Use of mixed duplex oligonucleotides to effect localized genetic changes in plants

Abstract

The invention concerns the use of duplex oligonucleotides about 25 to 30 base pairs to introduce site specific genetic alterations in plant cells. The oligonucleotides can be delivered by mechanical (biolistic) systems or by electroporation of plant protoplasts. Thereafter plants having the genetic alteration can be generated form the altered cells. In specific embodiments the invention concerns the alteration in the gene that encode acid invertase, UDP-glucose pyrophosphorylase, polyphenol oxidase, O-methyl transferase, cinnamyl alcohol dehydrogenase, ACC synthase and ACC oxidase or etr-1 or homolog of etr-1, and plants having isolated point mutations in such genes.

1 . A method of making a localized mutation in a target gene of a plastid of a plant cell which comprises the steps of: 
 (a) Introducing a recombinagenic oligonucleobase, which contains a first homologous region which has a sequence identical to the sequence of at lease 6 base pairs of a first fragment of the target gene and a second homologous region which has a sequence identical to the sequence of at least 6 base pairs of a second fragment of the target gene, and an intervening region which contains at least 1 nucleobase heterologous tot eh target gene, which intervening region connects the first homologous region and the second homologous region;    (b) Identifying a cell having a mutation in the region between the first and second fragments of the target gene.    
     
     
         2 . The method of  claim 1 , wherein the recombinagenic oligonucleobase is a MDON and each of the homologous regions contains an RNA segment of at least 6 RNA-Type nucleotides.  
     
     
         3 . The method of  claim 2 , which further comprises culturing the identified cell so that a plant is generated.  
     
     
         4 . A method of making a localized, non-selectable mutation in a target gene in a plant cell comprising the steps of: 
 (a) introducing into the cells of a population of cells a mixture of a first recombinagenic oligonucleobase and a second recombinagenic oligonucleobase wherein: 
 (i) the first recombinagenic oligonucleobase contains a first homologous region which has a sequence identical to the sequence of at least 6 base pairs of a first fragment of a first target gene and a second homologous region which has a sequence identical to the sequence of at least 6 base pairs of a second fragment of the first target gene, and an intervening region which contains at least 1 nucleobase heterologous to the target gene, which intervening region connects the first homologous region and the second homologous region, and  
 (ii) the second recombinagenic oligonucleobase contains a first homologous region which has a sequence identical to the sequence of at least 6 base pairs of a first fragment of a second target gene and a second homologous region which has a sequence identical to the sequence of at least 6 base pairs of a second fragment of the second target gene, and an intervening region which contains at least 1 nucleobase heterologous to the target gene, which intervening region connects the first homologous region and the second homologous region;  
   (b) selecting cells from the population having selectable mutation located between the first and second fragments of the first target gene from the population; and    (c) identifying a selected cell having a non-selectable mutation located between the first fragment and the second fragment of the second target cell.    
     
     
         5 . The method of  claim 4 , wherein each V is a MDON and each of the homologous regions contains an RNA segment of at least 6 RNA-Type nucleotides.  
     
     
         6 . The method of  claim 5 , wherein the first target gene is a first ALS gene, a second ALS gene, a psbA gene, a threonine dehydratase gene, a dihydrodipicolinate synthase gene, or an S14.rp59 gene.  
     
     
         7 . The method of  claim 6 , wherein the plant fell is a maize, wheat, rice or lettuce cell.  
     
     
         8 . The method of  claim 6 , wherein the plant cell is a potato, tomato, canola, soybean or cotton cell.  
     
     
         9 . The method of  claim 6 , wherein the second target gene is selected from the group consisting of the genes encoding acid invertase, UDP-glucose pyrophosphorylase, polyphenol oxidase, O-methyl transferase, cinnamyl alcohol dehydrogenase, etr-1 or a homolog thereof, ACC synthase and ACC oxidase.  
     
     
         10 . The method of  claim 9 , wherein the plant cell is a maize, wheat, rice or lettuce cell.  
     
     
         11 . The method of  claim 9 , wherein the plant cell is a potato, tomato, canola, soybean or cotton cell.  
     
     
         12 . The method of  claim 6 , which further comprises culturing the identified cell such that a plant is generated.  
     
     
         13 . The method of  claim 9 , which further comprises making seeds from the plant or from progeny of the plant.  
     
     
         14 . The method of  claim 4 , wherein the second recombinagenic oligonucleobase is a heteroduplex recombinagenic oligonucleobase and each of the homologous regions of the second recombinagenic oligonucleobase contains a RNA segment of at least 6 RNA-Type nucleotides.  
     
     
         15 . The method of  claim 14 , wherein the first target gene is a first ALS gene, a second ALS gene a psbA gene, or an S14/RP59 gene.  
     
     
         16 . The method of  claim 15 , wherein the plant cell is a maize, wheat, rice or lettuce cell.  
     
     
         17 . The method of  claim 15 , wherein the plant cell is a potato, tomato, canola, soybean or cotton cell.  
     
