Tissue cultures and viticultural applications

by | Jan 1, 2016 | Winetech Technical, Viticulture research

Introduction

Plant cells are considered totipotent, meaning that all plant cells have the capacity to regenerate into a complete new individual. Totipotency, in combination with the ability to differentiate (cells/tissues/organs develop to acquire a specialised function) and dedifferentiate (cells/tissues/organs undergo changes to revert to cells that are without specialised function and meristematic in nature) underlies the ability to manipulate plants in tissue culture. More than a century ago these findings sparked the field known today as plant tissue culture. Plant tissue culture research and knowledge has progressed rapidly in the last 40 years and is now recognised as a major discipline playing an important role in fundamental plant biological studies and product orientated research.

Grapevine tissue culture uses

The first grapevine tissue cultures were reported in 1944, with the objective of establishing sterile in vitro tissue cultures. Since then several advances have been made in manipulating grapevine explants in culture (the term for the starting material used in establishing a tissue culture). Several motivating factors propelled the research in grapevine tissue cultures and include:

  1. The development of micro propagation protocols for grapevine materials (Harris & Stevenson, 1982; Torregrosa et al., 2001);
  2. The elimination of viruses from plant material using the meristem cultures and somatic embryogenesis (Harris & Stevenson, 1979; Goussard et al., 1991);
  3. Development of methods to perform embryo-rescue in breeding programs that target seedless grapevine cultivars (Perl et al., 2000; Burger et al., 2003; Tian & Wang, 2008; Ji et al., 2013);
  4. Development of culturing systems to support genetic manipulation of grapevine cultivars (Martinelli & Mandolino, 1994; Kikkert et al., 1996; Perl et al., 1996; Vidal et al., 2003);
  5. The use of cell-based cultures to study-specific aspects of grapevine development and/or berry ripening (Symons et al., 2006; Ramaschandra et al., 2011), as well as regulation of metabolic pathways (Cakir et al., 2003; Ferri et al., 2011); and
  6. The production of valuable metabolites for grapevine suspension cultures (Calderón et al., 1993; Gagné et al., 2011). Lately, grapevine organ-specific cultures, such as berry cultures, are being evaluated as model systems to overcome specific problems with the study of grapevine organs, specifically grapevine berries in field conditions.
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Grapevine is difficult to manipulate in culture

Grapevine is considered recalcitrant to tissue cultures, mostly because significant optimisations are needed to implement efficient protocols for different grapevine explants and cultivars. Although a range of tissue culture methods and techniques have been published (Martinelli & Gribaudo, 2009), considerable optimisations per experiment, explants, variety, cultivar and/or clone are often needed, making the work time-consuming, technically demanding and not generally available to all labs working with grapevines. The initial drive to develop grapevine tissue culture and transformation technologies also suffered significant setbacks with consumer resistance against genetically modified foods. Notwithstanding, the technologies developed and are available in a few laboratories around the world and some countries and industries invested resources and efforts in implementing genetic improvement. Research groups typically use the technologies to provide proof-of-principle results (Terrier et al., 2009, Lashbrooke et al., 2013). In vitro grapevine plants can also be used for transient transformation experiments using Agrobacterium infiltration (Du Preez et al., 2010), a technique widely applicable in functional studies for crop improvement.

Somatic vs embryogenic grapevine tissue cultures – what’s the difference?

Callus is defined as unorganised, undifferentiated plant cells forming as a result of a wound response. Grapevine cultures can be established to form either embryogenic or non-embryogenic callus types (Stander et al., 2009). Embryogenic callus consists of small, tightly packed meristematic cells which have excellent regeneration potential. In other words, somatic embryogenic callus develop from ordinary plant body (somatic) tissues, but can develop into reproductive embryo structures, ultimately germinating in culture to form plantlets. Non-embryogenic callus, on the other hand, are not maintained for the purpose of regeneration into plants. These cells consist of highly prolific, thin walled cells containing mostly vacuoles and are typically used to study cellular and metabolic processes in culture. Here the use of non-embryogenic berry cultures will be considered further.

