly isolated cells expressed numerous markers for rodent SSCs,and they were cultured for 14 days.The expression of numerous SSC markers was maintained during the cultivation of GFRAl-enriched spermatogonia.RNA sequencing reflected a 97.3%similarity in global gene profiles between 0 day and 14 days of culture.The xenotransplantation assay indicated that the GFRAl-enriched spermatogonia formed colonies and proliferated in vivo in the recipient c-Kitw/w(W)mutant mice.Collectively,GFRAl-enriched spermatogonia are monkey SSCs phenotypically both in vitro and in vivo.This study suggests that monkey might provide an alternative to human SSCs for basic research and application in human diseases.

Isolation and cultivation of porcine hepatocytes for extracorporeal artificial liver support system

Objective To develop procedures for the successful harvesting of large quantities of viable and functional pig liver cells from abattoir organs.Methods The procedure included partial liver lobe retrograde perfusion and mechanical/enzymatic digestion of the liver tissue, followed by separation of the hepatocytes, based on size and density, from contaminating cell types.Results Digestion of the partial liver lobe resulted in an average yield of 1.39×109 cells (9.9×106 cells/g liver) with an average viability of 92.5%. The yield and viability of cells were improved by dispase/collagenase resultant digestion. The emergence of blebby cells was blocked by supplying oxygen to the cell isolation buffers. Isolated hepatocytes seeded onto polystyrene surfaces remained viable and functional at a level comparable to that of rat hepatocytes, although their function decreased over time.Conclusions Adult pig hepatocytes can be harvested with high yields and retain viability and differentiated function using this method. Abattoir pig livers can be an excellent source of hepatocytes for use as the biological component of artificial liver assist devices.