Zinc biofortification through seed nutri-priming using alternative zinc sources and concentration levels in pea and sunflower microgreens

**Background:** Zinc deficiency affects an estimated 17.1% of the global population and is a major contributor to 'hidden hunger,' particularly in low- and middle-income countries. Agronomic biofortification through seed nutri-priming—soaking seeds in nutrient solutions before sowing—offers a simple, scalable approach to enhance micronutrient content in crops. Microgreens are especially suitable for this strategy due to their short growth cycle, high nutrient density, and low antinutrient content. This study investigated the potential of Zn biofortification via seed nutri-priming in pea and sunflower microgreens using three Zn sources (ZnSO₄, Zn-EDTA, ZnO nanoparticles) at four concentrations (25, 50, 100, 200 mg/L).

**Methods:** The experiment was conducted in spring 2021 in a glasshouse at Pennsylvania State University. Seeds of 'Dwarf grey sugar' peas (96% germination) and 'Black oil' sunflower (90% germination) were soaked for 12 hours in aerated Zn solutions or deionized water (control). Seeds were sown in peat-perlite mix at 1 seed/cm², grown under supplemental LED light (6:00 AM–9:00 PM), and harvested at 9 days (sunflower) and 10 days (pea). Yield components, mineral profiles (ICP-OES), photosynthetic pigments, total phenols (Folin-Ciocalteu), flavonoids (AlCl₃ colorimetric), anthocyanins, antioxidant activity (DPPH), and phytic acid (Megazyme kit) were measured. Data were analyzed using ANOVA with Fisher LSD (α=0.05) and linear contrasts vs. control.

**Key Results:** In pea microgreens, ZnSO₄ at 200 ppm produced the highest Zn accumulation (182.33 mg/kg DW), a 126% increase over control (80.67 mg/kg DW), followed by ZnO at 200 ppm (149.00 mg/kg DW, +84.7%). Zn-EDTA did not significantly increase Zn content at any rate. However, ZnSO₄ at 200 ppm reduced fresh weight, chlorophyll a, N, and Fe content, with visible chlorosis at the highest rate. ZnO maintained yield and chlorophyll levels better than ZnSO₄. An antagonistic effect on Fe, Mn, and Cu was observed with increasing Zn accumulation from ZnSO₄ and ZnO. Total phenols and antioxidant activity increased with Zn accumulation from ZnSO₄ and ZnO. Phytic acid in pea seeds decreased by 13.7% after soaking, and microgreens had 38.7% less phytic acid than unsoaked seeds. The phytic acid/Zn molar ratio decreased significantly with ZnSO₄ and ZnO treatments, indicating improved Zn bioavailability.

In sunflower microgreens, ZnSO₄ at 200 ppm achieved the highest Zn content (3.32 times control), reaching 229.7% increase over control (70.67 mg/kg DW). ZnO at 200 ppm produced a 118.4% increase. Zn-EDTA only increased Zn at 200 ppm. No yield reduction or phytotoxicity was observed at any treatment level, consistent with sunflower's known Zn hyperaccumulator capacity. Unlike peas, no antagonistic effects on Fe, Mn, Cu, or B were observed. Chlorophyll and carotenoid levels were higher with ZnO and Zn-EDTA than ZnSO₄. No treatment effects on total phenols or antioxidant activity were found in sunflower. Phytic acid decreased by 12.64% after soaking, and microgreens had 80.6% less phytic acid than unsoaked seeds. The phytic acid/Zn molar ratio decreased with ZnSO₄ and ZnO treatments.

**Clinical Implications:** Consumption of 100 g fresh pea microgreens biofortified with 200 ppm ZnSO₄ or ZnO could provide 21.3% and 17.6% of the adult RDA for Zn (8 mg), respectively. Similarly, sunflower microgreens could provide 21.5% (ZnSO₄) and 13.6% (ZnO) of the RDA. Under FDA criteria, these would qualify as a 'high source' (≥20% RDA) or 'good source' (10–19% RDA) of Zn. The reduced phytic acid/Zn molar ratio in biofortified microgreens suggests improved Zn bioavailability compared to untreated microgreens. However, the trade-off between Zn enrichment and Fe reduction in peas (but not sunflower) must be considered. Further research is needed on in vitro/in vivo Zn bioavailability, simultaneous Fe+Zn biofortification, and safety assessment of ZnO nanoparticle and Zn-EDTA residues before commercial implementation.