Name: Infection of Cotyledons with Agrobacterium rhizogenes Strain NCPPB2659 (K599), Selection, and Harvesting of Hairy Roots.
Uploaded: 2023-05-05T14:40:00
Description: Watch this Scientific Journal Video about Infection of Cotyledons with Agrobacterium rhizogenes Strain NCPPB2659 K599, Selection, and Harvesting of Hairy Roots. at JoVE.com

infection of cotyledons with agrobacterium rhizogenes strain ncppb2659 (k599), selection, and harvesting of hairy roots.

Högeffektiv produktion av transgena håriga sojabönsrötter

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.

Författare i fokus: Sojaböna hårig rotomvandling för analys av genfunktion  

Soybean Hairy Root Transformation för analys av genfunktion

Our soybean hairy root transformation method is sufficient to simultaneously study the functions of several genes or networks, and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches. Soybean hairy root transformation is a useful tool for the analysis of gene function in soybean plants. The technology that are currently used, include CRISPR-Cas9 genome editing, RNA interference, transcriptomics and proteomics, and imaging technologies.The major challenge in the model era of functional genomics is sufficient analyzing the function of numerous genes. Most genes are polygenic, being achieved by the interaction of many genes. Our hairy root protocol will facilitate gene function analysis with sufficient throughput, so that polygenic networks can begin to be understood.We have established the importance of the Ti plasmid in agrobacterium rhizogene strains. We found that without screening for the Ti plasmid, it increased the risk for the transformed soybean cotyledon, to fail to produce any colors. As a result, screening for the plasmid became a vital step before the transformation.Compared to other gene expression techniques, our hair root expression system is easy to handle, less time consuming, and cost effective. Also, our new protocol has improved hairy root transformation rate up to 50%by checking for the Ti plasmid and agrobacterium prior to cotyledon transformation. By characterizing the function of more than a dozen transcription factor genes, our recent results have paved the way for multi-gene engineering studies, aimed at unlocking the biosynthesis of valuable phytoalexin metabolites in plants.In the future, we will use this hairy root transformation method to dissect the transcription factor gene networks that regulate the biosynthesis of phytoalexins. The approach could be used to enhance the production of these specialized metabolites for pharmaceutical and agricultural industries.

Watch this Scientific Journal Video about Infection of Cotyledons with Agrobacterium rhizogenes Strain NCPPB2659 K599, Selection, and Harvesting of Hairy Roots. at JoVE.com

Infection of Cotyledons with Agrobacterium rhizogenes Strain NCPPB2659 (K599), Selection, and Harvesting of Hairy Roots.  

Infektion av kotyledoner med Agrobacterium rhizogenes stam NCPPB2659 (K599), urval och skörd av håriga rötter.

För att infektera sojabönkotyledoner med Agrobacterium rhizogenes, designa primers för att detektera TI-plasmidgenen virD2 med hjälp av sekvensen av Agrobacterium rhizogenes PRI2659 plasmid. Efter transformation, testa Agrobacterium-kolonierna för retention av virD2 med PCR med hjälp av ett PCR-kit. Efter att ha valt kolonierna av Agrobacterium rhizogenes innehållande både virD2 och genen av intresse, stryk några kolonier på LB-plattorna innehållande 100 milligram per liter spektinomycin för plasmiden av intresse.Följande dag, med hjälp av en P200-pipettspets, skrapa bort en 1,5 centimeter lång av Agrobacterium från LB-plattan och resuspendera den i en milliliter fosfatbuffert. Späd den återsuspenderade cellsuspensionen och steriliserat ultrarent vatten i acetosyringone. Mät absorbansen med ett kyvettrör med en optisk densitet på 600 nanometer.Därefter, i ett biosäkerhetsskåp, doppa en steriliserad skalpell i Agrobacterium-lösningen och gör ett millimeter djupt snitt längs den inre ytan av cotyledon. Placera sex till åtta kotyledoner skurna nedåt på en petriskål som innehåller filterpapper mättat med groning och odlingsmedium med acetosyringon. Inkubera plattorna vid rumstemperatur i tre dagar under en 16 timmars fotoperiod.Efter tre dagar, överför de infekterade cotyledonerna till hårig rottillväxt eller HRG-plattor. Inkubera plattorna i tillväxtkamrarna inställda på 22 grader Celsius och en ljusintensitet på 100 mikromol under en 16 timmars fotoperiod tills primära rötter med sekundära rötter två till tre centimeter långa observeras. Efter tre till fyra veckor, skörda primära rötter som växer från callus och innehåller sekundära rötter med en steril skalpell och pincett.Överför rötterna till utvalda HRG-plattor som innehåller lämpliga antibiotika och låt dem växa i ytterligare fem dagar. På dag fem, skörda transgena håriga rötter med sekundära rötter som är tre till sex centimeter långa. Om du observerar fluorescerande proteiner, se till att sekundärrötterna har liten autofluorescens.Därefter, för att utföra elicitor eller kemiska behandlingar, skär sekundärrötterna i en centimeter bitar och placera cirka 100 milligram på HRG-agar i en hög. Mätt sedan högen med 80 mikroliter lämplig behandlingslösning och låt plattan inkubera vid rumstemperatur. Efter 24 timmar, för RNA-extraktion, torka snabbt rötterna torrt på en steriliserad pappershandduk och skörda dem direkt i ett två milliliter mikrocentrifugrör.Försegla omedelbart toppen av röret med parafilm och gör två små hål med spetsiga pincett. Kolonins PCR-resultat från den transformerade Agrobacterium visas. De positiva kolonierna i PCR indikerade genen av intresse.En tredjedel till hälften av kolonierna var dock negativa för virD2-genscreeningen. Fluorescensmikroskopi demonstrerar den subcellulära lokaliseringen av GFP-GmJAZ1-6. Genuttrycksanalys bekräftade överuttrycket av glyceollintranskriptionsfaktorn GmHSF6-1 och RNAi-tystning av GmMYB2912 i Williams 82 harryrötter.

Research

JoVE Journal

Bioengineering

Infection of Cotyledons with Agrobacterium rhizogenes Strain NCPPB2659 (K599), Selection, and Harvesting of Hairy Roots.

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 ...