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Plant C ell Reports (1991) 10:349-353

Plant eU

Reports

9 Springer-Verlag1991

nhanced codeine and m orph ine production in suspended

apaver somniferum

ultures after removal of exogenous hormones

C h u i L i S ia h a nd P a u f i n e M D o r a n

Departm ent of Biotechnology, University of N ew So uth Wales, P.O. Box 1, Kensington NSW 2033, Australia

Received Ma rch 12, 1991/Revised version received June 3, 1991 - Com municated by M. Tabata

S u m m a r y

Morph ine and codeine accumula t ion in

P a p a v e r s o m n i f e r u m suspens ion cu l tu res increased

marked ly a f te r removal o f ho rmones f rom the med ium.

Cul tu res deve loped hormone se l f -su ff ic iency wi thou t

o r g a n o g e n e s i s o r d e v e l o p m e n t o f m e r i s te m o i d s ;

enhanced syn thes i s o f morph inan a lka lo ids was no t

dependen t on fo rmat ion o f shoo ts , roo ts o r embryos .

Wi thou t exogenous ho rmones , max imum codeine and

morph ine concen t ra t ions were 3 .0 mg g -1 d ry weigh t

and 2 .5 mg g -1 d ry weigh t respec t ive ly , up to th ree

t imes higher than in cul tures supplied with hormones.

Ho rm o n e -d e p r i v e d c e l l s p ro d u c e d a h i g h e r r a t i o o f

codeine:m orphine than cul tures supplied with auxin and

cytokinin . Imp roved alkaloid production was correlated

with s lower overal l grow th rate.

I n t r o d u c t i o n

Latex f rom the op ium poppy , Papaver somniferum, is a

commerc ia l sou rce o f the ana lges ics , morph ine and

c o d e i n e . C a l l u s a n d s u s p e n s i o n c u lt u r e s o f P .

s o m n i f e r u m a r e being inves t iga ted as an a l t e rna t ive

means fo r p roduct ion o f these compounds . Amounts o f

theba ine , morph ine and codeine in morpho log ica l ly

undifferentiated cultures hav e been repo rted in the range

0 gg per 100 g d ry weigh t (Yosh ikaw a and Furuya

1985) to 1.5 mg g-1 dry weight (Tam et al. 1980); this

can be compared wi th 1 .4 mg g -] d ry weigh t in l eaf

t issue and 200 m g gq in dried latex (Constabel 1985).

New strategies for improving synthesis of second-

ary metabo l i t es a re a imed a t mimick ing cond i t ions in

the wh ole plant . The environme nt experienc ed by cel ls

in culture is significantly differe nt from that in viv o and

is responsible for diminished product levels. Conditions

affect ing metabolism of suspended plant cel ls include:

( i ) reduced ce l l -ce l l con tac t ; ii) fewer chemica l and

e lec t rochemica l g rad ien ts ; iii) absence o f microb ia l

con tamina t ion ; and iv) presence o f h igh leve l s o f

exoge nous hormones. All of these factors are know n to

a f f e c t d i f fe r e n t i a t i o n a n d s e c o n d a r y m e t a b o l i s m

(L indsey and Yeom an 1983). Immobi l i sa t ion o f p lan t

ce l l s has been used to res to re ce l l -ce l l con tac t and

produc e gradients; el ici tors are being tested with several

species , including P. somni ferum (Heinstein 1985), to

s imula te micr ob ia l a t t ack on p lan t s . Bo th these

techn iques have led to improvemen ts in secondary

metaboli te production.

