Allelism test for two threonine over-producing mutants

Green and Phillips (Crop Sci. 14:827-830, 1974) first suggested a method for selecting amino acid over-producing mutants of the aspartate pathway The addition of lysine plus threonine to corn tissue culture media inhibits callus growth by feedback inhibiting enzymes in the aspartate pathway. The inhibitory effect of lysine plus threonine can be overcome by the addition of methionine.

Two mutants resistant to increased levels of lysine plus threonine were isolated from tissue culture. The first mutant was designated LT19 (K.A. Hibberd and C.E. Green, Proc. Nat. Acad. Sci. 79:559-563, 1982). A second mutant, LT20, was isolated by Diedrick (Ph.D. thesis UM-St. Paul, 1984). Both mutants essentially behave as though controlled by single dominant genes and have elevated free threonine levels (20- to 100-fold) in mature kernels.

The objective of this study was to determine whether LT19 and LT20 are allelic forms of the same gene. F1 plants obtained from crossing homozygous LT19 and LT20 lines were selfed and testcrossed to A619 wildtype plants. F2 and testcross kernels were classified for free threonine concentration by TLC separation of 5% TCA extracts of whole kernels. Segregation of mutant and wildtype kernels was determined by scoring for the intensity of the threonine spot.

Wildtype F2 segregants were found in a frequency of 30 wildtype to 620 mutants. This ratio deviates slightly (p=.10) from the 15:1 ratio expected for independent duplicate dominant genes. The expected number of wildtype kernels was 41; their deficiency could be from misclassification due to threonine contributed by the maternal F1 plant.

The testcross kernels segregated 127 mutants to 73 wildtype. This ratio is significantly different (p<.005) from the 3:1 ratio expected for independent duplicate dominant genes. This ratio is not significantly different from a 2:1 ratio, suggesting that pollen transmission of one or more mutant gametes might be affected. The reciprocal cross has not been analysed yet.

This study shows that the LT19 and LT20 mutants have two non-allelic genes conditioning the same phenotype. It remains to be seen if the two genes affect different enzymes or code for alternate isozymes of the aspartate pathway.

David A. Frisch and Burle G. Gengenbach
 
 

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