ELSEVIER SCIENTIFIC PUBLISHERS IRELAND Plant Science 92 (1993) 1-12

plan ience

Taxol formation in y e w - Taxus

Gary A. Strobel a, Andrea Stierle a, W.M. Hess b

aDepartment of Plant Pathology Montana State University, Bozeman, MT 59717, USA bDepartment of Botany and Range Science, Brigham Young University, Provo, UT 84601, USA

(Received 5 February 1993; revision received 9 April 1993; accepted 12 April 1993)

Abstract

In Pacific yew, Taxus brevifolia, taxol is found most abundantly in the vascular cambial region followed by the phloem, sapwood and heartwood. It is in this order that 'in vitro' taxol biosynthetic activity was also demonstrated using [l-14C]acetate as a precursor. However, only traces of taxol biosynthesis could be demonstrated in the xylem (sapwood-heartwood) suggesting that taxol may be mobilized from its place of greatest biosynthetic activity (vascular cambial region) to the xylem; perhaps via ray parenchyma. Taxol could also be effectively trapped under 'bark flaps', over a period of several weeks, using silica gel powder as an adsorbant. In vitro taxol biosynthesis using [l- taClacetate in outer bark samples (cambium-phloem) could only be demonstrated in Taxus brevifolia, Taxusfloridana and Taxus canadensis, among 10 of the 11 native Taxus species tested.

Key words: [taC]Taxol; Xylem; Phloem; Cambial area; Cancer; Taxus spp.

1. Introduction

The ancient Greeks named the yew Toxus after two important aspects of this tree: Taxon = bow and toxikon = poison [1]. The Irish Druids, also held the tree in high regard because it was believed to be efficacious against fairies and witches in magical ceremonies [1]. For modern man, the yew has once again taken on special importance. It appears to be the sole source of the extremely important anticancer drug - - taxol [2,3]. This complex diterpenoid, and its related taxanes (Fig. 1), are found in the bark and/or leaves of all 11 of

* Corresponding author.

the native species of Taxus (Dr. Kenneth Snader, National Cancer Institute, personal communica- tion). However, currently the most important source of taxol is the bark (phloem and cambium) of the mature Pacific yew, Taxus brevifolia, which is harvested in the Pacific Northwest [4].

In order to study the influence of various factors such as inhibitors, growth regulators, microbes and environmental changes on taxol biosynthesis, we devised an in vitro bioassay system for taxol production [5]. The system employs incubation of a solution of dithiothreitol with small pieces of plant material and [1-14C]acetate'or other appro- priate precursors such as [UL-14C]phenylalanine followed by extraction and chromatographic sepa-

0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0168-9452(93)03647-E

G.A. Strobe/et al./Plant Sol 92 (1993) 1-12

0

" ~ 0 0 OH

o

O ~ 0-" ~

Fig. I. The structure of taxol.

ration of [14C]taxol [5]. Using this system, we have examined those specific tissues in the Pacific yew in which [l-14C]acetate serves as a precursor to [lac]taxol. In particular, this study reports the presence of taxol in the xylem (sapwood and heart- wood) of Pacific yew and then focuses on which stem tissues carry out taxol biosynthesis as measured by the in vitro assay [5]. The in vitro assay has also been used with bark pieces of 10 of the 11 known native species of Taxus in order to determine which of them successfully converts [1- lac]acetate to taxol. Once the tissues that are the most biosynthetically active in taxol production were identified, it was possible to devise an 'open bark flap' technique to trap taxol 'in vivo'.

2. Materials and methods

2.1. Taxus spp. The native species of Taxus were generally sent

to us as stem sections approx. 1-5 cm diam. × 20 cm, length (parafilm sealed ends) from friends, col- leagues and herbaria in various parts of the world (see Acknowledgements). Taxus brevifolia, indige- nous to Montana, was harvested from mature undergrowths in the Flathead National Forest. All specimens were used within 3 weeks of harvest and all could be successfully stored in dark, moist, cool (4°C) storage for several months without loss of biological activity [5].

