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Plant Cell, Tissue and Organ Culture 54: 173–182, 1998.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands. 173
Establishment of cell suspension cultures of Morinda elliptica for the
production of anthraquinones
M.A. Abdullah1,A.M.Ali
1, M. Marziah2,N.H.Lajis
3&A.B.Ariff
1∗
1Department of Biotechnology, 2Department of Biochemistry, 3Department of Chemistry, Universiti Putra
Malaysia, 43400 UPM Serdang, Selangor, Malaysia (∗requests for offprints; fax: 603 9423552; e-mail:
arbarif@fsb.upm.edu.my)
Received 24 June 1998; accepted in revised form 12 October 1998
Key words: anthraquinones, auxin, cytokinin, inoculum age, medium optimization, Morinda elliptica
Abstract
Morinda elliptica (Rubiaceae) cell suspension cultures were established in shake flask system for the production
of anthraquinones. The optimized medium formulation for cell growth and anthraquinone production is proposed.
Murashige and Skoog’s basal medium (MS) was found to be the best medium, used in combination with 0.5 mg
l−1NAA and 0.5 mg l−1kinetin. At the range of sucrose concentration tested (3–8% w/v), 8% was the best in
enhancing both cell growth and anthraquinoneproduction. A strategy to formulate growth and productionmedium
by manipulating culture age and inoculumage, the type of medium formulation used to grow inoculum, incubation
temperature and light intensity was established. By using 18 month old culture and 7 day old inoculum at incubation
temperature of 27 ±3◦C, anthraquinone yield of 2.9 g l−1and 4.5 g l−1, under illumination of 1200 lux and in
the dark was obtained, respectively.
Abbreviations: AQ – anthraquinones; BAP – 6-benzylaminopurine; B5 – Gamborg et al. (1968); 2,4-Dor D –
2,4-dichlorophenoxyacetic acid; NAA – α-naphthaleneacetic acid; DW – dry weight; IAA – indole-3-acetic acid;
K – 6-furfurylaminopurine (kinetin); MS – Murashige & Skoog (1962); SH – Schenk & Hildebrandt (1972); W –
White (1963); Z – zeatin
Introduction
The genus Morinda of Rubiaceae family is believed
to include about 80 species, mostly of Old World Ori-
gin (Morton, 1992). In Peninsular Malaysia, Morinda
comprises nine species which include three species of
trees (M. citrifolia, M. elliptica and M. corneri)andsix
species of climbers (M. lacunosa, M. rigida, M. calci-
phila, M. scortechinii, M. umbellata and M. ridleyi)
(Wong, 1984).
The natural pigment from Morinda species has
been used traditionally as dyes (Burkill, 1935). An-
thraquinones (AQ) extracted from the root of M.
elliptica have shown antibacterial, antifungal and an-
tileukemic properties (Ismail et al.,1997). Other
Morinda species have been reported to show antibac-
terial, analgesic, anticongestive, hypotensive, seda-
tive and insecticidal activities such as in M. citrifo-
lia (Younos et al., 1990; Dittmar, 1993; Legal and
Plawecki, 1995), antifungal, antiprotozoal, antimalar-
ial and hypoglycaemic activities in M. lucida (Kouma-
glo et al., 1992; Kamanyi et al., 1994; Makinde et
al., 1994; Rath et al., 1995), and anti-tumour and
anti-leukemic activities in M. parvifolia (Chang et al.,
1982; Chang and Lee, 1985; Khanapure and Biehl,
1989). In pharmaceutical industry, AQ glycosides are
used in the production of Pyralvexrto treat gingivitis,
stomatitis, mouth ulcers, inflammatory oral mucosa
and periodontal conditions. Another member of the
AQ group, senna, is used in the preparation of stim-
ulant laxative drugs Senokotr, to treat constipation or
for bowel evacuation prior to abdominal radiological
procedures (DIMS, 1992; British National Formulary,
1994).
