FROOTS was structured into three scientific Working Packages (WP) aimed at the research objectives, with the inclusion of WP4, dedicated to the dissemination, communication, and exploitation of results, and WP5, designed for training in teaching and supervision. Throughout the course of the fellowship, Milestones have served as essential control points. At Milestone 1 enabled us to make informed decisions regarding the most reliable drought resistance indicators. This milestone carries great importance since it served as the pivotal point for deciding the allocation of effort within WP3. Milestone 2, which aims the development of the "micrografting" technique allowed the start of WP3.
Work Package 1: Drought resistance indicators in grafted plants
Several methods have been tested to identify drought tolerance in olive plants. This WP presented some difficulties at the start due to the variability in plant material from different nurseries, plant propagation methods, etc. After some initial experiments, the experiments that provided the best methodology to discriminate tolerance to drought in olive plants were the minimum leaf conductance and the biomass partitioning experiments. These procedures have allowed to determine, together with the literature, the most suitable olive cultivars to use an controls in water deficit experiments.
Work Package 2: Rapid “micrografting” technique
The initial results of the project have enabled the establishment of grafting techniques in olive seedlings. Grafting in olive seedlings has been successful under specific conditions of plant health, humidity, temperature, and light. Grafting techniques in olive seedlings have been similar to those used in herbaceous plants. The age of the plant also influences the success rate, although some success has been achieved in very young plants shortly after germination. In addition, various types of plant material have been successfully grafted, including seedling, micropropagated plants. The determination has been conducted using a diverse range of plant materials from WP1 and WP3, which vary in terms of age, juvenility stage, and the method used for their reproduction. The reconnection of vascular tissues after grafting, necessary for the survival of the grafted plant, has been demonstrated using a fluorescent marker applied to the root and visualized in the grafted part of the plant using a microscope to identify whether reconnection of xylem tissue has occurred (Figure 1). Currently, experiments are underway to determine the precise timing of these vascular reconnection processes, which would provide valuable information for both seedling grafting and the determination of incompatibility mechanisms at early stages, as well as for understanding the grafting process in olive and other woody species. The development of these procedures represent Milestone 2.
Work Package 3: Drought resistance in rootstock breeding populations
This work package has worked on implementing the most effective method identified in WP1 for high-throughput phenotyping of a significant number of sexually reproduced plants resulting from controlled crosses. Germplasm resources chosen for evaluation in this WP were selected based on documented drought tolerance as well as from the results obtained in WP1 (see previous section). This way, cultivars ‘Cornicabra’ and ‘Hojiblanca’ were selected as tolerant and cultivars ‘Cornezuelo de Jaén’ and ‘Manzanilla de Sevilla’ were selected as susceptible. Three test breeding populations were targeted for their development with the mentioned parents: Tolerant (T) x T, T x Sensitive (S), and S x S. To ensure accurate comparisons, the selected parents were asexually propagated for proper assessment alongside seedling plants. Seeds from those combinations were germinated and will be evaluated soon.
