Methods for simultaneous control of lignin content and composition, and cellulose content in plants

Abstract

The present invention relates to a method of concurrently introducing multiple genes into plants and trees is provided. The method includes simultaneous transformation of plants with multiple genes from the phenylpropanoid pathways including 4CL, CAld5H, AldOMT, SAD and CAD genes and combinations thereof to produce various lines of transgenic plants displaying altered agronomic traits. The agronomic traits of the plants are regulated by the orientation of the specific genes and the selected gene combinations, which are incorporated into the plant genome.

What is claimed is:  
     
         1 . A method of genetically transforming a plant simultaneously with multiple genes from the phenylpropanoid pathways, comprising incorporating into the genome of the plant a plurality of genes, the genes selected from the group consisting of 4CL, CAld5H, AldOMT, CAD, and SAD, substantially similar fragments thereof, and combinations thereof to produce plants displaying altered agronomic traits.  
     
     
         2 . The method of  claim 1  wherein the genes incorporated into the genome are CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof or combinations thereof in sense orientation, to produce increased syringyl lignin in the plant compared to a non-transformed plant.  
     
     
         3 . The method of  claim 1 , wherein the genes incorporated into its genome are 4CL gene or substantially similar fragments thereof in a sense or antisense orientation and CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof or combinations thereof in sense orientation to downregulate 4CL gene expression in the plant compared to a non-transformed plant.  
     
     
         4 . The method of  claim 3  wherein the down regulation of 4CL correlates with decreased lignin content, increased syringyl/guaiacyl (S/G) lignin ratio and increased cellulose content compared to a non-transformed plant.  
     
     
         5 . The method of  claim 1 , wherein the genes incorporated into its genome are 4CL and CAD genes or substantially similar fragments thereof in sense or antisense orientation and CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof or combinations thereof in sense orientation to downregulate 4CL and CAD gene expression in the plant compared to a non-transformed plant.  
     
     
         6 . The method of  claim 5  wherein the down regulation of 4CL and CAD correlates with decreased lignin content, increased S/G lignin ratio and increased cellulose content as compared to a non-transformed plant.  
     
     
         7 . The method of  claim 1  wherein the gene incorporated into the genome of the plant is 4CL gene or substantially similar fragments thereof in the sense orientation, to upregulate 4CL gene expression in the plant compared to a non-transformed plant.  
     
     
         8 . The method of  claim 7 , wherein the upregulation of 4CL gene correlates to increased lignin content in the plant compared to a non-transformed plant.  
     
     
         9 . A method of  claim 1  wherein the genes incorporated into the genome of the plant are 4CL, CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof or combinations thereof in the sense orientation, to upregulate 4CL, CAld5H, AldOMT, and SAD gene expression  
     
     
         10 . The method of  claim 9  wherein the upregulation of 4CL, CAld5H, AldOMT, and SAD genes correlates to an increased lignin content and increased S/G ratio compared to a non-transformed plant.  
     
     
         11 . The method of  claim 1  wherein the genes incorporated into the genome of the plant 4CL, CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof or combinations thereof in the sense orientation and the CAD gene or substantially similar fragments thereof in the antisense orientation, to upregulate 4CL, CAld5H, AldOMT, and SAD gene expression and to downregulate CAD gene expression in the plant compared to a non-transformed plant.  
     
     
         12 . The method of  claim 11 , wherein the upregulation of 4CL, CAld5H, AldOMT, and SAD gene expression and the downregulation of CAD gene expression correlate with an increased lignin content and an increased S/G ratio compared to a non-transformed plant.  
     
     
         13 . The method of  claim 2  wherein the plant is an angiosperm or a gymnosperm.  
     
     
         14 . The method of  claim 3  wherein the plant is an angiosperm or a gymnosperm.  
     
     
         15 . The method of  claim 5  wherein the plant is an angiosperm or a gymnosperm.  
     