2.2. T a x o l - isolation from wood (sapwood and heartwood)

The fine sawdust (10 g) (1 × 2 mm pieces) of sapwood (outer 1-2 cm of white xylem tissue) and heartwood of Pacific yew of various ages was ex- haustively extracted in chloroform/methanol 10:1 (v/v), using a fresh weight to solvent volume ratio of 10:1 (g/v) (Fig. 2). After solvent removal, the re- sidue was redissolved in chloroform and applied to a 1 cm × 10 cm column of silica gel (60-200 mesh), rinsed with 20 ml of chloroform followed by 20 ml of acetonitrile. Taxol was isolated from the acetonitrile eluant and subjected to spectral and quantitative measurements.

2.3. Taxol - - chromatographic separation and quantitation

Taxol from plant extracts was chromatographed on prepared Merck silica gel thin layer chroma- tography (TLC) plates (20 cm x 20 cm) either 0.5 or 0.25 mm in thickness, depending upon the sam- ple load. The solvent systems employed were: (A) chloroform/methanol (7:1, v/v); (B) chloroform/ acetonitrile (7:3, v/v); (C) ethylacetate/isopropanol (95:5, v/v); (D) methylene chloride/tetrahydro- furan (6:2, v/v). Reverse phase chromatography was done on Whatman KCI8F plates (0.2 mm) using solvent; (E) hexane/isopropanol/acetone (10:0.5:0.5, by vol.).

Taxol was detected on TLC by its characteristic

G.A. Strobel et al./Plant Sci. 92 (1993) 1-12 3

Fig. 2. Cross-section of a ca. 120 yr old stem of Pacific yew used in the taxol biosynthesis studies. A = phloem (inner bark) tissue, B = cambial tissue, C = xylem-sapwood, D = young heartwood 20-30 yrs. old, E = old heartwood (70-120 yrs. old). The stem is ca.

12 cm top to bottom.

4 G.A. Strohel et al . /Plant Sci, 92 (1993) 1-12

blue color when sprayed with a 1% (w/v) vanillin in conc. sulfuric acid followed by gentle heating [6]. Also, taxol quenches short wavelength UV light (254 nm) [2].

Taxol was quantitated by its mMolar absor- bance of 1.7 (1.0 cm light path) at 273 nm [2]. It was also separated and quantitated by analytical high performance liquid chromatography (HPLC) on phenyl-bonded silica (4.6 mm x 25 cm i.d.) [7].

2.4. In vitro [14C]taxol assay This assay was done precisely as stated in an

earlier study [5]. [1-14C]Acetate (20 #Ci) with a specific activity of 54 mCi/mmol was administered to 0.4 g fresh wt. of stem tissues (small pieces; 2 x 4 ram), under the prescribed conditions for 3 days [5]. The reaction mixture was extracted with an equal volume of chloroform/methanol (10:1, v/v), dried under N 2 and dissolved in 200 ~1 of acetonitrile. An aliquot (25 ~1) was then streaked over 5 cm on a 0.25 mm silica gel TLC plate and chromatography performed in solvent A, using standard taxol as a reference compound. The sol- vent system was allowed to move to within 1 cm of the top of the plate. A narrow band containing taxol was scraped from the plate and eluted with acetonitrile. After solvent evaporation, 12 ml of aquasol was added to the sample vial and the radioactivity determined.

To determine if [t4C]taxol was being produced in the tissues of Pacific yew as well as of the other Taxus spp. studied, the acetonitrile solution (25-200 ~1) was first chromatographed in solvent A. The narrow band corresponding to taxol was eluted, followed by subsequent two dimensional (2-D) chromatography on TLC silica gel in sol- vents C and D. The 2-D step was performed using authentic taxol as an internal standard. The 2-D TLC plate was then overlayed with Kodak X Omat film for 1 week and developed. The TLC plate was subsequently sprayed with the vanillin/sulfuric acid reagent to detect taxol and to allow comparison of the size, shape and location of the spot on the film relative to that on the plate [6]. Cochromatography in all other systems, in- cluding reverse phase TLC and HPLC, provided further evidence for the identity of [14C]taxol. However, in all of the tabular data shown, up to

20% of the radioactivity indicated for taxol are due to other taxanes which have approximately the same RF as taxol in solvent system A, such as cephalomannine and baccatin III [5].

