MOSP - Macao Scientific Publishers

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Manual Cloning of Mammals: An Innovative Methodology

Adil Salim l Elsheikh

Abstract: Handmade embryo reconstitution (HMER) has been used to study the parameters of nuclear transfer experiments such as fusion process, nuclear remodeling, nuclear reprogramming, biochemical processes, and biological processes during embryogenesis. These parameters have been widely investigated using the micromanipulator-based cloning technique (MBCT). This technique is a tedious, multi-step, time consuming and complicated procedure that utilizes expensive equipment. The HMER has emerged as an alternative for the MBCT. If the HMER is used to produce cloned animals it is known as handmade cloning (HMC). The HMC will allow the scientists to produce cloned animals with simple non-expensive equipment. Consequently, enormous data concerning all the facets of the nuclear transplantation experiments could be retrieved from various laboratories. This will allow a better future application of the cloning technique for the welfare of human, through production of animals with high genetic traits, rescue of endangered animal species and production of transgenic animals that can produce medicine for certain human diseases.[...] Read More.

Keywords: Cloning, chemical enucleation, oocyte bisection, mouse.

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Incorporation and Preservation of Newcastle Disease Virus on Filter Papers with Detection of Viral RNA via a Single-Tube RT-PCR Assay

P. N. Wambura*

Advanced Journal of Microbiology Research ISSN 2736-1756 Vol. 19 (2), pp. 001-004, February, 2025. Available online at www.internationalscholarsjournals.org © International Scholars Journals

Full Length Research Paper

Incorporation and Preservation of Newcastle Disease Virus on Filter Papers with Detection of Viral RNA via a Single-Tube RT-PCR Assay

P. N. Wambura*

School of Veterinary Science, Faculty of Natural Resources, Agriculture and Veterinary Science, University of Queensland, Brisbane, QLD 4072, Australia. E-mail: p.wambura@mailbox.uq.edu.ac, phil_wambura@yahoo.com, pwambura@suanet.ac.tz.

Accepted 21 October, 2024

Suitability of storing infected allantoic fluid (AF) and cell culture supernatants (CCS) with strain I-2 of Newcastle disease virus (NDV) on Whatman filter papers at room temperature (22-25°C) and 37°C was determined. RNA was extracted from filter papers or liquid aliquots and subjected to reverse transcriptase- polymerase chain reactions (RT-PCR). The results showed that filter papers soaked with NDV infected AF or CCS stored at 37°C yielded amplicons with intensity similar to that kept at room temperature for up to 150 days. The study demonstrates that NDV infected samples can be soaked onto filter papers, stored and subsequently detected by RT-PCR. This method might be safely used for storage and transportation of NDV samples to the designated laboratories for molecular studies without the need for cooling.

Key words: Allantoic fluid, chicken embryo fibroblast, Newcastle diseases virus, polymerase chain reaction, RNA storage onto filter paper, strain I-2.

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Somatic embryogenesis in two Iranian date palm cultivars

Parisa Eshraghi, Reza Zarghami*, and Mitra Mirabdulbaghi

Abstract: Shoot tips were removed from 3 to 4 year-old offshoots of adult date palm (Phoenix dactylifera L. cv. Khanizi and Mordarsing) and were cultured on medium that consisted of Murashige and Skoog basal salts medium. After 12 months they were transferred to three different treatments of growth regulators. Five months later, cv. Khanizi produced embryogenic callus on a medium containing 453 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 15 µM N2- (2-isopentenyl) adenine (2ip), and 13 µM 6-benzyl amino purine. This callus was subcultured to another medium that was supplemented with 54 µM NAA and 148 µM 2ip. At this stage the embryos grew into plantlets. The cv. Mordarsing explants, on a medium containing 679 µM 2,4-D and 15 µM 2ip, produced embryogenic callus but the embryos remained in the globular stage.[...] Read More.

Keywords: Callus, in vitro tissue culture, offshoot, Phoenix dactylifera, regenerant.

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In Vitro Regeneration of Hybrid Cashew Plantlets (Anacardium occidentale L.) via Embryo Culture Techniques

Aliyu, O. M.* and Awopetu J.A.

