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Salt Tolerance in Cotton Callus Tissue Transformed with Ascorbate Peroxidase, Glutathione Reductase, and Superoxide Dismutase

Dalton R. Gossett, Stephen W. Banks, Lisa Jones, Randy Allen, and Paxton Payton

ABSTRACT

Exposure to high levels of NaCl has been shown to induce an oxidative stress in cotton (Gossypium hirsutum) callus tissue. In addition, it has been demonstrated that cotton cell lines which are capable of up-regulating their antioxidant enzyme activities are more tolerant to NaCl stress than cell lines which do not increase the activities of these enzymes. This experiment was designed to determine if cotton callus tissue which had been genetically transformed with cDNA which encodes for antioxidant enzymes was more tolerant to NaCl stress than a NaCl-sensitive cell line and a NaCl-tolerant cell line selected to grow on elevated NaCl levels. Callus from a NaCl-sensitive cell line, a NaCl-tolerant cell line manually selected to grow at higher NaCl concentrations, a transgenic cell line transformed with glutathione reductase (GR+), a transgenic cell line transformed with ascorbate peroxidase (APX+), and a transgenic cell line transformed with superoxide dismutase (SOD+) was grown on media amended with 0, 150 mM NaCl, or 250 mM NaCl. After 28 days, the callus was harvested, weighed, and analyzed for catalase, peroxidase, GR, APX, and SOD activities. The growth studies showed that the transgenic cell lines grew better at both the 0 and the 150 mM NaCl levels than did either the NaCl-sensitive or the NaCl-tolerant cell lines. At the 250 mM NaCl level, the transgenic cell lines were more tolerant than the NaCl-sensitive cell line which died before the end of 28 days; however, the NaCl-tolerant cell line exhibited significantly more NaCl tolerance than any of the transgenics. An analysis of the antioxidant enzymes revealed that while each of the transgenics over expressed its respective antioxidant enzyme, the activities of the other enzymes remained relatively low, and in some cases the activity was actually lower than the activity expressed by the NaCl-sensitive cell line. On the other hand, the NaCl-tolerant cell line expressed significantly higher activities of all five antioxidant enzymes when grown on high NaCl than did the NaCl-sensitive cell line. These data suggest that in order to achieve a significant level of NaCl tolerance, the activities of several of the antioxidant enzyme must be up-regulated simultaneously.





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Document last modified 04/27/04