Methods for distinguishing and identifying plant varieties

Abstract

Methods are disclosed for distinguishing and identifying plants by measuring partial hydrolysis of polysaccharides on account of polysaccharide-hydrolyzing enzyme activity at pre-determined incubation times and temperatures. Methods also are disclosed for identifying the source organism of a heterologous polysaccharide-hydrolyzing enzyme in a plant by measuring partial hydrolysis of polysaccharides on account of polysaccharide-hydrolyzing enzyme activity at pre-determined incubation times and temperatures. The reaction mixture has unique chemical and physical properties that can be used to construct viscosity curves for measuring polysaccharide-hydrolyzing enzyme activity. The viscosity curves can be compared among plants to distinguish or identify the plants from one another. Likewise, viscosity curves can be compared among source organisms to identify the source organism of the heterologous polysaccharide-hydrolyzing enzyme in the plant.

1 - 6 . (canceled) 
     
     
         7 . A method of distinguishing a first plant from a second plant, the method comprising the steps of:
 a. heating a solution of a milled plant pan from the first plant that comprises a polysaccharide-hydrolyzing enzyme and polysaccharide substrate to a temperature above a gelatinization temperature of the polysaccharide substrate;   b. measuring viscosity changes of the solution for about 10 seconds to about 2 minutes;   c. obtaining a viscosity curve for the first plant; and   d. comparing the viscosity curve from the first plant to a viscosity curve obtained in the same manner from the second plant, wherein differences in the slopes of the viscosity curves distinguish the first plant from the second plant   
     
     
         8 . The method of  claim 7 , wherein the temperature is between about 60° C. to about 100° C. 
     
     
         9 . The method of  claim 7 , wherein the temperature is at least 80° C. 
     
     
         10 . The method of  claim 7 , wherein the temperature is about 95° C. 
     
     
         11 . The method of  claim 7 , wherein viscosity changes are measured from about 10 seconds to about 60 seconds. 
     
     
         12 . The method of  claim 7 , wherein viscosity changes are measured at about 60 seconds. 
     
     
         13 . The method of  claim 7 , wherein viscosity changes are measured at about 45 seconds. 
     
     
         14 . The method of  claim 7 , wherein viscosity changes are measured at about 30 seconds. 
     
     
         15 . The method of  claim 7 , wherein viscosity changes are measured at about 10 seconds. 
     
     
         16 . The method of  claim 7 , wherein the polysaccharide substrate is starch. 
     
     
         17 . The method of  claim 7 , wherein the polysaccharide-hydrolyzing enzyme is amylase. 
     
     
         18 . The method of  claim 7 , wherein the polysaccharide-hydrolyzing enzyme is a thermotolerant amylase. 
     
     
         19 . The method of  claim 18 , wherein the thermotolerant amylase is 797GL3 or D45. 
     
     
         20 . The method of  claim 7 , wherein the plant part is from maize. 
     
     
         21 . The method of  claim 20 , wherein the maize is a maize seed. 
     
     
         22 . The method of claim  1 , wherein step a is performed for about 1 minute to about 3 minutes. 
     
     
         23 . A method of identifying a maize hybrid comprising a polysaccharide-hydrolyzing enzyme, the method comprising the steps of:
 a. milling seed of the maize hybrid to create a flour;   b. adding water to the flour, wherein the water results in a 10% to 50% dry solids corn slurry;   c. heating the corn slurry of step b to a temperature above a gelatinization temperature of the polysaccharide substrate;   d. mixing the corn slurry of step c;   e. measuring viscosity of the corn slurry of step d at a time point between 10 seconds to 100 seconds;   f. measuring activity of the polysaccharide-hydrolyzing enzyme;   g. extrapolating the viscosity data and activity data of steps c and f onto a database of standard curves, wherein the database of standard curves contains standard curves of known maize hybrids, and the standard curve of the maize hybrid is plotted in relation to polysaccharide-hydrolyzing enzyme activity and viscosity at a time point between 10 seconds to 100 seconds; and   h. comparing the standard curve of the maize hybrid to the standard curves of the known maize hybrids, wherein differences in the slopes of the standard curves can be used to identify the maize hybrid.   
     
     
         24 . A method of distinguishing a first maize plant from a second maize plant, the method comprising the steps of:
 a. milling a seed of the first maize hybrid plant to create a flour;   b. adding water to the flour of step a, wherein the water results in a 10% to 50% dry solids corn slurry;   c. heating the corn slurry of step b to a temperature above a gelatinization temperature in the presence of a thermotolerant amylase;   d. mixing the corn slurry of step c;   e. measuring viscosity of the corn slurry of step d at a time point, between about 10 seconds to about 100 seconds;   f. measuring the glucose level of the corn slurry of step d at a time point between about 10 seconds to about 100 seconds, wherein said glucose level can be correlated to thermotolerant amylase activity;   g. obtaining a standard curve for the first maize plant; and   h. comparing the standard curve of the maize plant to a standard curve obtained in the same manner for the second maize plant, wherein differences in the slopes of the viscosity curves distinguish the first plant from the second plant.