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: Biolistic bombardment; Co-expression of GFP and mCherry; In vitro endosperm culture.
© 2021 The Authors. Published by Elsevier B.V.