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Biology

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

Published: June 30th, 2023

DOI:

10.3791/65688

1College of Agriculture, Liaocheng University, 2College of Life Science, Liaocheng University
* These authors contributed equally

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 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.

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 ....

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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.......

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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

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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.......

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

....

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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

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