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 - 32 . cancelled.  
     
     
         33 . A population of transgenic plants producing a desired product wherein less than about 20% of said transgenic plants undergo transgene silencing, said population of transgenic plants comprising a minimal transgene expression cassette, said minimal transgene expression cassette consisting of a nucleic acid molecule encoding the desired product wherein no more than about 50 nucleotide pairs of sequences in the nucleic acid molecule are sequences that do not encode or regulate expression of the desired gene product.  
     
     
         34 . The population of transgenic plants of  claim 33 , wherein at least about 70% 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.  
     
     
         35 . The population of transgenic plants of  claim 34 , wherein the simple integration pattern is characterized by the minimal transgene expression cassettes integrated in at least one and no more than three integration sites in the genome of said transgenic plants.  
     
     
         36 . The population of transgenic plants of  claim 35 , wherein the integration sites contain the minimal transgene expression cassette at low copy number.  
     
     
         37 . The population of transgenic plants of  claim 36 , wherein low copy number is at least one but no more than five copies of the minimal transgene expression cassette integrated at each of said integration sites.  
     
     
         38 . The population of transgenic plants of  claim 33 , 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.  
     
     
         39 . The population of transgenic plants of  claim 33 , wherein less than about 5% of said population of transgenic plants undergoes transgene silencing.  
     
     
         40 . The population of transgenic plants of  claim 33 , wherein none of the population of transgenic plants undergoes transgene silencing.  
     
     
         41 . The population of transgenic plants of  claim 33 , wherein the minimal transgene expression cassette comprises no more than about 20 nucleotide pairs that are sequences that do not encode or regulate expression of the desired gene product.  
     
     
         42 . The population of transgenic plants of  claim 33 , wherein the minimal transgene expression cassette comprises only sequences that encode or regulate expression of the desired gene product.  
     
     
         43 . The population of transgenic plants of  claim 33 , wherein the minimal transgene expression cassette comprises a genetic marker.  
     
     
         44 . The population of transgenic plants of  claim 33 , wherein the transgenic plants comprise a first minimal transgene expression cassette comprising a genetic marker and a second minimal transgene expression cassette comprising a transgene that is not the genetic marker in the first transgene expression cassette.  
     
     
         45 . The population of transgenic plants of  claim 43 , wherein the genetic marker encodes a product that confers resistance to an antibiotic, an herbicide, methotrexate, glyphosate or a metabolic inhibitor.  
     
     
         46 . The population of transgenic plants of  claim 45 , 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.  
     
     
         47 . The population of transgenic plants of  claim 43 , wherein the transgene of the first minimal transgene expression cassette is a genetic marker selected from the group consisting of hpt, bar, neo, luc, gus and Gfp.  
     
     
         48 . The population of transgenic plants of  claim 33 , wherein the transgene of the minimal transgene expression cassette encodes a seed storage protein.  
     
     
         49 . The population of transgenic plants of  claim 33 , wherein the transgene of the minimal transgene expression cassette encodes an antibody.  
     
     
         50 . The population of transgenic plants of  claim 33 , wherein the transgene of the minimal transgene expression cassette encodes an antibody fragment.  
     
     
         51 . The population of transgenic plants of  claim 50 , 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.  
     
     
         52 . The population of transgenic plants of  claim 33 , wherein the transgene of the expression cassette encodes an antisense RNA.  
     
     
         53 . The population of transgenic plants of  claim 33 , wherein the transgenic plants are monocotyledonous or a dicotyledonous transgenic plants.  
     
     
         54 . The population of transgenic plants of  claim 33  produced by transforming intact plant cells with one, or more, minimal transgene expression cassette encoding the desired product by direct DNA transfer techniques and regenerating a population of transgenic plants from said transformed plant cells.  
     
     
         55 . The method of  claim 54 , wherein the direct DNA transfer technique is selected from the group consisting of particle bombardment, microinjection, silicon carbide fiber transformation, the vortexing method and liposome mediated DNA uptake.  
     
     
         56 . The method of  claim 55 , wherein the direct DNA transfer technique is particle bombardment.