Agriculture Reference
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leaves used for the measurements were pre-conditioned in the dark for 30 minutes, and
illuminated for 5 seconds to induce fluorescence. The initial fluorescence (F0) and the
maximum fluorescence (Fm) were measured, further, the variable fluorescence (Fv = Fm -
F0) and the Fv/Fm ratio were calculated. Leaf area (LA) per plant were measured with copy
method described by Tao (2006), and leaf chlorophyll contents for the upper second fully
expanded leaves were estimated according to Zhang (1992). At the same time, starch and
sucrose contents in leaves were determined according to the description by Verma and Dubey
(2001) and Hashimoto el al. (2004) respectively. Then, plants of each treatment were
harvested and washed thoroughly with distilled water for 2 hours, leaves were separated and
weighted as leaf fresh weight (LFW), then dried at 80°C four 48h and weighted as leaf dry
weight (LDW). Then, leaf water content (LWC = (LFW - LDW) / LFW) were calculated.
All data presented are the mean values of three replicates. Statistical assays were carried
out by one-way ANOVA using Student's t-test to evaluate whether the means were
significantly different.
3. R ESULTS AND D ISCUSSION
3.1. Leaf Traits
Leaf traits in terms of leaf area (LA), leaf fresh and dry weight (LFW and LDW), and the
estimated data leaf water content (LWC) all significantly decreased when plants were
exposed to stress medium including low pH alone, especially for the ternary metals
combination of Al + Cd + Cu in Shang 70-119 (table 1). Inhibition of leaf expansion in
stressed plants was in agreement with the previous report by Florence et al. (2002) who
detected a reduction in cucumber leaf area of young expanding leaves under 10 µg g −1 Cu
supplementary in sand, but not for mature leaves. Similarly, Gichner et al. (2006) showed that
the average tobacco leaf area significantly reduced in plants growing on the combined heavy
metals (Cd, Cu, Pb and Zn) polluted soil. Reduction in leaf expansion in this investigation is
likely related to direct metals inhibition of cell growth as observed in Cu-stressed bean
seedlings (Maksymiec et al., 1995). In addition, the noticeable decreases in fresh and dry
weight detected in stressed barley plants were in good coincidence with the results obtained
on tobacco plants (Gorinova et al., 2007). Further, compared with the Al alone treatment,
binary metals combinations resulted in a severe inhibition than that of Al alone treatment for
both barley genotypes, indicating the existence of notable synergistic interactions between Al
and Cu or Al and Cd. However, for ternary metals combination of Al + Cu + Cd, it produced
different toxic results in two genotypes. In Gebeina, the toxicity caused by ternary metals
mixture was slighter than that caused by binary metals combinations, but severer than that
caused by Al alone treatment, while for Shang 70-119, noticeable synergistic effect was
detected in the three metals. Our previous study (Guo et al., 2007) also demonstrated that
whole plant growth of barley seedlings was dramatically inhibited by the treatments of Al,
Cd, Cu individual, binary and ternary combinations, and the toxicity was highly correlated
with Al, Cd and Cu contents in tissues. Likewise, the inhibition was more severe under Al +
Cd and Al + Cu treatments than Al alone treatment, but the ternary metals combination
induced the different toxic results in two genotypes.
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