Methods for distinguishing and identifying plant varieties
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. A method of distinguishing a first transgenic maize plant from a second maize plant, the method comprising the steps of:
a. heating a solution of a milled transgenic maize plant part from a first transgenic maize plant comprising event 3272 expressing a heterologous thermotolerant 797GL3 alpha-amylase enzyme to a temperature above a gelatinization temperature of starch, wherein the temperature is between about 60° C. to about 100° C.;
b. continuously measuring viscosity changes of the solution for a time period of about 10 seconds to about 2 minutes, which results in only partial hydrolysis of starch;
c. obtaining a viscosity curve for the first transgenic maize plant based on the measurements of step (b); and
d. comparing the viscosity curve from the first transgenic maize plant to a viscosity curve obtained in the same manner from a second maize plant, wherein differences in the slopes of the viscosity curves distinguish the first transgenic maize plant from the second maize plant.
2. The method of claim 1 , wherein the temperature is at least 80° C.
3. The method of claim 2 , wherein the temperature is about 95° C.
4. The method of claim 1 , wherein viscosity changes are measured from about 10 seconds to about 60 seconds.
5. The method of claim 1 , wherein viscosity changes are measured at about 60 seconds.
6. The method of claim 1 , wherein viscosity changes are measured at about 45 seconds.
7. The method of claim 1 , wherein viscosity changes are measured at about 30 seconds.
8. The method of claim 1 , wherein viscosity changes are measured at about 10 seconds.