Structure elucidation, enantioselective analysis, and biogenesis of nerol oxide in Pelargonium species

Article abstract of DOI:10.1021/jf981245m

A novel enantioselective synthesis of nerol oxide (3,6-dihydro-4-methyl- 2-(2-methyl-1-propenyl)2H-pyran) was used for the determination of the absolute configuration at C-2. The order of elution of the enantiomers on octakis-(2,3-di-O-butyryl-6-O-tert-butyldimethylsilyl)-γ-cyclodextrin in OV 1701-vi as the chiral stationary phase in enantioselective GC was determined as (2R) before (2S). Enantioselective multidimensional GC/MS (enantio- MDGC/MS) was used for the determination of the enantiomeric ratios of nerol oxide in different geranium oils. As a result, in all investigated oils nerol oxide occurs as a racemate. The biogenesis of nerol oxide in Pelargonium species was investigated by feeding experiments using deuterium-labeled neryl glucoside as the precursor. The Pelargonium plants were able to convert the fed precursor into racemic nerol oxide, which has to be considered as a 'natural racemate'.

Full text of DOI:10.1021/jf981245m

J. Agric. Food Chem. 1999, 47, 3145−3150
3145
Str u ctu r e Elu cid a tion , En a n tioselective An a lysis, a n d Biogen esis of
Ner ol Oxid e in P ela r gon iu m Sp ecies
Matthias Wu¨st, J u¨rgen Reindl, Sabine Fuchs, Thomas Beck, and Armin Mosandl*
Institut fu¨r Lebensmittelchemie, Biozentrum, J ohann Wolfgang Goethe-Universita¨t, Marie-Curie Strasse 9,
D-60439 Frankfurt, Germany
A novel enantioselective synthesis of nerol oxide (3,6-dihydro-4-methyl-2-(2-methyl-1-propenyl)-
2H-pyran) was used for the determination of the absolute configuration at C-2. The order of elution
of the enantiomers on octakis-(2,3-di-O-butyryl-6-O-tert-butyldimethylsilyl)-γ-cyclodextrin in OV
1701-vi as the chiral stationary phase in enantioselective GC was determined as (2R) before (2S).
Enantioselective multidimensional GC/MS (enantio-MDGC/MS) was used for the determination of
the enantiomeric ratios of nerol oxide in different geranium oils. As a result, in all investigated oils
nerol oxide occurs as a racemate. The biogenesis of nerol oxide in Pelargonium species was
investigated by feeding experiments using deuterium-labeled neryl glucoside as the precursor. The
Pelargonium plants were able to convert the fed precursor into racemic nerol oxide, which has to be
considered as a “natural racemate”.
Keyw or d s: Pelargonium graveolens; Geraniaceae; stable isotope labeling; nerol oxide; biogenesis;
geranium oil
INTRODUCTION
graveolens and an unknown Pelargonium hybrid (see above).
The oils were isolated using steam distillation (officinal
apparatus DAB 9).
Nerol oxide (3,6-dihydro-4-methyl-2-(2-methyl-1-pro-
penyl)-2H-pyran, 9a /b, see Figure 1) is a chiral mono-
terpenoid ether. It was isolated from Bulgarian rose oil
in 1965 by E. sz. Kova´ts (Ohloff et al., 1980) and is
nowadays a valuable base material in perfumery. In
1974, nerol oxide was detected in wine from several
grape varieties where it contributes to the unique flavor
(Schreier and Drawert, 1974). Since that time, nerol
oxide has been detected in numerous plants as in
Pelargonium species, which are used for the production
of the so-called geranium oil. The olfactory properties
of the enantiomers were investigated by an enantiose-
lective synthesis (Ohloff et al., 1980). The first enanti-
omer separation of nerol oxide was performed by
complexation GC (Schurig, 1987). In 1993, nerol oxide
enantiomers were separated using a modified cyclodex-
trin derivative as the chiral stationary phase (Lindstro¨m
et al., 1993). However, the elution order of nerol oxide
enantiomers on modified cyclodextrin phases is still
unknown. Moreover, biogenetic studies on the biogen-
esis of nerol oxide using labeled precursors have not
been performed yet. Hence, this paper deals with a novel
enantioselective synthesis of nerol oxide, the elucidation
of the absolute configuration of its optical antipodes, the
investigation of its biogenesis in Pelargonium plants
using deuterium-labeled precursors, and its enantiose-
lective analysis in different geranium oils.
