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...

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

<ol>
	<li>Chilton, M. 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. Application in Lotus corniculatus transformation.</a> Methods in Molecular Biology. 49, 49-61 (1995).</li><li>Kereszt, 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=Agrobacterium+rhizogenes-mediated+transformation+of+soybean+to+study+root+biology.">Agrobacterium rhizogenes-mediated transformation of soybean to study root biology.</a> Nature Protocols. 2 (4), 948-952 (2007).</li><li>Ho-Pl&#225;garo, T., Huertas, R., Tamayo-Navarrete, M. I., Ocampo, J. A., Garc&#237;a-Garrido, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+Agrobacterium+rhizogenes-mediated+transformation+of+tomato+suitable+for+the+study+of+arbuscular+mycorrhizal+symbiosis.">An improved method for Agrobacterium rhizogenes-mediated transformation of tomato suitable for the study of arbuscular mycorrhizal symbiosis.</a> Plant Methods. 14, 34 (2018).</li><li>Yu, 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=Overexpression+of+phosphatidylserine+synthase+IbPSS1+affords+cellular+Na++homeostasis+and+salt+tolerance+by+activating+plasma+membrane+Na+/H++antiport+activity+in+sweet+potato+roots.">Overexpression of phosphatidylserine synthase IbPSS1 affords cellular Na+ homeostasis and salt tolerance by activating plasma membrane Na+/H+ antiport activity in sweet potato roots.</a> Horticulture Research. 7, 131 (2020).</li><li>Boisson-Dernier, 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=Agrobacterium+rhizogenes-transformed+roots+of+Medicago+truncatula+for+the+study+of+nitrogen-fixing+and+endomycorrhizal+symbiotic+associations.">Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations.</a> Molecular Plant-Microbe Interactions: MPMI. 14 (6), 695-700 (2001).</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=One-step+generation+of+composite+soybean+plants+with+transgenic+roots+by+Agrobacterium+rhizogenes-mediated+transformation.">One-step generation of composite soybean plants with transgenic roots by Agrobacterium rhizogenes-mediated transformation.</a> BMC Plant Biology. 20 (1), 208 (2020).</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=Anthocyanin,+a+novel+and+user-friendly+reporter+for+convenient,+non-destructive,+low+cost,+directly+visual+selection+of+transgenic+hairy+roots+in+the+study+of+rhizobia-legume+symbiosis.">Anthocyanin, a novel and user-friendly reporter for convenient, non-destructive, low cost, directly visual selection of transgenic hairy roots in the study of rhizobia-legume symbiosis.</a> Plant Methods. 16, 94 (2020).</li><li>Wang, X., 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=Application+of+AtMYB75+as+a+reporter+gene+in+the+study+of+symbiosis+between+tomato+and+Funneliformis+mosseae.">Application of AtMYB75 as a reporter gene in the study of symbiosis between tomato and Funneliformis mosseae.</a> Mycorrhiza. 33 (3), 181-185 (2023).</li><li>Wang, X., 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=Development+of+a+set+of+novel+binary+expression+vectors+for+plant+gene+function+analysis+and+genetic+transformation.">Development of a set of novel binary expression vectors for plant gene function analysis and genetic transformation.</a> Frontiers in Plant Science. 13, 1104905 (2023).</li><li>Li, Q. Q., 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=Phytochrome+B+inhibits+darkness-induced+hypocotyl+adventitious+root+formation+by+stabilizing+IAA14+and+suppressing+ARF7+and+ARF19.">Phytochrome B inhibits darkness-induced hypocotyl adventitious root formation by stabilizing IAA14 and suppressing ARF7 and ARF19.</a> The Plant Journal: For Cell and Molecular Biology. 105 (6), 1689-1702 (2021).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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.

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

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

Our team focuses on Agrobacterium rhizogenes-based hairy root transformation. Hairy root transformation is crucial in studying plant gene function, metabolic engineering, and interactions between roots and Rhizosphere microorganisms. This article provides a one-step transformation method, mediated by Agrobacterium rhizogenes.Hairy root transformation is established in various dicotyledons and monocotyledons species, and is almost independent with the genotype. The one-step method mediated by Agrobacterium rhizogenes for inducing hairy roots is simpler and more efficient for creating composite plants compared to the hypocotyl injection method. The hypocotyl injection method is inefficient and is likely to cause the death of young or tiny hypocotyl plants.The one-step Agrobacterium rhizogenes-mediated method is highly efficient and requires less time to produce hairy roots. Moreover, there is no need for transplanting past the formation of hairy roots. Alpha-sign regulation genes and beta-sign synthesis genes were used as viral screen markers in hairy root transformation mediated by Agrobacterium rhizogenes.

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...

Watch this Scientific Journal Video about Streamlining Composite Plant Production via Agrobacterium rhizogenes-Mediated Hairy Root Transformation at JoVE.com

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 ...

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Biology

Production of Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

Production of Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation  

Begin by collecting seeds of wild soybean, S.Americanum, and the local variety of pumpkin. Sow the seeds in vermiculite at a depth of one centimeter and water them thoroughly. Cultivate them in a growth chamber at approximately 70%relative humidity.Remove the A.rhizogenes strain K599 from the 80 degrees Celsius freezer and activate it on a solid LB medium at 28 degrees Celsius for 48 hours. Then select a single clone of strain K599 and culture it in one milliliter of liquid antibiotic containing LB medium for 12 hours. Spread 500 microliters of the bacterial suspension evenly on a solid antibiotic containing LB medium and incubate at 28 degrees Celsius for 24 hours.After seven days, when seedling cotyledons are unfolded, use a sterilized sharp scalpel to cut approximately 0.5 to one centimeter of the hypocotyl. Discard the primary root and a portion of the hypocotyl. Coat the hypocotyl incision with K599 bacteria.Plant seedlings in moist vermiculite and infect 30 plants of each species using A.rhizogenes-mediated hairy root transformation. Then water the plants with five milliliters of resuspended K599 bacterial suspension in quarter-strength Gamborg B5 basic medium. Cover the pots with a transparent plastic bag and place them in a growth chamber.Grow the plants for approximately 12 days post-inoculation. At this time, new hairy roots should be generated at the incision site, typically reaching lengths of two to five centimeters. Using a CAMI luminescence imaging system with green excitation light at 540 nanometers and emission at 600 nanometers, detect the expression of the reporter gene DsRed2.Roots of composite plants of wild soybean, S.Americanum, and pumpkin under natural and green excitation light are visualized.

1.4K Views.  Liaocheng University.  Here, we provide the detailed protocol of a one-step transformation method mediated by Agrobacterium tumefaciens to produce composite plants.

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

Summary