Genetically engineered duckweed

Abstract

Methods and compositions for the efficient transformation of duckweed are provided. Preferably, the methods involve transformation by either ballistic bombardment or Agrobacterium . In this manner, any gene or nucleic acid of interest can be introduced and expressed in duckweed plants. Transformed duckweed plants, cells, tissues are also provided. Transformed duckweed plant tissue culture and methods of producing recombinant proteins and peptides from transformed duckweed plants are also disclosed.

1 . A stably transformed duckweed tissue culture produced according to a method comprising the steps of: 
 (a) providing a duckweed tissue target, the cells of the duckweed tissue culture including cell walls; and    (b) propelling a heterologous nucleotide sequence of interest at the duckweed tissue target at a velocity sufficient to pierce the cell walls and deposit the heterologous nucleotide sequence within a cell of the tissue,     wherein the heterologous nucleotide sequence is carried by a microprojectile; and     wherein the heterologous nucleotide sequence is propelled at the tissue by propelling the microprojectile at the tissue; and    (c) producing a stably transformed duckweed tissue culture.    
   
   
       2 . The stably transformed duckweed tissue culture according to  claim 1 , wherein the nucleotide sequence comprises at least one expression cassette comprising a gene which confers resistance to a selection agent.  
   
   
       3 . The stably transformed tissue culture according to  claim 2  further comprising the step of culturing the stably transformed tissue culture with the selection agent.  
   
   
       4 . The stably transformed duckweed tissue culture according to  claim 2 , wherein the gene which confers resistance to a selection agent is selected from the group consisting of neo, bar, pat, ALS, HPH, HYG, EPSP and Hml.  
   
   
       5 . The stably transformed duckweed tissue culture according to  claim 1 , wherein the nucleotide sequence comprises two genes of interest.  
   
   
       6 . The stably transformed duckweed tissue culture according to  claim 1 , wherein the nucleotide sequence encodes a protein or peptide selected from the group consisting of insulin, growth hormone, α-interferon, β-glucocerebrosidase, retinoblastoma protein, p53 protein, angiostatin, leptin, and serum albumin.  
   
   
       7 . The stably transformed duckweed tissue culture according to  claim 1 , wherein the nucleotide sequence encodes at least one protein or peptide subunit of a multimeric protein selected from the group consisting of hemoglobin, collagen, P450 oxidase, and a monoclonal antibody.  
   
   
       8 . The stably transformed duckweed tissue culture according to  claim 1 , wherein the nucleotide sequence encodes a secreted protein or peptide.  
   
   
       9 . The stably transformed duckweed tissue culture according to  claim 1 , wherein the duckweed tissue culture is a callus tissue culture.  
   
   
       10 . The stably transformed duckweed callus tissue culture according to  claim 9 , wherein the duckweed callus tissue culture is a Type I callus tissue culture.  
   
   
       11 . The stably transformed duckweed tissue culture according to  claim 1 , wherein the duckweed tissue culture is a  Spirodela, Wolffia, Wolfiella , or  Lemna  tissue culture.  
   
   
       12 . The stably transformed duckweed tissue culture according to  claim 11 , wherein the duckweed tissue culture is a  Lemna  tissue culture.  
   
   
       13 . The stably transformed duckweed tissue culture according to  claim 12 , wherein the duckweed tissue culture is a  Lemna minor, Lemna miniscula , or  Lemna gibba  tissue culture.  
   
   
       14 . The stably transformed duckweed tissue culture according to  claim 13 , wherein the duckweed tissue culture is a  Lemna minor  tissue culture.  
   
   
       15 . A stably transformed duckweed tissue culture comprising a chimeric nucleotide sequence of interest, said chimeric nucleotide sequence comprising a coding sequence operably linked to a transcription initiation region that is heterologous to said coding sequence, the method comprising the steps of: 
 (a) providing a duckweed tissue culture target, the cells of the duckweed tissue culture including cell walls; and    (b) propelling a chimeric nucleotide sequence of interest at the duckweed tissue culture target at a velocity sufficient to pierce the cell walls and deposit the chimeric nucleotide sequence within a cell of the tissue, 
 wherein the chimeric nucleotide sequence is carried by a microprojectile;  
 and wherein the chimeric nucleotide sequence is propelled at the tissue by propelling the microprojectile at the tissue; and  
   (c) producing a stably transformed duckweed tissue culture.    
   
   
       16 . The stably transformed duckweed tissue culture according to  claim 15 , wherein the chimeric nucleotide sequence comprises a duckweed coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.  
   
   
       17 . The stably transformed duckweed tissue culture according to  claim 15 , wherein the chimeric nucleotide sequence of interest comprises at least one expression cassette comprising a gene which confers resistance to a selection agent.  
   
   
       18 . The stably transformed tissue culture according to  claim 17  further comprising the step of culturing the stably transformed tissue culture with the selection agent.  
   
   
       19 . The stably transformed duckweed tissue culture according to  claim 17 , wherein the gene which confers resistance to a selection agent is selected from the group consisting of neo, bar, pat, ALS, HPH, HYG, EPSP and Hml.  
   
   
       20 . The stably transformed duckweed tissue culture according to  claim 15 , wherein the chimeric nucleotide sequence comprises two genes of interest.  
   
   
       21 . The stably transformed duckweed tissue culture according to  claim 15 , wherein the chimeric nucleotide sequence encodes a protein or peptide selected from the group consisting of insulin, growth hormone, α-interferon, β-glucocerebrosidase, retinoblastoma protein, p53 protein, angiostatin, leptin, and serum albumin.  
   
   
       22 . The stably transformed duckweed tissue culture according to  claim 15 , wherein the chimeric nucleotide sequence encodes at least one protein or peptide subunit of a multimeric protein selected from the group consisting of hemoglobin, collagen, P450 oxidase, and a monoclonal antibody.  
   
   
       23 . The stably transformed duckweed tissue culture according to  claim 15 , wherein the chimeric nucleotide sequence encodes a secreted protein or peptide.  
   
   
       24 . The stably transformed duckweed tissue culture according to  claim 15 , wherein the duckweed tissue culture is a callus tissue culture.  
   
   
       25 . The stably transformed duckweed callus tissue culture according to  claim 24 , wherein the duckweed callus tissue culture is a Type I callus tissue culture.  
   
   
       26 . The stably transformed duckweed tissue culture according to  claim 15 , wherein the duckweed tissue culture is a  Spirodela, Wolffia, Wolfiella , or  Lemna  tissue culture.  
   
   
       27 . The stably transformed duckweed tissue culture according to  claim 26 , wherein the duckweed tissue culture is a  Lemna  tissue culture.  
   
   
       28 . The stably transformed duckweed tissue culture according to  claim 27 , wherein the duckweed tissue culture is a  Lemna minor, Lemna miniscula , or  Lemna gibba  tissue culture.  
   
   
       29 . The stably transformed duckweed tissue culture according to  claim 28 , wherein the duckweed tissue culture is a  Lemna minor  tissue culture.