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Hematoxylin Staining: A Potential Tool for Screening of Aluminium Tolerance in Crop Plants



In India, 49 million hectares of land is affected by soil acidity of which 24 million hectares have pH below 5.5. In the north-eastern region of India, more than 95% area is affected by soil acidity.

The productivity potential of acidic soils is estimated to range from 25% to 80% less than normal soil. In acidic soils, poor crop productivity and low soil fertility are mainly due to the combination of aluminium and manganese toxicities coupled with nutrient deficiencies (P, Ca, Mg and K).

Among these problems, aluminium toxicity has been identified as a major growth limiting factor in acidic soils. The main effect of aluminum in higher plants is the inhibition of root growth and the degree of Al-induced root growth inhibition is therefore used to screen plants at seedling stage for their relative sensitivity to aluminum.

To breed genotypes with improved Al tolerance, reliable, efficient screening method must be available to the breeder. There have been various basic approaches to screen germplasm for tolerance to Al: hematoxylin staining (solution culture), soil culture, sand culture, in vitro, microplots and field evaluation.

Hematoxylin screening technique for Al tolerance is widely used because they are quick, highly accurate, non-destructive and can be applied at early developmental stages.

Hematoxylin staining method differentiates between aluminium tolerant and aluminium sensitive genotypes on the basis of the staining pattern. Visual evaluation of stained roots can be used to detect aluminium accumulation in root tissues.

This is a simple method based on the staining pattern in which aluminium binds to form a purple complex in the root tips of sensitive genotypes and the absence of colour in root tips of the aluminium tolerant genotypes indicates that these genotypes either exclude aluminium or bind aluminium in complexes that are unavailable to hematoxylin.

This reaction occurs by the oxidation of hematoxylin to hematyn, in the presence of sodium iodate (NaIO3) which in the presence of aluminium produces nucleic acid coloration. The hematoxylin method is very common for the evaluation of aluminium tolerance in pea, french bean, chickpea, corn, okra etc.

Hematoxylin staining is amenable for screening germplasm in large numbers as compared to other screening method. Hematoxylin root staining technique is quite effective but the mechanisms behind the selectivity of hematoxylin staining need to be understood for effective juvenile screening.


Genotypes without aluminium treatment did not take up any stain. Results indicated that as the aluminium concentration is increased from lower to higher aluminium concentrations, the staining of the seedlings roots also increased.

This is an indication of differential ability of genotypes to take in toxic aluminium. The correlation between growth parameters and hematoxylin staining for all genotypes showed a negative trend. It could be explained by the fact that sensitive seedlings have poor growth as a consequence of high quantities of accumulated aluminium (Al) in the root cap and therefore, these genotypes showed higher hematoxylin staining.

On the other hand, tolerant seedlings have some mechanisms to avoid aluminium toxicity therefore, they present higher growth and low hematoxylin staining. At higher concentration, tolerant genotypes are non stained/partial stained whereas sensitive genotypes are deeply stained in crop plants (Plate 1).


Roots of tolerant genotypes are non-stained or partial stained at higher level indicating that they tend to exclude aluminium from the root tips. The high pH immobilizes aluminium and thus protects the plants from aluminium toxicity. Roots of sensitive genotypes are deeply stained at higher aluminium concentration indicating that they accumulate and/or binding of aluminium by root tissues.

Results suggested that hematoxylin staining is a reliable screening technique of plants in particularly pea, chickpea, pigeonpea, okra etc. which allow the rapid evaluation of a large number of genotypes for their aluminium (Al) tolerance. Further test is required, especially for comprehensive information about yield and growth of the tolerant plants.

Contributed By:

Rajendra Singh and Dharmendra Singh

Division of Soil Science and Agricultural Chemistry, Indian Agricultural Research Institute, New Delhi-110 012

Division of Genetics, Indian Agricultural Research Institute, New Delhi-110 012