| Heavy metal pollution of water bodies has become an increasingly serious environmental problem. Understand the principles of heavy metal contamination and heavy metals in water treatment have become a topic that must be addressed. Heavy metals can cause serious health hazards to animals and humans through the food chain due to the refractory, easy accumulation, toxicity and other characteristics. Therefore, much attention has been devoted to the studies on the mechanisms of heavy metal tolerance in plants and how to improve heavy metal tolerance of plants. Although antioxidative reactions of plants subjected to Pb2+ have been studied by several authors, there are less reports on the research of callus response to heavy metals.At present, the plants of natural growth were used as the object of Pb2+ poisoning study at home and abroad, mainly of the impact of Pb2+ on plant growth and development and plant physiological processes, but also a small amount of literature related to Pb2+ on the impact of plant genes and amino acids. However? the study of aquatic plant callus under heavy metals stress is also no detailed reports. In view of this, callus of Alternanthera philoxeroides was cultured to overcome the uncontrollability of light, temperature, moisture and plant growth and development in the natural state, so that the experimental data is more repeated and scientific. In this paper, callus of Alternanthera philoxeroides was cultured by tissue culture technique, which was used as experimental material, Pb2+ was selected as the stress factor. The callus was cultivated in solution containing different concentrations of Pb2+ under lab condition. The mechanisms for the protection of aquatic plant callus from the toxic effects of heavy metals as well as heavy metal accumulation in callus and the cellular localization and effect on the absorption of mineral elements were systemically studied. The results are as follow:(1) This paper discussed the induction of Alternanthera philoxeroides callus. The results showed that:The induction rate of stems was higher than leaves. At the same time, the callus of stems displayed close texture, larger volume and number, stems were still green, but the callus of leaves had loose texture, smaller and the leaves became brown. Bacterial contamination rate of the callus was the lowest,16.7% in control, and the survival rate was 63.3% after the disinfection of 70% ethanol 30 s, 5% sodium hypochlorite 10 min and 0.1% mercuric chloride 10 min, sterilization consequent was the best.1/2 MS medium including 6-BA (3.0 mg/L) and NAA (0.2 mg/L) was the most suitable for the growth of callus, the induction rate was 86.7%. Stems were used as explants, which were cultured in the temperature of 25℃, day light 16 h, light intensity 1200-1500 lx by the best sterilization methods and hormones. After about 10 d, both ends of the cells in the stems grew rapidly and proliferated dense green callus of Alternanthera philoxeroides.(2) Under different concentrations (0,0.2,0.4,0.8 and 1.6 mmol/L) of Pb2+ stress, the results indicated that, with the increasing of Pb2+ concentration, the contents of photosynthetic pigments and soluble protein decreased gradually; the contents of O2 and H2O2 increased gradually, while the content of MDA increased at first and then decreased; the changes of SOD and POD activities were the same as MDA, but CAT activity decreased gradually. Ultrastructure showed:grana and stroma thylakoids of chloroplast were swollen, disorder and gradually reduced the number in tending to vacuole; cristae were disorder and some of them appeared vacuolization; nuclear envelope break down, chromatin and nucleoplasm almost disappeared and nucleolus disintegrated. It was concluded that Pb2+ stress induced not only destruction of physiological function and structural foundation of Alternanthera philoxeroides callus, but also the disorder of physiological metabolism, which led to the death of the callus at last.(3) This paper studied that the toxic effects of Pb2+ gradient concentrations (0, 0.2,0.4,0.8 and 1.6 mmol/L) on the accumulation, subcellular distribution, ultrastructural localization and mineral elements were studied. The results indicated that, with the increasing of Pb2+ concentration, Alternanthera philoxeroides callus was able to accumulate lead, the bioconcentration factor was 2341—2681. Subcellular fraction analysis revealed that the contents of Pb2+ increased gradually in cell wall, organelles and soluble fraction, but the distribution ratio was obviously unequal, and the Pb2+ levels occurred in different parts of callus cell with the following sequence: cell wall〉organelles〉soluble fraction. Ultrastructural localization observation showed that Pb2+ mainly appeared in cell wall, and was also found in cell membrane structure and cell matrix. The effects of lead on mineral elements in three subcellular fractions were different, of which the absorption of macroelements P, K, Mg and microelements Na, Zn, Mn were inhibitted; the content of Ca increased at first and then decreased in cell wall, decreased gradually in organelles, increased gradually in soluble fraction; Fe and B increased at first and then decreased in subcellular fractions; the content of Cu in cell wall and organelles displayed with increase trends, increased at first and then decreased in soluble fraction; the content of Si in cell wall decreased gradually, increased at first and then decreased in organelles, while it was not able to be measured in soluble fraction. It could clearly be seen that the toxic symptoms of callus showed an evident correlation between dose and effect; the ultrastructural damage was closely related to the distribution of Pb2+; the ion equilibrium in subcellular fractions was disrupted under Pb2+ stress, resulting in disorder of physiological metabolism. All of these were major performance of lead on callus of Alternanthera philoxeroides. |
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