The best evidence for the identity of [14C]taxol in all of the biological systems in this report comes from the cochromatography of [14C]taxol with authentic taxol. More convincingly, however, is the successful cocrystallization of the biologically generated [14C]taxol with 1-2 mg of authentic taxol in 0.5-1.0 ml of methanol. Crystals of taxot formed after the slow addition of an equal volume of H20 at 23°C over 30 min. The taxol was recrystallized to a constant specific radioactivity after at least 3 cycles of recrystallization.

2.5. In vivo taxol trapping Freshly harvested yew logs (approx. 10 cm

diam. x 70 cm in length) used for this portion of the study were cut at 1-2 meters from ground level, which appears to be the location on the stem of the greatest taxol content and synthesizing ac- tivity [5]. A series o f 'ba rk flaps' were cut (6 cm x 6 cm x 6 cm) into each log and peeled back. Then, 2 g of silica gel (60-200 mesh) was placed under each flap and the cut areas sealed with tape to the log surface. The logs were incubated at 6°C for varying times.

The silica gel was carefully removed and was dried over P205, rinsed with chloroform and the taxol was then eluted with 10-20 ml of acetonitrile. Taxol was quantitated by analytical HPLC [7]. In addition, the tip of the bark flap, the central part of the flap, and the uncut bark of the two logs were assayed for in vitro taxol biosynthe- sis using the standard assay at the termination of the 4 week experiment [5].

2.6. Light microscopy Tissues were processed using the procedures

described by Upadhyay et al. [8] which consisted of fixing tissues in 2% (v/v) glutaraldehyde and 3% (v/v) acrolein in 0.1 M sodium cacodylate buffer (pH 7.2-7.4) for 2 h at room temperature. After fixation, samples were washed 6 times with 1:1 (v/v) water cacodylate buffer solution and post fixed and stained with buffered 1% (w/v) osmium tetroxide for 2 h at 0-4°C. The samples were then

G.A. Strobel et al./Plant Sci. 92 (1993) 1-12 5

stained overnight with aqueous 0.5% uranyl ace- tate, dehydrated in a graded series of ethanol and embedded in Spurr's resin. Sections (2-4 #m) were cut with a Sorvall JB-4 microtome and were stain- ed in a solution containing aqueous 1% (w/v) toluidine blue 0, 1% (w/v) azure II, and 1% (w/v) sodium carbonate. Stained samples were washed with distilled water and dried in an oven at 60°C. After the slides were dried, they were placed in xy- lene and coverslips were sealed with Permount. Photographs were taken on Polaroid P/N 55 film with a Zeiss Laser Scanning Microscope (LSM) using argon (488 and 514 nm) and HeNe (633 and 1152 nm) lasers.

2. 7. Spectral determinations Nuclear magnetic resonance (NMR) spec-

troscopy was done with a Bruker AC300 instru- ment in 100% deuterated chloroform. Electrospray mass spectrometry was done with a VG trio-2 quadrapole instrument in methanol/H20/acetic acid (80:19:1, by vol.).

2.8. Radioactivity measurements All samples for radioactivity determinations

were counted in New England Nuclear's 'aquasol' (12 ml). The taxol samples were dissolved in 0.1-1.0 ml of methanol and cooled before count- ing. The pulse-height shift method was used to cor- rect counts/min in samples to dpm.

2.9. Experimental confidence All studies, if possible, were done at least twice

(limited by availability of certain plant materials). It is to be noted that taxol synthesizing ability of tree tissues varies markedly from tree to tree and from one region of the tree to the other [5]. How- ever, where possible we present the results of more than one study or present data with appropriate confidence limits.

3. Results and discussion

3.1. In vitro [14C]taxol assay A question that is central to [14C]taxol biosyn-

thetic studies is the relative abilities of the native Taxus spp. to effectively produce [14C]taxol in the standard [l-14C]acetate assay [5]. For this reason,

