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 phototoxic 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 for stably transforming a plastid genome of a solanaceous plant species where plant growth is inhibited by an antibiotic-free phytotoxic agent, wherein said integration and expression plastid vector comprises an expression cassette which comprises as operably joined components a 5′ end of a plastid DNA spacer sequence, a DNA sequence encoding a detoxifying enzyme, where said detoxifying enzyme is capable of detoxifying said phytotoxic agent in a cell to the corresponding nontoxic compound, a transcription termination region functional in said plastid, and a 3′ end of a plastid DNA spacer sequence, wherein the antibiotic-free phytotoxic agent is a phytotoxic aldehyde and the detoxifying enzyme is a plant betaine aldehyde dehydrogenase capable of detoxifying said phytotoxic aldehyde. 
     
     
         2 . The vector of  claim 1 , wherein said vector further comprises at least one heterologous DNA sequence coding for at least one molecular of interest wherein said at least one heterologous DNA sequence is heterologous to said vector, and wherein said heterologous DNA sequence is located between said 5′ and said 3′ end. 
     
     
         3 . The vector of  claim 1 , wherein said vector further comprises a ribosome binding site and a 5′ untranslated region (5′ UTR), located upstream of said DNA sequence coding for the detoxifying enzyme. 
     
     
         4 . The vector of  claim 1 , wherein the antibiotic-free phytotoxic agent is a phytotoxic aldehyde and the detoxifying enzyme is an aldehyde dehydrogenase capable of detoxifying said phytotoxic aldehyde. 
     
     
         5 . The vector of  claim 2 , wherein said at least one molecule of interest is a polypeptide. 
     
     
         6 . The vector of  claim 3 , wherein said plastid is a plant chloroplast. 
     
     
         7 . The vector of  claim 1 , wherein said phytotoxic aldehyde is selected from the group consisting of acetaldehyde, formaldehyde, butyraldehyde, and betaine aldehyde. 
     
     
         8 . An integration and expression plastid vector for stably transforming a plastid genome of a solanaceous plant species wherein plant growth is inhibited by betaine aldehyde, wherein said integration and expression plastid vector comprises an expression cassette which comprises as operably joined components, a 5′ end of a plastid DNA 16S-23S spacer sequence, a promoter operative in said plastid genome, a DNA sequence encoding a plant betaine aldehyde dehydrogenase (BADH) as a selectable marker which is capable of detoxifying betaine aldehyde in said cells to the corresponding non-toxic compound, a DNA sequence hereterologous to said vector which codes for a molecule of interest, a transcription termination region functional in said plastic genome, and a 3′ end of a plastid DNA the spacer sequence. 
     
     
         9 . A stably transformed plant of a plant species that is naturally inhibited by betaine aldehyde, wherein said plant comprises a plastid and a plastid genome that has been stably transformed with an integration and expression vector, wherein said integration and expression vector comprises operably joined components, a 5′ end of plastid DNA spacer sequence, a 5′ regulatory region having a ribosome binding site operative in said plastid genome, a DNA sequence encoding a plant betaine aldehyde dehydrogenase (BADH) which is capable of detoxifying betaine aldehyde, a 3′ regulatory region function in said plastid genome and a 3′ end of a plastid DNA spacer sequence. 
     
     
         10 . The stably transformed plant of  claim 9 , wherein the plant is a solanaceous plant edible for a mammal. 
     
     
         11 . The stably transformed plant of  claim 9 , wherein the plant is a monocotyledonous plant, selected from the group consisting of rice, wheat, grass, rye, barley, oat and maize. 
     
     
         12 . The stably transformed plant of  claim 9 , wherein the plant is a dicotyledonous plant selected from the group consisting of soybean, peanut, grape, sweet potato, pea canola, tobacco tomato, and cotton. 
     
     
         13 . The stably transformed plant of  claim 9 , wherein the plant is a tobacco, tomato, potato, rice,  brassica , cotton, maize, or soybean plant. 
     
     
         14 . The stably transformed plant of  claim 9 , wherein the plant is a homoplasmic plant. 
     
     
         15 . The vector of  claim 2  wherein the expression of said detoxifying enzyme is driven by a promoter selected from the group consisting of 16rRNA promoter, the psbA promoter, the atpBpromoter, and or the accD promoter. 
     
     
         16 . An integration and expression plastid vector competent for stably transforming a tobacco plastid genome, said vector comprising an expression cassette which comprises as operably joined components, a 5′ part of the plastid DNA spacer sequence, a promoter operative in said tobacco plastid genome, a DNA sequence encoding spinach betaine aldehyde dehydrogenase (BADH) which is capable of detoxifying said phytotoxic aldehyde in the cells to glycine betaine, a heterologous DNA sequence which codes for a molecule of interest and is heterologous to said vector, a transcription termination region function in said tobacco plastid genome, and a 3′ part of a plastid DNAa the spacer sequence. 
     
     
         17 . A progeny plant of the stably transformed plant of  claim 9 , said progeny plant comprising chloroplasts comprising genomes comprising said DNA sequence encoding said BADH. 
     
     
         18 . The vector of  claim 1 , wherein said plant betaine aldehyde dehydrogenase is a spinach betaine aldehyde dehydrogenase. 
     
     
         19 . The vector of  claim 8 , wherein said plant betaine aldehyde dehydrogenase is a betaine aldehyde dehydrogenase. 
     
     
         20 . The vector of  claim 1  wherein said plant species is tobacco. 
     
     
         21 . The vector of  claim 8 , wherein said plant species is tobacco. 
     
     
         22 . A transformed plant comprising a plurality of plastids comprising a plastid genome and plant transgene is said plastid genome, wherein expression of said plant transgene in said plastids renders the plant resistant to a non-antibiotic phytotoxic agent, wherein said non-antibiotic phytotoxic agent is a lethal aldehyde.