Development of novel and sustainable approaches to improve seed germination and mineral content in rice (Oryza sativa L.)

Water stress brought about by climate change and the prevalence of micronutrient deficiency are among the major global concerns threatening food security and public health. Rice is a staple food among low-income countries where there is a simultaneous prevalence of micronutrient deficiency and increasing demand for food due to a high population index. To address these concerns, the current work proposed to develop techniques directed at promoting rice resilience to water stress during germination and enhancing iron content in grains by implementing specific seed priming treatments or genome editing approaches. To address rice biofortification, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) was used to induce 4 base pair deletions in the OsNAS2 (nicotianamine synthase 2) gene promoter sequence that disrupted cis-regulatory elements associated with inhibitors of iron- and zinc-related transport genes. This led to elevated grain zinc concentration and increased spikelet number. To address water stress resilience during germination, popular Italian rice varieties were subjected to innovative priming treatments using poly-gamma-glutamic acid (PGA), degraded PGA, and iron pulsing, and were grown both under control and water stress conditions imposed through polyethylene glycol (PEG). In a parallel experiment, biofortified lines (developed through conventional breeding, transgenic approach, and genome editing) and Italian rice cultivars were germinated in soil with water levels at field capacity and mild drought. Results from selected germination parameters and histochemical analysis on PEG and soil set-up showed that PGA buffers water stress, while degraded PGA (dPGA) provides an additional food source for the seed. Iron pulsing resulted in enhanced iron uptake during germination in a genotype-specific manner. Gene expression data related to DNA damage repair, antioxidant defense mechanism, and iron and zinc homeostasis were investigated in two varieties with different sensitivity to the imposed treatments to understand the germination behavior at a molecular level. The gene expression patterns showed contrastive and similarities between Carnaroli (responsive to all the priming methods) and Cerere (responsive mainly to iron pulsing). Seeds from both varieties had elevated expressions of genes involved in stress and amino acid transport, after 16 hours of soaking in water or WS. Carnaroli seeds soaked in PGA had elevated expression on the majority of genes while a similar pattern was observed in dPGA-treated Cerere seeds rather than those under PGA treatment. At three days after germination, both varieties have low gene expression detected in CTRL, but Cerere seed treated with PGA, dPGA, and Fe showed elevated gene expression compared with Carnaroli seeds treated with similar priming methods. At seven days after germination, Carnaroli primed seeds showed more genes with elevated expression compared to Cerere. This study has evidenced that the expression of specific genes can be induced or repressed by these treatments in a time- and treatment-dependent manner. Nonetheless, further investigation is required to expand the understanding of the molecular mechanisms related to seed germination and priming. This will ultimately aid in providing faster and more sustainable solutions to increase rice productivity under water stress, highly induced by the context of the climate change scenario.