Reliability of a microbial assay assessing lysine and methionine concentrations in maize (Zea mays L.) kernels
It has been recognized that the genetic constitution of the maize kernel may have an impact on its chemical composition. Maize has been shown to be limiting in several of the essential amino acids required by monogastric animals, specifically lysine and methionine. Plant breeders are attempting to screen maize populations for lines containing higher essential amino acid levels and incorporate those traits into elite lines. However, in order for this to happen, the plant breeders need to have a reliable screening procedure. One such approach is to utilize an auxotrophic microbial assay to assess the specific amino acid concentration. In this study, a statistical review of the assay procedure was conducted along with a simulation program to help project the most practical, repeatable assay protocol. Subsets of fourteen maize samples were each ground to a fine powder before they were allocated in a RCB design on a 96-well microplate. A plate was comprised of three blocks each containing the same ground subset. Each grind was replicated across three plates for a total of nine plates. The individual plates were then measured three times. The conclusions of this study were that this assay is highly repeatable and consistent. The microbial assay was reviewed and found to be reliable for amino acid measurement. The repeatability of this assay was extremely high, with most of the repeatability values being above 90%. The assay was sensitive enough to detect genotypic differences between the entries. Only one replication of each field sample was necessary. In a simulation program, three plates is the practical number to replicate entries over in this assay. The block, grind, and plate by grind interaction effects were all determined to not be a significant source of variation in this assay. Any plant breeder can take comfort in this assay that the entries submitted would be consistently analyzed.