Potatoes with reduced cold-induced sweetening

Abstract

Materials and methods are provided for making plants (e.g., Solanum varieties) with decreased accumulation of reducing sugars and acrylamide in cold-stored potatoes, specifically, by making TALE-nuclease-induced mutations in genes encoding vacuolar invertase.

1 - 41 . (canceled) 
     
     
         42 . A method for generating a plant comprising at least two mutations, the method comprising:
 (a) providing protoplasts;   (b) transforming the protoplasts with a nucleic acid encoding a rare-cutting endonuclease, wherein the rare-cutting endonuclease targets a protein coding region endogenous to the protoplasts;   (c) culturing the protoplasts to generate plant lines; and   (d) identifying a plant line comprising the at least two mutations.   
     
     
         43 . The method of  claim 42 , wherein the plant is a  Solanum tuberosum  plant and the protoplasts are  S. tuberosum  protoplasts. 
     
     
         44 . The method of  claim 42 , wherein each mutation is a deletion of more than one nucleotide base pair. 
     
     
         45 . The method of  claim 44 , wherein at least two VInv alleles exhibit a deletion of more than one nucleotide base pair. 
     
     
         46 . The method of  claim 44 , wherein each VInv allele exhibits removal of an endogenous nucleic acid and does not include any exogenous nucleic acid. 
     
     
         47 . The method of  claim 44 , wherein each deletion is at a target sequence as set forth in SEQ ID NO:27, or at a target sequence having at least 95 percent sequence identity to SEQ ID NO:27. 
     
     
         48 . The method of  claim 44 , wherein each deletion is at a target sequence as set forth in SEQ ID NO: 1, or at a target sequence having at least 95 percent sequence identity to SEQ ID NO: 1. 
     
     
         49 . The method of  claim 45 , wherein the at least two VInv alleles comprise a deletion selected from a −4 bp deletion as shown in SEQ ID NO:32, a −4 bp deletion as shown in SEQ ID NO:33, or a −17 bp deletion as shown in SEQ ID NO:34. 
     
     
         50 . The method of  claim 49 , wherein the plant comprises three deletions, and wherein the deletions comprise a −4 bp deletion as shown in SEQ ID NO: 32, a −4 bp deletion as shown in SEQ ID NO:33, and a −17 bp deletion as shown in SEQ ID NO:34. 
     
     
         51 . The method of  claim 42 , wherein the rare-cutting endonuclease is a transcription activator-like effector (TALE) nuclease or an RNA-guided Cas9 nuclease. 
     
     
         52 . The method of  claim 51 , wherein the rare-cutting endonuclease is a TALE-nuclease. 
     
     
         53 . The method of  claim 52 , wherein the TALE-nuclease binds to a sequence as set forth in any of SEQ ID NOS:18-23. 
     
     
         54 . The method of  claim 42 , wherein the transforming is mediated by PEG.