we tested pieces of relatively freshly harvested inner bark (phloem and cambial tissues) of 9 of 11 commonly reported wild species of Taxus [9]. In- formation of this type would allow us to do more comprehensive studies on taxol biosynthesis in various tissues subsequently and to begin to understand the mobilization of taxol in the tree. Confirmation of the identity of [14C]taxol from each assay that was positive was obtained by 2-D TLC in solvents C and D (Fig. 3) and by recrystallization to constant specific radioactivity. Traces of at least 3 other 14C-labeled compounds appeared on the X-ray film after 2-D TLC. How- ever, the contribution of these compounds to the total radioactivity in the 'Taxol' sample was <20% [5]. Interestingly, we noted that the T. brevifolia from Montana (Northern Rocky Moun- tains) made [14C]taxol in the [1-14C]acetate assay and the 7". brevifolia from the Pacific Northwest did not (Table 1). Also, only the Taxus species from the relatively narrow geographic locations of Florida (T. floridana), the Northern Rocky Moun- tains (T. brevifolia - - 'biotype'), and Eastern U.S. and Canada (T. canadensis) made [14C]taxol from [1-14C]acetate in our assay system while those from all other regions of the world did not.

We did not have access to T. celebica for our studies. But since this species is an Asiatic yew, and since it might be equivalent to T. mairei, T. chinensis, or T. sumatrana [1] we speculate that it too would not utilize [14C]acetate to produce [14C]taxol. Further studies on the utilization of other taxol precursors [5] by these different Taxus spp. may reveal other biochemical patterns that will prove useful in Taxus spp. classification. Fur- thermore, it is interesting to speculate why only 3 geographically related Taxus spp. could produce [14C]taxol from [l-14C]acetate (Table 1). For in- stance, it may be that there is more than one bio- chemical pathway leading to taxol, or some critical factor is missing in the non-[14C]taxol producers, or that the non-utilizers of [1-~4C]acetate have en- zymes involved in taxol synthesis that are labile in our system? Alternatively, the ecological setting of the tree may influence taxol biosynthesis from [1- ~4C]acetate.

Thus, even though all native Taxus spp. were shown to produce taxol, for this study we focused

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G.A. Strobel et al . /Plant Sci. 92 (1993) 1-12

Table 1 [1-]4C]Acetate incorporation into [14C]taxol 'in vitro' by various yew species

Species of Taxus Total taxol Total 14C in Taxol (/~g) chloroform solubles (dpm)

(dpm)

T. canadensis 75 749 633 76 850 T. brevifolia (Montana) 77 665 400 56 540 T. brevifolia (Pacific Coast) 92 62 250 0 T. floridana 42 779 533 79 390 T. globosa 87 432 033 0 T. baccata (England) 70 1 874 166 0 T. wallichiana 52 369 736 0 T. chinensis 67 69 200 0 7". baccata (Germany) 50 558 533 0 T. cuspidata 65 904 466 0 T. maieri 62 212 325 0 T. sumatrana 75 726 950 0

The standard in vitro assay for taxol biosynthesis from [1- ]4C]acetate was employed for each of the 9 wild species of Taxus [5]. Each assay involved 0.4 g fresh wt. of inner bark material, and a 3-day incubation period.

TAXOL FROM SAPWOOD A

e.o r.s z.o 6'.S S'.0 S'.s s'.o 41S 4:0 3'.s 3'.0 ='.s 2'.0 1.s 1.o o.s PPM

_b

TAXOL (AUTHENTIC)

' e'.0 7'.s r'.0 s'.s S'.0 S'.s s'.o 4'.S 4'.0 £S s'.0 2'.s 21o ~:s i'.0 ols PPM

on T. brevifolia, not only because of its immediate availability to us in Montana, but because of its ability to form [14C]taxol in the in vitro assay [5].

3.2. Taxol from the xylem tissues Inasmuch as specific parenchymatous cells, ray

parenchyma, connect the phloem (inner bark where taxol is located) to the xylem, it seems logical that taxol may also be found in the xylem of the tree. Taxol was isolated from the organic solvent extract of sawdust prepared from the sap- wood (outer whitish wood, recent 1-20 years of growth) of an approx. 120-year-old Pacific yew tree (see C in Fig. 2). After flash silica gel column chromatography followed by preparative TLC in solvents A, B, C and E, using authentic taxol as a standard, the compound yielded an NMR spec- trum identical to taxol (Fig. 4). It also yielded an electrospray mass spectrum with a primary peak at m/e 853 + H ÷ of 854, which was identical to authentic taxol.

Some sawdust from the heartwood of the tree

Fig. 4. NMR spectrum of taxol from Pacific yew sapwood (A) and authentic taxol (B). The x axis is represented as ppm rela- tive to the tetramethylsilane peak at 0 ppm, whereas the y axis

is the relative peak intensity.