R efer en ce Com p ou n d s. cis-(2R,4S)/trans-(2S,4S)-Rose
oxides (4-methyl-2-(2-methyl-1-propenyl)-tetrahydropyran, enan-
tioselective GC: ee ) 65%, cis/trans ) 75/25) were obtained
from Dragoco, Holzminden, Germany. Nerol oxide (3,6-dihy-
dro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran, 9a /b) was ob-
tained from Roth, Karlsruhe, Germany.
1H NMR. The spectra were recorded on a Bruker AMX 500
or ARX 300, CDCl3/TMS. NMR assignments were clarified by
1H/1H-COSY.
En a n tio-MDGC-MS. Enantio-MDGC-MS (enantio-multi-
dimensional gas chromatography-mass spectrometry) analy-
sis of the SPME (solid-phase microextraction) headspace
extracts, synthetic products, essential oils, and reference
compounds was performed with a Siemens SICHROMAT 2,
equipped with independent column oven programs and a live
T-switching device. The main column was coupled to the
transfer line of a Finnigan MAT ITD 800, using an open split
interface; transfer line 250 °C; open split interface 250 °C;
helium sweeping flow 1 mL/min; ion trap manifold 230 °C; EI
70 eV. The reference compounds and synthetic products were
analyzed in full scan mode (40-250 amu).
Column Combination for the Separation of cis-/ trans-Rose
Oxide (10,11). Precolumn conditions: Duran glass capillary
(28 m × 0.23 mm) coated with a 0.23 µm film of PS-268; carrier
gas hydrogen 125 kPa; split 25 mL/min; injector temperature
220 °C; detector FID 250 °C; oven temperature 110 °C (60 min
isothermal), then 2 °C/min to 250 °C. Main column condi-
tions: Duran glass capillary (30 m × 0.23 mm), coated with a
0.23 µm film of 50% heptakis(2,3-di-O-acetyl-6-O-tert-bu-
tyldimethylsilyl)-â-cyclodextrin in PS-268 polysiloxane deriva-
tive, column was self-prepared according to Dietrich et al.
(1995); carrier gas hydrogen at 100 kPa; oven temperature 65
°C (20 min isothermal), then 1 °C/min to 150 °C; live-T-cutting
11.000-13.000 min.
EXPERIMENTAL PROCEDURES
P la n t Ma ter ia l. Young plants of Pelargonium graveolens
L’He´ritier and an unknown Pelargonium hybrid were kindly
provided by Gartenbau Stegmeier, Essingen, Germany.
Essen tia l Oils. Commercially available samples of gera-
nium oils were purchased from the German fragrance market.
Authentic samples were self-prepared oils from Pelargonium
Column Combination for the Separation of Nerol Oxide (9a /
b). Precolumn conditions: Duran glass capillary (30 m × 0.23
mm), coated with a 0.23 µm film of SE-52; carrier gas hydrogen
at 121 kPa; split 25 mL/min; injector temperature 220 °C;
detector FID 250 °C; oven temperature 60 °C (5 min isother-
mal), then 5 °C/min to 250 °C. Main column conditions: Duran
* To whom correspondence should be addressed (e-mail
Mosandl@em.uni-frankfurt.de).
10.1021/jf981245m CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/03/1999

3146 J. Agric. Food Chem., Vol. 47, No. 8, 1999
Wu¨st et al.
Sch em e 1. En a n tioselective Syn th esis of (S)-Ner ol Oxid e 9a a n d cis- a n d tr a n s-Rose Oxid e 10, 11
glass capillary (30 m × 0.23 mm) coated with a 0.23 µm film
of 50% octakis(2,3-di-O-butyryl-6-O-tert-butyldimethylsilyl)-γ-
cyclodextrin in OV 1701-vi, column was self-prepared accord-
ing to Schmarr (1992); carrier gas hydrogen at 100 kPa; oven
temperature 60 °C (25 min isothermal), then 1.5 °C/min to 150
°C; live-T-cutting 23.500-24.500 min.
2H), 4.22-4.36 (m, H-6, 2H), 5.44 (m, H-2, 1H), 9.35 (s, br,
COOH, 1H).
