Method for modifying lignin composition and increasing in vivo digestibility of forages

Abstract

Methods for transforming forage legumes or woody plants with a DNA construct comprising at least one open reading frame encoding for a caffeoyl CoA 3-O-methyltransferase enzyme or a Medicago sativa caffeic acid 3-O-methyltransferase enzyme or a fragment thereof in either a sense or antisense orientation under a lignification-associated tissue specific promoter have been found, resulting in the down-regulation of the corresponding homologous gene either through antisense inhibition or sense suppression, as well as reduced lignin content and modified lignin composition in the transgenic plants. The expression of the caffeoyl CoA 3-O-methyltransferase transgene produces an increased syringyl lignin to guaiacyl lignin ratio in the transformed plant, and greatly improved forage digestibility.

We claim:  
     
         1 . A method for modulating the lignin content of a forage legume comprising 
 transforming a forage legume cell with a vector comprising a lignification-associated tissue specific promoter functionally linked to a DNA construct comprising at least one open reading frame encoding for a caffeoyl CoA 3-O-methyltransferase enzyme or a fragment thereof; and    generating plants from said transformed forage legume cell    
     
     
         2 . The method of  claim 1 , wherein said forage legume cell is co-transformed with a vector comprising a lignification-associated tissue specific promoter functionally linked to a DNA construct comprising at least one open reading frame encoding for a  Medicago sativa  caffeic acid 3-O-methyltransferase enzyme or a fragment thereof.  
     
     
         3 . A method for modulating the lignin content of a forage legume comprising 
 transforming a forage legume cell with a vector comprising a lignification-associated tissue specific promoter functionally linked to a DNA construct comprising at least one open reading frame encoding for a  Medicago sativa  caffeic acid 3-O-methyltransferase enzyme or a fragment thereof; and    generating plants from said transformed forage legume cell.    
     
     
         4 . A method for modulating the lignin content of a forage legume comprising 
 transforming a forage legume cell with a vector comprising a DNA construct comprising in tandem at least one open reading frame encoding for a caffeoyl CoA 3-O-methyltransferase enzyme or a fragment thereof under expression control of a first lignification-associated tissue specific promoter and at least one open reading frame encoding for a caffeic acid 3-O-methyltransferase enzyme or a fragment thereof under expression control of a second lignification-associated tissue specific promoter, wherein said first and second lignification-associated tissue specific promoter can be the same or different; and    generating plants from said transformed forage legume cell.    
     
     
         5 . A method for producing a forage legume having altered lignin composition comprising 
 transforming a forage legume cell with a DNA construct comprising at least one open reading frame encoding for a caffeoyl CoA 3-O-methyltransferase enzyme or a fragment thereof under expression control of a lignification-associated tissue specific promoter to form a transgenic cell; and    cultivating said transgenic cell under conditions conducive to regeneration and plant growth.    
     
     
         6 . The method of  claim 5 , wherein said forage legume cell is co-transformed with a DNA construct comprising at least one open reading frame encoding for a  Medicago sativa  caffeic acid 3-O-methyltransferase enzyme or a fragment thereof under expression control of a lignification-associated tissue specific promoter.  
     
     
         7 . A method for producing a forage legume having altered lignin composition comprising 
 transforming a forage legume cell with a DNA construct comprising at least one open reading frame encoding for a  Medicago sativa  caffeic acid 3-O-methyltransferase enzyme or a fragment thereof under expression control of a lignification-associated tissue specific promoter to form a transgenic cell; and    cultivating said transgenic cell under conditions conducive to regeneration and plant growth.    
     
     
         8 . A method for producing a forage legume having altered lignin composition comprising 
 transforming a forage legume cell with a DNA construct comprising in tandem at least one open reading frame encoding for a caffeoyl CoA 3-O-methyltransferase enzyme or a fragment thereof under expression control of a first lignification-associated tissue specific promoter and at least one open reading frame encoding for a caffeic acid 3-O-methyltransferase enzyme or a fragment thereof under expression control of a second lignification-associated tissue specific promoter to form a transgenic cell, wherein said first and second lignification-associated tissue specific promoter can be the same or different; and    cultivating said transgenic cell under conditions conducive to regeneration and plant growth.    
     
