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

High-Efficiency Production of Transgenic Soybean Hairy Roots

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.

Author Spotlight: Soybean Hairy Root Transformation for the Analysis of Gene Function  

Soybean Hairy Root Transformation for the Analysis of Gene Function

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.  

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

To infect soybean cotyledons with Agrobacterium rhizogenes, design primers to detect the TI plasmid gene virD2 using the sequence of the Agrobacterium rhizogenes PRI2659 plasmid. Following transformation, test the Agrobacterium colonies for the retention of virD2 by PCR using a PCR kit. After selecting the Agrobacterium rhizogenes colonies containing both virD2 and the gene of interest, streak some colonies onto the LB plates containing 100 milligrams per liter of spectinomycin for the plasmid of interest.The following day, using a P200 pipette tip, scrape off a 1.5 centimeter length of the Agrobacterium from the LB plate and resuspend it in one milliliter of phosphate buffer. Dilute the resuspended cell suspension and sterilized ultrapure water in acetosyringone. Measure the absorbance using a cuvette tube at an optical density of 600 nanometers.Next, in a biosafety cabinet, dip a sterilized scalpel in the Agrobacterium solution and make a one millimeter deep cut along the inner surface of the cotyledon. Place six to eight cotyledons cut side down on a Petri dish containing filter paper saturated with germination and cultivation medium with acetosyringone. Incubate the plates at room temperature for three days under a 16 hour photo period.After three days, transfer the infected cotyledons to hairy root growth, or HRG plates. Incubate the plates in the growth chambers set to 22 degrees Celsius and a light intensity of 100 micromoles on a 16 hour photo period until primary roots with secondary roots two to three centimeters in length are observed. After three to four weeks, harvest primary roots that grow from the callus and contain secondary roots using a sterile scalpel and forceps.Transfer the roots to selection HRG plates containing appropriate antibiotics and allow them to grow for an additional five days. On day five, harvest transgenic hairy roots with secondary roots that are three to six centimeters in length. If observing fluorescent proteins, ensure that the secondary roots have little autofluorescence.Next, to perform elicitor or chemical treatments, cut the secondary roots into one centimeter pieces and place approximately 100 milligrams on HRG agar in a pile. Then saturate the pile with 80 microliters of the appropriate treatment solution and allow the plate to incubate at room temperature. After 24 hours, for RNA extraction, rapidly dab the roots dry on a sterilized paper towel and harvest them directly into a two milliliter microcentrifuge tube.Immediately seal the top of the tube using parafilm and make two small holes using pointed forceps. The colony PCR results of the transformed Agrobacterium are shown. The positive colonies in PCR indicated the gene of interest.However, one third to one half of the colonies were negative for the virD2 gene screening. Fluorescence microscopy demonstrates the subcellular localization of GFP-GmJAZ1-6. Gene expression analysis confirmed the overexpression of the glyceollin transcription factor GmHSF6-1 and RNAi silencing of GmMYB2912 in Williams 82 harry roots.

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Research

JoVE Journal

Bioengineering

295 vistas.  York University. Para infectar cotiledones de soja con Agrobacterium rhizogenes, diseñe cebadores para detectar el gen del plásmido TI virD2 utilizando la secuencia del plásmido Agrobacterium rhizogenes PRI2659 plásmido. Después de la transformación, pruebe las colonias de Agrobacterium para la retención de virD2 por PCR utilizando un kit de PCR. Después de seleccionar las colonias de Agrobacterium rhizogenes que contienen tanto virD2 como el gen de interés, raye algunas colonias en las placas LB que...