Stable tetraploid olive (Olea europaea L.) plants derived from embryogenic cultures show altered growth and improved water retention

The olive tree ( Olea europaea L.) faces increasing challenges to sustainability and genetic improvement due to climate-change-driven water scarcity and heightened biotic stress from emerging pests and diseases. These challenges are exacerbated by the shift towards high-density and super-intensive production systems. Its prolonged juvenile phase and narrow genetic base further complicates its situation. In this context, artificial polyploidization, particularly autotetraploid induction, represents a powerful biotechnological strategy to enhance plant plasticity, architectural compactness, and stress resilience. A chromosome-doubling protocol was established and optimized using embryogenic callus cultures (cv. ‘Picual’, line P1), from which somatic embryos were exposed to 0.1% colchicine for 1–6 days to induce autotetraploidy. Ploidy levels were assessed by flow cytometry and confirmed by chromosome counting, and stable tetraploid lines were characterized morpho-anatomically and physiologically under in vitro conditions and after 6 months and three-four years of greenhouse growth. The optimal treatment (2–3 days) enabled efficient recovery of stable, non-chimeric tetraploids (4n = 92). Tetraploid plants exhibited a pronounced gigas effect, with wider, thicker leaves and increased cell size across tissues, along with reduced stomatal density. Morphologically, tetraploids exhibited a more compact growth habit and slower development than diploids. At the physiological level, they maintained stable photosynthetic performance and displayed improved leaf water retention in desiccation experiments. Our results demonstrate, for the first time, that embryogenic cultures provide an efficient platform for the recovery of stable, non-chimeric tetraploid olive plants. These plants represent a valuable genetic resource for investigating the effects of genome duplication and may facilitate the future development of cultivars and rootstocks with enhanced resilience to environmental stress.