Liquid Culture Systems for in vitro Plant Propagation

298 M. Vágner et al.
maturation period, during which the cultures are exposed to high exogenous
ABA concentration, is supposed to be crucial for further ‘normal’ seedling
development. No difference in germination frequency was observed in cell
lines with higher embryogenic capacity (Figure 2). Germination frequency
of cell lines with low embryogenic capacity increased. Probably, this
phenomenon could be ascribed to the better quality of somatic embryos
grown on membrane rafts.
Compared to solidified media, routine passage of the cultures grown on
membrane rafts is less time consuming, as the cultures are not transferred to
fresh media individually, but with the whole raft. Moreover, there is
probably a better exchange at the medium – embryogenic tissue interface in
this system, because the cultures could be transferred at slightly longer
subcultivation intervals. The relatively high price of membrane rafts
together with their low durability remained the only drawback of this
system.
The ability of different cell lines to proliferate in liquid medium varied
markedly; a few of them did not change morphological appearance even
after a long cultivation in liquid medium. On the other hand, cultivation of
all embryogenic cell lines in liquid maturation medium resulted in severe
decrease of the number of developed somatic embryos, which, after
desiccation, were able to germinate.
3.2 Pre-maturation (PGR-free) phase
Insertion of a pre-maturation phase (no PGR in the medium) (Bozhkov et
al., 2002) between proliferation (+ 2,4-D, BAP, and kinetin) and maturation
phase (+ ABA) resulted in further improvement of synchronization, the
enhancement of speed of somatic embryo development, and an increase in
the number of developed embryos (Figure 3). This improvement was more
pronounced in cultures grown on membrane rafts compared to solidified
media. We suppose that after pre-maturation phase the endogenous levels of
auxins (2,4-D, IAA) could be lower in cultures grown on rafts due to
facilitated flow from the culture to the liquid medium. In a similar way after
pre-maturation stage, the endogenous levels of cytokinins in embryogenic
tissue grown on membrane rafts were found to be lower compared to
cultures grown on solidified medium (data not shown). The ABA is thus
supplied to the embryogenic cultures with lower levels of endogenous
cytokinins and IAA in the moment of ABA application. These endogenous
hormonal levels (high level of ABA, low levels of auxins and cytokinins)
seem to be beneficial for embryo development.