The test used in our experiments is based on the
assumption that inhibition of plant growth by Al is not observed before root
systems develop. Since inhibition of root elongation is the first visible
symptom of Al injury, direct reference to this process in selection seems to be
a reasonable approach.
The pulse test is based on
exposure of roots to short Al shock after which the effect on root elongation
is recorded. In the method used in our laboratory, the Al pulse principle of
MOORE ET AL.1976 was combined with the staining technique developed by POLLE ET
ALL 1978. Seeds were sterilized with 0.1%Hg2 Cl2 aqueous
solution for 10 min., rinsed thoroughly with water, and germinated overnight on
filter paper in Petri dishes. Sprouted seeds were sown the next day on
polyethylene net fixed in lucite frames. Styrofoam blocks were attached to the
frames with rubber bands and floated on the surface of a vigorously aerated
nutrient solution. Containers with nutrient solution were placed in water bath
at 25oC under continuous light (12 w/m). Nutrient solution of the
following composition was used: 0.4 mom calcium chloride, 0.65 mom potassium
nitrate, 0.25 mom magnesium chloride, 0.01 mom ammonium sulfate and 0.04 mom
ammonium nitrate. Four day-o1d seedlings were transferred to the same nutrient
medium supplied with Al, in the form of aluminum chloride, at concentration
indicated in the experiments. After 24 hours of incubation in the medium
containing Al, seedlings were thoroughly washed for 2-3 min. in running water and
transferred to nutrient solution without Al for 48 h. Seedlings were removed
from nutrient solution, again washed with tap water, and stained with 0.1 %
aqueous solution of Eriochrome cyanine R for 10 min. The excess dye was washed
after staining with tap water. The root regrowth after Al shock (or additional
root growth) was easily assessed. Seedlings with apical meristems, damaged by a
given Al pulse, had intensively stained root tips while those not damaged by Al
had a stained section of root followed by white root tip which developed after
Al shock. The dye is nontoxic to roots at the concentration used and at the
time stain was applied. During all stages of growth, and particularly during Al
treatment, nutrient solution was maintained at pH 4.5, adjusted with 0.1 M HCl.
At the ratio of approximately 20 ml of nutrient solution per seedling, changes
of pH of the medium were minimal during 24 h of Al treatment. Seedlings after
the test were still viable and could be transplanted for seed increase. Aluminum
concentration in nutrient solution causing irreversible damage to root apical
meristem during pulse treatment is a measure of tolerance of tested genotype.
The described test was used for
screening parental cultivars and hybrid populations. The results correspond
very well with cultivars of known Al resistance. Selected seedlings were
transplanted to the field for seed increase or further crossing. The test gives
reproducible results provided that proper conditions (temperature, pH, Al
concentration, time of exposure to Al) are controlled.
This test was used successfully in
our program directed at development of winter wheat strains with introduced
Al-tolerance from Brazilian, extensive spring cultivar BH 1146. We can now
offer to the wheat breeders a set of strains with agronomic characters at the
level of top cultivars on the list and Al-tolerance similar to BH 1146.
Several major genes control Al
tolerance in hexaploid wheat and the character is dominant and heritable,
therefore, selection for Al tolerance in wheat would be effective.
The described screening test does
not allow the discrimination between Al-tolerant homozygotes and heterozygotes.
Moreover, Al-tolerance in hexaploid wheat is a genetically complex character
and sensitive segregants were observed in many generations after cross. This
obstacle was solved by development of DH lines in F3 /F4
Moore D P, Kronstad W E and Metzger R J 1976. Screening wheat
for aluminum tolerance. In: Plant Adaptation to Mineral Stress in
Problem Soils. Ed. M J Wright. Cornell Univ Agric.
Polle E, Konzak A F and Kittric J A 1978 Visual detection of aluminum tolerance
levels in wheat by hematoxylin staining of seedling
roots. Crop Sci. 18, 823-827.