Enabling technologies and genetic resources

Abstract

Genetic transformation of grapevines is an essential tool for understanding gene function, which builds our understanding of how traits are controlled. In Australia, the capability to transform grapevines is unique to CSIRO and this project served to maintain this enabling technology along with the germplasm collection at Irymple. Transformed plants were produced to assist the understanding of genes involved in salt tolerance and to test fast breeding systems involving rapid flowering and floral sex. The optimisation of exciting new methods to produce improved clones of existing varieties using DNA-free gene editing was also progressed.

Summary

Traits are controlled, in large part, by genes. Therefore, any fundamental understanding of important traits that will improve the profitability and/or the sustainability of growing grapes and making wine requires knowledge of the underlying genes. Genetic mutants are the best way to study the relationship between genes and traits. This is the reason why so much research has been conducted on the model plant Arabidopsis due to its short life cycle, small stature and ease of transformation. In non-model plants, especially perennial crops, the production of a large collection of mutated plants is not possible. A means to fill this void is to alter the expression of genes suspected of being involved in certain traits using genetic transformation methods. This is not an easy task and only a few laboratories around the world can currently transform winegrape cultivars. Researchers at CSIRO developed Agrobacterium-based grapevine transformation methods in the late 1990s (Franks et al. 1998; Iocco et al. 2001) and have been maintaining this capability in Australia ever since. We also have access to CSIRO-developed RNAi technology for the suppression of gene expression or can over-express genes using constitutive promoters (e.g. CaMV 35S). Both techniques assist in the characterisation of genes whose functions are unknown.

This grapevine transformation capability was used to assist other projects to characterise candidate genes for traits targeted in the various lines of research. For example, transporter genes predicted to be involved in salt tolerance, putatively identified through genetic mapping strategies, were tested in transgenic vines that were engineered to either over-express genes or have their expression suppressed by RNAi. This provided evidence that certain candidate genes did indeed play a role in salt exclusion. In addition, this project was involved in the production of plants with editing copies of a gene suspected of being involved in the production of six-carbon aldehyde and alcohols in grape berries, which contribute to the green character of wine. The assessment of these plants is ongoing.

Transformation technology was also used to demonstrate our ability to develop rapid flowering versions of scions and rootstocks. The original ‘microvine’ was derived from the outer cell layer of Pinot Meunier (Boss and Thomas 2002) and as such the genetic background of any rapid breeding strategy using this plant has to consider that Pinot-like traits may be predominant in the progeny. The mutated Vvgai1 gene was introduced into Shiraz and 140 Ruggeri to test whether this is a viable strategy to reduce the stature of the vine and accelerate flowering. When expressed constitutively or under its own promoter, the Vvgai1 gene was able to accelerate flowering in dwarfed Shiraz vines, but interestingly, only the endogenous promoter could produce the same effect in 140 Ruggeri. Such plants could be used in rapid breeding provided that the transgene is segregated away in the final selections.

Flower sex is also an important part of any breeding strategy, as female plants are much easier to use for pollinations. While the flower sex locus was mapped to a small region of grapevine chromosome 2 some years ago (Marguerit et al. 2009; Fechter et al. 2012; Battilana et al. 2013), the causal gene had not been identified. 

Understanding how this trait is controlled allows for greater efficiencies in a breeding program. Gene editing, using CRISPR/Cas9 technology introduced via Agrobacterium stable transformation, was used to demonstrate that the VvPLATZ1 gene causes normal stamen production and when mutated, results in the production of flowers with reflex stamens (Iocco-Corena et al. 2021). This information allows the development of strategies to produce clones of cultivars or breeding lines with female flowers to aid pollination and makes breeding more efficient. These experiments also aided the development of some fundamental tools required for the application of gene editing in grapevine. 

While gene editing of grapevine had been demonstrated with the VvPLATZ1 flower sex experiments, the plants produced were transgenic and would require the removal of the transgene through a sexual reproduction step if they were to be grown commercially. The genomes of winegrapes are highly heterozygous, meaning that any sexual reproduction step will mean that a new cultivar is formed which is unlikely to be similar to the parent(s). An exciting new methodology allowing the introduction of the gene editing machinery to plant protoplasts via ribonucleoproteins enables the production of edited plants that are not regarded as genetically modified organisms. This enables the production of new clones of existing premium varieties with improved traits. Experiments conducted as part of this project demonstrated that we could produce protoplasts from embryogenic callus and then regenerate plants from the isolated protoplasts. We also showed that we could transfect grape protoplasts with nucleic acid and maintain their viability under mock transfection conditions. These developments provide a sound basis for the future application of DNA-free gene editing in grapevine scions to generate improved clones of existing cultivars that are not regarded as genetically modified.