Agrobacterium‐Mediated Transformation of Oilseed Rape (Brassica napus)

Ruth Bates1, Melanie Craze1, Emma J. Wallington1

1 The John Bingham Laboratory, National Institute of Agricultural Botany, Cambridge
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20060
Online Posting Date:  December, 2017
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Oilseed rape (Brassica napus) is a commercially important member of the Brassicacea family. It is grown for its edible and industrial oils as well as for animal feed. Genetic transformation technology has been used to study gene function and produce oilseed rape with improved agronomic characteristics. This protocol describes a method for the Agrobacterium tumefaciens–mediated transformation of oilseed rape cotyledonary petioles. The method is reproducible and has been used to transform both spring and winter cultivars. Modifications have been made to the rooting stage, which have reduced the vitrification of shoots. This has not only increased the number of phenotypically normal shoots but has also resulted in an increase in transformation efficiency, concomitant with a dramatic reduction in the number of escapes regenerated. Transformation frequencies typically range from 5% to 10%, with an average of 12% using doubled haploid model varieties, but a maximum efficiency of 20% has been achieved. © 2017 by John Wiley & Sons, Inc.

Keywords: agrobacterium‐mediated transformation; cotyledonary petioles; oilseed rape

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Table of Contents

  • Introduction
  • Basic Protocol 1: Agrobacterium‐ Mediated Transformation of Oilseed Rape Cotyledonary Petioles
  • Support Protocol 1: Preparation of Agrobacterium Stock Cultures Harboring Binary Plasmid Constructs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Agrobacterium‐ Mediated Transformation of Oilseed Rape Cotyledonary Petioles

  • Oilseed rape seed [e.g., doubled haploid Westar derivatives DH12075 (AAFC, Agriculture and AgriFood Canada) or BGRV4 (Biogemma SAS), etc.]
  • 70% (v/v) ethanol solution
  • 1–5% (v/v) sodium hypochlorite
  • Tween 20
  • Sterile, de‐ionized water
  • R1 germination medium (see recipe)
  • Agrobacterium tumefaciens strain harboring constructs (see protocol 2Support Protocol)
  • LB plates (see recipe)
  • R2 co‐cultivation medium (see recipe)
  • R3 selection medium (see recipe)
  • R4 selection medium (see recipe)
  • TRM rooting medium with selection (see recipe)
  • M2 Compost (Levingtons)
  • Osmocote Exact standard slow release fertilizer 5 to 6 M (ICL)
  • Dymax 5 “air tweezers” (Charles Austen Pumps Ltd.)
  • Tea infuser
  • Sterilized 250‐ml round glass Beatson jars (Richardsons of Leicester)
  • 9‐cm sterile plastic petri dishes
  • 1‐cm and 2.5‐cm medical sealing tape (e.g., Micropore)
  • Growth chamber, 25.5°C day, 23.5°C night, 16‐hr photoperiod, 80 to 100 μmol/m2/sec (MLR‐351, Sanyo)
  • Culture incubator, 28°C
  • Scalpel with no. 11 blades (e.g., Swann‐ Morton, Sheffield)
  • 100 × 20 mm sterile untreated deep plastic Petri dishes (e.g., Corning)
  • 100‐ml metal lidded containers (e.g., Sterilin)
  • 5‐cm diameter, single vent, 18 mm deep petri dish, (e.g., Sterilin)
  • 42‐mm Jiffy‐7 peat pellets, rehydrated with 50 ml/Jiffy tap water (Jiffy Products)
  • Magenta GA‐7 Vessel and lid (Sigma Aldrich)
  • Growth chamber, 20°C day, 15°C night, 16‐hr photoperiod, 400 to 450 μmol/m2/sec (PGR15, Conviron)
  • 9‐cm plant pots
  • Plant trays lined with Aqua mat capillary matting (LBS Horticulture)
  • Canes for support
  • Plant ties
  • Micro perforated bread bags (Starlight Packaging/Dlan Ltd.)

