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A Technical Guide for Wine Producers
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Proving more versatile than just 'strawberries and cream'?
The use of strawberries in the genetic manipulation of grapevine fruit metabolism
Genetic improvement of grapevines offers great potential for the future competitiveness of the South African wine industry. This study addresses two of the factors currently limiting the realisation of this enormous potential.
With just over one percent of the world's vineyards - and an annual output at some 8 million hectolitre - South Africa ranks about 16th in area under vines and is the world's 8th largest wine producer. Apart from being an important primary source of economical growth and development (specifically in the Western Cape region where most of the vineyards are located), it has been shown that the South African wine industry has an exceptional ability to create prosperity. Nevertheless, with recent trends in consumer preference and consumption, steep increases in production costs and minimal increases in the final product prices, it is evident that the industry is under pressure to stay economically viable and competitive. In addition, the value and competitive nature of the grape and wine industry led to an increasing emphasis on producing quality fruit for the production of quality wines. Already faced, in certain inland regions, with the challenging task to counteract low total acidity, high pH and excessive accumulation of sugars generally associated with hot grape producing regions, the industry's strategies to improve fruit and wine quality and to develop novel products, are most challenging.
The molecular era opened new avenues to improve our understanding of the physiological and biochemical processes involved in plant development, but moreover, paved the way for the development of genetic engineering as a powerful tool for plant improvement. Despite large international interest in plant improvement through genetic manipulation, major obstacles prevent successful application of this technology in the manipulation of fruit ripening and quality. Specifically in grapevine two of the major obstacles are a lack of fundamental knowledge of the biochemistry and molecular biology of the fruit ripening process (Botha, 1999; Boss and Davies, 2001), and an understanding of, and mechanisms to regulate transgene expression to specific stages of development, or to specific tissues and/or cells.
The genetic manipulation of grapevine fruit metabolism To address these obstacles in grapevine, a programme on the genetic manipulation of fruit metabolism was initiated in 1998 at the Institute for Plant Biotechnology (IPB), Stellenbosch University. The objective of the programme was two-fold, viz. 1) the studying of gene expression in ripening grape berries to contributing towards a better understanding of the molecular processes involved in grape berry ripening, and to identify and isolate genes which could possibly be used to manipulate fruit quality in ripening wine grapes, and 2) the isolation of promoters to regulate the expression of these genes in transgenic grapevine.
Indeed, since 1998 several potentially useful genes and promoters were isolated at the IPB. However, final verification of the functionality and usefulness of these genes and promoters can only be achieved in plants in which the sequence(s) have been stably integrated. Due to the low genetic transformation efficiency, slow regeneration (about 18 months) and long reproductive life cycle of grapevine, the use of an alternative system for the evaluation of these genetic elements was investigated. Important considerations for such an evaluation system included: 1) a relatively short reproductive life cycle; 2) genetically closely related to grapevine, displaying a non-climacteric fruit ripening like grapevine; 3) the availability of a system for genetic transformation and regeneration of genetically transformed plants; and 4) a relatively short reproductive life cycle.
Since a system for the genetic transformation and regeneration of strawberries was available at the Biotechnology division of ARC Infruitec-Nietvoorbij (and strawberries also meeting the other considerations), a project to investigate the usefulness of strawberries for the evaluation of genes and promoters from grapevine, was initiated in 2002. Since then three promoters from grapevine and a gene which can possibly be used to manipulate sugar and acid metabolism in ripening grape berries, were introduced into strawberries. Promoter activity in transgenic strawberry plants and their ripening fruit was studied by fusing each of the promoters to a gene which encodes an easily assayed protein, the Green Fluorescent Protein (GFP) reporter gene. This allowed studying promoter activity in living strawberry cells in a non-destructive manner by GFP fluorescence microscopy. In Figure 1, the ripening-induced expression of the GFP reporter gene under control of the ripening-related promoter, mrip1 (Merlot ripening-induced protein 1) is illustrated. Accumulation of GFP in half ripe (1/2R) and full ripe (R) strawberries is used as an indication of the ripening-related nature of the mrip1 promoter. Moreover, the ripening-induced accumulation of GFP in the ripening strawberries compares well with the ripening-induced accumulation of the mrip1 transcript in grape berries (shown in Figure 2 A & B). Figure 2 also shows that the mrip1 promoter limits gene expression to the ripening berries, maturing seeds and bunch stems of the grapevine. Future applications
Boss, P.K. and Davies, C. 2001. Molecular biology of sugar and anthocyanin accumulation in grape berries. In: K.A. Roubelakis-Angelakis (Ed.): Molecular biology and Biotechnology of the grapevine, 1-33. Kluwer Academic Publishers, London. Botha, F.C. 1999. Struikelblokke in die weg van die benutting van genetiese manipulering vir die verbetering van druiwe. Wynboer, August: 54-56. |
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