MON-95275-7 - Coleoptera-protected maize | BCH-LMO-SCBD-263095 | Living Modified Organism | Biosafety Clearing-House

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Living Modified Organism (LMO)
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Decisions on the LMO Risk Assessments  
last updated: 06 Feb 2023
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.
Coleoptera-protected maize
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MON95275
Yes
MON-95275-7
  • - Person: Bayer CropScience Bayer CropScience | BCH-CON-US-7091-2
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    Bayer CropScience Bayer CropScience
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The maize (Zea mays) was modified for resistance to Coleopteran insect pests through the expression of insecticidal proteins and double-stranded RNA molecules. For broad proteins against Coleoptera, the maize expresses Brevibacillus laterosporus Mpp75Aa1.1 and Bacillus thuringiensis Vpb4Da2 proteins, which have pore-forming mode of action in the midgut of feeding larvae. To specifically target Western corn rootworm (Diabrotica virgifera virgifera), the maize contains an RNA interference cassette that specifically targets expression of Snf7, an essential cellular component of endosomal sorting complex required for transport. The hairpin RNA expressed triggers an RNAi response, which targets the mRNA for degradation, resulting in gene silencing and in turn death of the feeding larvae. The modified maize is "marker-free" as the selectable marker (glyphosate resistance) was crossed out of the development of the line.
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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.
Line LH244
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Characteristics of the modification process
PV-ZMIR525664
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  • Agrobacterium-mediated DNA transfer
Some of these genetic elements may be present as fragments or truncated forms. Please see notes below, where applicable.
  • BCH-GENE-SCBD-101877-5 rbcS-E9 gene terminator | Pisum sativum (Garden pea, PEA)
    Terminator
  • BCH-GENE-SCBD-108875-2 Snf7 coding sequence | Diabrotica virgifera virgifera (Western corn rootworm, DIAVI)
    Protein coding sequence | Resistance to diseases and pests (Insects, Coleoptera (beetles), Western corn rootworm (Diabrotica virgifera))
  • BCH-GENE-SCBD-100359-7 Hsp70 intron | Zea mays (Maize, Corn, MAIZE)
    Intron
  • BCH-GENE-SCBD-100287-7 CaMV 35S promoter | Cauliflower mosaic virus (CaMV)
    Promoter
  • BCH-GENE-SCBD-263094-1 Physical impedance induced protein enhancer | Zea mays (Maize, Corn, MAIZE)
    Enhancer
  • BCH-GENE-SCBD-263018-2 Dahlia mosaic virus enhancer | Dahlia mosaic virus (DMV, 9VIRU, DaMV)
    Enhancer
  • BCH-GENE-SCBD-263035-1 RCc3 leader | Tripsacum dactyloides (Eastern gamagrass, TRIDA, Gama grass)
    Leader
  • BCH-GENE-SCBD-263036-1 14-3-3C intron | Setaria italica (Foxtail millet, Italian millet)
    Intron
  • BCH-GENE-SCBD-263044-2 Mpp75Aa1.1 | Brevibacillus laterosporus (BRELA)
    Protein coding sequence | Resistance to diseases and pests (Insects, Coleoptera (beetles), Colorado potato beetle (Leptinotarsa decemlineata), Western corn rootworm (Diabrotica virgifera), Northern corn rootworm (Diabrotica barberi))
  • BCH-GENE-SCBD-263046-1 Heat shock protein 16.9 terminator | Coix lacryma-jobi (Job's tears, Adlay millet)
    Terminator
  • BCH-GENE-SCBD-263047-1 S-adenosylmethionine synthase 1 terminator | Setaria italica (Foxtail millet, Italian millet)
    Terminator
  • BCH-GENE-SCBD-263048-1 Vpb4Da2 | Bacillus thuringiensis (Bt, Bacillus, BACTU)
    Protein coding sequence | Resistance to diseases and pests (Insects, Coleoptera (beetles), Colorado potato beetle (Leptinotarsa decemlineata), Western corn rootworm (Diabrotica virgifera), Northern corn rootworm (Diabrotica barberi))
  • BCH-GENE-SCBD-263049-1 Actin 4 intron | Setaria italica (Foxtail millet, Italian millet)
    Intron
  • BCH-GENE-SCBD-263057-1 Lipid transfer protein 1 promoter | Zea mays (Maize, Corn, MAIZE)
    Promoter
  • BCH-GENE-SCBD-263058-1 Open reading frame 6 enhancer | Dahlia mosaic virus (DMV, 9VIRU, DaMV)
    Enhancer
  • BCH-GENE-SCBD-101415-9 Ti plasmid left border repeat | Agrobacterium tumefaciens (Agrobacterium)
    Plasmid vector
  • BCH-GENE-SCBD-101416-6 Ti plasmid right border repeat | Agrobacterium tumefaciens (Agrobacterium)
    Plasmid vector
  • BCH-GENE-SCBD-263034-1 RCc3 promoter | Tripsacum dactyloides (Eastern gamagrass, TRIDA, Gama grass)
    Promoter
The modified maize contains three gene cassettes: RNA interference (RNAi) cassette targeting Diabrotica virgifera ESCRT-III complex Snf7, a Brevibacillus laterosporus mpp75Aa1.1 cassette and a Bacillus thuringiensis vpb4Da2 cassette.

