Method of producing transgenic maize using direct transformation of commercially important genotypes

Abstract

Methods for transformation of maize with nucleic acid sequences of interest are disclosed. The method involves subjecting immature zygotic embryos or Type I callus to high velocity microprojectile bombardment. The method is capable of producing transformed maize lines of commercial importance and their hybrid combinations.

1 - 82 . (canceled)  
     
     
         82 . A fertile transgenic  Zea mays  plant comprising a preselected DNA sequence encoding a  Bacillus thuringiensis  endotoxin, wherein the preselected DNA sequence is adjusted to be more efficiently expressed in maize than the native B. thuringiensis DNA sequence encoding said endotoxin, and wherein said preselected DNA is heritable.  
     
     
         83 . The transgenic Zea mays plant of  claim 82  wherein the preselected DNA sequence comprises an increased G+C content of the degenerate third base of the codons.  
     
     
         84 . The transgenic  Zea mays  plant of  claim 82  wherein the preselected DNA sequence comprises a sequence encoding the HD73 endotoxin of Bacillus thuringiensis.  
     
     
         85 . A seed produced by the transgenic  Zea mays  plant of  claim 82 , 83 or 84 which comprises said preselected DNA sequence.  
     
     
         86 . The transgenic  Zea mays  plant of  claim 82  wherein the preselected DNA sequence encodes a truncated  Bacillus thuringiensis  endotoxin.  
     
     
         87 . The transgenic  Zea mays  plant of  claim 82  wherein the preselected DNA sequence comprises a promoter.  
     
     
         88 . A fertile inbred transgenic  Zea mays  plant comprising a preselected DNA sequence encoding a  Bacillus thuringiensis  endotoxin, wherein the preselected DNA sequence is adjusted to be more efficiently expressed in maize than the native  B. thuringiensis  DNA sequence encoding said endotoxin and wherein the preselected DNA sequence is heritable.  
     
     
         89 . A fertile hybrid transgenic  Zea mays  plant comprising a preselected DNA sequence encoding a  Bacillus thuringiensis  endotoxin, wherein the preselected DNA sequence is adjusted to be more efficiently expressed in maize than the native  B. thuringiensis  DNA sequence encoding said endotoxin, and wherein the preselected DNA sequence is heritable.  
     
     
         90 . A fertile transgenic  Zea mays  plant comprising a preselected heritable DNA sequence encoding a  Bacillus thuringiensis  endotoxin, wherein the preselected DNA sequence is adjusted to be more efficiently expressed in maize than the native  B. thuringiensis  DNA sequence encoding said endotoxin, and wherein the preselected DNA sequence further comprises a selectable marker gene or a reporter gene.  
     
     
         91 . The transgenic  Zea mays  plant of  claim 90  wherein the preselected DNA sequence comprises a sequence encoding the HD73 endotoxin of  Bacillus thuringiensis.    
     
     
         92 . The transgenic  Zea mays  plant of  claim 90  wherein the preselected DNA sequence comprises a sequence encoding the HD1 endotoxin of  Bacillus thuringiensis.    
     
     
         93 . A seed produced by the transgenic  Zea mays  plant of  claim 90 , 91 or 92 which comprises said preselected DNA sequence.  
     
     
         94 . The transgenic  Zea mays  plant of  claim 90  wherein the DNA sequence encodes a truncated  Bacillus thuringiensis  endotoxin.  
     
     
         95 . The transgenic  Zea mays  plant of  claim 90  wherein the preselected DNA sequence further comprises a promoter operably linked to said DNA sequence encoding said endotoxin and a promoter operably linked to said selectable marker gene.  
     
     
         96 . A fertile inbred transgenic  Zea mays  plant comprising a preselected heritable DNA sequence encoding a  Bacillus thuringiensis  endotoxin, wherein the preselected DNA sequence is adjusted to be more efficiently expressed in maize than the native B. thuringiensis DNA sequence encoding said endotoxin, and wherein the preselected DNA sequence further comprises a selectable marker gene or a reporter gene.  
     
