Liquid Culture Systems for in vitro Plant Propagation

Experimental use of a novel temporary immersion system 265
agar addition and autoclaving. Cultures were kept at 22±2 o C under a 16-h
photoperiod of cool white fluorescent light (40 µmol m -2 s -1 ) and subcultured
every 8 weeks (Grigoriadou et al., 2002).
2.2 Effect of temperature
A preliminary experiment on agar-solidified medium to estimate a
favourable temperature for microshoot development was carried out. For this
purpose the effect of high (28±2 o C) and low (22±2 o C) temperatures was
studied in test tubes (100 x 20 mm) containing 10 ml of the medium
mentioned above. All other conditions remained the same. The culture
continued till all plant material had senesced (105 days). Every 15 days the
number of new microshoots and their length were recorded. At the end of the
culture period the number of nodes per microshoot and internode length
were also determined.
2.3 Liquid medium culture
The effect of liquid medium on olive microshoot formation using several
systems designed for this purpose was examined. The devices compared
were:
– LifeReactor © , the bioreactor system produced by Osmotek Ltd, Israel.
Disposable plastic vessel of 1.5-litre working volume filled with 1-litre of
medium was used (Ziv et al., 1998) (Figure 3a).
– Erlenmeyer flasks of 100 ml, filled with 30 ml of liquid medium, shaken
on a rotary shaker under 100 rpm (Figure 4a).
– Test tubes equipped with filter paper bridges, containing 10 ml of liquid
medium (Figure 4c).
– A novel TIS (Figures 5a and 5b). Immersion period was 15 min every 8
hours (Escalona et al., 1999; Damiano et al., 2000; Welander et al.,
2001). Three periodical sequences of culture were examined: a) 10 days
in TIS followed by 20 days in test tubes containing 10 ml of agar
solidified medium, b) 20 days in TIS followed by 10 days in agar
solidified medium and c) 30 days continuous culture in TIS.
– Cultures of explants in test tubes in agar solidified medium (0.5% w/v)
were used as control.
Cultures were kept at 28±2 o C, which proved to be an adequate temperature
for in vitro olive microshoot growth. The experiments lasted for 30 days. At
the end of the culture period the number of new microshoots per explant and
the shoot length were measured, while the percentage of buds that developed
into microshoots was determined.