Reliable Transient Transformation of Intact Maize Leaf Cells for Functional Genomics and Experimental Study

Abstract

AbstractMaize (Zea mays) transformation routinely produces stable transgenic lines essential for functional genomics; however, transient expression of target proteins in maize cells is not yet routine. Such techniques are critical for rapid testing of transgene constructs and for experimental studies. Here, we report bombardment methods that depend on leaf developmental stage and result in successful expression with broad applications. Fluorescent marker genes were constructed and bombarded into five developmental regions in a growing maize leaf. Expression efficiency was highest in the basal-most 3 cm above the ligule of an approximately 50-cm growing adult leaf. Straightforward dissection procedures provide access to the receptive leaf regions, increasing efficiency from less than one transformant per cm2 to over 21 transformants per cm2. Successful expression was routine for proteins from full genomic sequences driven by native regulatory regions and from complementary DNA sequences driven by the constitutive maize polyubiquitin promoter and a heterologous terminator. Four tested fusion proteins, maize PROTEIN DISULFIDE ISOMERASE-Yellow Fluorescent Protein, GLOSSY8a-monomeric Red Fluorescent Protein and maize XYLOSYLTRANSFERASE, and maize Rho-of-Plants7-monomeric Teal Fluorescent Protein, localized as predicted in the endoplasmic reticulum, Golgi, and plasma membrane, respectively. Localization patterns were similar between transient and stable modes of expression, and cotransformation was equally successful. Coexpression was also demonstrated by transiently transforming cells in a stable line expressing a second marker protein, thus increasing the utility of a single stable transformant. Given the ease of dissection procedures, this method replaces heterologous expression assays with a more direct, native, and informative system, and the techniques will be useful for localization, colocalization, and functional studies.