Liquid Culture Systems for in vitro Plant Propagation

286 Sara von Arnold et al.
and cytokinin permits both multiplication by new PEM I formation and
successive growth to more advanced levels (PEMI to PEMII and PEMII to
PEMIII). In contrast, the predominant response of PEMs plated at low
density in well aerated liquid medium with high levels of auxin and
cytokinin is unequal division of embryogenic cells with dense cytoplasm
leading to the restart of the process from the PEMI level. When auxin and
cytokinin are being gradually depleted, the average level of the whole
system is biased forward to PEMII or PEMIII levels. This occurs towards
the end of the liquid culture passage.
Comprising a link between the unorganised proliferation phase (i.e.
PEMs) and highly organised embryonic development phase, and at the same
time holding them apart, PEM-to-embryo transition plays a pivotal role in
Norway spruce somatic embryogenesis (Filonova et al., 2000a) just as it
does during somatic embryogenesis of angiosperms. It seems likely that the
inability of many embryogenic cell lines to form well-developed
cotyledonary embryos is in large part associated with disturbed or arrested
PEM-to-embryo transition that might be a consequence of inappropriate
culture conditions.
Developmental dynamics experiments performed with whole suspension
cultures have strengthened the significance of PEM-to-embryo transition
(Bozhkov et al., 2002). Three major points to consider for efficient
regulation of somatic embryogenesis were identified. First, PEM-to-embryo
transition occurs within a short time after withdrawal of PGRs. The time for
this process can probably vary for different cell lines but usually the switch
from proliferation to development occurs after about 24 hours. Second, ABA
is incapable of inducing PEM-to-embryo transition. This may be responsible
for the failure to stop proliferation once embryogenic cultures are directly
transferred from medium containing auxin and/or cytokinin to ABAcontaining
medium. Third, since newly formed somatic embryos could
develop in PGR-free medium for at least 7 days and thereafter retain the
ability to respond to ABA with continuing development until the
cotyledonary stage, it is evident that early somatic embryogeny does not
require exogenous ABA which, however, is important for promoting late
embryogeny. This provides the possibilities to avoid prolonged contact with
ABA during somatic embryo maturation, which otherwise inhibits the
growth of somatic embryo plants (Bozhkov and von Arnold, 1998; Högberg
et al., 2001). Taken together, these three points have significantly improved
the biotechnology of somatic embryogenesis in terms of yield and quality of
somatic embryos.
Ex vitro growth of somatic embryo plants is under a cumulative influence
of a number of previously applied treatments. The time of contact with ABA
during somatic embryo maturation and the duration of continuous growth
during the first growth period strongly affect the height growth during two