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Soybean Hairy Root Transformation for the Analysis of Gene Function

Published: May 5th, 2023



1Department of Biology, York University, 2Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology

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 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.

Soybean (Glycine max) is among the most valuable crops in agriculture. It has thousands of commercial and industrial uses, such as food, animal feed, oil, and as a source of raw materials for manufacturing1. Its ability to form a symbiotic relationship with nitrogen-fixing soil microorganisms, namely rhizobia, further elevates the importance of studying soybean genetics2. For instance, fine-tuning nitrogen fixation properties in soybean roots can lead to the reduction of carbon emissions and greatly reduce requirements for nitrogen fertilizer3. Thus, understanding the genetics that contro....

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NOTE: It is recommended that all of the proceeding steps be conducted under sterile conditions.

1. Soybean seed sterilization

  1. In a biosafety cabinet, place 16-20 round Williams 82 soybean seeds in pristine condition (i.e., no cracks or blemishes) in a 50 mL centrifuge tube.
  2. Add 30 mL of 70% isopropyl alcohol, gently shake for 30 s, and then decant the alcohol.
  3. Gently shake the seeds with 30 mL of 10% bleach for 10 s and allow the seeds to sit in.......

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The representative results are from the published data19,20. The colony PCR (cPCR) results of the transformed K599 Agrobacterium are shown in Figure 1. As indicated by the positive colonies in Figure 1, the gene of interest was detected by cPCR (Figure 1A). However, one-third to one-half of the colonies were negative for the VirD2 gene screening (

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During the past decade, the soybean HR method has been developed as a powerful tool to study genes involved in nitrogen fixation22,23, biotic and abiotic stress tolerance24,25, and metabolite biosynthetic pathways26,27. The knowledge of how plants produce metabolites has a plethora of benefits for agricultural production and the pharmaceutical ind.......

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This research was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) grant number RGPIN-2020-06111 and by a generous donation from Brad Lace. We would like to thank Wayne Parrott (University of Georgia) for the K599 Agrobacterium and the preliminary protocol, and the Nakagawa & Hachiya lab (Shimane University) for the pGWB2, pGWB6, and pANDA35HK empty vectors.


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Name Company Catalog Number Comments
Acetosyringone Cayman 23224
Bleach lavo 21124
DMSO Fisher bioreagents 195679
Gelzan Phytotech HYY3251089A
Hygromycin Phytotech HHA0397050B
Isopropyl alcohol Fisher chemical 206462
Kanamycin Phytotech SQS0378007G
LB powder Fisher bioreagents 200318
MS powder Caisson labs 2210001
Na2HPO4 Fisher bioreagents 194171
NaCl Fisher chemical 192946
Petri dishes Fisherbrand 08-757-11 100 mm x 25 mm
Phosphinothricin Cedarlane P034-250MG
REDExtract-N-Amp PCR Kit Sigma R4775
Sucrose Bioshop 2D76475
Timentin Caisson labs 12222002
Vitamins Caisson labs 2211010

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