PlantCeU

Reports

Plant Cell Reports (1991) 10:349-353

9 Springer-Verlag1991

Enhanced codeine and morphine production in suspended

Papaver somniferum cultures after removal of exogenous hormones
ChuiLi Siah and PaufineM. Doran
Department of Biotechnology, University of New South Wales, P.O. Box 1, Kensington NSW 2033, Australia
Received March 12, 1991/Revisedversion received June 3, 1991 - Communicated by M. Tabata

Summary. Morphine and codeine accumulation in

Papaver somniferum suspension cultures increased

markedly after removal of hormones from the medium.
Cultures developed hormone self-sufficiency without
organogenesis or development of meristemoids;
enhanced synthesis of morphinan alkaloids was not
dependent on formation of shoots, roots or embryos.
Without exogenous hormones, maximum codeine and
morphine concentrations were 3.0 mg g-1 dry weight
and 2.5 mg g-1 dry weight respectively, up to three
times higher than in cultures supplied with hormones.
Hormone-deprived cells produced a higher ratio of
codeine:morphine than cultures supplied with auxin and
cytokinin. Improved alkaloid production was correlated
with slower overall growth rate.

Introduction
Latex from the opium poppy, Papaver somniferum, is a
commercial source of the analgesics, morphine and
codeine. Callus and suspension cultures of P.
somniferum are being investigated as an alternative
means for production of these compounds. Amounts of
thebaine, morphine and codeine in morphologically
undifferentiated cultures have been reported in the range
0 gg per 100 g dry weight (Yoshikawa and Furuya
1985) to 1.5 mg g-1 dry weight (Tam et al. 1980); this
can be compared with 1.4 mg g-] dry weight in leaf
tissue and 200 mg gq in dried latex (Constabel 1985).
New strategies for improving synthesis of secondary metabolites are aimed at mimicking conditions in
the whole plant. The environment experienced by cells
in culture is significantly different from that in vivo and
is responsible for diminished product levels. Conditions
affecting metabolism of suspended plant cells include:
(i) reduced cell-cell contact; (ii) fewer chemical and
electrochemical gradients; (iii) absence of microbial
contamination; and (iv) presence of high levels of
exogenous hormones. All of these factors are known to

Offprint requests to: P.M. Doran

affect differentiation and secondary metabolism

(Lindsey and Yeoman 1983). Immobilisation of plant
cells has been used to restore cell-cell contact and
produce gradients; elicitors are being tested with several
species, including P. somniferum (Heinstein 1985), to
simulate microbial attack on plants. Both these
techniques have led to improvements in secondary
metabolite production.
Exogenous phytohormones are normally required
for tissue-culture initiation and to promote growth in
vitro. Choice of hormones also has a profound effect on
the profile of morphinan alkaloid accumulation in P.
somniferum cells (Hodges and Rapoport 1982; Kamo et
al. 1982). However, hormone levels higher than those
in intact plants may suppress secondary metabolism at
the same time as stimulating growth. Growth regulators
such as 2,4-D (dichlorophenoxyacetic acid) and NAA
(naphthalene acetic acid) have been found to produce
chromosomal aberrations in plant cells (Sunderland
1977; Bayliss 1980), and are particularly suspect as
mutagenic agents. Future regulatory guidelines for
tissue-culture products could restrict use of these
substances in production of pharmaceutical- and foodgrade products. Bayliss (1980) has suggested that the
concentrations of hormones used in tissue culture are
too low to produce a significant effect; however, in view
of the high frequency of chromosomal changes observed
in many cultures and the toxic nature of 2,4-D, it would
be preferable to avoid use of hormones altogether.
Removal of exogenous hormones from largescale culture systems could be implemented using a
two-stage process strategy. After high cell densities are
reached with the aid of exogenous hormones, hormonefree medium would then be applied to stimulate
secondary production. Several studies into the effects of
elicitors and cell immobilisation have used hormonefree medium (Brodelius et al. 1979; Shuler et al. 1983;
Barnabas and David 1988; Cline and Coscia 1988); the
extent to which hormone deprivation contributed to the
observed improvements in secondary synthesis is
difficult to define. This study is aimed at determining

350
whether removal of hormones from Papaver
somniferum suspensions could be used to stimulate in

Results

vitro production of morphinan alkaloids.

