Name: Streamlining Composite Plant Production via Agrobacterium rhizogenes-Mediated Hairy Root Transformation (Video) | JoVE
Uploaded: 2023-06-30T14:20:00
Description: Watch this Scientific Journal Video about Streamlining Composite Plant Production via Agrobacterium rhizogenes-Mediated Hairy Root Transformation at JoVE.com

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

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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.

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

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

Streamlining Composite Plant Production via Agrobacterium rhizogenes-Mediated Hairy Root Transformation (Video) | JoVE

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 ...