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TRANSGENIC GRAPEVINE CULTIVARS AIMED AT DISEASE RESISTANCE
Introduction
Within the context of genetic improvement of plant species, the word transformation implies the transfer of genetic material to a target organism. This involves a targeted plant improvement process that is dependent on tissue culture systems and the availability of useful genes and promotors as additional components of the process. In a recent issue of Wynboer (August 1999) the advantages, the areas targeted for manipulation as well as some obstacles typically encountered in the generation of transgenic grapevines were highlighted and will consequently not be discussed again. In this article the focus falls on the multidisciplinary approach employed by the Institute for Wine Biotechnology (IWBT) to manipulate disease resistance in Vitis vinifera, especially against fungal infections, with the aid of genetic manipulation techniques.
Click to see Figure 2: The steps followed in the transformation and regeneration of grape
cultivars.
Disease resistance
Any factor (abiotic and/or biotic) that has a negative influence on the quantity and/or quality of the crop of the grapevine and its processed products, is of direct economic importance. Natural pathogens, including fungi, bacteria and viruses, play a crucial role in this regard as they may have a negative influence on all stages of growth and berry development.
The disease control mechanisms currently used to prevent and control fungal infections mostly involve repeated spraying of chemicals combined with the labour intensive practices of canopy management. The use of chemicals is undesirable on many levels, it is inter alia very expensive, must be regularly repeated and causes a build-up of resistant strains in the pathogens population. Moreover, the application of chemicals is becoming increasingly undesirable to a growing number of consumers who insist on healthier and more naturally produced products. Furthermore, the long-term adverse effects of agricultural chemicals on the environment are well-known and it is generally accepted now that the agricultural community should scale down their dependency on these products.
In the light of the above background, this research programme focuses mainly on increasing the resistance of grapevines to fungal infections by using recombinant DNA technology. The strategy used relies on the strengthening of the plant's natural defence mechanisms against pathogen attacks. Plants have developed a variety of mechanisms to limit and/or resist pathogen attack. Of these the first line of defence is formed by structural obstacles, such as waxy layers or strategically positioned hydrolysing enzymes and/or antimicrobial compounds to stop the initial colonisation by the pathogen. In addition to these pre-formed defence elements, the plant is also able to orchestrate an induced response (known as active defence) as soon as the structural obstacles have been breached. These active defence responses may be initiated by all classes of plant pathogens in almost all living cells of a plant. This type of defence response typically exhibits a cascade effect with the initial response occurring in the cells that are in direct contact with the pathogen (frequently causing programmed cell death). The primary reaction usually gives rise to diffusible precursor molecules that spread to surrounding tissue where further induced resistance responses take place. The response spreads systemically through the plant due to a hormonally induced mechanism. From this it is clear that plants use complex mechanisms to observe pathogens and activate and co-ordinate their resistance responses, first in the cells that are in direct contact with the pathogen, then in surrounding cells and eventually throughout the plant.
Our approach is aimed at increasing the efficacy and sensitivity of the defence response in grapevines. One example is the expression of heterologous chitinase and glucanase encoding genes in grapevines to reinforce the primary defence responses. Various studies have confirmed that these genes and their protein products are important role players in the defence responses of plants. Both proteins act as hydrolytic enzymes and are targeted to the plant cell surface where the initial contact occurs between pathogen and host. The levels of chitinase and glucanase increase dramatically as soon as a pathogen attack occurs. Both these enzymes are responsible for disrupting the fungal cell wall and/or prevention of hyphal growth, therefore preventing the pathogen from further colonising the plant tissue. Overexpression of these genes will therefore cause higher levels of the enzymes on the plant cell surface, which might lead to a faster and more effective interaction with and neutralisation of the invading pathogen.
Another strategy that is based on the same principle involves the expression of antifungal peptides that show specific activity against a pathogen such as Botrytis cinerea on the plant cell surface. Antimicrobial peptides are found in most plant species and a specific peptide often provides resistance to one or more pathogens. The mechanism of their action often entails the prevention of hyphal growth, once again limiting the infecting pathogen to the initial point of infection. One such peptide, Hs-AFP1, was isolated from Heuchera sanuinea and shows high activity against B. cinerea in particular; it causes excessive branching of the fungus, which prevents spore tube germination and limitation of the subsequent hyphal growth (see Fig. 1).
In a complementary project an attempt is made to prevent the fungal infection process directly by means of polygalacturonase inhibiting proteins (PGIPs) which occur commonly in dicotyledonous plants and have been proven to show antifungal activity. These proteins also have structural characteristics typical of proteins that function as receptors in the relaying of signals (such as the resistance response) in cells. The isolation and characterisation of the genes and their products will therefore directly complement our understanding, and eventual manipulation of resistance response, of grapevines.
Transgenic grapevines
Somatic embryos of Chardonnay, Merlot and Sultana have recently been successfully transformed by the IWBT; a reporter gene (ß-glucuronidase) was integrated into the plant genome. Selected transgenic embryos were regenerated into mature and hardened-off plants. The steps followed are indicated in Fig. 2. The success of these transformations is measured against the stable expression of the transgene in the plant tissue, as indicated by genetic analysis (by means of Southern blot analysis) and the development of a blue colour in the tissue after histochemical staining (Fig. 3). The successful recovery of a useful number of transformants (on average 25-35 transformants per gram of somatic embryos) may be directly ascribed to the intrinsic regenerability of somatic embryos - the availability of such tissue is the key to the success of grapevine transformation projects. The somatic embryos used in our transformation experiments originated from immature anther filaments manipulated by means of tissue culture practices to generate embryogenic cell lines and ultimately, somatic embryos (Fig. 4). The regenerated transgenics are currently subjected to further long-term analyses.
In spite of a few reported successes employing genetic improvements of grape cultivars world-wide, somatic embryogenesis and transformations of grapevines are still not routine procedures and significant cultivar differences complicate the application of the established protocols to all cultivars. Our successful transformation and regeneration of a few cultivars, however, offer a much-needed point of departure to the intended disease resistance programme where an attempt is made to combat fungal infections of grapevines in a targeted and eco-friendly manner.
Acknowledgements
This work is financially supported by Winetech and is undertaken in collaboration with Proff. Pretorius (IWBT), Goussard (Department of Viticulture and Oenology), Botha (Institute for Plant Biotechnology), Bellstedt (Biochemistry) and Dr Burger (Genetics). Hs-AFP1 was kindly supplied by Prof. Broekaert, Catholic University, Leuven, as part of a transfer agreement.
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