Compositions and methods for improving plastid transformation efficiency in higher plants

Abstract

Compositions and methods for improving plastid transformation in difficult to transform plants are disclosed.

1 . A method for increasing plastid transformation efficiency in plastids of a  Brassica  ssp. plant, comprising;
 a) providing a plant comprising a nonfunctional or defective ACC2 nuclear gene;   b) introducing one or more plastid transformation vectors into the plastids in cells from said plant, said one or more vectors comprising an aadA spectinomycin resistance marker sequence and a nucleic acid sequence encoding a protein of interest;   c) contacting said cells with spectinomycin and selecting plant cells which are resistant to spectinomycin and accumulate said protein of interest in said plastids; and   d) culturing said plant cells under conditions suitable to regenerate a transplastomic plant therefrom.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein said protein of interest is green fluorescent protein. 
     
     
         4 . The method of  claim 1 , wherein the plant of step a) is a naturally occurring mutant which encodes non-functional or defective ACC2. 
     
     
         5 . The method of  claim 1 , wherein said ACC2 gene is inactivated in said plant using CRISPR/Cas prior to plastid transformation. 
     
     
         6 .- 7 . (canceled) 
     
     
         8 . The method of  claim 1 , further comprising excising said aadA spectinomycin resistance marker sequence from said plant. 
     
     
         9 . The method of  claim 5 , wherein said protein of interest is selected from the group consisting of a protein conferring herbicide resistance, a protein conferring insect resistance, a vaccine, an antibody, regulatory RNA, dsRNA, siRNA, shRNA and insecticidal proteins. 
     
     
         10 . A method for seed-specific plastid expression comprising:
 a) introducing a nuclear expression vector encoding a modified PPR10 binding protein driven by a seed-specific promoter and   b) a plastid expression vector encoding a gene of interest linked to an upstream PPR10 binding site, wherein nuclear-expressed PPR10 is imported into plastids and binds said PPR10 binding site to drive expression of the gene of interest in seed plastids.   
     
     
         11 . The method of  claim 10 , wherein said vector comprises a seed specific promoter selected from a napin or a phaseolin gene promoter. 
     
     
         12 . The method of  claim 10 , wherein said modified PPR10 binding protein is PPR10 GG  encoded by SEQ ID NO: 265. 
     
     
         13 . The method of  claim 10 , wherein said PPR10 binding site encoded by SEQ ID NO: 261. 
     
     
         14 . The method of  claim 10 , further comprising plastid expression of an aadA spectinomycin resistance gene. 
     
     
         15 . The method of  claim 10 , wherein the plastid expressed gene of interest is linked to an upstream sequence encoding a maize atpH gene and/or tRNA sequence in said plastid vector. 
     
     
         16 . A method for increasing plastid transformation efficiency in plastids of a  Brassica  ssp. plant recalcitrant to plastid transformation, comprising;
 a) providing a plant comprising a nonfunctional ACC2 nuclear gene;   b) introducing one or more plastid transformation vectors into the plastids in cells from said plant, said one or more vectors comprising a nucleic acid sequence conferring resistance to said plastid translation inhibitor, and a nucleic acid sequence encoding a protein of interest;   c) contacting said cells with said inhibitor and selecting plant cells which are resistant to said inhibitor and accumulate said protein of interest in said plastids; and   d) culturing said plant cells under conditions suitable to regenerate a transplastomic plant therefrom.   
     
     
         17 . The method of  claim 16 , wherein said plastid translation inhibitor is selected from the group consisting of kanamycin, chloramphenicol, tobramycin and gentamycin. 
     
     
         18 . The method of  claim 17 , wherein inhibitor is kanamycin. 
     
     
         19 . The method of  claim 17 , wherein said inhibitor is chloramphenicol and said nucleic acid encodes chloramphenicol acetyl transferase. 
     
     
         20 . The method of  claim 17 , wherein said inhibitor is tobramycin. 
     
     
         21 . The method of  claim 17 , wherein said inhibitor is gentamycin.