TRANSIENT MARKER-GENE EXPRESSION DURING ZYGOTIC IN-VITRO EMBRYOGENESIS OF BRASSICA-JUNCEA (INDIAN MUSTARD) FOLLOWING PARTICLE BOMBARDMENT

A method has been established that allows the transfer of genes into s ingle cells of excised globular-stage zygotic Brassica juncea L. embry os. The fate of single, genetically marked cells was followed during i n-vitro embryogenesis. A simple and defined embryo culture medium has been designed on which zygotic B. juncea embryos, excised at the globu lar or at later stages, develop normally into mature, fully grown embr yos. The smallest embryos which can be efficiently cultured are 30 mu m long (embryo proper without suspensor) and are comprised of less tha n 20 cells. The embryos grow on the surface of solid medium without em bedding and are freely accessible to microprojectile bombardment. Shoo ting at globular, transition and early heart-shaped embryos using both a particle inflow gun and a micro-targeting particle accelerator resu lted in transient expression of genes encoding visible markers. For bo th particle-acceleration devices the shooting conditions have been opt imised based on transient beta-glucuronidase (GUS) expression. Bombard ing embryos under optimal conditions had no deleterious effects on in- vitro embryogenesis. Multicellular GUS-expressing sectors were obtaine d, showing that cells can survive receiving a particle and resume norm al development. The examination of these sectors has provided new info rmation about the cell division patterns characterising early B. junce a embryogenesis. To be able to follow the development of particular ge netically marked sectors, we tried to identify reporter genes that, in contrast to the uid A gene (which encodes GUS), can be non-destructiv ely assayed in embryonic cells. Preliminary data has shown that expres sion of the firefly luciferase gene (Luc) can be detected in bombarded embryos without affecting their viability.