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Doctoral Degrees (Soil Science)

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Browsing Doctoral Degrees (Soil Science) by Author "Cooke, John Anthony."

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    Investigations into aluminium toxicity and resistance in cynodon dactylon using invitro techniques.
    (2004) Ramgareeb, Sumita.; Cooke, John Anthony.; Watt, Maria Paula Mousaco Deoliveira.
    Aluminium toxicity is a significant limiting factor to agricultural crop production globally, promoting the need for plants that are resistant to low pH and high Al3 +, Current literature suggests that Al3 + inhibits plant growth by stopping root elongation. Although considerable research has been directed towards Al3+-inhibited root growth, the initial cellular targets and primary mechanisms of Al3+ toxicity still remain unclear. The present study, therefore, considered an alternate approach to investigating Al3 + toxicity and Al3 + resistance. Callus, a group of actively dividing meristematic cells, was exposed to Al3 + and the influence of Al3 + on callus growth was investigated. In South Africa, gold mining results in the production of wastes that require vegetation cover resistant to low pH and high Al3+, in order to promote stabilisation and prevent erosion. Cynodon dactylon was considered a key species for such a purpose since small populations of this grass were found growing on the acidic gold tailings. Different C. dactylon genotypes were exposed to Al3 + and the feasibility of using differences in callus growth to identify potential Al3 +-resistant individuals was assessed. An in vitro method for indirect somatic embryogenesis was concurrently established to regenerate whole plants from such calli. Embryogenic calli were initiated from young leaf segments, using 2,4-D. Somatic embryo maturation and plant regeneration were achieved on a hormone-free Murashige ~d Skoog (MS) nutrient medium. In addition to this protocol for micropropagation via indirect somatic embryogenesis, nodal cuttings, on a single hormone-free MS nutrient medium, were shown to be suitable explants for micropropagation via direct organogenesis, albeit resulting in low plantlet yields (1 plant/explant). In the investigation of Al3 + resistance, each of the three parameters tested (genotype, Al3+ concentration and exposure time) had a significant influence on callus growth rate. The nutrient medium supporting callus growth was modified in order to ensure known concentrations of free Al3 + ions (0.08-2.3 mM). This was achieved through the use of a chemical speclatlOn model (MINTEQA2). Fresh callus mass measurements for three genotypes were recorded at two-weekly intervals for a total of 8 weeks. Significant differences in callus growth rate were used to identify the genotypes as Al3+-sensitive (AlS), moderately Al3+-resistant and Al3+-resistant (Al-R), suggesting that it is feasible to use undifferentiated meristematic callus cells to screen for resistance to Al3+. In addition to callus growth rate, it was also possible to differentiate between the Al-S and the Al-R genotype using differences in cell numbers. Exposure to 0.8 mM AI3+ for 2 weeks resulted in an 88% reduction in the Al-S meristematic cell number whereas no Al3 + concentration tested had a significant inhibitory effect on the Al-R cell number. Aluminium was detected inside the callus cells, with the Al-S cells accumulating three times more Al in the nucleus than did the Al-R cells. It is suggested, therefore, that Al3 + inhibited meristematic cell number in the Al-S genotype by interfering with cell division. Two possible mechanisms by which the Al-R genotype was able to exclude Al3+ from its cells were investigated. The Al-R callus was able to maintain a higher extracellular pH (4.34 in Al-R and 4.08 in Al-S) and immobilise more Al in the cell wall (33% more in the Al-R) than the Al-S genotype. The present study has developed a valuable tool for investigating the physiological effects of Al3 + on actively dividing meristematic cells. In addition, the somatic embryogenesis route allows for the concurrent in vitro selection and plantlet regeneration of genotypes of interest. Future work is necessary to confirm that the properties of undifferentiated cells in culture are maintained by the ex vitro whole mature plants.