Insect resistant cotton plants and methods for identifying same
AssigneeBayer BioScience N.V.
InventorsLinda Trolinder, Sofie Moens, Dimitri Paelinck, Veerle Habex, Hans Van Herck
Life of this patent
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Abstract
The invention provides specific transgenic cotton plants, plant material and seeds, characterized in that these products harbor a specific transformation event at a specific location in the cotton genome. Tools are also provided which allow rapid and unequivocal identification of the event in biological samples.
Claims27
A transgenic cotton plant, cells, parts, seed, or progeny thereof, each comprising elite event EE-GH5 in its genome, reference seed comprising said event having being deposited at the ATCC under deposit number PTA-8171.
A seed comprising elite event EE-GH5 deposited at the ATCC under deposit number PTA-8171 or derivatives therefrom.
A cotton plant, seed, cells, or tissues thereof, each comprising elite event EE-GH5 in its genome, obtained by propagation of and/or breeding with a cotton plant grown from the seed deposited at the ATCC under deposit number PTA-8171.
A cotton seed comprising elite event EE-GH5, reference seed comprising said event having been deposited at the ATCC under deposit number PTA-8171.
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A method for identifying elite event EE-GH5 in biological samples comprises amplifying a DNA fragment of between 100 and 500 by from a nucleic acid present in said biological samples using a polymerase chain reaction with at least two primers, one of said primers recognizing the 5′ flanking region of EE-GH5, said 5′ flanking region comprising the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 98 or the nucleotide sequence of SEQ ID No 16 from nucleotide 1 to nucleotide 563 or the 3′ flanking region of EE-GH5, said 3′ flanking region comprising the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 41 to nucleotide 452, the other primer of said primers recognizing a sequence within the foreign DNA comprising the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 99 to nucleotide 334 or the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 40.
A kit for identifying elite event EE-GH5 in biological samples comprising one primer recognizing the 5′ flanking region of EE-GH5, said 5′ flanking region comprising the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 98 or the nucleotide sequence of SEQ ID No 16 from nucleotide 1 to nucleotide 563 or one primer recognizing the 3′ flanking region of EE-GH5, said 3′ flanking region comprising the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 41 to nucleotide 452, and one primer recognizing a sequence within the foreign DNA, said foreign DNA comprising the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 99 to nucleotide 334 or the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 40.
21-35. (canceled)
A method for confirming seed purity comprising detection of an EE-GH5 specific region with a specific primer or probe which specifically recognizes the 5′ or 3′ flanking region of EE-GH5, in samples of seed lots.
A method for screening seeds for the presence of EE-GH5 comprising detection of an EE-GH5 specific region with a specific primer or probe which specifically recognizes the 5′ or 3′ flanking region of EE-GH5, in samples of seed lots.
A method for determining the zygosity status of a plant, plant material or seed comprising elite event EE-GH5 comprising amplifying DNA fragments of between 100 and 500 by from a nucleic acid present in said biological samples using a polymerase chain reaction with at least three primers, two of said primers specifically recognizing pre-insertion plant DNA, such as the 5′ flanking region of EE-GH5, said 5′ flanking region comprising the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 98 or the nucleotide sequence of SEQ ID No 16 from nucleotide 1 to nucleotide 563, or the 3′ flanking region of EE-GH5, said 3′ flanking region comprising the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 41 to nucleotide 452 or the pre-insertion DNA comprising the nucleotide sequence of SEQ ID No 15 or the complement thereof, the third of said primers recognizing a sequence within the foreign DNA comprising the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 99 to nucleotide 334 or the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 40.
A method of detecting the presence of elite event EE-GH5 in biological samples through hybridization with a substantially complementary labeled nucleic acid probe in which the probe:target nucleic acid ratio is amplified through recycling of the target nucleic acid sequence comprising:
- a) hybridizing said target nucleic acid sequence to a first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No 1 from nucleotide 99 to nucleotide 116 or its complement or said first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No 2 from nucleotide 23 to nucleotide 40 or its complement;
- b) hybridizing said target nucleic acid sequence to a second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No 1 from nucleotide 81 to nucleotide 98 or its complement or said labeled nucleic acid probe comprising the nucleotide sequence of SEQ ID No 2 from nucleotide 41 to nucleotide 58 or its complement, wherein said first and second oligonucleotide overlap by at least one nucleotide and wherein either said first or said second oligonucleotide is labeled to be said labeled nucleic acid probe;
- c) cleaving only the labeled probe within the probe:target nucleic acid sequence duplex with an enzyme which causes selective probe cleavage resulting in duplex disassociation, leaving the target sequence intact;
- d) recycling of the target nucleic acid sequence by repeating steps (a) to (c);
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- e) detecting cleaved labeled probe, thereby determining the presence of said target nucleic acid sequence.
41-45. (canceled)
Dependencies are inferred from the claim text.