Soybean Hairy Root Transformation for the Analysis of Gene Function

Summary

Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

Abstract

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.

Introduction

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....

Protocol

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.......

Discussion

During the past decade, the soybean HR method has been developed as a powerful tool to study genes involved in nitrogen fixation^22,23, biotic and abiotic stress tolerance^24,25, and metabolite biosynthetic pathways^26,27. The knowledge of how plants produce metabolites has a plethora of benefits for agricultural production and the pharmaceutical ind.......

Materials

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