Cytokinin-mediated enhancement of multi-kernel seed formation and productivity in rice revealed using non-destructive X-ray imaging

The increasing demand for rice necessitates strategies to enhance grain number and seed productivity. The multi-kernel rice genotype Jugal, characterized by multiple carpels within a single spikelet, exhibits a unique trait that increases kernel number. However, the external similarity between single- and multi-kernel seeds limits efficient identification. In this study, a non-destructive X-ray imaging approach was employed to identify multi-kernel seeds and evaluate the effect of phytohormones on their formation and associated productivity traits. Digital radiography reliably distinguished internal kernel structure without affecting seed germination and showed comparable accuracy to soft X-ray computed tomography-based imaging. Field experiments conducted across dry and wet seasons revealed a strong cytokinin effect on multi-kernel formation. Zeatin application at 45 µM produced the highest response, with up to a 9.48-fold increase over the control, while kinetin showed moderate effects and gibberellic acid and 2, 4-D were less effective. Cytokinin treatments also improved seed filling, 1000-seed weight, and seedling vigour. Multi-kernel seeds exhibited high germination (> 97%), and 14.1% produced twin seedlings. These findings demonstrate that cytokinin-mediated regulation enhances multi-kernel formation and associated yield traits, while X-ray imaging provides a rapid and reliable tool for seed phenotyping in rice.