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

is less indicative of phenotype since iodine binding is reduced in lines with corneous endosperm (Cagampang Visual classification of sorghum [Sorghum bicolor (L.) Moench] and Kirleis, 1985). However, such sorghum starch can grain for the waxy phenotype is subjective and can be confounded be made more amenable to iodine staining by gelatinizaby genetic background, maturity, environment, and experience of the classifier. Rapid iodine staining methods for identifying the waxy tion. Our objective was to adapt iodine staining techphenotype in sorghum grain and waxy genotypes in sorghum pollen niques to sorghum grain and pollen for rapid and objecwere developed. Mature single sorghum seeds were placed in 48-well tive identification of waxy and wild-type phenotypes. 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 MATERIALS AND METHODS sample throughput. Sorghum pollen was analyzed for waxy genotype Iodine Staining of Sorghum Grain by mixing isolated pollen with iodine solution and viewing under a microscope at 40 . Waxy pollen was visually distinguishable from Single sorghum seeds of known waxy (BTx630, TxAGR1, wild-type pollen using freshly collected as well as aged pollen. These RTx2907, B9307, B94C274) and wild-type (RTx430, Wheatmethods will allow large-scale screening of both mature sorghum land, Redlan) genotypes grown at the University of Nebraska grains as well as sorghum pollen for waxy characteristics. Field Laboratory at Ithaca, NE, in 2002 were placed in Costar 48-well micro-plates (Corning Inc., Corning, NY) and coarsely crushed with a HyPure Seed Crusher (PerkinElmer Wallac T waxy phenotype in grains is associated with Inc., Norton, OH). One milliliter H2O was added to each cell and the samples were heated in a 95 C oven for 1 h followed endosperm containing little or no amylose. Starch by cooling to room temperature to gelatinize the starch. Fifty in waxy grain is nearly pure amylopectin and the endomicroliters of iodine stain solution (2.5 g KI, 250 mg I2, 125 mL sperm in sorghum exhibits the appearance of candle H2O) was added to each well and the color reaction scored wax, hence the designation “waxy” (Rooney and Miller, in 10 to 60 s. Wells developing dark blue were scored as wild 1982). The waxy trait has been recognized in sorghum type, and wells developing various shades of magenta were since at least 1933 when the gene symbol wx was asscored as waxy. The protocol was repeated in a second laborasigned to this trait (Karper, 1933). Classification of sortory to verify results. ghum lines as waxy or wild type is often a visual assessment of endosperm fracture patterns. Such classification is subjective and can be confounded by genetic backIodine Staining of Sorghum Pollen ground, grain maturity, environment, and by the experiFresh Pollen ence of the classifier. It is also limited in application to Fresh pollen was collected from greenhouse-grown known mature grain. waxy (BTx630, TxAGR1) and wild-type (RTx430, WheatWaxy and wild-type grain phenotypes of other grains land) genotypes at pollen dehiscence (0800–1000 h). Pollen can be readily distinguished by iodine staining (Nakawas collected by shaking a panicle on a clean sheet of paper, mura et al., 1995; Neuffer et al., 1997). Wild-type grain pouring the pollen into a 1.5-mL microcentrifuge tube, and usually stains deep blue because of the presence of amycovering the pollen with 0.5 mL 70% (v/v) ethanol. Samples lose, and waxy phenotypes usually stain reddish brown. were stored in a refrigerator at 4 C. Waxy and wild-type genotypes can also be identified in Before subsampling for staining, pollen was suspended in maize (Zea mays L.) pollen by iodine staining, with the ethanol solution by shaking the microcentrifuge tube, and wild-type pollen staining dark brown and waxy pollen 5 L transferred to another microcentrifuge tube. Ten microstaining light tan (MaizeDB, 2003). In sorghum, iodine liters of the iodine stain solution (2.5 g KI, 250 mg I2, 125 mL H2O) was added to the 5 L of pollen suspension, and mixed. The stained suspension was placed on a microscope slide and J.F. Pedersen, D.L. Funnell, and R.A. Graybosch, USDA-ARS, NPA examined at 40 with a high resolution dissecting microscope Wheat, Sorghum and Forage Research, 344 Keim Hall, University of with illumination provided from above and below the slide. Nebraska-Lincoln, Lincoln, NE 68583-0937; S.R. Bean, USDA-ARS, Pollen staining dark brown was scored as wild type. Pollen NPA, GMPRC, 1515 College Avenue, Manhattan, KS, 66502. Joint staining light tan or yellow was scored as waxy. contribution of the USDA-ARS and the Univ. of Nebraska Agric. Exp. Stn. as Paper no. 14242, Journal Series, Nebraska Agric. Exp. Stn. Mention of a trademark, proprietary product, or a vendor does Aged Pollen not constitute a guarantee or warranty of the USDA or the University of Nebraska and does not imply its approval to the exclusion of other Aged pollen was collected from closed pollination bags that products or vendors that may also be suitable. Received 8 September had been placed on greenhouse-grown known waxy (BTx630) 2003. *Corresponding author ([email protected]). and wild-type (RTx430, Wheatland, BTx623, and Redlan) genotypes 38 to 42 d before pollen collection. Aged pollen was Published in Crop Sci. 44:764–767 (2004). stored and stained as above. Staining of both fresh and aged  Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA pollen was repeated in a second laboratory to verify results.