8 G.A. Strobel et a l . /Plant Sci. 92 (19t)3) 1-1-'

(dark wood areas D and E in Fig. 2) was also prepared in an identical manner as the sapwood sawdust (above). It too yielded a compound whose spectral characteristics were identical to taxol. Comparative yields of extractable taxol in the sap- wood per gram dry weight varied from 15-50% of that in the inner bark (phloem), while that of the older heartwood varied from 1-10%. Although the amounts of taxol recoverable from the xylem tissues varied from tree to tree, it was always possi- ble to demonstrate its presence in each of the 5 Pacific yew trees that were sampled.

Presently, only taxol from the inner bark of the Pacific yew is approved for drug use in the USA. Since the bark conservatively represents only about 4% of the weight of the tree, and the re- mainder is xylem (sapwood and heartwood), it is obvious that significant amounts of xylem-taxol is being discarded.

3.3. Taxol biosynthesis in the xylem Since taxol can be consistently found in the

xylem of Pacific yew, we examined the most likely origins of this compound. Our initial approach

was to apply the in vitro assay to xylem tissues utilizing [l-14C]acetate as a precursor with the subsequent isolation and determination of [14C]taxol. The outer phloem (the pink tissues) of the inner bark was an active [lac]taxol producer, although, the vascular cambium region (thin- whitish tissue adjacent to sapwood, see B in Fig. 2) was more active than all other tissues in synthe- sizing [t4C]taxol (Fig. 2) (Table 2). An earlier study utilizing [1-13C]acetate, demonstrated un- equivocally that taxol derived from [1-13C]acetate in this system resulted in detectable label through- out the taxane skeleton, as well as the acetyl func- tional groups on the molecule [5]. This indicates that the labeling we have observed does not simply represent single acetate group exchange to pro- duce [14C]taxol in these assays. Our data also show that in both the sapwood and the heartwood a diminishingly small amount of [14C]taxol is pro- duced from [l-14C]acetate (Table 2). Thus, although active acetate uptake and metabolic con- version is occurring in the sapwood and younger heartwood it is not being converted to taxol in such significant amounts as was previously sug-

Table 2 [l-14C]Acetate labeling of taxol in a log from aprox. 120-year-old Pacific yew log

Tissue Total taxol Total Label in ttg chloroform solubles

(dpm) a

Total taxol (dpm)

Outer phloem 57.7 778 100 (pink tissue) (area A Fig. 2)

Cambial tissue 72.7 934 400 (white) (area B Fig. 2)

Sapwood 47.6 239 433

(white wood) (area C Fig. 2)

Heartwood 40.0 197 966 (20-30 years) (area D Fig. 2)

Heartwood 8.0 366 (70-120 years) (area E Fig. 2)

51 660

57 073

250

42

The standard in vitro assay for taxol biosynthesis from [1-14C]acetate was applied to 0.4 g (fresh wt) of each of these tissues from

the tree-log shown in Fig. 2 [5]. aThe expt was repeated 3 times with samples taken from relatively the same area on the log shown in Fig. 2. The values shown are

an average of 3 determinations and have a S.E. of +9%

G.A. Strobel et al . /Plant Sci. 92 (1993) 1-12 9

Fig. 5. Light micrographs (X, Y, Z) of Pacific yew (T. brevifolia) stems. (X) Oblique section showing the vascular cambial region (VC), and ray cells (R). (Y) Cross section of 30-40 year-old wood showing growth rings (GR) and ray cells (R). (Z) Cross section

of 70-120-year-old wood showing a growth ring (GR) and ray cells (R). Bar = 100 #m for X, Y and Z.

10 G . A Strohel et a / Pkmt Sci. 92 (1993; 1 -12

i Fig. 6. A diagrammatic illustration of bark flaps on a Pacific yew log (10-15 cm, diam.) used to trap taxol. Approximately 2 g of silica gel (60-120 mesh) were placed under each flap and the flap sealed with tape. At the end of various time of incubation, the silica gel was carefully removed, dried, rinsed with chloroform and eluted with acetonitrile. Taxol was eventually recovered from the residue remaining after removal of the acetonitrile. The flap areas a (tip of flap) and b (center of flap) 1 month after the flap were

made was assayed for [14C]taxol production in the in vitro assay. Area c (regular bark) also assayed. See Table 4.