Exogenous phy tohormones a re no rmal ly requ i red

fo r t i s sue-cu l tu re in i t i a t ion and to p romote g rowth in

vi tro . Choice of hormones also has a profound effect on

the p ro f i l e o f morph inan a lka lo id accumula t ion in P .

somniferum ce l ls (Hodges and Rap opor t 1982; Kam o e t

al . 1982). Ho we ver, horm one levels h igher than those

in in tac t p lan ts may suppress secondary m etabo l ism a t

the sam e time as stimulating growth. Gro wth regulators

such as 2 ,4 -D (d ich lo rophenoxyace t ic ac id ) and NAA

(naph tha lene ace t ic ac id ) have been found to p roduce

chromosomal aberra t ions in p lan t ce l l s (Sunder land

1977; Ba yliss 1980), and are part icularly s uspec t as

mutagen ic agen ts . Fu tu re regu la to ry gu ide l ines fo r

t i s sue-cu l tu re p roducts cou ld res t r i c t u se o f these

substances in production o f pharma ceutical- and foo d-

grade products . Bay liss (1980) has suggested that the

concen t ra t ions o f ho rmones used in t i s sue cu l tu re a re

too low to produc e a s ignificant effect ; howeve r, in view

of the h igh f requency o f ch romosomal changes observed

in many cul tures and the toxic nature of 2 ,4-D, i t would

be preferab le to avoid use o f hormones al together.

Removal o f exogenous ho rmones f rom la rge-

sca le cu l tu re sys tems cou ld be implemen ted us ing a

two -stag e proc ess strategy. A fter high cell densities are

reached with the aid of exogenous hormones, hormone-

f r e e m e d i u m wo u l d t h e n b e a p p l i e d t o s t i m u l a t e

secondary production. Several s tudies in to the effects of

e l ic i to rs and ce l l immobi l i sa t ion have used hormone-

free medium (Brode lius et al. 1979; Shuler et al. 1983;

Barnabas and David 1988; Cline and Coscia 1988); the

extent to which hormone deprivat ion contributed to the

o b s e rv e d i m p ro v e m e n t s i n s e c o n d a ry s y n t h e s i s i s

difficul t to define. This s tud y is aimed at determining

Offprint req uests to:

P.M. Doran

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350

whether removal of hormones from

P a p a v e r

somniferum suspensions could be used to stimulate in

vitro production of morphinan alkaloids.

a t e r ia l s a n d m e t h o d s

Cell suspension cultures

S e e d s o f

Papaver somniferum

L . c u l t i va r

1 4 - 1 4 w e r e k i n d l y d o n a t e d b y T a s m a n i a n A l k a l o i d s P t y L t d ,

A u s t r al i a . C a l l u s i n i ti a t e d fr o m s e e d l i n g h y p o c o t y l s w a s m a i n t a i n e d

o n n u t r i e n t m e d i u m c o n t a i n in g M u r a s h i g e a n d S k o o g ( 1 9 62 ) s a lt s , 3

s u c r o s e , 2 . 5 m g 1-1 c y s t e i n e . H C 1 , 0 . 1 m g 1-1 2 , 4 - D , 2 m g

l t

k i n e t i n , a n d 1 a g a r . T w o y e a r s a f t e r c a l l u s i n i t i a t i o n , c e l l

s u s p e n s i o n s w e r e d e v e l o p e d u s i n g t h e s a m e m e d i u m w i t h ou t a g a r.

S u s p e n s i o n c u l t u r e s w e r e m a i n t a i n e d in s h a k e f l a s k s a t 2 5 ~ o n

o r b i t a l s h a k e r s u n d e r l o w l i g h t i n t e n s i t y ( 2 0 0 l u x ) f o r a p p r o x . 4

m o n t h s b e f o r e t h e s t a r t o f t h e e x p e r i m e n t s d e s c r i b e d b e l o w .

Flas k experiments

T w e l ve 1 - l i tr e E r l e nm e ye r f l a sks e a c h c on t a i n i ng

2 5 0 m l c e l l s u s p e n s i o n w e r e u s e d i n t h e e x p e ri m e n t s . S i x w e r e

s u b c u l t u r e d e v e r y 7 d u s i n g t h e l i q u id m e d i u m d e s c r i b e d a b o v e ; t h e

r e m a i n i n g 6 f l a s k s w e r e s u b c u l t u r e d u s i n g t h e s a m e m e d i u m w i t h o u t

h o r m o n e s . A t e a c h s u b c u l tu r e , 2 0 0 m l f r e s h m e d i u m w a s p l a c e d i n t o

a s te r i l e f l a s k a n d 5 0 m l c e l l s u s p e n s i o n a d d e d . S a m p l e s w e r e

r e m o v e d e v e r y 2 - 3 d o v e r a p e r io d o f 5 6 d . A t t h e e n d o f 5 6 d th e

e x p e r i m e n t w a s c o n t i n u e d f o r a f u r t h e r 1 2 d w i t h o u t s u b c u l t u r i n g to

o b t a i n b a t c h g r o w t h d a t a .