     
         9 . A method for improving the digestibility of forage legumes comprising stably incorporating into the genome of said forage legume a DNA construct comprising at least one open reading frame encoding for a 3-O-methyltransferase enzyme or a fragment thereof from the lignin biosynthetic pathway under expression control of a lignification-associated tissue specific promoter, wherein expression of said enzyme or fragment produces a change in lignin composition in said forage legume.  
     
     
         10 . The method of  claim 9 , wherein said enzyme is caffeoyl CoA 3-O-methyltransferase.  
     
     
         11 . The method of  claim 10 , wherein said forage legume is co-transformed with at least one open reading frame encoding for a caffeic acid 3-O-methyltransferase enzyme or fragment thereof under expression control of a lignification-associated tissue specific promoter.  
     
     
         12 . The method of  claim 9 , wherein said DNA construct comprising in tandem at least one open reading frame encoding for a caffeoyl CoA 3-O-methyltransferase enzyme or a fragment thereof under expression control of a first lignification-associated tissue specific promoter and at least one open reading frame encoding for a caffeic acid 3-O-methyltransferase enzyme or a fragment thereof under expression control of a second lignification-associated tissue specific promoter, wherein said first and second lignification-associated tissue specific promoter can be the same or different.  
     
     
         13 . The method of  claim 9 ,  10 ,  11  or  12 , wherein guaiacyl lignin content is reduced.  
     
     
         14 . A method for producing a woody plant having altered lignin composition comprising 
 transforming a woody plant cell with a DNA construct comprising at least one open reading frame encoding for a  Medicago sativa  caffeoyl CoA 3-O-methyltransferase enzyme or fragment thereof under expression control of a lignification-associated tissue specific promoter to form a transgenic cell; and    cultivating said transgenic cell under conditions conducive to regeneration and plant growth.    
     
     
         15 . The method of  claim 14 , wherein said woody plant cell is co-transformed with a DNA construct comprising at least one open reading frame encoding for a  Medicago sativa  caffeic acid 3-O-methyltransferase enzyme or a fragment thereof under expression control of a lignification-associated tissue specific promoter.  
     
     
         16 . A method for producing a woody plant having altered lignin composition comprising 
 transforming a woody plant cell with a DNA construct comprising at least one open reading frame encoding for a  Medicago sativa  caffeic acid 3-O-methyltransferase enzyme or fragment thereof under expression control of a lignification-associated tissue specific promoter to form a transgenic cell; and    cultivating said transgenic cell under conditions conducive to regeneration and plant growth.    
     
     
         17 . A method for producing a woody plant having altered lignin composition comprising 
 transforming a woody plant cell with a DNA construct comprising in tandem at least one open reading frame encoding for a  Medicago sativa  caffeoyl CoA 3-O-methyltransferase enzyme or fragment thereof under expression control of a first lignification-associated tissue specific promoter and at least one open reading frame encoding for a  Medicago sativa  caffeic acid 3-O-methyltransferase enzyme or fragment thereof under expression control of a second lignification-associated tissue specific promoter to form a transgenic cell, wherein said first and second lignification-associated tissue specific promoter can be the same or different; and    cultivating said transgenic cell under conditions conducive to regeneration and plant growth.    
     
     
         18 . A method for modulating the lignin content of a woody plant comprising 
 transforming a woody plant cell with a DNA construct comprising at least one open reading frame encoding for a  Medicago saliva  caffeoyl CoA 3-O-methyltransferase enzyme or fragment thereof under expression control of a lignification-associated tissue specific promoter to form a transgenic cell; and    cultivating said transgenic cell under conditions conducive to regeneration and plant growth.    
     