Syn th esis of 3,6-Dih yd r o-4-m eth yl-2H-p yr a n -2-ca r box-
ylic Acid Ch lor id e (4). Compound 4 was prepared according
to Becker et al. (1993). A 2.7 mmol amount of 3 yielded 1.7
mmol (63%) of 4 after distillation (Kugelrohr, 95 °C, 0.12
mbar). Because of its instability, compound 4 was used for the
next step without GC/MS analysis.
GC-MS. The GC-MS analysis of the synthetic products and
the reference compounds was performed with a Fisons Instru-
ments GC 8065 coupled to a Fisons Instruments MD 800 mass
spectrometer, equipped with a HTFS capillary column (30 m
× 0.25 mm; coated with SE-52; film thickness 0.5 µm; column
was self-prepared according to Grob (1986)). GC conditions:
carrier gas helium 70 kPa; split 30 mL/min; injector temper-
ature 230 °C; oven temperature 40 °C (5 min isothermal), then
3 °C/min to 250 °C (30 min isothermal); ion source temperature
200 °C; mass range 40-250 amu; electron energy 70 eV. The
molecular ions (M+) and fragment ions are given as m/z with
relative peak intensities in % of the most abundant peaks.
Ad m in istr a tion of th e La beled P r ecu r sor . A solution
of 16 with a concentration of 1.0 mg/mL was prepared by
dissolving the same amount of 16 and Tween 20 in water. A
100 µL amount of this solution and two pieces of the leaf blade
of P. graveolens (weight 100-200 mg) were incubated at room
temperature for 24 h with exclusion of light in a sealed 2 mL
vial. After incubation, the essential oil evaporating from the
glandular trichomes was analyzed by headspace SPME.
SP ME. A SPME fiber holder for manual use equipped with
a fused silica fiber coated with poly(dimethylsiloxane) (film
thickness 100 µm) was used (both SUPELCO, Munich, Ger-
many). For headspace sampling, the septum of the vial
containing the pieces of leaf blade was pierced and the fiber
exposed for 3 min to the headspace. For thermal desorption,
the SPME fiber remained in the injector for 3 min. Splitless
injection mode was used, the split valve being opened after 2
min.
Syn th esis of (R)- a n d (S)-3,6-Dih yd r o-4-m eth yl-2H-
p yr a n -2-ca r boxylic Acid (S)-P h en ylglycin ol Am id e (5,6).
The diastereomeric amides 5 and 6 were prepared according
to Helmchen et al. (1979). A 1.7 mmol amount of 4 gave 0.7
mmol (44%) of (R)-5 and 0.4 mmol (25%) of (S)-6 after
separation by flash chromatography (ethyl acetate/pentane 70/
30 (v/v)). 1H NMR (δ) of 5: 1.74 (s, H-7, 3H), 2.19-2.36 (m,
H-3, 2H), 2.98-3.00 (m, OH, 1H), 3.83-3.91 (m, H-11, 2H),
4.01 (dd, J ) 3.9/10.9 Hz, H-2, 1H), 4.17-4.27 (m, H-6, 2H),
5.05-5.09 (m, H-10, 1H), 5.41 (s, br, H-5, 1H), 7.24-7.38 (m,
phenyl-H, H-9, 6H). 1H NMR (δ) of 6: 1.72 (s, H-7, 3H), 2.10-
2.33 (m, H-3, 2H), 2.71-2.74 (m, OH, 1H), 3.90-3.92 (m, H-11,
2H), 4.07 (dd, J ) 3.9/10.9 Hz, H-2, 1H), 4.20-4.28 (m, H-6,
2H), 5.08-5.11 (m, H-10, 2H), 5.41 (s, br, H-5, 1H), 7.26-7.38
(m, phenyl-H, H-9, 6H).
Syn th esis of (S)-3,6-Dih yd r o-4-m eth yl-2H-p yr a n -2-ca r -
boxylic Acid Meth yl Ester (7). A 0.4 mmol amount of
compound 6 was dissolved in 1 mL of THF and 25 mL of
methanol, and 0.4 mmol of sulfuric acid was added. The
mixture was refluxed for 18 h. The solvents were removed
under reduced pressure, and the residue was suspended in
water. It was extracted 3 times with portions of 30 mL of
diethyl ether, and the combined extracts were dried over
anhydrous sodium sulfate. After filtration over a layer of silica
gel, the solvent was removed under reduced pressure to afford
0.24 mmol (56%) of 7. GC-MS: 156 (M+, 5), 141 (15), 138 (34),
128 (31), 97 (100), 69 (77), 41 (53).
