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Living Modified Organism
(LMO)
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.
Maruca-resistant cowpea
EN
DPS08501
Yes
DPS-Ø85Ø1-8
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Organization:Donald Danforth Plant Science Center ()Non-governmental organization (NGO)975 N Warson Rd.St. Louis, Missouri
63132, United States of AmericaPhone: +1 314.587.1000, +1 314.406.4287,Fax:Email: kroeber@danforthcenter.org,Website: https://www.danforthcenter.org/,
Cowpea (Vigna unguiculata) was modified to express a modified crystal delta endotoxin Cry1Ca for resistance to Lepidoptera pests, particularly maruca pod borer (Maruca vitrata). The modified Cry1Ca protein has a similar pore forming mode of action to other crystal proteins. The protein interacts with receptors present on the cell membrane of the gut epithelial cells in the insect larvae to form pores. These pores act as non-selective ion channels causing cell swelling and lysis, leading to eventual death of the feeding larvae. Further, the modified cowpea is does not express any selectable markers as these were removed during product development (also see the section “Notes regarding the genetic elements present in this LMO” 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.
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BCH-ORGA-SCBD-103617-3 Organism Vigna unguiculata (Cowpea, Black eyed pea)Crops
EN
pDIICI-085
EN
- Agrobacterium-mediated DNA transfer
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0.025 kb
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1.673 kb
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0.234 kb
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3.495 kb
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0.372 kb
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0.034 kb
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0.115 kb
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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-101416-7 Ti plasmid right border repeat | Agrobacterium tumefaciens (Agrobacterium)Plasmid vector
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BCH-GENE-SCBD-104517-2 Actin 2 promoter | Arabidopsis thaliana (Thale cress, Mouse-ear cress, Arabidopsis, ARATH)Promoter
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BCH-GENE-SCBD-103899-3 Chloroplast Transit Peptide 4 | Petunia hybrida (Petunia, PETHY)Transit signal
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BCH-GENE-SCBD-269933-1 Modified Cry1Ca | Bacillus thuringiensis (Bt, Bacillus, BACTU)Protein coding sequence | Resistance to diseases and pests (Insects, Lepidoptera (butterflies and moths), Cotton bollworm (Helicoverpa spp.), European corn borer (Ostrinia nubilalis), Fall armyworm (Spodoptera frugiperda))
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BCH-GENE-SCBD-115757-2 Actin 2 terminator | Arabidopsis thaliana (Thale cress, Mouse-ear cress, Arabidopsis, ARATH)Terminator
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BCH-GENE-SCBD-103069-3 loxP recombination site | Bacteriophage P1 (Phage P1)recombination site
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BCH-GENE-SCBD-101415-9 Ti plasmid left border repeat | Agrobacterium tumefaciens (Agrobacterium)Plasmid vector
The modified cowpea expresses a singular gene cassette for the synthetic crystal protein Cry1Ca (mcry1Ca).
Transcription of mcry1Ca is controlled by an Arabidopsis thaliana actin 2 promoter and terminator. A Petunia hybrida chloroplast transit peptide 4 N-terminal sequence was fused in frame with the mcry1Ca sequence to direct the translated peptide to the chloroplasts.
Note:
- The A. thaliana actin 2 promoter sequence contains the first intron of the actin 2 gene.
- The modified Cry1Ca protein was originally derived from Bacillus thuringiensis.
- During development of the modified cowpea, the initial T-DNA insertion contained three additional gene cassettes between two loxP sites:
- Glycine max heat shock protein 17.3B promoter (0.772 kb) – Bacteriophage P1 recombinase cre (1.221 kb) – Solanum tuberosum proteinase inhibitor II terminator (0.311 kb);
- Glycine max ubiquitin promoter + intron (1.946 kb) – synthetic chloroplast transit peptide (0.165 kb) – Streptomyces spectabilis spectinomycin phosphotransferase (spcN; 0.996 kb) – A. thaliana ubiquitin 14 terminator (0.902 kb); and
- Glycine max elongation factor 1 alpha promoter + intron (1.501 kb) – Discosoma sp. DsRed2 (0.678 kb) – A. thaliana ubiquitin 3 terminator (1.088 kb).
- The additional gene cassettes were removed using heat shock to induce the expression of recombinase cre, which excised the three gene cassettes between the two loxP sites. The excised cassettes were used as fluorescent (DsRed2) and spectinomycin (spcN) selectable markers during the initial transformation of the cowpea.
EN
- Feed
- Food
EN
Vector development
A derivative of the pCAMBIA2300 binary vector, p5000, was modified by removing the neomycin phosphotransferase II plant-selectable marker expression cassette and replacing it with the recombinase cre, the spectinomycin resistance and the DsRed2 gene cassettes. In addition, the base vector was designed to include restriction endonuclease sites and sequences to facilitate Golden Gate cloning of the gene-of-interest expression cassette for production of mCry1Ca to create the transformation plasmid pDIICI-085. Agrobacterium tumefaciens strain LBA4404 was used to transform cowpea embryonic axis explants using established protocols.
A derivative of the pCAMBIA2300 binary vector, p5000, was modified by removing the neomycin phosphotransferase II plant-selectable marker expression cassette and replacing it with the recombinase cre, the spectinomycin resistance and the DsRed2 gene cassettes. In addition, the base vector was designed to include restriction endonuclease sites and sequences to facilitate Golden Gate cloning of the gene-of-interest expression cassette for production of mCry1Ca to create the transformation plasmid pDIICI-085. Agrobacterium tumefaciens strain LBA4404 was used to transform cowpea embryonic axis explants using established protocols.
| Record type | Field | Record(s) | |
|---|---|---|---|
| Risk Assessment generated by a regulatory process | Living modified organism(s) | 5 | |
| Country's Decision or any other Communication | LMO identification | 3 | |
