Liquid Culture Systems for in vitro Plant Propagation

276 Ch. Wawrosch et al.
possesses strong immunoregulatory activities (Terness et al., 2001). For
various reasons large scale collection of wild-growing plants can not be
recommended and field cultivation would therefore be the preferred way for
the production of crude drug for further processing. However, the vegetative
multiplication by bulb offsets is very low (Van Horn and Domingo, 1950).
In vitro propagation of Charybdis sp. has been performed e.g. through
bulblet induction (El Grari and Backhaus, 1987) or by adventitious shoot
formation (Stojakowska, 1993).
Nodules, cell aggregates with a high regenerative potential, have been
first described for poplar by McCown et al. (1988) and have since then been
reported for a few other plants, too. Micropropagation through nodule
culture is also an interesting alternative for Charybdis sp., although the use
of liquid medium, as one of the advantages of nodule culture, is restricted
due to hyperhydration (Wawrosch et al., 2001). In the present
communication the results obtained on semisolid and in submerged liquid
culture are compared to those achieved in temporary immersion systems.
2. Materials and methods
Shoot cultures were established from adult bulbs and the formation of
meristematic nodules was induced on leaf cuttings (Wawrosch et al., 2001).
Nodules of the clone UN26 (Charybdis numidica) were used for the
experiments (Figure 1A). Nodules were multiplied in liquid MS basal
medium (Murashige and Skoog, 1962) with 3 % sucrose and 20 µmol BA
(50 ml of medium in 250 ml Erlenmeyer flasks) in the dark. Shoot
regeneration was studied in the following three systems:
As semisolid medium (AM) we used MS basal medium with 0.8 % agar
(Merck) and 1 % sucrose. 40 ml of medium were dispensed in baby food jars
(9.5 cm height and 6 cm diameter) closed with Magenta B-caps ® . The nodule
inoculum weight was approximatily 1 g. Submerged liquid culture (LM) was
carried out in 250 ml Erlenmeyer flasks containing 50 ml of MS basal
medium with 1 % sucrose. The bottles were closed with cellulose plugs, plug
and bottleneck were wrapped with aluminium foil. Flasks were inoculated
with ca. 2 g of nodules and kept on a gyratory shaker at 80 rpm. The
temporary immersion (TI) system used in our studies was based on the
simple device described by Damiano et al. (2001). Basically, two 1000 ml
Schott Duran � bottles were connected through glass and silicone tubings.
One bottle contained 500 ml of the nutrient medium (MS with 1 % sucrose),
the other one approximately 10 g of nodules on top of a layer of glass beads.
The medium was moved periodically by applying compressed air, which was
sterilized through a 0.2 µm filter. Two of these units were operated serially