Evaluation of Aluminum Tolerance by Hematoxylin Root Staining
Aluminum toxicity tolerance is largely expressed by the exclusion of aluminum from roots or its binding, thereby avoiding absorption and toxification. For details see 'Mineral Toxicity' under our 'The Stresses' page. Polle et al. (1978) established a method to evaluate for this tolerance mechanism in cereal crop plants by hematoxylin root staining. Hematoxylin binds aluminum to produce a purple complex. The absence of the color in root tips treated by hematoxylin indicates that these plants either exclude or bind aluminum in complexes that are unavailable to hematoxylin. Since that publication, the method has been used extensively, with or without slight modifications, for quick evaluation of and screening for aluminum tolerance.
A typical protocol begins by growing germinated seedlings in nutrient solution (preferably aerated) set to pH 4.5. After 2-3 days the nutrient solution is replaced with an identical one which also includes 50 mM AlCl3. Other concentrations can be used depending on the specific study. A preliminary test with several aluminum concentrations is recommended before a large scale screening is embarked upon. Use of standard tolerant and susceptible cultivars is important for scaling the results.
After 24h of exposure to aluminum the seedlings are washed with de-ionized water for 30 min (with several changes of water) after which the roots are exposed for 30 min to a solution of hematoxylin. Following staining the roots are washed again for 30 minutes. The different levels of root tip staining (see example in Fig.1) can be visually scored and/or photographed.
The hematoxylin stain is prepared by placing 0.2 g of hematoxylin and 0.02 g of KI03 in 100 ml of water and keeping the solution overnight to dissolve the hematoxylin.
Fig.1. Differential hematoxylin staining of root tips in different barley cultivars. Left- tolerant; right-susceptible. (from Tang et al., 2000, Crop Sci.40:778-782)
Polle E., Konzak C.F. and Kittrick J.A. 1978. Visual detection of aluminum tolerance levels in wheat by hematoxylin staining. Crop Sci 18: 823-827
Tang Y., Sorrells M.E., Kochian L.V. and Garvin D.F. 2000. Identification of RFLP markers linked to the barley aluminum tolerance gene Alp Crop Sci.40:778-782.