Generation of Soybean (Glycine max) Transient Transgenic Roots

Katalin Tóth1, Josef Batek1, Gary Stacey2

1 University of Missouri, Division of Plant Sciences, Columbia, Missouri, 2 University of Missouri, Divisions of Plant Sciences and Biochemistry, National Center for Soybean Biotechnology, Columbia, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20017
Online Posting Date:  May, 2016
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Abstract

Legumes—because of their nitrogen‐fixing capacity—have both ecological and agronomic importance, and are also the major plant protein source for animal consumption. The model legume species are Lotus japonicus, Medicago truncatula, and soybean (Glycine max). These species have sequenced genomes and are amenable to genetic manipulation, as well as to various functional genomic and cell biology approaches. Plant transformation mediated by Agrobacterium is one of the most powerful methods in plant biotechnology. Using the traditional Agrobacterium tumefaciens method, stable transgenic plants take 6 to 12 months to create, depending on species. Besides being time consuming, this approach is often quite laborious. Hence, there is a need for more rapid methods to create transgenic tissues. In the case of roots, this can be done using hairy root transformation mediated by Agrobacterium rhizogenes. This protocol describes a method to generate transgenic soybean roots in as little as 3 weeks. © 2016 by John Wiley & Sons, Inc.

Keywords: legume; soybean; agrobacterium‐mediated transformation; root

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Soybean Agrobacterium rhizogenes–Mediated Hairy Root Transformation
  • Basic Protocol 2: Nodulation Assays
  • Support Protocol 1: Agrobacterium Culture Preparation
  • Support Protocol 2: Preparation of Pouches with Solid and Liquid Fahraeus Medium
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Soybean Agrobacterium rhizogenes–Mediated Hairy Root Transformation

  Materials
  • Soybean seeds
  • 10% (v/v) household bleach
  • 0.03 N HCl: mix ∼825 μl concentrated HCl with 1 liter sterile distilled H 2O
  • Ethanol
  • Plate containing “lawn” of agrobacteria ( protocol 3)
  • Square Fahraeus plates (see recipe)
  • Pouches containing Fahraeus medium with 1% agar, 1 mM CaCl 2, and 1 to 2 mM KNO 3 ( protocol 4)
  • Pouches containing liquid Fahraeus medium with 1 mM CaCl 2 and 1mM KNO 3 ( protocol 4)
  • Fahraeus medium (see recipe) with 1 mM CaCl 2 and 1mM KNO 3 for replenishing pouches
  • Germination paper (Anchor Seed Germination Paper, ∼38 × 25–cm; Anchor Paper Co.)
  • Sterile glass and plastic beakers
  • Plastic wrap
  • Aluminum foil
  • Conviron Growth Chamber PGR15: settings 26°C/16 hr light and 22°C/8 hr dark, 80% humidity (used for seed germination)
  • Growth chamber, settings 21°C 16 hr/light, 8 hr/dark; humidity not controllable (Percival Scientific; used after transformation)
  • Square Petri dish (120 × 120 × 17, with vents; Greiner Bio‐One, cat. no. 688102)
  • Forceps, scalpel, razor blades
  • Aluminum foil
  • Stereomicroscope

Basic Protocol 2: Nodulation Assays

  Materials
  • Plant examined for transgenic roots ( protocol 1)
  • Fahraeus medium (see recipe)
  • 3:1 vermiculite:perlite, autoclaved
  • Bradyrhizobium japonicum USDA110 (USDA‐ARS culture collection, NRRL B‐4361)
  • Liquid HM (HEPES‐MES) medium (see recipe)
  • Planting pots (∼8.5 cm × 8.5 cm; Hummert International)
  • Sorvall RC5C Plus centrifuge with fixed‐angle F‐13S rotor (or equivalent centrifuge/rotor combination)
  • Greenhouse (30°C/16 hr light; 26°C/8 hr dark) or in walk‐in growth chamber (25°C/16 hr light; 22°C/8 hr dark)

