Liquid Culture Systems for in vitro Plant Propagation

Propagation of Norway Spruce 289
5. Conditioning factors regulating somatic embryogenesis
It has long been known that conditioned medium from embryogenic
cultures can promote embryogenesis. The ability of conditioned medium to
sustain or stimulate somatic embryogenesis implies that secreted soluble
signal molecules play an important role. Several components in conditioned
medium have been found to promote somatic embryogenesis. In Norway
spruce we have shown that extracellular chitinases (Egertsdotter et al., 1993;
Dyachok et al., 2000), arabinogalactan proteins (AGPs) (Egertsdotter and
von Arnold 1995) and lipo-chitooligosaccharides (LCOs) (Dyachok et al.,
2000, 2002) affect somatic embryogenesis.
Chitinases from sugar beet and Streptomyces griseus stimulate early
development of somatic embryos in Norway spruce (Egertsdotter and von
Arnold 1998; Dyachok et al., 2002). However, we have also shown that
chitinases from S. griseus degrade LCOs (Dyachok et al., 2002). Taken
together, our results suggest that chitinases can regulate embryogenesis in
different ways, both by degrading LCOs and by formation of LCOs. It has
previously been shown that enzymes that form and degrade oligosaccharides
are largely responsible for when and where oligosaccharides are active in the
plant tissue (Albertsheim et al., 1994). Chitinases might therefore be a part
of such a regulatory mechanism involving production and degradation of
LCOs.
AGPs are a heterogeneous group of structurally complex macromolecules
composed of a polypeptide and a large branched glycan chain (Majewska-
Sawka and Notnagel 2000). Some AGPs also have a lipid chain. AGPs
isolated from seeds of Norway spruce promote formation of more developed
somatic embryos in Norway spruce (Egertsdotter and von Arnold, 1995).
LCOs are a class of signalling molecules that promote division of plant
cells. Nod factors, LCOs, produced by different Rhizobium species
uniformly consist of an oligosaccharide backbone of 1,4-linked N-acetyl-Dglucosamine
residues varying in length between 3 and 5 sugar units, and
always carry an N-acyl chain at the non-reducing terminus. This basic
structure is essential for the infection leading to formation of nitrogen-fixing
nodules. At the same time, several lines of evidence suggest the involvement
of LCOs in regulating somatic embryo development. Extracts of media
conditioned by embryogenic cultures stimulate development of PEMs in
auxin-deficient media in Norway spruce. Partial characterisation of the
conditioning factor has shown that it is a lipophilic, low molecular weight
molecule, which is sensitive to chitinase and contains GlcNAc residues
(Dyachok et al., 2002). Our conclusion is that the conditioning factor is a
LCO. The amount of LCO correlates to the developmental stages of PEMs
and somatic embryos, with the highest level in media conditioned by
developmentally blocked cultures. LCOs are not present in non-embryogenic