This work was supported by the Research Fund of Liaocheng University (318012028) and the Natural Science Foundation of Shandong Province (ZR2020MC034).

1. Plant growth conditions and A. rhizogenes culture
<ol>
	<li>Seed sowing 
	NOTE: Wild soybean seeds were collected in Yanggu County, Liaocheng, China; seeds of S. americanum and pumpkin local variety Yinsu were purchased from a market.
	<ol>
		<li>Collect seeds of wild soybean, S. americanum, and the local variety of pumpkin, sow them in vermiculite at a depth of 1 cm, and thoroughly water them. Plant 20 seeds in 8 cm x 11 cm x 9 cm plastic boxes. Cultivate the plants in a growth chamber at 24 &#177; 2 &#176;C with a 16 h light/8 h dark cycle at approximately 70% relative humidity. 
		NOTE: Coats of wild soybean seeds must be broken before sowing. If the research question focuses on interactions between plants and microorganisms, seeds, vermiculite, plastic boxes, and water must be sterilized before use.</li>
		<li>Activation and culture of A. rhizogenes K599 
		NOTE: The A. rhizogenes K599 strain had one binary vector pRed13052 carrying a red fluorescent reporter gene DsRed2.
		<ol>
			<li>Remove the A. rhizogenes strain K599 from a -80 &#176;C freezer, and activate the bacteria on solid LB medium (with 50 mg/L kanamycin and 50 mg/L streptomycin) at 28 &#176;C for 48 h.</li>
			<li>Pick out a single clone of strain K599 and culture it in 1 mL of liquid antibiotic-containing LB medium for 12 h.</li>
			<li>Evenly spread 500 &#181;L of the bacterial suspension on solid antibiotic-containing LB medium, followed by incubation at 28 &#176;C for 24 h.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. One-step A. rhizogenes-mediated hairy root transformation method
<ol>
	<li>Hypocotyl incision
	<ol>
		<li>After 7 days (define the sowing of the seeds as day 0), seedling cotyledons have just unfolded (Figure 1A), use a sterilized, sharp scalpel to cut approximately 0.5-1 cm of the hypocotyl (Figure 1B). Discard the primary root and some of the partial hypocotyl. 
		NOTE: Use the scalpel with care.</li>
	</ol>
	</li>
	<li>K599 inoculation
	<ol>
		<li>Coat the hypocotyl incision with K599 bacteria (Figure 1C).</li>
		<li>Plant the seedlings in moist vermiculite (Figure 1D).</li>
		<li>Infect 30 plants of each species via A. rhizogenes-mediated hairy root transformation. Water each plant with 5 mL of resuspended K599 bacterial suspension (OD600 = 0.5-0.6) in a quarter strength (0.25x) Gamborg B-5 basic medium (Figure 1E).</li>
		<li>Cover the pots with a highly transparent plastic bag (Figure 1F) and place them in a growth chamber.</li>
	</ol>
	</li>
</ol>
3. Hairy root production
<ol>
	<li>Grow the plants for ~12 days post inoculation, new hairy roots will be induced and generated at the incision site. After 15 days, the hairy root lengths typically reach 2-5 cm (Figure 2).</li>
	<li>To determine whether the hairy roots produced are transgenic, examine the expression of reporter genes dependent on the transformed vector in K599.
	<ol>
		<li>Detect the expression of the reporter gene DsRed2 using a chemiluminescence imaging system with green excitation light at 540 nm and emission at 600 nm (Figure 2B, Figure 2D, and Figure 2F).</li>
	</ol>
	</li>
</ol>

