In vitro secondary metabolite production in Scoparia...

Chapter II

In vitro secondary metabolite production in Scoparia dulcis Linn.

2.1 INTRODUCTION

Most of the medicinally important plant products are secondary

metabolites which are synthesized at variied periods of plant growth and

development. Secondary metabolites are biologically active compounds.

Since they are produced in low amounts, substantial quantity of raw

plant materials is required to extract a few grams of the useful product.

Therefore novel methods have to be adopted to enhance the

biosynthesis of secondary products so that the prevention of further loss

of raw plant materials can be avoided. During the period between 1950

and 1970, production of secondary metabolites like Ginseng saponins

(Furuya et al., 1973) and diosgenin (Kaul et al., 1969) were commercialized

at low yield. After 1973, a turning point in cell culture was demonstrated

with increased yield and synthesis of secondary metabolites by the

application of modern techniques of in vitro culture (Butcher, 1977; Staba,

1977, Constabel et al., 1974.).

Plant cell culture offers many advantages over intact plants for

secondary metabolite production and their biosynthetic studies. One of the

main reasons is that they can be kept under strictly controlled nutritional

and environmental conditions. Hence the uncertainty of changes in climate

and soil are avoided and also cells can be cultured aseptically. Many

compounds belonging to about fifty categories of products have been

found to be produced by plant cell culture (Staba,1980; Fowler,1986).

Some of them were isolated in concentrations equal to or higher than in

parent plant. Plant tissue culture may be used for biotransformation for

dispersing biochemicals and some enzymes. Products may accumulate in

media as well in cells (Bajaj, 1986). Suspension cultures are good source

for physiological studies and artificial seed production because the

population of cells is more uniform than in callus and also these are ideal

for cryopreservation.

48 Chapter II

Secondary metabolism is the synthesis and metabolism of

endogenous compounds by specialised proteins and the products formed

as a result of the processes are called secondary metabolites (Luckner,

1971). Plant cell cultures are poor producers of natural compounds

(secondary metabolites) if they grow in an optimal environment. When the

compounds in the medium become exhausted and environmental

conditions become limited, production of secondary metabolites

commences in plant cell cultures (Berlin, 1986). There is a wide range of

compounds in plants which can be synthesized via the first metabolic

pathway. Even though the function of many metabolites is not clear, it is

presumed that they are used against predators and also to attract

pollinators. To some extend they help in resisting infections and diseases.

The secondary compounds include alkaloids, terpeniods, steroids,

anthocyanins, anthroquinones and volatile oils. Specific compounds

accumulate only in restricted range of species and within specific stage of

development. Metabolites produced by plant cell culture have the

advantage of having products of uniform quality that can be stably

produced without being affected by weather and other conditions.

Compounds like proteins and peptides with high molecular weights and

distinct biological activity being the fore runners of new therapeutic era are

paving way for the blooming up of a new “biosociety” (Hownik et al., 1985).

Attempts are being made on the research of producing

phytochemicals and also to discover new compounds from plants for using

as pharmaceuticals. Hence production of compounds at a lower cost than

extraction from intact plants has to be targeted. Suspension cell culture

offers an effective way of incorporating precursor which is often difficult to

administer to the plants growing naturally, and it is an efficient way of

producing commercially important metabolites. Production in plant cell

culture is economically feasible for certain compounds provided that cell

In vitro secondary metabolite production in Scoparia dulcis 49

culture also produces them. Suspension culture offers advantage of faster

growth than that of callus culture (George 1993).

A cell suspension culture originates with a random critical event

occurring during the early exposure of plant cells in liquid medium. When

a plant material is first placed in to the medium there is initial lag period

prior to any sign of cell division followed by an exponential rise in cell

population. Gradually deceleration in division rate occurs resulting in a

stationary or non dividing stages with a sigmoid growth curve (Dodds and

Roberts, 1995). Cells undergoing their transition in metabolism and growth

rate produce a cell line (King, 1980). Secondary metabolite production has

been closely related to growth cycle in which the rate of cell division has

slowed down at the end of the log phase and at the beginning of stationary

phase. As the plant cells slow down their rate of growth production of

some type of secondary metabolite appear to increase concomitantly with

an increase in cell and tissue differentiation as in Atropa belladonna

(Lindsey and Yeoman, 1979). Plant metabolites like shikonin from cell

cultures of Lithospermum erythrorhizon (Tabata and Fujita, 1985) and

Berberine from Coscinium fenestratum (Fujita and Tabata, 1986) were

commercially produced in large scale.

