Transformation method and transgenic plants produced thereby

Abstract

This invention relates to methods for producing, at a high frequency, transgenic plants that contain little if any vector sequences, have simple integration patterns, contain few copies of the transgene at each locus, express the transgene at all stages of development and do not exhibit transgene silencing. The method comprises introducing minimal transgene expression cassettes, which are substantially or totally devoid of vector sequences, by direct DNA transfer, preferably by particle or microprojectile bombardment This invention also relates to transformed plant cells, the transgenic plants regenerated therefrom, and subparts of the transgenic plants produced by the methods of this invention. The invention also includes all progeny and subsequent progeny (i.e., all subsequent generations) derived from primary transformants through selfing or crossing.

1 - 24 . (canceled)  
     
     
         25 . A population of transgenic plants produced by transforming intact plant cells with one, or more, minimal transgene expression cassette encoding a desired product by direct DNA transfer techniques and regenerating a population of transgenic plants from said transformed plant cells, wherein less than about 20% of said transgenic plants undergo transgene silencing, wherein said minimal transgene expression cassette is a nucleic acid molecule encoding the desired product and wherein no more than about 10-15% of the nucleic acid molecule are sequences that do not encode or regulate expression of the desired product.  
     
     
         26 . The population of transgenic plants of  claim 25 , wherein the minimal transgene expression cassette is integrated in at least one and no more than three integration sites in the genome of 70% of said transgenic plants.  
     
     
         27 . The population of transgenic plants of  claim 26 , wherein the integration sites contain the minimal transgene expression cassette at low copy number.  
     
     
         28 . The population of transgenic plants of  claim 27 , wherein at least one but no more than five copies of the minimal transgene expression cassette is integrated at each of said integration sites.  
     
     
         29 . The population of transgenic plants of  claim 25 , wherein at least about 80% of said population of transgenic plants have at least one minimal transgene expression cassette integrated into the genomes of said transgenic plants in a simple integration pattern.  
     
     
         30 . A population of transgenic plants wherein less than about 20% of said population of transgenic plants undergoes transgene silencing, said transgenic plants comprising a minimal transgene expression cassette, wherein said cassette is a nucleic acid molecule encoding a desired product and wherein no more than about 10-15% of said nucleic acid molecule are sequences that do not encode or regulate expression of the desired product.  
     
     
         31 . The population of transgenic plants of  claim 25  or  30 , wherein less than about 5% of said population of transgenic plants undergoes transgene silencing.  
     
     
         32 . The population of transgenic plants of claims  25  or  30 , wherein none of the population of transgenic plants undergoes transgene silencing.  
     
     
         33 . The population of transgenic plants of  claim 25  or  30 , wherein the minimal transgene expression cassette comprises a genetic marker.  
     
     
         34 . The population of transgenic plants of  claim 33  wherein the genetic marker encodes a product that confers resistance to an antibiotic, an herbicide, methotrexate, glyphosate or a metabolic inhibitor.  
     
     
         35 . The population of transgenic plants of  claim 33 , wherein the genetic marker encodes an enzyme selected from the group consisting of hygromycin phosphotransferase, phosphophinothricin acetyltransferase, neomycin phosphotransferase, kanamycin phosphotransferase, epsp synthase, acetolactate synthase, and mannose isomerase.  
     
     
         36 . The population of transgenic plants of  claim 25  or  30 , wherein the minimal transgene expression cassette encodes a seed storage protein.  
     
     
         37 . The population of transgenic plants of  claim 25  or  30 , wherein the minimal transgene expression cassette encodes an antibody.  
     
     
         38 . The population of transgenic plants of  claim 25  or  30 , wherein the minimal transgene expression cassette encodes an antibody fragment.  
     
     
         39 . The population of transgenic plants of  claim 38 , wherein the antibody fragment is selected from the group consisting of Fab′, F(ab′) 2 , single chain Fv fragments, bispecific single chain Fv fragments and diabodies.  
     
     
         40 . The population of transgenic plants of  claim 25  or  30 , wherein the minimal transgene expression cassette encodes an antisense RNA.  
     
     
         41 . A transgenic plant produced by transforming at least one intact plant cell with one or more minimal transgene expression cassette(s) encoding a desired product by direct DNA transfer techniques and regenerating at least one transgenic plant from said at least one transformed plant cell , wherein said minimal transgene expression cassette is a nucleic acid molecule encoding the desired product and wherein less than about  10-15% of the nucleotide sequences of said minimal transgene expression cassette are vector sequences.  
     
     
         42 . The transgenic plant according to  claim 41  wherein no more than about 50 base pairs of the minimal transgene expression cassette are vector sequences.  
     
     
         43 . A transgenic plant cell produced by transforming at least one intact plant cell with one or more minimal transgene expression cassette(s) encoding a desired product by direct DNA transfer techniques, wherein said minimal transgene expression cassette is a nucleic acid molecule encoding the desired product and wherein less than about 10-15% of the nucleotide sequences of said minimal transgene expression cassette are vector sequences.