soybean seed sterilization

Producción de alta eficiencia de raíces pilosas de soja transgénica

Soybean (Glycine max) is among the most valuable crops in agriculture. It has thousands of commercial and industrial uses, such as food, animal feed, oil, and as a source of raw materials for manufacturing1. Its ability to form a symbiotic relationship with nitrogen-fixing soil microorganisms, namely rhizobia, further elevates the importance of studying soybean genetics2. For instance, fine-tuning nitrogen fixation properties in soybean roots can lead to the reduction of carbon emissions and greatly reduce requirements for nitrogen fertilizer3. Thus, understanding the genetics that controls aspects of soybean root biology, in particular, has wide applications in agriculture and industry. Considering these benefits, it is important to have a reliable protocol to analyze the function of soybean genes.
Agrobacterium tumefaciens is perhaps the most commonly used tool for plant genetic transformation as it has the capability of integrating transfer DNA (T-DNA) into the nuclear genome of many plant species. When Agrobacterium infects a plant, it transfers the tumor-inducing (Ti) plasmid into the host chromosome, leading to the formation of a tumor at the infection site. The Agrobacterium-mediated transformation has been widely used for decades for gene functional analysis and in order to modify crop traits4. Although any gene of interest can be readily transferred into host plant cells through A. tumefaciens-mediated transformation, this method has several drawbacks; it is time-consuming, expensive, and requires extensive expertise for many plant species, such as soybean. Although a few varieties of soybean can be transformed by the cotyledonary node approach using A. tumefaciens, the inefficiency of this approach necessitates the need for an alternative genetic transformation technology that is rapid and highly efficient4,5. Even a non-expert can use this Agrobacterium rhizogenes-mediated hairy root (HR) transformation method to overcome these disadvantages.
HR transformation is a relatively fast tool, not only for analyzing gene function, but also for biotechnological applications, such as the production of specialized metabolites and fine chemicals, and complex bioactive glycoproteins6. The production of soybean HRs does not require extensive expertise, as they can be generated by wounding the surfaces of cotyledons, followed by inoculation with Agrobacterium rhizogenes7. A. rhizogenes expresses virulence (Vir) genes encoded by its Ti plasmid that transfer, carry, and integrate its T-DNA segment into the genome of plant cells while simultaneously stimulating ectopic root growth8.
Compared to other soybean gene&#160;expression systems, such as biolistics or A. tumefaciens-based transformation of tissue, cell, and organ culture, the HR expression system exhibits several advantages. Firstly, HRs are genetically stable and produced quickly on hormone-free media1,9,10. Additionally, HRs can produce specialized metabolites in amounts equivalent to or greater than native roots11,12. These advantages make HRs a desirable biotechnological tool for plant species that are incompatible with A. tumefaciens or that require special tissue culture conditions to form compatible tissues. The HR method is an efficient approach for analyzing protein-protein interactions, protein subcellular localization, recombinant protein production, phytoremediation, mutagenesis, and genome wide effects using RNA sequencing13,14,15. It can also be used to study the production of specialized metabolites that have value in the industry, including glyceollins, which medicate soybean&#39;s defense against the important microbial pathogen Phytophthora sojae and have impressive anticancer and neuroprotective activities in humans16,17.
This report demonstrates an easy, efficient protocol to produce soybean HRs. Compared to previous HR transformation methods, this protocol provides a significant (33%-50%) improvement in the rate of HR formation by prescreening A. rhizogenes transformants for the presence of the Ti plasmid prior to inoculating soybean cotyledons. We demonstrate the applicability of this protocol by transforming several binary vectors that overexpress or silence soybean transcription factor genes.

Autor destacado: Transformación de la raíz peluda de la soja para el análisis de la función de los genes  

Transformación de la raíz peluda de soja para el análisis de la función génica

Watch this Scientific Journal Video about Soybean Seed Sterilization at JoVE.com

Soybean Seed Sterilization  

Esterilización de semillas de soja

Para comenzar, coloque de 16 a 20 semillas de soja Williams 82 redondas prístinas en un tubo de centrífuga de 50 mililitros dentro de un gabinete de bioseguridad. Agregue 30 mililitros de alcohol isopropílico al 70% al tubo y agite durante 30 segundos. Decantar el alcohol.A continuación, agite suavemente las semillas con 30 mililitros de blanqueador al 10% durante 10 segundos y deje que las semillas reposen en la solución durante 5 minutos a temperatura ambiente. Después de 5 minutos, drene el blanqueador y luego enjuague las semillas tres veces con 30 mililitros de agua ultrapura estéril durante 1 minuto por enjuague y deseche el agua entre cada enjuague. Coloque las semillas esterilizadas en papel de filtro saturado con 5 mililitros de germinación y medio de cultivo conjunto en una placa de Petri estéril.Coloque las placas en la oscuridad a temperatura ambiente durante 3 días antes de transferirlas a 22 grados centígrados bajo luces fluorescentes T5 blancas frías de 16 horas durante 4 días para permitir que las semillas germinen.

Research

JoVE Journal

Bioengineering

Soybean Seed Sterilization

603 vistas. Para comenzar, coloque de 16 a 20 semillas de soja Williams 82 redondas prístinas en un tubo de centrífuga de 50 mililitros dentro de un gabinete de bioseguridad. Agregue 30 mililitros de alcohol isopropílico al 70% al tubo y agite durante 30 segundos. Decantar el alcohol.A continuación, agite suavemente las semillas con 30 mililitros de blanqueador al 10% durante 10 segundos y deje que las semillas reposen en la solución durante 5 minutos a temperatura ambiente. Después de 5 min...

Video: Soybean Seed Sterilization  

Soybean Hairy Root Transformation for the Analysis of Gene Function

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with soil-borne microbes that form symbiosis to fix nitrogen and pathogens, which makes research involving soybean root genetics of prime importance to improve its agricultural production. The genetic transformation of soybean hairy roots (HRs) is mediated by the Agrobacterium rhizogenes strain NCPPB2659 (K599) and is an efficient tool for studying gene function in soybean roots, taking only 2 months from start to finish. Here, we provide a detailed protocol that outlines the method for overexpressing and silencing a gene of interest in soybean HRs. This methodology includes soybean seed sterilization, infection of cotyledons with K599, and the selection and harvesting of genetically transformed HRs for RNA isolation and, if warranted, metabolite analyses. The throughput of the approach is sufficient to simultaneously study several genes or networks and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches.

Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with ...