Biology Methods and Protocols

Abstract In this protocol for obtaining doubled haploids plants (DH), we propose a new method for microspore isolation. This method is useful for genotypes of the Brassicaceae family with low responsiveness to DH technology. For such crops, it allows increasing the embryo yield several times and sometimes obtaining embryos for the first time. This method of microspore isolation reduces the mechanical impact on the bud tissue, which minimizes somatic cell destruction and reduce to get it into the preparation through the filter, thus increasing its purity. The new isolation method also increases the relative concentration of embryogenic microspores in the preparation. This is possible because the anther tissues are not destroyed during the isolation process. Therefore, the anther retains its structure and microspores of early and late stages are trapped by the anther tissue, thus the anther acts as a sieve. Late stages are trapped because of their larger size, while early stages are trapped because they are even more tightly bound to the anther tissue. Together, these factors increase the efficiency of the technology for DH production in vitro microspore culture. This protocol article provides a detailed experimental protocol to the method presented in the experimental article (E.V. Kozar, E.G. Kozar, E.A. Domblides. Effect of the Method of Microspore Isolation on the Efficiency of Isolated Microspore Culture In Vitro for Brassicaceae Family. Horticulturae. – 2022. – Vol. 8, No. 10. – P. 864. – DOI 10.3390/horticulturae8100864) but does not repeat all the results documenting the efficacy of the actual method.

Elena V Kozar

Elena A Domblides

Protocol for obtaining doubled haploids in isolated microspore culture <i>in vitro</i> for poorly responsive genotypes of brassicaceae family

甘蓝型油菜低反应基因型花粉离体培养获得双单倍体的操作规程

Abstract Intravenous injection provides a direct, rapid, and efficient route for delivering drugs or other substances, particularly for compounds with poor intestinal absorption or molecules (e.g., proteins) that are prone to structural changes and degradation within the digestive system. While Drosophila larvae represent a well-established genetic model for studying developmental and physiological pathways, as well as human diseases, their use in analyzing the molecular effects of substance exposure remains limited. In this study, we present a highly efficient injection method for Drosophila first- and second-instar larvae. Despite causing a slight developmental delay, this method achieves a high survival rate and offers a quick, easily adjustable protocol. The process requires 3–5 hours to inject 150–300 larvae, depending on the microcapillary needle, microinjection system, and the compound being administered. As proof of concept, we compared the effects of injecting ferritin protein into Fer1HCH00451 mutant first instar larvae with those of dietary ferritin administration. Our results show that ferritin injection rescues Fer1HCH mutants, a result that cannot be achieved through dietary delivery. This approach is particularly valuable for delivery of complex compounds in cases where oral administration is impaired or limited by the digestive system.

Sattar Soltani

Nhan Huynh

Kirst King-Jones

An Efficient Injection Protocol for <i>Drosophila</i> Larvae

一种用于果蝇幼虫的高效注射方案

Abstract The main objective of the ZebraCool programme was to create a positive attitude and curiosity towards science by bringing experimental activities within schools using an introductory cognitive and sensory approach. This innovative programme was offered at all levels of primary and secondary education including vocational high schools. Thematic workshops can be carried out on various themes such as comparative anatomy and embryology, molecular biology and evolution, or toxicology and endocrine disruptors. They were on an ad hoc basis or as part of an annual school project using zebrafish as a model. This animal was a very attractive entry point for the educator to motivate students to appreciate biology, in particular in the field of molecular biology and evolution. For each practical workshop, the student was an actor in his/her learning, which was intended to arouse the curiosity and desire to understand and learn. The programme was based on close collaboration between class teachers and programme educators to adapt workshops’ content to the school curriculum. Students conducted their own experiments, formulated and tested hypotheses, learned laboratory techniques, collected and analysed data. ZebraCool scientific activities fell within a conceptual framework of evolutionary biology through which participants perceived their own inner fish through the comparison of biological processes between humans and zebrafish.

Laure M Bourcier

Patrick J Babin

A cognitive and sensory approach based on workshops using the zebrafish model promotes the discovery of life sciences in the classroom