Syn th esis of 3,6-Dih yd r o-4-m eth yl-2H-p yr a n -2-ca r box-
ylic Acid (3). Compound 3 was prepared according to Lu-
bineau et al. (1994). The crude acid was purified by flash
chromatography (eluent: pentane/diethyl ether/formic acid 70/
25/5 (v/v/v)). 1H NMR (δ): 1.75 (s, H-7, 3H), 2.27-2.36 (m, H-3,
Syn th esis of (S)-3,6-Dih yd r o-4-m eth yl-2H-p yr a n -2-ca r -
ba ld eh yd e (8). Compound 8 was prepared according to
Zakharkin and Khorlina (1962) by reduction of 7 using DIBAL.
The crude product was used for the next step without further

Biogenesis of Nerol Oxide
J. Agric. Food Chem., Vol. 47, No. 8, 1999 3147
Ta ble 1. En a n tiom er ic Ra tio of Gen u in e a n d La beled Ner ol Oxid e fr om Differ en t Ger a n iu m Oils in P er cen t
geranium oils
self-prepared
commercial samples
Pelargonium
graveolens
Pelargonium
unknown spp.
compound
Re´union
China
Egypt
(+)-(R)-nerol oxide
(-)-(S)-nerol oxide
(+)-(R)-d2-nerol oxide
(-)-(S)-d2-nerol oxide
9b
9a
15b
15a
50.6
49.4
48.6
51.4
49.7
50.3
47.9
52.1
48.5
51.5
49.5
50.5
48.6
51.4
purification. GC-MS: 126 (M+, 0.4), 97 (76), 80 (8), 69 (28), 53
(10), 41 (100).
Syn th esis of (S)-3,6-Dih yd r o-4-m eth yl-2-(2-m eth yl-1-
p r op en yl)-2H-p yr a n (9a ). Compound 9a was prepared ac-
cording to Ogawa et al. (1978) using the Wittig reaction. A
0.01 mmol amount of 9a was obtained after purification by
flash chromatography (pentane/ether 90/7 (v/v). GC-MS: 152
(M+, 4), 109 (5), 96 (9), 83 (62), 68 (80), 67 (100), 53 (32).
Syn th esis of (2S,4R)-4-Meth yl-2-(2-m eth yl-1-p r op en yl)-
tetr a h yd r op yr a n (10) a n d (2S,4S)-4-Meth yl-2-(2-m eth yl-
1-p r op en yl)tetr a h yd r op yr a n (11). Compounds 10 and 11
were prepared by partial reduction of 9a in a micromole scale
according to Ohloff et al. (1964). MS data are in agreement
with the data given by Snowden et al. (1987).
Syn th esis of (Z)-Meth yl-3,7-d im eth yl-2,6-octa d ien oa te
(13). Compound 13 was prepared according to Corey et al.
(1968). A 3.3 mmol amount of (Z)-3,7-dimethyl-2,6-octadienal
yielded 2.5 mmol (75%) of crude product. The product was used
for the next step without further purification. GC-MS: 182
(M+, 3), 123 (17), 114 (25), 83 (29), 69 (79), 41 (100).
Syn th esis of (Z)-3,7-Dim eth yl-2,6-(1,1-2H2)octa d ien -1-
ol (14). Compound 14 was prepared according to Becker et
al. (1993) using lithium aluminum deuteride (>99% atom D;
Fluka, Deisenhofen, Germany). A 2.6 mmol amount of 13
yielded 0.3 mmol (11%) of 14 after purification by column
chromatography (silica gel, pentane/diethyl ether 2/5 (v/v)).
Purity >95% (GC). Deuterium content: d2 > 97% (1H NMR).
GC-MS: 156 (M+, 0), 138 (15), 123 (21), 122 (6), 95 (80), 69
F igu r e 1. Separation of nerol oxide enantiomers on octakis-
(2,3-di-O-butyryl-6-O-tert-butyldimethylsilyl)-γ-cyclodextrin in
OV 1701-vi.
