Plants can regenerate new tissues, organs, and even entire organisms as part of their normal growth or in response to wounding. This regenerative ability has been utilised for various biotechnology and breeding purposes. Plant regeneration protocols rely on the application of exogenous growth regulators and/or abiotic stresses. These protocols are usually developed in a trial-and-error approach on a species- or genotype-by-genotype basis, which makes protocol development extensive and time-consuming. In recent years, autocrine-derived plant peptides, including PSK and REF1, have been identified that promote plant regeneration. These peptides have been utilised to enhance existing regeneration protocols, often promoting regeneration across species. Although peptides can be powerful regeneration tools, their application in tissue culture has been underexploited. This is due in part due to 1) the small number of peptides identified as regeneration-promoting peptides, 2) their variable effect, and 3) their predominant application in callus-based regeneration systems. To bridge this gap, we will utilise recently developed mass spectrometry-based ‘peptidomics’ methodologies for the proteome-wide identification of novel peptides that facilitate plant regeneration. We will identify a set of regeneration-promoting peptides by comparing responsive and non-responsive genotypes from both crops and model plants and across different regeneration systems. Using this method, we aim to find both universal and system-specific regeneration peptides. Discovering the receptors for these peptides will help us uncover the underlying signalling pathways that promote regeneration, which will then provide additional tools for modulating plant regeneration.

- Objective 1: High-throughput identification of the small secreted peptides with potential plant regeneration activity. | The activities linked to Objective 1 take place in WP1.
- Objective 2: Validation of the peptides with potential regeneration activity using various regeneration systems and plant species. | The activities linked to Objective 2 take place in WP2.
- Objective 3: Identification of peptide recognition and signal transduction mechanisms for universal regeneration-promoting peptides. | The activities linked to Objective 3 take place in WP3.

This project contributes to the ST2 subprogram, through 1) its focus on ‘Innovative Breeding Methods’ and the development of ‘Efficient Transformation and Regeneration Methods’ and 2) through its focus on ‘Bioinformatics and Big Data Technologies’, through its application of proteome-wide mapping of (non-conventional) peptides and development of ML bioinformatics tools to identify regeneration-promoting peptides and optimise peptide function.

• Well-established and broadly applicable pipelines to identify and quantify conventional and non-conventional SSP. (For academic and company partners)
• Database of SSP expressed across model and crop species and regeneration systems. (For academic and company partners)
• Identification of new peptides that promote efficient regeneration across species and regeneration systems. (For academic and company partners)
• Development of ML tools to identify SSPs and optimise their interaction with receptors to enhance plant regeneration. (For academic and company partners)
• Identification of new regulatory pathways that serve as a starting point for deepening our mechanistic understanding of plant regeneration. (For academic and company partners)
• New regeneration pathway genes for gene editing or breeding. (For company partners)
• Bioinformatics training in the analysis of peptidomics data. (For company partners)