A mutant line with increased lysine levels designated B37LTI (lysine-threonine inhibited) was developed through EMS mutagenesis of B37 seed (Muenchrath, D.A. and R.L. Phillips, Crop Sci. 33:1095-1099, 1993). The region of chromosome 9 encompassing the high-methionine 10 kD structural gene, dzs10, from B37LTI has been introgressed into A619, A632, A679 and A682 using RFLP markers. Within the A679 and A682 genetic backgrounds, two independent BC1 plants were used to develop heterozygous BC3 individuals. From these heterozygous BC3 plants, seven BC3S2 ears homozygous for the donor parent allele (DP) and seven BC3S2 ears homozygous for the recurrent parent allele (RP) were derived. Identical backcross strategies were utilized in the A619 and A632 genetic backgrounds; however in the A619 background, BC2S2 ears were analyzed, while in the A632 background, BC1S2 ears were used.

From each ear, approximately 50 kernels were ground to pass a 1 mm screen. Total protein was extracted from 35 mg meal with 1.7 mL of a 1% SDS, 0.0125 M Na borate, 2% b-mercaptoethanol, pH 10 solution (Wallace, J.C., M.A. Lopes, E. Paiva, and B.A. Larkins, Plant Physiol. 92:191-196, 1990). Norleucine was used as an internal standard. Following extraction, samples were centrifuged at 16000 x g for 15 min and 7.5 µL protein extract from each sample was placed in a 6 x 50 mm glass tube. Samples were dried under vacuum and 50 µL 6 N HCl was added to each tube. Thirteen sample tubes were placed together in a Waters hydrolysis vial. Hydrolysis vials were alternately evacuated and flushed with N₂ four times and sealed prior to hydrolysis at 150 C for 1 h. Following hydrolysis, samples were allowed to cool and then dried under vacuum.

Derivitization, separation and quantitation of amino acids followed procedures by Knect and Chang (Knect, R. and J. Chang, Anal. Chem., 58:2375-2379, 1986). Dried hydrolysate was resuspended in 32 µL 50 mM NaHCO₃. Thirty microliters of resuspended hydrolysate was transferred to a 12 x 32 mm HPLC vial (Chrom Tech). Derivitization was accomplished by adding 60 mL Dabsyl-Cl solution (1.3 mg Dabsyl Cl / 1 mL acetonitrile) and heating at 70 C until all precipitates had dissolved. Following derivitization, 410 µL of a 1:1 95% ethanol : 50 mM Na₂HPO₄·7H₂O pH 7.5 solution was added. Amino acid separation and quantitation were achieved using High Performance Liquid Chromatography (HPLC). All HPLC columns used were Spherisorb S5ODS2 analytical columns (Chrom Tech). Derivitized amino acids were detected at 436 nm.

Plants were considered completely random in the field. A randomized complete block design was employed during laboratory analysis to control vial-to-vial variability during acid hydrolysis of proteins prior to HPLC quantitation of amino acid levels. The randomized complete block design had seven replications with eight treatments. All four of the recurrent parents and the donor parent (B37LTI) were included in four of the replications. Analysis of variance was performed for each of the amino acids measured both as a percent of the total recovered amino acids and as a percent of the dry meal. Within each genetic background, contrasts were used to compare backcross-derived plants homozygous for the B37LTI chromosome 9 region with backcross-derived plants homozygous for the recurrent parent chromosome 9 region, recurrent parent plants, and B37LTI plants.

In the A679 genetic background, backcross-derived individuals carrying the introgressed segment (A679+DP) were significantly higher in methionine than backcross-derived A679 individuals not carrying the B37LTI chromosome 9 region (A679-DP) or A679 individuals on a percent of total recovered amino acid basis (Table 1). A679+DP ears were 49.6% higher in methionine than A679-DP ears and 43.8% higher in methionine than A679. A679+DP exhibited a significant 63.8% increase in methionine over A679-DP on a dry matter basis (Table 2). Similar increases in methionine attributed to the B37LTI chromosome 9 introgression were not observed in the other three genetic backgrounds.

A considerable change in the amino acid profile of A679 was associated with the introgression of the B37LTI chromosome 9 segment. There was a relatively large and highly significant increase in methionine associated with the B37LTI chromosome 9 segment. A679 was the only genetic background in which a methionine increase was contributed by the introgressed segment. This change in methionine could be attributed to the replacement of the A679 dzs10 allele with the B37LTI dzs10 allele. The dzs10 gene is a structural gene encoding a storage protein which is 23% methionine (Kirihara, J.A., J.B. Petri, and J.W. Messing., Gene 71:359-370, 1988). Interference of normal expression of this gene in A679 may contribute to the low level of methionine in this inbred line.

Table 1. Comparison of backcross-derived plants homozygous for the B37LTI chromosome 9 segment (+DP), backcross-derived plants homozygous for the recurrent parent chromosome 9 segment (-DP), recurrent parent plants (RP) and B37LTI for methionine levels on a percent of total recovered amino acids basis.
 

	mg methionine/100mg total recovered amino acids
	+DP	-DP	RP	B37LTI
A679	1.87	1.25**	1.30*	2.24
A682	1.67	1.59	1.16	2.24*
A619	1.87	1.89	1.71	2.24
A632	1.91	1.88	1.91	2.24

*,** Significantly different from A679+DP at the a=0.05 and 0.01 levels respectively.

Table 2. Comparison of backcross-derived plants homozygous for the B37LTI chromosome 9 segment (+DP), backcross-derived plants homozygous for the recurrent parent chromosome 9 segment (-DP), recurrent parent plants (RP) and B37LTI for methionine levels on a dry matter basis.
 

	mg methionine/g dry meal
	+DP	-DP	RP	B37LTI
A679	1.54	0.94*	1.07	2.14
A682	1.58	1.50	1.21	2.14
A619	1.70	1.66	1.58	2.14
A632	2.05	1.82	1.91	2.14

*Significantly different from A679+DP at the a=0.05 level.
 

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