Optimizing Transformation for Transient Expression of Exogenous Genes in Areca Palm via Microparticle Bombardment

Areca palm (Areca catechu L.) is an economically significant crop in Southeast Asia. Yellow leaf disease (YLD) severely threatens its cultivation due to lacking resistant cultivars. Agrobacterium-mediated transformation have been proven to face significant challenges in areca palm. This study aimed to achieve transient expression of exogenous genes and establish VIGS (virus-induced gene silencing) in areca palm. Parameters such as DNA loading ratio (DLR), helium pressure, and bombardment distance were first optimized with vectors expressing reporter genes. The highest DNA loading efficiency was achieved with a DNA concentration of 50 ng/μL, spermine at 0.016 μmol/L, and CaCl₂ at 1 mol/L. Optimal transformation conditions were identified as a helium pressure of 870 psi and a bombardment distance of 1 cm. Under these conditions, exogenous genes, including GUS (β-glucuronidase, 2023 bp) and RUBY (betalain synthesis gene, 3951 bp), were successfully expressed transiently in areca palm leaves. Additionally, a GFP-labeled infectious clone for a mild isolate of areca palm necrotic spindle-spot virus (ANSSVm-GFP) was constructed and successfully transformed into Nicotiana benthamiana. Although the transformation of ANSSVm-GFP in areca palm has not achieved, this study establishes a reliable method for foreign gene expression, laying the groundwork for the development of a VIGS system in areca palm. This has significant implications for gene characterization and breeding disease-resistant areca palm varieties.