EFFECT OF TWO-DIMENSIONAL NANOMATERIALS (2D-NMs) ON SEED PLANT REPRODUCTION PROCESSES

After the discovery of graphene in 2004, which is a two-dimensional nanomaterial (2D-NMs), Many other synthetic 2D-NMs have also been developed, such as hexagonal boron nitride (hBN) and molybdenum disulfide (MoS2). Due to their extraordinary physical and chemical properties such as these are thinnest, strongest consist huge surface area, mechanical strength, which are derived from their 2D structure these are used in electronics and sensors to coatings, biomedicine, and nano enabled agrochemicals. During their life cycle like manufacturing, use, weathering, and end of life, however, unintentional releases could occur. Due to their ultralight weight and 2D morphology, these materials may travel long distances and fall on vegetation. Flowers of wind pollinated species are efficient biological collectors of airborne particulates, which can affect the pollen–stigma interface highly exposed and particularly sensitive. Yet, while nanomaterial effects on vegetative parts like roots, stems, and leaves are well documented, impacts on sexual reproduction remain poorly resolved. This thesis aimed to investigate whether four chemically distinct 2D-NMs, graphene oxide (GO), hBN, MoS2, and muscovite mica (natural benchmark) interfere with the earliest steps of pollination (pollen adhesion, hydration, and germination) in three anemophilous and economically relevant species: Cannabis sativa, Corylus avellana, and Zea mays. Simulated atmospheric deposition was applied to receptive stigmas using two dry deposition scenarios at the same nominal concentration (0.1 mg mL−1): (i) aerosol/spray to mimic environmentally realistic dispersion and (ii) brush to simulate high coverage accumulation. Pollen was deposited after 1.5, 6, and 24 hours to capture time-dependent responses. Environmental scanning electron microscopy (ESEM) and transmission electron microscopy (TEM) were used to characterize material adhesion, surface integrity, and potential internalization, including assessments of membrane continuity and cytoplasmic leakage in pollens and stigmatic surfaces. SEM showed material adhesion of nanosheets to stigmatic papillae and to the pollen exine, forming partial to continuous lamellar coatings. TEM demonstrated that all four 2D-NMs remained extracellular under our conditions: papillar cell walls, plasma membranes, vacuoles, and organelles appeared intact, with no evidence of penetration into intercellular spaces or uptake by papillar or pollen cells. Thus, the materials acted primarily as external physical stressors rather than as intracellular toxicants. Despite the absence of internalization or ultrastructural damage, biologically meaningful functional impairment was evident. Pollen germination declined in a material and time-dependent manner, with the magnitude of inhibition consistently greater under the brush (high coverage) than spray (realistic) exposure. In C. sativa, germination decreased from ~89% (controls) to ~82% after 24 hours of spray exposure, and from ~73% to ~42–46% after 24 hours under brush. In C. avellana, sensitivity was higher: germination fell from ~70% to ~50–60% (spray) and from ~62% to ~38–40% (brush) by 24 hours. Across species, GO and mica generally produced the strongest inhibition, with MoS2 and hBN exerting intermediate but significant effects. The comparable impact of mica, a natural lamellar mineral, indicates that flake geometry and surface persistence, rather than engineered provenance alone, are key determinants of interference at the pollen–stigma interface. Mechanistically, adhered 2D-NMs can disrupt the finely tuned process that precedes tube emergence by (i) impeding water fluxes that drive pollen hydration, (ii) altering surface wettability and microcapillarity across papillar cuticles, and (iii) perturbing biochemical signaling at the stigma surface that is required to activate pollen metabolism.

After the discovery of graphene in 2004, which is a two-dimensional nanomaterial (2D-NMs), Many other synthetic 2D-NMs have also been developed, such as hexagonal boron nitride (hBN) and molybdenum disulfide (MoS2). Due to their extraordinary physical and chemical properties such as these are thinnest, strongest consist huge surface area, mechanical strength, which are derived from their 2D structure these are used in electronics and sensors to coatings, biomedicine, and nano enabled agrochemicals. During their life cycle like manufacturing, use, weathering, and end of life, however, unintentional releases could occur. Due to their ultralight weight and 2D morphology, these materials may travel long distances and fall on vegetation. Flowers of wind pollinated species are efficient biological collectors of airborne particulates, which can affect the pollen–stigma interface highly exposed and particularly sensitive. Yet, while nanomaterial effects on vegetative parts like roots, stems, and leaves are well documented, impacts on sexual reproduction remain poorly resolved. This thesis aimed to investigate whether four chemically distinct 2D-NMs, graphene oxide (GO), hBN, MoS2, and muscovite mica (natural benchmark) interfere with the earliest steps of pollination (pollen adhesion, hydration, and germination) in three anemophilous and economically relevant species: Cannabis sativa, Corylus avellana, and Zea mays. Simulated atmospheric deposition was applied to receptive stigmas using two dry deposition scenarios at the same nominal concentration (0.1 mg mL−1): (i) aerosol/spray to mimic environmentally realistic dispersion and (ii) brush to simulate high coverage accumulation. Pollen was deposited after 1.5, 6, and 24 hours to capture time-dependent responses. Environmental scanning electron microscopy (ESEM) and transmission electron microscopy (TEM) were used to characterize material adhesion, surface integrity, and potential internalization, including assessments of membrane continuity and cytoplasmic leakage in pollens and stigmatic surfaces. SEM showed material adhesion of nanosheets to stigmatic papillae and to the pollen exine, forming partial to continuous lamellar coatings. TEM demonstrated that all four 2D-NMs remained extracellular under our conditions: papillar cell walls, plasma membranes, vacuoles, and organelles appeared intact, with no evidence of penetration into intercellular spaces or uptake by papillar or pollen cells. Thus, the materials acted primarily as external physical stressors rather than as intracellular toxicants. Despite the absence of internalization or ultrastructural damage, biologically meaningful functional impairment was evident. Pollen germination declined in a material and time-dependent manner, with the magnitude of inhibition consistently greater under the brush (high coverage) than spray (realistic) exposure. In C. sativa, germination decreased from ~89% (controls) to ~82% after 24 hours of spray exposure, and from ~73% to ~42–46% after 24 hours under brush. In C. avellana, sensitivity was higher: germination fell from ~70% to ~50–60% (spray) and from ~62% to ~38–40% (brush) by 24 hours. Across species, GO and mica generally produced the strongest inhibition, with MoS2 and hBN exerting intermediate but significant effects. The comparable impact of mica, a natural lamellar mineral, indicates that flake geometry and surface persistence, rather than engineered provenance alone, are key determinants of interference at the pollen–stigma interface. Mechanistically, adhered 2D-NMs can disrupt the finely tuned process that precedes tube emergence by (i) impeding water fluxes that drive pollen hydration, (ii) altering surface wettability and microcapillarity across papillar cuticles, and (iii) perturbing biochemical signaling at the stigma surface that is required to activate pollen metabolism.