     
         18 . The method of  claim 14  wherein the second target gene is selected from the group consisting of the genes encoding acid invertase, UDP-glucose pyrophosphorylase, polyphenol oxidase, O-methyl transferase, cinnamyl alcohol dehydrogenase, etr-1 or a homolog thereof, ACC synthase and ACC oxidase.  
     
     
         19 . The method of claims  9  and  18 , wherein the second target gene is from maize, wheat, rice or lettuce plant.  
     
     
         20 . The method of claims  9  and  18 , wherein the second target gene is from a potato, tomato, canola, soybean or cotton plant.  
     
     
         21 . The method of  claim 14 , which further comprises culturing the identified cell such that a plant is generated.  
     
     
         22 . The method of  claim 21 , which further comprises making seeds from the plant or from progeny of the plant.  
     
     
         23 . A method of making a localized mutation in a target gene in a plant cell comprising the steps of: 
 (a) digesting a plant part with cellulose such that plant cell protoplasts are formed;    (b) suspending the protoplasts in a solution comprising a recombinagenic oligonucleobase which contains a first homologous region which has a sequence identical to the sequence of at least 6 base pairs of a first fragment of the target gene and a second homologous region which has a sequence identical to the sequence of at least 6 base pairs of a second fragment of the target gene, and an intervening region which contains at least 1 nucleobase heterologous to the target gene, which intervening gene connects the first homologous region and the second homologous region;    (c) electroporating the suspension such that the recombinagenic oligonucleobase enters a protoplast of the suspension;    (d) culturing the protoplast; and    (e) identifying a progeny of the protoplast having a mutation located between the first and second fragments of the target gene.    
     
     
         24 . The method of  claim 23 , which further comprises the step of culturing the identified progeny such that a plant is generated.  
     
     
         25 . The method of  claim 23 , wherein the recombinagenic oligonucleobase is a MDON and each of the homologous regions contains an RNA segment of at least 6 RNA-Type nucleotides.  
     
     
         26 . The method of  claim 23 , wherein the recombinagenic oligonucleobase is a heteroduplex recombinagenic oligonucleobase.  
     
     
         27 . A plant or seed having a point mutation in a gene is in its wild type genetic position, which gene is selected from the group consisting of the genes encoding acid invertase, UDP-glucose pyrophosphorylase, polyphenol oxidase, O-methyl transferase, cinnamyl alcohol dehydrogenase, ACC synthase and ACC oxidase or etr-1 or a homolog of etr-1, and the sequence of the genomic DNA within 23 KB of the mutation is the sequence of the wild type DNA, and the point mutation forms a stop codon or is a frameshift mutation.  
     
     
         28 . The plant or seed of  claim 27 , in which the point mutation forms a stop codon.  
     
     
         29 . The plant or seed of  claim 28 , in which the sequence of the genomic DNA within 40 KB of the selectable mutation is the sequence of the wild type DNA.  
     
     
         30 . The plant or seed of  claim 28 , in which the sequence of the genomic DNA within 100 KB of the selectable mutation is the sequence of the wild type DNA.  
     
     
         31 . The plant or seed of  claim 28 , in which the point mutation is a single base pair mutation.  
     
     
         32 . The plant or seed of  claim 28 , which is a maize, wheat, rice, or lettuce plant or seed.  
     
     
         33 . The plant or seed of  claim 28 , which is a potato, tomato, canola, soybean or cotton plant or seed.  
     
     
         34 . The plant or seed of  claim 28 , further having a selectable point mutation in a second gene and the sequence of the genomic DNA within 23 KB of the selectable point mutation is the sequence of the wild type DNA.  
     
     
         35 . The plant or seed of  claim 34 , in which the sequence of the genomic DNA within 40 KB of the selectable mutation is the sequence of the wild type DNA.  
     
     
         36 . The plant or seed of  claim 34 , in which the sequence of the genomic DNA within 100 KB of the selectable mutation is the sequence of the wild type DNA.  
     
     
         37 . The plant or seed of  claim 27 , in which the point mutation is a frameshift mutation.  
     
     
         38 . The plant or seed of  claim 37 , in which the sequence of the genomic DNA within 40 KB of the selectable mutation is the sequence of the wild type DNA.  
     
     
         39 . The plant or seed of  claim 37 , in which the sequence of the genomic DNA within 100 KB of the selectable mutation is the sequence of the wild type DNA.  
     
     
         40 . The plant or seed of  claim 37 , in which the point mutation is a single base pair mutation.  
     
     
         41 . The plant or seed of  claim 37 , which is a maize, wheat, rice, or lettuce plant or seed.  
     
     
         42 . The plant or seed of  claim 37 , which is a potato, tomato, canola, soybean or cotton plant or seed.  
     
     
         43 . The plant or seed of  claim 37 , further having a selectable point mutation in a second gene and the sequence of the genomic DNA within 23 KB of the selectable point mutation is the sequence of the wild type DNA.  
     
     
         44 . The plant or seed of  claim 43 , in which the sequence of the genomic DNA within 40 KB of the selectable mutation is the sequence of the wild type DNA.  
     
     
         45 . The plant or seed of  claim 43 , in which the sequence of the genomic DNA within 100 KB of the selectable mutation is the sequence of the wild type DNA.