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- SAMMBA is a high-throughput pipeline for isolating and phenotyping macroalgal strainsPublication . Alves-Lima, Cicero; de Matos Barreto, Luís António; Monico, Carina; Gouvêa, Lidiane; Félix de Azeredo Pinto e Melo, Francisca; Varga, Brigitta; Filipe, Joana; Camacho, Rita; Lymperaki, Myrsini; Alberto, Filipe; Rörig, Leonardo R.; Engelen, Aschwin; Serrao, Ester A.; Pearson, Gareth Anthony; Martins, NeusaDespite successful preservation efforts, macroalgal diversity remains under-represented in global biobanks. A major limitation is the extreme morphological diversity of seaweed thalli, which hinders standardized isolation and phenotyping and often requires taxon-specific protocols. Here we present SAMMBA (Seaweed Automatable Microplate Microscopy for Breeding Approaches), an end-to-end pipeline for the high-throughput isolation, phenotyping and storage of macroalgal propagules in 384-well plates. By optimizing live-cell manipulation for chlorophyll autofluorescence (CAF) imaging and segmentation, multiple unialgal propagules can be isolated by dilution-based workflows. In a single plate, we obtained 68 singlet gametophyte fragments of Laminaria ochroleuca (17.7%) and 60 meiospores of Phyllariopsis purpurascens (31.25%). We demonstrated taxonomic and morphological versatility by isolating 60 unialgal cultures from three distinct Rhodophyta morphotypes (filamentous, crustose and foliose) and 10 strains of Ulva sp., also in a single plate. Furthermore, CAF-based area increase over 30 days enabled high-precision estimates of specific growth rates, yielding 0.130 ± 0.006 and 0.117 ± 0.01 day− 1 for male and female L. ochroleuca gametophytes, respectively (n = 768; p = 1.27e− 53). This precision substantially increases experimental reproducibility and statistical power compared to conventional methods, supporting high-throughput recovery of unialgal strains without motorized platforms, while remaining fully compatible with automation. SAMMBA expands operational capacity for strain discovery and phenotyping, providing a scalable foundation for phenomics, domestication workflows, and standardized macroalgal biobanking. We outline how the platform can benefit multiple areas of phycological research and facilitate the development of improved strains that can support aquaculture and restoration efforts.
- High-throughput assessment of gametophyte survival and growth in the kelp Laminaria ochroleuca through autofluorescence analysisPublication . Félix de Azeredo Pinto e Melo, Francisca; Alves-Lima, Cicero; de Matos Barreto, Luís António; Monico, Carina; Serrao, Ester A.; Pearson, Gareth Anthony; Martins, NeusaThe viability of kelp microscopic gametophytes is currently analyzed using subjective visual methods based on bright-field (BF) microscope images. Fluorescence microscopy (FM) can be employed, but the dyes used can be toxic and blue/UV light intensity may induce gametogenesis. This study aimed to develop a non-invasive and accurate methodology for assessing gametophyte viability over time, using FM observation of chlorophyll autofluorescence emitted by live gametophytes. Six isolated gametophyte strains of Laminaria ochroleuca (Italy), maintained at CCMAR Biobank, were cultured in triplicate (female, male, and both sexes combined) in Petri dishes containing 10 mL of half-strength Provasoli’s enriched seawater medium (PES), under red light for 1 month. Survival and growth were assessed on days 1, 7 and 14 by photographing 20 fields-of-view, both in BF and FM. Then, cultures were transferred to white light to induce gametogenesis and test whether the autofluorescence analysis (AFA) method affected gametophyte reproduction. Image analysis was performed using FIJI software either by manually counting live gametophytes (survival) and measuring their area (growth), or by implementing a machine-learning (ML) model using FIJI’s WEKA segmentation plugin. Results revealed that both methods were correlated, validating our ML model. However, the AFA method was faster and more accurate than BF image analysis, especially for male gametophytes, and did not compromise reproductive capacity. After 9 days under white light, sporophytes were present in mixed cultures. The AFA method provides a reliable technique for assessing gametophyte viability without compromising gametogenesis.