US2004093649A1PendingUtilityA1

Assay for the detection of selectable marker expression in plants

58
Priority: Feb 26, 1999Filed: Jul 28, 2003Published: May 13, 2004
Est. expiryFeb 26, 2019(expired)· nominal 20-yr term from priority
Inventors:Arlene R. HowePaul C. C. Feng
C12N 15/8209C12N 15/8212
58
PatentIndex Score
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Claims

Abstract

Improved methods for the identification of transgenic plants containing NPTII protein are disclosed. Application of organosilicone surfactant in combination with kanamycin and/or paromomycin facilitates the identification of plants containing NPTII protein.

Claims

exact text as granted — not AI-modified
What is claimed is:  
     
         1 . A method for detecting the presence of a selectable marker gene product in a plant growing in an environment without selective pressure, said method comprising the steps of: 
 obtaining a plant suspected of comprising a selectable marker gene product;    contacting such plant with a composition comprising a selective agent corresponding to said selectable marker gene product and an organosilicone surfactant;    assessing the physical appearance of said plant for the presence of necrosis and/or bleaching of the contacted plant tissue wherein plant tissue exhibiting reduced or no necrosis or bleaching evidences the presence of a selectable marker gene product in said plant.    
     
     
         2 . The method of  claim 1  wherein the selectable marker gene product confers resistance to an antibiotic selective agent.  
     
     
         3 . The method of  claim 2  wherein the selectable marker gene product encodes a NPTII protein.  
     
     
         4 . The method of  claim 1  wherein the plant is a monocotyledonous plant.  
     
     
         5 . The method of  claim 1  wherein the plant is a member of the family Gramineae.  
     
     
         6 . The method of  claim 1  wherein the plant is corn.  
     
     
         7 . The method of  claim 2  wherein the selective agent is kanamycin, paromomycin, ribostamycin, butirosin, genenticin, or a combination thereof.  
     
     
         8 . The method of  claim 2  wherein the selective agent is kanamycin or paromomycin.  
     
     
         9 . The method of  claim 2 , wherein the selective agent is a combination of kanamycin and paromomycin.  
     
     
         10 . The method of  claim 1  wherein the organosilicone surfactant is dissolved in a solution.  
     
     
         11 . The method of  claim 10  wherein the organosilicone surfactant is SILWET L-77, SILWET 408, SILWET Y-12808, SILWET L-7607, SILWET L-7602, SILWET L-7210, SILWET L-7002, SILWET L-720, or SILWET L-7200.  
     
     
         12 . The method of  claim 11  wherein the organosilicone surfactant is SILWET L-77.  
     
     
         13 . The method of  claim 12  wherein the concentration of SILWET L-77 in the composition contacted with the plant is about 0.001% (v/v) to about 1.0% (v/v).  
     
     
         14 . The method of  claim 12  wherein the concentration of SILWET L-77 in the composition contacted with the plant is about 0.01% (v/v) to about 0.08% (v/v).  
     
     
         15 . The method of  claim 12  wherein the concentration of SILWET L-77 in the composition contacted with the plant is about 0.04% (v/v) to about 0.07% (v/v).  
     
     
         16 . The method of  claim 6  wherein said composition is contacted with the whorl of the corn plant.  
     
     
         17 . A method for detecting the presence of a selectable marker gene product in a plant growing in an environment without selective pressure, said method comprising the steps of: 
 obtaining a plant suspected of comprising a selectable marker gene product;    applying an effective amount of a selective agent corresponding to said selectable marker gene product and an effective amount of an organosilicone surfactant to said plant;    assessing the physical appearance of said plant for the presence of necrosis and/or bleaching of the contacted plant tissue wherein plant tissue exhibiting reduced or no necrosis or bleaching evidences the presence of a selectable marker gene product in said plant.    
     
     
         18 . The method of  claim 17  wherein the selective agent and organosilicone surfactant are applied to said plant sequentially.  
     
     
         19 . The method of  claim 17  wherein the selectable marker gene product confers resistance to an antibiotic selective agent.  
     
     
         20 . The method of  claim 19  wherein the selectable marker gene product encodes a NPTII protein.  
     
     
         21 . The method of  claim 17  wherein the plant is a monocotyledonous plant.  
     
     
         22 . The method of  claim 17  wherein the plant is a member of the family Gramineae.  
     
     
         23 . The method of  claim 17  wherein the plant is corn.  
     
     
         24 . The method of  claim 19  wherein the selective agent is kanamycin, paromomycin, ribostamycin, butirosin, genenticin, or a combination thereof.  
     
     
         25 . The method of  claim 19  wherein the selective agent is kanamycin or paromomycin.  
     
     
         26 . The method of  claim 19  wherein the selective agent is a combination of kanamycin and paromomycin.  
     
     
         27 . The method of  claim 17  wherein the organosilicone surfactant is dissolved in a solution.  
     
     
         28 . The method of  claim 27  wherein the organosilicone surfactant is SILWET L-77, SILWET 408, SILWET Y-12808, SILWET L-7607, SILWET L-7602, SILWET L-7210, SILWET L-7002, SILWET L-720, or SILWET L-7200.  
     
     
         29 . The method of  claim 28  wherein the organosilicone surfactant is SILWET L-77.  
     
     
         30 . The method of  claim 29  wherein the concentration of SILWET L-77 in the composition contacted with the plant is about 0.001% (v/v) to about 1.0% (v/v).  
     
     
         31 . The method of  claim 29  wherein the concentration of SILWET L-77 in the composition contacted with the plant is about 0.01% (v/v) to about 0.08% (v/v).  
     
     
         32 . The method of  claim 29  wherein the concentration of SILWET L-77 in the composition contacted with the plant is about 0.04% (v/v) to about 0.07% (v/v).  
     
     
         33 . The method of  claim 23  wherein said composition is contacted with the whorl of the corn plant.  
     
     
         34 . A kit to aid in the detection of transgenic plants containing NPTII protein, the kit comprising: 
 a first vessel containing an organosilicone surfactant; and    a second vessel containing one or more selective agents to which the NPTII protein confers resistance in plants.    
     
     
         35 . The kit of  claim 34  wherein the organosilicone surfactant is SILWET L-77, SILWET 408, SILWET Y-12808, SILWET L-7607, SILWET L-7602, SILWET L-7210, SILWET L-7002, SILWET L-720, or SILWET L-7200.  
     
     
         36 . A kit to aid in the detection of transgenic plants containing NPTII protein, the kit comprising a vessel containing an effective amount of an organosilicone surfactant and an effective amount of one or more selective agents to which the NPTII protein confers resistance in plants.  
     
     
         37 . The kit of  claim 36  wherein the organosilicone surfactant is SILWET L-77, SILWET 408, SILWET Y-12808, SILWET L-7607, SILWET L-7602, SILWET L-7210, SILWET L-7002, SILWET L-720, or SILWET L-7200.  
     
     
         38 . The method of  claim 1  wherein the plant is soybean.  
     
     
         39 . The method of  claim 17  wherein the plant is soybean.

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