     
         16 . A method of preparing plant cells having in their genome a plurality of DNA constructs, the method comprising 
 a) incorporating into the genome of the cells a plurality of DNA constructs to yield transformed cells, each construct comprising a polynucleotide sequence encoding a protein selected from the group consisting of 4CL, CAld5H, AldOMT, CAD, and SAD, substantially similar fragments thereof, and combinations thereof, operably linked to a promoter sequence functional in the cells, and a termination sequence    b) identifying the transformed plant cells, the genome of which is augmented with DNA from the different DNA constructs.    
     
     
         17 . The method of  claim 16  wherein the expression of the protein is associated with an agronomic trait in the plant cells.  
     
     
         18 . The method of  claim 17  wherein the trait is lignin biosynthesis, cellulose biosynthesis, growth, wood quality, stress resistance, sterility, grain yield or nutritional value.  
     
     
         19 . The method of  claim 16  further comprising regenerating the identified transformed plants cells to yield a transgenic plant.  
     
     
         20 . The method of  claim 16  wherein the plant cells are regenerable.  
     
     
         21 . The method of  claim 16  wherein the plant cells are tree cells.  
     
     
         22 . The method of  claim 16  wherein the plant cells are angiosperm cells.  
     
     
         23 . The method of  claim 16  wherein the plant cells are gymnosperm cells.  
     
     
         24 . A method of preparing transgenic plants having altered lignin and cellulose compositions, the method comprising 
 a) providing the genome of the plants with a plurality of DNA constructs to yield transformed plant cells, each construct comprising a polynucleotide sequence encoding a protein selected from the group consisting of 4CL, CAld5H, AldOMT, CAD, and SAD, substantially similar fragments thereof, and combinations thereof, the polynucleotide sequence operably linked to a promoter sequence functional in the plant cells, and a termination sequence;    b) regenerating the transformed plant cells to yield transgenic plants, the genome of which is augmented with DNA from different DNA constructs, and    c) expressing the DNA constructs in the cells of the transgenic plants in an amount effective to alter the lignin and cellulose composition in the plants.    
     
     
         25 . A plant of  claim 24  wherein the promoter sequence can be constitutive or tissue-specific.  
     
     
         26 . A plant of  claim 24  wherein the promoter sequence can be homologous or heterologous.  
     
     
         27 . A plant if  claim 24  wherein the promoter sequence provides for transcription in xylem.  
     
     
         28 . A method of  claim 24 , wherein the plant is a plant cell, plant organ, or an entire plant.  
     
     
         29 . A method of  claim 24 , wherein the plant is a plant fruit, seeds and progeny thereof.  
     
     
         30 . The method of  claim 24  wherein the plants are trees.  
     
     
         31 . The method of  claim 24  wherein the plants are angiosperms.  
     
     
         32 . The method of  claim 24  wherein the plants are gymnosperms.  
     
     
         33 . A transgenic plant prepared by the method of  claim 24 .  
     
     
         34 . A progeny plant of the transgenic plant of  claim 32 .  
     
     
         35 . A method of preparing a transgenic tree comprising 
 a) incorporating into the genome of the tree a plurality of desired DNA constructs to produce transformed tree cells, each construct comprising a polynucleotide sequence encoding a protein selected from the group consisting of 4CL, CAld5H, AldOMT, CAD, and SAD, substantially similar fragments thereof, and combinations thereof, operably linked to a promoter sequence functional in the cells, and a termination sequence;    b) regenerating the transformed tree cells to yield transgenic trees, the genome of which is augmented with the plurality of DNA constructs; and    c) expressing the DNA construct in the cells of the transgenic tree in an amount effective to alter the lignin and cellulose composition of the tree.    
     
     
         36 . A transgenic tree prepared by the method of  claim 34 .  
     
     
         37 . The method of  claim 34  wherein the transgenic tree is a  Populus tremuloides.    
     
     
         38 . A plant having incorporated into its genome a DNA construct comprising a polynucleotide sequence encoding a protein selected from the group consisting of 4CL, CAld5H, AldOMT, CAD, and SAD, substantially similar fragments thereof, and combinations thereof, operably linked to a promoter sequence, and a termination sequence.  
     
