Rapid and flexible assessment of gene functions in plant cells with particle bombardment and linear DNA

Abstract

Biolistic transformation is a versatile tool in plant science, yet high equipment costs and tissue damage from high-pressure gas remain significant barriers. Building on our previously developed "TSGMAC", a low-cost, helium-free biolistic system, we report three major advancements to enhance its throughput, delivery quality, and quantitative capability. First, a "guide barrel" assembled from commercial DIY fittings was developed; it effectively eliminates physical tissue damage and ensures uniform particle distribution, even in soft tissues like bok choy (Brassica rapa subsp. chinensis). Second, a rapid gene expression platform using PCR products was characterized. Results demonstrate that linear DNA constructs are efficiently circularized via non-homologous end joining (NHEJ) in plant cells, and protein expression is robust regardless of the relative positions of the promoter, coding sequence, and terminator. This system bypasses time-consuming cloning. Third, a cost-effective, highly sensitive dual-luciferase assay system utilizing teal Luc (teLuc) and inexpensive firefly luciferase (FLuc) inhibitors was established. This integrated workflow enables rapid, quantitative molecular biology using supermarket-obtained materials and standard PCR reagents. Our findings provide a practical foundation for plant scientists, synergistically accelerating gene functional analysis and genetic tool development.