Method for Modifying Plant Morphology, Biochemistry and Physiology Comprising Expression of Cytokinin Oxydase in the Seeds

Abstract

The present invention relates to methods and compositions for increasing seed yield of a plant. The methods comprise expression of a cytokinin oxidase in the aleurone and/or embryo of a seed. The invention also relates to vectors comprising a nucleic acid encoding a cytokinin oxidase that is operably linked to a promoter capable of driving expression in the aleurone and/or embryo of a seed, and to host cells, transgenic cells and plants comprising such sequences. The use of these sequences for increasing yield is also provided.

1 . A method for increasing seed yield in a plant, said method comprising increasing the level and/or activity of a cytokinin oxidase (CKX) in the embryo and/or aleurone of a plant seed, wherein said embryo and/or aleurone have increased levels and/or activity of cytokinin oxidase relative to other parts of said seed. 
     
     
         2 . The method according to  claim 1 , wherein said increased expression in the embryo and/or aleurone is effected by introducing a genetic modification. 
     
     
         3 . The method according to  claim 2 , wherein said genetic modification is effected by one of: T-DNA activation, TILLING, site-directed mutagenesis or directed evolution. 
     
     
         4 . A method for increasing seed yield in a plant, said method comprising introducing into a plant cell a genetic construct comprising an isolated nucleic acid molecule encoding a cytokinin oxidase wherein said isolated nucleic acid molecule is operably linked to a promoter capable of driving expression in the embryo and/or aleurone of a seed. 
     
     
         5 . A method for producing a plant having increased seed yield and increased expression of a cytokinin oxidase in the embryo and/or aleurone of a seed, said method comprising introducing into a plant cell a genetic construct comprising an isolated nucleic acid molecule encoding a cytokinin oxidase wherein said isolated nucleic acid molecule is operably linked to a promoter capable of driving expression in the embryo and/or aleurone of a seed. 
     
     
         6 . A method for producing a plant having increased seed yield and increased expression of a cytokinin oxidase in the embryo and/or aleurone of a seed, said method comprising:
 (a) introducing into a plant cell a genetic construct comprising an isolated nucleic acid molecule encoding a cytokinin oxidase, wherein said isolated nucleic acid molecule is operably linked to a promoter capable of driving expression in the embryo and/or aleurone of a seed;   (b) regenerating a plant therefrom;   (c) growing the regenerated plant to seed set; and   (d) selecting a plant with increased seed yield compared to a corresponding wild type plant.   
     
     
         7 . The method of any of  claim 4  wherein the promoter capable of driving expression in the embryo and/or aleurone comprises nucleotides 1-1256 of SEQ ID NO: 41. 
     
     
         8 . The method of  claim 2  wherein the nucleic acid molecule encoding a cytokinin oxidase is selected from the group consisting of:
 (a) nucleic acids comprising a DNA sequence as given in any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or the complement thereof,   (b) nucleic acids comprising the RNA sequences corresponding to any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or the complement thereof,   (c) nucleic acids specifically hybridizing to the complement of any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34,   (d) nucleic acids encoding a protein comprising the amino acid sequence as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or the complement thereof,   (e) nucleic acids as defined in any of (a) to (d) characterized in that said nucleic acid is DNA, genomic DNA, cDNA, synthetic DNA or RNA wherein T is replaced by U,   (f) nucleic acid which is degenerate compared to a nucleic acid as given in any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or which is degenerate compared to a nucleic acid as defined in any of (a) to (e) as a result of the genetic code,   (g) nucleic acids which are divergent from a nucleic acid encoding a protein as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or which is divergent from a nucleic acid as defined in any of (a) to (e), due to the differences in codon usage between the organisms,   (h) nucleic acids encoding a protein as given in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or nucleic acids as defined in (a) to (e) which are divergent due to the differences between alleles,   (i) nucleic acids encoding a protein as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38,   (j) functional fragments of nucleic acids as defined in any of (a) to (i) having the biological activity of a cytokinin oxidase, and   (k) nucleic acids encoding a plant cytokinin oxidase, comprising the consensus sequence hTDYLhholGGTLSssG and cLFxushGsLGQFGIIstA or comprising expression in seeds of a nucleic acid encoding a protein that reduces the level of active cytokinins in plants or plant parts.   
     