Table 3 Taxol recovered from yew bark flaps via silica gel

Length of exposure Taxol (~g/g silica gel)

1 day Log 1 0.21 Log 2 0.28

7 days Log 1 2.60 Log 2 2.81

14 days Log 1 13.24 Log 2 3.4

28 days Log 1 29.01 Log 2 23.40

Yew logs (10 cm, diam. × 70 cm, length) had bark flaps made in them according to Materials and methods. Two logs were studied. Approximately 2 g of silica gel was placed under each bark flap. Each flap was dedicated to 1 time period and the experiment was replicated on 2 logs (above). At the end of each period, the silica gel was removed, dried, extracted and its taxol content determined as per Materials and methods.

gested [5] (Table 2). One may conclude that, over time, this rate, although small, is adequate enough to account for all of the taxol located in the xylem. On the other hand, more reasonable explanations are that taxol, although formed in the vascular cambial region, is either mobilized via the ray parenchyma and translocated to the xylem (sap- wood) or is simply deposited in the xylem in the process of differentiation of cambial cells. Mor- phologically supportive evidence for the concept of taxol transport is presented in Fig. 5. Ray parenchyma (R) are seen connecting the phloem via the vascular cambium region (vc) to the sap- wood xylem (Fig. 5, X). In Fig. 5 (Y) the R is seen transversing the elements of younger xylem (heart- wood) 20-30 years old (D in Fig. 2), while in Fig. 5 (Z) the R in the older xylem (heartwood) shows visible signs of deterioration after 70-120 years (E in Fig. 2). Thus, little or no living tissue in this region of the plant could result in [l-14C]acetate incorporation into taxol (Table 2). Mobilization of taxol to these xylem tissues may occur via the transport of taxol xylosides which have been isola-

G.A. Strobel et al./Plant Sci. 92 (1993) 1-12

Table 4 Taxol synthesizing activity in 'flap' areas on yew logs after 4 weeks of silica gel taxol trapping (see Fig. 6 and Table 3)

II

Log flap area Total taxol Total label Total label ttg in chloroform solubles in taxol

(dpm) (dpm)

Expt. 1 a 210 1 147 433 56 285 b 190 726 066 80 966 c 130 602 833 39 096

Expt. 2 a 120 998 733 42 680 b 85 845 900 53 913 c 70 477000 48 120

The standard in vitro assay for taxol biosynthesis from [l-14C]acetate was applied in two separate experiments (2 separate logs) to areas a, b and c of the flap tissues (see Fig. 6).

ted and identified from the inner bark of Pacific yew [10].

3.4. Trapping of taxol 'in vivo' The tree tissues having both the greatest taxol

synthesizing content and biosynthetic capability are associated with the vascular cambium region (Fig. 5 and Table 2). If taxol is made in this tissue and is ultimately transported into the xylem, it seems logical that it might be possible to trap it. Silica gel is a reasonable trapping agent because it adsorbs taxol. The best taxol biosynthetic capa- bility in the tree stem is at about 1 meter from ground level [5]. Two yew logs cut from this region of two different trees were used to effectively demonstrate our ability to trap taxol under bark flaps (Fig. 6). Over the course of 1 month, increas- ing amounts of taxol were observed in the silica gel traps (Table 3). Since the amounts of taxol did increase it seems appropriate to conclude that taxol movement from the tissue into the silica gel is a dynamic process. Simple adsorption of taxol from wounded tissues would have resulted in max- imum taxol trapping early in the experiment, e.g. at 1 day, instead of a continuous increase as seen (Table 3). Furthermore, at the end of the harvest time (4 weeks), taxol biosynthetic activity remain- ed just as high or higher in the bark flaps (areas a and b in Fig. 6) as it was in the undisturbed area of the bark (area c in Fig. 6). Preliminary field studies done in the Flathead National Forest using

the bark flap technique to trap taxol have shown promise. It is not known if the technique has any practical applicability, or if the 'bark flapping' process has any detrimental affect on the tree. However, we feel that the silica gel trapping tech- nique may prove useful in 'in vivo' studies of the mobilization of taxanes in the tree.

