DP-915635-4 - Borer-resistant, herbicide-tolerant maize | BCH-LMO-SCBD-260914 | Living Modified Organism | Biosafety Clearing-House


Living Modified Organism (LMO)

Decisions on the LMO Risk Assessments  
last updated: 04 Jul 2022
Living Modified Organism identity
The image below identifies the LMO through its unique identifier, trade name and a link to this page of the BCH. Click on it to download a larger image on your computer. For help on how to use it go to the LMO quick-links page.
Borer-resistant, herbicide-tolerant maize
The maize was modified through a site-specific transformation protocol for insect resistance and herbicide tolerance. For resistance to Western corn rootworm (Diabrotica virgifera), the maize expresses Ophioderma pendulum insecticidal protein IPD079Ea, which has a pore-forming mode of action against feeding larvae. The protein binds receptors in the insect's midgut which are different from the receptors that Bt toxins interact with, allowing the maize to overcome Bt-resistance in Coleoptera pests. For tolerance to glufosinate, the maize expresses Streptomyces viridochromogenes phosphinothricin N-acetyltransferase, which inactivates the herbicidal compound through acetylation. In addition, the maize also contains an Escherichia coli phosphomannose isomerase cassette, which allows for modified plants to use mannose as a carbon source and thus is a selectable marker during transformation. The transformation protocol involved two steps to achieve a site specific integration into the maize genome. In the first step, four plasmids were introduced by microparticle bombardment, which introduced recombination sites at a specific location using transiently expressed CRISPR/Cas9. In the second step, Agrobacterium-mediated transformation was used to introduce the final gene cassettes through a recombination with sequences present in the first insertion site. More information regarding the two-step site-specific transformation is provided below.
The term “Recipient organism” refers to an organism (either already modified or non-modified) that was subjected to genetic modification, whereas “Parental organisms” refers to those that were involved in cross breeding or cell fusion.
Maize variety PHR03
Characteristics of the modification process
PHP73878 and PHP83175
  • Agrobacterium-mediated DNA transfer
  • Biolistic / Particle gun
  • Gene editing (e.g. CRISPR-Cas, etc.)
Some of these genetic elements may be present as fragments or truncated forms. Please see notes below, where applicable.
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    BCH-GENE-SCBD-260877-1 Flippase recombinase recognition targets | Saccharomyces cerevisiae (Yeast, YEASX)
    Recognition sequence
  • BCH-GENE-SCBD-15003-7 Phosphomannose Isomerase gene | Escherichia coli (ECOLX)
    Protein coding sequence | Mannose tolerance,Selectable marker genes and reporter genes
  • BCH-GENE-SCBD-100367-4 Proteinase inhibitor II gene terminator | Solanum tuberosum (Potato, SOLTU)
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    BCH-GENE-SCBD-116046-1 19-kDa zein gene terminator | Zea mays (Maize, Corn, MAIZE)
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    BCH-GENE-SCBD-100364-5 Rice actin 1 gene promoter | Oryza sativa (Rice, ORYSA)
  • BCH-GENE-SCBD-100355-6 Rice actin 1, intron | Oryza sativa (Rice, ORYSA)
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    BCH-GENE-SCBD-15002-4 Phosphinothricin N-acetyltransferase gene | Streptomyces viridochromogenes (STRVR)
    Protein coding sequence | Resistance to herbicides (Glufosinate)
  • BCH-GENE-SCBD-100290-6 CaMV 35S terminator | Cauliflower mosaic virus (CaMV)
  • BCH-GENE-SCBD-103069-3 loxP recombination site | Bacteriophage P1 (Phage P1)
    recombination site
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    BCH-GENE-SCBD-116047-2 Ubiquitin terminator | Sorghum bicolor (Sorghum)
  • BCH-GENE-SCBD-116062-1 Gamma kafarin terminator | Sorghum bicolor (Sorghum)
  • BCH-GENE-SCBD-260878-1 RCc3 enhancer | Sorghum bicolor (Sorghum)
  • BCH-GENE-SCBD-116052-1 Predicted calmodulin 5 gene intron | Zea mays (Maize, Corn, MAIZE)
  • BCH-GENE-SCBD-260880-1 PCOa promoter | Zea mays (Maize, Corn, MAIZE)
  • BCH-GENE-SCBD-260899-1 Insecticidal protein IPD079Ea | Ophioderma pendulum (Old World adder's-tongue, Daun rambu, OPHPE)
    Protein coding sequence | Resistance to diseases and pests (Insects, Coleoptera (beetles), Western corn rootworm (Diabrotica virgifera))
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    BCH-GENE-SCBD-260900-2 Subtilisin-chymotrypsin inhibitor 1B terminator | Sorghum bicolor (Sorghum)
  • BCH-GENE-SCBD-116051-1 27-kDA gamma zein terminator | Zea mays (Maize, Corn, MAIZE)
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    BCH-GENE-SCBD-105058-3 In2-1 Terminator | Zea mays (Maize, Corn, MAIZE)
The modified maize contains three gene cassettes: Escherichia coli phosphomannose isomerase (pmi);  Streptomyces viridochromogenes phosphinothricin N-acetyltransferase (pat) and Ophioderma pendulum insecticidal protein IPD079Ea (ipd079ea). 

