1. C. Stander, K. Vasanth, M. Korkie and M.A. Vivier
Introduction
Grapevine (Vitis vinifera L.) is considered the most important fruit crop globally. It is a woody perennial that has been proven to require extensive tissue culture interventions to facilitate successful genetic transformation and regeneration. Here we describe two in vitro systems that have been optimised to support research in grapevine molecular biology and biotechnology. Somatic embryogenic cultures are mainly used for genetic transformations. Non-embryogenic cell lines have also been established as model systems to study key processes in the grapevine, but have other applications, such as secondary metabolite production as well.
Methods, callus type and growth behavior
Somatic embryogenic cultures
Organ specific, non-embryogenic cultures
Somatic embryogenic cell lines were successfully established from immature anthers and converted to suspension cell lines for the cultivars Red Globe, Sultana, Merlot, Chardonnay and the rootstock R110 (Fig 1 A – C). The callus production is strongly influenced by the developmental stage of the anthers [1]. A B D C
Embryogenic callus cells consisted of small, tightly packed meristematic cells (Fig 1D) with excellent regeneration potential (Fig 2). Regeneration involves differentiation through typical embryo stages until a germinating embryo and regenerated plantlet is obtained.
Non-embryogenic callus was initiated from grape berries harvested during a range of developmental and ripening stages. The callus cultures were highly prolific (Fig 3 A – D). The thin walled non-embryogenic callus cells consist mostly of vacuoles (Fig 3 D).
Fig. 1. The development of somatic embryogenic cultures from immature anthers. A. Grapevine flower cluster. B. Immature anther. C. Embryogenic callus culture. D. Embryogenic cells in suspension.
Fig. 3: The development of berry derived callus cultures. A. Dauphine berries. B Berry explants on media for callus initiation,. C. Non embryogenic callus culture. D. Non-embryogenic cells in suspension.
Cell suspensions were weekly maintained by transferring 20 ml of cell culture into 80 ml of fresh media. Callus production was strongly influenced by the stage of the berry development and ripening (Fig 4). The cell suspensions exhibited a typical sigmoidal growth curve over a three week period (Fig 5).
Fig. 4. Callus production from berries during three developmental stages
Fig 5. Typical growth curve of a non -embryogenic callus in cell suspension
Fig. 2. Regeneration of somatic embryos. A. Germinating somatic embryos. B.Cotyledonary stage germinated embryo. C. Regenerated in vitro plantlet. D. Acclimatised regenerated grapevine plant.
In vitro culture systems to study and genetically improve grapevine (Vitis vinifera) cultivars
C. Stander, K. Vasanth, M. Korkie and M.A. Vivier
Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, Stellenbosch ZA-7600, South Africa
Targeted applications
Conclusion
Acknowledgements
References
Somatic embryogenic cultures are mainly used for genetic transformation (predominantly via Agrobacterium [2]). Targets for genetic improvement of cultivars include traits such as disease and pest resistance, tolerance to environmental stresses and improved product quality. It is integral to functional analysis of grapevine genes since the grapevine genome has been sequenced.
Although significant progress has been made to apply molecular tools to grapevine, a high-throughput transformation systems is still lacking. Moreover, it remains limiting to work with a seasonal crop where certain organs, are only present for a short period. Technologies needs to be developed for grapevine to overcome these problems. Here we report on the successful development of two callus cultures, the one as starting material for genetic transformations and the second as organ-specific cultures that could be used to study berry development and metabolism.
Cell cultures that mimic berry-specific developmental metabolism and gene expression could facilitate our understanding of key aspects of berry development and ripening. The concept has been well illustrated in studies with V. vinifera suspension cultures where simple changes in nutrient composition activated the biosynthesis and accumulation of PR proteins, stilbenes and anthocyanins [3].
1. Gribaudo, I., Gambino, G. and Vallania (2004). Somatic embryogenesis from grapevine anthers: the optimal developmental stage for collecting explants. Am. J. Enol. Vitic. 55, 427.
2. Iocco, P., Franks, T. and Thomas, M.R. (2001). Genetic transformation of major wine grape cultivars of Vitis vinifera L. Transgenic Research 10: 105 – 112.
3. Belhadj, A., Telef, N., Saigne, C., Cluzet, S., Barrieu, F., Hamdi, S. and Mérillon, J.M. (2008). Effect of methyl jasmonate in combination with carbohydrates on gene expression of PR proteins, stilbene and anthocyanin accumulation in grapevine cell cultures. Plant Physiology and Biochemistry 46 (4): 493 – 499.