The proportion and size of small cell aggregate vary according to the

plant variety and the medium in which the culture is grown. As cells tend to

divide more frequently in aggregates than in isolation, the size of cell

clusters increases during the phase of rapid cell division. As agitation

causes formation of single and small cells, the size of cell clusters

decreases in batch cultures as they approach a stationary growth phase.

Plant cells will not divide at a cell concentration below critical inoculum

development. For commencing a suspension culture, liquid cell culture

contains about 1-1.5 x 10 4cell m l-1 (George 1993).

50 Chapter II

The plant Scoparia dulcis is an ingredient of herbal medicine, which

is being used throughout the tropics where the plant is available. As

mentioned in the documented properties, this plant is having antibacterial

and antiviral properties. It is being exploited and hence often there is a

shortage due to the over use. Moreover, the plant being a perennial herb

disappears almost during the dry season and is available only during the

next on set of showers. Hence commercial availability of secondary

metabolite from nature is not possible all the year around.

In order to compensate the above problem a fast and economic

method is the production of secondary metabolite in vitro. This has been

done through cell suspension culture. Here depletion of natural resources

by permanent damage to the plant resources by over exploitation can be

prevented. Further, manipulation of the medium appropriately with suitable

hormones, precursors, elicitors, biosurfactants and immobilization

techniques may be done to enhance the production for the enhancement

of secondary metabolites.

2.1.1 Diterpenes

The diterpene compounds comprise a chemically heterogenous

group of compounds with C-20 carbon skeleton based on 4 isoprene

units. Diterpenes include seven skeletal forms. Labdane (bicyclic),

abietene, primarane, cassane (tricyclic, kaurine, gibberellins, bayerene

(tretracyclic) and trachylobane (pentacyclic) (Daniel, 1991). Geranyl

geranyl pyrophosphate is considered to be a general precursor of

diterpenoids. Diterpenes are usually separated by TLC, GLC, and HPLC

methods. Stevia gilleissii was reported to posses four types of diterpenes

in addition to sesquiterpenes, lactones, longipinanes (Meragelman et al.,

2004). Paclitaxel (Taxol) a highly functional diterpenoid was isolated from

bark of Taxus brevifolia which was successfully used for the therapy of

breast and ovarian cancer (Veerasham and Kokate, 1997). In Rosemary


(Rosemarinus officinalis) the main compound was a diterpene carnosic

acid which was responsible for antioxidant activity (Aruoma et al., 1992).

Scoparia dulcis posses a number of phytochemically important

compounds. They include diterpenes like dulciniol, scopadulciol,

scopadiol, scopadulcic acid A, scopadulcic acid B, scopadulin, scoparic

acid A, and scoparic acid B and cytotoxic diterpenes, iso–dulcinol,

4-episcopadulcic acid, B, dulcidiol and scopanolal. Flavones such as

acetatin, apigenin, cirsimarin, cristakaoside, hymenoxin, hymenoxin

linarin, leutolin, methyl ester scutellarin, scutellarin, vicenin 2, vitexin, iso

vitexinare are also present. Triterpenes including amyrin alpha,

betulinicacid, dulcoic acid, friedelin, glutinol ifflaoinic acid, glut-5-(6)en -3-

beta-ol and heterocyclic nitrogen benzoxazolinone, benzoxazolin -2-one,

6 methoxy, coixol are present in Scopaia dulcis. Steroids such as

daucosterol, beta sitosterol, stigmasterol and taraxerol were also

reported in Scoparia dulcis (Tropical plant database, 2004).

2.1.2 Properteis of Scopadulcic Acid B

Scopadulcic acid B was isolated as colourless prisms, with a

melting point 228-232 0 C α, β - 49.60 C (MeOH) from Scoparia dulcis.

The molecular formula of the compound was determined as C27 H34 O 5.

Infra red spectrum (IR) showed maximum absorption at 3200, 1730, 1710,

1580 cm-1, indicating the presence of hydroxyl, carbonyl, and phenyl

functions in molecules. The absorption bands for UV spectrum was UV

max 229 (3.99), 265 (2.81), 272, 280 (2.74). (Hayashi et al., 1990).

