types of antimicrobial isoprenoids using potato. The set of isogens and isoprenoids that are stimu-
lated by elicitation is identical to that appearing after pathogen attack, whereas the set of isogens
and isoprenoids stimulated by MJ is like those resulting from the wound response. These results
indicate a defined role of jasmonic acid derivatives in the activation of the wound response, whereas
defense responses are induced upon elicitation that simulates attack by some fungal pathogens [16].
Two types of b-1,4-linked glucosamine oligosac- charides, both potentially derived from the chitin
cell walls of pathogenic fungi, act as potent elici- tors in suspension cultured plant cells. The first
type, N-acetylchitooligosaccharides, induce phy- toalexin momilactones and oryzalexins forma-
tion in rice cells even in the nanomolar range [17]. Inhibition studies with various other oligosaccha-
rides show specificity of the binding site for oligosaccharides with degree of polymerization
DP greater than or equal to that of N-acetylchi- tohexaose [18]. Using alkalinization of extracellu-
lar medium as the assay, Felix et al. investigated a time and concentration-dependent saturation of
chitin oligosaccharide surface binding sites on tomato suspension-culture cells and document
desensitization of the primary defense response by repeated treatments with chitin oligosaccharides
[19].
The second type of oligosaccharide used as elic- itor is derived from chitosan, the deacetylated
form of chitin. Phytoalexin formation is not in- duced by this compound in the rice system [17],
but chitosan is an active elicitor in other plant culture systems. Anthraquininone biosynthesis is
stimulated in Morinda citrifolia by both chitin and chitosan [10]. The degree of acetylation of chitin
was found to be important in inducing defense responses. In actuality, the difference between
chitin and chitosan is a continuum of the degree of N-acetylation of the glucosamine residues in the
polymer [20]. Chitosan elicitors induce formation of phytoalexins in legumes soybean, chickpea,
bean, alfalfa, pea and solanaceous plants potato, sweet pepper [21].
Jasmonic acid arises in plants from linolenic acid via the octadecanoic pathway [22]. Rapid, but
transient, synthesis of cis-jasmonic acid has been demonstrated in both whole plants and suspension
cultures [8]. However, elicitor treatment as well as wounding leads to the induction of the jasmonic
acid biosynthesis [2,23]. Co-mediation of oligosaccharides and MJ has
been demonstrated for the induction of phy- toalexin in the rice system [5]. Exogenously ap-
plied MJ increases production of momilactone A in elicited cells to levels higher than those elicited
with N-acetylchitoheptaose alone. In suspension cultured cells of parsley, MJ potentiates elicitation
of phytoalexins using a cell wall derived elicitor of Phytophthora sojae Pmg elicitor [24]. These re-
sults suggest MJ primes the parsley suspension cells in a time dependent manner to become more
responsive to elicitation. Also using parsley sus- pension cultures, Ellard-Ivey and Douglas [25]
show the elicitor response can be partially mim- icked by MJ pretreatment in expression of phenyl-
propanoid genes.
Higher plants are suppliers of indispensable raw materials and drugs in the food and pharmaceuti-
cal industries and of phytoalexins for plant de- fense. Paclitaxel is a plant-derived drug used in the
treatment of breast, ovarian and lung cancers. Several papers now present results showing that
MJ enhances paclitaxel production from several Taxus species [13,26,27]. Chitin-derived oligosac-
charides mimic the effects of elicitation from some pathogenic microbes. The addition of cell extracts
and cultures filtrates of fungal cultures stimulate paclitaxel and other taxanes in Taxus sp. RO1-
M28 [28]. The contribution of this document is the study of the interaction of MJ with chitin and
chitosan-derived oligosaccharides to stimulate pa- clitaxel production. Combinations of both com-
pounds are used to analyze possible mutual influence. Experiments with T. canadensis cell cul-
ture systems are described to more generally show co-mediated oligosaccharide and MJ elicitation
may involve ethylene biosynthesis.
2. Experimental procedures
2
.
1
. Plant materials and maintenance The cell line is T. canadensis Marsh C93AD,
kindly provided by the laboratories of M.L. Shuler School of Chemical Engineering, Cornell Univer-
sity, Ithaca, NY and D.A. Gibson USDAARS Plant Nutrition Laboratory, Ithaca, NY. The
growth medium is according to Ketchum et al. [27]. The maintenance of cultures is carried out as
follows: 10 ml of 14-day-old suspensions are trans-
ferred by pipetting into 40 ml of fresh medium in 125-ml Erlenmeyer flasks. The culture flasks are
capped with 28 mm i.d. Bellco silicone closures Vineland, NJ and kept in a New Brunswick
Scientific Edison, NJ Model G-25 shaking incu- bator in the dark at 130 rpm and 25°C.
2
.
2
. Elicitor preparations Solutions of MJ Bedoukian Research, Dan-
bury, CT are prepared in ethanol in such a way that constant volume aliquots are added to each
well of the 12-well plate see below to make all suspensions 1 vv in ethanol. A slight but
reproducible increase in paclitaxel production be- cause of added ethanol has been noted [13]. At the
same time as MJ addition, equal volumes of aqueous solutions of N-acetylchitohexaose Seika-
gaku, Tokyo 103, Japan, chitosan hydrolysate AgriHouse Inc., Berthoud, CO or suspensions of
colloidal chitin are added to each well according to the experimental design. Preparation of col-
loidal chitin is based on the methanesulfonic acid method of Hirano and Nagao [29] using practical
grade chitin Sigma, St. Louis, MO that is milled to a 60 mesh flour. Concentrations are determined
by the phenol – sulfuric acid method using N- acetylglucosamine as standard [30].
