Abstract
A maize (Zea mays L.) endosperm cell culture has been shown to efficiently replicate DNA sequences derived from wheat dwarf virus (WDV), a monopartite monocot geminivirus. To analyze sequences necessary for viral replication and to verify their application for a plant gene expression vector, we have developed a 3.7 kilobase pairs Escherichia coli--plant cell shuttle vector, pWI-11. The p15A origin of replication, functional in E. coli, was introduced into the viral sequences. We have replaced the coding region of the coat protein gene by that of bacterial neomycin phosphotransferase II (NPT II) gene. The resulting NPT II gene fusion can serve as a selectable marker in both plant and E. coli systems. Into a unique cloning site in this pWI-11 vector, we introduced a gene fusion carrying the bacterial beta-glucuronidase (GUS) coding region under control of the cauliflower mosaic virus 35S (CaMV35S) gene promoter and terminator. By transferring these viral sequences into protoplasts derived from maize endosperm cell cultures, we have demonstrated that the plasmid pWI-11 can replicate in maize endosperm cells, that the GUS reporter gene introduced into pWI-11 can be expressed at high level in the transformed cells, and that the replicating viral DNA can be rescued from endosperm cells by transforming E. coli in the presence of kanamycin. The level of GUS gene expression increased progressively in transformed endosperm cells during a prolonged culture period, coinciding with replication of the viral sequences in these cells.