Analyses

C e l l c o n c e n t r a t i o n w a s m e a s u r e d a s d r y w e i g h t a f t e r t h e

s a m p l e s w e r e f i l t e r e d u n d e r v a c u u m , t h e s u p e r n a t a n t r e m o v e d f o r

s u g a r a n d a l k a l o i d a n a l y s i s , a n d t h e r e m a i n i n g s o l i d s f r e e z e - d r i e d

o v e r n i g h t . S u c r o s e , f ru c t o s e a n d g l u c o s e l e v e l s w e r e m e a s u r e d a s

de sc r i be d p r e v i ous l y ( S ha r p a nd D or a n 1990) .

A l k a l o id a n a l y s e s w e r e c a r r i e d o u t o n b o t h c e l l s a n d l i q u i d m e d i u m .

C e l l s w e r e g r o u n d w i t h a c i d - w a s h e d s a n d , w a s h e d w i t h m e t h a n o l a n d

r e f lu x e d f o r 2 h r. T h e s a m p l e w a s t h e n fi l te r e d th r o u g h W h a t m a n N o

1 fi l t e r pa p e r t o r e m ove c e l l de b r i s , a nd w a sh e d f u r t he r w i t h me t ha no l .

T h e m e t h a n o l w a s r e m o v e d f r o m t h e f i l t ra t e u s i n g a r o t a ry e v a p o r a t or .

T h e r e s i d u e w a s r e - d i s s o l v e d i n 1 M H C 1 , a n d c h l o r o f o r m a d d e d . T h e

a que ou s a nd o r ga n i c pha se s w e r e s e pa r a t e d i n a s e pa r a t ing f unne l ; t he

a q u e o u s l a y e r w a s c o l l e c t e d a n d t h e p H a d j u s t e d t o 9 .5 w i t h N H 4 O H .

T h i s s o l u t io n w a s e x t r a c t e d f u r t h e r w i t h 7 5 c h l o r o f o r m - 2 5 i s o -

p r o p a n o l . T h e h e a v i e r o r g a n i c p h a s e w a s d r a i n e d an d r e t u r n e d to a

s e p a r a ti n g f u n n e l f o r b a c k - w a s h i n g w i t h N H 4 O H ; t h e o r g a n i c p h a s e

w a s t h e n c o U e c t e d a n d e v a p o r a t e d . T h e r e s i d u e w a s d i s s o l v e d i n

c h l o r o f o r m , t h e v o l u m e a d j u s t ed t o 4 m l , a n d t h e c h l o r o f o r m

e v a p o r a t e d . T h e r e s i d u e w a s t h e n d i s s o l v e d i n 1 0 m M N a H z P O 4

b u f f e r a t p H 2 . 1 c o n t a i n i n g 1 m M d o d e c y l s u l p h a t e a n d 5 0

a c e t on i t r i l e . Be f o r e H P L C a na l ys i s t he s a m pl e w a s f i lt e r e d t h r ough a

0.22 ~tm filter .

