Developmental and transgenic analysis of two tomato fruit enhanced genes.

Tomato fruit development is characterized by distinct developmental stages: fruit set, periods of rapid cell division and cell expansion, and the period where processes associated with ripening are dominant. During each of these stages, different aspects of cellular metabolism are favored. Accompanying these developmental changes are dramatic differences in gene expression, with a subset of genes being expressed early and a subset being expressed later in development. We have isolated and characterized several sequences from tomato that are expressed primarily in immature green fruit. Two of these genes (Tfm7 and Tfm5) have been characterized more extensively and their sequence indicates that they encode proteins corresponding to a proline-rich protein (PRP) and a glycine-rich protein (GRP). RNA blot analysis indicates that the transcripts from these genes are present at the earliest stages of fruit development, and continue to be expressed throughout the growth period of the fruit. Expression analysis during development indicates that the gene encoding the PRP may be down-regulated by ethylene. As a means to understanding the functional significance and the transcriptional contribution of these tissue-limited proteins during development, we constructed promoter-reporter gene fusions to identify which cell types express each of these sequences. GUS protein produced in transgenic plants by both promoter-reporter gene constructs was detected in most tissues of the fruit including the pericarp, columella, and placental tissues of young immature fruit through the mature green stage. However, only one of the promoter sequences conferred expression in the fruit locular tissue.

Glyphosate-tolerant CP4 and GOX genes as a selectable marker in wheat transformation.

The lack of alternative selectable markers in crop transformation has been a substantial barrier for commercial application of agricultural biotechnology. We have developed an efficient selection system for wheat transformation using glyphosate-tolerant CP4 and GOX genes as a selectable marker. Immature embryos of the wheat cultivar Bobwhite were bombarded with two separate plasmids harboring the CP4/GOX and GUS genes. After a 1 week delay, the bombarded embryos were transferred to a selection medium containing 2 mM glyphosate. Embryo-derived calli were subcultured onto the same selection medium every 3 weeks consecutively for 9-12 weeks, and were then regenerated and rooted on selection media with lower glyphosate concentrations. Transgenic plants tolerant to glyphosate were recovered. ELISA assay confirmed expression of the CP4 and GOX genes in R0 plants. Southern blot analysis demonstrated that the transgenes were integrated into the wheat genomes and transmitted to the following generation. The use of CP4 and GOX genes as a selectable marker provides an efficient, effective, and alternative transformation selection system for wheat.