Liquid Culture Systems for in vitro Plant Propagation

240 Tino Hempfling & Walter Preil
4.1 Effect of medium substitution every two or four weeks
The release of phenolics by Phalaenopsis cultured in vitro is a common
phenomenon. The accumulation depends on genotype and biomass increase
in the culture vessel. The addition of activated charcoal or various
antioxidants has been tested to overcome accumulation of phenolics in
orchid cultures (for references see Arditti and Ernst, 1993). Park et al. (2000)
reported that filtering of the liquid medium in TIS bioreactor strongly
enhanced PLB growth.
Medium substitution every two weeks in TIS cultures of Phalaenopsis
cv. Jaunina resulted in shoot multiplication rates of 25.4, i.e. 70 shoots used
as inoculum gave rise to 1,780 adventitious shoots after twelve weeks.
Medium substitution at four-week intervals decreased the multiplication rate
to 14.5, i.e. only 1,015 shoots were harvested (= 57 % of two-week interval
experiment).
Surprisingly, the fresh weight increase was less affected by accumulated
phenolics than the shoot multiplication rate. Starting with 20 g inoculum,
medium substitution at two-week intervals resulted in 20.5-fold increase of
fresh weigth (= 410 g biomass) and at four-week intervals gave rise to 18.5
fold increase (370 g biomass; = 90 % of two-week interval experiment).
Therefore, phenolic compounds more strongly inhibit initiation of
adventitious shoots than biomass production.
4.2 Effect of TDZ
Thidiazuron (TDZ), a cytokinin-like compound, was originally registered
as a cotton defoliant (Arndt et al., 1976). Later TDZ was used for in vitro
culture of recalcitrant species. Many of them are woody plants (Huetteman
and Preece, 1993). Ernst (1994) described that Phalaenopsis flower stem
sections developed multiple shoots on TDZ-containing medium, and with
higher levels also PLBs were initiated. Protocorm proliferation increased
with increasing TDZ-concentration in the range of 0.23 – 1.14 µmol.
Highest efficiency in adventitious bud induction was in the range of 5-10
µmol TDZ (Chen and Piluek, 1995). Protocorm-like bodies were formed in
Phalaenopsis callus cultures when the medium was supplemented with
0.45–4.52 µmol TDZ (0.1 – 1.0 mg l -1 TDZ) (Chen et al., 2000).
Twelve-week TIS-culture on 0.5 mg l -1 (2.27 µmol) TDZ-containing
medium resulted in doubling of shoot multiplication rate to 25.4 (Figure 2)
compared with cultures grown for seven weeks on TDZ-medium and
afterwards five weeks on TDZ-free medium, giving rise to a multiplication
rate of 12.0.