Regeneration of Fertile Barley Plants from Mechanically Isolated Protoplasts of the Fertilized Egg Cell.

Abstract

A simple procedure is described for the mechanical isolation of protoplasts of unfertilized and fertilized barley egg cells from dissected ovules. Viable protoplasts were isolated from m75% of the dissected ovules. Unfertilized protoplasts did not divide, whereas almost all fertilized protoplasts developed into microcalli. These degenerated when grown in medium only. When cocultivated with barley microspores undergoing microspore embryogenesis, the protoplasts of the fertilized egg cells developed into embryo-like structures that gave rise to fully fertile plants. On average, 75% of cocultivated protoplasts of fertilized egg cells developed into embryo-like structures. Fully fertile plants were regenerated from 4 0 % of the embryo-like structures. The isolation-regeneration techniques may be largely genotype independent, because similar frequencies were obtained in two different barley varieties with very different performance in anther and microspore culture. Protoplasts of unfertilized and fertilized eggs of wheat were isolated by the same procedure, and a fully fertile wheat plant was regenerated by cocultivation with barley microspores.

Dynamics of Nuclear DNA Quantities during Zygote Development in Barley.

“…These data will contribute to our basic understanding of sexual reproduction in flowering plants and will serve as a guide for ongoing studies on genetic transformation in barley via the microinjection method. Furthermore, these data will be used as a comparative base for similar studies on barley zygotes cultured in vitro (Holm et al, 1994).…”

“…The plants used for this study, the growing conditions, and the procedures for egg and zygote protoplast isolation have been described in detail by Holm et al (1994).…”

“…Such a study would provide information on the probable time course of DNA synthesis and would, in turn, be important from a fundamental as well as a practical standpoint. Concerning the latter aspect, knowing the timing of zygotic DNA synthesis would be valuable when conducting experiments involving the microinjection of exogenous DNA into isolated zygotes with the goal of effecting stable genetic transformations followed by regeneration of fertile plants (Holm et al, 1994). To avoid some of the potential pitfalls of the Feulgen technique, we used the DNA-specific fluorochrome 4;6-diamidino9-phenylindole (DAPI) and microspectrofluorometric analysis (Coleman et al, 1981;Coleman and Goff, 1985;Friedman, 1991) to estimate changes in nuclear DNA quantities during zygote development in two cultivars of barley-Alexis, a spring variety, and Igri, a winter variety.…”

Double Fertilization in Gnetum gnemon: The Relationship between the Cell Cycle and Sexual Reproduction.

“…With the advent of in vitro fertilization techniques (Kranz et al, 1991;Lorz, 1993, 1994;Faure et al, 1994aFaure et al, , 1994b and regeneration of genetically engineered zygotes and embryos (Sangwan et al, 1991;Holm et al, 1994), there is renewed interest in the relationship between the cell cycle and sexual reproduction in higher plants (Friedman, 1991;Mogensen and Holm, 1995;Sherwood, 1995). Unfortunately, reproductive cell cycles have not been adequately investigated in most groups of seed plants, and, with one exception (Friedman, 1991), relationships between cell cycle patterns and sexual reproduction have been derived exclusively from a small number of studies on angiosperms (Taylor and McMaster, 1954;Woodard, 1956;DAmato et al, 1965;Woodcock and Bell, 1968;Mericle, 1970, 1973;Hesemann, 1973;Bennett and Smith, 1976;Ermakov et al, 1980;Bino et al, 1990;Mogensen and Holm, 1995;Sherwood, 1995).…”

High efficiency transient and stable transformation by optimized DNA microinjection intoNicotiana tabacumprotoplasts

“…Making use of these advantages, interesting work has been done with plant material, including the analysis of visible marker gene expression in meristematic cells and derived cell lineages (Simmonds et al, 1992;Lusardi et al, 1994) as well as the partial elucidation of signal transduction pathways involved in the hght-regulation of plant gene expression . Progress in the culture of isolated plant zygotes has recently been reported (Kranz and Lorz, 1993;Holm et al, 1994). DNA delivery by microinjection into isolated zygotes might emerge as an important technique for plant transformation.…”