Abstract: Embryos from immature nuts of cashew (Anacardium occidentale L.) were cultured in vitro to regenerate improved hybrid plantlets. Explants (embryo) were excised from developing F1 hybrid immature nuts derived from diallel cross and harvested at 2-, 4-, 6- and 8-weeks after pollination (WAPo) for in vitro culture. The explants were surface sterilized, aseptically dissected and cultured into pure basal Murashige and Skoog (MS) agar medium and MS medium supplemented with 1 mM each of naphthaleneacetic acid (NAA), benzyladenine (BA) and gibberellic acid (GA3) and subsequently observed for germination and survival rates until successful ones were transferred to the field. Age of explants was found to significantly influence both the germination and survival rates. Explants of 6 weeks old and above were found to give better germination rate and highest survival percentage in this study. Only MS medium supplemented with 1 mM of gibberellic acid (MS+GA3) supported germination and growth at 2-WAPo, suggesting the essentiality of GA3 as a growth regulator to a very young cashew embryo. Analysis also showed that factors such as medium composition, age of embryo and genotype (accession) significantly influence the germination rate of cashew embryo. It was observed that cashew embryos were found to be autonomy of growth regulator as the age increases and medium composition is only critical at very young age of the embryo. Successful germinated explants simultaneously produced shoot and root and were ready for transfer to field and acclimatization, between 90 and 112 days after inoculation.[...] Read More.

Keywords: Anacardium occidentale, in vitro culture, explant, embryo.

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Exploring Callus Proliferation and Somatic Embryogenesis in Gossypium hirsutum L.

Ikram-ul-Haq

Abstract: Somatic embryogenesis and plant regeneration are fundamental to tissue culture biotechnology in cotton (Gossypium hirsutum L.) cv. Coker 312. Callus proliferation was considered best on MS1a (2.0 mg/L NAA; 0.1 mg/L ZT; 0.1 mg/L KT) when 6 weeks old callus was cultured from MS1b (0.1 mg/L 2, 4- D; 0.5 mg/L KT) medium, there is no need to select embryogenic calli for somatic embryogenesis, as all of them were converted to somatic embryos. NH4NO3 play an important role in differentiation of callus into somatic embryos but is lethal for embryos just after two weeks. However, KNO 3 is less efficient for somatic embryo induction but is best for embryo maturation. By this procedure 56.51% cotyledenary embryos were developed within 5 weeks. Of that, 82.05% cotyledenary embryos were developed not only into normal plantlets, but rooted simultaneously when cultured on MS (with 0.05 mg/L GA3) medium. A complete plant of Cocker-312 could be regenerated through somatic embryogenesis within 4 to 5 months.[...] Read More.

Keywords: Gossypium hirsutum L,plant regeneration, Coker 312, callus induction, somatic embryogenesis, in vitro regeneration.

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Evaluating Growth Regulator Effects on Two Differently Cultured Explants of Carapa guianensis in vitro

Larisse Lobo de Oliveira, Anaíze Borges Henriques, Andrea Furtado Macedo,*

Abstract: Carapa guianensis Aubl. (Meliaceae), known locally as andiroba, is a multi-use species from Amazonia. Andiroba oil is considered an important natural product in the Brazilian market, and international demand is increasing due to its cosmetic and pharmaceutical potential. C. guianensis trees produce seed irregularly over different harvest periods, leading to inconsistent oil production and difficulties with supply. No management plans or protocols have been developed for in vitro or clonal production of Carapa seedlings and the maintenance of genetic resources. The objective of this study was to assess the effect of growth regulators on explants (young leaves, old leaves and apical buds). Explants consisting of leaf segments 1 cm on a side were cultivated in MS medium with and without growth regulators. Evaluation was based on fresh and dry weight of the explants after 20 days. In the media with 2,4-dichlorophenoxyacetic acid (5, 15, 35 or 45 µM), changes were observed in weight and explant appearance (callus). Bud breakage and development of shoots were achieved using 5 µM of 6-benzylaminopurine. Overall, the results showed that 2,4-dichlorophenoxyacetic acid stimulates callus formation on andiroba foliar explants, while 6-benzylaminopurine was superior to thidiazuron for the initial development of shoots.[...] Read More.

Keywords: Growth regulators, Carapa guianensis, in vitro, tissue culture, organogenesis.