2
.
3
. Characterization of oligosaccharide elicitors The supernatants of the chitosan oligosaccha-
ride preparations are analyzed using a Waters Associates Ultrahydrogel Linear Column Med-
ford, MA; eluant for this gel permeation chem- istry is 0.5 mM sodium sulfate in distilled water at
25°C flowing at 0.6 ml min
− 1
. Overlaying the chromatogram of N-acetylchitohexaose with that
of the chitosan hydrolysate allows identification of the degrees of polymerization of the oligosaccha-
rides in the chitosan hydrolysate preparation based on retention times. The majority of material
is pentasaccharide, and a lesser amount is tetrasac- charide. The quantity of oligosaccharide with DP
6 in the undiluted chitosan preparation is esti- mated as 8.0 mg ml
− 1
, based on standard curve quantitation using a N-acetylchitohexaose stan-
dard curve in the HPLC analysis. Since a 1000- fold dilution of the chitosan preparation was used
in the 75 mg l
− 1
elicitation of the culture, the chitosan hexasaccharide concentration could be
estimated to be 8 mg l
− 1
. The degree of acetylation of the chitosan hy-
drolysate is analyzed by proton NMR spectra obtained at the analytical service center in the
Department of Chemistry at Colorado State Uni- versity. The – NH, – CH and – CH
3
proton intensi- ties from 400 MHz spectra obtained from a 5 mM
N-acetylchitohexaose solution in 10 vv D
2
O are compared with those of the unknown chitosan
hydrolysate solution. The relative methyl proton intensities from the chitosan hydrolysate are ap-
proximately 20 as great as those from the fully acetylated N-acetylchitohexaose spectrum. The re-
sults indicate that an average of 1 in 5 of the glucosamine residues in the chitosan hydrolysate is
acetylated.
2
.
4
. Experimental conditions For cell culture studies of the interdependence
of MJ with chitin and chitosan derived elicitors on formation of paclitaxel, experiments are conducted
using 3.0 ml of 8-day-old suspensions in individual wells of Falcon Lincoln Park, NJ 12-well plates
[31]. After additions of carbohydrate and MJ solu- tions to replicated wells according to the experi-
mental
design, the
multi-well plates
are individually sealed in their original paper wrappers
and incubated in the dark at 25°C with 130 rpm shaking. Except in the kinetics study, samples are
taken for analyses of paclitaxel, sucrose, glucose and fructose 13 days after elicitation.
2
.
5
. Analysis of paclitaxel and sugars in cell culture media
Paclitaxel is measured on a Waters Associates Model 501 HPLC system equipped with a Waters
Associates Model 486 UV detector at 228 nm using a Phenomenex Curosil G 250 × 4.6 mm
column. The mobile phase is acetonitrile – water 45:55 at 1.0 ml min
− 1
. Extracellular paclitaxel is measured in 2.0 ml of a supernatant from each
sample that is filtered through a 0.20 mm Gelman nylon filter [32]. Then, 150 ml of methanol is
passed through the filter to desorb paclitaxel from the membrane and is collected into 0.2 ml vials for
HPLC analysis. The analyzed data was qualified and quantified against standards Hauser Chemi-
cal, Boulder, CO, which were prepared in the same way as the samples. Verification of paclitaxel
is based on UV absorption spectra and electron-
spray mass spectrometer parent ion analyses. Su- crose, glucose and fructose in filtered, but unex-
tracted, medium samples are analyzed using a Waters Associates Model 6000 HPLC system
equipped with a Waters Associates RI detector using a BioRad HPX-87H column at 65°C with
0.008 N H
2
SO
4
at 0.6 ml min
− 1
.
2
.
6
. Analysis of ethylene produced by cell cultures These experiments are conducted such that eth-
ylene is not provided to the cultures as in earlier work [13,14,33]; however, most of the cultures
produce ethylene. For studies of ethylene produc- tion that results from elicitation with combina-
tions of MJ and oligosaccharides, the silicon cap closures on 125 ml maintenance flasks see above
are either covered with aluminum foil to impede diffusion of ethylene from the culture headspace
or are replaced by serum caps. Following elicita- tion 21 days after culture transfer, ethylene con-
centrations in culture headspace samples are determined and quantitated by GC as described
previously [33]. In addition to final concentrations of 10 mM CaCl
2
added to all flasks, various amounts of MJ and N-acetylchitohexaose are
transferred aseptically to the replicated 40 ml cul- tures. Headspace volume is 125 ml, from which
small amounts of ethylene 0.1 ppm could be reproducibly detected within 2 h by injecting 2.0
ml into the GC [33]. A hypodermic needle fitted with a sterile 0.45 m filter is inserted through the
serum cap while the 2.0 ml headspace samples are being withdrawn using a gas tight syringe. Such
precautions to admit air to cultures in aluminum foil covered flasks is not necessary, but ethylene
diffusion from the flasks is apparently significant because the ethylene headspace accumulation is
only 0.001 of that in the experiment using serum capped flasks. Subsequent duplicate analysis from
each flask was conducted daily. Ethylene accumu- lation is based on cell dry weight that is deter-
mined at the end of the experiment.
3. Results
