Overexpression of cryIAb gene for enhancing yellow stem borer (Scirpophaga incertulas) resistance using a marker-free double T-DNA system in Rojolele of Indonesia Rice cultivar.

The aromatic rice cultivar Rojolele (Oryza sativa L. cv. Rojolele), prized for its superior organoleptic qualities, is highly susceptible to yellow stem borer (Scirpophaga incertulas), a pest causing significant yield losses in Asia. To address this vulnerability, we developed marker-free transgenic Rojolele lines expressing the cry1Ab gene using a double T-DNA vector system, eliminating reliance on antibiotic resistance markers. Agrobacterium-mediated transformation yielded 21 independent T0 lines, with PCR and Southern blot analysis confirming stable integration of cry1Ab in seven lines by the T3 generation. Immunostrip assays verified functional Cry1Ab protein expression, while bioassays demonstrated complete resistance in lines I.AR.4 and III.AR.5.3. Susceptible lines (I.AR.2, III.AR.5.2) exhibited variable transgene expressions, highlighting the importance of event selection. The marker-free strategy aligns with biosafety regulations, and the retained agronomic traits ensure compatibility with commercial cultivation. This study provides a sustainable solution to protect high-value aromatic rice, combining insect resistance with consumer-preferred qualities. Field trials and resistance management strategies, such as gene pyramiding, are recommended for future deployment.