Monitoring individual development of isolated wheat zygotes: a novel approach to study early embryogenesis

A culture method has been established by which development of isolated whea t (Triticum aestivum L.) zygotes can be monitored individually until format ion of multicellular structures. As was shown recently these isolated zygot es have a high capacity to form differentiated embryos and normal plants, a nd thus constitute a suitable object to study early embryogenesis. After be ing isolated within 6 h alter pollination (hap), zygotes were immobilized i n an agarose droplet directly on a microscopic chamber slide, which allows for both subsequent development through co-culture with feeder aggregates, as well as detailed observation and photographic documentation of individua l behavior. Shortly after fertilization, the wheat zygote, like the unferti lized egg cell, is characterized by one conspicuous nucleolus. Typically, a second and a third nucleolus appeared between 5 and 8.5 hap. Between 7 and 15 hap,we observed nucleolar vacuolation indicating enhanced ribosomal act ivity. Continuous cell expansion with slight cell elongation was detected u ntil around 15 hap, followed by a period of transitory reduction in cell vo lume which roughly corresponded with mitosis. Mitotic prophase of a zygote could easily be detected by the disappearance of all nucleoli within a few minutes. The division plane was generally established perpendicular to the formerly established cell elongation axis. At cytokinesis, which was comple ted by 19 hap in 90% of the individuals observed, 2 or 3 nucleoli were dete cted again per daughter cell. The first cell division, including the establ ishment of a cleavage furrow with intercellular spaces, was completed in al l cases within 23 hap. Since this result is in accordance with what is know n from earlier studies based upon fixed material, and since the zygotes sub sequently continue embryogenesis, in vitro development is assumed to be ana logous to that: in planta. This experimental system constitutes a valuable experimental tool for further detailed research, both at the cellular and a t the molecular level.