ISOLATED MAIZE ZYGOTES MIMIC IN-VIVO EMBRYONIC-DEVELOPMENT AND EXPRESS MICROINJECTED GENES WHEN CULTURED IN-VITRO

We established conditions for the regeneration of natural maize zygote s isolated from pollinated plants with the goal of investigating the m olecular control of early embryogenesis in higher plants. Viable zygot es were excised from embryo sacs by minimal enzymatic digestion and mi crodissection. Viable zygotes transferred to coculture with androgenic microspores from barley developed into embryo-like structures in 61% of the cases. No development was observed when zygotes were cultured i n the presence of maize anthers undergoing androgenetic embryogenesis. Zygote-derived embryo-like structures regenerated into fertile plants through secondary embryogenesis when transferred to solid medium. The first zygotic division was asymmetrical and bipolar structures simila r to pretransitional embryos observed in planta were later produced as observed using light and electron microscopy. Conditions for efficien t microinjection of DNA into zygotes were established. Calcofluor and PATAG staining of zygotes showed that cell wall regeneration occurred as early as 20 min after enzymatic isolation and that after 2 hr, each zygote was bordered with cell wall material. Through quantitative mic rophotometry, DNA synthesis during the first cell cycle of the zygote was shown to occur between isolation and 12 hr of culture. Microinject ion of two types of reporter genes (GUS gene and anthocyanin regulator y genes) demonstrates transient expression in plant zygotes. On averag e, 3.5% of microinjected zygotes showed transgenic expression. Reporte r gene expression was observed in zygotes at different time points of their first cell cycle. (C) 1996 Academic Press, Inc.