174
Members of the Rubiaceae family are among the
few plant families, easily taken into cell cultures to
produce substantial amount of AQ (Zenk et al., 1975;
Suzuki et al., 1982). In Rubiaceae species belonging to
the genera Asperula, Galium, Rubia and Sherardia,17
out of 19 cases of AQ productionin optimized cell cul-
tures surpassed that of differentiated plants whereby
maximum AQ yield of 1.7 g l−1was observed in Gal-
ium verum cell cultures (Schulte et al., 1984). Cell
suspension cultures of M. citrifolia yielded 2.5 g l−1of
AQ under optimal conditions, which exceeded those
of differentiated tissue by a factor of 10 on a dry
weight basis (Zenk et al., 1975). The optimization of
medium and culture conditions for AQ production by
M. elliptica cell suspension cultures has not yet been
reported.
The objectives of this study were to establish cell
suspension cultures of M. elliptica for subsequent
use in AQ production by manipulating the different
medium formulations and different types of hormone
and their combination. The effect of culture and inocu-
lum age, incubation temperature and light intensity on
cell growth and AQ production was also studied.
Materials and methods
Callus cultures
Callus of M. elliptica was induced from young leaves
(Aziz et al., 1997). Cultures were maintained on MS
medium fortified with 30 g l−1sucrose, 2 mg l−12,4-
D,1mgl
−1kinetin and 2.5 g l−1Phytogel (Sigma
Chemical Co.), under 16-h photoperiod at illumination
of 2000 lux. The incubation temperature was main-
tained at 24 ±2◦C. Subculture of callus cultures was
made at a regular period of one month.
Suspension cultures
Suspension cultures were developed by inoculating
2–5 g of callus into liquid medium. Subculturing in-
volved the transfer of 5 ml batches of suspension
cultures into 35 ml of MS medium in 100 ml Erlen-
meyer flasks using the same formulation as for callus
cultures minus phytogel. In this report, the mainte-
nance medium formulation of suspension cultures will
be termed as an M medium. The flasks were sealed
with stoppers and placed on an orbital shaker (130
rpm) at 24 ±2◦C, at light intensity of 500 lux, for
16-h photoperiod. For the preparation of stock cul-
tures, 10 ml of suspended cells were inoculated into
90 ml medium in 250 ml Erlenmeyer flasks. After 9
to 12 days, the stock cultures could be used as in-
oculum. With increasing age of cultures, the stock
cultures were best subcultured after 6 to 8 days to
ensure healthy cells being used as inoculum. Unless
stated otherwise, all the experiments were conducted
using the M formulation and culture conditions.
Analytical procedures
The flasks were removed in triplicate at time intervals
for analysis. The cells were harvested by suction filtra-
tion via a Buchner funnel with a filter paper(Whatman
No. 4). The cells were dried in an oven at 70 ◦Cfor
24 h to obtain dry cell weight (DW). For AQ content
analysis, 0.020 ±0.001 g dry cells were extracted
in 2 ml of dichloromethane for several times until
the extractant in the final extraction became colour-
less. To speed up the release of AQ, the dried cells
were slightly wetted with deionized water (withoutex-
cess water) and left for a few minutes before adding
dichloromethane, and the samples were gently shaken
on a shaker at 90 rpm. The amount of AQ extracted
were measured spectrophotometrically at 420 nm us-
ing alizarin as a reference substance (Zenk et al.,
1975). All data presented in figures and tables are the
mean values with the standard errors.
Results and discussion
Establishment of callus and cell suspension cultures
The callus cultures of M. elliptica were friable, sticky
and yellowish in colour. Healthy and young cells
showed bright yellow colour but turned brownish as
they grew older and subsequently resulting in the
medium becoming increasingly yellowish due to the
release of AQ. The establishment of cell suspension
cultures was done after the callus cultures were about
one and a half year-old. After the inoculation of callus
cells into liquid medium, it took about two months
after several subcultures, for the suspended cells to
become more homogeneous and be free from callus
debris. The freely suspended cells were also yellowish
in colour and made up of very fine cells and visually
uniform in size. The clumpy aggregatesize of between
0.5–10 mm as being reported for Capsicum frutescens
(Mavituna & Park, 1987; Williams et al., 1988) and
Pilocarpus pennatifolius cell suspension cultures (Ab-
dullah, 1994), was not observed in the cultures of M.
