Liquid Culture Systems for in vitro Plant Propagation

Adaptions of the mineral composition of culture media 495
the cultures). For the experiments, two other mineral media were tested:
half-strength DKW medium and CAM medium (macronutrients adapted
according to the elemental analysis of Cymbidium-, see ‘Results and
Discussion’ section; plus ½ MS micronutrients). All compounds, and readymade
MS and DKW macro- and micronutrients were obtained from Duchefa
(Haarlem, Netherlands).
Gerbera plantlets were grown in glass jars (10 propagules per jar) on 150
ml medium with MS macro- and micronutrients with extra 50 µmol
NaFeEDTA, 3 % (w/v) sucrose, vitamins (300 mg l -1 thiamine HCl, 100 mg
l -1 nicotinic acid, 10 mg l -1 pyridoxine HCl and 1 g l -1 m-inositol), and 12
µmol kinetin, pH 5.8, 0.6 % (w/v) BBL Granulated (Becton Dickinson,
Aalst, Belgium) or Daishin (Brunschwig Chemie, Amsterdam) agar at 25 °C
and 16 h light (30 µmol m -2 s -1 ). Every 4 – 5 weeks, the clusters of plantlets
were weighed, separated into individual plantlets, counted and divided into
size classes (arbitrary units: 1: small, 2: medium and 3: large). Plants of
approximately the same size (mostly 3, occasionally 2) were used for the
next cycle. For experiments, the same composition was used except for the
minerals. The adapted media are presented in the ‘Results and Discussion’
section. For experiments with hydroponic adaptations in Gerbera, the
concentrations of two microelements were changed: extra Cu was added (1.8
µmol), whereas for Mn, a lower concentration was used (10 µmol).
2.3 Elemental analysis of plant tissues and of hydroponic
solutions
The elemental compositions of adult leaves of Gerbera and Cymbidium
are from Bergmann (1992). The concentrations of elements used for
hydroponic cultures are from data and advice from the IKC organisation
(Anonymous, 1999). For selected media and plantlets, elemental analysis
was performed by contracted service laboratories.
3. Results and discussion
3.1 Formulation of media
For the formulation of the adapted media, some initial choices had to be
made. First, the type of tissue used ‘for reference’ had to be decided upon:
whether for the whole plant or just a specific organ (e.g., leaf, embryo, root),
the age, the growing nutrient conditions of the reference-plants and their
source (field, greenhouse or tissue culture). We used the analyses of youngadult
leaves of healthy growing plants as reference material. This is the same