Non-embryogenic somatic berry callus cultures as research tools

Callus cells theoretically will grow and proliferate if provided with appropriate nutrients and a supportive environment. It is well-known that these callus cells can maintain metabolic functions characteristic from the organ they originate, but the extent of this “memory” needs to be experimentally proven and not assumed.

The process involved to develop such cultures from grapevine berries are outlined in Figure 1 and starts with berries being collected and surface sterilised after which the berries are cut into slices and pips are removed. The slices are placed on media optimised for callus formation. The callus cultures are rigorously selected and transferred to fresh medium until homogenous cultures are obtained after four to six selection and transfer cycles.

Sharathchandra et al. (2011) reported an analysis from these somatic grape berry cell cultures, comparing them to evaluate if the callus forming process would eliminate the characteristic features of the berry development stages (green to ripe). The first interesting and significant result was that the cultures from green, véraison and ripe berry explants formed different proteins, providing evidence that the process to initiate callus formation does not abolish “signatures” linked to the different developmental stages of the explants. Moreover, the results showed that soluble proteins produced by the cell lines were comparable to those of berries from the field in corresponding developmental stages. In the study reported by Sharathchandra et al. (2011) the different cell cultures were incubated in the dark, eliminating light as a modulating factor.

Interestingly, in a subsequent study the analyses of the green, véraison and ripe cultures were extended to evaluate how the cultures will respond to exposure to light (Noqobo, 2015). The results of this study confirmed that the berry cultures originating from explants in different developmental stages maintained characteristic differences in metabolite production (pigments and aroma compounds) when exposed to different light regimes. These berry cultures therefore retained the capacity to respond differentially to the changing light environment, providing support for their use as potential test systems when studying berry metabolism (Noqobo, 2015).

These berry suspension cultures were also used to study pathogen responses in grapevine cells (Mutawila et al., 2012; Mutawila, 2014). The suspensions were treated with elicitors (extracts from the fungi that will trigger a defence response in the plant cells) originating from Eutypa lata, a destructive wood-infecting pathogen, as well as with elicitors originating from a Trichoderma biocontrol agent that is used as a protective treatment on pruning wounds. The suspension cultures treated with these elicitors were used to study the early responses of the grapevine cells to the elicitors and to study the expression of defence-related genes and the development of protective metabolites such as polyphenols (Mutawila, 2014). These cultures showed some of the mechanisms the biocontrol agent employs to protect grapevines against this destructive pathogen, as well as the response that is triggered by the pathogen.

Maintaining intact berries in culture to study developmental and ripening processes

Berries comprise skin, pulp and seed tissues and several processes and metabolic changes are uniquely different in these tissue types. One of the problems with generating callus cultures of grapevine organs such as berries, is the fact that tissue-specificity is lost when initiating callus formation. Exciting techniques have however recently been developed to adapt intact grape berries to tissue cultures and maintain their development/ripening under controlled conditions in culture (Dai et al., 2014, 2015). These cultures allow the berries to be studied and sampled as intact organs, but with the possibility to control the media and environmental conditions.

These examples and literature illustrate that grapevine tissue culture techniques are important as study tools, but also important to support plant improvement strategies for cultivars and clones.

Summary

Tissue culture is a relatively old plant science, originated in the early 1900’s and still plays a crucial role in many modern technologies. It has contributed significantly to grapevine biology and biotechnology and is facilitating our understanding of how grapevine functions in terms of gene-expression, metabolism and development. Grapevine tissue culture techniques are continuously updated, establishing systems and protocols that allow these cultures to be used for exciting new applications.

This article originates from research funded by Winetech and the final report of project IWBT 4-10 (The establishment of stable and synchronous embryogenic cell lines of grapevine rootstock cultivars for use in transformation systems) and IWBT P 08/14 (Funding support for transformation and regeneration facility for grapevine) can be downloaded from: http://www.sawislibrary.co.za/dbtextimages/finalreport86.pdf and http://www.sawislibrary.co.za/dbtextimages/VivierM.pdf.

Acknowledgements

We would like to thank Winetech, National Research Foundation and THRIP for financial support for the tissue culture research conducted within the Institute for Wine Biotechnology’s Plant Tissue Culture and Transformation Platform.

References

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– For more information, contact Melané Vivier at mav@sun.ac.za.

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