Hormone self-sufficiency
Papaver somniferum cells adapted to the absence of

Materials and methods

Cell suspension cultures. Seeds of Papaver somniferum L. cultivar
14-14 were kindly donated by Tasmanian Alkaloids Pty Ltd,
Australia. Callus initiated from seedling hypocotyls was maintained
on nutrient medium containing Murashige and Skoog (1962) salts, 3%
sucrose, 2.5 mg 1-1 cysteine.HC1,0.1 mg 1-1 2,4-D, 2 mg l -t
kinetin, and 1% agar. Two years after callus initiation, cell
suspensions were developed using the same medium without agar.
Suspension cultures were maintained in shake flasks at 25~ on
orbital shakers under low light intensity (200 lux) for approx. 4
months before the start of the experiments described below.

Flask experiments. Twelve 1-litre Erlenmeyer flasks each containing

250 ml cell suspension were used in the experiments. Six were
subcultured every 7 d using the liquid medium described above; the
remaining 6 flasks were subcultured using the same medium without
hormones. At each subculture, 200 ml fresh medium was placed into
a sterile flask and 50 ml cell suspension added. Samples were
removed every 2-3 d over a period of 56 d. At the end of 56 d the
experiment was continued for a further 12 d without subculturing to
obtain batch growth data.

Analyses. Cell concentration was measured as dry weight after the

samples were filtered under vacuum, the supernatant removed for
sugar and alkaloid analysis, and the remaining solids freeze-dried
overnight. Sucrose, fructose and glucose levels were measured as
described previously (Sharp and Doran 1990).
Alkaloid analyses were carried out on both cells and liquid medium.
Cells were ground with acid-washed sand, washed with methanol and
refluxed for 2 hr. The sample was then filtered through Whatman No
1 filter paper to remove cell debris, and washed further with methanol.
The methanol was removed from the filtrate using a rotary evaporator.
The residue was re-dissolved in 1M HC1, and chloroform added. The
aqueous and organic phases were separated in a separating funnel; the
aqueous layer was collected and the pH adjusted to 9.5 with NH4OH.
This solution was extracted further with 75% chloroform-25% isopropanol. The heavier organic phase was drained and returned to a
separating funnel for back-washing with NH4OH; the organic phase
was then coUected and evaporated. The residue was dissolved in
chloroform, the volume adjusted to 4 ml, and the chloroform
evaporated. The residue was then dissolved in 10 m M NaHzPO 4
buffer at pH 2.1 containing 1 m M dodecyl sulphate and 50%
acetonitrile. Before HPLC analysis the sample was filtered through a
0.22 ~tm filter.
Medium filtrate from the cultures was evaporated using a
rotary evaporator, re-dissolved in 0.5 M ammonium sulphate, and the
pH adjusted to 9.3-9.5 with NH4OH. Samples were passed through a
Sep-Pak C18 cartridge (Waters Associates) after treatment of the
cartridge with methanol. The cartridge was washed with 5 mM
ammonium sulphate adjusted to pH 9.3-9.5 with NH4OH, and then
distilled water. Morphinan alkaloids were eluted with 50%
acetonitrile in 10 mM NaHzPO4 buffer at pH 2.1.
Alkaloids were separated at room temperature using a 30 cm x
3.9 mm i.d. Phenomenex Bondex Cls HPLC column containing 10
~ n packing. The mobile phase was l0 mM NaHzPOa buffer at pH
2.1 containing 1 mM dodecyl sulphate and 35% acetonitrile. The flow
rate was 1 ml min -1. Caffeine was used as internal standard.
Morphine, codeine and thebaine were detected at 254 nm with
retention times of 5.3 rain, 7.0 min and 18.4 min, respectively.
Amounts of morphine, codeine and thebaine in samples were
determined using standard curves.

exogenous hormones in liquid medium without

formation of shoots, roots, embryos or other organised
structures. Dispersed cultures with minimal aggregation
were maintained in hormone-free liquid medium for
over 9 months. Cultures without exogenous hormones
were not visibly different from the control cultures. As
shown in Fig. 1, both suspensions contained cells with
varied sizes and morphologies. The extent of clumping
and formation of giant cells was similar in both cultures.

