Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
417
1
Plant Material Selection and Preparation
1.1
Type and Source of Explant
The procedures described here were estab-
lished primarily from results obtained with
Taxus cuspidata
(Japanese yew), although
some studies were performed using
Taxus
canadensis
and
Taxus brevifolia
. In general
lines, variations of these procedures have
been successfully applied to a number of
other
Taxus
species. In the recent literature
of taxane production by yew cell culture,
preference has been given to
T. chinensis
,
T. media
,
T. cuspidata
,and
T. canadensis
.
There
spon
seo
fyewt
i
s
sueto
in vitro
cu
l
tu
r
ei
sd
ep
end
en
tonth
et
yp
eo
fe
x
-
plant used to initiate the culture, on the
physiological status of the donor plant,
and on its genetic makeup. Explants that
contain meristematic or parenchymal tis-
sue are preferable. These are the types of
tissue most likely to display cell prolifera-
tion in response to the growth regulators
in the culture medium, and this ten-
dency is attributed to these tissues’ low
degree of differentiation, which seems to
be associated with a certain developmental
flexibility in the presence of the appropri-
ate stimuli. Relatively more differentiated
tissues, such as sclerenchyma or xylem, are
less responsive to exogenous plant growth
regulators, possibly because of the genetic
commitment to a specialized developmen-
tal pathway.
Young stem segments (flexible, but bear-
ing dark green needles) are ideal for rapid
initiation of callus to be used in developing
cell cultures. Seed contents (megagame-
tophyte and embryo) are better suited for
establishing long-term maintenance of cal-
lus cultures of
Taxus brevifolia
. Needles
are also a suitable explant type for callus
initiation, although initial growth rates
tend to decrease more rapidly than those of
young stem segments. Arils (fleshy basal
covering of the seed) display different re-
sponses to
in vitro
culture depending on
their stage of development. Green arils
grow relatively fast for the Frst two weeks,
but from then on, the growth seems to
stop. Calli derived from green arils are rel-
atively hard and not friable. Mature red
arils, not surprisingly, fail to grow in cul-
ture, possibly as a result of the advanced
degree of cell lysis at this later stage of
their development (Table 1).
The physiological condition of the donor
p
l
a
n
tm
u
s
tb
es
t
a
b
l
ea
n
df
a
v
o
r
a
b
l
et
o
growth.
Outdoor-grown
plants
display
higher contamination rates and tissue pig-
mentation when cultures are started in
the winter months in Canada (Novem-
ber to March) relative to those started
in other seasons. Genetic differences be-
tween plants of the same species are also
factors to be considered; it may be nec-
essary to test different donor plants to
develop
in vitro
cultures. Donor plants
of
the
same
species,
although
grown
under similar environmental conditions,
may display signiFcant differences in the
Tab. 1
Effect of different explant sources of
T. cuspidata
cultivated in B5 medium
(containing 1 mg L
1
2,4-D) on callus formation
and growth.
Explant
Growth
a
Friability
Green aril
+
No
Red aril
Seed content
++
Yes
Young stem
b
+++
Yes
Needle
b
++
Yes
a
+
, directly proportional to growth;
, poor
growth.
b
Also tested with
T. Canadensis
.
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