Liquid Culture Systems for in vitro Plant Propagation

Propagation of Norway Spruce 287
successive growth periods. In both cases longer treatments exerted negative
effects (Högberg et al., 2001). The maturation step can be shortened (not
exceeding 5 weeks) and synchronised by giving the cultures a prematuration
treatment in growth regulator-free medium. The period of
continuous growth during the first growth period can be shortened by a twophase
germination treatment, first on solidified medium and then in liquid
medium. Another advantage of the two-phase germination treatment is a
better developed root system possessing lateral roots. Somatic embryo plants
produced according to this method can be transferred directly to the
greenhouse (Högberg et al., 2001).
4. Programmed cell death during somatic embryogenesis
Two successive waves of programmed cell death (PCD) occur during
formation and development of somatic embryos of Norway spruce (Filonova
et al., 2000b). The first wave of PCD is responsible for the degradation of
PEMs when they give rise to somatic embryos. The second wave of PCD
eliminates terminally differentiated embryo-suspensor cells at the end of
early embryogeny. During dismantling phase of PCD, PEMs and embryosuspensor
cells exhibit progressive autophagy, resulting in the formation of a
large central vacuole. Autolytic degradation of the cytoplasm is
accompanied by lobing and budding-like segmentation of the nucleus.
Nuclear DNA undergoes fragmentation into both large fragments of about
50 kb and multiples of approximately 180 bp. The tonoplast rupture is
delayed until lysis of the cytoplasm and organelles, including the nucleus, is
almost complete. The protoplasm then disappears, leaving a cellular corpse
represented by only the cell wall.
Programmed cell death (PCD) is an important component of PEM-toembryo
transition in Norway spruce (Filonova et al., 2000b). Triggered by
withdrawal of PGRs, somatic embryo formation is accompanied by massive
cell death in PEMs. Furthermore, strong positive correlation has been shown
between the frequency of somatic embryo formation and the percentage of
PEM cells fragmenting DNA suggesting that PCD in PEMs and somatic
embryo formation are closely interlinked processes both stimulated upon
withdrawal or partial depletion of PGRs (Filonova et al., 2000b). This type
of PCD is also accompanied by a marked decrease in extracellular pH
(Bozhkov et al., 2002). If extracellular acidification is artificially abolished
by buffering PGR-free medium, PEM-to-embryo transition together with
concomitant PCD is inhibited. Our results point to a rigid pH-control in
developmental PCD associated with plant embryogenesis.