THE PLANT CELL, Vol 6, Issue 4 531-543, Copyright © 1994 by American Society of Plant Biologists
Regeneration of Fertile Barley Plants from Mechanically Isolated Protoplasts of the Fertilized Egg Cell
P. B. Holm, S. Knudsen, P. Mouritzen, D. Negri, F. L. Olsen and C. Roue
Carlsberg Research Laboratory, 10, Gamle Carlsberg Vej, DK-2500 Copenhagen, Denmark
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 ~75% 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 ~50% 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.
