Multiplication of plants through cuttings or tissue culture forms the basis numerous plant breeding and propagation applications. These technologies are applied sector-wide, but many genotypes and even entire species are recalcitrant for regeneration or regenerate too inefficiently to be commercially propagated. The molecular-genetic factors that define this genotype-dependent response are not known. In this project we will dissect the genetic variation that underlies one type of plant regeneration, somatic embryogenesis. Phenotypic screening of the Arabidopsis thaliana HapMap population combined with genome-wide association (GWAS) mapping will be used to identify the quantitative trait loci (QTL) that are significantly associated with the competence or recalcitrance for somatic embryogenesis. In a novel approach, identification of the causal loci underlying these QTL will be facilitated by the identification of gene expression QTL (eQTL) and protein abundance QTL (pQTL) in the same population, and by identification of chemical modulators of somatic embryogenesis. Omics analyses in diverse crop plants will be used to prioritize the list of candidate genes for functional analysis. These approaches will allow us to identify a core set of conserved somatic embryogenesis regeneration genes across plant species. The project will deliver gene variants that can be incorporated into a breeding program and targets for gene editing and small molecule intervention, all of which can be used to promote highly efficient somatic embryogenesis in crop genotypes

Objective 1: Identify the causal loci and underlying pathways associated with competence for somatic embryogenesis.
We will focus on three key steps/bottlenecks in somatic embryogenesis: 1) the initiation of primary somatic embryos; 2) the formation of callus from primary somatic embryos, and 3) the development of somatic embryos from callus (Figure 1, Annex 2). GWAS mapping will be used to unveil QTL in the genome that are significantly associated with the competence or recalcitrance for somatic embryogenesis at each step. Bulk transcriptomics and quantitative proteomics will be used to generate respectively, eQTL and pQTL, which will be integrated with the GWAS-identified regions to narrow down the genes controlling the phenotypic variation for these traits and to understand how these loci mechanistically affect somatic embryogenesis. Finally, chemical genomics will be used to identify small molecules that perturb somatic embryogenesis, providing a bridge between the protein that is targeted by the small molecule and the candidate QTL and/or QTL-regulated pathways. Knowledge on the proteins targeted by these small molecules provides another layer of information that can be integrated with the information from the QTL analyses to identify causal loci and regulatory pathways. This research will be performed in the model plant Arabidopsis thaliana (arabidopsis).
Objective 2: Identify key factors driving the different steps in somatic embryogenesis across systems.
To strengthen our ability to identify common loci across plant species, we will perform proteomics and transcriptomics in a few responsive/recalcitrant genotypes of the private partners crops of interest. These analyses will be performed at the same development time points outlined above. Additionally, each company will test the small molecules identified as part of Objective 1. This will allow us to prioritize the list of candidate genes from Objective 1 to identify a common set of core regulatory gene variants and associated downstream processes.

This proposal falls under the ST2 Biotechnology and Breeding Key Technology program. The major aim of the ST2 program is to develop knowledge, concepts and supporting technologies for optimal plant propagation. This aim is fully in line with the main objective of this proposal: to improve somatic embryogenesis-based plant propagation through a better understanding of the genetic variation that underlies this process. Somatic embryogenesis is a clonal plant propagation technique that is used in a wide range of cultivated plants, from ferns to trees and from ornamentals to field crops(Park et al. 1998; Mujib 2015; Loyola-Vargas and Ochoa-Alejo 2016; Etienne et al. 2018; Mikula et al. 2018). It is a valuable technique for clonally propagating breeding lines and commercial plant lines with low fertility, long life cycles or that suffer from inbreeding depression. This technique is also used to generate embryos for the rapidly advancing synthetic seed sector, to regenerate plants after transformation or gene editing, and to bypass the problems in F1 hybrid production. The forestry and ornamental sectors alone deploy millions of somatic embryo-derived plantlets per year. Somatic embryogenesis is generally preferred over other clonal propagation methods like cuttings and organogenesis due to the shorter time frame and reduced labour input required to obtain plantlets, due to its potential for unlimited multiplication capacity, and the possibilities for automation in bioreactors and immortalization of juvenile cultures and mature embryos by cryopreservation. The use of somatic embryogenesis as a clonal propagation tool has led to significant increases in production efficiency and -uniformity, and in the quality of cultivated plant germplasm.
This project contributes to both the ST2 Plant Reproduction subprogram, through its focus on in vitro propagation, and to the ST2 Bioinformatics and Big data subprogram, through its use of genetic mapping and high-throughput -omics technologies and the accompanying bioinformatics tools. To our knowledge, this is the first example where GWAS mapping, proteomics, transcriptomics, and chemical screening will be used together in plants to identify candidate genes and gene variants that control complex phenotypic traits. When successful, the novel approach that will be developed in this proposal has the potential to act as a disruptive factor in the plant genomics, plant breeding and academic plant biology fields.
The research proposed here can only be performed in the framework of a collaborative TKI T& U public private partnership project. The academic partners contribute expertise, knowledge and powerful model systems not found in the private companies. The private partners provide crop germplasm, somatic embryogenesis systems, and (potentially) proprietary mapping data for prioritization of candidate genes, all of which is likewise not available to the academic partners. Together both parties will work together to evaluate the utility of the identified candidates in their systems of interest,

Deliverables
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Private Partners: New loci/nucleotide polymorphisms that can be integrated in a breeding program to obtain germplasm with a high capacity for the three key steps in somatic embryogenesis.
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Private Partners: New genes and proteins that can be transiently modified by respectively, genome editing and small molecule intervention, to induce highly efficient somatic embryogenesis.
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Private Partners and Academic Partners: Proof-of-concept for the genome-wide multi-omics approach for mapping phenotypic variation for complex traits in at the population level.
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Academic Partners: Identification of new regulatory pathways that serve as a starting point for deepening our mechanistic understanding of plant regeneration.
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Academic Partners: Knowledge-based concepts that can be converted into novel applications for plant breeding.