     
         19 . The method of  claim 18 , wherein said woody plant cell is co-transformed with a DNA construct comprising at least one open reading frame encoding for a  Medicago sativa  caffeic acid 3-O-methyltransferase enzyme or a fragment thereof under expression control of a lignification-associated tissue specific promoter.  
     
     
         20 . A method for modulating the lignin content of a woody plant comprising 
 transforming a woody plant cell with a DNA construct comprising at least one open reading frame encoding for a  Medicago saliva  caffeic acid 3-O-methyltransferase enzyme or fragment thereof under expression control of a lignification-associated tissue specific promoter to form a transgenic cell; and    cultivating said transgenic cell under conditions conducive to regeneration and plant growth.    
     
     
         21 . A method for modulating the lignin content of a woody plant comprising 
 transforming a woody plant cell with a DNA construct comprising in tandem at least one open reading frame encoding for a  Medicago sativa  caffeoyl CoA 3-O-methyltransferase enzyme or fragment thereof under expression control of a first lignification-associated tissue specific promoter or fragment thereof and at least one open reading frame encoding for a  Medicago sativa  caffeic acid 3-O-methyltransferase enzyme or fragment thereof under expression control of a second lignification-associated tissue specific promoter to form a transgenic cell, wherein said first and second lignification-associated tissue specific promoter can be the same or different; and    cultivating said transgenic cell under conditions conducive to regeneration and plant growth.    
     
     
         22 . A method for making lignins with altered dimer bonding patterns comprising transforming a plant cell with a DNA construct comprising at least one open reading frame encoding for a caffeoyl CoA 3-O-methyltransferase enzyme or fragment thereof under expression control of a lignification-associated tissue specific promoter to form a transgenic cell; and 
 cultivating said transgenic cell under conditions conducive to regeneration and plant growth.    
     
     
         23 . The method of  claim 22 , wherein said plant cell is co-transformed with a DNA construct comprising at least one open reading frame encoding for a  Medicago sativa  caffeic acid 3-O-methyltransferase enzyme or a fragment thereof under expression control of a lignification-associated tissue specific promoter.  
     
     
         24 . A method for making lignins with altered dimer bonding patterns comprising transforming a plant cell with a DNA construct comprising at least one open reading frame encoding for a caffeic acid 3-O-methyltransferase enzyme or fragment thereof under expression control of a lignification-associated tissue specific promoter to form a transgenic cell; and 
 cultivating said transgenic cell under conditions conducive to regeneration and plant growth.    
     
     
         25 . A method for making lignins with altered dimer bonding patterns comprising transforming a plant cell with a DNA construct comprising in tandem at least one open reading frame encoding for a caffeoyl CoA 3-O-methyltransferase enzyme or fragment thereof under expression control of a first lignification-associated tissue specific promoter and at least one open reading frame encoding for a caffeic acid 3-O-methyltransferase enzyme or fragment thereof under expression control of a second lignification-associated tissue specific promoterto form a transgenic cell, wherein said first and second lignification-associated tissue specific promoter can be the same or different; and 
 cultivating said transgenic cell under conditions conducive to regeneration and plant growth.    
     
     
         26 . The method of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 ,  13 ,  14 ,  15 ,  16 ,  17 ,  18 ,  19 ,  20 ,  21 ,  22 ,  23 ,  24  or  25 , wherein the open reading frame is in a sense orientation.  
     
     
         27 . The method of  claim 1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  11 ,  12 ,  13 ,  14 ,  15 ,  16 ,  17 ,  18 ,  19 ,  20 ,  21 ,  22 ,  23 ,  24  or  25 , wherein the open reading frame is in an antisense orientation.  
     
     
         28 . The method of  claim 26 , wherein said promoter is a bean PAL2 promoter.  
     
     
         29 . The method of  claim 27 , wherein said promoter is a bean PAL2 promoter.  
     
     
         30 . A plant transformed by the method of  claim 26 .  
     
     
         31 . A plant transformed by the method of  claim 27 .  
     
     
         32 . A plant transformed by the method of  claim 28 .  
     
     
         33 . A plant transformed by the method of  claim 29.