Low titre of agroinoculum with prolonged incubation period and low auxin concentration in the regeneration media are the key to high frequency of transformation in climate-resilient Aus-type rice genotype Nagina 22.

Abstract

Developing an efficient and reproducible regeneration protocol holds paramount significance for advancing genetic transformation technologies in rice, facilitating their utilisation in crop improvement. Nagina 22 (N22), a climate-resilient Aus-type rice genotype known for its tolerance against multiple stresses, lacks a standardised transformation protocol, limiting its utilisation as a background for genetic transformation. This study reports, for the first time, a highly efficient transformation and regeneration protocol for N22 using a CRISPR/Cas9 vector. Mature seeds were used to induce embryogenic calli on CHU(N6)-based callus induction media (CIM) with varying concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). The highest callus induction efficiency (~ 94%) was achieved using 3 mgL^-1 2,4-D. For regeneration, calli were transferred to different regeneration media-I (RM-Ia to RM-Ie), where a combination of 5 mgL^-1 6-benzylaminopurine (BAP) and 0.02 mgL^-1 naphthalene acetic acid (NAA) resulted in ~ 44% regeneration frequency. Subsequent optimisation of regeneration media-II (RM-II) with low NAA concentration enhanced shoot elongation and root development. Furthermore, reducing basal salt concentration in the resuspension media significantly enhanced transformation efficiency to 44%, achieved, by only using sterile distilled water (SDW) with 150 mM acetosyringone for calli infection. The optimised protocol was successfully validated using CRISPR/Cas9 vector, facilitating targeted gene knockouts for functional genomic studies. This approach addresses a critical gap in N22 genetic transformation, providing a reliable protocol for advancing rice improvement through gene editing. It offers valuable insights for future research and practical applications in genetic transformation of this elite rice genotype for various agronomic and scientific purposes.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-025-04210-y.