175
Figure 1. Time course of dry weight and anthraquinone produc-
tion of Morinda elliptica cell suspension cultures in different me-
dia formulations. (A) dry weight; (B) anthraquinone content; (C)
anthraquinone yield. (♦)MS;()SH;(1)B5;()W.
elliptica. This renders the cultures several advantages
for their application in bioreactors.
Effect of medium formulation
Effect of different medium formulations on growth
and AQ production of M. elliptica cell cultures is
shown in Figure 1. Except for White’s (W), the other
medium formulations tested were capable of support-
ing the growth of cells. A lag phase of about 6 days
was observed, followed by rapid growth thereafter. In
all cases, except forSH, growthattained its maximum
on day 12. The maximum cell concentration on day
10 obtained in SH medium was about 24% and 50%
higher than in MS and B5 media, respectively. A sig-
nificant increase in AQ production during the early
stages of growth was only observed for cultures in MS
and W media. In both cases, the AQ production grad-
ually increased during the early slow-growing phase
before dropping sharply during exponential phase and
reached a steady-state during death phase. This ob-
servation is consistent with the suggestion that plant
secondary metabolites are produced at higher con-
centrations in slow-growing or non-growing cultures
(Kurtz and Constabel, 1985: Rokem and Goldberg,
1985). Even though the AQ yield in SH was the
highest at 0.05 g l−1on day 10, it was incapable of
promoting AQ content above 4 mg g−1DW. On the
other hand, MS medium was effective in promoting
both cell growth and AQ content. Hence in this study,
MS medium was chosen as the medium to undergo
further investigations.
Effect of sugar concentration
Sucrose or its component monosaccharides, glucose or
fructose, were the best carbon sources for the growth
of most plant cell cultures (Maretzki et al., 1974).
Out of 14 carbohydrates tested at 2% concentration
on M. citrifolia cell cultures, sucrose gave the high-
est yield of AQ (Zenk et al., 1975). However, 5%
glucose produced higher AQ yield than 5% sucrose
in Rubia cordifolia cell cultures (Suzuki et al., 1984).
In our studies, sucrose was superior to glucose, fruc-
tose and mannitol in promoting growth of M. elliptica
cell cultures when tested at 3% and 5% concentra-
tion (data not shown). Although higher AQ content
was obtained at 5% glucose (4.0 mg g−1DW) than
at 5% sucrose (3.4 mg g−1DW), maximum DW at
5% glucose (11.5 g l−1) was only half of that obtained
at 5% sucrose (23.7 g l−1). Thus, the AQ yield at 5%
sucrose (0.081 g l−1) was almost 2-fold higher than at
5% glucose (0.045 g l−1).
The effect of sucrose concentration on growth and
AQ production of M. elliptica cell cultures is shown
in Figure 2. The highest cell concentration on day 12
was obtained at 5% and 6% sucrose which was around
25gl
−1but AQ content at 7% and 8% sucrose was
176
Figure 2. Effect of sucrose concentration on growth and an-
thraquinone production of Morinda elliptica cell suspension cul-
tures. () dry weight; () anthraquinone content.
higher than at 3% sucrose by 1.6 and 1.4 times, re-
spectively. This result resembles that obtained by Zenk
et al. (1975) and Suzuki et al. (1984) for M. citrifolia
and R. cordifolia cell suspension cultures, respectively,
where 5% sucrose gives maximal growth and 7% su-
crose gives maximal total AQ production. As to the
lower DW recorded for 8% sucrose than that at 5, 6
and 7%, the maximum DW could have been achieved
at later days of culture instead of on day 12. The spe-
cific growth rate of Nicotiana tabacum var. Xanthi,
for example, decreased at high sucrose concentration
(Yasuda et al., 1972). This therefore increased the dou-
bling time and resulted in maximum DW attained on
later days. Later on, we observed that cultures in 8%
sucrose obtained highest concentration between day
15 and 18 of culture period.