52 Chapter II

2.1.3 Structure of Scopadulcic acid B

2.1.4 Objectives

The objectives of the suspension culture studies include

(1) Standardization of the medium for the production of suspension

cultures.

(2) To study the growth of cells in the suspension medium.

(3) To study the biochemical changes during the suspension culture.

(4) To study the effects of different growth regulators on suspension

culture.

(5) Detection of scopadulcic acid B (SDB) in vitro and in vivo.


2.2 REVIEW OF LITERATURE

For a long time it was regarded that growing suspension cultures

were the only relevant system for biotechnological processes. In actively

growing suspension cultures, it was found that inorganic phosphate rapidly

utilized and consequently it became a limiting factor (Erikkson 1965). A cell

culture exists as a heterogeneous population of cells in different

physiological state and may react differently to an external event such as

medium variation. This may be the reason for presence of many different

secondary metabolites accumulating in cells (Bajaj, 1986).

The degree of cell dispersion in suspension culture is particularly

susceptible to concentration of growth regulators in culture medium. Auxin

growth regulators increased specific activity of enzymes, which bring about

the dissolution of middle lamella of plant cell walls. Thus by using high

concentration of auxins and low concentration of cytokinins in culture medium

it was possible to increase cell dispersion (Torrey and Reinert ,1961).

6-methoxy-2-benzoxazolinone (MBOA) a plant resistant factor

(diterpene) was extracted from the entire plant of S.dulcis. It was

produced from hairy root cultures by A.rhizogenes along with other

compounds like acetin, glutinol, and scopadulciol (Hayashi et al., 1994).

Suspension cultures technique was employed for the production of

scopadulciol by cultured tissues of Scoparia dulcis (Hayashi et al., 1996).

For production of Scopadulcic acid B, leaf organ cultures were kept

continuously in reciprocal shakers under illumination (Hayashi et al., 1997).

An efficient production of biologically active diterpenoid scopadiol by leaf

organ culture was established by Hayashi et al., (1997) in S. dulcis.

Continuous supply of light for six days along with the presence of the

phytohormone Methyl jasmonate, enhanced SDB production in Scoparia

dulcis (Kasidimoko et al., 2005).

54 Chapter II

2.2.1 Medium Composition

The effects of nutrients on the production of secondary metabolites

have been stressed by several workers. Addition of nutrients, growth

hormones, vitamins etc. in culture medium is primarily used to increase cell

growth in culture conditions. It has been reported that certain nutrients in

culture medium increased secondary metabolites while others showed

inhibitory effect. A large amount of medium was usually necessary to

initiate suspension (Dixon and Gonzales, 1994).

Sugars are important carbon source through out all stages of plant

life cycle. Lowering of concentration of sugar increased production of

ubiquenones in tobacco cell culture (Ikeda et al., 1976). Addition of

sucrose in culture medium above ordinary level (1-5%) increased Shikonin

in callus cultures of Lithospermum erythrorhizon (Mizukami et al., 1977).

Different sugars like maltose, glucose, fructose and sucrose used in medium

increased the growth of callus (Mezzeti et al., 1991). In culturing plant

tissues, a continuous supply of sugars from medium was necessary since

the photosynthetic activity of the in vitro tissues was reduced because of low

transportation, limited gas exchange and high relative humidity (Kozai,

1991). Sucrose had a positive effect on Salidrose synthesis (Xu et al.,

1999). In cultures of Symphytum officinale suspended cell cultures showed

a decrease in concentration of sucrose from 30 mg/l to zero (Kerner et al.,

2000). Effects of sucrose, IAA and BA concentrations on cell growth of

suspension cultures of Centella asiatica were studied by Omar et al.,

(2004). Callus aggregates were some times formed by cell culture as

spherical, coherent cellular units as in Sassurea medusa. Production of

secondary metabolite was correlated with size of compact cell aggregate

(CCA) and cellular viability. Under favourable conditions, some CCA got

into continuous proliferation while maintaining their morphological integrity


and were regarded as self immobilized cellular clumps similar to

immobilized cell cultures (Fu et al., 2005).

