Planta

In vitro regeneration of Citrus plants is a widely used method, however, induction of adventitious roots from regenerated shoots remains a major bottleneck, limiting the recovery of healthy plants for commercial production and genomic research for crop improvement. We established an in vitro regeneration system producing profuse, healthy roots for sweet orange (Citrus sinensis cv. Benyenda) by optimising combinations and concentrations of auxins. Prior to optimising the rooting media (RTMs), we obtained a shoot regeneration rate of 90.6% from sweet orange epicotyl explants using a cytokinin, 6-benzylaminopurine (BAP). Across twelve auxin-supplemented RTMs containing different concentrations of indole-3-butyric acid (IBA) and/or 1-naphthaleneacetic acid (NAA), rooting percentages ranged from 8 - 87.5%. The combination of IBA 1.0 mg L-1 and NAA 0.1 mg L-1 promoted the best overall performance, 75 {+/-} 7.2% rooting percentage with healthy, callus-free roots ([≥]5 cm in length), whereas other RTMs with other auxin combinations induced callus and limited root elongation. The best-performing SRM and RTM were subsequently used for selection and recovery of transgenic sweet orange lines carrying an empty CRISPR/Cas9 construct, resulting in an 4.8% transformation efficiency. Both transgenic and non-transgenic rooted plantlets were successfully acclimatised under glasshouse conditions with a survival rate of 90%. This enhanced regeneration system overcomes rooting bottleneck and improves plant survival,enabling faster recovery of transgenic citrus lines within four months. It supports accelerated development for commercial applications and advances in citrus genetic improvement.

Ovate Family Proteins (OFPs) are a class of plant-specific, negative nuclear transcriptional regulators characterized by conserved C-terminal OVATE domain. This study on comparative functional characterization of two head-to-head arranged OFPs - AtOFP2 (Ovate-OFP with full ovate domain) and AtOFP17 (Ovate-Like OFP with partial ovate domain) provides critical insight into how structural variations in ovate domain leads to functional divergence. Detailed phenotypic analysis of 28 physical and physiological traits of loss- and gain-of-function mutants revealed that both genes act as broad, pleotropic repressors of plant growth and development. Removal of repression in knock-down mutants of both genes exhibited reduced duration of seed dormancy, faster rate of germination and growth, bigger plants and significantly higher seed yield. In contrast, constitutive over-expression showed a generalized repressive nature of both genes, with nuanced differences for fine tuning of specific traits. For example, both genes showed antagonistic behaviours on root hair architecture. AtOFP2 act as a strong repressor of root hair development whereas AtOFP17 is a stronger repressor of hypocotyl and root cell architecture. AtOFP17 owing to partial ovate domain exerts a mild level of repression throughout life span as indicated by smaller plants and lesser yield in knock-down AtOFP17 mutants. On the contrary, AtOFP2 exerted a much stronger repressor effect in which > 90% over-expression mutants died at the juvenile stage ; the survival of remaining 10% is probably owing to activation of dosage-dependent feedback loop mechanism as indicated by normal growth of mature plants, and is also evident by transcriptome data. Transcriptome analysis of roots of 7-day old seedling of knock-down and over-expression mutants of AtOFP2 showed downregulation of OFP2 in over-expressed mutants. However, severely stunted phenotype indicated presence of stable OFP2 protein to exert effects. Analysis of DEGs in OFP2 mutants revealed that it acts as an important regulator working at intersection of hormonal signalling affecting critical genes required for auxin, cytokinin, GA, BR and ABA functioning. Perturbations across hormonal signalling pathways affects cell wall remodelling factors such as EXPANSINS, Xyloglucan hydrolases (XTHs) and cellulose synthases (CSLs) causing overall stunted growth; and epidermal patterning genes such as WER, GL1, EGL3, TTG1 leading to severely reduced root length and root hairs. Significantly, functional analysis of this master regulator highlighted a significant economic potential. Knockdown of both these genes relieves their natural repression on reproductive traits, leading to longer siliques, bigger and heavier seeds, and substantially increased overall seed yield, positioning AtOFP2 and AtOFP17 as highly valuable targets for agricultural crop improvement.