基于斑马鱼模型的认知与感知工作坊方法促进课堂生命科学探索

Abstract Peroxidase DNAzymes are single-stranded, stable G-quadruplexes structures that exhibit catalytic activity with cofactor hemin. This class of DNAzymes offers several advantages over traditional protein and RNA catalysts, including thermal stability, resistance to hydrolysis, and easy of synthesis in the laboratory. However, their use in medicine, biology, and chemistry is limited due to their low catalytic rates. Selecting and evolving for higher catalytic rates has been challenging due to limitations in selection methodology which generally use affinity as the selection pressure instead of kinetics. We previously evolved a new peroxidase DNAzyme (mSBDZ-X-3) through a directed evolution method, which was subsequently used for proximity labelling in a proteomic experiment in cell culture. Herein, we present a detailed protocol for this function-based laboratory evolution method to evolve peroxidase DNAzymes for future laboratory implementation. This approach is based on capturing self-biotinylated DNA, which is catalyzed by intrinsic peroxidase activity to select for DNAzyme molecules. The selection method uses fluorescence-based real-time monitoring of the DNA pools, allowing for the enrichment of catalytic activity and capture of catalytic DNA across evolutionary selection rounds. The evolved mSBDZ-X-3 DNAzyme attributes parallel G-quadruplex structure and demonstrates better catalytic properties than DNAzyme variants evolved previously. The influence of critical reaction parameters is outlined. This protocol enables discovery of improved peroxidase DNAzyme/RNAzyme variants from natural or chemical-modified nucleotide libraries. The approach could be applicable for the selection of catalytic activities in a variety of directed molecular evolution contexts.

Soubhagya K Bhuyan

Weisi He

Jingyu Cui

Julian A Tanner

Directed evolution of peroxidase DNAzymes by a function-based approach

基于功能方法的过氧化物酶DNAzyme的定向进化

Abstract The COVID-19 pandemic underscored the necessity for rapid and efficient diagnostic testing to mitigate outbreaks and control disease transmission. While RT-qPCR has been the gold standard due to its high sensitivity and specificity, its logistical complexities and extended turnaround times highlighted the need for alternative molecular methods and non-standard equipment and consumables not subject to supply chain pressure. Loop-mediated isothermal amplification (LAMP) offers several advantages over RT-qPCR, including faster processing time, assay flexibility and cost-effectiveness. During the pandemic, LAMP was successfully demonstrated as a viable alternative to RT-qPCR for SARS-CoV-2 detection. However, due to a 100 to 1,000-fold increase in testing volumes, there was an imminent need for automating and scaling up existing LAMP testing workflows leveraging a robotic infrastructure, while retaining analytical performance and cost-effectiveness. In 2020 the Foundation TOMi started the “TOMi corona initiative” to develop and validate a high-throughput, end-to-end, automated, scalable single-step RNA purification and LAMP-based COVID-19 testing system called SMART-LAMP (Scalable Molecular Automation for Rapid Testing using LAMP) that can process up to 40,000 samples per day using existing laboratory equipment infrastructure with sensitivity comparable to RT-qPCR. This system provides a rapid and scalable diagnostic solution for future pandemics, capable of processing over 40,000 samples per day. In addition, the system is designed to minimise consumable costs and reduce the overall use of plastics to align with increasingly strict sustainability goals that will be imposed over the coming years. Importantly, this system and public-private partnerships in the TOMi corona initiative has the potential to serve as a baseline to enhance pandemic preparedness and response capabilities.

Rinie van Beuningen

Kin Ki Jim

Maikel Boot

Michel Ossendrijver

Bart J F Keijser

Jeroen H B van de Bovenkamp

Willem J G Melchers

Tim Kievits

Development of a large-scale rapid LAMP diagnostic testing platform for pandemic preparedness and outbreak response

大规模快速LAMP诊断测试平台的发展：为大流行准备和疫情响应

短名	Biol. Methods. Protoc.
Journal Impact	2.70
国际分区	BIOCHEMICAL RESEARCH METHODS(Q3)

ISSN	2396-8923
h-index	13

涉及主题	geneticsbiochemistrymolecular biologyecologybiological sciences
出版信息	出版商: Oxford University Press，出版周期: ，期刊类型: journal，开源期刊: 是
基本数据	创刊年份: 2016，原创研究文献占比: ，自引率:， Gold OA占比: 92.59
期刊费用	$1067详情
平均审稿周期	19 weeks

Biology Methods and Protocols

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Protocol for obtaining doubled haploids in isolated microspore culture in vitro for poorly responsive genotypes of brassicaceae family

An Efficient Injection Protocol for Drosophila Larvae

A cognitive and sensory approach based on workshops using the zebrafish model promotes the discovery of life sciences in the classroom

Directed evolution of peroxidase DNAzymes by a function-based approach

Development of a large-scale rapid LAMP diagnostic testing platform for pandemic preparedness and outbreak response

Directed evolution of peroxidase DNAzymes by a function-based approach

An Efficient Injection Protocol for Drosophila Larvae

A cognitive and sensory approach based on workshops using the zebrafish model promotes the discovery of life sciences in the classroom

Protocol for obtaining doubled haploids in isolated microspore culture in vitro for poorly responsive genotypes of brassicaceae family

Development of a large-scale rapid LAMP diagnostic testing platform for pandemic preparedness and outbreak response