This research was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) grant number RGPIN-2020-06111 and by a generous donation from Brad Lace. We would like to thank Wayne Parrott (University of Georgia) for the K599 Agrobacterium and the preliminary protocol, and the Nakagawa &#38; Hachiya lab (Shimane University) for the pGWB2, pGWB6, and pANDA35HK empty vectors.

NOTE: It is recommended that all of the proceeding steps be conducted under sterile conditions.
1. Soybean seed sterilization
<ol>
	<li>In a biosafety cabinet, place 16-20 round Williams 82 soybean seeds in pristine condition (i.e., no cracks or blemishes) in a 50 mL centrifuge tube.</li>
	<li>Add 30 mL of 70% isopropyl alcohol, gently shake for 30 s, and then decant the alcohol.</li>
	<li>Gently shake the seeds with 30 mL of 10% bleach for 10 s and allow the seeds to sit in the solution for 5 min at room temperature (RT, 25 &#176;C). After 5 min, drain the bleach.</li>
	<li>Repeat the shaking three times with 30 mL of sterile ultrapure H2O for 1 min per rinse and discard the H2O between each rinse.</li>
	<li>Place the sterilized seeds on filter paper saturated with 5 mL of germination and co-cultivation (GC) medium (autoclave half-strength liquid Murashige and Skoog [MS] medium with 1% sucrose [pH = 5.8] and then add 2.5 mL/L vitamins) in sterile Petri dishes.</li>
	<li>Place the plates in the dark at RT (25 &#176;C) for 3 days before transferring the plates at 22 &#176;C under 16 h cool-white T5 fluorescent lights (100 &#181;E m-2&#183;s-1) for 4 days to allow the seeds to germinate. 
	&#8203;NOTE: Discard seeds that are wrinkled or cracked. The best seeds are those that are large and uniformly yellow. Sterilize at least double the number of seeds required for the experiment to select seeds of good quality, as some seeds may not be optimal due to damage, diseases, or failure to germinate.</li>
</ol>
2. Infection of cotyledons with K599
NOTE: Use pGWB series vectors, as their dual selection ensures genomic integration of the entire T-DNA cassette. Electroporation was used to transform a binary vector harboring the gene of interest into A. rhizogenes pRi265918.
<ol>
	<li>Based on the sequence of the A. rhizogenes pRi2659 plasmid18, design primers to detect the Ti plasmid gene VirD2 (VirD2 forward: 5&#39;-CCCGATCGAGCTCAAGTTAT-3&#39;; VirD2 reverse: 5&#39;-TCGTCTGGCTGACTTTCGT-3&#39;; expected amplification size: 221 bp). Following transformation, test the Agrobacterium colonies for the retention of VirD2 by polymerase chain reaction (PCR) using a PCR Kit (see Table of Materials). PCR cycle: 94 &#176;C for 3 min; 35 cycles (94 &#176;C for 1 min, 58 &#176;C for 30 sec, 72 &#176;C for 1 min); and 72 &#176;C for 10 min.</li>
	<li>Following the selection of the A. rhizogenes colony that contains both VirD2 and the gene of interest (GOI), streak out some&#160;onto Luria-Bertani (LB) medium plates containing the appropriate antibiotics (50 mg/L) for the plasmid of interest and incubate overnight at 30 &#176;C. If using spectinomycin, add a concentration of 100 mg/L.</li>
	<li>Use a P200 pipette tip to scrape off a ~1.5 cm length of the K599 Agrobacterium from the LB plates and resuspend the cells in 1 mL of phosphate buffer (PB; 0.01 M Na2HPO4, 0.15 M NaCl, pH 7.5).</li>
	<li>Dilute the Agrobacterium in sterilized, ultrapure H2O (v/v = 1:1) and acetosyringone (AS; 100 mM stock in dimethyl sulfoxide [DMSO], v/v = 1:1000). Measure the absorbance using a cuvette tube at an optical density of 600 nm (OD600). The expected optimal range is between 0.5 and 0.8.</li>
	<li>In a biosafety cabinet, dip a sterilized scalpel in the K599 Agrobacterium solution and make several 1 mm deep cuts along the inner surface (adaxial, flat side) of the cotyledon. During the inoculation, use sterilized forceps to stabilize the cotyledon.</li>
	<li>Place about 6-8 cotyledons cut side-down on a Petri dish containing filter paper saturated with GC medium with AS. 
	NOTE: To prepare 6 plates, 50 mL of GC medium and 50 &#181;L of 100 mM AS to achieve a final concentration of 100 &#181;M is sufficient.</li>
	<li>Incubate the plates at RT (25 &#176;C) for 3 days under a 16 h photoperiod (~65 &#181;E).</li>
	<li>Transfer the infected cotyledons to hairy root growth (HRG) plates (autoclave half-strength MS with 3% sucrose [pH = 5.8] and 2.6 g/L Gelzan, then add 2.5 mL/L vitamins mixture and 500 mg/L timentin). 
	NOTE: There were some issues with timentin from some alternative vendors, of which the K599 was not eliminated. It is recommended to use taller Petri dish plates (100 mm x 25 mm) for HRG medium.</li>
	<li>Incubate the HRG plates at 22 &#176;C in a growth chamber with the parameters set to 100 &#181;E light on a 16 h photoperiod until primary roots with secondary roots 2-3 cm in length are observed (~3-4 weeks).</li>
</ol>
3. Selection and harvesting of HRs
<ol>
	<li>Harvest primary roots (5-7 cm in length) that grow from the callus and contain secondary roots (2-3 cm in length) using a sterile scalpel and forceps. Transfer to selection HRG plates containing the appropriate antibiotics. Let the HRs grow for 5 more days on the selection HRG plates. 
	NOTE: Kanamycin (50 mg/L), hygromycin (50 mg/L), or phosphinothricin (10 mg/L) are typically used for selection. For expression vectors, pGWB2 is used for overexpression, pANDA35HK is used for RNAi silencing, pGWB6 is used for subcellular localization, and pMDC7 is used for inducible expression.</li>
	<li>On day 5, harvest transgenic HRs with secondary roots 3-6 cm in length. If observing fluorescent proteins, the secondary roots have little autofluorescence. If performing elicitor or chemical treatments, cut the secondary roots into 1 cm pieces and place ~100 mg on HRG agar in a pile. Then, saturate the piles with 80 &#181;L of the appropriate treatment and allow the plates to incubate at RT (25 &#176;C).</li>
	<li>After the desired treatment time, proceed with RNA or metabolite extractions.</li>
	<li>For RNA, rapidly dab the roots dry on a sterilized paper towel and harvest them directly into a 2 mL microcentrifuge tube.</li>
	<li>Immediately seal the top of the tube using parafilm, make two small holes using pointed forceps, and submerge the tubes in liquid nitrogen. Lyophilize for 3 days, then store the samples at -80 &#176;C. 
	NOTE: It is essential to select HRs that are white. After 5 days of growth on the selective plate, transgenic HRs will remain white, but non-transgenic roots will turn brown.</li>
</ol>

