Liquid Culture Systems for in vitro Plant Propagation

570 K.Bru�áková et al.
3. Results and Discussion
All parts of young seedlings were more responsive to cell proliferation on
callus-induction media as compared with young stems of adult trees.
Following the first subculture all calli grew slowly. After 1-2 years of
culture some of them improved their growth. Two callus cultures (labelled
V/Kle and VI/Ha) were selected for subsequent study.
3.1 Growth characteristics
The fast-growing white-coloured and friable V/Kle callus culture was
initiated from cotyledons of embryo-derived seedlings. After 42 days of
culture, GI was 7.96�0.49 (n=9; the 24 th subculture) with the doubling time
estimated in the range of 12-14 days (Figure 2). The majority of callus
samples originating from white-coloured calli released a red-coloured
compound into the solid or liquid medium, grew at a slow rate and did not
survive in subsequent subcultures. According to Wickremesinhe and Arteca
(1994) the production of these red-coloured exudates – possibly phenolics –
is a totally random and unpredictable process, which eventually leads to cell
death. V/Kle suspension culture was pale yellow with the presence of
individual oval- or oblong-shaped cells and cell aggregates ranging between
0.5-2.0 mm in diameter. Methyl-blue staining revealed the high viability of
cells with relatively large vacuoles and a thin layer of cytoplasm.
The slowly growing, light-brown and more aggregated VI/Ha callus
culture was initiated from hypocotyls of embryo-derived seedling. Unlike
V/Kle, callus exudates, no red-coloured compound was produced in the
presence of a phenolic-binding compound (PVP). GI was 3.45�0.21 (n=9;
the 23 rd subculture) (Figure 2). The relatively slow growth rate and the
doubling time in the range of 14-16 days are comparable with other slowgrowing
woody species (Fett-Neto and DiCosmo, 1997).
3.2 Taxane yield
The concentrations of paclitaxel in callus cells reached the maximum of
0.0109 ± 0.0037 % of the extracted dry weight in slow-growing VI/Ha callus
culture. This amount of paclitaxel is comparable to that found in the bark of
the intact plant of various yew species (Fett-Neto et al., 1992; Vance et al.,
1994; Jaziri et al., 1991). The highest value obtained from V/Kle callus
selected for faster growth represented a paclitaxel yield only of
0.00006 ± 0.00003 % of the extracted dry weight. Both callus cultures have