Liquid Culture Systems for in vitro Plant Propagation

572 K.Bru�áková et al.
been in culture for more than 20 months. The amount of important
paclitaxel precursors 10-deacetyl baccatin III and cephalomannine was also
investigated during the same subculture (Table 1). The amount of paclitaxel
and 10-deacetylbaccatin III peaked after approximately 62 days of culture
and cephalomannine after 52 days of culture.
The study on the growth kinetics and taxane production indicated an
inverse relationship between growth and taxane accumulation (Sakuta and
Komamine, 1987). The pattern of accumulation of all taxanes was similar
and mostly had two peaks (Figure 2). The first one appeared in the
exponential growth phase of the callus and the second one was typical for
the phase of progressive deceleration or stationary phase. As mentioned by
Fett-Neto and DiCosmo (1997), the early release of paclitaxel into the
medium of cell suspensions of T. cuspidata could be a response to the
transfer into fresh medium due to absorption of ammonium and a second
peak occurring at a stationary phase could result from excretion by the cells
that reached their highest level of paclitaxel.
Our observations are consistent with the suggestion that in comparison
with the younger pale-yellow callus containing most mitotically active cells,
the older brown callus with more non-dividing cells contains a higher
amount of paclitaxel (Wickremesinhe and Arteca, 1993). As mentioned by
these authors the selection for friable and faster-growing callus may lead to
the selection of cells that produce lower amounts of secondary metabolites.
Table 1: Taxane yield from two different in vitro cultures of T. baccata L.
Callus Culture
Paclitaxel
(% E.D.W.)
10-Deacetyl
baccatin III
(% E.D.W.)
Cephalomannine
(% E.D.W.)
V/Kle 0.00006 ± 0.00003 — —
VI/Ha 0.0109 ± 0.0037 0.0011 ± 0.0002 0.0059 ± 0.0013
4. Conclusion
Stable-growing seedling-derived callus and suspension cultures were
established using callus-induction Gamborg’s B5 medium. We have
demonstrated the ability to produce paclitaxel and its analogues from two
stable-growing callus lines with different growth characteristics. The amount
of paclitaxel was comparable to that found in the bark of the intact plant of