Improved materials and methods for transformation

Abstract

Disclosed herein are novel methods and materials directed to transforming a host cell and expressing exogenous RNA therein. Specifically disclosed are DNA-launching platforms used to introduce a replicating viral segment attached to an exogenous polynucleotide into a cell, whereby the exogenous polynucleotide is expressed in said cell and confers a detectable trait.

1 . A DNA-launching platform comprising: 
 a) a polynucleotide molecule encoding a modified viral RNA molecule; and    b) a DNA dependent RNA polymerase promoter.    
     
     
         2 . The DNA-launching platform of  claim 1  further comprising a sequence encoding at least one cis-acting element.  
     
     
         3 . The DNA-launching platform of  claim 1  further comprising a ribozyme sequence.  
     
     
         4 . The DNA-launching platform of  claim 1  further comprising a termination sequence.  
     
     
         5 . The DNA-launching platform of  claim 1  further comprising a restriction site.  
     
     
         6 . The DNA-launching platform of  claim 1  wherein said modified RNA molecule comprises an exogenous RNA segment.  
     
     
         7 . The DNA-launching platform of  claim 1  wherein said DNA dependent RNA polymerase promoter is capable of functioning in a plant cell.  
     
     
         8 . A method of genotypically or phenotypically modifying one or more cells comprising the following steps: 
 a) obtaining a DNA-launching platform comprising a polynucleotide molecule encoding a modified viral RNA; and    b) transfecting said one or more cells with said DNA-launching platform, wherein said polynucleotide molecule is transcribed thereby forming a replicatable RNA transcript.    
     
     
         9 . The method of  claim 8  further comprising pre-transforming said cell with at least one polynucleotide molecule encoding at least one trans-acting factor.  
     
     
         10 . The method of  claim 8  further comprising introducing a trans-acting factor.  
     
     
         11 . The method of  claim 10  wherein said introducing a trans-acting factor comprises co-transfection of an expression plasmid comprising a nucleotide sequence encoding said trans-acting factor.  
     
     
         12 . The method of  claim 10  wherein said introducing a trans-acting factor comprises co-transfection of an RNA transcript encoding said trans-acting factor.  
     
     
         13 . The method of  claim 10  wherein said trans-acting factor is stably expressed.  
     
     
         14 . The method of  claim 8  wherein said modified viral RNA comprises an exogenous RNA segment.  
     
     
         15 . The method of  claim 8  wherein said DNA-launching platform comprises a ribozyme sequence.  
     
     
         16 . The method of  claim 8  wherein said DNA-launching platform comprises a promoter.  
     
     
         17 . The method of  claim 8  wherein said DNA-launching platform comprises a termination sequence.  
     
     
         18 . The method of  claim 8  wherein said DNA-launching platform comprises a restriction site.  
     
     
         19 . The modified cell produced by the method of  claim 8 .  
     
     
         20 . A method of producing a plant or plant tissue comprising at least one genotypically or phenotypically modified cell, said method comprising transfecting cells of said plant or plant tissue with a DNA-launching platform, wherein said DNA-launching platform comprises a polynucleotide encoding a modified RNA molecule, such that said polynucleotide molecule is transcribed to form a replicatable RNA transcript.  
     
     
         21 . The method of  claim 20  wherein said modified RNA molecule comprises an exogenous RNA segment.  
     
     
         22 . The method of  claim 20  wherein said DNA-launching platform comprises a ribozyme sequence.  
     
     
         23 . The method of  claim 20  wherein said DNA-launching platform comprises a promoter.  
     
     
         24 . The method of  claim 20  wherein said DNA-launching platform comprises a termination sequence.  
     
     
         25 . The method of  claim 20  wherein said DNA-launching platform comprises a restriction site.  
     
     
         26 . A method of producing a genotypically or phenotypically modified plant comprising obtaining at least one modified cell produced by the method of  claim 8;  and subjecting said modified cell to conditions whereby a plant is regenerated therefrom.  
     
     
         27 . A plant produced by the method of  claim 26 .  
     
     
         28 . A plant descended from the plant of  claim 27 .  
     
     
         29 . The method of  claim 20 , wherein said plant or plant tissue comprises one or more cells transformed with a polynucleotide molecule encoding at least one trans-acting factor, wherein said polynucleotide molecule is expressed.  
     
     
         30 . The method of  claim 29 , wherein said modified viral RNA molecule is capable of replication only in said one or more cells transformed with a polynucleotide molecule encoding at least one trans-acting factor.