Liquid Culture Systems for in vitro Plant Propagation

296 M. Vágner et al.
1995). In liquid proliferation medium, cultures usually grow even more
rapidly than on agar-solidified medium. Maturation of somatic embryos is
considered as the more problematic step. The ability to produce mature
somatic embryos in liquid medium differs widely across species and even
across cell lines. On the other hand, the use of somatic embryogenesis as a
large-scale micropropagation tool in commercial applications depends
mainly on reduction of labour costs through the automation of the process.
Automated techniques clearly demand protocols based on liquid media
(Paques et al., 1992).
There are a few examples of successful use of liquid cultures for somatic
embryo production of Norway spruce (Paques et al., 1992, Gorbatenko and
Hakman, 2001). The use of liquid medium is usually limited to the
proliferation phase, maturation cannot be easily achieved in the liquid
medium (Paques et al., 1995). Many trials thus have been conducted to
combine proliferation in liquid medium with maturation on a solidified one.
Although the production of mature embryos and emblings was successful in
a few cases, these protocols are rather complicated to be automated.
Successful use of the polypropylene membrane rafts (LifeRafts,
Osmotec, Israel) was published for a number of tissue cultures (Luckett et
al., 1991, Watad et al., 1995, Paek et al., 2001). Cultivation on membrane
rafts floating on liquid medium could have a great potential to improve the
development of coniferous somatic embryos. Beside the numerous problems
with the cultivation of Norway spruce embryogenic cultures in liquid media,
long vacuolated suspensor cells of embryogenic cultures are sensitive to the
damage made by forceps during transfer to fresh medium. Therefore, as
entire rafts with cultures are transferred to fresh liquid medium,
embryogenic cultures are not touched from the proliferation stage untill the
stage of mature embryos. Moreover, membrane rafts could combine the
advantages both of solidified and liquid media: the cultures are sufficiently
aerated, the exchange of compounds on the tissue-medium interface is
enhanced, and medium can be rapidly replenished or replaced with minimal
disturbance of embryogenic tissue.
Firstly, this work focused on the possibility of the use of polypropylene
membrane rafts for the cultivation of Norway spruce embryogenic cultures
and the production of mature somatic embryos. Different cultivation
techniques were compared. The cultures grown on membrane rafts were
further used for the evaluation of benefits and drawbacks of the insertion of
a pre-maturation phase on PGR-free medium. Finally, we studied the effects
of PEG in maturation medium on the embryo yield, quality and germination
frequency.