Liquid Culture Systems for in vitro Plant Propagation

Shoot regeneration from nodules of Charybdis 277
(Figure 1). In the first setup (TI 1) the time of immersion was 5 min every 24
hours while in the second setup (TI 2) the frequency of immersion was
doubled, i.e. 5 min every 12 hours. During the 5 min immersion a constant
air flow was kept in order to remove gaseous metabolites and to supply fresh
air (Damiano et al., 2001). The cultures were kept at 25±1 °C under
continuous light of 60 µmol m -2 s -1 (cool white fluorescent tubes) for 4 (LM,
TI 1, TI 2) and 8 weeks (AM), respectively. Subsequently, the number of
regenerated shoots was counted on every single nodule, differing between
shoots (length > 2 mm) and buds. Hyperhydration was rated with a score of
0 (no hyperhydration) to 3 (all shoots hyperhydrated). Statistical analysis
(ANOVA followed by Duncan´s multiple range test) was performed using
the software Statistica ® v5.0 by StatSoft, Inc.
Figure 1: Shoot regeneration from nodules of Charybdis numidica clone UN26. (A) Typical
nodules prior to inoculation in regeneration systems (bar=10 mm). (B) Shoot regeneration on
MS medium after 8 weeks of culture under continuous light (bar=10 mm). (C) Hyperhydrated
shoots regenerated in submerged culture in MS medium after 4 weeks under continuous light
(bar=10 mm). (D) Two serially coupled temporary immersion systems after Damiano et al.
(2001) (bar=5 cm). (E) Mostly normal shoots regenerated after 4 weeks with 5 min immersion
with MS medium every 24 hours (bar=10 mm). (F) Hyperhydrated shoots regenerated after 4
weeks with 5 min immersion with MS medium every 12 hours (bar=10 mm).