Rapid Iodine Staining Techniques for Identifying the Waxy Phenotype in Sorghum Grain and Waxy Genotype in Sorghum Pollen

Visual classification of sorghum [Sorghum bicolor (L.) Moench] grain for the waxy phenotype is subjective and can be confounded by genetic background, maturity, environment, and experience of the classifier. Rapid iodine staining methods for identifying the waxy phenotype in sorghum grain and waxy genotypes in sorghum pollen were developed. Mature single sorghum seeds were placed in 48-well micro-plates and crushed. Water was added and the mixture heated to 95°C for 1 h to gelatinize the starch. After cooling, iodine solution was added and color scored after 10 to 60 s allowing for very high sample throughput. Sorghum pollen was analyzed for waxy genotype by mixing isolated pollen with iodine solution and viewing under a microscope at 40x. Waxy pollen was visually distinguishable from wild-type pollen using freshly collected as well as aged pollen. These methods will allow large-scale screening of both mature sorghum grains as well as sorghum pollen for waxy characteristics

The maize brown midrib2 (bm2) gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation

Citation: Tang, H., . . . & Schnable, P. (2013). The maize brown midrib2 (bm2) gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation. The Plant Journal, 77(3), 380-392. https://doi.org/10.1111/tpj.12394The midribs of maize brown midrib (bm) mutants exhibit a reddish-brown color associated with reductions in lignin concentration and alterations in lignin composition. Here, we report the mapping, cloning, and functional and biochemical analyses of the bm2 gene. The bm2 gene was mapped to a small region of chromosome 1 that contains a putative methylenetetrahydrofolate reductase (MTHFR) gene, which is down-regulated in bm2 mutant plants. Analyses of multiple Mu-induced bm2-Mu mutant alleles confirmed that this constitutively expressed gene is bm2. Yeast complementation experiments and a previously published biochemical characterization show that the bm2 gene encodes a functional MTHFR. Quantitative RT-PCR analyses demonstrated that the bm2 mutants accumulate substantially reduced levels of bm2 transcript. Alteration of MTHFR function is expected to influence accumulation of the methyl donor S-adenosyl-l-methionine (SAM). Because SAM is consumed by two methyltransferases in the lignin pathway (Ye et al., 1994), the finding that bm2 encodes a functional MTHFR is consistent with its lignin phenotype. Consistent with this functional assignment of bm2, the expression patterns of genes in a variety of SAM-dependent or -related pathways, including lignin biosynthesis, are altered in the bm2 mutant. Biochemical assays confirmed that bm2 mutants accumulate reduced levels of lignin with altered composition compared to wild-type. Hence, this study demonstrates a role for MTHFR in lignin biosynthesis