Transient expression of fluorescently tagged proteins in developing maize aleurone cells

Abstract

Expressing transgenes in the endosperm of cereals by developing stably transformed lines is an expensive and labor-intensive process. An alternative that is less expensive and faster is to express the transgenes transiently. We describe here a detailed protocol to express transiently genes in maize aleurone cells by biolistic bombardment of in vitro cultured developing endosperms. Maize endosperms are isolated from kernels at 6–8 days after pollination and placed on culture medium plates for 1–2 days. Afterwards, the endosperms can be transfected with either a single gene or multiple transgenes simultaneously. Microparticles coated with the selected plasmids are delivered into the aleurone cells by biolistic bombardment. As a demonstration, we co-expressed two transgenes simultaneously, one tagged by GFP and the other tagged by mCherry. Our transfection efficiency is comparable to that obtained with Agrobacterium-mediated transformation, but requires a shorter time for gene expression after transfection. We provide optimized conditions and parameters for key steps in this procedure. • Small, non-binary plasmids can be used to drive expression of fluorescent proteins. • Optimized distribution of DNA-coated microparticles maximizes transfection of in vitro grown maize endosperms while minimizing cellular damage. • Transgene expression can be detected as early as one day after bombardment.

Keywords

• In vitro endosperm culture
• Biolistic bombardment
• Co-expression of GFP and mCherry