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Optimizing Blastocyst Formation from Cryopreserved Mouse Two-Cell Embryos: The Role of Fibroblast and Hepatocyte Growth Factors

Fatemeh Ghasemian, Mahnaz Azarnia, Abtin Heidarzadeh, Shervin Ghadarjani and Mohammad Hadi Bahadori*

Abstract: There is great need to improve our understanding of what increases an embryo’s development potential, after vitrification-thawing processes. For this subject, 358 two-cell stage embryos were collected from oviduct of pregnant two-day old mice and vitrified. After thawing, embryos were cultured in Tyrode's (T6) medium supplemented with different doses of fibroblast growth factor (FGF; 0, 10, 20, 50 and 100 ng/ml) and hepatocyte growth factor (HGF; 0, 10, 20, 50 and 100 ng/ml) until the blastocyst stage. To determine quality of blastocysts, blastocysts were stained with hoechst and propidium iodide. After culture for 24 h, 92.65% of treated embryos with 20 ng/ml of FGF had higher (P64 cells had a significantly higher inner cell mass (ICM) in comparison to the control group (P< 0.01). In conclusion, in this experiment, addition of growth factors in the culture had favorable effects on post-thawed cleavage of vitrified 2-cell embryos and blastocyst quality. Keywords: Blastocyst quality, growth factors, preimplantation development, vitrification. [...] Read More.

Keywords: Blastocyst quality, growth factors, preimplantation development, vitrification.

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Direct Detection of Salmonella InvA, SpiC, and SipC in Clinical Samples Using Dot Blot Hybridization

Hang’ombe Bernard Mudenda*, Ulaya William, Mwansa James C. L., Mubita Charles, Isogai Nayuta, Mulenga Evans, Moonga Ladslav, Isogai Hiroshi and Isogai Emiko

Abstract: Pathogenesis of Salmonella depends upon a large number of factors controlled by an array of genes that synergise into actual virulence. The goal of this study was to detect Salmonella invA, spiC and sipC directly from clinical specimens, using the dot blot hybridization assay. We detected invA , spiC and sipC as a one combination from 4.5% (95% CI: 2.21 to 8.64) human feacal and 35.2% (95% CI: 26.4 to 45.0) poultry samples after enrichment. Furthermore the dot blot method had a higher sensitivity than routine culture, before and after enrichment. These results indicate that dot blot hybridization may be used to directly detect Salmonella invA, spiC and sipC in clinical samples.[...] Read More.

Keywords: Salmonella, dot-blot hybridization, spiC, sipC, invA, clinical-samples.

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Genetic Variability of Helicobacter pylori in Iran: Clarithromycin Resistance and 23S rRNA Mutations

Mohammad Kargar*, Maryam Baghernejad, Abbas Doosti and Sadegh Ghorbani-Dalini,

Abstract: To determine the 23S rRNA point mutations in clarithromycin resistance of Helicobacter pylori strains isolated from southwest, Iran. This was a cross-sectional survey, which was done on 263 patients who referred to endoscopy department of Shehrekord university of medical sciences. According to gram stain, urease, catalase, oxidase and polymerase chain reaction (PCR) H. pylori identified. Standard National Committee for Clinical Laboratory Standard (NCCLS) method used for assessment of clarithromycin resistance. Specific primers and restriction enzymes BsaI and MboII by PCR-RFLP were used for analysis of A2143G and A2142G mutations. So for the detection of A2142C, specific primers and PCR method were used. 84 strains of H. pylori (31.94%) determined by PCR method. Of 19 (22.62%) clarithromycin resistant strains 13 (68.40%), 3 (15.78%), 2 (10.52%) had A2143G, A2142G, A2142C respectively and one unknown mutation in 23S rRNA gene. Because of considerable resistance to clarithromycin, direct diagnosis of this mutation by molecular approach in other parts of the country is necessary.[...] Read More.

Keywords: Polymerase chain reaction, clarithromycin, resistance, 23S rRNA, polymerase chain reaction - restriction fragment length polymorphism.

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Rapid and Accurate Detection of Agrobacterium tumefaciens in Soil Using PCR

Wei Yang, Lei Ji, Li-Rong Tan, Shi-Mo Li, Yan Wang, Hong-Xia Liu*, and Yu-Ming Luo*

Abstract: One pair of primers was designed based on the sequence of tmr locus for specific and sensitive detection of Agrobacterium tumefaciens. Only the A. tumefaciens strain can produce the 236bp target fragment among the fourteen bacterial species that tested. The sensitivity of the specific PCR system was determined by a nested-PCR amplification which can numbered the copies of the template DNA. According to the results, it can give positive band when only 100 copies were in the template. The protocol was carried out for detection A. tumefaciens of twelve soil samples collected from six different gardens in Shanghai where crown gall happened. Two of the samples which collected from symptomless gardens also give the positive band. Based on the results we can make a conclusion that this pair of primers can be a useful tool in detecting A. tumefaciens, especially in detecting latent infection of this devastating pathogen.[...] Read More.

Keywords: Agrobacterium tumefaciens, detection, polymerase chain reaction (PCR).

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