Promotion of somatic embryogenesis in plants by wuschel gene expression

Abstract

The present invention relates to methods for promoting somatic embryogenesis from a tissue or organ of a plant, by overexpressing a Wuschel gene in said tissue or organ. In one embodiment, such overexpression can be used as a silent selectable marker for transgenic plants. In another embodiment, such expression can be used to confer apomixis to a plant. In another embodiment, such overexpression can be used to create haploid plants, which can be used to produce dihaploid plants.

What is claimed is:  
     
         1 . A method to promote somatic embryogenesis from a tissue or organ of a plant, said method comprising overexpressing a Wuschel gene in said tissue or organ.  
     
     
         2 . The method of  claim 1  wherein said method is performed in the absence of external plant hormones.  
     
     
         3 . The method of  claim 1  wherein said method comprises a step of culturing in the presence of an auxin.  
     
     
         4 . The method of  claim 1  wherein said tissue or organ comprises a promoter controlling expression of said Wuschel gene.  
     
     
         5 . The method of  claim 4  wherein said tissue or organ is transgenic for said promoter.  
     
     
         6 . The method of  claim 4  wherein said promoter is inducible.  
     
     
         7 . The method of  claim 1  wherein said Wuschel gene which is overexpressed is in a vector.  
     
     
         8 . The method of  claim 7  wherein said vector comprises an inducible promoter controlling expression of said Wuschel gene.  
     
     
         9 . The method of  claim 8  wherein said inducible promoter is inducible by an estrogen.  
     
     
         10 . The method of  claim 9  wherein said estrogen is 17-β-estradiol.  
     
     
         11 . The method of  claim 7  wherein said vector comprises a tissue specific promoter.  
     
     
         12 . The method of  claim 1  wherein said plant is selected from the group consisting of trees and crops.  
     
     
         13 . A method to generate plant somatic embryos wherein said method comprises overexpressing a Wuschel gene in a tissue or organ of a plant.  
     
     
         14 . The method of  claim 13  wherein the method is performed in the absence of external plant hormones.  
     
     
         15 . The method of  claim 13  wherein said method comprises a step of culturing in the presence of an auxin.  
     
     
         16 . The method of  claim 13  wherein said tissue or organ comprises a promoter controlling expression of said Wuschel gene.  
     
     
         17 . The method of  claim 16  wherein said tissue or organ is transgenic for said promoter.  
     
     
         18 . The method of  claim 16  wherein said promoter is inducible.  
     
     
         19 . The method of  claim 13  wherein said Wuschel gene which is overexpressed is in a vector.  
     
     
         20 . The method of  claim 19  wherein said vector comprises an inducible promoter controlling expression of said Wuschel gene.  
     
     
         21 . The method of  claim 20  wherein said inducible promoter is inducible by an estrogen.  
     
     
         22 . The method of  claim 21  wherein said estrogen is 17-β-estradiol.  
     
     
         23 . The method of  claim 19  wherein said vector comprises a tissue specific promoter.  
     
     
         24 . The method of  claim 13  wherein said plant is selected from the group consisting of trees and crops.  
     
     
         25 . A method for generating shoots from a tissue or organ of a plant, said method comprising overexpressing a Wuschel gene in said tissue or organ.  
     
     
         26 . The method of  claim 25  wherein said method is performed in the absence of external plant hormones.  
     
     
         27 . The method of  claim 25  wherein said method comprises a step of culturing in the presence of an auxin.  
     
     
         28 . The method of  claim 25  wherein said vegetative tissue or organ comprises a promoter controlling expression of said Wuschel gene.  
     
     
         29 . The method of  claim 28  wherein said vegetative tissue or organ is transgenic for said promoter.  
     
     
         30 . The method of  claim 28  wherein said promoter is inducible.  
     
     
         31 . The method of  claim 25  wherein said Wuschel gene which is overexpressed is in a vector.  
     
     
         32 . The method of  claim 31  wherein said vector comprises an inducible promoter controlling expression of said Wuschel gene.  
     
     
         33 . The method of  claim 32  wherein said inducible promoter is inducible by an estrogen.  
     
     
         34 . The method of  claim 33  wherein said estrogen is 17-β-estradiol.  
     
     
         35 . The method of  claim 31  wherein said vector comprises a tissue specific promoter.  
     
     
         36 . The method of  claim 25  wherein said plant is selected from the group consisting of trees and crops.  
     
     
         37 . A method of selecting plants transformed with a vector comprising a silent selectable marker, said silent selectable marker being a Wuschel gene, said method comprising 
 a) transforming a plant, plant tissue or plant organ with a vector comprising said Wuschel gene under the control of an inducible promoter;    b) culturing said plant, plant tissue or plant organ in the presence of an inducer of said inducible promoter;    c) selecting somatic embryos which form; and    d) growing said somatic embryos in the absence of said inducer to develop plants,    wherein said plants which develop are transformed with said vector.    
     
