Methods for generating transgenic plants

• US 10,443,063 B2
• Filed: 06/06/2014
• Issued: 10/15/2019
• Est. Priority Date: 06/11/2013
• Status: Active Grant

• Current Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Original Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Inventors: Li, Xianggan, Elumalai, Sivamani
• Primary Examiner(s): Ibrahim, Medina A
• Assistant Examiner(s): Zhong, Wayne
• Application Number:
  US14/298,328
• Publication Number:
  US 20140366223A1
• Time in Patent Office: 1,957 Days
• Field of Search: None
• CPC Class Codes: C12N 15/8201 Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
• CPC Class Codes: C12N 15/8205 Agrobacterium mediated transformation
• CPC Class Codes: C12N 15/8209 Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers

Abstract

This invention provides a method for generating transgenic plants with a low copy number. Plant cells are transformed with polynucleotides containing transcriptional cassettes designed to trigger silencing of a gene which is essential for the plant cell to survive the transformation and regeneration process. The present invention enables the recovery of an increased number of transgenic plants which have only one copy of each desired transcriptional cassette.

Claims

1. An Agrobacterium-mediated transformation method for producing transgenic plant cells comprising only one copy of each transcriptional cassette of interest, comprising:

(a) providing a first T-DNA polynucleotide comprising at least two transcriptional cassettes of interest, wherein a first transcriptional cassette comprises a polynucleotide that encodes for a positive selectable marker, wherein said positive selectable marker provides selection in the presence of a selection agent, wherein the selectable marker is PMI, and at least one transcriptional cassette comprises a trait gene, wherein the first T-DNA polynucleotide further comprises a first right T-DNA border and a first left T-DNA border;

(b) providing a second T-DNA polynucleotide comprising a transcriptional cassette, wherein the transcriptional cassette consists of a polynucleotide encoding a double-stranded RNA molecule, wherein the polynucleotide comprises a polynucleotide fragment at least 200 nucleotides in length and at least 95% identical to the polynucleotide sequence that encodes for the selectable marker of step (a), wherein the polynucleotide sequence encoding for the selectable marker is SEQ ID NO;

13, operably linked to a polynucleotide linker, operably linked to a second polynucleotide fragment at least 200 nucleotides in length and complementary to the first polynucleotide fragment, wherein said transcriptional cassette is transcribed into a double stranded RNA capable of inducing reduced expression or silencing of said polynucleotide that encodes for the positive selectable marker of step (a), wherein the second T-DNA polynucleotide further comprises a second right T-DNA border and a second left T-DNA border distinct from the T-DNA borders of the first T-DNA polynucleotide of step (a);

(c) providing a DNA plasmid for Agrobacterium-mediated transformation, comprising said first T-DNA polynucleotide and said second T-DNA polynucleotide;

(d) performing Agrobacterium-mediated transformation on a plurality of cells to produce a plurality of transgenic plant cells;

(e) selecting for transgenic plant cells which only contain said first T-DNA polynucleotide by use of the selection agent of step (a); and

(f) growing transgenic tissues from said transgenic plant cells of step (e),

wherein transgenic plant cells with the second T-DNA polynucleotide of step (b) have reduced survivability through the transformation and regeneration process, and an increased percentage of said transgenic cells carrying only one copy of each transcriptional cassette within the first T-DNA polynucleotide of step (a) is generated compared to methods without step (b).

2. The method of claim 1, wherein said transcriptional cassette of step (b) comprises SEQ ID NO:

16.

4. The method of claim 1, further comprising the step of determining the copy number of each transcriptional cassette of interest comprised in said transgenic tissues.

5. The method of claim 4, further comprising the step of selecting a transgenic tissue comprising a single copy of the transcriptional cassettes and growing the plant tissue into a plant.

6. A plant, plant tissue, or plant cell made by the method of claim 1, wherein the plant, plant tissue, or plant cell comprises the DNA plasmid comprising the first T-DNA polynucleotide and the second T-DNA polynucleotide.

3. A DNA plasmid comprising:

(a) a first T-DNA polynucleotide comprising at least two transcriptional cassettes of interest, wherein a first transcriptional cassette comprises a polynucleotide that encodes for a positive selectable marker, wherein the selectable marker is PMI, and at least one transcriptional cassette comprises a trait gene, wherein the first T-DNA polynucleotide further comprises a first right T-DNA border and a first left T-DNA border;

(b) a second T-DNA polynucleotide comprising a transcriptional cassette, wherein the transcriptional cassette consists of a polynucleotide encoding a double-stranded RNA molecule, wherein the polynucleotide comprises a polynucleotide fragment of at least 200 nucleotides in length and at least 95% identical to the polynucleotide sequence that encodes for the selectable marker of (a), wherein the polynucleotide sequence encoding for the selectable marker is SEQ ID NO;

13, operably linked to a polynucleotide linker, operably linked to a second polynucleotide fragment of at least 200 nucleotides in length and complementary to the first polynucleotide fragment, wherein said transcriptional cassette is transcribed into a double stranded RNA capable of inducing reduced expression or silencing of said polynucleotide that encodes for the positive selectable marker of (a), wherein the second T-DNA polynucleotide further comprises a second right T-DNA border and a second left T-DNA border distinct from the T-DNA borders of the first T-DNA polynucleotide of (a).