Liquid Culture Systems for in vitro Plant Propagation

530 Dirk Wilken et al.
described a retention time of 3.8 min (�-citral) and 4.2 min (�-citral) was
observed.
2.5.5 Sample preparation and determination of oleanolic acid
Samples of approximately 190-210 mg of dry material were extracted
with 90 ml dichloromethane in a Soxhlet apparatus for 30 min. The organic
phase was filtered and taken to dryness under reduced pressure. Percentage
recovery was estimated by spiking an inert support with a known amount of
oleanolic acid. The oleanolic acid content of the samples was determined by
HPLC using calibration curves as reported by Halkes (1998). A Merck-
Hitachi (Darmstadt, F.R.G.) HPLC equipment with UV detector (Model
Series L-4000, Column: LiChrocart 5 �m RP 18 Select B, 250 mm) and a
data processor were used. The mobile phase consisted of acetonitrile, water
and acetic acid (70:30:0.5; v/v), the flow rate was 1.0 ml min -1 . The signal
was detected at 220 nm. The identity of oleanolic acid was checked by coinjection
of a reference sample isolated from Fabiana. Under the working
conditions described, the retention time for oleanolic acid was 14.1 – 14.5
min.
3. Results
The multiplication rates after four weeks for shoot and callus cultures in
microcontainers as well as for shoots in TIS are given in table 1. Although
the increase of biomass differed considerably between the four species
investigated, shoot growth was always highest in the TIS compared to
culture in microcontainers.
Multiplication rates of the callus cultures ranged from 3.1 within four
weeks for Cymbopogon to 8.6 for Lavandula (Table 1). These callus cultures
were used to establish cell suspensions which in turn were used as inoculum
for Lavandula and Hypericum bioreactor cultures. The growth curves are
given in figure 1.
Contents of bioactive compounds for all in vitro cultures in comparison
with plants grown in the field are shown in table 2. For Lavandula the
content of rosmarinic acid was higher for shoots and cells grown in any
tissue culture system compared to field grown plants (containing 4.8 mg
rosmarinic acid per g dry weight). The highest concentration of rosmarinic
acid (108 mg g -1 dry weight) was found in callus cultures. For Lavandula
also the composition of the essential oil from field grown as well as from in
vitro grown shoots was investigated (Figure 2). This analysis revealed that