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

Summary

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

Abstract

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 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.

Introduction

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 ....

Protocol

1. Plant growth conditions and A. rhizogenes culture

1. 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.
   1. 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 ± 2 °C with a 16 h light/8 h dark cycle at approximately 70% relative humidity.
      NOTE: Coats of wild soybe....

Results

Highly efficient one-step A. rhizogenes-mediated 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,.......

Discussion

The one-step A. rhizogenes-mediated 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
kanamycin	Sangon Biotech (Shanghai) Co., Ltd.	A506636
LB medium	Sangon Biotech (Shanghai) Co., Ltd.	B540113
plastic box	LiaoSu		8 cm x 11 cm x 9 cm
pumpkin			local variety Yinsu
streptomycin	Sangon Biotech (Shanghai) Co., Ltd.	A610494
Tanon-5200Multi machine	Tanon Co., Ltd., China	5200Multi	chemiluminescence imaging system
tomato			local variety Zhongshu4
wild soybean			collected in Yanggu County, Liaocheng, China