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

Abstract

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 40×. 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.

Characterization of waxy grain sorghum lines in relation to granule-bound starch synthase

“…The F 1 plants were visually confirmed as hybrids, and F 2 generation pollen and seed collected for use in testing segregation ratios and for fine mapping. Pollen and seed were stained with potassium iodine as per Pedersen et al (2004), and presence (+) or absence (−) of GBSS was determined using SDS-PAGE as above (Gaines et al, 2000).…”

“…Recent improvements in staining methodologies to detect the presence or absence of amylose in sorghum grain and pollen (Pedersen et al, 2004) encourage reexamination of the hypothesis that the waxy trait is controlled by a single allele, wx, in sorghum. The report of an induced waxy mutation in sorghum with reduced granule-bound starch synthase (GBSS) activity (Hsieh et al, 1996a,b) and a preliminary report of existence of waxy sorghum lines differing in presence or absence of GBSS (McIntyre, 1998) strongly suggest that variants of the waxy gene exist in sorghum as in other cereals.…”

Evaluation of Waxy Grain Sorghum for Ethanol Production

“…tum its color dark blue; waxy grains contain no or little amylose and will tum reddish brown (Pedersen et al 2004). The iodinestaining test showed that amylose contents were low in most waxy sorghum samples and slightly higher in a few other samples.…”

“…Total starch and amylose contents were measured through colorimetric procedures (Megazyme total starch and amylose/amylopectin kits; procedures are available at http:// secure.megazyme.comldownloads/enidatalK-TSTA.pdf and http:// secure.megazyme.comldownloads/en/datalK-AMYL.pdf). The presence of amylose in the waxy sorghum kernels was also qualitatively examined through iodine-staining techniques (Pedersen et al 2004). FAN was analyzed through the European Brewery Convention method (EBC 1987) with modification.…”

Modification of Starch Composition Using RNAi Targeting Soluble Starch Synthase I in Japonica Rice

“…range from 11.6 to 15.6%. To visually determine the change in amylose content of the transgenic plants, T2 transgenic seeds of RNAi-SSSI were subjected to Lugol's solution or iodine staining (Pedersen et al 2004). Iodine reacts in the presence of starch, which turned into dark purple or even black.…”