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 METHODSEstablishment 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 productionA 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 inoculum

(g)

Packed Cell Volume (%) MEAN12 days 24

days36

days48

days60

days72

days90

daysT1 0.5 0.56 0.62 0.72 0.79 0.72 0.65 0.59 0.66T2 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.

LITERATURE CITED

1.Heijden, D.V.R, Robert Verpoorte and Hens J.G.Ten Hoppen. 1989. Cell and tissue cultures of

Catharanthus roseus (L.) G. Don: a literature survey. Plant Cell, Tissue and Organ Culture,

18: 231 – 280.

2. Helgeson, J.P. 1979. Tissue and cell suspension culture. pp. 52-59. In: Nicotiana. Procedures for experimental use, (Ed.). R.D.Durbin., Washington, D.C.: U.S. Department of Agriculture Tech. Bull. No.1586.

3. King, P.J. 1980. Cell proliferation and growth in suspension cultures. Int. Rev. Cytol. Supplement.,

11A: 25-54.

4. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue Cultures. Physiol plant. , 15: 437 – 497.

5. Rashid, A. 1988. Cell physiology and genetics of higher plants, vol I. CRC Press, Baco Raton, FL ,

pp.1-38.

6. Siripong, P., B. Kongkathip, K. Preechanukool, P. Picha, K. Tunsuwan and W.C. Taylor.1992.

Cytotoxic diterpenoid constituents from Andrographis paniculata Nees. leaves, J. Sci. Soc.,

Thailand 18: 187 - 194.

7. Street, H.E.1969. Growth in organized and unorganized systems. Knowledge gained by cultures of

organs and tissue explants. pp 3-224. In: Plant Physiol. A treatise, vol.VB. (Ed.). F.C.Steward,

New York: Academic Press.

8. Yeomen, M.M. and A.J.Mcleod. 1977. In: Plant Tissue and Cell Culture. (Ed.). H.E.Street Uni.Calif.

Press, Los Angeles.