Effect of hormone combination and concentration
Our studies with 6 month old cultures (data not shown)
utilising various auxins and cytokinins at 2:1 ratio,
showed that AQ content was promoted whenever 2,4-
Dwas in the medium. Cell growth was however, lower
in the presence of 2,4-Dthan in the presence of NAA
and IAA. For cytokinins, the presence of zeatin and
kinetin improved cell growth while the incorporation
of BAP suppressed cell growth. In all the cytokinins
tested, little variation on AQ content was observed, as
compared to when different auxins were utilised. After
one year of culture in M medium, the effect of 2,4-Don
AQ content was not far different from IAA and NAA
but the cell concentration remained slightly lower, in
Figure 3. Effect of different combinations of NAA and kinetin on
growth of Morinda elliptica cell suspension cultures.
the presence of the former than in the latter two aux-
ins. Similar observation was made in R. cordifolia
cell cultures where inclusion of 2,4-Ddoes not inhibit
AQ formation (Suzuki et al., 1984). In M. citrifolia
cell cultures however, at none of the concentrations of
2,4-Dtested was AQ biosynthesis induced. AQ were
only produced when NAA was included in the basal
medium (Zenk et al., 1975; Hagendoorn et al., 1994;
Van der Plas, 1995). NAA has been suggested to be
able to switch on the expression of genes responsible
for the various enzymes necessary for the production
of AQ (Hagendoornet al., 1994). The absence of pro-
duction with 2,4-Dwas proposed due to either a lack
of induction or repression/inhibition of these enzymes;
and also due to the lack of sufficient carbon-skeletons
for the secondary metabolite pathway which might
prevent an ‘overflow’ in the direction of the synthesis
of AQ. This suggestion appears to hold little truth in
the cell cultures of M. elliptica and R. cordifolia.
Effect of 2,4-Dand NAA with kinetin at various
concentrations are shown in Figures 3 and 4. Cell
growth was generally higher at 0 and 0.5 mg l−1NAA
and in the absence of 2,4-D, in combination with all
tested concentrations of kinetin. This observation was
similar to R. cordifolia cell cultures where NAA con-
centration between 0–0.6 mg l−1was favourable for
cell growth and AQ production (Suzuki et al., 1984).
Although cell growth of M. elliptica in NAA between
0.5to5mgl
−1remained high around 16–17 g l−1
(Figure 3), NAA between 2 to 5 mg l−1in R. cordifolia
cell cultures, was inhibitory to cell growth and even
ineffective for AQ formation. In the hormone-free
177
Table 1. Effect of sucrose concentration and different hormone combina-
tions on growth and anthraquinone production of Melliptica cell suspension
cultures.
Sucrose concentration Hormone Dry weight AQ content
(% w/v) combination (g l−1)(mggDW
−1)
3 0.5D,0.5K 16.4 ±0.3 2.51 ±0.19
0.5N,0.5K 16.6 ±0.1 2.46 ±0.10
0.5I,0.5Z 16.3 ±0.3 2.67 ±0.03
2D,0.5K 16.4 ±0.4 2.53 ±0.21
2N,0.5K 18.3 ±0.4 2.88 ±0.13
2I,0.5Z 17.7 ±0.6 2.78 ±0.36
2D, 1K 14.7 ±0.7 2.74 ±0.08
5 Hormone-free 33.0 ±0.4 1.69 ±0.06
2N,0.5K 32.0 ±0.1 2.20 ±0.10
7 0.5D,0.5K 24.8 ±0.7 2.16 ±0.06
0.5N,0.5K 38.0 ±3.3 3.87 ±0.38
2D,0.5K 19.3 ±0.8 2.68 ±0.12
2N,0.5K 27.6 ±0.5 3.13 ±0.06
2D,1K 18.9 ±1.2 2.98 ±0.21
8 0.5N,0.5K 30.9 ±1.1 6.15 ±0.46
2D,1K 21.2 ±0.1 4.97 ±0.42
xA, yB–xmg l−1of A and ymg l−1of B (A is auxins and B is cytokinins).
Values are means of three replicates with ±standard errors.
Figure 4. Effect of different combinations of 2,4-Dand kinetin on
growth of Morinda elliptica cell suspension cultures.
medium, growth of around 17 g l−1was obtained in
M. elliptica cell cultures, but the AQ content (data not
shown) was lower than when hormones were incor-
porated (as also confirmed at 5% sucrose in Table 1).