Background and AimsThe development of unisexual flowers relies on the tight coordination of flower organ identity and sex determination. The genus Quercus is typically considered strictly monoecious, bearing fully segregated male and female flowers within the same individual tree. However, several reports of atypical flowering across the genus challenge this canonical view, suggesting that flowering in oaks may be more flexible than traditionally assumed. In this work, the dynamics of flower development in Quercus orocantabrica were examined to correlate contrasting floral morphologies with divergent molecular profiles. MethodsThe flowering phenology of Q. orocantabrica trees was closely monitored over several individuals and years, together with a detailed floral morphological analysis of male, female and atypical flowers. Key floral homeotic gene homologues were identified, and their expression assayed in the development of different flowers. Key ResultsRecurrent and widespread hermaphroditic flowering was detected in several Q. orocantabrica trees, frequently associated with unseasonal flowering events. Gene expression analysis of male, female and hermaphroditic flowers revealed a sex-biased expression of Q. orocantabrica B- and C-class genes, with the B-class gene QoPI in particular being tightly associated with the presence of fully-developed stamens. In addition, the expression of the C-class gene QoSHP contrasted with reports in other Fagaceae, highlighting a potential functional divergence of the C/D-class lineage within the family. ConclusionsThe results here depicted indicate that the dynamics of floral sex identity in oaks are more plastic than traditionally assumed, supporting a reinterpretation of oak reproductive biology based on a versatile and resilient framework responsive to different developmental contexts.

Background and AimsPaspalum notatum reproduces through either sexuality or apomixis, two pathways that may coexist within the same individual and are regulated by interconnected molecular networks responsive to environmental cues. Here, we characterized the transcript structure and expression of BUD SITE SELECTION PROTEIN 13 (BUD13), a component of the RES spliceosomal complex previously reported as differentially expressed in florets of sexual and apomictic plants, as a first step toward testing its involvement in the molecular regulation of the apomixis-sexuality switch. MethodsPreviously generated floral and leaf transcriptomes from sexual and apomictic Paspalum notatum plants, including Oxford Nanopore long-read data, were mined to characterize BUD13 transcript structure and expression. Phylogenetic analyses and in silico mapping were conducted to infer evolutionary relationships and determine the origin of the transcripts. Differential expression was validated by RT-qPCR, while in situ hybridization was used to reveal cell-specific ovule expression patterns. Key resultsBUD13 is expressed in Paspalum notatum florets as a truncated isoform (SHORT) encoding a small protein lacking part of the herpes simplex virus regulatory protein (ICP4) domain. Two SHORT transcripts, SHORT1 and SHORT2, with different 5' untranslated region (UTR) regions, were identified in flowers. SHORT1 was consistently upregulated in apomictic ovules from premeiosis to anthesis. Both transcripts originated from a single genomic locus located in the subtelomeric region of the short arm of chromosome 6. SHORT isoforms with variable structures were detected in other monocots. In situ hybridization showed that, whereas BUD13 was expressed throughout sexual ovules, expression was absent from the female germline of apomictic ovules. A consistent expression was observed in somatic proembryos of aposporous embryo sacs. ConclusionsOur findings reveal structural, spatial and temporal divergence in BUD13 expression between sexual and apomictic reproductive programs, providing new insights into the molecular regulation of asexual seed formation.

Heavy metals act as essential metalloprotein cofactors in numerous physiological processes but can become toxic when non-essential metals accumulate or when essential metals are in excess. As plants continuously encounter heavy metals through their roots, they have evolved complex homeostatic mechanisms to regulate metal uptake and distribution. The Heavy Metal ATPase (HMA) gene family encodes a group of heavy metal transporting P-type ATPases that have been linked to stress resistance and nutrient supply. Here, we used a bioinformatics approach to identify and characterise 13 HMA genes containing characteristic P1B-type ATPase domains and motifs in the barley Morex V3 reference genome. The genes are located on five of the seven barley chromosomes. Phylogenetic analysis revealed that they cluster into five sub-clades, including one clade unique to barley. Expression profiling across multiple datasets showed distinct temporal and tissue-specific expression patterns among HvHMAs, with several members exhibiting significant transcriptional responses to specific biotic and abiotic stresses. By utilising recently available pan-transcriptomic and pan-genomic resources, we have identified substantial allelic diversity and inter-accession variation in HvHMAs. Our findings suggest that HvHMAs have functions extending beyond canonical heavy metal homeostasis and warrant further investigation for their potential roles in broader physiological and stress-related processes.

Transgenerational plasticity can shape plant phenotype and influence plant response to environmental changes in interaction with the current conditions. While how past stress interact with either current optimal or stress conditions is increasingly documented within a single plant, transgenerational plasticity remains particularly poorly understood especially at the metabolome level. In our study, we investigated whether heat stress induces transgenerational metabolic and phenotypic modifications along two successive clonal ramet generations of the sub-Antarctic aquatic plant Limosella australis. We performed untargeted metabolomic approaches and measured morphologic and performance traits, to assess both transgenerational plasticity of the metabolome and the phenotype. We found that heat stress remodelled the metabolic profile and influenced the foraging strategy of our clonal plant, and that some of these metabolic changes persisted into the first clonal generation. This one therefore adopted an intermediate growth strategy, even though culture conditions were optimal. By comparing differentially accumulated features between daughter ramets from heat stressed mother ramets and from unstressed mother ramets, we identified common and specific metabolites accumulation to heat stress response, belonging to diverse compound families. However, we did not observe any adaptative advantage and any metabolic imprint during another heat stress applied on the second clonal generation. This work provides especially new clues into how plant metabolome integrates and transfers previous stressed clonal generation's information.

Bacterial canker remains a major constraint affecting apricot production in South-East of France. It is primarily caused by Pseudomonas syringae, a Gram-negative bacterium, many strains of which exhibit ice nucleation activity. By promoting ice formation at relatively high subzero temperatures, ice nucleation-active bacteria may facilitate tissue disruption and pathogen entry. Concurrently, climate-driven shifts toward warmer winter-spring periods have advanced flowering phenology, increasing exposure to late frost events. Despite breeders having developed less susceptible varieties to bacterial canker and early flowering varieties, the link between these traits and frost sensitivity, an emerging risk in this location, remains unresolved. Here, we have evaluated the links between canker susceptibility and frost sensitivity using three cultivar pairs contrasting in disease response and flowering time. Ice nucleation temperature was measured in excised buds under controlled conditions throughout the frost-risk period, alongside field-based diameter variation monitoring over two years. Disease susceptibility (P < 0.001), phenology (P = 0.003), varieties (P < 0.001), locations (P < 0.001), and sampling date (P < 0.001) significantly affected nucleation temperature, whereas epiphytic bacterial abundance and xylem vessel diameter did not. Trees froze at higher temperatures in situ than in laboratory assays (1 to -2{degrees}C versus -3 to 4{degrees}C, respectively), indicating strong environmental modulation of freezing processes beyond Psy-like bacterial activity, which is reflected in contrasting disease susceptibilities (P < 0.001) and precocities (P < 0.001). These results shed light on the complexity of the freezing process in trees under natural conditions. We discuss the potential roles of microclimatic conditions and alternative ice nucleation sources beyond Psy-like bacteria in driving these physiological processes.

Plant regeneration is a major determinant of transformation and genome-editing efficiency, yet the endogenous regulatory networks controlling regenerative competence in horticultural crops remain incompletely understood. The miR319-TCP module regulates multiple developmental processes in plants, but its function in lettuce regeneration has not been defined. Here, we performed a genome-wide analysis of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) gene family in lettuce (Lactuca sativa). Thirty-three LsTCP genes were identified and classified into Class I/PCF, Class II/CIN, and Class II/CYC/TB1 groups. Five CIN-class genes, LsTCP2, LsTCP3, LsTCP4, LsTCP10, and LsTCP24, were predicted as high-confidence miR319 targets and supported by degradome-based cleavage evidence. MIR319-overexpression (OX319) explants showed enhanced de novo shoot regeneration, with 94.5% regeneration efficiency and 1.92 shoots per explant, whereas STTM-miR319 suppression (S319) explants showed reduced regeneration, with 28.5% regeneration efficiency and 0.36 shoots per explant. These phenotypes were associated with altered expression of several miR319-targeted CIN-TCP genes, particularly LsTCP4, LsTCP10, and LsTCP24. Disruption of LsTCP4 increased regeneration efficiency to 91.4% and shoot production to 2.05 shoots per explant, resembling the regeneration-enhancing effect of miR319 overexpression. In contrast, disruption of the non-target CIN gene LsTCP17 did not significantly affect regeneration under the tested conditions. Together, these results identify LsTCP4 as a key miR319-responsive negative regulator of de novo shoot regeneration and highlight miR319-mediated repression of LsTCP4 as a potential endogenous strategy for improving lettuce regeneration.

AbstractTomato production faces a persistent challenge from the tomato leaf miner, Phthorimaea absoluta, a pest that severely limits yields while effective resistance in cultivated varieties remains scarce. To address this gap, wild tomato relatives represent a promising reservoir of resistance traits. In this study, 24 tomato accessions, including both cultivated types and wild species, were evaluated under greenhouse (no-choice) and tunnel (choice) conditions. Resistance mechanisms were characterized through measures of antibiosis such as leaflet lesion type, proportion of attacked leaflets, and mine density. The results revealed substantial variation between and within species, allowing classification of accessions into resistant, intermediate, and susceptible groups through multivariate analysis. Notably, the wild accession Solanum habrochaites PI248707 exhibited strong resistance, in contrast to susceptible cultivated varieties such as Rose de Berne. Under choice conditions, PI248707 sustained limited damage and disrupted larval development, with early instar larvae present but few reaching advanced stages, indicating an inhibitory defense response. Untargeted metabolomic profiling further highlighted pronounced constitutive differences between wild and cultivated accessions, with S. pennellii and S. habrochaites displaying higher metabolic diversity. By integrating phenotypic and metabolic data, specific metabolite classes associated with resistance were identified. These findings underscore the potential of wild tomato germplasm in breeding programs, with PI248707 standing out as a strong candidate for resistance introgression.

Far-red light (FR; 700-750 nm) is increasingly incorporated into controlled-environment lighting because it can improve photosynthetic efficiency when combined with comparatively shorter wavelengths. In long-day leafy crops such as spinach, however, FR may also promote the transition from vegetative to reproductive growth and thereby reduce marketable yield. Most studies have evaluated FR fraction, intensity or end-of-day exposure, whereas the developmental timing of FR has rarely been tested, particularly in spinach. Here, we evaluated six commercial spinach cultivars (Amador, Harp, Renegade, Responder, Rubino and Santa Cruz) in an indoor vertical farm under a common red-green-blue background (PPFD 260-264 {micro}mol m-{superscript 2} s-{superscript 1}, 12 h photoperiod, 24 {degrees}C) and four FR timing treatments: no FR (Control), FR throughout production (FullFR), FR during early development only (EarlyFR), and FR during late development only (LateFR). LateFR increased marketable fresh weight relative to Control (244 vs 224 g) and reduced flowering incidence, whereas far-red supplied during early development reduced fresh weight (158 g) and increased flowering. The magnitude of the timing response differed among cultivars: switching from EarlyFR to LateFR recovered 0 % fresh weight in Amador but 107 % in Renegade and Rubino, with the largest penalties occurring in otherwise bolt-resistant cultivars. EarlyFR also increased total chlorophyll and reduced the chlorophyll a:b ratio. These results show that FR response in spinach is strongly conditioned by developmental stage and cultivar. Although LateFR received more total far-red than EarlyFR, it behaved like the Control, indicating that the penalty was set by far-red timing rather than dose. Treatment differences in bolting and yield tracked an estimated phytochrome photostationary-state deficit during early development: a phytochrome-deficit model markedly outperformed a cumulative-dose model ({Delta}AIC = 441), and the deficit x cultivar interaction was strong (p < 0.001), with bolt-resistant cultivars losing most yield when far-red coincided with the early developmental window. We therefore propose that FR should be treated as a genotype-dependent management variable rather than as a fixed spectral input, with late application and bolt-resistant cultivars offering the most favourable combination for vertical-farm spinach production. Framed within the breeders equation, the close match between the trial and production environment and the scope for shorter breeding cycles indoors suggest that genotype and far-red timing can be optimised jointly to accelerate genetic gain.

Crop photoperiodism models assume that flowering time is primarily controlled by daylength, yet many field observations contradict this view. We previously proposed an alternative framework integrating daily changes in sunrise and sunset times (dSR and dSS). Variety trials in Madagascar and in Argentina supported this concept: mid-late sorghum varieties from the northern hemisphere flowered late or very late when sown in November and December, consistent with the higher dSR/dSS values of the southern hemisphere summer. One Malian variety, sown monthly over six years in West Africa, exhibited high interannual variability in flowering time when sown between November and February. This revealed that up to four photoperiodic responses -- two quantitative and two qualitative, occurring at different times of the year -- may coexist within a single late photoperiod sensitive variety. All responses use only dSR and dSS cues. The qualitative responses are triggered by an internal phasic coincidence, which is set by a linear relationship between dSR and dSS at the onset of plant photoperiod sensitivity, and between dSR+dSS at panicle initiation. The research model fitted data from 28 varieties grown in Mali well. It also accurately fitted the duration to PI observed in three varieties sown at tropical and temperate latitudes. HighlightThe seasonal photoperiodic adaptation of flowering time in sorghum plants may rely on several signal transduction pathways regulated by sunrise and sunset times rather than day length.

Rice (Oryza sativa) is highly sensitive to salinity, yet the metabolic mechanisms underlying salt tolerance remains incompletely understood. In this study, we performed leaf tissue-specific untargeted metabolomic profiling of the salt-tolerant introgression line JN100 (JN), its donor parent Nona Bokra (NB), and its recurrent parent Jupiter (JU) to characterize metabolic responses to salt stress. Comparative analysis identified differentially accumulated metabolites (DAMs) spanning diverse chemical classes, including amino acids, sugars and carbohydrates, lipids, organic acids, cofactors, electron carriers, and nucleotides. Under salt stress (SS), 201 DAMs (89 upregulated and 112 downregulated) were detected in JN relative to JU. Notably, metabolites such as allantoin, glycitin, nicotinamide ribotide, D-arabinono-1,4-lactone, violanthin, L-methionine S-oxide, ribitol, lysine, rutin, glutamine, pantothenic acid, and quinic acid, showed significant differential accumulation. Pathway enrichment analysis revealed significant enrichment of arginine biosynthesis, purine metabolism, and alanine, aspartate, and glutamate metabolism, indicating extensive reprogramming of nitrogen and energy-associated metabolic pathways under salinity stress. Integration of transcriptomic and metabolomic datasets from the SS experiments further identified ten differentially expressed genes (DEGs) associated with the metabolite network in the JN vs. JU comparison. Among these, OsDHQDT/SDH, OsFd-GOGAT, phenylalanyl-tRNA synthetase, OsP5CS1, OsP5CS2, and a pyridoxal phosphate-dependent transferase were linked to metabolites involved in shikimate, amino acid, and proline metabolism. Collectively, these results demonstrate that salinity tolerance in rice is associated with coordinated transcriptional and metabolic reprogramming that supports oxidative stress mitigation and adaptive stress responses.

Elongating rice internodes present a developmental gradient from dividing meristem to mature cells, providing an elegant pseudo-time course for study of plant vegetative development. We tested the hypothesis that DNA methylation regulates gene expression during rice internode development by integrating RNA-seq and bisulfite DNA sequencing across eight internode segments. Previously described topologically associated chromatin domain borders aligned with transcription start sites of constitutive expressed genes. CpG and CHG differential methylation was enriched in young segments, consistent with maintenance methylation; whereas CHH methylation showed similar differential abundance in young and old segments. CHH and CHG methylation in upstream regions, CpG methylation within gene bodies, and any methylation in 5' and 3' untranslated regions were permissive of moderate to high gene expression. Very low expression was associated with CpG methylation upstream, CHG and CHH methylation within gene bodies, and CpG and CHG methylation downstream. A nonrandom subset of genes, including cell wall-related glycoside hydrolases, lignin and tricin biosynthesis enzymes, and WD40 proteins, showed methylation-expression correlations, with expression changes enriched in triple-marked elements. These results suggest that internode phenotypes of DNA methylation machinery mutants relate to alteration of specific target genes, opening approaches for grass culm improvement for lodging resistance and biomass production.

