BiBAC Modification and Stable Transfer into Maize (Zea mays) Hi‐II Immature Embryos via Agrobacterium‐Mediated Transformation

Jon P. Cody1, Nathaniel D. Graham1, James A. Birchler1

1 Division of Biological Sciences, Tucker Hall, University of Missouri, Columbia, Missouri
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20061
Online Posting Date:  December, 2017
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Abstract

Binary Bacterial Artificial Chromosomes (BiBAC) are large insert cloning vectors that contain the necessary features required for Agrobacterium‐mediated transformation. However, the large size of BiBACs and low‐copy number in Escherichia coli (DH10B) and Agrobacterium tumefaciens make cloning experiments more difficult than other available binary vector systems. Therefore, a protocol that outlines preparation, modification, and transformation of high‐molecular weight (HMW) constructs is advantageous for researchers looking to use BiBACs in plant genomics research. This unit does not cover the cloning of HMW DNA into BiBAC vectors. Researchers looking to clone HMW DNA into BiBACs can refer to Zhang et al. (2012; doi: 10.1038/nprot.2011.456). © 2017 by John Wiley & Sons, Inc.

Keywords: Agrobacterium tumefaciens; BiBAC; genetic engineering; transgenes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: High‐Yield Purification of BiBAC DNA from Escherichia coli
  • Basic Protocol 2: Modification of BiBAC Constructs
  • Basic Protocol 3: Transformation into Agrobacterium Cells
  • Basic Protocol 4: Transformation of BIBAC Constructs into Immature Maize Embryos
  • Support Protocol 1: Producing HI‐II Immature Maize Embryos
  • Support Protocol 2: Preparation of Agrobacterium
  • Support Protocol 3: Preparation of Phi–B Cocultivation Medium
  • Reagents and Solutions
  • Commentary
  • Literature cited
  • Figures
     
 
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Materials

Basic Protocol 1: High‐Yield Purification of BiBAC DNA from Escherichia coli

  Materials
  • −80°C E. coli BiBAC freezer stock (CCTEC; http://www.ctl.cornell.edu)
  • 150 × 15–cm LB agar plates with appropriate antibiotics
  • Appropriate antibiotics in sterile ddH 2O
  • LB medium (see recipe)
  • 500 ml of autoclaved LB medium in 1000‐ml Erlenmeyer flask
  • Nucleobond Xtra BAC Kit (Macherey‐Nagel, cat. no. 740436.10)
  • Centrifuge
  • Inoculation loops (Fisherbrand, cat. no. 22363602)
  • Shaking Incubator: setting 37°C; 250 rpm/min
  • 17 × 100–mm polystyrene culture test tubes (Thermo Fisher Scientific, cat. no. 14‐956‐6D)
  • Electronic pipette
  • Incubator: setting 37°C
  • Aluminum foil

Basic Protocol 2: Modification of BiBAC Constructs

  Materials
  • Purified BiBAC vector ( protocol 1)
  • Sterile ddH 2O
  • Antarctic Phosphatase (New England Biolabs, cat no. M0289S)
  • Restriction enzymes to generate complementary overhangs between vector and insert (New England Biolabs, Sigma Aldrich, Thermo Fisher Scientific, etc.)
  • Insert DNA suspended in sterile H 2O
  • T4 DNA ligase (New England Biolabs, cat no. M0202S)
  • Electrocompetent DH10B cells (Thermo Fisher Scientific, cat. no 18297010)
  • Insulated bucket filled with ice
  • SOC medium (Thermo Fisher Scientific, cat. no. 15544034)
  • 150 × 15–cm LB agar plates with appropriate antibiotics
  • 0.6‐ml microcentrifuge tubes
  • Micropipettes
  • Incubator: set to 37°C
  • 80°C heat block
  • Electroporator
  • Electroporation cuvettes
  • 17 × 100–mm polystyrene culture tubes (Thermo Fisher Scientific, cat. no. 14‐956‐6D)
  • Shaking incubator: set to 37°C
  • Additional reagents and equipment for ethanol precipitation (Green and Sambrook, )

Basic Protocol 3: Transformation into Agrobacterium Cells

  Materials
  • Electrocompetent Agrobacterium tumefaciens EHA105 cells (Hood, Gelvin, Melchers, & Hoekema, )
  • Ice
  • Plasmid pCH32 (Cornell CCTEC: ctl.cornell.edu)
  • SOC medium (Thermo Fisher Scientific, cat. no. 15544034)
  • LB plates containing 5 mg/ml tetracycline and 25 mg/ml rifampicin
  • LB growth medium (Thermo Fisher Scientific, cat. no. BP9723‐2)
  • Sterile autoclaved water
  • BiBAC construct DNA ( protocol 2)
  • Sterile inoculation loops
  • Electroporation cuvettes
  • Pipettes
  • Electroporator
  • 17 × 100–mm polystyrene culture tubes (Thermo Fisher Scientific, cat. no. 14‐956‐6D)
  • 28°C shaker
  • 28°C incubator
  • 2‐ml tubes
  • Centrifuge