1
(69), 41 (100). H NMR (δ): 1.62 (s, H-8 or H-10, 3H), 1.71 (s,
H-8 or H-10, 3H), 1.77 (s, H-9, 3H), 2.09-2.12 (m, H-4, H-5,
4H), 5.10-5.15 (m, H-6, 1H), 5.46 (s, H-2, 1H).
Syn th esis of 3,6-Dih yd r o-4-m eth yl-2-(2-m eth yl-1-p r o-
p en yl)-2H-(6,6-2H2)p yr a n (15a /b). Compound 15a /b was
prepared according to Taneja et al. (1978) in a micromole scale
with some modifications. A 25 mg amount of 14 and a catalytic
amount of N-iodosuccinimide (NIS) were taken up in 1 mL of
CCl4 in a GC vial. The vial was sealed and heated at 90 °C for
25 min. The dark violet solution was shaken several times with
an aqueous solution of Na2S2O3 until the iodine was completely
removed. The reaction mixture was subjected to preparative
TLC (Polygram SIL G/UV254, Machery-Nagel, Du¨ren, Ger-
many; eluent pentane/ether 9/1 (v/v)). The nerol oxide fraction
was removed from the TLC plate and extracted with ether.
GC-MS: 154 (M+, 5), 111 (5), 96 (10), 83 (83), 70 (97), 69 (100),
55 (36).
tioselective synthesis of nerol oxide was elaborated (see
Scheme 1). To determine the absolute configuration of
the stereocenter at C-2, enantiopure nerol oxide was
hydrogenated to afford a mixture of enantiopure cis- and
trans-rose oxide (10,11).
The absolute configurations at C-2 and C-4 of 10 and
11 were determined by enantioselective GC analysis
using heptakis-(2,3-di-O-acetyl-6-O-tert-butyldimethyl-
silyl)-â-cyclodextrin as the chiral stationary phase. All
four stereoisomers of rose oxide can be separated on this
phase with known order of elution (Wu¨st et al., 1998a).
Hence, the absolute configuration of the stereocenter at
C-2 of nerol oxide can be deduced. The elution order of
the nerol oxide enantiomers on an octakis(2,3-di-O-
butyryl-6-O-tert-butyldimethylsilyl)-γ-cyclodextrin phase
was determined as (R)-9b before (S)-9a by injection of
(S)-enriched nerol oxide (see Figure 1).
The same cyclodextrin phase column was used in a
MDGC/MS system for the determination of the enan-
tiomeric ratios of nerol oxide in different geranium oils
(see Table 1). In all investigated oils, nerol oxide occurs
as a racemate. This result is in agreement with the
result of Kaiser (1984) who measured the optical rota-
tion power of nerol oxide isolated from Reunion gera-
nium oil and with the result of Werkhoff et al., who
determined the enantiomeric ratio of nerol oxide in a
geranium oil of undefined origin by enantioselective GC.
Racemic nerol oxide was also found in rose oil (Ohloff
Syn th esis of (Z)-3,7-Dim eth yl-2,6-(1,1-2H2)octa d ien -1-
yl-â-D-glu cop yr a n osid e (16). Compound 16 was prepared
according to Paulsen et al. (1985). A 0.7 mmol amount of 14
yielded 0.13 mmol (19%) of 16 after purification by column
chromatography (silica gel, ethyl acetate/pentane/methanol
1
5/5/1 (v/v/v)). H NMR (δ): 1.61 (s, H-8 or H-10, 3H), 1.70 (s,
H-8 or H-10, 3H), 1.77 (s, H-9, 3H), 2.0-2.2 (m, H-4, H-5, 4H),
3.3-3.9 (m, 11H), 4.36 (d, J ) 7.6 Hz, H-1’, 1H), 5.0-5.2 (m,
H-6, 1H), 5.37 (s, H-2, 1H).
RESULTS AND DISCUSSION
The first enantioselective synthesis of both nerol oxide
enantiomers was performed by Ohloff et al. (1980) using
(R)-linalool as the chiral building block. However, the
elucidation of the absolute configuration of the optical
antipodes remained unclear. Therefore, a novel enan-

3148 J. Agric. Food Chem., Vol. 47, No. 8, 1999
Wu¨st et al.
