Precise breeding-simultaneous silencing

Abstract

The present invention relates to a new plant breeding process. The process improves the agronomic performance of crop plants by using genetic material that is also used in classical breeding. Instead of sexually recombining entire genomes at random, as is done in classical breeding, specific genetic elements are rearranged in vitro and inserted back into individual plant cells. Plants obtained through this new plant breeding process do not contain foreign nucleic acid but only contain nucleic acid from the plant species selected for transformation or plants that are sexually compatible with the selected plant species. Plants developed through this new plant breeding process are provided. In particular, potato plants displaying improved tuber storage and health characteristics are provided.

1 . A method of modifying a trait of a selected plant, comprising (a) stably integrating into a plant cell a desired polynucleotide that comprises (i) a sense copy and an antisense copy of a polyphenol oxidase gene sequence and (ii) a sense copy and an antisense copy of at least one of an R1 gene sequence and a phosphorylase-L gene sequence, and (b) obtaining a stably transformed plant from transformed plant cells, wherein the stably transformed plant contains the desired polynucleotide stably integrated into its genome and exhibits a trait that is modified in comparison to a plant that does not comprise the desired polynucleotide. 
     
     
         2 . The method of  claim 1 , wherein the polyphenol oxidase gene sequence and the R1 and/or phosphorylase-L gene sequence are 5′- or 3′-untranslated sequences. 
     
     
         3 . The method of  claim 1 , wherein the R1 gene comprises a nucleotide sequence that has at least 75% sequence identity to SEQ ID NO: 8 or 16. 
     
     
         4 . The method of  claim 1 , wherein the phosphorylase-L gene comprises a nucleotide sequence that has at least 75% sequence identity to SEQ ID NO: 21 or 22. 
     
     
         5 . The method of  claim 1 , wherein the polyphenol oxidase gene comprises a nucleotide sequence that has at least 75% sequence identity to SEQ ID NO: 27 or 29. 
     
     
         6 . The method of  claim 1 , wherein the desired polynucleotide comprises two copies of at least part of the 5′- or 3′-untranslated region sequences of two or more genes selected from the group consisting of (i) the R1 gene, (ii) the phosphorylase-L gene, and (iii) the polyphenol oxidase gene. 
     
     
         7 . The method of  claim 1 , wherein expression of the desired polynucleotide modifies a trait in a plant regenerated from the transformed plant cell, wherein the trait is at least two of (i) lower levels of acrylamide, (ii) reduced black-spot bruising, (iii) reduced cold-induced sweetening, and (iv) reduced starch phosphate levels compared to a plant that does not comprise a cell that expresses the desired polynucleotide. 
     
     
         8 . The method of  claim 1 , wherein the desired polynucleotide is introduced into the plant cell by  Agrobacterium -mediated transformation. 
     
     
         9 . The method of  claim 1 , wherein a product that is made from the transformed plant contains lower levels of a mutagenic, carcinogenic or cytotoxic compound after heat-processing than a product made from an untransformed plant of the same variety. 
     
     
         10 . The method of  claim 1 , wherein a product that is made from the transformed plant displays enhanced health and nutritional characteristics, improved storage, improved starch composition, improved taste, improved texture, or decreased phosphate content. 
     
     
         11 . The method of  claim 9 , wherein the mutagenic, carcinogenic or cytotoxic compound is acrylamide. 
     
     
         12 . The method of  claim 9 , wherein the product is a French fry, potato chip, or hash brown. 
     
     
         13 . A plant comprising in its genome a desired polynucleotide, wherein the desired polynucleotide comprises (i) a sense copy and an antisense copy of a polyphenol oxidase gene sequence and (ii) a sense copy and an antisense copy of at least one of an R1 gene sequence and a phosphorylase-L gene sequence. 
     
     
         14 . The plant of  claim 13 , wherein the polyphenol oxidase gene sequence and the R1 and/or phosphorylase-L gene sequence are 5′- or 3′-untranslated sequences. 
     
     
         15 . The plant of  claim 13 , wherein the plant is a tuber-bearing plant. 
     
     
         16 . (canceled) 
     
     
         17 . An isolated nucleotide sequence, comprising (i) a sense copy and an antisense copy of a polyphenol oxidase gene sequence and (ii) a sense copy and an antisense copy of at least one of an R1 gene sequence and phosphorylase-L gene sequence. 
     
     
         18 . The isolated nucleotide sequence of  claim 17 , wherein the nucleotide sequence is positioned within an  Agrobacterium  T-DNA or a P-DNA. 
     
     
         19 . A vector, comprising the isolated nucleotide sequence of  claim 18 . 
     
     
         20 . A method of transforming a plant, comprising transforming the plant with the vector of  claim 19 . 
     
     
         21 . A plant transformed with the vector of  claim 20 . 
     
     
         22 - 24 . (canceled)