Support Protocol 1: Agrobacterium Culture Preparation

  Materials
  • A. rhizogenes K599: National Collection of Plant Pathogenic Bacteria (UK), NCPPB No. 2659), carrying the construct of interest
  • Luria broth (LB) medium and agar plates (Elbing and Brent, )
  • Appropriate selection antibiotic
  • Bacterial spreader

Support Protocol 2: Preparation of Pouches with Solid and Liquid Fahraeus Medium

  Materials
  • Fahraeus medium with and without 1% agar (see recipe)
  • Germination pouch (cyg seed germination pouch; Mega International)
  • Potassium nitrate (KNO 3)
  • Calcium chloride (CaCl 2)
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Figures

Videos

Literature Cited

Literature Cited
   Collier, R. , Fuchs, B. , Walter, N. , Lutke, W.K. , and Taylor, C.G. 2005. Ex vitro composite plants: An inexpensive, rapid method for root biology. Plant J. 43: 449‐457.
   Elbing, K. and Brent, R. 2002. Media preparation and bacteriological tools. Curr. Protoc. Mol. Biol. 59:1.1.1‐1.1.7.
   Fahraeus, G. 1957. The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J. Gen. Microbiol. 16:374‐381.
   Gelvin, S.B. 2003. Agrobacterium‐mediated plant transformation: The biology behind the “Gene‐jockeying” tool. Microbiol. Mol. Biol. Rev. 67:16‐37.
   Kandoth, P.K. , Heinz, R. , Yeckel, G. , Gross, N.W. , Juvale, P.S. , Hill, J. , Whitham, S.A. , Baum, T.J. , and Mitchum, M.G. 2013. A virus‐induced gene silencing method to study soybean cyst nematode parasitism in Glycine max . BMC Res. Notes 6:255.
   Kereszt, A. , Li, D. , Indrasumunar, A. , Nguyen, C.D. , Nontachaiyapoom, S. , Kinkema, M. , and Gresshoff, P.M. 2007. Agrobacterium rhizogenes–mediated transformation of soybean to study root biology. Nat. Protoc. 2:948‐952.
   Mankin, S.L. , Hill, D.S. , Olhoft, P.M. , Toren, E. , Wenck, A.R. , Nea, L. , Xing, L. , Brown, J.A. , Fu, H. , Ireland, L. , Jia, H. , Hillebrand, H. , Jones, T. , and Song, H.S. 2007. Disarming and sequencing of Agrobacterium rhizogenes strain K599 (NCPPB2659) plasmid pRi2659. In Vitro Cell. Dev. Biol. Plant 43:521‐535.
   Márquez, A.J. , Stougaard, J. , Udvardi, M. , Parniske, M. , Spaink, H. , Saalbach, G. , Webb, J. , Chiurazzi, M. , and Márquez, A.J. (eds.) 2005. Lotus japonicus Handbook, XXII, Springer, New York.
   Mathesius, U. , Journet, E.P. , and Sumner, L.W. (eds.) 2006. The Medicago truncatula Handbook. The Samuel Roberts Noble Foundation. Available at http://www.noble.org/MedicagoHandbook/.
   Nagel, R. , Elliott, A. , Masel, A. , Birch, R.G. , and Manners, J.M. 1990. Electroporation of binary Ti plasmid vector into Agrobacterium tumefaciens and Agrobacterium rhizogenes . FEMS Microbiol. Lett. 67:325‐328.
   Runo, S. , Macharia, S. , Alakonya, A. , Machuka, J. , Sinha, N. and Scholes, J. 2012. Striga parasitizes transgenic hairy roots of Zea mays and provides a tool for studying plant‐plant interactions. Plant Methods 8:20.
Key Reference
   Gelvin , 2003. See above.
  An excellent review on Agrobacterium‐mediated transformation that introduces different species of Agrobacteria, the mechanism of T‐DNA transfer, manipulation of Agrobacterium T‐DNA and its integration into plant genome, and detection of T‐DNA in the plant genome.
Internet Resources
   http://www.noble.org/MedicagoHandbook
  Medicago truncatula handbook (free access).
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