The pmi coding sequence was inserted near an endogenous promoter and thus is still expected to have sufficient activity for expression. Transcription is terminated by a Solanum tuberosum proteinase inhibitor II (pinII) terminator. A second terminator, maize 19-kDa zein gene terminator, was included to prevent transcription beyond the gene cassette (limits read through/'leaky' expression of the adjacent gene cassette).

The pat coding sequence is under control of an Oryza sativa actin promoter and Cauliflower mosaic virus 35S terminator. The promoter contains an intron from the rice actin gene for enhanced expression. Two additional terminators, Sorghum bicolor ubiquitin and gamma-kafarin, are included to isolate the gene cassette, preventing transcription beyond the gene cassette (limits read through/'leaky' expression of the adjacent gene cassette).

The ipd079ea coding sequence under control of a maize PCOa promoter and S. bicolor subtilisin-chymotrypsin inhibitor 1B terminator. The promoter is enhanced by S. bicolor RCc3 enhancers and promotes root-specific expression. An additional two terminators, maize 27-kDa gamma zein and ln2-1, were included to prevent transcription beyond the gene cassette.

  • Sequencing analysis indicated that the maize contains a single, intact insertion of the expected sequences into chromosome 1 of the maize genome. The sequences were not rearranged or truncated. The analysis also indicated that PHP73878 and PHP83175 vector backbone sequences were absent.
  • Sequencing analysis indicated that PHP70605, PHP21139 and PHP21875 were absent from the maize genome (also see below).

Transformation of the maize:
The transformation of the maize (site-specific integration) was performed in two steps to insert the transgene cassettes into chromosome 1 in a controlled manner.
  1. The first transformation (microparticle bombardment)
    • PHP73878 (for integration into maize genome)
      • Contains sequences for CRISPR/Cas9 mediated recombination, loxP site for Cre recombination, FRT1 site for FLP recombination, nptII, pinII terminator and FRT site.
    • PHP70605 (facilitates recombination; not inserted into the maize genome)
      • Contains T3 promoter, maize ubi promoter, nuclear locating signal from SV40, cas9 exon 1, intron 1 from potato LS1, cas9 exon 2, nuclear locating signal from A. tumefaciens virD2, pinII terminator, maize polIII U6 promoter, guide RNA (gRNA) for Cas9 and maize polIII U6 
    • PHP21139 (aids in regeneration during tissue culture; not inserted into genome)
      • Contains maize Wuschel 2 (wus2)
    • PHP21875 (aids in the regeneration of plants in vitro; not inserted into genome)
      • Contains coding optimized ovule development protein 2 (odp2)
    • Following microparticle bombardment, the gRNA and Cas9 are transiently expressed. The gRNA targets sequences on chromosome 1 (which are also present in PHP73878) to cause a site specific integration of PHP73878 into chromosome 1.
    • Plants were regenerated using tissue culture and selected for using kanamycin (from the introduced E. coli neomycin phosphotransferase II cassette).
  2. The second transformation (Agrobacterium tumefaciens-mediated)
    • PHP83175:
      • Integrated into genome: pmipat and ipd079ea.
      • Not integrated into genome (transiently expressed): wus2, odp2 and yeast flippase.
      • Following introduction of the plasmid into host cells by Agrobacterium tumefaciens, expression of flippase directs a recombination between the FRT sites in PHP73878 and PHP83175, resulting in the replacement of the nptII cassette with pmi, pat and ipd079ea cassettes. Transient expression of wus2 and odp2 facilitate the regeneration and tissue culture of transformed plants.

For more information, kindly refer to the documents attached in the 'Additional information' section of this record.
LMO characteristics
  • Feed
  • Food
Detection method(s)