Cells grown in the hormone-free medium were dead
after third passage of subculture which confirms the
suggestion on the importance of incorporating auxins
in the medium for continuous growth of cells (Van der
Plas et al., 1995). In addition, we proposed the im-
portance of incorporating hormones in the medium to
accentuate AQ production.
After screening different hormone combinations at
sucrose 3, 5, 7 and 8% (Table 1), we observed that
both growth (>30 g l−1) and AQ content (>6mgg
−1
DW) were promoted at 8% sucrose and 0.5N, 0.5K
combination. AQ content in all other combinations of
hormones at lower sucrose concentrations remained
below 4 mg g−1DW. In 3% sucrose, at none of the
combination of 2,4-Dand NAA (between 0 to 5 mg
l−1) with kinetin (between 0 to 2 mg l−1), was there
any significant increase in AQ production (data not
shown). However, with 8% sucrose, both 2,4-Dand
NAA promoted growth and AQ content (Table 1). In
M. citrifolia cell cultures, where inclusion of 2,4-D
switches off AQ production but appeared to induce
AQ production in hormone-free medium, NAA may
just play a ‘permissive’ role by allowing AQ produc-
tion without actually having an active role of activating
transcription in the induction of AQ-synthesis pathway
(Van der Plas, 1995). Our studies showed that carbon
source and its concentration rather than hormones, ex-
178
erts far greater influence in promoting cell growth and
AQ production. This seems to justify the ‘permissive’
role of hormones in AQ production.
Formulation of growth and production medium
Zenk et al. (1975) suggested that optimal growth can
be separated from optimal production which there-
fore made the formulation of growth and production
medium possible. Fujita et al. (1982) concluded that
a culture grown in a combination of two media might
produce both shikonin derivatives and cell growth in
Lithospermum erythrorhizon cell cultures because the
use of multiple media would compensate for a sin-
gle medium’s deficiencies. In our studies, three types
of formulation were proposed – maintenance medium
(M), growth medium (G) and production medium (P).
M formulation was as described before while G and P
formulation was MS medium fortified with 80 g l−1
sucrose, 0.5 mg l−1NAA and 0.5 mg l−1kinetin. The
only difference between G and P is that the inoculum
for G is grown in M, while inoculum for P is grown in
G. Upon harvesting, cells in M and G were yellowish
in colour while cells in P were reddish brown. Under
microscope, we observed trace of yellow colours in M
cells, pale yellow colours in G cells and high yellow
colour intensity in P cells, which respectively indi-
cated the concentration of AQ within the cells. Further
discussion on G and P formulation can be found in
later part of the report.
Our studies with 7% sucrose at 2D, 0.5K hormone
combination (data not shown) indicated that 12, 10
and 7-days old inoculum resulted in maximum cell
concentration of 4.6, 14.0 and 19.3 g l−1, respectively.
When tested in G and P (Table 2), younger inoculum
age gave higher cell concentration and AQ production.
Inoculum cultured in G up to 10–14 g l−1survived
well in P, while lower concentration in G upon inoc-
ulation into P may end up dead or had its cell growth
or AQ production retarded. Inoculum age became an
even more importantconsideration in our studies with
antifoaming agent (data not shown). With the addition
of 0.025% (v/v) silicone antifoaming agent, inoculum
cultured in M up to 7–11 g l−1survived well in G
(comparable to control), while inoculum in M at 11.8 g
l−1andat14.7gl
−1had cell growth in G, respec-
tively, suppressed by 50% and completely dead. The
concentrations of inocula which survived upon inocu-
lation into G or P actually correspond to the stage of
early exponential phases in the growth cycle of cells
in M and G which is between day 5–7 in M and day
6–8 in G. In our studies with 6 month-old culture
in M, utilizing different inoculum densities between
10–14.3% v/v (data not shown), cultures from 14.3%
(v/v) inoculum size grew fastest, followed by 12.5%,
11.1% and 10% (v/v). While the rest achieved max-
imum concentrations on day 12, cultures from 10%
(v/v) inoculum reached its maximum on day 15 in-
stead. AQ content in all cultures achieved maximum
values on day 6 but start to drop thereafter (same pro-
file as in Figure 1). However, the AQ profile of 10%
(v/v) inoculum showed that its content was lower than
the rest on day 4 and higher than the rest on day 6
till day 12 and 15. Therefore, it can be misleading
to describe that excess inoculation inhibited the pro-
duction rather than increased the shikonin yield (Fujita
and Hara, 1985). By harvesting all the cultures at the
same time interval of 14 days, cultures from lower in-
oculum density may be only in their late exponential
phase while those from higher inoculum density, could
have already been in their declining phase. Hence,
lower shikonin contentin Lithospermum erythrorhizon
cell cultures obtained from using higher inoculum size
(Fujita and Hara, 1985), could have resulted from at-
tainment of maximum cell growth or production much
earlier than day 14. At this stage, the effect of inocu-
lum age can therefore also be seen as the effect of
inoculum density.
