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><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>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</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.

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

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

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

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

Research

JoVE Journal

Bioengineering

3.4K Views.  York University. Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

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

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience 1, 2. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces 3. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence2, 4.
The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads 2. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration 5. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum)

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant secondary metabolites such as flavonoids, especially rutin. Agrobacterium rhizogenes have been gradually used worldwide to induce hairy roots in medicinal plants to investigate gene functions and increase the yield of secondary metabolites. In this study, we have described a detailed method to generate A. rhizogenes-mediated hairy roots in TB. Cotyledons and hypocotyledonary axis at 7&#8211;10 days were selected as explants and infected with A. rhizogenes carrying a binary vector, which induced adventitious hairy roots that appeared after 1 week. The generated hairy root transformation was identified based on morphology, resistance selection (kanamycin), and reporter gene expression (green fluorescent protein). Subsequently, the transformed hairy roots were self-propagated as required. Meanwhile, a myeloblastosis (MYB) transcription factor, FtMYB116, was transformed into the TB genome using the A. rhizogenes-mediated hairy roots to verify the role of FtMYB116 in synthesizing flavonoids. The results showed that the expression of flavonoid-related genes and the yield of flavonoid compounds (rutin and quercetin) were significantly (p &#60; 0.01) promoted by FtMYB116, indicating that A. rhizogenes-mediated hairy roots can be used as an effective alternative tool to investigate gene functions and the production of secondary metabolites. The detailed step-by-step protocol described in this study for generating hairy roots can be adopted for any genetic transformation or other medicinal plants after adjustment.

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat Fagopyrum tataricum

We describe a method of inducing hairy roots by Agrobacterium rhizogenes-mediated transformation in Tartary buckwheat (Fagopyrum tataricum). This can be used to investigate gene functions and production of secondary metabolites in Tartary buckwheat, be adopted for any genetic transformation, or used for other medicinal plants after improvement.

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant ...

Genetics

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Here we present an efficient and stable transformation system for the functional analysis of the CcCIPK14 gene as an example, providing a technical basis for studying the metabolism of non-model plants.

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an ...

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter &#946;-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates &#8805;2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

Promoter expression analyses are crucial to improving the understanding of gene regulation and the spatiotemporal expression of target genes. Herein we present a protocol to identify, isolate, and clone a plant promoter. Further, we describe the characterization of the nodule-specific promoter in the common bean hairy roots.

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...

Biology

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

One-Step Hairy Root Transformation Method for Advancing Root-Related Biology

An Efficient and Reproducible Method for Producing Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a powerful tool to study root-related biology. Hairy root transformation is established in a wide range of dicotyledons and in several monocotyledon species and is almost independent of the genotype. The traditional method of hypocotyl injection with A. rhizogenes to obtain composite plants is inefficient, time-consuming, laborious, and frequently causes the death of tender and tiny hypocotyl plants. A highly efficient, one-step hairy root transformation mediated by A. rhizogenes was established previously, which eliminates the need for transplanting after producing hairy roots. In this study, a partial hypocotyl and primary root were removed, the hypocotyl incision site was coated with A. rhizogenes, and&#160;then hypocotyls were planted in sterile vermiculite. After 12 days of cultivation, the hypocotyl incision expanded and new hairy roots were induced. This article provides the detailed protocol of a one-step transformation method mediated by A. rhizogenes, with its effectiveness demonstrated by producing composite plants of wild soybean, Solanum americanum, and pumpkin.

Author Spotlight: Streamlining Composite Plant Production via Agrobacterium rhizogenes-Mediated Hairy Root Transformation 

Here, we provide the detailed protocol of a one-step transformation method mediated by Agrobacterium tumefaciens to produce composite plants.

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a ...

Agrobacterium-Mediated Hairy Root Induction and Regeneration in Plants

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus

Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a Root-inducing (Ri) plasmid induces the formation of hairy roots at the wounding site after the transfer of T-DNA from the Ri plasmid into the plant genome. The protocol describes in detail the procedure of the injection-based hairy root induction in Brassica napus DH12075 and Arabidopsis thaliana Col-0. The hairy roots may be used to analyze a transgene of interest or processed for the generation of transgenic plants. Regeneration medium containing cytokinin 6-benzylaminopurine (5 mg/L) and auxin 1-naphthaleneacetic acid (8 mg/L) successfully elicits shoot formation in both species. The protocol covers the genotyping and selection of regenerants and T1 plants to obtain plants carrying a transgene of interest and free of T-DNA from the Ri plasmid. An alternative process leading to the formation of a composite plant is also depicted. In this case, hairy roots are kept on the shoot (instead of the natural roots), which enables the study of a transgene in hairy root cultures in the context of the whole plant.

Author Spotlight: Optimizing Hairy Root-Based Transformation Protocols for Enhanced Efficiency in Brassicaceae

The protocol describes hairy root induction using Arabidopsis primary inflorescence stems and Brassica napus hypocotyls. The hairy roots can be cultured and used as explants to regenerate transgenic plants.

Soybean Hairy Root Transformation for the Analysis of Gene Function

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Chapters in this video

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum)

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

An Efficient and Reproducible Method for Producing Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus