A comprehensive phenotypic and genotypic evaluation of Spanish groundnuts from diverse crosses to identify superior and stable donors for fresh seed dormancy.

Breeding high yielding groundnut cultivars with 2-3 weeks of fresh seed dormancy, particularly in Spanish-type cultivars, enhances the sustainability of agriculture in groundnuts. In this context, we conducted a comprehensive phenotypic and genotypic evaluation of advanced breeding lines developed in the genetic background of Spanish types. By employing multi-phenotyping and marker data, we identified PBS 15044, 16004, 16013, 16015, 16016, 16017, 16020, 16021, 16026, 16031, 16035, 16037, 16038, 16039, 16041, and 16042 with 2-3 weeks dormancy (> 90%).The various parametric and non-parametric estimates identified the stable fresh dormant genotypes with one or more superior economic trait. PBS 16021, 15044, 16038, and 16039 identified with high hundred pod weight (HPW) were also reported having high intensity of dormancy (> 90% for up to 3 weeks); PBS 15044, 16016, PBS 16038 and PBS 16039 with high hundred kernel weight (HKW) also reported with up to 3 weeks fresh seed dormancy; and PBS 16013, 16031, and 16038 with up to 3 weeks fresh seed dormancy had high shelling percentage (SP). They can be used to develop lines with the desired level of dormancy, and high yields, by designing appropriate breeding strategies.

Optimizing water relations, gas exchange parameters, biochemical attributes and yield of water-stressed maize plants through seed priming with iron oxide nanoparticles.

Drought poses significant risks to maize cultivation by impairing plant growth, water uptake and yield; nano priming offers a promising avenue to mitigate these effects by enhancing plant water relations, stress tolerance and overall productivity. In the current experiment, we tested a hypothesis that seed priming with iron oxide nanoparticles (n-Fe2O3) can improve maize performance under water stress by improving its growth, water relations, yield and biochemical attributes. The experiment was conducted on a one main plot bisected into two subplots corresponding to the water and drought environments. Within each subplot, maize plants were raised from n-Fe2O3 primed seeds corresponding to 0 mg. L- 1 (as control treatment), 25, 50, 75, and 100 mg. L- 1 (as trial treatments). Seed priming with n-Fe2O3 at a concentration of 75 mg. L- 1 improved the leaf relative water content, water potential, photosynthetic water use efficiency, and leaf intrinsic water use efficiency of maize plants by 13%, 44%, 64% and 17%, respectively compared to control under drought stress. The same treatments improved plant biochemical attributes such as total chlorophyll content, total flavonoids and ascorbic acid by 37%, 22%, and 36%, respectively. Seed priming with n-Fe2O3 accelerated the functioning of antioxidant enzymes such as SOD and POD and depressed the levels of leaf malondialdehyde and hydrogen peroxide significantly. Seed priming with n-Fe2O3 at a concentration of 75 mg. L- 1 improved cob length, number of kernel rows per cob, and 100 kernel weight by 59%, 27% and 33%, respectively, under drought stress. Seed priming with n-Fe2O3 can be used to increase maize production under limited water scenarios.

Salt stress amelioration and nutrient strengthening in spinach (Spinacia oleracea L.) via biochar amendment and zinc fortification: seed priming versus foliar application.

Soil salinity is a major nutritional challenge with poor agriculture production characterized by high sodium (Na+) ions in the soil. Zinc oxide nanoparticles (ZnO NPs) and biochar have received attention as a sustainable strategy to reduce biotic and abiotic stress. However, there is a lack of information regarding the incorporation of ZnO NPs with biochar to ameliorate the salinity stress (0, 50,100 mM). Therefore, the current study aimed to investigate the potentials of ZnO NPs application (priming and foliar) alone and with a combination of biochar on the growth and nutrient availability of spinach plants under salinity stress. Results demonstrated that salinity stress at a higher rate (100 mM) showed maximum growth retardation by inducing oxidative stress, resulted in reduced photosynthetic rate and nutrient availability. ZnO NPs (priming and foliar) alone enhanced growth, chlorophyll contents and gas exchange parameters by improving the antioxidant enzymes activity of spinach under salinity stress. While, a significant and more pronounced effect was observed at combined treatments of ZnO NPs with biochar amendment. More importantly, ZnO NPs foliar application with biochar significantly reduced the Na+ contents in root 57.69%, and leaves 61.27% of spinach as compared to the respective control. Furthermore, higher nutrient contents were also found at the combined treatment of ZnO NPs foliar application with biochar. Overall, ZnO NPs combined application with biochar proved to be an efficient and sustainable strategy to alleviate salinity stress and improve crop nutritional quality under salinity stress. We inferred that ZnO NPs foliar application with a combination of biochar is more effectual in improving crop nutritional status and salinity mitigation than priming treatments with a combination of biochar.

Genome-wide identification of the sorghum OVATE gene family and revelation of its expression characteristics in sorghum seeds and leaves.

The OVATE gene family plays an important role in regulating the development of plant organs and resisting stress, but its expression characteristics and functions in sorghum have not been revealed. In this study, we identified 26 OVATE genes in the sorghum BTx623 genome, which were divided into four groups and distributed unevenly across 9 chromosomes. Evolutionary analysis showed that after differentiation between sorghum and Arabidopsis, the OVATE gene family may have experienced unique expansion events, and all OVATE family members were negatively selected. Transcriptome sequencing and RT-qPCR results showed that OVATE genes in sorghum showed diverse expression characteristics, such as gene SORBl_3001G468900 and SORBl_3009G173400 were significantly expressed in seeds, while SORBI_3005G042700 and SORBI_3002G417700 were only highly expressed in L1. Meantime, in the promoter region, a large number of hormone-associated cis-acting elements were identified, and these results suggest that members of the OVATE gene family may be involved in regulating specific development of sorghum leaves and seeds. This study improves the understanding of the OVATE gene family of sorghum and provides important clues for further exploration of the function of the OVATE gene family.

Exploring selection signatures in the divergence and evolution of lipid droplet (LD) associated genes in major oilseed crops.

BACKGROUND: Oil bodies or lipid droplets (LDs) in the cytosol are the subcellular storage compartments of seeds and the sites of lipid metabolism providing energy to the germinating seeds. Major LD-associated proteins are lipoxygenases, phospholipaseD, oleosins, TAG-lipases, steroleosins, caleosins and SEIPINs; involved in facilitating germination and enhancing peroxidation resulting in off-flavours. However, how natural selection is balancing contradictory processes in lipid-rich seeds remains evasive. The present study was aimed at the prediction of selection signatures among orthologous clades in major oilseeds and the correlation of selection effect with gene expression. RESULTS: The LD-associated genes from the major oil-bearing crops were analyzed to predict natural selection signatures in phylogenetically close-knit ortholog clusters to understand adaptive evolution. Positive selection was the major force driving the evolution and diversification of orthologs in a lineage-specific manner. Significant positive selection effects were found in 94 genes particularly in oleosin and TAG-lipases, purifying with excess of non-synonymous substitution in 44 genes while 35 genes were neutral to selection effects. No significant selection impact was noticed in Brassicaceae as against LOX genes of oil palm. A heavy load of deleterious mutations affecting selection signatures was detected in T-lineage oleosins and LOX genes of Arachis hypogaea. The T-lineage oleosin genes were involved in mainly anther, tapetum and anther wall morphogenesis. In Ricinus communis and Sesamum indicum > 85% of PLD genes were under selection whereas selection pressures were low in Brassica juncea and Helianthus annuus. Steroleosin, caleosin and SEIPINs with large roles in lipid droplet organization expressed mostly in seeds and were under considerable positive selection pressures. Expression divergence was evident among paralogs and homeologs with one gene attaining functional superiority compared to the other. The LOX gene Glyma.13g347500 associated with off-flavor was not expressed during germination, rather its paralog Glyma.13g347600 showed expression in Glycine max. PLD-α genes were expressed on all the tissues except the seed,δ genes in seed and meristem while ß and γ genes expressed in the leaf. CONCLUSIONS: The genes involved in seed germination and lipid metabolism were under strong positive selection, although species differences were discernable. The present study identifies suitable candidate genes enhancing seed oil content and germination wherein directional selection can become more fruitful.

Enhancing drought stress tolerance and growth promotion in chiltepin pepper (Capsicum annuum var. glabriusculum) through native Bacillus spp.

The drought can cause a decrease in food production and loss of biodiversity. In northern Mexico, an arid region, the chiltepin grows as a semi-domesticated crop that has been affected in its productivity and yield. An alternative to mitigate the effect of drought and aid in its conservation could be using Plant Growth-Promoting Bacteria (PGPB). The present study evaluated the capacity of native Bacillus spp., isolated from arid soils, as PGPBs and drought stress tolerance inducers in chiltepin under controlled conditions. Chiltepin seeds and seedlings were inoculated with native strains of Bacillus spp. isolated from arid soils, evaluating germination, vegetative, and drought stress tolerance parameters. The PGPBs improved vegetative parameters such as height, stem diameter, root length, and slenderness index in vitro. B. cereus (Bc25-7) improved in vitro survival of stressed seedlings by 68% at -1.02 MPa. Under greenhouse conditions, seedlings treated with PGPBs exhibited increases in root length (9.6%), stem diameter (13.68%), leaf fresh weight (69.87%), and chlorophyll content (38.15%). Bc25-7 alleviated severe water stress symptoms (7 days of water retention stress), and isolates B. thuringiensis (Bt24-4) and B. cereus (Bc25-7, and Bc30-2) increased Relative Water Content (RWC) by 51%. Additionally, the treated seeds showed improved germination parameters with a 46.42% increase in Germination Rate (GR). These findings suggest that using PGPBs could be an alternative to mitigate the effect of drought on chiltepin.

CaLAP1 and CaLAP2 orchestrate anthocyanin biosynthesis in the seed coat of Cicer arietinum.

MAIN CONCLUSION: Our findings shed light on the regulation of anthocyanin and proanthocyanidin biosynthesis in chickpea seed coats. Expression of R2R3-MYB transcription factors CaLAP1 and CaLAP2 enhanced the anthocyanins and proanthocyanidins content in chickpea. The seed coat color is a major economic trait in leguminous crop chickpea (Cicer arietinum). Anthocyanins and proanthocyanidins (PAs) are two classes of flavonoids that mainly contribute to the flower, seed coat and color of Desi chickpea cultivars. Throughout the land plant lineage, the accumulation of anthocyanins and PAs is regulated by MYB and bHLH transcription factors (TFs), which form an MBW (MYB, bHLH, and WD40) complex. Here, we report two R2R3-MYB TFs in chickpea belonging to the anthocyanin-specific subgroup-6, CaLAP1 (Legume Anthocyanin Production 1), and CaLAP2 (Legume Anthocyanin Production 2), which are mainly expressed in the flowers and developmental stages of the seeds. CaLAP1 and CaLAP2 interact with TT8-like CabHLH1 and WD40, forming the MBW complex, and bind to the promoter sequences of anthocyanin- and PA biosynthetic genes CaCHS6, CaDFR2, CaANS, and CaANR, leading to anthocyanins and PA accumulation in the seed coat of chickpea. Moreover, these CaLAPs partially complement the anthocyanin-deficient phenotype in the Arabidopsis thaliana sextuple mutant seedlings. Overexpression of CaLAPs in chickpea resulted in significantly higher expression of anthocyanin and PA biosynthetic genes leading to a darker seed coat color with higher accumulation of anthocyanin and PA. Our findings show that CaLAPs positively modulate anthocyanin and PA content in seed coats, which might influence plant development and resistance to various biotic and abiotic stresses.

Effects of dispersing media on the rheological and tribological properties of basil seed mucilage-based thickened liquids.

The development of thickening powders for the management of dysphagia is imperative due to the rapid growth of aging population and prevalence of the dysphagia. One promising thickening agent that can be used to formulate dysphagia diets is basil seed mucilage (BSM). This work investigates the effects of dispersing media, including water, milk, skim milk, and apple juice, on the rheological and tribological properties of the BSM-thickened liquids. Shear rheology results revealed that the thickening ability of BSM in these media in ascending order is milk < skim milk ≈ apple juice < water. On the other hand, extensional rheology demonstrated that the longest filament breakup time was observed when BSM was dissolved in milk, followed by skim milk, water, and apple juice. Furthermore, tribological measurements showed varying lubrication behavior, depending on the BSM concentration and dispersing media. Dissolution of BSM in apple juice resulted in the most superior lubrication property compared with that in other dispersing media. Overall, this study provides insights on BSM's application as a novel gum-based thickening powder in a range of beverages and emphasizes how important it is for consumers to have clear guidance for the use of BSM in dysphagia management.

Unlocking the therapeutic potential of Nigella sativa extract: phytochemical analysis and revealing antimicrobial and antioxidant marvels.

The growing global threat of antimicrobial resistance endangers both human and animal life, necessitating the urgent discovery of novel antimicrobial solutions. Medicinal plants hold promise as sources of potential antimicrobial compounds. In this study, we investigated the phytochemical constituents and microbicidal capabilities of the ethanolic extract from Nigella sativa (black seed). Gas chromatography analysis (GC) identified 11 compounds, among them thymoquinone, and thymol, contributing to antimicrobial and antioxidant properties. Antimicrobial assays demonstrated notable inhibition zones against broad spectra of bacteria, including Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Staphylococcus aureus, Enterobacter, and Bacillus subtilis, along with potent antifungal activity against Aspergillus niger, Penicillium, and Candida albicans. Notably, when combined with antibiotics, the extract displayed exceptional synergistic antimicrobial efficacy. The black seed extract demonstrated membrane-damaging activity and disrupted virulence factors that protect microbes from antimicrobial agents, including the formation of bacterial biofilm and protease secretion. Thymoquinone, the primary active constituent of the extract, exhibited similar antimicrobial and ant virulence properties. In silico analysis targeting key regulators of quorum sensing and biofilm formation in P. aeruginosa, such as RhlG, LasR, and PqsR, showed a remarkable affinity of thymol and thymoquinone for these targets. Moreover, the N. sativa extract exhibited dose-dependent cytotoxicity against both the promastigote and amastigote forms of Leishmania tropica parasites, hinting at potential antiparasitic activity. In addition to its antimicrobial properties, the extract displayed potential antioxidant activity at a concentration of 400 µg/mL.

Comparison of four rheological models for estimating viscosity and rheological parameters of microwave treated Basil seed gum.

Dispersion of Basil seed gum has high viscosity and exhibits shear-thinning behavior. This study aimed to analyze the influence of microwave treatment (MT) at various time intervals (0, 1, 2, and 3 min) on the viscosity and rheological behavior of Basil seed gum dispersion (0.5%, w/v). The finding of this study revealed that the apparent viscosity of Basil seed gum dispersion (non-treated dispersion) reduced from 0.330 Pa.s to 0.068 Pa.s as the shear rate (SR) increased from 12.2 s-1 to 171.2 s-1. Additionally, the apparent viscosity of the Basil seed gum dispersion reduced from 0.173 Pa.s to 0.100 Pa.s as the MT time increased from 0 to 3 min (SR = 61 s-1). The rheological properties of gum dispersion were successfully modeled using Power law (PL), Bingham, Herschel-Bulkley (HB), and Casson models, and the PL model was the best one for describing the behavior of Basil seed gum dispersion. The PL model showed an excellent performance with the maximum r-value (mean r-value = 0.942) and the minimum sum of squared error (SSE) values (mean SSE value = 5.265) and root mean square error (RMSE) values (mean RMSE value = 0.624) for all gum dispersion. MT had a considerable effect on the changes in the consistency coefficient (k-value) and flow behavior index (n-value) of Basil seed gum dispersion (p < 0.05). The k-value of Basil seed gum dispersion decreased significantly from 3.149 Pa.sn to 1.153 Pa.sn (p < 0.05) with increasing MT time from 0 to 3 min. The n-value of Basil seed gum dispersion increased significantly from 0.25 to 0.42 (p < 0.05) as the MT time increased. The Bingham plastic viscosity of Basil seed gum dispersion increased significantly from 0.029 Pa.s to 0.039 Pa.s (p < 0.05) while the duration of MT increased. The Casson yield stress of Basil seed gum dispersion notably reduced from 5.010 Pa to 2.165 Pa (p < 0.05) with increasing MT time from 0 to 3 min.

Optimisation of indole acetic acid production by Neopestalotiopsis aotearoa endophyte isolated from Thymus vulgaris and its impact on seed germination of Ocimum basilicum.

BACKGROUND: Microbial growth during plant tissue culture is a common problem that causes significant losses in the plant micro-propagation system. Most of these endophytic microbes have the ability to propagate through horizontal and vertical transmission. On the one hand, these microbes provide a rich source of several beneficial metabolites. RESULTS: The present study reports on the isolation of fungal species from different in vitro medicinal plants (i.e., Breynia disticha major, Breynia disticha, Duranta plumieri, Thymus vulgaris, Salvia officinalis, Rosmarinus officinalis, and Ocimum basilicum l) cultures. These species were tested for their indole acetic acid (IAA) production capability. The most effective species for IAA production was that isolated from Thymus vulgaris plant (11.16 µg/mL) followed by that isolated from sweet basil plant (8.78 µg/mL). On screening for maximum IAA productivity, medium, "MOS + tryptophan" was chosen that gave 18.02 µg/mL. The macroscopic, microscopic examination and the 18S rRNA sequence analysis indicated that the isolate that given code T4 was identified as Neopestalotiopsis aotearoa (T4). The production of IAA by N. aotearoa was statistically modeled using the Box-Behnken design and optimized for maximum level, reaching 63.13 µg/mL. Also, IAA extract was administered to sweet basil seeds in vitro to determine its effect on plant growth traits. All concentrations of IAA extract boosted germination parameters as compared to controls, and 100 ppm of IAA extract exhibited a significant growth promotion effect for all seed germination measurements. CONCLUSIONS: The IAA produced from N. aotearoa (T4) demonstrated an essential role in the enhancement of sweet basil (Ocimum basilicum) growth, suggesting that it can be employed to promote the plant development while lowering the deleterious effect of using synthetic compounds in the environment.

Assessing Seed Germination Response of Parasitic Plant Striga hermonthica with Small-Molecule Probes.

Seed germination of a parasitic plant Striga hermonthica is elicited by strigolactones which are exuded from roots of host plants. Here, we describe a high-throughput germination assay and a method for visualizing in vivo strigolactone receptor functions with a fluorogenic probe.

Germination Assays for Comparable Seed Dormancy Depth and Distinction of Seed Color by Multiplex PCR in Wheat.

Seed dormancy is an important trait in cereal breeding, as it prevents preharvest sprouting (PHS). Although seed dormancy is a multifactorial trait, seed color has been demonstrated to be a major dormancy-related factor controlled by few genes. The R-1 gene is a seed color regulator that encodes a MYB-type transcription factor in wheat. A set of genetic markers designed against R-1 can provide a powerful tool for swift wheat breeding. Depth of seed dormancy varies not only among lines but also during seed development in each line. In this chapter, we describe how developmental seeds can be collected to perform germination tests, how seed color can be observed after NaOH staining, and how to genotype wheat R-1 genes using multiplex PCR.

Chromatin Immunoprecipitation Using Imbibed Seeds of Arabidopsis thaliana.

Chromatin immunoprecipitation (ChIP) is used to analyze the targeting of a protein to a specific region of chromatin in vivo. Here, we present an instructive ChIP protocol for Arabidopsis imbibed seeds. The protocol covers all steps, from the sampling of imbibed seeds to the reverse crosslinking of immunoprecipitated protein-DNA complexes, and includes experimental tips and notes. The targeting of the protein to DNA is determined by quantitative PCR (qPCR) using reverse crosslinked DNA. The protocol can be further scaled up for ChIP-sequencing (ChIP-seq) analysis. As an example of the protocol, we include a ChIP-quantitative PCR (ChIP-qPCR) analysis demonstrating the targeting of PIF1 to the ABI5 promoter.

Regulation of Seed Dormancy Genes in Triticeae Species.

Wild progenitors of Triticeae crops generally have long dormancy periods. Domesticated crops inherited these longer dormancy alleles from their wild progenitors, which have since been modified and selected during cultivation and utilization by humans. Thus, allelic combinations at different seed dormancy loci are currently represented in Triticeae germplasm preserved in seed repositories and gene banks as accessions and materials of breeding programs. Methods to evaluate seed dormancy are key to explore, analyze, and exploit optimal alleles in dormancy genes. Recent developments in genomics have accelerated the identification and analysis of seed dormancy loci in Triticeae species. Transgenic experiments have been conducted to validate if candidate genes affect seed dormancy and more recently have yielded an array of mutations derived from genome editing for practical applications. The information gathered on these seed dormancy loci provides a deeper knowledge of germplasm diversity and offers strategies to control seed dormancy in breeding programs in Triticeae crops.

Confocal Imaging of Seeds.

In flowering plants, proper seed development is achieved through the constant interplay of fertilization products, embryo and endosperm, and maternal tissues. Understanding such a complex biological process requires microscopy techniques able to unveil the seed internal morphological structure. Seed thickness and relatively low permeability make conventional tissue staining techniques impractical unless combined with time-consuming dissecting methods. Here, we describe two techniques to imaging the three-dimensional structure of Arabidopsis seeds by confocal laser scanning microscopy. Both procedures, while differing in their time of execution and resolution, are based on cell wall staining of seed tissues with fluorescent dyes.

Assessment of Dormancy Level and Germination Ability of Seeds with Physiological Dormancy.

Germination test is fundamental and commonly used technique for seed dormancy and germination studies, and proper assessment of dormancy level and germination ability of a given set of seeds is prerequisite for most of the studies. However, germination is very sensitive to imbibition conditions, and dormancy development is also sensitive to growth conditions of the mother plants. In this chapter, we describe tips for plant growth and germination test mainly for physiological and molecular genetic studies with Arabidopsis. This protocol can be applied for other plant species with relatively small seeds and for various studies to analyze the effect of light, phytohormones, and other chemicals in seed germination.

Map-Based Cloning of the Causal Gene for a Seed Dormancy Quantitative Trait Locus in Barley.

Seed dormancy is an important agronomic trait in cereal crops. Throughout the domestication of cereals, seed dormancy has been reduced to obtain uniform germination. However, grain crops must retain moderate levels of seed dormancy to prevent problems such as preharvest sprouting in wheat (Triticum aestivum) and barley (Hordeum vulgare). To produce modern cultivars with the appropriate seed dormancy levels, it is important to identify the genes responsible for seed dormancy. With recent advances in sequencing technology, several causal genes for seed dormancy quantitative trait loci (QTLs) have been identified in barley and wheat. Here, we present a method to identify causal genes for seed dormancy QTLs in barley, a method that is also applicable to other cereals.

Functional Analysis of Seed Dormancy Genes by Biolistic Transient Gene Expression in Immature Embryos of Wheat.

Seed dormancy genes typically suppress germination and cell division. Therefore, overexpressing these genes can negatively affect tissue culture, interfering with the generation of transgenic plants and thus hampering the analysis of gene function. Transient expression in target cells is a useful approach for studying the function of seed dormancy genes. Here, we describe a protocol for transiently expressing genes related to seed dormancy in the scutellum of immature wheat (Triticum aestivum) embryos to analyze their effects on germination.