Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
419
inhibition, and eventual loss of viability
ofthetissue.Theinclusioninthemedium
of phenolic-binding compounds, such as
polyvinylpolypyrrolidone (PVP), improves
the growth of the explants. The inclusion
of activated charcoal in the media can
also reduce darkening, although it may
also diminish explant growth as a result
of binding of medium components. In
addition, transfers to fresh medium (or
rotation of the explants to new positions in
the culture vessel containing portions of
unused medium) at approximately 15-day
intervals help prevent growth inhibition
due to phenolic oxidation.
More frequent transfers may be required
in the early period of cultivation or when
the donor plant is more prone to dark-
ening upon
in vitro
culture. After several
subcultures and growth stabilization of the
cultures, the need for phenolic-binding ad-
juvants may decrease or even disappear.
Following an initial screening and eval-
uation of some standard medium formu-
lations, the basic medium B5bPVP was
established for callus initiation in yew; it
consists of B5 medium salts and organic
supplements, 3.0% (w/v) sucrose, 1.5%
(w/v) insoluble PVP, 0.8% (w/v) agar, and
1mgL
1
of
2,4-dichlorophenoxyacetic
acid (2,4-D). The media are prepared with
distilled and deionized water, plant cell cul-
ture or reagent-grade chemicals, adjusted
to pH 5.8 with NaOH and HCl (after the
addition of PVP and prior to the addition
of agar) and autoclaved (1.1 kg cm
2
at
121
C for 20 min).
Preferably, the glassware used for plant
cell culture should be washed with neutral
detergent,
abundantly
rinsed
with
tap
water, and then rinsed with distilled water.
The cultures can be grown in plastic
petri dishes (9 cm in diameter) containing
30 mL
of
medium
(3–4
explants
per
plate)
and
sealed
with
para±lm
or
air permeable tape, or in 30 mL glass
vials with polypropylene or aluminum
foil lids containing 10 mL of medium
each (1 explant per vial). The cultures
are incubated at 25
Cind
a
rk
.V
a
r
iou
s
other media formulations based on MS,
WPM, SH and B5 salts and organics
supplemented with auxins and cytokinins
have been effectively used for several
Taxus
species.
2.2
Medium Optimization
Additional improvements on the basic
medium formulation for yew tissue culture
can
be
accomplished
by
investigating
several medium components (e.g. plant
growth regulator balance, salt and organic
supplement composition) that promote
the growth of related species, or seem
likely to have a stimulatory effect on the
putative biosynthetic pathway to Taxol
.
The effect of the medium components
when included in various amounts in the
basic formulation, or when replacing some
of its original constituents, can be analyzed
relative to growth and Taxol
yield in
developing yew calli. Medium factors with
a positive effect on growth and Taxol
yield are combined in new formulations to
investigate the possibility of synergistic or
antagonistic interactions.
A great deal of work is entailed in
the high number of treatments and re-
spective replicates needed for accurate
statistical analysis, but these comprehen-
sive experiments allow the identi±cation
of limiting factors for growth and Taxol
production. The overall analysis of the re-
sults also shows the relationship between
growth and Taxol
production. From such
a series of experiments, two improved me-
dia formulations were devised: B5bPhe
(B5bPVP with 0.1 mM of phenylalanine)
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