Markerless transformation
View Patent ↗Methods for identification of successful transformation without a need for in vitro selection are provided. Direct detection of nucleotide sequences of interest is described which eliminate the need for use of ancillary nucleotide sequences.
1. A method for producing a transgenic plant comprising a nucleotide sequence of interest inserted into its genome, where said method consists essentially of:
a) transforming a plant tissue or plant cell with said nucleotide sequence of interest, which nucleotide sequence of interest comprises a gene of interest; wherein said plant tissue or plant cell is not transformed with an ancillary nucleotide sequence comprising a reporter sequence or a selectable marker sequence;
b) regenerating said plant tissue or plant cell without selection for a reporter or selectable marker following said transforming, to produce a transgenic plant; and
c) directly detecting the presence or absence in said transgenic plant of said nucleotide sequence of interest, an encoded product of said nucleotide sequence of interest, or a metabolic product of said encoded product;
wherein said transgenic plant comprises said nucleotide sequence of interest introduced by said transforming;
wherein said transforming does not introduce a transposable element into said plant tissue or plant cell; and
wherein said nucleotide sequence of interest encodes one or more products selected from the group consisting of a hybrid Cry1Ac-1Acf Bacillus thuringiensis (Bt) protein, a Cry1Bb Bt protein, and a Cry1Ac Bt protein.
2. The method of claim 1 , wherein directly detecting comprises the use of a method selected from the group consisting of southern hybridization, polymerase chain reaction, and western hybridization for the nucleotide sequence or encoded product; and HPLC for a metabolic product of the encoded product.
3. The method of claim 2 , wherein directly detecting comprises the use of a method selected from the group consisting of southern hybridization for the nucleotide sequence of interest and HPLC for a metabolic product of the encoded product.
4. The method of claim 2 , wherein directly detecting comprises the use of a method selected from the group consisting of polymerase chain reaction for the nucleotide sequence of interest and HPLC for a metabolic product of the encoded product.
5. The method of claim 2 , wherein directly detecting comprises the use of a method selected from the group consisting of western hybridization for the encoded product and HPLC for a metabolic product of the encoded product.
6. The method of claim 1 , wherein directly detecting comprises the use of antibody binding to the encoded product or HPLC for a metabolic product of the encoded product.
7. The method of claim 1 , wherein said directly detecting comprises pooling samples from a group of plants.
8. The method of claim 1 , wherein said transgenic plant is selected from the group consisting of tomato, lettuce, and cabbage.
9. The method of claim 1 , wherein said plant tissue or plant cell is from a cotyledon explant.
10. A method for producing a transgenic plant comprising a nucleotide sequence of interest inserted into its genome, where said method comprises:
a) transforming a plant tissue or plant cell with said nucleotide sequence of interest, which nucleotide sequence of interest comprises a gene of interest; wherein said plant tissue or plant cell is not transformed with an ancillary nucleotide sequence comprising a reporter sequence or a selectable marker sequence;
b) regenerating said plant tissue or plant cell without screening for said nucleotide sequence of interest, an encoded product of said nucleotide sequence of interest, or a metabolic product of said encoded product; and without selection for a reporter or selectable marker following said transforming, to produce a transgenic plant; and
c) directly detecting the presence or absence in said transgenic plant of said nucleotide sequence of interest, said encoded product of said nucleotide sequence of interest, or said metabolic product of said encoded product;
wherein said transgenic plant comprises said nucleotide sequence of interest introduced by said transforming; and
wherein said nucleotide sequence of interest encodes one or more products selected from the group consisting of a hybrid Cry1Ac-1Acf Bacillus thuringiensis (Bt) protein, a Cry1Bb Bt protein, and a Cry1Ac Bt protein.
11. The method of claim 10 , wherein said transforming does not introduce a transposable element into said plant tissue or plant cell.
12. The method of claim 10 , wherein directly detecting comprises the use of a method selected from the group consisting of southern hybridization, polymerase chain reaction, and western hybridization for the nucleotide sequence or encoded product; and HPLC for a metabolic product of the encoded product.
13. The method of claim 10 , wherein directly detecting comprises the use of antibody binding to the encoded product or HPLC for a metabolic product of the encoded product.
14. The method of claim 10 , wherein said directly detecting comprises pooling samples from a group of plants.
15. The method of claim 10 , wherein said transgenic plant is selected from the group consisting of tomato, lettuce, and cabbage.
16. The method of claim 10 , wherein said plant tissue or plant cell is from a cotyledon explant.
17. The method of claim 1 , wherein said transforming involves Agrobacterium.
18. The method of claim 10 , wherein said transforming involves Agrobacterium.