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

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium+rhizogenes+inserts+T-DNA+into+the+genome+of+the+host+plant+root+cells.">Agrobacterium rhizogenes inserts T-DNA into the genome of the host plant root cells.</a> Nature. 295, 432-434 (1982).</li><li>Fan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fast,+simple,+high+efficient+and+one-step+generation+of+composite+cucumber+plants+with+transgenic+roots+by+Agrobacterium+rhizogenes-mediated+transformation.">A fast, simple, high efficient and one-step generation of composite cucumber plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation.</a> Plant Cell, Tissue and Organ Culture (PCTOC). 141, 207-216 (2020).</li><li>Du, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeted+mutagenesis+in+soybean+by+TALENs+and+CRISPR/Cas9.">Efficient targeted mutagenesis in soybean by TALENs and CRISPR/Cas9.</a> Journal of Biotechnology. 217, 90-97 (2016).</li><li>Nguyen, D. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+hairy+root+system+for+validation+of+plant+transformation+vector+and+CRISPR/Cas+construct+activities+in+cucumber+(Cucumis+sativus+L.).">An efficient hairy root system for validation of plant transformation vector and CRISPR/Cas construct activities in cucumber (Cucumis sativus L.).</a> Frontiers in Plant Science. 12, 770062 (2022).</li><li>Liu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtGCS+promoter-driven+clustered+regularly+interspaced+short+palindromic+repeats/Cas9+highly+efficiently+generates+homozygous/biallelic+mutations+in+the+transformed+roots+by+Agrobacterium+rhizogenes-mediated+transformation.">AtGCS promoter-driven clustered regularly interspaced short palindromic repeats/Cas9 highly efficiently generates homozygous/biallelic mutations in the transformed roots by Agrobacterium rhizogenes-mediated transformation.</a> Frontiers in Plant Science. 13, 952428 (2022).</li><li>Irigoyen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+hairy+roots+enable+high+throughput+identification+of+antimicrobials+against+Candidatus+Liberibacter+spp.">Plant hairy roots enable high throughput identification of antimicrobials against Candidatus Liberibacter spp.</a> Nature Communications. 11 (1), 5802 (2020).</li><li>Plasencia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eucalyptus+hairy+roots,+a+fast,+efficient+and+versatile+tool+to+explore+function+and+expression+of+genes+involved+in+wood+formation.">Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation.</a> Plant Biotechnology Journal. 14 (6), 1381-1393 (2016).</li><li>Gutierrez-Valdes, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hairy+root+cultures-a+versatile+tool+with+multiple+applications.">Hairy root cultures-a versatile tool with multiple applications.</a> Frontiers in Plant Science. 11, 33 (2020).</li><li>Stougaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium+rhizogenes+as+a+vector+for+transforming+higher+plants.+Application+in+Lotus+corniculatus+transformation.">Agrobacterium rhizogenes as a vector for transforming higher plants. 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The authors have no conflicts of interest to declare.

The one-step A. rhizogenes-mediated&#160;hairy root method is a simpler and more efficient method for producing composite plants than the hypocotyl-injection method. The one-step ARM method significantly improves the efficiency of hairy root transformation, shortens the time to produce hairy roots, increases the number of hairy roots, and reduces the amount of work involved. The improved transformation protocol is optimal for studies on symbioses between leguminous plants and rhizobia and between plants and arbu...

