Marker free transgenic plants engineering the chloroplast genome without the use of antibiotic selection

Abstract

The present invention provides for a method to circumvent the problem of using antibiotic resistant selectable markers. In particular, target plants are transformed using a plastid vector which contains heterologous DNA sequences coding for a phytotoxin detoxifying enzyme or protein. The selection process involves converting an antibiotic-free phytotoxic agent by the expressed phytotoxin detoxifying enzyme or protein to yield a nontoxic compound. The invention provides for various methods to use antibiotic-free selection in chloroplast transformation.

1 . An integration and expression plastid vector competent for stably transforming the plastid genome where growth is inhibited by an antibiotic-free phytotoxic agent which comprises an expression cassette which comprises as operably joined components, a 5′ part of the plastid DNA sequence inclusive of a spacer sequence, a promoter operative in said plastid, a DNA sequence encoding a detoxifying enzyme or protein acting as a selectable marker which is capable of detoxifying said antibiotic-free phytotoxic agent in the cells to the corresponding nontoxic compound, at least one restriction site for the insertion of a heterologous target gene, a transcription termination region functional in said plastid, and the 3′ part of a plastid DNA sequence inclusive of the spacer sequence.  
     
     
         2 . The vector of  claim 1  wherein a heterologous DNA sequence coding for a molecule of interest is inserted in one of the restriction sites.  
     
     
         3 . The vector of  claim 1  wherein said vector further comprises a ribosome binding site and a 5′ untranslated region (5′ UTR).  
     
     
         4 . A vector of  claim 1 , wherein the antibiotic-free phytotoxic agent is a phytotoxic aldehyde and the detoxifying enzyme or protein is a aldehyde dehydrogenase capable of detoxifying said phytotoxic aldehyde.  
     
     
         5 . A chloroplast vector of  claim 2  wherein the molecule of interest is a polypeptide.  
     
     
         6 . A vector of  claim 3  or  claim 4 , wherein plastid is tobacco chloroplast.  
     
     
         7 . A chloroplast vector which is described in FIG. 1 
     
     
         8 . A vector of  claim 4  competent for stably transforming the chloroplast genome where growth is inhibited by a phytotoxic aldehyde which is selected from the group consisting of acetaldehyde, formaldehyde, propronaldehyde, bytyraldehyde and betaine aldehyde.  
     
     
         9 . An integration and expression plastid vector competent for stably transforming the plastid genome where growth is inhibited by a phytotoxic aldehyde which comprises an expression cassette which comprises as operably joined components, a 5′ part of the plastid DNA sequence inclusive of a spacer sequence, a promoter operative in said plastid, a DNA sequence encoding betaine aldehyde dehydrogenase (BADM) as a selectable marker which is capable of detoxifyng said phytotoxic aldehyde in the cells to glycine betaine, a heterologous DNA sequence which codes for a molecule of interest, a transcription termination region functional in said plastid, and a 3′ part of a plastid DNA sequence inclusive of the spacer sequence.  
     
     
         10 . A stably transformed plant which comprises a chloroplast which has been stably transformed with a vector of  claim 8  or  claim 9 , or the progeny thereof.  
     
     
         11 . The stably transformed plant of  claim 10 , wherein the plant is a solanaceous plant edible for a mammal.  
     
     
         12 . The stably transformed plant of  claim 10 , wherein the plant is a crop plant edible for a mammal.  
     
     
         13 . A stably transformed plant of either  claim 11  or  claim 12 , wherein the mammal is a human.  
     
     
         14 . A stably transformed plant of  claim 10 , wherein the plant is a monocotyledonous plant, selected from the group of rice, wheat, grass, rye, barley, oat, or maize.  
     
     
         14 . A stably transformed plant of  claim 10 , wherein the plant is a dicotyledonous plant, selected from the group of soybean, peanut, grape, sweet potato, pea, canola, tobacco, tomato or cotton.  
     
     
         15 . A stable transformed plant of  claim 10 , wherein the plant is a tobacco, tomato, potato, rice, brassica, cotton, maize or soybean.  
     
     
         16 . A stable transformed plant of  claim 10 , wherein the plant is a homoplasmic plant.  
     
     
         17 . A vector of any one of claims  2 - 9 , wherein the selectable marker is driven by a promoter in green and non-green tissues selected from the group consisting of the 16SrRNA promoter, the psbA promoter, the alpB promoter, or the accD promoter.  
     
     
         18 . A method for transforming the plastid genome of a plant cell, which method does not require selection for successful transformants by the detection of antibiotic resistance, said method comprising introducing into cells of a plant species whose growth is inhibited by an antibiotic-free phytotoxic agent, an expression cassette which comprises as operably linked components, a 5′ part of a plastid DNA sequence inclusive of a spacer sequence, a promoter operative in said plastid, a DNA sequence encoding a detoxifying enzyme or protein acting as a selectable marker for transgenic plant cells and capable of detoxifying said phytotoxic agent in the cells to the corresponding nonoxic compound, a heterologous target DNA sequence, a transcription termination region functional in said plant chloroplast cells, and the 3′ part of the plastid DNA sequence inclusive of a spacer sequence.  
     
     
         19 . The method of  claim 18  wherein the heterologous target DNA sequence codes for a molecule of interest.  
     
     
         20 . The method of  claim 18  wherein the antibiotic-free phytotoxic agent codes for a phytotoxic aldehyde and the detoxifying enzyme or protein is a aldehyde dehydrogenase capable of detoxifying said phytotoxic aldehyde.  
     
     
         21 . The method of  claim 18  wherein the phytotoxic aldehyde which is selected from the group consisting of acetaldehyde, formaldehyde, propronaldehyde, bytyraldehyde and betaine aldehyde.  
     
     
         22 . A method of  claim 18 , wherein said method further comprises culturing said plant in a plant growth medium comprising said phytotoxic aldehyde, and selecting transformed plant cells capable of growth in the presence of said phytotoxic aldehyde.  
     
     
         23 . A method of  claim 22 , wherein said method further comprises regenerating a transformed plant from said transformed plant cells.  
     
     
         24 . A method of  claim 20  wherein said phytotoxic aldehyde is betaine aldehyde and the aldehyde dehydrogenase is betaine aldehyde dehydrogenase (BADH).  
     
     
         25 . A method of  claim 24 , wherein said DNA sequence is from plants such as sugar beet, or spinach.  
     
     
         26 . A method of  claim 24 , wherein said DNA sequence is from a microorganism such as  E. coli.    
     
     
         27 . A method of  claim 18 , wherein the promoter is selected from a group consisting of 16SrRNA, psbA, accD and alpB promoters.  
     
     
         28 . A vector of  claim 2 ,  3  or  17 , wherein the phytotoxic agent is selected from a group consisting of an herbicide listed in Table  18 . 4  of Molecular Biotechnology by Glick, light, betaine aldehyde and polyethylene glycol, and wherein the detoxifying agent is selected from a group consisting of an enzyme or protein capable of detoxifying said herbicide, the ch1B gene, betaine aldehyde dehydrogenase, or the TSP1 gene.  
     
     
         29 . A method of any one of claims  18 - 27 , where the expression cassette farther comprises a ribosome binding site (rbs) and a 5′ untranslated `region (5′UTR) to enhance expression.