| Zoysia matrella, namely manilagrass, is perennial herb of Gramineae. It’s an excellent warm-season turfgrass species for its low, green quality and long green period. As Zoysia matrella is widely used in golf courses, ground track fields and parks in most region of southern China, more and more people pay attention to its resistance and ornamental characteristics. But Zoysia matrella propagates asexually by stolons in nature, so conventional hybridization breeding procedure can’t be applied for its genetic improvement. Thus modern biological technology such as somaclonal variation is an effective way to improve its resistance.Copper is widespread pollutant in nature, mainly derived from industrial and agricultural activities, such as pigments, minerals, smelting, electroplating, etc. Copper is not only a necessary element for plant growth, but composition of some enzymes. However, copper, as other toxic heavy metals, is poisonous to plants when its content is more than suitable growth range. An important feature of copper poisoning is that it can cause oxidation reaction in plants. In order to adapt or to overcome copper stress, the activities of one or more intracellular antioxidant enzymes in Zoysia matrella tend to rise, to eliminate or reduce the harm of copper stress.In this experiment, the embryonic callus of Zoysia matrella subcultured for9years was used to study its responses to copper stress during callus growth and plant regeneration. The effects of different concentrations of copper on callus diameter, growth rate of fresh weight and regeneration rate, the activities of antioxidant enzymes (CAT, POD, SOD and APX), the contents of copper, MDA in the callus, the leaf and root of the plantlets in the courses of callus growth and plant regeneration were evaluated. The different concentration of copper in callus culture respectively were0.1,1.0,10.0,50.0,100.0,200.0,300.0,500.0and1000.0μM. The different concentration of copper in callus regeneration medium respectively were0.1,1.0,10.0,50.0,100.0,200.0,300.0and500.0μM. The effects of long-term and short-term treatment on physiological characteristics of callus were also studied. Furthermore, variants with improved resistance to copper stress were selected, and we got a small amount of copper-resistance plants. The primary were summarized as follows:1. The diameter and the growth rate of fresh weight of callus increased after touching different concentrations of copper for12weeks, and reached the maximum when the concentration of copper was at10.0μM. As the concentration of copper continued to increase, the average diameter and the growth rate of fresh weight of callus significantly reduced. At10.0μM, the average diameter of callus was twice as large as the diameter in500.0μM. So in callus culture, the concentration of copper in MS was not optimal for the growth of callus. The critical concentrations of copper for callus growth was about500.0μM.2. The callus grew rapidly after treated with different concentrations. The regeneration rate of callus increased after touching high concentrations of copper compare to control group of0.1μM. The regeneration rate reached the maximum when the concentration of copper was at10.0μM. With the increasing of concentration of copper, The regeneration rate reduced. So the concentration of copper in the MS was not optimal for callus regeneration. The critical concentrations of copper for callus regeneration was between50.0μM and100.0μM.3. With the increasing of the concentration of copper in the medium, the contents of copper in callus, leaves and roots increased. This study found that the resistance of callus to copper stress was better than regenerated plantlets. And the variation of copper content in callus was similar with the variation trend in root and leaf. So callus can be used in copper-resistance plant screening by screening copper-resistance callus under the stress of high concentration of copper.4. The activities of SOD, POD, APX increased continuously in short-term treated groups. The CAT activity increased at first, then decreased when the concentration of copper continued to increase. In the long-term treated groups, the enzymes of SOD, POD, CAT and APX of callus in all treatment were lower than the control group when the concentration of copper was at10.0μM. The activities of CAT, SOD, POD and APX increased continuously when the concentration of copper was higher than10.0μM, while decreased when the concentration of copper was at200.0μM.5. The accumulation of copper caused lipid oxidation reaction in Zoysia matrella. The contents of MDA in callus after short-term treatment and in roots of regenerated plants increased with the increasing of the concentration of copper. But in the long-term treatment, the content of MDA in callus was lowest when the concentration of copper was at10.0μM. The contents of MDA in the leaves of regenerated plants in different concentration of copper had no significant differences. And the content of MDA in roots was much lower than the MDA content in leaves.6.In the study of screening copper-resistance Zoysia matrella, we found that the mechanism of resistance to copper indeed existed in callus. Adding copper solution into the medium during the process of callus growth or regeneration can improve the resistance of callus to copper stress. And the degree of enhanced resistance was associated with the stages of adding copper solution. This experiment found that the callus resistance enhanced significantly when adding copper solution in the callus culture process. |
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