Plant selectable marker and plant transformation method
AssigneeApplied Phytologics, Inc.
InventorsRaymond L Rodriguez, Ning Huang
Life of this patent
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Abstract
A plant transformation expression cassette for transforming host plant cells with a selectable marker gene is disclosed. The cassette includes, operatively linked in sequence in a 5′ to 3′ direction, (i) a DNA promoter sequence from the rice beta-glucanase 9 (gns9) gene; (ii) a selectable marker gene, and (iii) a 3′ untranslated terminator region. Also disclosed are a expression cassette pair containing the transformation vector, a method of obtaining transformed monocots whose seeds produce a selected heterologous protein during sed germination, and a plant whose cells are transformed with the chimeric selectable marker gene.
Claims10
A method of transforming rice plants with one or more heterologous nucleic acid coding sequences capable of producing heterologous proteins in the rice, under selected induction conditions, comprising
- cotransforming rice callus cells with a set of two or more expression cassettes, said set comprising:
- (a) a chimeric selectable marker expression cassette having, operatively linked in sequence in a 5′ to 3′ direction, (i) a transcriptional regulatory region which expresses in transformed callus cells at a significantly higher level than in seed tissue, and hybridizes under high stringency conditions with the rice β-glucanase gene (Gns9) promoter identified by SEQ ID NO:1;
- (ii) a hygromycin phosphotransferase encoding gene and (iii) a 3′ untranslated terminator region;
- and
- (b) at least one heterologous gene expression cassette, having operatively linked in sequence in a 5′ to 3′ direction, (i) a transcriptional regulatory region that is expressed, induced or inducible in plant seeds, (ii) a first DNA sequence encoding a selected heterologous protein, and (iii) a 3′ untranslated terminator region, wherein the transcriptional regulatory region in said heterologous-gene expression cassette is induced during seed maturation or seed germination;
- culturing the callus cells in the presence of a selection agent effective to block growth of callus cells in the absence of expression of the hygromycin phosphotransferase encoding gene;
- selecting those callus cells that express hygromycin phosphotransferase, as evidenced by their growth in the presence of the selection agent;
- and
- regenerating the selected callus cells into transgenic plants under non-selection conditions.
A plant transformation expression cassette for transforming rice plant cells with a chimeric selectable marker gene, said cassette comprising, in a 5′ to 3′ direction:
- (i) a transcriptional regulatory region comprising a sequence which hybridizes under high stringency conditions with the rice β-glucanase gene promoter identified by SEQ ID NO:1, and which expresses in callus cells at a significantly higher level than in a selected target tissue,
- (ii) a hygromycin phosphotransferase encoding selectable marker coding sequence, and
- (iii) a 3′ untranslated terminator region.
Transgenic rice seed comprising:
- a chimeric selectable marker gene including a hygromycin phosphotransferase selectable marker coding sequence under the control of a transcriptional regulatory region that is induced in callus tissue at a significantly higher level than in seed tissue and hybridizes under high stringency conditions with the rice β-glucanase gene (Gns9) promoter identified by SEQ ID NO:1, and
- a heterologous protein coding sequence under the control of a transcriptional regulatory region that is induced or inducible during seed maturation or germination.
Dependencies are inferred from the claim text.