     
         39 . A plant of  claim 38  wherein the promoter sequence can be constitutive or tissue-specific.  
     
     
         40 . A plant of  claim 38  wherein the promoter sequence can be homologous or heterologous.  
     
     
         41 . A plant of  claim 38  wherein the promoter sequence provides for transcription in xylem.  
     
     
         42 . The plant of  claim 38  which is a tree.  
     
     
         43 . A plant of  claim 38  wherein the plant is a gymnosperm.  
     
     
         44 . The plant of  claim 43  wherein the nucleotide sequence encodes CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof and combinations thereof, in sense orientation, yielding increased syringyl lignin as compared to a non-transformed plant.  
     
     
         45 . The plant of  claim 43  wherein the nucleotide sequences encodes 4CL gene or substantially similar fragments thereof in sense or antisense orientation and CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof, and combinations thereof, in sense orientation, yielding decreased lignin content, increased syringyl/guaiacyl (S/G) lignin ratio and increased cellulose content as compared to a non-transformed plant.  
     
     
         46 . The plant of  claim 43  wherein the nucleotide sequences encodes 4CL and CAD genes or substantially similar fragments thereof in sense or antisense orientation and CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof, and combinations thereof, in sense orientation, yielding decreased lignin content, increased S/G lignin ratio and increased cellulose content as compared to a non-transformed plant.  
     
     
         47 . The plant of  claim 43  wherein the nucleotide sequences encodes 4CL gene or substantially similar fragments thereof in the sense orientation yielding increased lignin content as compared to a non-transformed plant.  
     
     
         48 . The plant of  claim 43  wherein the polynucleotide sequences incorporated into the genome are 4CL, CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof, and combinations thereof, in the sense orientation, yielding increased lignin content and increased S/G ratio as compared to a non-transformed plant.  
     
     
         49 . The plant of  claim 43  wherein the polynucleotide sequences encodes 4CL, CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof and combinations thereof in the sense orientation and the CAD gene, or substantially similar fragments thereof in the antisense orientation, yielding increased lignin content and increased S/G ratio as compared to a non-transformed plant.  
     
     
         50 . A plant of  claim 38  wherein the plant is an angiosperm.  
     
     
         51 . The plant of  claim 50  wherein the polynucleotide sequences encodes CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof and combinations thereof, in sense orientation, yielding increased S/G lignin ratio as compared to a non-transformed plant.  
     
     
         52 . The plant of  claim 51  wherein the polynucleotide sequences encode the 4CL gene or substantially similar fragments thereof in sense or antisense orientation, and CAld5H, AldOMT, and SAD genes, substantially similar fragment thereof, and combinations thereof in sense orientation, yielding decreased lignin content, increased S/G lignin ratio and increased cellulose content as compared to a non-transformed plant.  
     
     
         53 . The plant of  claim 50  wherein the nucleotide sequences encodes 4CL and CAD genes or substantially similar fragments thereof, and combinations thereof by sense or antisense orientation and CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof, and combinations thereof in sense orientation, yielding decreased lignin content, increased S/G lignin ratio and increased cellulose content as compared to a non-transformed plant.  
     
     
         54 . The plant of  claim 50  wherein the nucleotide sequences encodes 4CL gene or substantially similar fragment thereof, by sense orientation, yielding increased lignin content as compared to a non-transformed plant.  
     
     
         55 . The plant of  claim 50  wherein the nucleotide sequences encodes 4CL, CAld5H, AldOMT, and SAD genes, substantially similar fragment thereof, and combinations thereof, in sense orientation, yielding increased lignin content and increased S/G ratio as compared to a non-transformed plant.  
     
     
         56 . The plant of  claim 50  wherein the nucleotide sequences encode 4CL CAld5H, AldOMT, and SAD genes, substantially similar fragment thereof, and combinations thereof, in sense orientation, and CAD gene, substantially similar fragment thereof, and combinations thereof in antisense orientation, yielding increased lignin content and increased S/G ratio as compared to a non-transformed plant.  
     
