Thesis (MSc (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2002. / Pathogen devastation of food products has been the topic of extensive research efforts
worldwide. Fungal infections are foremost amongst these pests, contributing not only to
losses in product yield, but also significantly affecting the quality thereof. It is not surprising
then that producers of these foodstuffs and their derived products continually strive
towards the highest possible product quality. Therefore, it remains imperative that
satisfactory methods are implemented to control these fungal pathogens. The current
strategies are all hampered by drawbacks, and severe crop losses are still experienced.
New technologies are being explored; one such technology is the genetic
transformation of plant species. This method has enabled scientists to introduce foreign
genes, with known functions and predictable outcomes, into plants. Genes identified to be
involved in disease resistance have become the focus of numerous research efforts
concerned with the improvement of the plant's innate defence response. This study aimed
to enhance disease resistance to fungal pathogens by means of the genetic transformation
of two genes previously shown to be involved in disease resistance. These genes encode
polygalacturonase-inhibiting proteins (PGIPs) from Phaseolus vulgaris and resveratrol
synthase from Vitis vinifera. PGIPs specifically inhibit the action of fungal
polygalacturonases (PGs), which are enzymes responsible for the hydrolytic breakdown of
plant cell walls. These enzymes were also found to be the first enzymes that are secreted
by fungal pathogens during infection of the host plant. Additionally, PGIP-PG interaction
results in the existence of molecules involved in the activation of plant defence responses.
Resveratrol, the product of resveratrol synthase, exerts its antifungal action by destruction
of the microbial cellular membranes. These mentioned genes were transformed alone, and
in combination, into Nicotiana tabacum and the resultant transgenic lines were evaluated
for enhanced disease resistance and for possible synergistic effects between the
transgenes.
Several independent transgenic lines were regenerated with genes integrated into the
tobacco genome. Almost all the plants harbouring only pgip or vst1 genes also expressed
these genes at a high frequency. Some non-expressing lines were identified from the
transgenic plants that had integrated both genes, but several lines were obtained
expressing both transgenes. Good correlations were observed between transgene product
activity and enhanced resistance to the fungus Botrytis cinerea in an antifungal in planta
assay. Lines showing the highest PGIP activity and resveratrollevels were more resistant
to the pathogen, leading to disease resistance of up to 80% seven days after inoculation in
comparison to an untransformed control. These lines maintained their strong inhibition,
even three weeks post-inoculation, showing a complete halt in disease development and
fungal growth. These results provide good indications of the efficacy of these transgenes
in the upregulation of plant defence. However, the study will have to be expanded to include even more transgenic lines to elucidate the possible synergistic effects of these
genes.
In an additional pilot study, genes encoding for precursors and for the formation of
resveratrol were introduced into the yeast Saccharomyces cerevisiae. The resultant
recombinant yeast strains were evaluated for their ability to produce the phenolic
substance, resveratrol. This compound has been implicated in beneficial aspects relating
to human health, including positive effects on atherosclerosis and platelet aggregation as a
direct result of its antioxidant and anti-inflammatory activities.
Recombinant yeast strains were constructed that expressed genes coding for
coenzyme A ligase and resveratrol synthase. These strains were shown to be able to
produce the phenolic compound resveratrol from the precursors present in the yeast as
well as from the products introduced with the transformation. The resveratrol was
complexed with an added glucose moiety. These results are extremely positive,
considering the possibility of manipulating wine yeasts to produce resveratrol during the
wine fermentation, thereby adding to the health aspects of both red and white wine. This is
the first report of the production of this compound by the introduction of genes necessary
for its biosynthesis in a foreign host.
This study has confirmed the importance of PGIPs and resveratrol in the effort to
enhance disease resistance in plants through genetic transformation technology. It has
also shown that the health benefits of resveratrol could be exploited more optimally in the
wine industry, by producing wine yeasts with the ability to synthesise this important
antioxidant.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/2949 |
| Date | 03 1900 |
| Creators | Becker, John van Wyk |
| Contributors | Vivier, M. A., Pretorius, I. S., University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. |
| Publisher | Stellenbosch : University of Stellenbosch, University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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