Vacuum Infiltration Enables Efficient Transient T-DNA Delivery in Oil Palm (Elaeis guineensis) Seedlings: A Pilot Study

Stable genetic transformation, involving the incorporation of DNA or plasmids, is widely used to develop plant varieties with improved traits. However, transient transformation remains essential for evaluating genetic constructs prior to stable T-DNA integration. In oil palm (Elaeis guineensis), the transformation process faces challenges such as low insertion efficiency and limited callus availability, highlighting the need for rapid and scalable alternatives. In this pilot study, we evaluated vacuum infiltration as a method for transient T-DNA delivery using Agrobacterium tumefaciens strain C5C81 harboring the OST2-Cas9 construct in PPKS DxP 718 oil palm seedlings. Three incubation durations (1, 5, and 10 minutes) were tested under vacuum pressures of 100 and 200 mbar. Seeds were germinated for approximately 90 days until the first leaf emerged, after which they were surface sterilized and immersed in an Agrobacterium-plasmid buffer solution. Following infiltration, seedlings were replanted in sterile soil, incubated at 25 °C for 24 hours, and then transferred to a greenhouse. PCR analysis targeting Cas9 and the NPTII marker at 1, 7, and 30 days post-infiltration (DPI) demonstrated a 100% detection rate of T-DNA. Histological analysis at 1 DPI revealed that infiltration at 100 mbar for 5 minutes significantly expanded multiple tissues, particularly vascular regions, indicating efficient infiltration and potential for molecular transport. These findings suggest that short-duration vacuum infiltration under low pressure can effectively mediate transient T-DNA delivery in oil palm seedlings, offering a promising tool for genetic research and transformation development in this species.