Cell growth
Cell concentration data measured after 56 d hormonefree culture are plotted in Fig. 2 for comparison of
growth rates. By this time, carry-over of exogenous
hormones in the hormone-deprived cultures can be
assumed negligible. Each datum point represents
the average of at least three measurements. The cell
specific growth rate without hormones was approx. 20%
slower at 0.056 d -1 (doubling time 12.4 d) compared
with 0.071 d -1 (doubling time 9.8 d) in the control
cultures.

Sugar consumption
Sugar consumption rates and patterns were similar in
cultures with and without hormones. In both cases,
sucrose was hydrolysed to glucose and fructose within
the first 3 d after subculturing; glucose was then taken
up preferentially. Biomass yield from sugars was not
affected by hormone removal and was approximately
0.2g g-1.

Production of morphinan alkaloids

Morphine and codeine levels were monitored during the
first 56 d after transfer to hormone-free medium as
shown in Figs. 3 and 4. Subculturing was carried out
every 7 d so that the response of the ceils could be
followed for an extended period, longer than the
duration of a normal batch culture. The time between
successive passages was less than one doubling period.
Accordingly, the results of Figs. 3 and 4 can be
interpreted as those of an extended draw/fill culture,
during which a large proportion of broth was withdrawn
every 7 d and replaced with fresh medium. Sugar was
not exhausted during each passage; from a maximum
concentration of 30 g 11, total sugar levels at the end of
each 7-d period varied between 21 g 1-1 and 13 g 1-1.
Accumulation of alkaloids was therefore measured
under conditions of prolonged exposure to relatively
high sugar concentration.
Results for alkaloid content of the cultures are reported
in Figs. 3 and 4 as mg per g dry weight of biomass
present at the time of sampling, and as mg per 1 of

351

F i g . 1. Photomicrographs of P. somniferum suspensions 63 days after ini.tiation of hormone-deprived cultures, a, b: cultured witla
exogenous hormones, c, d: cultured without exogenous hormones. Both suspensions consisted mainly of individual cells and small aggregates
with varied size and morphology; giant cells were also present. The bar shown in each photograph represents 100 ~an.

6
"

5
4

"o

10

Time (d)

Fig. 2. CeU concentration during batch culture of P. somniferum

with (11) and without (O) exogenous hormones. The measurements
were started 56 d or 7 passages after initiation of the hormonedeprived culture when the cells had adapted to hormone-free
conditions.

culture fluid. Each datum point represents the average

of at least three measurements. After 14-21 d,
accumulation of morphinan alkaloids in hormonedeprived cultures was greater than when hormones were
provided, and increased with each passage. Levels of
both codeine and morphine were less than the detection
limit for the first 1.4-21 d of culture.
From Fig. 3, morphine levels were up to 2.3
times higher in the culture deprived of hormones; the
maximum specific morphine concentration was 2.5 mg
g-] dry weight compared with 1.1 mg g-I dry weight in
the control culture. Morphine concentration was
approximately constant after 35 d in the culture
supplied with hormones. In contrast, the culture
adapted to hormone-free medium continued to
accumulate morphine up to day 49; after this time the
concentration dropped rapidly presumably due to
further metabolism or morphine degradation.
As shown in Fig. 4, removal of exogenous hormones
increased codeine levels by a factor of about three; the
maximum specific codeine concentration without
exogenous hormones was 3.0 mg g-1 dry weight
compared with 1.0 mg g-1 dry weight with hormones.
Removal of hormones from the medium affected the
proportions of codeine and morphine accumulated in
the cultures. With added hormones, the mass ratio of

352
3

16

16

14

14

J~

12

~2
Y
b~
~

.."

..... ...... e-.....