Agrobacterium rhizogenes is a gram-negative soil bacterium from the family Rhizobiaceae. A. rhizogenes can infect almost all dicotyledons, a few monocotyledons, and individual gymnosperms through wounds, producing hairy roots in infected plants. The bacterium carries the Ri (root-inducing) plasmid, and the T-DNA of the Ri plasmid carries the opine synthesis gene and rol genes (root locus genes). After the T-DNA of the Ri plasmid enters a plant cell and is integrated into a host chromosome, the expression of the rol genes induces production of hairy roots1. A plant binary expression vector carrying a target gene is transformed into A. rhizogenes, and the transformed A. rhizogenes is used to infect a plant. Transgenic roots can be induced in infected plants, producing composite plants containing transgenic roots and nontransgenic stems and buds. Generally, a composite plant can be obtained within 14-20 days. A. rhizogenes-mediated hairy root transformation is generally not limited by genotype in dicotyledonous plants2. The hairy roots produced by A. rhizogenes-infected plants are characterized by a fast growth rate, stable inheritance, and easy operation. Hairy root transformation mediated by A. rhizogenes is currently widely used to study root-related biology. Furthermore, the transformation of hairy roots can also be used to validate and optimize the target-editing efficiency of the CRISPR/Cas9 system3,4,5 and protein subcellular localization. Therefore, hairy root transformation is an important tool in research on plant gene function, metabolic engineering, and interactions between roots and rhizosphere microorganisms6,7,8.
Composite plants containing transgenic roots obtained through hairy root transformation have been widely produced in dicotyledonous plants, especially in legumes. The traditional method of injecting the hypocotyl with A. rhizogenes has been used to produce composite Lotus corniculatus9, soybeans10, tomato11, sweet potatoes12,&#160;and many other plants5,8. The hypocotyl-injection method is inefficient and is likely to cause the death of young or tiny hypocotyl plants. Therefore, the method was improved by cutting off the embryonic roots, coating the seedling incision with A. rhizogenes, and then placing the hypocotyl on sterile culture medium for rooting cultivation13. However, those steps are performed in a sterile environment, and the operation steps are relatively cumbersome. In particular, the resulting composite plants need to be transplanted, which increases the amount of work. In previous work, one-step A. rhizogenes-mediated (ARM) hairy root transformation was established in cucumber, soybean, Lotus japonicus, Medicago truncatula,&#160;and tomato2,14,15,16,17. The primary root and partial hypocotyl were removed, the incision site of the remaining hypocotyl was coated with transformed A. rhizogenes, and the seedling was then planted into moist sterile vermiculite. After 12 days of cultivation, hairy roots were produced at the incision site. The one-step ARM method is highly efficient and requires less time to produce hairy roots. Transplanting after forming hairy roots is also not necessary. Because microbial contamination can be avoided without transplantation, the one-step ARM method can be particularly useful when studying interactions between plants and microorganisms, such as symbiotic nitrogen fixation between leguminous plants and Rhizobia, and symbioses between plants and arbuscular mycorrhizal fungi. In this paper, a detailed one-step A. rhizogenes-mediated&#160;hairy root transformation protocol is provided with examples of composite plants produced in wild soybean, Solanum americanum, and pumpkin. With the protocol, researchers can smoothly perform the one-step ARM transformation.

<table><tbody><tr><td>kanamycin</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A506636</td><td></td></tr><tr><td>LB medium</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>B540113</td><td></td></tr><tr><td>plastic box</td><td>LiaoSu</td><td></td><td>8 cm x 11 cm x 9 cm</td></tr><tr><td>pumpkin</td><td></td><td></td><td>local variety Yinsu</td></tr><tr><td>streptomycin</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A610494&amp;nbsp;</td><td></td></tr><tr><td>Tanon-5200Multi machine</td><td>Tanon Co., Ltd., China</td><td>5200Multi</td><td>chemiluminescence imaging system</td></tr><tr><td>tomato</td><td></td><td></td><td>local variety Zhongshu4</td></tr><tr><td>wild soybean</td><td></td><td></td><td>collected in Yanggu County, Liaocheng, China</td></tr></tbody></table>

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.

Highly efficient one-step A. rhizogenes-mediated&#160;hairy root transformation 
Hairy roots were produced at the hypocotyl incision site 12 days after inoculation with engineered K599. Transgenic hairy roots were determined based on the expression of the reporter gene contained in the binary vector. Transgenic roots transformed with the reporter gene DsRed2 of composite wild soybean, S. americanum, and pumpkin were observed under natural (Figure 2A</s...

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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.

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 ...

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Biology

2.0K Views.  Liaocheng University. Here, we provide the detailed protocol of a one-step transformation method mediated by Agrobacterium tumefaciens to produce composite plants.

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

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

Environment

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 ...

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1. Due to its capacity to undergo efficient mitotic homologous recombination, Physcomitrella patens has emerged as an important model system in recent years2. This capacity allows high frequencies of gene targeting3-9, which is not seen in other model plants such as Arabidopsis. To take full advantage of this system, we need an effective and easy method to deliver DNA into moss cells. The most common ways to transform this moss are particle bombardment10 and PEG-mediated DNA uptake11. Although particle bombardment can produce a high transformation efficiency12, gene guns are not readily available to many laboratories and the protocol is difficult to standardize. On the other hand, PEG mediated transformation does not require specialized equipments, and can be performed in any laboratory with a sterile hood. Here, we show a simple and highly efficient method for transformation of moss protoplasts. This method can generate more than 120 transient transformants per microgram of DNA, which is an improvement from the most efficient protocol previously reported13. Because of its simplicity, efficiency, and reproducibility, this method can be applied to projects requiring large number of transformants as well as for routine transformation.

Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1.

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella ...

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula.

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

We demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species Medicago truncatula.

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

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 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  

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 ...

Bioengineering

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

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.

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

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Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus