Generation of Transgenic Soybean (Glycine max) via Particle Bombardment of Embryogenic Cultures

John J. Finer1

1 Department of Horticulture and Crop Science, OARDC/The Ohio State University, Wooster, Ohio
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20039
Online Posting Date:  December, 2016
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Abstract

This protocol describes one method for generating transgenic soybean (Glycine max) using particle bombardment of embryogenic cultures. Embryogenic cultures consist of proliferating masses of somatic embryos and are initially obtained from the cotyledons of immature seeds. Embryogenic cultures are bombarded with small DNA‐coated particles and the cells that receive, incorporate and express the DNA are selected, based on a co‐delivered antibiotic resistance gene. For recovery of whole plants from the antibiotic resistant embryogenic tissues, the tissues are first placed on a medium conducive to embryo development. Mature embryos are then dried for a short period and placed on a germination medium. Plantlets are then gradually exposed to ambient light and humidity conditions, prior to transfer to a greenhouse. Operators need to be constantly monitoring the protocol and observant of all outcomes. These procedures also are mostly descriptive and need to be precisely followed to be successful. © 2016 by John Wiley & Sons, Inc.

Keywords: soybean transformation; particle bombardment; embryogenic cultures; transgenic

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Soybean Transformation via Particle Bombardment of Embryogenic Tissues
  • Support Protocol 1: Use of the Particle Inflow Gun
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Soybean Transformation via Particle Bombardment of Embryogenic Tissues

  Materials
  • Flowering soybean plants, producing pods (e.g., cultivar “Jack”)
  • 10% (v/v) household bleach
  • Detergent (e.g., Tween‐20)
  • Autoclaved water
  • D20 medium (see recipe)
  • Hygromycin (Sigma‐Aldrich)
  • M6AC medium (see recipe)
  • OMS medium (see recipe)
  • Greenhouse
  • Laminar air flow hood containing stereo dissecting microscope
  • Sterilizing containers: 50‐ml screw‐top polypropylene centrifuge tubes or screw cap bottles
  • Forceps (200 mm, BDR232R, Aesculap, https://www.aesculapusa.com)
  • Scalpel with no. 11 surgical blade
  • Petri dishes (100‐mm × 15‐mm for most applications; 100‐mm × 25‐mm for embryo germination)
  • Lighted culture shelves in environmentally controlled growth room and/or lighted incubators (∼40 μE m−2 sec−1) at >16 hr day photoperiod, 23°C and 25°C
  • Saran wrap, cut into 1‐in. sections with ratcheting pipe cutter, to wrap around and seal petri dishes
  • Particle bombardment device and all components/consumables (see protocol 2Support Protocol)
  • Magenta GA7 containers (Sigma‐Aldrich)

Support Protocol 1: Use of the Particle Inflow Gun

  Materials
  • M10 (average size 1 μm) tungsten particles (Sylvania/LEDVANCE)
  • 95% (v/v) ethanol
  • Sterile water
  • Helium
  • DNA 2.5 M CaCl 2
  • 100 mM spermidine
  • Ice
  • Petri dish containing proliferative embryogenic tissues (see Basic Protocol 1)
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • Vortex mixer
  • Gene gun
  • Microcentrifuge tube rack
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Figures

Videos

Literature Cited

  Finer, J.J. 1988. Apical proliferation of embryogenic tissue of soybean [Glycine max (L.) Merrill]. Plant Cell Rep. 7:238‐241.doi: 10.1007/BF00272532.
  Finer, J.J. and McMullen, M.D. 1991. Transformation of soybean via particle bombardment of embryogenic suspension culture tissue. In Vitro Cell Dev. Biol. Plant 27P:175‐182. doi: 10.1007/BF02632213.
  Finer, J.J., Vain, P., Jones, M.W., and McMullen, M.D. 1992. Development of the Particle Inflow Gun for DNA delivery to plant cells. Plant Cell Rep. 11:232‐238.doi: 10.1007/BF00233358.
  Gamborg, O.L., Miller, R.A., and Ojima, K. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50:151‐158. doi: 10.1016/0014‐4827(68)90403‐5.
  Hadi, M.Z., McMullen, M.D., and Finer, J.J. 1996. Transformation of 12 different plasmids into soybean via particle bombardment. Plant Cell Rep. 15:500‐505.doi: 10.1007/BF00232982.
  Hinchee, M.A.W., Connor‐Ward, D.V., Newell, C.A., McDonnell, R.E., Sato, S.J., Gasser, C.S., Fischhoff, D.A., Re, D.B., Fraley, R.T., and Horsch, R.B. 1988. Production of transgenic soybean plants using Agrobacterium‐mediated DNA transfer. Bio/Technol. 6:915‐922. doi: 10.1038/nbt0888‐915.
  McCabe, D.E., Swain, W.F., Martinell, B.J., and Christou, P. 1988. Stable transformation of soybean (Glycine max) by particle acceleration. Bio/Technol. 6:923‐926. doi: 10.1038/nbt0888‐923.
  Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Pl. 15:473‐497. doi: 10.1111/j.1399‐3054.1962.tb08052.x.
  Santarém, E.R. and Finer, J.J. 1999. Transformation of soybean (Glycine max (L.) Merrill) using proliferative embryogenic tissue maintained on semi‐solid medium. In Vitro Cell Dev. Biol. Plant 35:451‐455.doi: 10.1007/s11627‐999‐0067‐0.
  Trick, H.N. and Finer, J.J. 1998.Sonication‐assisted Agrobacterium‐mediated transformation of soybean (Glycine max [L.]Merrill) embryogenic suspension culture tissue. Plant Cell Rep. 17:482‐488.doi: 10.1007/s002990050429.
  Vain P., Keen N, Murillo J., Rathus C., Nemes C., and Finer, J.J. 1993. Development of the Particle Inflow Gun. Plant Cell Tiss. Org. Cult. 33:237‐246. doi: 10.1007/BF02319007.
Key References
  Santarém and Finer, 1999. See above.
  This protocol was developed by Santarém and Finer, 1999 (see above), which is a more user‐friendly modification of an earlier method described in Finer and McMullen, 1991 (see above).
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