Effect of incubation temperature and light intensity
Incubation at 25 ±1◦C and 27 ±3◦Cshowedbet-
ter cell growth than at lower incubation temperature
(Table 3). At these incubation temperatures, presence
or absence of light made no significant difference on
cell concentrations, though in general cell concentra-
tion in the dark cultures was slightly lower. However,
absence of light in 18 month old cultures at 27 ±3◦C
promoted AQ content in g and P by 70% and 100%
respectively, when compared to cultures under illumi-
nation. There was no influence of light illumination on
M. citrifolia cell cultures (Zenk et al., 1975).
Utilizing these findings, we had grown cells for
24 days duration under optimized culture conditions
and their performances are givenin Table 4. The max-
imum AQ yield under illumination and in the dark
was2.9gl
−1and4.5gl
1, respectively. This gave
an overall AQ productivity of 0.0058 g l−1h−1and
0.0089 g l−1h−1, respectively. Based on the report by
Stöckigt et al. (1995), these values could be among
the highest for the natural products formed by plant
cell suspension cultures, ranging between 2.5 g l−1
179
Table 2. Effect of culture and inoculum age on growth and anthraquinone production of M.
elliptica cell suspension cultures in growth (G) and production (P) medium.
Culture age (months) Inoculum age Inoculum age Dry weight AQ content
in M (days) in G (days) (g l−1) (mggDW
−1)
Gmedium
15 – 12.2 ±0.1 6.10 ±0.91
6 10 – 27.4 ±3.3 6.80 ±0.34
7 – 41.2 ±0.5 6.03 ±0.19
Pmedium
6 101134.7±0.4 32.7 ±1.17
71131.8±2.1 70.7 ±13.4
18 7 11 33.9 ±1.1 38.0 ±0.94
7 8 56.4 ±0.9 53.8 ±1.13
Values are means of three replicates with ±standard errors.
Table 3. Effect of incubation temperature, light intensity and culture age on growth and
anthraquinone production of M. elliptica cell cultures in growth (G) and production (P)
medium.
Culture age (months) ◦C Light intensity Dry weight AQ content
(lux) (g l−1)(mggDW
−1)
Gmedium
623±2 500 lux 20.8 ±2.2 7.67 ±0.40
25 ±1 500 lux 41.2 ±0.5 5.97 ±0.13
18 23 ±2 500 lux 29.7 ±0.8 6.35 ±0.57
25 ±1 1200 lux 52.3 ±1.0 7.06 ±0.65
Dark 51.8 ±0.1 8.02 ±0.65
27 ±3 1200 lux 53.5 ±0.4 7.19 ±0.30
Dark 47.7 ±4.9 12.1 ±0.10
Pmedium
623±2 500 lux 18.5 ±3.7 81.6 ±7.9
Dark 18.8 ±3.6 67.6 ±11.9
25 ±1 1200 lux 22.5 ±0.3 58.2 ±7.4
Dark 16.9 ±1.7 64.2 ±12.3
27 ±3 1200 lux 31.8 ±2.1 72.7 ±7.9
Dark 29.3 ±5.8 81.6 ±1.6
18 23 ±2 500 lux 25.1 ±1.0 23.6 ±2.2
25 ±1 1200 lux 33.9 ±1.1 38.0 ±0.9
27 ±3 1200 lux 50.4 ±0.4 32.7 ±1.3
Dark 52.8 ±1.6 67.3 ±6.0
Values are means of three replicates with ±standard errors.
