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pORE: a modular binary vector series suited for both monocot and dicot plant transformation

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Abstract

We present a series of 14 binary vectors suitable for Agrobacterium-mediated transformation of dicotyledonous plants and adaptable for biolistic transformation of monocotyledonous plants. The vector size has been minimized by eliminating all non-essential elements from the vector backbone and T-DNA regions while maintaining the ability to replicate independently. The smallest of the vector series is 6.3 kb and possesses an extensive multiple cloning site with 21 unique restriction endonuclease sites that are compatible with common cloning, protein expression, yeast two-hybrid and other binary vectors. The T-DNA region was engineered using a synthetic designer oligonucleotide resulting in an entirely modular system whereby any vector element can be independently exchanged. The high copy number ColE1 origin of replication has been included to enhance plasmid yield in Escherichia coli. FRT recombination sites flank the selectable marker cassette regions and allow for in planta excision by FLP recombinase. The pORE series consists of three basic types; an ‘open’ set for general plant transformation, a ‘reporter’ set for promoter analysis and an ‘expression’ set for constitutive expression of transgenes. The sets comprise various combinations of promoters (P HPL, P ENTCUP2 and P TAPADH), selectable markers (nptII and pat) and reporter genes (gusA and smgfp).

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Abbreviations

bp:

Base pair

DNA:

Deoxyribonucleic acid

dsDNA:

Double stranded DNA

gusA :

β-Glucuronidase

LB:

Left T-DNA border

MCS:

Multiple cloning site

nptII:

Neomycin phosphotransferase II

OxOx:

Oxalate oxidase

pat :

Phosphinothricin acetyltransferase

P ENTCUP2 :

Tobacco cryptic constitutive promoter

PCR:

Polymerase chain reaction

P HPL :

Arabidopsis thaliana hydroperoxide lyase promoter

P TAP :

Triticum aestivum lipid transfer protein promoter

P TAPADH :

Triticum aestivum lipid transfer protein promoter fused to an alcohol dehydrogenase intron

RB:

Right T-DNA border

SDM:

Site directed mutagenesis

smgfp :

soluble modified green fluorescent protein

T-DNA:

Transferred DNA

Ti:

Tumour inducing

T NOS :

Polyadenylation signal from the nopaline synthase gene

Vir :

Virulence region of the Ti plasmid

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Acknowledgments

This research was made possible with funds from the Agriculture and Agri-Food Canada Crop Genomics Initiative

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Authors and Affiliations

  1. Agriculture and Agrifood Canada, Saskatoon Research Centre, 107 Science Place, Saskatoon, SK, Canada, S7N 0X2

    Catherine Coutu & Dwayne D. Hegedus

  2. Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Center, 1391 Sandford Street, London, ON, Canada, N5V 4T3

    James Brandle, Dan Brown & Kirk Brown

  3. Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Centre, K.W. Neatby Building, 960 Carling Ave, Ottawa, ON, Canada, K1A 0C6

    Brian Miki & John Simmonds

Authors
  1. Catherine Coutu
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  2. James Brandle
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  3. Dan Brown
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  4. Kirk Brown
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  5. Brian Miki
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  6. John Simmonds
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  7. Dwayne D. Hegedus
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Correspondence to Dwayne D. Hegedus.

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Coutu, C., Brandle, J., Brown, D. et al. pORE: a modular binary vector series suited for both monocot and dicot plant transformation. Transgenic Res 16, 771–781 (2007). https://doi.org/10.1007/s11248-007-9066-2

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  • DOI: https://doi.org/10.1007/s11248-007-9066-2

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