Support Protocol 1: Preparation of Agrobacterium Stock Cultures Harboring Binary Plasmid Constructs

  • Agrobacterium tumefaciens strain (e.g., Agl1, LBA4404 or C58pMP90)
  • Binary plasmid construct containing the nptII selectable marker cassette and gene(s) of interest
  • DYT (see recipe)
  • Sterile 10% (v/v) glycerol
  • SOC medium (see recipe)
  • Wizard Plus SV Minipreps DNA Purification System (Promega Corporation), containing:
  • Resuspension solution
  • Lysis solution
  • Alkaline protease solution
  • Neutralization solution
  • Spin column
  • 2‐ml collection tube
  • Wash solution
  • 20 mg/ml lysozyme (freshly made)
  • TE buffer, pH 8.0 (see recipe)
  • 3 M sodium acetate, pH 5.2
  • Propan‐2‐ol
  • LB/15% (v/v) glycerol (see recipe)
  • Sterile 30‐ml universal tubes (Sterilin)
  • Sterilized 250‐ml conical flask
  • Shaking incubator 28°C
  • Spectrophotometer
  • Sterile 50‐ml conical centrifuge tubes
  • Refrigerated benchtop centrifuge
  • BioRad MicroPulser
  • 0.1‐cm electroporation cuvettes (BioRad or Sigma Aldrich)
  • 9‐cm petri dishes
  • 2‐ml cryotubes (Nunc)
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Literature Cited

Literature Cited
  Braatz, J., Harloff, H. J., Mascher, M., Stein, N., Himmelbach, A., & Jung, C. (2017). CRISPR‐Cas9 induced mutations in polyploid oilseed rape. Plant Physiology, 174(2):935‐942. doi: 10.1104/pp.17.00426.
  De Block, M., De Brouwer, D., & Tenning, P. (1989). Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in the transgenic plants. Plant Physiology, 91(2), 694–701. doi: 10.1104/pp.91.2.694.
  Figurski, D. H., & Helinski, D. R. (1979). Replication of an origin‐containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proceedings of the National Academy of Sciences, 76(4), 1648–1652. doi: 10.1073/pnas.76.4.1648.
  Fry, J., Barnason, A., & Horsch, R. B. (1987). Transformation of Brassica napus with Agrobacterium tumefaciens based vectors. Plant Cell Reports, 6(5), 321–325. doi: 10.1007/BF00269550.
  Guerche, P., Jouanin, L., Tepfer, D., & Pelletier, G. (1987). Genetic transformation of oilseed rape (Brassica napus) by the Ri T‐DNA of Agrobacterium rhizogenes and analysis of inheritance of the transformed phenotype. Molecular and General Genetics MGG, 206(3), 382–386. doi: 10.1007/BF00428875.
  Moloney, M. M., Walker, J. M., & Sharma, K. K. (1989). High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Reports, 8(4), 238–242. doi: 10.1007/BF00778542.
  Pechan, P. M. (1989). Successful cocultivation of Brassica napus microspores and proembryos with Agrobacterium. Plant Cell Reports, 8(7), 387–390. doi: 10.1007/BF00270075.
  Poulsen, G. B. (1996). Genetic transformation of Brassica. Plant Breeding, 115(4), 209–225. doi: 10.1111/j.1439‐0523.1996.tb00907.x.
  Radke, S. E., Andrews, B. M., Moloney, M. M., Crouch, M. L., Kridl, J. C., & Knauf, V. C. (1988). Transformation of Brassica napus L. using Agrobacterium tumefaciens: Developmentally regulated expression of a reintroduced napin gene. TAG Theoretical and Applied Genetics, 75(5), 685–694. doi: 10.1007/BF00265588.
  Rupert, R. A., & Bottino, P. J. (1999) Promega Notes 70, 23–24.
  Tang, G. X., Knecht, K., Yang, X. F., Qin, Y. B., Zhou, W. J., & Cai, D. (2011). A two‐step protocol for shoot regeneration from hypocotyl explants of oilseed rape and its application for Agrobacterium‐mediated transformation. Biologia Plantarum, 55(1), 21–26. doi: 10.1007/s10535‐011‐0003‐0.
  Van Haute, E., Joos, H., Maes, M., Warren, G., Van Montagu, M., & Schell, J. (1983). Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: A novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. The EMBO Journal, 2(3), 411‐7.
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