Transcription of the RNAi cassette commences from the Cauliflower mosaic virus 35S promoter and terminates at the Pisum sativum ribulose bisphosphate carboxylase small chain 2 terminator. The transcript initially contains a Zea mays heat shock protein 70 intron, which contributes to enhanced expression in vegetative tissues of the plant, and two partial coding sequences of the D. virgifera virgifera snf7 gene, which encodes the SNF7 subunit of the ESCRT-III complex. The two snf7 sequences are in an inverted orientation, separated by a 150-nucleotide intervening sequence, which allows base pairing between the inverted sequences and hairpin RNA formation post-transcription. The presence of double-stranded RNA structure then triggers an RNAi response. Due to RNAi processing, small interfering RNA molecules (roughly 21-23 nucleotides in length) will be produced and thus no translation into protein will occur from this cassette. An intergenic spacer is present between this cassette and the mpp75Aa1.1 cassette to insulate the gene cassettes (minimize effects of genetic elements between the two cassettes).

Transcription of the mpp75Aa1.1 coding sequence is under control of a Tripsacum dactyloides RCc3 promoter + leader and Coix lacryma-jobi heat shock protein 16.9 terminator. Transcription is enhanced by Setaria italica 14-3-3C intron and a dahlia mosaic virus (DMV) enhancer. Transcription is expected in all plant tissues (no tissue-specificity).

Transcription of vpb4Da2 is under control Zea mays lipid transfer protein 1 promoter and S. italica S-adenosylmethionine synthase 1 terminator. Expression is enhanced by S. italica actin 4 intron and DMV open reading frame 6 enhancer. The promoter is not expected to be active in pollen tissues (very low or no expression).

Note:
  • The T-DNA that was initially inserted contained an Agrobacterium tumefaciens 5-enolpyruvulshikimate-3-phosphate synthase (cp4-epsps) selectable marker cassette (Oryza sativa tubulin A terminator, cp4-epsps, Arabidopsis thaliana chloroplast transit peptide 2, and O. sativa tubulin A promoter) flanked by lox recombination sites. This cassette allows for glyphosate selection after the initial transformation. After MON 95275 was selected as a successful transformant, the selectable marker cassette was excised by crossing MON 95275 with a Cre recombinase expressing line (the Cre line was transformed with the vector PV-ZMOO513642). Subsequently, traditional breeding, segregation, selection and screening were used to isolate those plants that contain the Snf7.1 RNAi suppression cassette, as well as the mpp75Aa1.1 and vpb4Da2 expression cassettes, while also lacking any sequence from PV-ZMOO513642.
  • Sequencing and bioinformatic analysis indicated that the maize contained a single T-DNA insertion without vector backbone sequences or the PV-ZMOO513642 plasmid.
  • The mpp75Aa1.1 and vpb4Da2 sequences were codon-optimized for expression in plant cells.
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LMO characteristics
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  • Feed
  • Food
Detection method(s)
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Additional Information
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