Exocyst was initially uncovered in yeast genetic sec-screen as a tethering complex for exocytotic vesicles and this function was later found to be evolutionarily conserved in other eukaryotes including plants. Later however, a surprising engagement of the exocyst complex in autophagy was observed in animals, plants and recently also in yeast. Using the genetic approach we observed EXO70E2 exocyst complex subunit engagement in the defence response to Pseudomonas syringae attack linked to the autophagy pathway. CRISPR/CAS LOF mutant of EXO70E2 is more sensitive to Pseudomonas infection (both virulent as well as T3SS mutant) and autophagy flux monitored by NBR1 antibody is compromised in comparison to WT. We conclude that the plant exocyst complex linked to the EXO70E2 subunit participates in defence against Pseudomonas bacteria in conjunction with the autophagy pathway. HighlightArabidopsis exocyst subunit EXO70E2 affects selective autophagic flux monitored by NBR1 and is participating in defense against Pseudomonas syringae infection.

Potato leafroll virus (PLRV) is an economically important viral disease of potato (S. tuberosum). Genetic resistance to this phloem-limited virus is rare, and no cloned resistance (R) genes have been reported. Rladg confers resistance to PLRV in an Andean potato landrace, LOP-868 (Velasquez et al. 2007). We identified the functional Rladg gene as a homolog of the tomato TIR-NLR-encoding Bs4. Rladg interacts with the serine protease domain of the PLRV protein P1, which is essential for virus replication. This recognition is independent of the proteases enzymatic activity, and the Rladg immune receptor oligomerizes upon direct association with the protease. Like PLRV, many poleroviruses contain a serine protease. Despite their diverse amino acid sequences, these proteases are predicted to share similar structures. Rladg recognizes all ten tested polerovirus proteases, suggesting a conserved structural recognition mechanism. We propose that Rladgs broad recognition capacity could enable resistance to poleroviruses in many crop species. Rladg is the first R-gene reported to confer resistance to a phloem-limited pathogen and could provide enhanced resistance to many economically important poleroviruses.

Leaf senescence directly affects lettuce quality and postharvest shelf life, but the regulatory roles of miR319-targeted and non-target CIN-TCP transcription factors remain unclear. Here, we examined whether the miR319-TCP module controls lettuce leaf senescence through separable genetic branches. MIR319 overexpression delayed dark-induced senescence, whereas STTM-mediated miR319 suppression accelerated chlorophyll loss, photosynthetic decline, and senescence-marker activation. Disruption of the miR319-targeted gene LsTCP4 phenocopied MIR319 overexpression, supporting LsTCP4 as a pro-senescence factor downstream of miR319. We further found that the miR319 non-target CIN gene LsTCP17 also promoted senescence, as tcp17 leaves retained more chlorophyll than wild type during dark treatment. Genetic combinations showed that tcp17 enhanced chlorophyll retention in the OX319 background and partially rescued the accelerated senescence phenotype of S319, indicating that LsTCP17 acts through a route separable from the miR319-targeted branch. Together, these results reveal a split CIN-TCP architecture in which miR319-targeted LsTCP4 and non-target LsTCP17 provide parallel pro-senescence inputs, offering a genetic framework for targeted improvement of lettuce quality.

Nitrogen-use efficiency (NUE) is a pivotal breeding target in tomato (Solanum lycopersicum L.) to sustain production under reduced N inputs. Here, we leveraged a recently developed tomato multi-parent advanced generation inter-cross (ToMAGIC) population to identify lines with superior performance under reduced N availability. The eight founders and a core subset of 118 ToMAGIC lines were characterized with 10,684 SNP markers and evaluated under optimal (opN, 15 mM) and suboptimal (subN, 8 mM) N supply in an experiment totalling 1,576 plants, generating 48,068 data points across 61 phenotypic variables. Under both N treatments, ToMAGIC lines exhibited transgressive segregation for most traits, confirming the value of this population as a reservoir of untapped variation. Notably, under subN conditions, harvest index (Hi) increased by 29-44%, suggesting adaptive resource redistribution toward reproductive sinks. Variance partitioning revealed that agronomic and NUE-related traits were largely under genetic control, with heritability estimates frequently above 0.80 and broadly conserved across N treatments. Multivariate trait analysis identified fruit yield N concentration (NUE component, CN,y), shoot biomass N content (NAb), and shoot growth-related traits as the main drivers of treatment differentiation. Finally, proxy traits were prioritized by integrating response magnitude, heritability, trait correlations, and treatment-discriminatory power into multi-trait selection indices. This strategy generated favorable predicted genetic gains, reaching 158% for high-performance lines and 170% for subN-adapted lines, and consistently identified lines 402, 428, 518, 800, and 816 as promising pre-breeding materials. Overall, this study supports ToMAGIC as a powerful resource for developing N-efficient cultivars suited for sustainable agriculture.

