Test specificity for the L-1, L-2 or L-3 test is based on the pH of the test solution for which the corresponding isozyme has optimum activity.
Samples were left undisturbed for at least 10 min for the L-1 or the L-2 test or at least 30 min for the L-3 test. After the soaking period, 2 mL of the appropriate test solution was added.
The L-3 test solution was prepared by adding 1.25 mL of L-3 buffer, 0.25 mL of dye solution, and 0.5 mL of substrate solution for each sample.
An aliquot of 1.2 [micro]L of polyoxyethylenesorbitan mono-laurate (Tween 20) per sample for the L-1 or L-2 test or 2.4 [micro]L per sample for the L-3 test was added by using a pipette tip with the tip cut off.
The minimum amount of dye made for the L-3 test was for 20 samples to ensure pipetting accuracy.
The L-3 test was the slowest and the least stable of the three tests.
Evaluation of a bulk of seed chips did not provide satisfactory results for the L-3 test. Bleaching of the dye did not consistently occur when at least one normal seed chip was mixed with lx3lx3 null chips.
The cost of the test solutions was approximately $0.01 per sample for the L-1 test, $0.09 for the L-2 test, and $0.02 for the L-3 test. Because of the tight coupling-phase linkage between lx1 and lx2, the use of the L-2 test is optional because a seed or plant that is lx1lx1 should be lx2lx2, if it was derived from a triple-null X normal cross.