Basic Protocol 4: Transformation of BIBAC Constructs into Immature Maize Embryos

  Materials
  • Hi‐II A × B ear(s) ( protocol 5 should be started 9 to 12 days prior to infection)
  • Bleach
  • 2 × 1.8 liters of autoclaved H 2O in 2‐liter Erlenmeyer flask
  • TWEEN 20
  • Phi–A infection medium (see recipe)
  • Agrobacterium infection medium (see protocol 6)
  • Phi–B cocultivation medium plates (see protocol 7 and recipe)
  • Phi–C callus induction medium plates (see recipe)
  • Phi–D callus selection medium plates (see recipe)
  • Phi–F root regeneration medium tubes (see recipe)
  • Soil
  • Phi–E shoot regeneration medium plates (see recipe)
  • Sterile laminar flow hood
  • Forceps, sterile
  • Spectrophotometer
  • 1000‐ to 2000‐ml glass beakers
  • #11 scalpel blade (Feather, cat. no. 504170)
  • Spatula, sterile
  • 1.7‐ml microcentrifuge tubes
  • Micropipettes
  • Parafilm
  • Incubator
  • Growth chamber #1: Set day to 6 am, 24°C, and 2/3 strength light; set night to 12 am, 24°C, and lights off
  • Growth chamber #2: set day to 6 am, 24°C, and high light; set night to 12 am, 24°, and lights off
  • 2‐gallon pots

Support Protocol 1: Producing HI‐II Immature Maize Embryos

  Materials
  • Hi‐II A seed
  • Hi‐II B seed
  • Osmocote Plus 16‐9‐12 slow release fertilizer (ICL‐SF, product code A903206)
  • Peter's Professional 20‐20‐20 fertilizer (ICL‐SF, product code G99290)
  • Iron chelate fertilizer (Carl Pool)
  • Small germination pots
  • 2‐gallon pots

Support Protocol 2: Preparation of Agrobacterium

  Additional Materials (also see protocol 4)
  • BiBAC Agrobacterium stock
  • 150 × 15–cm LB agar plates with appropriate antibiotics
  • Phi–A infection medium (see recipe)
  • Acetosyringone stock (see recipe)
  • N6 vitamin stock (see recipe)
  • Inoculation loop
  • Spectrophotometer
  • Shaker set to 120 rpm

Support Protocol 3: Preparation of Phi–B Cocultivation Medium

  Materials
  • 250 ml aliquot of Phi–B cocultivation medium (see recipe)
  • N6 vitamin stock (see recipe)
  • Silver nitrate stock (see recipe)
  • Acetosyringone stock (see recipe)
  • Microwave
  • Sterile laminar flow hood
  • Micropipettes
  • 100 × 15–cm polystyrene plates
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Figures

Videos

Literature Cited

Literature cited
  Armstrong, C. L., Green, C. E., & Phillips, R. L. (1991). Development and availability of germplasm with high Type II culture formation response. Maize Genetics Cooperation Newsletter, 92–93.
  Frary, A., & Hamilton, C. M. (2001). Efficiency and stability of high molecular weight DNA transformation: An analysis in tomato. Transgenic Research, 10, 121–132. doi: 10.1023/A:1008924726270.
  Green, M. R., & Sambrook, J. (2012). Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Laboratory Press.
  Hamilton, C. M., Frary, a, Lewis, C., & Tanksley, S. D. (1996). Stable transfer of intact high molecular weight DNA into plant chromosomes. Proceedings of the National Academy of Sciences of the United States of America, 93, 9975–9979. doi: 10.1073/pnas.93.18.9975.
  Hamilton, C. M. (1997). A binary‐BAC system for plant transformation with high‐molecular‐weight DNA. Gene, 200, 107–116. doi: 10.1016/S0378‐1119(97)00388‐0.
  Hood, E., Gelvin, S., Melchers, L., & Hoekema, A. (1993). New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Research, 2, 208–218. doi: 10.1007/BF01977351.
  Shizuya, H., Birren, B., Kim, U. J., Mancino, V., Slepak, T., Tachiiri, Y., & Simon, M. (1992). Cloning and stable maintenance of 300‐kilobase‐pair fragments of human DNA in Escherichia coli using an F‐factor‐based vector. Proceedings of the National Academy of Sciences of the United States of America, 89, 8794–8797. doi: 10.1073/pnas.89.18.8794.
  Sinnett, D., & Montpetit, A. (2003). Isolation of Cosmid and BAC DNA from E. coli. Methods in Molecular Biology, 235, 99–102. doi: 10.1385/1‐59259‐409‐3:99.
  Vega, J. M., Yu, W., Kennon, A. R., Chen, X., & Zhang, Z. J. (2008). Improvement of Agrobacterium‐mediated transformation in Hi‐II maize (Zea mays) using standard binary vectors. Plant Cell Reports, 27, 297–305. doi: 10.1007/s00299‐007‐0463‐z.
  Zhang, H., Scheuring, C. F., Zhang, M., Zhang, Y., Wu, C., Dong, J. J., & Li, Y. (2012). Construction of BiBAC and BAC Libraries form a Variety of Organisms for Advanced Genomics Research. Nature Protocols, 7, 479–499. doi: 10.1038/nprot.2011.456.
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