Plastid transformation of Brassica
A method is provided for transforming Brassica plants to express DNA sequences of interest from the plant cell plastid. The method allows the transformation of Brassica plant tissue with heterologous DNA constructs. Such DNA constructs comprise, in the 5′ to 3′ direction of transcription, a promoter region functional in a plant plastid and a DNA sequence of interest. The invention further provides for Brassica cells in which the plastids contain heterologous DNA constructs.
1. A method for the transformation of a Brassica plant cell comprising:
introducing into plastids of said Brassica plant cell a construct comprising a promoter functional in a Brassica plant plastid operably associated with a nucleic acid sequence from Bacillus thuringiensis encoding a crystal protein providing for resistance to insects and a transcriptional terminator functional in a Brassica plant cell plastid, and flanking regions of homology to the Brassica plastid genome comprised of a pair of regions of a Brassica chloroplast genome of a size from about 1 kb to about 2.5 kb and which comprise or reside within a region identified by PCR amplification at 94 degrees C. for 4 minutes, 30 cycles of 94 degrees C. for 30 seconds, 50 degrees C. for 30 seconds, 72 degrees C. for 1 minute 45 seconds, with a final extension at 72 degrees C. for 7 minutes using SEQ ID NOS 3-4 and SEQ ID NOS 5-6 as primers, whereby said flanking regions cause the integration of the construct into the plastid genome of said Brassica plant cell, and
selecting Brassica plant cells having plastids containing said construct.
2. A method for the transformation of a Brassica plant cell comprising:
introducing into plastids of said Brassica plant cell a construct comprising a promoter functional in a Brassica plant plastid operably associated with a nucleic acid sequence encoding a human biological protein and a transcriptional terminator functional in a Brassica plant cell plastid, and flanking regions of homology to the Brassica plastid genome comprised of a pair of regions of a Brassica chloroplast genome of a size from about 1 kb to about 2.5 kb and which comprise or reside within a region identified by PCR amplification at 94 degrees C. for 4 minutes, 30 cycles of 94 degrees C. for 30 seconds, 50 degrees C. for 30 seconds, 72 degrees C. for 1 minute 45 seconds, with a final extension at 72 degrees C. for 7 minutes using SEQ ID NOS 3-4 and SEQ ID NOS 5-6 as primers, whereby said flanking regions cause the integration of the construct into the plastid genome of said Brassica plant cell, and
selecting Brassica plant cells having plastids containing said construct.
3. The method according to claim 1 further comprising regenerating a mature Brassica plant containing transformed plastids from said Brassica plant cells.
4. The method according to claim 1 wherein said Brassica plant cell is a Brassica napus plant cell.
5. The method according to claim 1 wherein said Brassica plant cell comprises a leaf cell.
6. A Brassica plant cell obtained by the method of claim 1 .
7. A Brassica plant, plant seed or plant part, or progeny thereof, containing a Brassica plant cell according to claim 6 .
8. A fertile Brassica plant having plastids containing a heterologous construct, wherein said construct comprises as operably associated components in the 5′ to 3′ direction of transcription, a promoter functional in a plant cell plastid, a nucleic acid sequence from Bacillus thuringiensis encoding a crystal protein providing for resistance to insects and a transcriptional terminator functional in a plant cell plastid whereby said construct is integrated into the plastid genome of said Brassica plant through regions of homology to the Brassica plastid genome flanking the heterologous construct, said regions of homology comprised of a pair of regions of a Brassica chloroplast genome of a size from about 1 kb to about 2.5 kb and which comprise or reside within a region identified by PCR amplification at 94 degrees C. for 4 minutes, 30 cycles of 94 degrees C. for 30 seconds, 50 degrees C. for 30 seconds, 72 degrees C. for 1 minute 45 seconds, with a final extension at 72 degrees C. for 7 minutes using SEQ ID NOS 3-4 and SEQ ID NOS 5-6 as primers.
9. The Brassica plant according to claim 8 wherein said Brassica plant is Brassica napus.
10. The Brassica plant according to claim 8 wherein said plastids containing the heterologous construct are present in the leaf cells of said Brassica plant.
11. A Brassica seed or Brassica plant part, or progeny thereof, derived from a Brassica plant according to claim 8 , each containing said heterologous construct.
12. The Brassica plant of claim 8 wherein the regions of homology include the trnV-rps7/12 intergenic region and the heterologous construct is integrated into the corresponding location in the Brassica plastid genome.
13. The Brassica plant of claim 8 wherein the regions of homology are nucleotides 1-2432 and 4960-7454 of SEQ ID NO:19.
14. The method according to claim 2 further comprising regenerating a mature Brassica plant containing transformed plastids from said Brassica plant cells.
15. The method according to claim 2 wherein said Brassica plant cell is a Brassica napus plant cell.
16. The method according to claim 2 wherein said Brassica plant cell comprises a leaf cell.
17. A Brassica plant cell obtained by the method of claim 2 .
18. A Brassica plant, plant seed or plant part, or progeny thereof, containing a Brassica plant cell according to claim 17 .
19. A fertile Brassica plant having plastids containing a heterologous construct, wherein said construct comprises as operably associated components in the 5′ to 3′ direction of transcription, a promoter functional in a plant cell plastid, a nucleic acid sequence encoding a human biological protein and a transcriptional terminator functional in a plant cell plastid whereby said construct is integrated into the plastid genome of said Brassica plant through regions of homology to the Brassica plastid genome flanking the heterologous construct, said regions of homology comprised of a pair of regions of a Brassica chloroplast genome of a size from about 1 kb to about 2.5 kb and which comprise or reside within a region identified by PCR amplification at 94 degrees C. for 4 minutes, 30 cycles of 94 degrees C. for 30 seconds, 50 degrees C. for 30 seconds, 72 degrees C. for 1 minute 45 seconds, with a final extension at 72 degrees C. for 7 minutes using SEQ ID NOS 3-4 and SEQ ID NOS 5-6 as primers.
20. The Brassica plant according to claim 19 wherein said Brassica plant is Brassica napus.
21. The Brassica plant according to claim 19 wherein said plastids containing the heterologous construct are present in the leaf cells of said Brassica plant.
22. A Brassica seed or Brassica plant part, or progeny thereof, derived from a Brassica plant according to claim 19 , each containing said heterologous construct.
23. The Brassica plant of claim 19 wherein the regions of homology include the tmV-rps7/12 intergenic region and the heterologous construct is integrated into the corresponding location in the Brassica plastid genome.
24. The Brassica plant of claim 19 wherein the regions of homology are nucleotides 1-2432 and 4960-7454 of SEQ ID NO:19.