'\

2
-

=
7

0
14

21

28

35

42

49

56

Time (d)

Fig. 3. Morphine levels in suspended P. somniferura culture with

( El: mg g-1 dry wt; O :mg 1-z ) and without ( II: mg g-I dry wt; O:
mg 1-z) exogenous hormones,

codeine:morphine averaged 0.4 over the 56 d period;

when hormones were withdrawn from the medium this
ratio rose to about 1.3.
Thebaine was detected in only one sample: 1.8 mg g-1
intracellular thebaine was measured in hormone-free
cultures after 21 d. This amount was reduced to zero in
subsequent samples as codeine and morphine synthesis
commenced.

Discussion

Removal of hormones is ordinarily used in plant tissue

culture to induce embryogenesis and promote
differentiation. Previous reports have described
meristemoid (Nessler and Mahlberg 1979) formation,
somatic embryogenesis, and regeneration of plants after
Papaver somniferum cultures were transferred to auxinfree medium (Schuchmann and Wellmann 1983).
Under the conditions employed in this work, removal of
hormones from P. somniferum suspensions did not
cause development of shoots, roots or meristemoids.
Instead, the culture retained its dispersed character and
became habituated to the absence of exogenous
hormones. Hormone-deprived suspensions were
maintained for over 9 months. Development of
hormone self-sufficiency in tissue culture has been
reported for other plant species such as Daucus carota
(Hamilton et al. 1984) and Nicotiana tabacum (Hallsby
and Shuler 1986).
Whether or not morphological differentiation
is required for in vitro production of morphinan
alkaloids has been the subject of considerable debate.
In previous work, thebaine synthesis was shown to
increase with the number of serial passages when
hormones were removed from cultures of P. somniferum
(Schuchmann and Wellmann 1983) and P. bracteatum

.~

~
0

12

~2

~-

2
0

14

21

28

35

42

49

56

Time (d)

Fig. 4. Codeine levels in suspended P. somniferum oalture with

( I:l:rng g"z drywt; O : m g l q ) a n d
mg l-z) exogenous hormones.

without( II:mg g-1 dry wt; O:

(Kamimura et al. 1976; Kutchan et al. 1983). However,

thebaine content was correlated with formation of
embryos in the case of P. somniferum, and formation of
aggregates with bud-like protrusions, shoots and
meristemoids in the case of P. bracteatum. In the
present study, elevated levels of morphine and codeine
were produced in hormone-free medium in the absence
of organ or embryoid development. These results show
that the effect of hormone removal on morphinan
alkaloid production does not depend entirely on
differentiation into organised structures.
Although removal of exogenous hormones alone was
sufficient to elicit a 2-3 fold increase in alkaloid
accumulation, further improvement could be possible if
regeneration were allowed to occur. Kamo et al. (1982)
reported that shoots of P. somniferum contained about
10 times more alkaloid than the callus from which they
were derived; Yoshikawa and Furuya (1985) determined
a 10-25 fold increase in total morphinan alkaloids when
green callus of P. somniferum produced buds.
Disappearance of morphine as codeine and thebaine
levels rise has been demonstrated by several workers
(Fairbairn et al. 1964; Fairbairn and Wassel 1964) even
though the reaction sequence thebaine ---> codeine --->
morphine is considered irreversible in whole plants. If
morphine levels tend to drop during culture, time of cell
harvest becomes an important parameter. The rapid
decline in morphine concentration during the 8th week
of hormone-free draw-fill culture suggests that some
morphine was catabolised after synthesis.
Removal of hormones and enhancement of morphinan
alkaloid production was accompanied by slower growth.
This inverse correlation between growth rate and
alkaloid synthesis confirms the findings of Kamo et al.
(1982) with P. somniferum cultured using various
hormone combinations. In the present work removal of
hormones increased the ratio of codeine:morphine found

353
in the cultures by a factor of about 3.3; higher
percentages of codeine were also found by Kamo et al.
(1982) in slower growing callus.
Acknowledgements. We

are grateful to Russell Cail for assisting with

the ttPLC analyses, and to Malcolm Noble for the photography. This
work was funded by the Australian Research Council (ARC).

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