of anthraquinones in M. citrifolia and7.0gl
−1of
berberine in Coptis japonica cell cultures. Although
maximum cell concentration in G is comparable to P,
AQ production in the former is low (see Tables 3 and
4). In contrast, both cell growth and AQ production
are high in P. Thus apart from the conventional idea
of defining growth medium (high growth, low produc-
tion) and production medium (high production, low
growth), we are proposing here another perspective in
looking at the M, G and P formulation. We are focus-
180
Table 4. Cell growth and anthraquinone production of M.elliptica cell suspension cultures in growth (G) and production medium
(P) under optimized culture conditions.
Medium and Maximum Maximum Maximum Yield of cell Yield of AQ Overall AQ
culture conditions dry cell weight AQ content AQ yield based on sucrose based on sucrose productivity
(g l−1)(mggDW
−1)(gl
−1) hydrolysed hydrolysed (g l−1h−1)
(g g−1)(gl
−1h−1)
Gmedium
Illumination
(1200 lux) 54.8 ±0.8 7.23 ±0.13 0.35 0.85 0.003 0.0009
Dark 49.0 ±0.5 13.6 ±0.10 0.65 0.63 0.004 0.0013
Pmedium
Illumination
(1200 lux) 58.8 ±1.0 52.9 ±6.25 2.92 0.96 0.038 0.0058
Dark 59.7 ±0.6 80.4 ±1.00 4.48 1.11 0.067 0.0089
Values are means of three replicates with ±standard errors.
ing on the differences between their growth rates and
production capacities together. The cell doubling time
in M, G and P are 54 h, 52 h and 76 h, respectively,
while maximum AQ content are approximately 6, 8
and90mgg
−1DW, respectively. Thus, based on this
suggestion, it is possible for G and P to have compara-
ble maximum cell concentration but the time interval
for cultures to achieve their maximum concentration
will be shorter in G than in P. However, the production
capacity remains higher in P.
In G and P, both high cell growth and AQ pro-
duction are obtained during the stationary phase. As
growth and secondary metabolite production both
compete for the same pools of substrates, these
processes often are separated in time: productionstarts
after completion of the growth phase (Hagendoorn et
al., 1994). Thus in G and P, although cell growth
and production of AQ both take place at the same
time, substantial amount of substrates could have been
channelled towards promoting the cell growth, with
smaller proportion being used for AQ production.
Once growth ceases, the precursors and energy devel-
oped during growth phase will be focussed at greater
intensity on AQ production. Ironically, the opposite
seems to be true in M where maximum AQ content is
obtained during the late lag phase or early exponential
phase and drops sharply as cells enter the exponential
phase (Figure 1). This shows that AQ accumulation
actually starts as soon as cells entered the medium
whether in M, G or P. Therefore, the different produc-
tion capacities in each medium formulation can bestbe
explained in terms of the extent of osmotic stress ex-
perienced by the cells. Soluble sugars, mainly glucose,
fructose and sucrose are often referred to as the sub-
stances responsible for osmotic adjustment in tissues
under osmotic stress (Irigoyen et al., 1992; Prema-
chandra et al., 1992). Increase in the concentration of
sugar in the medium results in a more negative water
potential of the medium, which might inhibit growth.
However, it is highly probable that the growth of plants
in vitro supplied with exogenous sugars, is stimulated
by the influx of sugars and at the same time is inhib-
ited by low water potential of the medium (Lipavska
and Vreugdenhil, 1996). Thus in P, the cells are ex-
periencing ‘double’ osmotic stress that is as inoculum
grown in g and as cell cultures in P, due to high su-
crose concentration in both formulations. This slow
growth rate may have favoured more AQ accumula-
tion as have also been demonstrated in tissue cultures
of Capsicum frutescens where the production of cap-
saicin is inversely related to the culture growth rate
(Lindsey and Yeoman, 1984; Lindsey, 1985, 1986).
