The ultimate goal of RECROP is to propose and implement different strategies for the improvement of crops resilience under different stress scenarios. For this purpose, experts from different fields will propose the most up do date methods for crop improvement via (I) Breeding, (II) Gene editing techniques such as CRISPR and (III) transgenic lines. We foresee that at least some of the proposed strategies can be implemented by members of RECROP. In this task, plant breeding companies will be encouraged to be involved in the strategic planning as well as in the generation of new lines through collaborations with research groups from institutes. Upon publication, the lines will be deposited in the online repository. We will encourage the simultaneous testing of new lines by multiple partners, for cross-validation of the stress resilience, using the methods described in WG1-2. We will further encourage the evaluation of the performance of these lines in combination with chemical treatments described in WG2. Currently, several research groups involved in RECROP have in hand lines from breeding populations, mutants and transgenic plants from different crops that show enhanced tolerance to abiotic stresses. RECROP members will encourage the sharing of this valuable genetic material in the frame of bi- or multilateral collaborations for further improvement. To stimulate material exchange and progress, RECROP will create a platform where a description of the genetic background and the 13 traits for each line will be displayed. We believe that this step will accelerate crop improvement efforts for high yield under suboptimal conditions. TASK 3.1: Create a repository with stress tolerant genotypes. RECROP members are engaged to scientific exchange and research progress. A key aspect for RECROP community is that scientists bring in complementary skills and come from laboratories with a variety of expertise in different aspects that are relevant and essential to achieve the goals of RECROP. Several members of RECROP possess varieties and hybrids, mutants and transgenic lines from different crops that exhibit enhanced resilience to abiotic stresses. RECROP will encourage the sharing of this material, by creating an online repository, in which different lines will be presented along with their genetic background, description of important traits and contact information with the research group that own the material. By this, we will stimulate the phenotypic and molecular characterization of promising lines using the approaches described in WG1 and 2. We also envision that such lines can be used as starting material for pre-breeding but can be also be subjected to further improvement by genetic manipulation, e.g. to reduce trade-offs. We will encourage through bilateral agreements the use of this material by breeding and biotechnology companies and in collaboration with the research groups to exploit them as basis for crop improvement. The website will be integrated within the RECROP webpage. TASK 3.2: Enhancing reproductive success under different stress scenarios using state-of-the art genomics-based breeding approaches The development of climate-resilient crops can be accelerated by the utilization of integrative genomics approaches. RECROP will take advantage of the existence of seed collection as well the rich genomic resources for major crops (e.g. wheat, rice, barley, maize, tomato) to mine stress resilience related traits with the use of ML. Members of the WG3 will design adaptive introgression of genes from crop wild relatives, that may have been lost during long period of conventional breeding, a source that is rich for adaptive traits, to elite cultivars. Furthermore, we will explore the possible use of available pangenomes to link traits related to reproductive resilience to abiotic stresses to single nucleotide polymorphisms (SNPs), mutations and genes harbouring structural variants (SVs). SVs although they represent complex variations that are difficult to detect and therefore largely unexplored compared to SNPs, they are considered as important genomics resources for developing new crops with desired adaptive traits. We will encourage the active involvement of experts in Deep Learning genomics mining, to create high confidence pipelines for the discovery of SVs. TASK 3.3: Design and implement strategies to improve crop reproductive resilience by gene editing approaches. Gene editing is a powerful tool to study gene function and targeted mutations in specific genes have been proven to have stimulatory effects of stress resilience. Currently CRISPR- based mutagenesis is considered as a fast-forward molecular engineering technique for basic research as commercial use due to European legislation is still regulated. However, using CRISPR or other gene editing techniques such as TALENs can provide information on genetic variations and gene functions that can be valuable for the development of breeding strategies. Gene editing can accelerate de novo domestication of a wild crop relative. Therefore, based on information derived from WG2, RECROP will create a list of genes that their mutation can cause enhanced resilience of reproductive tissues to different stress scenarios. Several of these genes will be mutated in different crops by members of RECROP and their resilience will be evaluated by the methods presented in WG1. TASK 3.4: Using synthetic biology to introduce novel entities for trait improvement. Stress resilience often is associated with trade-offs, as typically stress response mechanisms can interfere with growth and development, and it is frequently manifested as disturbances in reproductive development. This can be a major obstacle for the generation of stress resilient germplasm through breeding approaches or even gene editing. To overcome this problem, synthetic biology approaches can be implemented which allow the tight regulation of gene expression in time and space (e.g. specific cell types) and genes can be modified in order to produce proteins with customized activities. Members of WG2 and 3 will design strategies for synthetic genes that could target the improvement of specific traits. Several of these genes will be transformed in different crops to evaluate the validity of the proposed strategy. TASK 3.5: Identify exogenous treatments with chemicals that can stimulate the genetically inherited capacity of crops to tolerate stress Exogenous chemical treatments with plant-based biostimulants can reduce the negative impacts of stresses in crops and have a positive impact in yield [9]. WG3 will establish guidelines for the exogenous application of such compounds, their benefits for sustainable agriculture and their suitability for impacting yield under different stress scenarios. Furthermore, WG3 will examine the effects of these treatments on plant reproductive tissues and whether they can reduce the negative impacts of stresses on specific traits. We envision that the genetically imprinted tolerance limits of even resilient varieties and hybrids can be further boosted when combined with such exogenous treatments, and therefore this can be a powerful tool for the farmers in 14 the future. This task will be carried out in cooperation with private enterprises and growers to ensure that the guidelines reflect the newest advances in agricultural practices and therefore can be exploited by farmers. WG3 members will test these treatments in different crops and under different scenarios using the commonly established protocols (WG1, Task 1.2) and the results will be presented in MC meetings, conferences, meetings with various stakeholders including private companies and farmer associations.
Milestones WG3: M3.1 (every year month 1) Determine meetings and topics to discuss for WG3 members; M3.2 (Y2M9) Platform with genetic repository online; M3.3 (Y3M9) Define breeding strategies for crop improvement; M3.4 (Y3M9) Determine specific strategies to enhance reproductive resilience through gene editing approaches; M3.5 (Y3M9) Define synthetic biology approaches to improve stress resilience of different crops; M3.6 (Y3M9) Define specific treatments and agricultural practices to improve stress resilience.

WG3 Contact: Jan Fila - fila@ueb.cas.cz