BackgroundElectron microscopy (EM) techniques enable identification of the main accumulations of lead (Pb) in cells and cellular organelles and observations of changes in cell ultrastructure. Although there is extensive literature relating to studies on the influence of heavy metals on plants, Pb tolerance strategies of plants have not yet been fully explained. Allium sativum L. is a potential plant for absorption and accumulation of heavy metals. In previous investigations the effects of different concentrations (10-5 to 10-3 M) of Pb were investigated in A. sativum, indicating a significant inhibitory effect on shoot and root growth at 10-3 to 10-4 M Pb. In the present study, we used EM and cytochemistry to investigate ultrastructural alterations, identify the synthesis and distribution of cysteine-rich proteins induced by Pb and explain the possible mechanisms of the Pb-induced cellular defense system in A. sativum.ResultsAfter 1 h of Pb treatment, dictyosomes were accompanied by numerous vesicles within cytoplasm. The endoplasm reticulum (ER) with swollen cisternae was arranged along the cell wall after 2 h. Some flattened cisternae were broken up into small closed vesicles and the nuclear envelope was generally more dilated after 4 h. During 24-36 h, phenomena appeared such as high vacuolization of cytoplasm and electron-dense granules in cell walls, vacuoles, cytoplasm and mitochondrial membranes. Other changes included mitochondrial swelling and loss of cristae, and vacuolization of ER and dictyosomes during 48-72 h. In the Pb-treatment groups, silver grains were observed in cell walls and in cytoplasm, suggesting the Gomori-Swift reaction can indirectly evaluate the Pb effects on plant cells.ConclusionsCell walls can immobilize some Pb ions. Cysteine-rich proteins in cell walls were confirmed by the Gomori-Swift reaction. The morphological alterations in plasma membrane, dictyosomes and ER reflect the features of detoxification and tolerance under Pb stress. Vacuoles are ultimately one of main storage sites of Pb. Root meristematic cells of A. sativum exposed to lower Pb have a rapid and effective defense system, but with the increased level of Pb in the cytosol, cells were seriously injured.

A general report on the use of the Allium test as cytotoxicological and genotoxicological assay is proposed, with particular emphasis about the standardization of the test in several common applications. The intraspecific variation in Allium cepa has been overlooked, as in most investigations no mention is made about origin and denomination of the onion cultivar used. A standardization of the used material would allow a better generalization of the results, since we cannot be sure that all cultivars would give the same response. A more frequent use of transmission electron microscopy (TEM) investigation is proposed. Even if relatively time consuming and not available in all laboratories, it may help to better understand the mechanism of cytotoxicity, since many morphological characters may appear similar but be arisen from different processes observable only with TEM. About statistical testing, tests other than chi-squared may be used in case of a lower amount of data. The most commonly used statistical tests are the parametric tests ANOVA and Student's t, and the nonparametric tests Kruskal-Wallis and Mann-Whitney U, for analysis of variance. Tests should be used also to assess the minimal sample dimension for obtaining significance, since data collection (microscope observation) appears to be one of the main bottle necks of the test. Also the use of the Allium test for testing liposomes and other nanovectors for drug delivery is proposed, in order to assess the cytotoxicity of these types of medium and the possible increase in cytotoxicity of the associated drug.

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The effects of different concentrations of Pb on growth of Allium sativum L, Pb uptake and accumulation, antioxidant enzyme activity and malondialdehyde content were investigated. The results indicated that shoot growth at high concentration of Pb (10(-3) M) and roots growth at 10(-3) M and 10(-4) M Pb were significantly inhibited. Lead ions were accumulated mainly in the roots and only small amounts were translocated to bulbs and shoots. SOD activities in shoot and roots exposed to 10(-3) M Pb were observed to be high. Plants exposed to 10(-3) M Pb showed a significant increase in POD activity in roots versus the control and other Pb treatments. In roots, CAT activity and MDA concentration at 10(-3) M Pb is high significantly. The mechanisms of Pb toxicity and tolerance in garlic are briefly discussed.

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