Development of novel germplasm using segregates from transgenic crosses

Abstract

This invention provides a method for the development of novel plant germplasm using segregates from a transgenic line combined with PCR-based zygosity testing and, optionally, Southern blot analysis.

1. A method for developing an elite crop variety, comprising:
 a) crossing a first elite line comprising a transgene that encodes a useful trait in said first elite line, and that exhibits at least one elite trait, with a second line that exhibits an elite trait;  
 b) obtaining multiple progeny from the cross;  
 c) selecting at least one individual from said multiple progeny that exhibits said elite trait derived from the first elite line or second line;  
 d) producing at least one further progeny population from the selected individual;  
 e) screening said further progeny population for said one of the additional elite trait of the first elite line and for the presence of the transgene;  
 f) identifying at least one individual from said further progeny population that lacks the transgene of the first elite line; and  
 (g) using the individual identified in step (f) to produce at least one additional progeny population.  
 
     
     
       2. The method of  claim 1  wherein the screening process of step (e) comprises a zygosity assay or ELISA assay. 
     
     
       3. The method of  claim 1  wherein the individual identified in step (f) further comprises the elite trait of the second line. 
     
     
       4. The method of  claim 1  wherein the useful trait of the first elite line or the elite trait of the second line is herbicide tolerance. 
     
     
       5. The method of  claim 4  wherein the herbicide tolerance is glyphosate tolerance. 
     
     
       6. The method of  claim 5  wherein the glyphosate tolerance is provided by expression of glyphosate tolerant (CP 4  EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), EPSPS, GOX (glyphosate oxidoreductase), or GAT (glyphosate-N-acetyl transferase). 
     
     
       7. The method of  claim 4  wherein the useful trait of the first elite line and the elite trait of the second line both comprise herbicide tolerance. 
     
     
       8. The method of  claim 1  wherein the crop is selected from the group consisting of soybean, corn, cotton, rice, wheat, canola, alfalfas turfgrass, flax, sugar beet, potato, and chicory. 
     
     
       9. The method of  claim 8 , wherein the crop is soybean and the first elite line comprises glyphosate tolerant soybean event  40 - 3 - 2 . 
     
     
       10. The method of  claim 8 , wherein the crop is corn and the first elite line comprises event GA 21 . 
     
     
       11. The method of  claim 1 , wherein the second line comprises a transgene. 
     
     
       12. The method of  claim 1 , wherein said identified individual is crossed with a glyphosate tolerant plant to produce said additional progeny population, and wherein the method further comprises:
 h) selecting from said additional progeny population at least one individual plant that exhibits glyphosate tolerance;  
 i) deriving at least one further generation from the individual selected in step (h);  
 j) screening the further generation for the presence of the at least one additional elite trait of the first elite line and for glyphosate tolerance; and  
 k) selecting at least one individual from the generation screened in step (j) that exhibits glyphosate tolerance.  
 
     
     
       13. The method of  claim 12 , wherein the glyphosate tolerance is provided by expression of a CP 4  EPSPS, EPSPS, GOX, or GAT. 
     
     
       14. The method of  claim 12 , wherein the crop is soybean and the glyphosate tolerant plant is soybean event  781 . 
     
     
       15. A method for producing an elite crop variety, comprising:
 a) crossing an elite line that (i) comprises a transgene encoding a useful trait in said elite line and (ii) exhibits one or more transgenically- derived elite traits and one or more conventionally-derived elite traits, with a conventional non-transgenic line;  
 b) obtaining individual F 1  hybrid lines;  
 c) selecting at least one F 1  hybrid individual exhibiting at least one elite characteristic of the elite line;  
 d) deriving at least one further progeny generation from the selected F 1  hybrid individual;  
 e) screening F 2  or later progeny exhibiting at least one of the conventionally-derived elite traits of the elite line for the presence of said transgene;  
 f) selecting at least one individual from the progeny screened in step (e) that lacks DNA sequences of said transgene in its genome; and  
 g) using the individual selected in step (f) to produce at least one additional progeny population.  
 
     
     
       16. The method of  claim 15 , wherein the elite line comprises soybean event  40 - 3 - 2 . 
     
     
       17. The method of  claim 16 , wherein the elite line comprising soybean event  40 - 3 - 2  has been exposed to a mutagenizing agent to develop a unique genetic profile. 
     
     
       18. The method of  claim 15 , wherein the elite line exhibits one or more traits selected from the group consisting of: herbicide tolerance, insect resistance, and male sterility. 
     
     
       19. The method of  claim 15 , further comprising:
 h) selecting from said additional progeny population at least one individual plant that exhibits one or more elite traits derived from either of its parents;  
 i) deriving at least one further generation from the individual selected in step (h);  
 j) screening the further generation for the presence of elite traits; and  
 k) selecting at least one individual from the generation screened in step (j) that exhibits one or more elite traits.  
 
     
     
       20. A method for producing null segregants for a transgene in a plant breeding program, comprising:
 a) crossing a first line containing a transgene that encodes a useful trait in said first line with a second line;  
 b) obtaining one or more F 1  hybrid individuals;  
 c) deriving at least one further progeny generation from the one or more F 1  individuals;  
 d) screening the further progeny generation for the presence of the transgene;  
 e) selecting progeny that do not have the transgene; and  
 f) deriving at least one further generation from the progeny selected in step (e).  
 
     
     
       21. The method of  claim 20 , wherein the transgene encodes herbicide tolerant. 
     
     
       22. The method of  claim 21 , wherein the transgene encodes glyphosate tolerance. 
     
     
       23. The method of  claim 22 , wherein the transgene encodes CP 4  EPSPS, EPSPS, GOX, or GAT. 
     
     
       24. The method of  claim 20 , wherein the transgene encodes an insecticidal toxin derive from  Bacilli thuringiensis.    
     
     
       25. The method of  claim 23 , wherein the transgene encoding CP 4  EPSPS comprises a plant DNA virus promoter; a sequence encoding a chloroplast transit peptide, a sequence encoding a CP 4  EPSPS; and a NOS terminator. 
     
     
       26. The method of  claim 20 , wherein screening step (d) comprises performing zygosity analysis. 
     
     
       27. The method of  claim 26 , wherein screening step (d) further comprises performing Southern blot analysis or western blot analysis. 
     
     
       28. The method of  claim 20 , wherein the removal of the transgene is not caused by the presence of a transgenic recombinase. 
     
     
       29. The method of  claim 20 , wherein the method further comprises selecting at least one F 1  hybrid individual obtained in step (b) that exhibits at least one characteristic of the first line. 
     
     
       30. The method of  claim 11 , wherein the individual identified in step (f) comprises the transgene of the second line.