Screening large populations for recessive bx1 genotypes; a variation of the FeCl3 root-tip squash assay
--Kevin D. Simcox

The qualitative methods most commonly used to identify bx1/bx1 genotypes are variations of the FeCl3 root-tip squash method of Hamilton (Weeds 12:27-30, 1964). This procedure involves squashing root-tips of germinated kernels in a drop of 0.1M FeCl3. Formation of a dark blue-purple coloration indicates the presence of DIMBOA and other related 1,4-benzoxazinones. Homozygous recessive bx1 genotypes either lack or accumulate very small quantities of DIMBOA and subsequently do not produce the dark blue-purple coloration. This procedure was used on several thousand kernels to map the bx1 gene to the short arm of chromosome four using monosomic analysis and B-A translocations (Simcox and Weber, D, Crop Sci. 25:827-830, 1985). Although this procedure identifies very few false negatives, application of this procedure to screen large populations for mutations in the DIMBOA biosynthetic pathway (i.e., transposon tagging or EMS mutagenesis) would be laborious and time consuming. I have used the following variation of the FeCl3 root-tip squash procedure to screen approximately 30,000 seedlings for Mutator-induced mutations at the bx1 locus.

Kernels are planted in either greenhouse seedling beds or in dark-colored Tupperware tubs containing vermiculite. To obtain even germination, use a shallow planting depth and heat beds or tubs above 72 C. Cover the beds with a black plastic sheet; I use landscaping plastic, or cover the tubs with aluminum foil. After four days, the etiolated coleoptiles (etiolated coleoptiles are used because chlorophyll will react with FeCl3) are sprayed with an alcoholic FeCl3 solution (50g FeCl3 € 6H2O dissolved in 500ml of 95% ethanol, 0.1N HCl; Tipton et al., Biochemistry 6:2866-2870, 1967), using a plant mister. After 15 min, rinse the coleoptiles with tap water. Coleoptiles heterozygous or homozygous dominant Bx1 will form dark blue-purple longitudinal streaks on the side of the coleoptile opposite from the direction of the spray (droplets of the solution form on the opposite side of the coleoptile, soaking into the tissue). Recessive bx1 genotypes will not react to the FeCl3 solution, but may form slight brown discoloration due to the HCl in the solution. To confirm the negative reaction, seedlings are sprayed again with the FeCl3 solution, and if still negative, seedlings are removed from the planting media and the root-tips are squashed on a filter paper saturated with the FeCl3 solution.

Using this procedure I have identified 4 putative Mutator-induced bx1 mutants out of 30,000 F1 seedlings. Approximately 45 min is needed to screen 2,000 seedlings and seedling beds or tubs can be replanted very 5 days. Care should be taken in selecting what dominant Bx1 source is used as a parent. There is a great deal of variation between inbred lines in the amount of DIMBOA that accumulates in seedlings and adult plants (Klun and Brindley, J. Econ. Entomol. 59:711-718, 1966; Dunn et al., Can. J. Plant Sci. 61:583-593, 1981). It is also important to screen seedlings prior to the emergence of the initial leaf from the coleoptile. Once the leaf emerges, the fully expanded coleoptile becomes unresponsive to treatment and variable results are obtained scoring the emerging leaf blade. Since cyclic hydroxamates are primarily localized in meristematic regions and vascular tissue, it is important not to use adult tissue in screening procedures. 

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