418
Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
amount of growth
in vitro
as a result of
genetic variability. It may also be beneF-
cial to begin
in vitro
culture using plants
shown to produce high yields of Taxol
.
High taxane yields
in vitro
, however, are
not always warranted by this phenotype in
explant donor plants.
1.2
Sterilization Procedure
Sterilization is accomplished using stan-
dard plant tissue culture techniques. The
tissue is washed with distilled water to re-
move dust and solid particles, immersed
in 70% ethanol for one minute, immersed
with a few drops of Tween 20 (surfactant
agent) in NaClO (1.5% v/v) for 30 minutes,
and rinsed three times with sterile distilled
and deionized water. During the steriliza-
tion period, the tissue must be kept fully
immersed and under agitation. Steriliza-
tion times may have to be reduced for
more sensitive and tender tissues. Our ex-
perience has yielded typical contamination
rates using outdoor-grown donor plants
that are less than 3% of the total number of
explants used. In winter, however, the in-
cidence of contamination can be 10 times
the regular rate. In certain cases, a par-
ticular plant may be extensively infested
with endophytes that are not destroyed
by surface-sterilization procedures; in this
situation, contamination can be very high
and a change of donor plant is advisable.
1.3
Explant Size and Final Preparation
±ollowing sterilization, the exposed cut
por
t
ionso
fthet
issuetha
tweredamaged
by
the
ethanol
and
NaClO
solutions
are excised, and the healthy tissue is
appropriately trimmed and placed in the
growth
medium.
Excision
of
explants
should be made smoothly, with a relatively
new blade, to minimize the production
of phenolics in the cut areas. In the
case of stem segments, approximately
1-cm long segments give good results.
Stem segments can be cultured at an
approximate angle of 45
to the agar
surface,
with
one
of
the
transversely
sectioned ends immersed in the medium
and
the
other
partly
exposed
to
the
air of the culture vessel. Alternatively,
longitudinally cut stem segments can be
cultured with their cut portions facing
the surface of the medium. The contents
of
longitudinally
halved seeds can be
placed on the medium after separation
from
the
seed
coat
with
a
spatula.
Isolated embryos can be used instead,
in order to avoid calli derived from the
haploid megagametophyte tissue; haploid
tissues, on the other hand, may be useful
for
mutant
generation
and
screening.
Hypocotyl segments or other portions of
aseptically germinated seedlings are also
good sources of explants. Yew tissues
seem to be very sensitive to heat, relative
to
tissues
of
other
woody plants.
All
tools used in explant preparation and cell
transfer should be sterilized in ethanol,
flamed briefly to remove ethanol residue,
and cooled to room temperature before
touching the plant material.
2
Establishment of Callus and Cell Cultures
2.1
General Procedure
Cultures of yew are relatively difFcult to
establish because growth rates are slow
and the explants produce and release
phenolics; in turn, the oxidation of the
phenolics
leads
to
darkening,
growth
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