In vitro establishment of Cell Suspension Culture for Secondary Metabolite Production in

Andrographis paniculata Nees.

HARI PRIYA.S, M.KANNAN and K.MANIAN

Department of Floriculture and Landscaping, Horticultural College and Research Institute,

Tamil Nadu Agricultural University, Coimbatore - 641 003

Andrographis paniculata Nees. (Acanthaceae), universally known as “King of Bitters” or

“ The Creat ” is accredited worldwide for its anti-inflammatory, anti-hepatitic, anti-diabetic and anti–
HIV activity, apart from a renowned immune booster. The major constituents of Andrographis are
diterpene lactones (free and glycosidic forms) including andrographolide, deoxy-andrographolide,
11,12 - di dehydro-14 -deoxy-andrographolide,neo-andrographolide, deoxy-andrographiside,
andrographiside and andropanoside (6). Among these, andrographolide, a colourless, extremely bitter,
crystalline compound, was considered as the drug of commerce. These compounds are mostly organ
and tissue specific, thus an investigation was carried out for producing the secondary metabolites
responsible for these activities under in vitro condition employing plant tissue culture techniques. Plant
cells and tissue cultures hold great promise for controlled production of a myriad of useful secondary
metabolites in demand.

MATERIALS AND METHODS

Establishment of cell suspension culture

Establishment of cell suspension includes initiation and maintenance of suspension culture in

Andrographis paniculata Nees.

Initiation of suspension culture

The callus obtained from vegetative phase (30 - 45 days old seedlings) leaf bits of Andrographis was
used to initiate the suspension culture for secondary metabolite production. The vegetative phase leaf

bits (0.5 - 1.0 cm2 ) inoculated in full strength solid MS medium (4) fortified with 3 % sucrose and
plant growth regulator combination of 2,4-Dichlorophenoxy acetic acid (1 mg/l) and Naphthalene
acetic acid (1 mg/l) was found to be the best medium for initiation and maintenance of callus culture
specifically for in vitro production of secondary metabolite.

The callus cells of 0.50 g and 1.00 g fresh weights were carefully transferred to 100ml Erlenmyer flask,
containing 30 ml of the liquid callus induction medium i.e., full strength liquid MS medium containing
3 % sucrose fortified with 2,4-D (1 mg/l) and NAA (1 mg/l). The transfer was aseptically carried out in

laminar flood hood. The cells were then cultured at 120 rpm on a gyratory platform shaker at 25+ 2 0C
with an orbital motion stroke of 2-4 cm in continuous light of less than 2000 lux on gyratory platform
shakers for 15 days.

Sub culturing of suspension culture

To maintain the batch culture, the primary cell suspensions were sub cultured at 15 days interval by
transferring half the amount of cell suspension to fresh suspension media to make up the final volume
to 30 ml. The transfer was aseptically carried out in laminar flow hood and other procedures are as
same as mentioned above. The culture was maintained until sufficient amount of cell suspensions were
obtained for carrying further analysis.

Growth assessment of suspension culture

The growth analysis of cells in cell suspension culture was assessed through packed cell volume
(PCV). A known volume of uniformly dispersed suspension culture was transferred to 15 ml graduated
plastic centrifuge tube and centrifuged at 2000 rpm for 5 minutes. After centrifuging, the supernatant
was thoroughly decanted to measure the volume of the cell. PCV is the volume of the pellet as a
function of the volume of the culture and is usually expressed as percentage. The observations for the
weight of the initial inoculum and PCV were recorded at regular intervals.

RESULTS AND DISCUSSION

Secondary metabolite production

A plant cell in an advanced state of specialization would undergo differentiation
i.e., embryonisation and provide new cells that may have potentials to exhibit other types of
specialization. Callus and cells in suspension are the least specialized and relatively homogenous, thus
amenable to physio-chemical manipulations to arrive at a meaningful interpretation of cell
differentiation. Product accumulation is a result of secondary metabolism. Secondary metabolites are
synthesized mostly at the time of differentiation. It includes product formation, storage, excretion,
uptake and degradation in each individual cell (1).

Initiation of suspension culture

The Callus tissues retain the characteristics of the plant from which they are derived in their
biosynthetic and regenerative potential. In order to prevent the callus cells from developing further, a
cell suspension culture was established, where cell multiplication was the prime focus. Transfer of
callus pieces in to flasks with a liquid callus induction medium initiates the cell suspensions, where the
growth rate of the suspension-cultured cells in liquid medium is generally higher than that of the solid
medium. The liquid MS medium containing 3 % sucrose fortified with plant growth regulators
combination of 2,4-D (1 mg/l) and NAA (1 mg/l) was found to be the best culture medium for
establishing the suspension culture.

The suspension was then placed on gyratory shaker to provide aeration to the cells and uniform
dispersion of cells in suspension (5). A culture consisting of a high percentage of single cells and small
clusters of cells was considered as a “good suspension” (3). Suspensions of free cells being devoid of
external constraints and practically free of chemical gradients associated with growth centers, the cells
undergo division in all planes indicating randomness and ease of cell division, thus amenable to
manipulation by external applications. Immediately on culturing, depending on the cell density, cell
division is resumed after a lag phase leading to the exponential or logarithmic growth phase, during
which there is an increase of biomass.

