Improving oil palm drought tolerance through CRISPR/Cas9-OST2 and silica supplementation: biochemical insights under in vitro conditions

Abstract Developing drought-tolerant oil palm through conventional breeding is time-consuming. Genome editing techniques, such as CRISPR/Cas9, offer a faster alternative, while silica application is known to enhance plant drought tolerance. This study combined genome editing and silica treatment to improve drought tolerance in oil palm plantlets via vacuum-assisted agroinfiltration. Plantlets were placed in a desiccator and immersed in MMA buffer containing Agrobacterium harboring the CRISPR/Cas9-OST2 plasmid (OD600 = 0.01). Agroinfiltration was performed at vacuum pressures of 100 and 200 mbar for 1, 5 and 10 minutes. The treated plantlets were then cultured on Murashige and Skoog (MS) medium containing antibiotics under a 12-hour photoperiod. After two weeks, drought stress was induced using 4% PEG-6000, followed by silica application (Na 2 SiO 3 and CaSiO 3 at 3, 5, and 7 mg/L). Biochemical parameters, including chlorophyll, carotenoids, proline, hydrogen peroxide (H 2 O 2 ), and malondialdehyde (MDA), were measured. The results showed that vacuum-infiltration caused no significant morphological damage. Under drought stress, plantlets infiltrated with the CRISPR/Cas9-OST2 plasmid and treated with 7 mg/L Na 2 SiO 3 exhibited significantly higher levels of chlorophyll A, chlorophyll B, total chlorophyll, carotenoids, and proline compared to wild-type controls. They also showed reduced levels of H 2 O 2 and MDA, indicating enhanced oxidative stress tolerance and decreased membrane damage. These findings suggest that CRISPR/Cas9-OST2 plasmid delivery via vacuum agroinfiltration, combined with silica supplementation, effectively enhances drought tolerance in oil palm plantlets through coordinated physiological and biochemical adaptations.