Optimization of Agrobacterium-mediated transformation procedure for an indica rice variety-Khang Dan 18

Abstract

In addition to traditional methods, advanced biotechnologies, especially CRISPR/Cas9-mediated genome editing, have emerged as effective tools for improving important agronomic traits in rice. However, the critical step for utilizing these systems is to develope an effective system for rice transformation and regeneration. This study was performed to establish procedures for Agrobacterium-mediated transformation and regeneration of rice cultivar Khang Dan 18 (KD18) – a popular indica variety in the North of Vietnam. The tissue cultures procedure with optimized medium compositions showed high frequencies of callus induction and shoot regeneration, at 94.7% and 45.3% respectively. We found that 30 mg/L of hygromycin was an effective concentration for transgenic KD18 selection. Light-yellow friable calli were used as the starting material for transformation mediated by A. tumefaciens strain AGL1 harbouring pHUE411 vector containing gus intron and hygromycin resistance gene (hptII). Important factors related to transformation procedure had been optimized in this study. The high transformation frequency (12.8%) was achieved by using the optimized procedure for KD18 cultivar. In which, bacterial density (OD600), infection time and co-cultivation period were performed at 0.1, 20 minutes and 3 days, respectively. PCR analysis using specific primers and the histological GUS staining demonstrated the presence of hptII and gus genes in transgenic rice lines. This result provides a potential protocol to transfer genes of interest into KD18 as well as other indica rice cultivars.

Development of CRISPR/Cas9 systems to induce targeted mutations in the promoter region of the OsSRFP1 gene in rice

“…The Agrobacterium-mediated method as described by our previous reports (Thao et al, 2022) was used for rice transformation. Transgenic rice lines were selected and transferred into the greenhouse condition (16:8 h, light/dark photoperiod; temperature and humidity depend on environmental conditions).…”