4. Acknowledgements

The authors wish to thank Dr. George Strunz, Canadian Forestry Fredericton, N.B., for the sam- ples of T. canadensis, Dr. Andres M. Cano of Mex- ico for T. globosa, Mr. Paul Strobel, Massillon, Ohio for T. cuspidata and the ARS, US National Arboretum, Washington, DC for T. chinensis, and T. wallichiana (originally from Mr. John Silba ob- tained from the Sino-American botanical expedi- tion to China, 1980). We are especially grateful to Mr. Mark Garland of the Florida Dept. of Nat. Resources for his efforts in initially acquiring the relatively rare samples of T. floridana in its ex- tremely limited native range and to the Nature Conservancy of Florida for helping us to collect additional samples of T. floridana. T. baccata sam- ples were provided by Drs. Ulrich Matern, Freiburg, Germany and Richard Strange, London, UK. T. sumatrana samples were obtained in Luzon, Philippines, by Mr. Rob Nicholson, Smith College, Northampton, MA. Dr. Xianshu Yang of the Chinese Academy of Medical Sciences, Beijing,

12 G.A. Strobel et al./Plant Sci. 92 (1993J 1-12

China, and his b ro ther - in - law most graciously

sacrificed t ime and effort to acquire and send sam- ples o f T. mairei. 7". brevifolia f rom the State o f Wash ing ton was suppl ied by Drs. Heinz Floss and Ursula Mocek. Dr. Dav id Bailey and col leagues of

Hauser, Co., Boulder, Co lo rado , p rovided assis- tance in quant i t a t ing taxol in some o f our samples.

The au thors also acknowledge Suzan St robe l ' s help in collecting samples of T. brevifolia in Mon- tana and doing the yew log d rawing (Fig. 6). Dr. Scott Strobel is t hanked for his d rawing o f the taxol structure. Authen t ic samples o f taxol were provided by both the Na t iona l Cancer Ins t i tu te and Bristol Myers Squibb Co. Mr. A1 Chr i s toph- ersen, Distr ic t Ranger on the F l a thead Na t iona l Forest , Mon tana , p rov ided advice and permits for Taxus collection. Mr. Dennis Bodily o f Bozeman,

Montana , assisted in collect ing yew samples and in prepar ing samples for examina t ion . F inanc ia l suppor t from the Na t iona l Science F o u n d a t i o n , Na t iona l Cancer Inst i tute, the M o n t a n a Science and Technology All iance, the Amer ican Cancer Society, and the M o n t a n a Agr icu l tu ra l Experi-

ment Sta t ion is also apprec ia ted .

5. References

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2 M.C. Wani, H.L. Taylor, M.E. Wall, P. Coggon and A.T. McPhail, Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J. Am. Chem. Soc., 93 (1971) 2325-2327.

3 E.K. Rowinsky, L.A. Cazenave and F.C. Donehower, Taxol: a novel investigational antimicrotuble agent. J. Natl. Cancer Inst., 82 (1990) 1247-1259.

4 G.M. Cragg and K.M. Snader, Taxol: the supply issue. Cancer Cells, 3 (1991) 233-235.

5 G.A. Strobel, A. Stierle and F.J.G.M. VanKuijk, Factors influencing the in vitro production of radiolabeled taxol by Pacific yew, Taxus brevifolia. Plant Sci., 84 (1992) 65-74.

6 J.H. Cardellina, HPLC separation of taxol and cephalomannine. J. Liquid Chromatogr., 14 (1991) 659-665.

7 N.C. Wheeler, K. Jech, S. Masters, S. Brobst, A.B. Alvarado and A.J. Hoover, Effects of genetic, epigenetic and environmental factors on taxol content in Taxus brevifolia and related species. J. Nat. Prod., 55 (1992) 432-440.

8 R.V. Upadhyay, G.A. Strobel and W.M. Hess, Morpho- genesis and ultrastructure of the conidiomata of Ascochyta cypericola. Mycol. Res., 95 (1991) 785-791.

9 R.W. Spjut, A taxonomic key to the species of Taxus. 2nd NCI workshop on taxol and taxus. NCI, Bethesda. MD, 1992.

10 D.G.I. Kingston, The chemistry of taxol. Pharmacol. Ther., 52 (1991) 1-34.