M e d i u m f i l tr a t e f r o m t h e c u l t u r e s w a s e v a p o r a t e d u s i n g a

r o t a ry e v a p o r a t o r , r e - d is s o l v e d in 0 . 5 M a m m o n i u m s u l p h a t e, a n d t h e

p H a d j u s t e d t o 9 .3 - 9 . 5 w i t h N H 4 O H . S a m p l e s w e r e p a s s e d t h r o u g h a

S e p - P a k C 1 8 c a r tr i d g e ( W a t e r s A s s o c i a t e s ) a f t e r t r e a t m e n t o f t h e

c a r t r i d g e w i t h m e t h a n o l . T h e c a r t r id g e w a s w a s h e d w i t h 5 m M

a m m o n i u m s u l p h a t e a d j u s t e d to p H 9 . 3 - 9 .5 w i t h N H 4 O H , a n d t h e n

d i s t i ll e d w a te r . M o r p h i n a n a l k a l o i d s w e r e e l u t e d w i t h 5 0

a c e t on i t r i l e i n 10 m M N a H z P O 4 buf f e r a t pH 2 . 1 .

A l k a l o i d s w e r e s e p a r a t e d a t r o o m t e m p e r a t u r e u s i n g a 3 0 c m x

3 .9 m m i .d . P h e n o m e n e x B o n d e x C l s H P L C c o l u m n c o n t a in i n g 1 0

~ n p a c k in g . T h e m o b i l e p h as e w a s l 0 m M N a H z P O a b u f f e r a t p H

2 .1 c on t a i n i ng 1 m M dode c y l su l pha t e a nd 35 a c e t on i t r i le . T he f l ow

r a t e w a s 1 m l m i n - 1. C a f f e i n e w a s u s e d a s i n t e r n a l s t a n d a r d .

M o r p h i n e , c o d e i n e a n d t h e b a i n e w e r e d e t e c t e d a t 2 5 4 n m w i t h

r e t e n t i o n t i m e s o f 5 . 3 r a i n , 7 . 0 m i n a n d 1 8 . 4 m i n , r e s p e c t i v e l y .

A m o u n t s o f m o r p h i n e , c o d e i n e a n d t h e b a i n e i n s a m p l e s w e r e

d e t e r m i n e d u s i n g s t a n d a rd c u r v e s .

R e s u l t s

Horm one self sufficiency

Papaver somniferum cells adapted to the absence of

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 hormone-

free 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 o f sampling, and as mg per 1 of

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351

Fi g. 1. Photomicrographs of P somniferum suspensions 63 days after ini.tiation of hor mone-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

o

o

o

0

i [

2 4 6 8 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 hormone-

deprived 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 hormone-

deprived 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 concent ration 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

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352

3 , , , 16

I 4

J~

~ 2

Y

b~

~ . . ' \

~ . . . . . . . . . . . e- . . . . 4

~

0 = 0

0 7 1 4 2 1 2 8 3 5 4 2 4 9 5 6

T i m e d )

Fig. 3. M o r p h i n e levels in suspended

P somniferura

c u l t u r e w i t h

( E l : m g g -1 d r y w t ; O : m g 1 z ) a n d w i t h o u t ( I I : m g g - I d r y w t ; O :

m g 1 z ) ex o g e n o u s h o r m o n e s ,

1 2

i i

~ 2

v

4

~-

0 : = 0

7 1 4 2 1 2 8 3 5 4 2 4 9 5 6

Time d )

Fig. 4. C o d e i n e l e v e l s i n s u s p e n d e d

P somniferum oal ture wi th

( I :l : rn g g z d r y w t ; O : m g l q ) a n d w i t h o u t ( I I : m g g -1 d r y w t ; O :

m g l z ) exogenous hormones

1 6

1 4

1 2

a .~

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.

D i s c u s s i o n

Removal o f 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 auxin-

free medium Schuchmann and Wellmann 1983).

Under the conditions employed in this work, removal o f

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 fo r 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

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 react ion 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

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i n the cu l tu res by a fac to r o f abou t 3 .3 ; h igher

percen tages o f code ine were a l so found by Kam o e t a l .

1982) in s lowe r growing cal lus .

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W e a r e g r a t e f u l to R u s s e l l C a i l f o r a s s i s t in g w i t h

t h e t t P L C a n a l y s e s , a n d t o M a l c o l m N o b l e f o r t h e p h o t o g r a p h y . T h i s

w o r k w a s f u n d e d b y t h e A u s t r a l ia n R e s e a r c h C o u n c i l A R C ) .

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