     
         57 . A plurality of DNA constructs, each construct comprising in the 5′-3′ direction: 
 a) a gene promoter sequence,  
 b) a gene termination sequence; and  
 c) a polynucleotide sequence encoding a protein selected from the group consisting of 4CL, CAld5H, AldOMT, CAD, and SAD, substantially similar fragments thereof, and combinations thereof involved in a lignin biosynthetic pathway, the polynucleotide sequence operably linked to the promoter and termination sequences.  
 
     
     
         58 . A plurality of DNA constructs of  claim 57  wherein the gene promoter sequence is a constitutive or tissue-specific promoter.  
     
     
         59 . A plurality of DNA constructs of  claim 57  wherein the gene promoter sequence is homologous or heterologous.  
     
     
         60 . A plurality of DNA constructs of  claim 57  wherein the gene promoter sequences are xylem-specific.  
     
     
         61 . A plurality of DNA constructs incorporated into the genome of gymnosperms, the constructs comprising nucleotide sequences encoding CAld5H, AldOMT, and SAD genes, a substantially similar fragments thereof and combinations thereof, in sense orientation, to produce syringyl lignin as compared to a non-transformed plant.  
     
     
         62 . A plurality of DNA constructs incorporated into the genome of gymnosperm, the constructs comprising 4CL gene or substantially similar fragments thereof in sense or antisense orientation and CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof and combination thereof in sense orientation to decrease lignin content, increase syringyl/guaiacyl (S/G) lignin ratio and increase cellulose content as compared to a non-transformed plant.  
     
     
         63 . A plurality of DNA constructs incorporated into the genome of gymnosperms, the constructs comprising 4CL and CAD genes or substantially similar fragments thereof in sense or antisense orientation and CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof and combination thereof in sense orientation to decrease lignin content, increase S/G lignin ratio and increase cellulose content as compared to a non-transformed plant.  
     
     
         64 . A DNA construct incorporated into the genome of plants, comprising 4CL gene or substantially similar fragments thereof in the sense orientation to increase lignin content compared to a non-transformed plant.  
     
     
         65 . A DNA construct of  claim 63  wherein the plant is an angiosperm or a gymnosperm.  
     
     
         66 . A plurality of DNA constructs incorporated into the genome of plants, the constructs comprising 4CL, CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof or combination thereof, in the sense orientation, to increase lignin content and increase S/G ratio compared to a non-transformed plant.  
     
     
         67 . A plurality of DNA constructs of  claim 65  wherein the plant is an angiosperm or a gymnosperm.  
     
     
         68 . A plurality of DNA constructs incorporated into the genome of plants, the constructs comprising 4CL, CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof and combination thereof in the sense orientation and CAD gene or substantially similar fragments thereof in the antisense orientation to increase lignin content and increase S/G ratio compared to a non-transformed plant.  
     
     
         69 . A plurality of DNA constructs of  claim 67  wherein the plant is an angiosperm or a gymnosperm.  
     
     
         70 . A plurality of DNA constructs incorporated into the genome of angiosperms, the constructs comprising CAld5H, AldOMT, and SAD genes, substantially similar fragments thereof and combinations thereof in sense orientation to engineer high S/G lignin ratio compared to a non-transformed plant.  
     
     
         71 . A plurality of DNA constructs incorporated incorporating into the genome of angiosperms, the constructs comprising 4CL gene or substantially similar fragment thereof in sense or antisense orientation and CAld5H, AldOMT, and SAD genes, substantially similar fragment thereof, and combinations thereof, in sense orientation, to decrease lignin content, increase S/G lignin ratio and increase cellulose content compared to a non-transformed plant.  
     
     
         72 . A plurality of DNA constructs incorporated into the genome of angiosperms, the constructs comprising 4CL and CAD genes, substantially similar fragments thereof, and combinations thereof by sense or antisense orientation and CAld5H, AldOMT, and SAD genes, or substantially similar fragments thereof, and combinations thereof, in sense orientation, to decrease lignin content, increase S/G lignin ratio and increase cellulose content compared to a non-transformed plant.