Tissue and Organ Culture 73: 167–176, 2003.
2003 Kluwer Academic Publishers. Printed in the Netherlands.
Proteins produced by barley microspores and their derived androgenic
structures promote in vitro zygotic maize embryo formation
A. Paire , P. Devaux , C. Laﬁtte , C. Dumas & E. Matthys-Rochon
Laboratoire de Reproduction et developpement des Plantes
CNRS/INRA /ENS /UNIV
Ecole Normale Superieure
Laboratoire de Biologie
Cellulaire et Moleculaire
Cappelle en Pevele
´´ ´´ ´
Biologie et de Physiologie Vegetales
UMR CNRS UPS
Pole de Biotechnologies Vegetales
Chemin de Borde Rouge
Received 3 April 2002; accepted in revised form 25 November 2002
arabinogalactan-proteins (AGPs), maize, oligosaccharides, proteins, zygote and microspore in vitro
culture, zygotic embryo
Maize zygotes formed in planta were isolated and co-cultured with barley microspores. Evidence suggests that
culture with microspores and / or conditioned media in barley promoted zygotic embryo formation. In order to
characterise active substances present in the conditioned media, we collected medium at various times after the
initiation of culture. We showed that proteins appeared over time and that their quantity increased during the
course of the culture. Some proteins were glycosylated as revealed by the ConA peroxidase test. The use of the
-glucosyl Yariv reagent has shown that arabinogalactan-proteins (AGPs) were present. In addition,
conditioned medium samples were analysed for their oligosaccharide content. New oligosaccharides appear in the
course of the culture but they do not seem to affect development. We discuss in details the results in the context of
understanding cell–cell interactions between embryo and nurse cells and the possible parallel with that occurs in
ovulo between endosperm and embryo.
Introduction given that in natural conditions the endosperm
nourishes the embryo.
In higher plants, the formation of the zygote and the Only a few experimental processes have been
development of the embryo occur deep in the mater- developed using nurse cells for the culture of zygotes.
nal tissues. In order to study the early steps of Kranz and Lorz (1993) have regenerated plants from
embryogenesis in maize, which is our plant model electrofused gametes (artiﬁcial zygotes) and Holm et
(Dumas and Mogensen, 1993), an in vitro culture al. (1994) have succeeded in developing embryos
system was established (Leduc et al., 1996). This from isolated, naturally fertilized barley and wheat
system allows direct access to very young embryos zygotes. It is known that at low density, cells in
and opens the way to molecular analysis at all stages culture do not divide and enter necrosis (Hari, 1980)
of embryogenesis. Zygotes are prepared from plants 1 or a programmed cell death pathway (Lennon et al.,
day after fertilization (Mol et al., 1994) and are 1991); proliferation can be induced by conditioned
cultured in the presence of barley microspores which medium prepared from high cell density cultures
act as nurse cells. Nurse cells are essential to sustain (Hari, 1980; McCabe et al., 1997). These data suggest
zygotic embryo development under in vitro condi- that cell-derived molecules are released or secreted
tions (Holm et al., 1994). It is thought that this culture into the culture medium and help to sustain further
system may mimic endosperm–embryo interrelations, cell development.