Factors Influencing Gene Delivery into Zea Mays Cells by High–Velocity Microprojectiles

Abstract

Factors influencing the efficiency of DNA delivery into suspension culture cells of Zea mays by the particle bombardment process were studied using a chimeric gene coding for the production of β–glucuronidase. Two days following bombardment with plasmid–coated microprojectiles, expression of the β–glucuronidase gene was detected with the synthetic substrate 5–bromo–4–chloro–3–indoyl–β–D–glucuronic acid, which, upon cleavage, forms a blue precipitate visually detectable within affected cells. The number of cells expressing β–glucuronidase was about 30 times greater when the cells were bombarded on a filter paper support than when they were bombarded while covered by liquid media without such a support. The efficiency of gene delivery was also significantly affected by the velocity of the microprojectile and the number of micro–projectiles used for bombardment, and the concentration of CaCl2 and spermidine used to adsorb DNA to the microprojectiles. In addition to cell cultures, the particle bombardment process was also used to deliver the β–glucuronidase gene to intact cells on the surface of excised Z. mays embryos. The results indicate that delivery by high–velocity microprojectiles may be useful for the stable transformation of monocot species.