Sch em e 2. Syn th esis of La beled Ner yl Glu cosid e 16
a n d Refer en ce Com p ou n d s 15a /b
F igu r e 3. Precolumn chromatogram obtained from P. gra-
veolens by headspace SPME.
et al., 1980; Kova´ts, 1987; Lindstro¨m et al., 1993),
labdanum oil, osmanthus oil, and lavender oil (Werkhoff
et al.).
These findings led to the hypothesis that nerol oxide
is not formed directly in the plant but from a precursor
which is preformed to a large extent. Nerol is discussed
as a potential precursor, and singlet oxygen might act
as an active agent for the introduction of a second allylic
hydroxy group (Ohloff et al., 1980). To verify this
F igu r e 4. Main column chromatogram obtained from P.
graveolens fed with 16.
hypothesis, deuterium-labeled nerol oxide 14 was pre-
pared (see Scheme 2).
Since Croteau et al. (1987) could show that monoter-
pene glucosides have a transport function in monoter-
pene catabolism, the labeled nerol 14 was glucosidated
and fed to the plants by incubating leaf disks with a
solution of glucoside 16 in septum-sealed 2 mL vials for
24 h in the dark. These conditions were chosen in order
to exclude a photooxygenation of labeled neryl glucoside
16 by singlet oxygen. After the incubation, the head-
space of the septum-sealed vial was analyzed using
solid-phase microextraction (SPME). The SPME ex-
tracts were analyzed by enantio-MDGC-MS. Figure 3
shows a precolumn chromatogram obtained from P.
graveolens which was fed with 16. Nerol oxide was
transferred to the chiral main cloumn and detected by
MS. Figure 4 shows the main column chromatogram.
As can be seen on mass lane m/z ) 68 and 70, genuine
and labeled nerol oxide occur as racemates. On mass
lane m/z ) 70, the labeled nerol oxide enantiomers are
mainly detected, as can be seen by the position of the
peak maxima on this mass lane: Because of the so-
called inverse isotope effect (Matucha, 1991), the deu-
terium-labeled nerol oxide isotopomers show a shift of
their peak maxima of approximately 3 s relative to the
unlabeled one. The presence of labeled nerol oxide is
evident from the mass spectra measured at the peak
maximum on mass lane m/z ) 70 (see Figure 2). The
base peak at m/z ) 69, which is shifted by two mass
units compared to unlabeled nerol oxide, is clearly
detectable. The fact that the cyclization of labeled nerol
also proceeds in the dark excludes a photooxygenation
F igu r e 2. Mass spectra of unlabeled (9) and labeled (reference
compound 15) nerol oxide in comparison with nerol oxide
obtained from P. graveolens fed with 16.

Biogenesis of Nerol Oxide
J. Agric. Food Chem., Vol. 47, No. 8, 1999 3149
Sch em e 3. P r op osed Biosyn th etic P a th w a y for th e
Con ver sion of La beled Ner yl Glu cosid e 16 in to Ner ol
Oxid e 9a /b in P ela r gon iu m Sp ecies
F. Dettmar for technical assistance, and Dipl.-Ing. S.
Bihler and Dr. H. Hanssum for recording the H-NMR
spectra.
1
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mechanism which was previously advanced. It shows
that the plant is able to convert the fed nerol into nerol
oxide. Since a photooxygenation mechanism can be
excluded, it is highly probable that nerol is enzymati-
cally oxydized in an allylic position to give one of the
diols in Scheme 3 (17 or 18), which are equivalent by
allylic rearrangement. 17/18 can easily cyclize to nerol
oxide by acid catalysis (Ohloff et al., 1964).
In petals of Rosa damascena, the diol 17 was recently
isolated and spectroscopically characterized (Knapp et
al., 1998), which supports the above-mentioned sugges-
tions concerning the cyclization mechanism.
CONCLUSIONS
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Since nerol oxide appears in all geranium oils as a
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catalysis. It is highly probable that the formation of 17/
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corresponding monoterpenol nerol in analogy to the
formation of rose oxide (Wu¨st et al., 1996, 1998a,b). A
photooxygenation mechanism is rather unlikely. Nerol
oxide has to be considered as a natural racemate.
ACKNOWLEDGMENT
We thank D. Stegmeier, Gartenbau Stegmeier, Essin-
gen, Germany, for providing authentic plant material,

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Received for review November 12, 1998. Revised manuscript
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