High-Efficiency Production of Transgenic Soybean Hairy Roots

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The authors have nothing to disclose.

During the past decade, the soybean HR method has been developed as a powerful tool to study genes involved in nitrogen fixation22,23, biotic and abiotic stress tolerance24,25, and metabolite biosynthetic pathways26,27. The knowledge of how plants produce metabolites has a plethora of benefits for agricultural production and the pharmaceutical ind...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetosyringone</td>
			<td>Cayman</td>
			<td>23224</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>lavo</td>
			<td>21124</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher bioreagents</td>
			<td>195679</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelzan</td>
			<td>Phytotech</td>
			<td>HYY3251089A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin</td>
			<td>Phytotech</td>
			<td>HHA0397050B</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>Fisher chemical</td>
			<td>206462</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Phytotech</td>
			<td>SQS0378007G</td>
			<td></td>
		</tr>
		<tr>
			<td>LB powder</td>
			<td>Fisher bioreagents</td>
			<td>200318</td>
			<td></td>
		</tr>
		<tr>
			<td>MS powder</td>
			<td>Caisson labs</td>
			<td>2210001</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Fisher bioreagents</td>
			<td>194171</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher chemical</td>
			<td>192946</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisherbrand</td>
			<td>08-757-11</td>
			<td>100 mm x 25 mm</td>
		</tr>
		<tr>
			<td>Phosphinothricin</td>
			<td>Cedarlane</td>
			<td>P034-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>REDExtract-N-Amp PCR Kit</td>
			<td>Sigma</td>
			<td>R4775</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Bioshop</td>
			<td>2D76475</td>
			<td></td>
		</tr>
		<tr>
			<td>Timentin</td>
			<td>Caisson labs</td>
			<td>12222002</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamins</td>
			<td>Caisson labs</td>
			<td>2211010</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

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.

The representative results are from the published data19,20. The colony PCR (cPCR) results of the transformed K599 Agrobacterium are shown in Figure 1. As indicated by the positive colonies in Figure 1, the gene of interest was detected by cPCR (Figure 1A). However, one-third to one-half of the colonies were negative for the VirD2 gene screening (<strong cl...

Watch this Scientific Journal Video about Soybean Hairy Root Transformation for the Analysis of Gene Function at JoVE.com

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 ...

soybean-hairy-root-transformation-for-the-analysis-of-gene-function

Research

JoVE Journal

Bioengineering

Soybean Seed Sterilization

To begin, place 16 to 20 pristine round Williams 82 soybean seeds in a 50 milliliter centrifuge tube inside a biosafety cabinet. Add 30 milliliters of 70%isopropyl alcohol to the tube and shake for 30 seconds. Decant the alcohol.Next, gently shake the seeds with 30 milliliters of 10%bleach for 10 seconds, and allow the seeds to sit in the solution for 5 minutes at room temperature. After 5 minutes, drain the bleach then rinse the seeds three times with 30 milliliters of sterile ultrapure water for 1 minute per rinse and discard the water between each rinse. Place the sterilized seeds on filter paper saturated with 5 milliliters of germination and co-cultivation medium in a sterile petri dish.Place the plates in the dark at room temperature for 3 days before transferring them at 22 degrees Celsius under 16 hour cool white T5 fluorescent lights for 4 days to allow the seeds to germinate.

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.

2.3K Views.  York University.  Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

High-Efficiency Production of Transgenic Soybean Hairy Roots

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