Applications of tissue culture are critical for Cannabis sativa L. (cannabis), supporting clonal propagation, germplasm preservation, pathogen elimination, among other biotechnological applications. However, extensive genetic diversity associated with cannabis results in highly variable responses to in vitro conditioning, and no consensus basal media formulation exists to support reproducible micropropagation across genotypes. To address these limitations, a hybridized ensemble-NSGA-II approach was employed for concurrent optimization of individual media components to create a species specific, cultivar inclusive basal salt formulation for cannabis micropropagation. The resulting PHJ media represents a unique formulation that overcomes recalcitrance across a wide array of cannabis cultivars, facilitating improved growth and uniformity for the nine cultivars used in its development and validation. These results remain consistent from explant initiation through multiple rounds of subculture. The ability of PHJ to overcome genotypic recalcitrance is telling of its potential applicability with an array of plant species beyond cannabis. Additionally, robust performance both with and without plant growth regulators underscores the plausible use of PHJ for diverse applications beyond standard micropropagation. Ultimately, this cultivar-inclusive basal medium demonstrates utility for both scientific research and industrial-scale operations.

O_LIFloral nectar plays a critical role in shaping the ecology and evolution of plant-pollinator interactions. Effective and efficient methods that allow for broad-scale sampling of nectar volume and sugar concentration across a diversity of taxa are needed to improve our understanding of many dimensions of mutualistic plant-pollinator interactions--including their basic ecology and evolution, their responses to environmental change, and their conservation and restoration. C_LIO_LIDespite the key importance of nectar for mediating plant-pollinator interactions, quantifying floral nectar in the field from many different plant species is challenging because there is often no one-size-fits-all sampling method that is effective across a diversity of floral structures and nectar traits. Different methods require different preparation, and sampling from many species involves a variety of logistical challenges. C_LIO_LIHere we provide a methodological roadmap for sampling floral nectar in the field from many different plant species. We describe our nectar collection methods in detail, including necessary equipment, calculations, and approaches appropriate for different floral morphologies. We also provide a troubleshooting guide for common problems encountered while collecting nectar in the field. To demonstrate the utility and effectiveness of our methods for collecting nectar from many different species, we present results on nectar trait variation from 53 species in an ecosystem. C_LIO_LIOur method illustrates that nectar traits vary considerably within and among plant species, indicating that large-scale nectar sampling projects are an important consideration for many basic and applied questions in pollination ecology and evolution. We hope that across many plant communities and ecosystems, our paper provides a practical roadmap for how to navigate the complexities of quantifying floral nectar traits. C_LI

Artificial light at night (ALAN) can alter the movement of nocturnal pollinators such as moths, with potential consequences for pollination services. However, most studies have focused on the presence or properties of lights, while the role of spatial lighting configuration remains poorly understood. We conducted a small-scale field experiment to test how different lighting configurations affect pollination success in moth-pollinated plants, using low-intensity LED garden lights. Potted phytometer plants of three moth-pollinated species were exposed to one of three treatments: no-light control, isolated LED point lights, or 25 m linear arrays of multiple LED lights. We quantified pollination success as both the probability of seed set and reproductive output through seed and capsule production. Experimental lighting affected plant reproductive success, with light arrays increasing seed-set probability, seed and seed capsule number relative to unlit controls, whereas point lights showed no clear effect. These results provide preliminary evidence that the spatial configuration of artificial lights may influence nocturnal pollination outcomes. Future work combining phytometer assays with direct tracking of moth movement is needed to assess whether light arrays facilitate or redirect the dispersal of nocturnal pollinators.