US2005132435A1PendingUtilityA1

Method for plant transformation based on the pollination-fecundation pathway and the products thereof

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Assignee: UNIV CARMEL HAIFA ECONOMIC CORPriority: Apr 19, 2000Filed: Oct 18, 2004Published: Jun 16, 2005
Est. expiryApr 19, 2020(expired)· nominal 20-yr term from priority
Inventors:Abraham KorolTzion FahimaEviator Nevo
C12N 15/8207C12N 15/8201
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Claims

Abstract

A genotype-independent method for efficiently carrying out pollen-mediated gene transformation of cereals uses pollen which is pretreated with silicon carbide, and transforming DNA coding for specific traits, whereby the method can produce transformed plants exhibiting useful traits, with high efficiency and reproducibility.

Claims

exact text as granted — not AI-modified
1 . A method for genetic transformation of any cereal plant species with sexual reproduction based on a pollination-fecundation process comprising, 
 (a) preparing a silicon carbide fibers solution; (b) preparing a pollen germination medium;    (c) preparing a DNA solution;    (d) preparing a mixture by mixing said silicon carbide fibers solution and said pollen germination medium with said DNA solution;    (e) adding fresh pollens into said mixture to form a paste;    (f) vortexing said paste for a time interval of 30-60 seconds;    (g) applying said paste for pollination; and    (h) selecting for transformants;    with the proviso that said cereal plant species is not maize.    
     
     
         2 . A method for genetic transformation of any cereal plant species with sexual reproduction based on a pollination-fecundation process according to  claim 1 , wherein said silicon carbide fibers are approximately 0.1-20 μm average diameter and 1-250 μm length.  
     
     
         3 . A method for genetic transformation of any cereal plant species with sexual reproduction based on a pollination-fecundation process according to  claim 1 , wherein the preferred size of said silicon carbide fibers is 1-2 μm diameter and 10-80 μm length.  
     
     
         4 . A method for genetic transformation of any cereal plant species with sexual reproduction based on a pollination-fecundation process according to  claim 1 , wherein an aqueous solution for silicon carbide fibers is prepared by adding sterile water or solvent to said fibers.  
     
     
         5 . A method for genetic transformation of any cereal plant species with sexual reproduction based on a pollination-fecundation process according to  claim 4 , wherein said solution is 5% to 25% aqueous solution.  
     
     
         6 . A method for genetic transformation of any cereal plant species with sexual reproduction based on a pollination-fecundation process according to  claim 1 , wherein said pollen germination medium is a solution containing about 5%-15% sucrose, 0.01%-1.0% H 3 BO 3 , 0.01%-1.0% Ca(NO 3 ) 2 4H 2 0 at pH 5.6.  
     
     
         7 . A method for genetic transformation of any cereal plant species with sexual reproduction based on a pollination-fecundation process according to  claim 1 , wherein said preferred pollen germination medium is a solution containing about 15% sucrose, 0.018% H 3 BO 3 , 0.04% Ca(NO 3 ) 2 4H 2 0 at pH 5.6.  
     
     
         8 . A method for genetic transformation of any cereal plant species with sexual reproduction based on a pollination-fecundation process according to  claim 1 , wherein said DNA is a plasmid DNA.  
     
     
         9 . A method for genetic transformation of any cereal plant species with sexual reproduction based on a pollination-fecundation process according to  claim 8 , wherein said plasmid DNA is dissolved in a TE solution.  
     
     
         10 . A method for genetic transformation of any cereal plant species with sexual reproduction based on a pollination-fecundation process according to  claim 1 , wherein said DNA solution is further incubated at about 20-25° C.  
     
     
         11 . A method for genetic transformation of any cereal plant species with sexual reproduction based on a pollination-fecundation process according to  claim 1 , wherein the selection of a transformate is performed by specific cloned selectable markers having a phenotypic expression or providing resistance to some drugs.  
     
     
         12 . A method for genetic transformation according to  claim 1 , wherein the selection of a transformate is performed by a specific closed selectable marker having a phenotypic expression is an anthocyanin regulator.  
     
     
         13 . A method for genetic transformation according to  claim 11 , wherein said selectable markers providing resistance to some drugs are antibiotics or herbicides.  
     
     
         14 . A method for genetic transformation according to  claim 13 , wherein said selectable markers providing resistance to antibiotics is neomycin phosphotransferase gene or kanamycin gene.  
     
     
         15 . A method for genetic transformation according to  claim 11 , wherein said selectable markers providing resistance to herbicides is phosphinothricin acetyltransferase gene.  
     
     
         17 . A method for genetic transformation of any cereal plant species with sexual reproduction based on a pollination-fecundation process according to  claim 1 , in any cereal plant species with sexual reproduction comprising rice, wheat, oats and barley.  
     
     
         17 . A transgenic cereal, inclusive of maize, having an antibiotic kanamycin resistant property prepared by the process of  claim 1 .  
     
     
         18 . A transgenic cereal, inclusive of maize, having a herbicide bialaphos resistant property prepared by the process of  claim 1 .  
     
     
         19 . A transgenic cereal, inclusive of maize, having an anthocyanin producing property prepared by the process of  claim 1 .  
     
     
         20 . A paste comprising a silicon carbide fiber, a pollen germination medium, and a purified and isolated DNA molecule.  
     
     
         21 . A paste as recited in  claim 20  wherein said silicon carbide fibers having 1-2 μm average diameter and 10-80 μm length.  
     
     
         22 . A paste as recited in  claim 20  wherein said silicon carbide fibers is a 5% aqueous solution.  
     
     
         23 . A paste as recited in  claim 20  wherein said pollen germination medium is a solution containing about 15% sucrose, 0.018% H 3 BO 3 , 0.04% Ca(NO 3 ) 2 4H 2 0 at pH 5.6.  
     
     
         24 . A paste as recited in  claim 20  wherein said DNA is a plasmid DNA.

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