Enabling technologies for production of improved clones of existing premium winegrape varieties using ‘DNA-free’ gene-editing
Summary
Objective
The Australian wine sector has a history of adopting innovative technologies to maintain a competitive advantage in the international wine market. The development of CRISPR/Cas9 gene-editing technology represents a step change in plant breeding, by offering the possibility of modifying specific traits in existing premium wine grape varieties, without these plants having to be labelled as genetic modified organisms. CSIRO is uniquely placed in Australia to realise the promise of this technology for the wine sector, having extensive experience with genetic modification of crop plants, and having partnered with Wine Australia for over 20 years in traditional grapevine breeding.
The main aim of this project is to develop a reliable and efficient protocol to apply new ‘DNA-free’ gene editing technology to the key winegrape varieties in Australia. This involves specialised techniques to firstly deliver the gene-editing machinery into grape protoplasts and then regenerate plants from the gene-edited protoplasts.
Once established, the protocol will be used to alter traits identified in the aligned project CSA 2301-2: Premium winegrape varieties with improved fungal pathogen tolerance and quality traits through gene-editing. Together, the program will provide a pipeline of superior clones of existing premium winegrape varieties, with improved agronomic and processing traits such as increased disease tolerance or improved fruit composition.
This project will also support the trait discovery team by maintaining stable transformation technologies for testing gene function and the efficiency of gene editing. The projects will work together to deliver improved germplasm to the Australian wine sector that should be easily adoptable into existing infrastructure and markets, while reducing costs associated with vineyard and winemaking inputs and improving sustainability.
Background
Traditional breeding methods have been used for centuries to generate new plant varieties with improved traits. While this process can take many years, the new varieties will have advantages over existing plantings. Because crossing is involved, the resultant plants cannot retain the original variety name, which is a challenge in winegrapes because of the perceived better marketability of varieties with a name familiar to consumers. This has stifled the uptake of novel winegrape germplasm from the CSIRO grapevine breeding program, despite the disease resistance traits they possess.
Recently, new gene-editing technology has been developed that allows for the specific alteration of DNA sequences in plant genomes. With certain gene-editing applications, referred to as ‘DNA-free’ gene-editing, it is possible to alter the plant DNA without the integration of any foreign DNA. In this case the resultant plant is no different from what would be observed if a mutation had arisen spontaneously, and is not classed as a genetically modified organism under current regulations. In addition, as cross-breeding is not involved, the new clone can retain the existing varietal name.
The method involves regeneration of plants from individual plant cells (protoplasts) which have undergone gene-editing. Plant regeneration is technically challenging and has only been achieved for a limited number of woody crop plants, with grapevine being particularly recalcitrant. However, overcoming the challenges with this specialist technology will make it possible to produce new clones of existing premium grapevine varieties that have improved traits.
This project will optimise DNA-free gene-editing methodology for key winegrape varieties to allow development of new clones to address challenges faced by the Australian wine industry.
Research approach
There are several steps in the development of a robust method for ‘DNA-free’ gene editing, each of which must be optimised experimentally. These include:
- Maintenance and initiation of cell cultures for target grapevine varieties
- Optimisation of methods for isolation of protoplasts (single plant cells without cell walls) and regeneration of plantlets
- Development of an efficient transfection method for delivery of the gene-editing machinery into protoplasts
- Development of techniques to gene-edit multiple target sequences in the grape genome.
Once the pipeline is established, it will be applied to specific gene targets identified and validated in project CSA 2301-2, to produce gene-edited clones of existing winegrape varieties.
This project will also maintain materials and capability associated with the portfolio of gene-editing projects. It will assist the gene discovery research by providing grapevine transformation capability and provide technical support and training for researchers in relevant tissue culture and transformation techniques.
Sector benefits
This project will, in combination with CSA 2301-2, provide the Australian wine industry with improved clones of existing wine grape varieties that will:
- benefit the sustainability and profitability of winegrape growing in Australia by minimising fungicide usage
- promote climate resilience by enhancing fruit composition to meet market specifications and preserve typicity in the face of a changing climate.