Dry weight composition of AQ-producing NAA-
cells was higher than non-AQ producing 2,4-D-cells
in M. citrifolia cell cultures (Hagendoorn et al., 1994)
due to higher amount of soluble sugars and AQ in
NAA-cells. On the other hand, 2,4-D-cells contained
higher amount of lipids and especially proteins but
lower sugar contents. High dry cell weight in both G
and P could be attributed to these high endogenous
sugar levels. Based on the growth rate of cells, em-
phasis in M and G appears to be laid upon growth
with high cell division rate and high metabolic activ-
ity but lower production; while emphasis in P is laid
on production, lower cell division rate and metabolic
activity.
181
Effect of culture age
Table 3 also shows the effect of culture age on growth
of M. elliptica and AQ production in different me-
dia. The cell concentration in M for the first three
months was low but started to pick up after 6 months
as the cells got acclimatized with the culture condi-
tions and becoming stable at 14–17 g l−1.However,
a steady decrease in AQ content was observed from
6–7 mg g−1DW to a mere 2–4 mg g−1DW through-
out 18 months culture period. After one year, we
also observed a shift of the day on which maximum
AQ were detected, from day 6 to day 3–4, though
the AQ content was maintained around 2–4 mg g−1
DW. Hence, with increasing age of cell cultures, 2,4-
Dno longer retained the capability to promote AQ
production, as was initially observed in 6 month-old
cultures. In G, cell concentration dropped from 45–
55gl
−1in 18 month-old culture to 35–45 g l−1
in 27 month-old culture while AQ content dropped
from 6–8 mg g−1DW to around 3–5 mg g−1DW.
In P, cell concentration dropped from 35–55 g l−1
after 18 months, to 30–40 g l−1after 27 months,
while AQ content dropped from 80–90 mg g−1DW
in 6 month-old culture, to 45–60 mg g−1DW after
18 months and to around 25–35 mg g−1DW after
27 months. Therefore there was a drop of 30% and
60% in AQ content after 18 and 27 months in P,
respectively.
The difficulties in maintaining the compound pro-
duction in plant cell cultures are widely recognized.
This phenomenon can be partly explained in terms
of the quality of the inoculum in M. As the culture
age is increasing, there is a gradual build-up of cell
concentration after every subculture due to improved
growth rate. This could be seen after 6 months, where
the frequency of subculture had to be reduced from
every 12–15 days, to every 8–10 days, and after one
year to every 5–7 days. This could have resulted in
the inoculum after 7 days in 18 month-old cultures,
and after 5 days in 27 month-old cultures, to be at the
same stage in the growth cycle. Hence, it becomes
imperative to look at the inoculum density together
with the inoculum age. We have reduced the inocu-
lum density in M from 12.5% to 7.9% (v/v) after
27 months of culture, and frequency of subcultur-
ing increased from 5 days interval to 7 days interval.
Manipulation of the quality of inoculum could be
the alternative answer, in addition to the manipula-
tion of culture medium and condition, to improve the
AQ yield. Research is going on now to improve the
inoculum quality further by using healthy, younger
inoculum age and appropriate inoculum density from
high-yielding cell-line.
Conclusions
Studies on the M. elliptica cells cultures in mainte-
nance medium showed that with increasing age of
cultures, a steady decrease in AQ yield was observed
whilst cell growth becoming increasingly stable. It be-
comes important to devise strategies to improve cell
growth and AQ production. This was done by using
younger inoculum age and combinationof 0.5 mg l−1
NAA, 0.5 mg l−1kinetin in 8% (w/v) sucrose. Cul-
tures grown at incubation temperature of 27 ±3◦C
under the influence of light and in the dark,resulted in
AQ yield of 2.9 g l−1and 4.5 g l−1, respectively. These
were among the highest yield ever being reported for
plant cell cultures.
Acknowledgements
This work was supported by Malaysian government
under IRPA program (Grant no: 03-02-04-0046). We
wish to thank Musa Al-Bakri and Rosli Mohd Din for
their assistance in some parts of the experiment.
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