Sub culturing of suspension culture

Cell suspensions are maintained by sequential sub culture during early stationary phase and at a time
when cell aggregation is maximal. The cells in culture may be genetically identical (homogenous
population) or may show some genetic variation (heterogenous population). In order to avoid the
genetic variation in suspension culture, the early stationary phase has to be identified for optimum sub
culturing. Based on the cell behaviour in suspension culture, the optimum stage of sub culturing was
identified to be within 12-14 days and 18-20 days is considered to be the critical stage for sub culturing
to get maximum secondary metabolite production.

Single cells do not readily divide, whereas cell division and incidence of mitosis were higher in cell
aggregates. Frequent sub culturing cause increased cell aggregation and stimulation of cell division.
The cell aggregates increase the biomass and coupled with viscosity of the medium, tend to rise
exponentially. Following the lag phase, the cells begin to grow at an increasing rate until growth
becomes exponential. It is this behaviour of limited duration, the population passes through a period of
declining growth rate until stationary phase is reached, where the net growth of the cell has ceased. The
decline in growth is due to the exhaustion of nutrients in the medium or by the formation of toxic
products of cell metabolism or both (8).
Growth assessment of suspension culture

The data on packed cell volume (Table.1) revealed that it increases with increased initial inoculum of
1.0 g of callus per 30 ml liquid medium and was maximum during the period between 36 to 72 days of
culturing.

After initiating the cell suspension with 0.5 g initial inoculum, the cell volume slowly increased by 12
% in 12 days, 16 % in 14 days and 20 % in 36 days respectively. Within 48 to 60 days duration, the
cells increased only by 6 % in volume and then gradually started to decrease by 14 % in 60 days, 14 %
in 72 days and 16 % 90 days. On an average, the cell volume increased by 32 % starting from the initial
inoculum at a duration of 90 days (Table.1).

In case of 1.0 g initial inoculum, the cell volume slowly increased by 12% in 12 days, 16% in 14 days
and 24% in 36 days regularly. Within 48 to 60 days duration, the cells remained only by 8% increase
and then gradually started to decrease by 7% in 60 days, 14% in 72 days and 12 % 90 days. On an
average, the cell volume increased by 35% from the initial inoculum at duration of 90 days (Table .1).

The initiation of a cell suspension culture requires a relatively large amount of callus to serve as the

inoculum i.e., 2 – 3 g for 100 cm3 (2). A continual increase in cell density will occur until the stationary
phase is reached. The period of exponential growth is prolonged if lower initial inoculum is used.

Table 1.Growth analysis of cell suspension culture under various sub cultures in Andrographis

paniculata Nees.

Treatment Weight of Packed Cell Volume (%) MEAN

inoculum 12 days 24 36 48 60 72 90
(g) days days days days days days
T1 0.5 0.56 0.62 0.72 0.79 0.72 0.65 0.59 0.66
T2 1.0 1.12 1.28 1.48 1.56 1.42 1.36 1.24 1.35
MEAN 0.84 0.95 1.10 1.18 1.07 1.01 0.91

Statistically Not analysed.

After two sub culturing, the auxin concentration in the medium was gradually reduced. The degree of
cell dispersion in suspension cultures is susceptible to the concentration of growth regulators in the
culture medium. Auxin growth regulators increase specific activity of the enzymes, which bring about
the dissolution of the cell walls. A positive correlation between the rate of cell division and ethylene
production has been observed and it is assumed that ethylene is a byproduct of actively dividing
suspension cells (7).

SUMMARY

Medicinal plants are sources of various Secondary metabolites quite essential to mankind for drug
treatment. The callus tissues specifically obtained from vegetative phase leaf bits of Andrographis
dispersed in full strength Liquid MS medium fortified with 3 % sucrose and plant growth regulator
combination of 2,4-D (1 mg/l) and NAA (1mg/l) was found to be the best medium for establishing
suspension culture for secondary metabolite production under in vitro condition. The cells in

suspension were cultured at 120 rpm on a gyratory shaker platform at 25+ 2 0C with an orbital motion
stroke of 2-4 cm in continuous light of less than 2000 lux for 15 days. Based on the behaviour of cells
in suspension, the early stationary phase was identified to be 12-14 days after inoculation, a time when
cell aggregation is maximum in suspension. Growth analysis of cell suspension under various sub
cultures revealed that the packed cell volume increases with increased initial inoculum of 1.0 g of
callus per 30 ml liquid medium and was maximum during the period between 36 to 72 days of
culturing. After two sub culturing, the auxin concentration in the medium was gradually reduced, in
order to avoid cell dissolution in the suspension. Thus resulting in maximizing the in vitro biomass
product yield efficiency both qualitatively and quantitatively, so that commercialization of this
technology in pharmaceutical industry can accomplish the rising demand in global market.

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New York: Academic Press.

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