     
         9 . The method of  claim 4 , wherein the isolated nucleic acid molecule operably linked to a promoter capable of driving expression in the embryo and/or aleurone comprises the nucleotide sequence as set forth in SEQ ID NO: 41. 
     
     
         10 . The method of  claim 1  wherein the increase in seed yield comprises at least one of: total weight of seeds, total number of seeds, total number of filled seeds, harvest index, and thousand kernel weight. 
     
     
         11 . An isolated nucleic acid molecule encoding a cytokinin oxidase operably linked to a promoter capable of driving overexpression of said nucleic acid molecule in the embryo and/or aleurone of a seed relative to other parts of said seed. 
     
     
         12 . The isolated nucleic acid molecule of  claim 11  wherein the nucleic acid molecule encoding a cytokinin oxidase is selected from the group consisting of:
 (a) nucleic acids comprising a DNA sequence as given in any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or the complement thereof,   (b) nucleic acids comprising the RNA sequences corresponding to any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or the complement thereof,   (c) nucleic acids specifically hybridizing to the complement of any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34,   (d) nucleic acids encoding a protein comprising the amino acid sequence as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or the complement thereof,   (e) nucleic acids as defined in any of (a) to (d) characterized in that said nucleic acid is DNA, genomic DNA, cDNA, synthetic DNA or RNA wherein T is replaced by U,   (f) nucleic acid which is degenerate compared to a nucleic acid as given in any of SEQ ID NOs: 42, 37, 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 30, 33 or 34, or which is degenerate compared to a nucleic acid as defined in any of (a) to (e) as a result of the genetic code,   (g) nucleic acids which are divergent from a nucleic acid encoding a protein as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38, or which is divergent from a nucleic acid as defined in any of (a) to (e), due to the differences in codon usage,   (h) nucleic acids encoding a protein as given in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38 or nucleic acids as defined in (a) to (e) which are divergent due to the differences between alleles,   (i) nucleic acids encoding a protein as given in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 35, 36 or 38,   (j) functional fragments of nucleic acids as defined in any of (a) to (i) having the biological activity of a cytokinin oxidase, and   (k) nucleic acids encoding a plant cytokinin oxidase, comprising the consensus sequence hTDYLhholGGTLSssG and cLFxushGsLGQFGIIstA or comprising expression, preferably in seeds, of a nucleic acid encoding a protein that reduces the level of active cytokinins in plants or plant parts.   
     
     
         13 . A vector comprising the isolated nucleic acid molecule of  claim 11 . 
     
     
         14 . A vector comprising the isolated nucleic acid molecule having the sequence set forth in SEQ ID NO: 41. 
     
     
         15 . A host cell comprising the isolated nucleic acid molecule of  claim 11 . 
     
     
         16 . A host cell comprising a vector of  claims 13 . 
     
     
         17 . The host cell of  claim 16 , which is a bacterial, fungal, or plant cell. 
     
     
         18 . Plant having increased seed yield, comprising a genetic modification, which genetic modification results in increased expression of cytokinin oxidase in the aleurone and/or embryo of a seed, relative to other parts of said seed. 
     
     
         19 . A transgenic plant, plant part, or plant tissue comprising plant cells of  claim 17 . 
     
     
         20 . The transgenic plant of  claim 19  which is a dicotyledonous or monocotyledonous plant. 
     
     
         21 . Transgenic plant according to  claim 20 , wherein said monocotyledonous plant is selected from a group consisting of sugarcane, rice, maize, wheat, barley, millet, rye, oats, and sorghum. 
     
     
         22 . A transgenic harvestable part, or a product directly derived from a transgenic harvestable part of the transgenic plant of  claim 19 . 
     
     
         23 . A transgenic harvestable part of the transgenic plant of  claim 22  selected from the group consisting of seeds, leaves, fruits, stem cultures, rhizomes, roots, tubers and bulbs. 
     
     
         24 . Transgenic progeny of the plant or plant part of  claim 19 . 
     
     
         25 . Harvestable parts of the plant according to  claim 18 , or products directly derived from said harvestable parts. 
     
     
         26 - 29 . (canceled) 
     
     
         30 . A method for increasing seed yield in a plant, said method comprises introducing into a plant cell the vector according to  claim 13 . 
     
     
         31 . The method according to  claim 1 , wherein said increased expression in the embryo and/or aleurone is effected by introducing a genetic modification in the locus of the cytokinin oxidase-encoding gene.