     
         38 . The method of  claim 37  wherein method is performed in the absence of external plant hormones.  
     
     
         39 . The method of  claim 37  wherein said method comprises a step of culturing in the presence of an auxin.  
     
     
         40 . The method of  claim 37  wherein said inducible promoter is inducible by an estrogen.  
     
     
         41 . The method of  claim 40  wherein said estrogen is 17-β-estradiol.  
     
     
         42 . The method of  claim 37  wherein said vector comprises a tissue specific promoter.  
     
     
         43 . The method of  claim 37  wherein said vector comprises a gene of interest.  
     
     
         44 . The method of  claim 43  wherein said gene of interest is under the control of a promoter.  
     
     
         45 . The method of  claim 44  wherein said promoter controlling the gene of interest is a constitutive promoter or an inducible promoter.  
     
     
         46 . The method of  claim 37  wherein said plant, plant tissue or plant organ is selected from the group consisting of trees or crops.  
     
     
         47 . The method of  claim 37  wherein said vector comprises means for deleting said Wuschel gene.  
     
     
         48 . A method for inducing apomixis in a plant cell comprising overexpressing Wuschel in said plant cell.  
     
     
         49 . The method of  claim 48  wherein said plant cell is transformed with a vector comprising Wuschel.  
     
     
         50 . The method of  claim 49  wherein Wuschel is under the control of an inducible promoter.  
     
     
         51 . The method of  claim 49  wherein said vector comprises a second gene in addition to said Wuschel.  
     
     
         52 . The method of  claim 51  wherein said second gene encodes a marker.  
     
     
         53 . The method of  claim 51  wherein said second gene confers a desired trait to said plant.  
     
     
         54 . An apomictic plant comprising Wuschel under the control of an inducible promoter.  
     
     
         55 . A method for conferring apomixis to a plant comprising 
 a. stably transforming a responsive plant cell with a DNA sequence comprising at least one Wuschel coding sequence under the control of a promoter to produce a transgenic plant cell, said promoter being selected from the group consisting of a tissue-specific promoter, an inducible promoter and a promoter that is both tissue-specific and inducible,    b. regenerating a transgenic plant from said transgenic plant cell,    whereby overexpression of a Wuschel polypeptide from the Wuschel coding sequence in the transgenic plant results in the formation of an apomictic embryo.    
     
     
         56 . The method of  claim 55  wherein the promoter is a tissue-specific promoter.  
     
     
         57 . The method of  claim 55  wherein the promoter is an inducible promoter.  
     
     
         58 . The method of  claim 55  wherein the promoter is both tissue-specific and inducible.  
     
     
         59 . The method of  claim 56  wherein the promoter is a nucellus-specific promoter.  
     
     
         60 . The method of  claim 56  wherein the transgenic plant cell comprises at least one additional heterologous DNA sequence in its genome.  
     
     
         61 . The method of  claim 55  wherein the Wuschel gene is PGA6.  
     
     
         62 . A plant produced by the method of  claim 55 .  
     
     
         63 . A method for producing a haploid plant comprising 
 a. stably transforming a plant cell with a DNA sequence comprising at least one Wuschel coding sequence under the control of a promoter to produce a transgenic plant cell, wherein the promoter is selected from the group consisting of a haploid tissue specific promoter, an inducible promoter and a promoter that is both haploid-tissue specific and inducible,    b. generating a transgenic plant from said transgenic plant cell,    c. overexpressing the Wuschel coding sequence in a haploid tissue of said transgenic plant to produce a haploid somatic embryo,    d. growing said embryo into a haploid plant.    
     
     
         64 . The method of  claim 63  wherein the promoter is a haploid tissue specific promoter.  
     
     
         65 . The method of  claim 64  wherein the promoter is a pollen-specific promoter, and the haploid tissue of the transgenic plant is pollen.  
     
     
         66 . The method of  claim 64  wherein the promoter is an ovule-specific promoter, and the haploid tissue of the transgenic plant is ovule tissue.  
     
     
         67 . The method of  claim 63  wherein the promoter is an inducible promoter.  
     
     
         68 . The method of  claim 63  wherein the promoter is both haploid-tissue specific and inducible.  
     
     
         69 . The method of  claim 67  wherein the promoter is pollen-specific, and the haploid plant cell is a pollen cell.  
     
     
         70 . The method of  claim 66  wherein the overexpressing step c. is achieved in excised haploid tissue cultured in the presence of the inducer specific for the inducible promoter, for a time sufficient to induce formation of the haploid somatic embryo, followed by withdrawal of the inducer, and further wherein the growing step d. is carried out in the absence of the inducer.