     
         97 . A fertile hybrid transgenic  Zea mays  plant comprising a preselected heritable DNA sequence encoding a  Bacillus thuringiensis  endotoxin, wherein the preselected DNA sequence is adjusted to be more efficiently expressed in maize than the native B. thuringiensis DNA sequence encoding said endotoxin, and wherein the preselected DNA sequence further comprises a selectable marker gene or a reporter gene.  
     
     
         98 . The transgenic  Zea mays  plant of  claim 90  wherein the selectable marker gene confers resistance or tolerance to a compound selected from the group consisting of hygromycin, sethoxydim, haloxyfop, glyphosate, methotrexate, imidazoline, sulfonylurea, triazolopyrimidine, s-triazine, bromoxynil, phosphinothricin, kanamycin, G418, 2,2-dichloropropionic acid and neomycin.  
     
     
         99 . The transgenic plant of  claim 98  wherein the compound is phosphinothricin.  
     
     
         100 . The transgenic plant of  claim 98  wherein the compound is glyphosate.  
     
     
         101 . The transgenic plant of  claim 98  wherein the compound is kanamycin.  
     
     
         102 . The transgenic plant of  claim 98  wherein the compound is hygromycin.  
     
     
         103 . The transgenic plant of  claim 90  wherein the DNA encoding the Bacillus thuringiensis endotoxin is fused in frame with said selectable marker or reporter gene.  
     
     
         104 . The inbred transgenic plant of  claim 96  wherein the DNA encodes a truncated  Bacillus thuringiensis  endotoxin.  
     
     
         105 . The hybrid transgenic plant of  claim 99  wherein the DNA encodes a truncated  Bacillus thuringiensis  endotoxin.  
     
     
         106 . The transgenic plant of  claim 86 ,  104  or  105  wherein the truncated Bacillus thuringiensis endotoxin comprises about the N-terminal 50% of the endotoxin.  
     
     
         107 . The transgenic plant of  claim 82  or  90  wherein the preselected DNA further encodes a protease inhibitor.  
     
     
         108 . The transgenic plant of  claim 87  or  95  wherein the preselected DNA sequence further comprises the maize AdhIS first intron or the maize Shrunken-2 first intron positioned between the promoter and the DNA encoding said endotoxin.  
     
     
         109 . The transgenic plant of  claim 87  or  95  wherein the preselected DNA sequence further comprises a manopine synthase promoter, a nopaline synthase promoter or an octopine synthase promoter.  
     
     
         110 . The transgenic plant of  claim 87  or  95  wherein the promoter is the CaMV 35S or 19S promoter.  
     
     
         111 . A population of plants obtained by breeding the transgenic plants of  claim 82  or 95 wherein the preselected DNA sequence is transmitted by Mendelian inheritance through both male and female parent plants.  
     
     
         112 . An inbred insect-resistant transgenic  Zea mays  plant prepared by a process comprising: 
 (a) crossing a fertile transgenic  Zea mays  plant comprising a preselected DNA sequence encoding a  Bacillus thuringiensis  endotoxin, wherein the preselected DNA sequence is adjusted to be more efficiently expressed in maize than the  B. thuringiensis  DNA sequence encoding said endotoxin, and wherein said DNA sequence is heritable, with a member of a second inbred Zea mays line;    (b) recovering insect-resistant transgenic progeny plants from said cross;    (c) back-crossing one of the transgenic progeny plants with a member of said second inbred line;    (d) recovering insect-resistant transgenic progeny plants from said cross; and    (e) repeating steps (b) and (c) to obtain said inbred plant.    
     
     
         113 . The inbred transgenic  Zea mays  plant of  claim 112  wherein said preselected DNA sequence encodes a truncated  Bacillus thuringiensis  endotoxin.  
     
     
         114 . A transgenic insect-resistant hybrid plant prepared by crossing the inbred plant of  claim 112  or  113  with an inbred  Zea mays  line, and recovering said hybrid plant.