Caffeine and chlorogenic acid in suspension cultures of Coffea

arabica accumulated intracellularly (Baumann and Rohrig, 1989). Indole

alkaloids were reported from Rauwolfia sellowii (Batista et al., 1996). Many

antifungal compounds like Spirostanol saponins are being produced from

plant cell suspension cultures in Solanum chrysotrichum (Villarreal et al.,

1997). Cell cultures of Rauwolfia sellowii in Gamborg B5 medium

supplemented with 2, 4 - D (1mg/l) and Kinetin (0.2mg/l) showed growth

and production of major alkaloids like sellowiine, vomilenine and picrinene

(Rech et al., 1998). Accumulation of chlorogenic acid in cell suspension

cultures of Eucommia ulmoides (Wang et al., 2000) showed that MS

medium when supplemented with 2,4 - D at 2mg/l concentration was

effective. A scale up study of suspension cultures of Taxus chinensis cells

for production of Taxane diterpenes was reported by Pan et al; (2000).

Napthoquinone were reported to be produced in in vitro cultured plants and

cell suspensions of Dioanea muscipula (Venus fly trap) and Drosera

(Sundew) species on MS media supplemented with 7-methyljuglone (Hook,

2001). The accumulation of indole alkaloid content in Catheranthus roseus

was increased by the addition of precursors loganin and tryptamine along

with phytohormones 2,4 - D, salicyclic acid, methyl jasmonate and

abscicic acid (Magdi and Verporte, 2002). Release of berberrine and its

crystallisation in liquid medium of cell suspension was reported in cultures

of Coscinium fenestratum (Narasimhan and Nair, 2004). Secondary

metabolites which did not exist in plant parts were identified in suspension

cultures of Camptotheca acuminata. In this plant two alkaloids

isocamptothecin A (ICPTA) and isocamptothecin B (ICPTB) were already

reported (Yu et al., 2005). Rosmaric acid was produced by Lavendula vera

56 Chapter II

in Lingonour and Skooge medium supplemented with 2,4 -D at 0.2mg/l

concentration (Atanas et al., 2005).

2.2.2 Role of auxins and cytokinins in suspension culture In cell suspension cultures, manipulation of auxins and cytokinin

ratio achieved better dispersion of cells. For tobacco, increasing the

concentration of 2,4 - D from 0.3 - 2mg/l in the media with the addition of

vitamins and ceasin hydrolysate increased the biomass production

(Reynolds and Murashige,1979). High level of auxins added to liquid media

prevented morphogenesis but induced embryogenesis (George, 1993).

Phytohormones regulated secondary metabolism in plant cell culture and

thereby influenced both culture growth and secondary metabolite

production (Arvey et al., 1994).

Cytokinins promoted formation of chlorophyll in cells and in

suspension culture where auxins were inhibitory Hildebrant et al., (1963).The

effects of cytokinins in tissue culture showed varied effect. Addition of 2, 4 - D

and yeast extract (Negutice et al., 1977) and small amount of hydrolytic

enzyme, cellulase and pectinase (Street, 1977) had promoting effects on

dispersion of cells. In Oxalis dispar callus was found to turn green only when

auxin was reduced to 1/10, and the concentration of 2, 4 - D from 1.0 to 0.1

mg/l or NAA from 10 to 1 mg/l (Meyer and Staden, 1995). The effect of

elicitation on the peroxide activity in some cell suspension cultures of

Humulus lupulus were reported by Trevesian et al., (1997). Auxins promoted

cell dispersion in suspension cultures while cytokinins tend to cause cell

aggregation. Improved menthol production from chitosan elicited suspension

culture of Mentha piperita was reported by Chang et al., (1998).

In callus cultures of pea, tomato and potato, a reduction in chlorophyll

formation in the presence of 2,4 - D was reported by Hildebrant et al., (1963).

Suspension cultures of callus in MS medium when transferred to Gamborg

B5 liquid medium supplemented with 5µm NAA and1µm BA yielded


Scoparic acid A and Scopadulcic acid B (Hayashi et al., 1993). Elicitation

of anthocyanin production in callus cultures of Dacus carota and

involvement of calcium channel modulators were also reported (Sudha and

Ravisasnkar, 2003).

Due to differential uptake of anions and cations in plant tissues, pH of

medium does not usually stay constant but the plants absorb, as ions and

compounds. Plant cells usually tolerate a pH at the range of 4 -7.2. The

best results are in slightly acidic medium with an average pH of 5.6. A pH

of 5.6-5.8 supported growth of meristem tips of most plants in culture

where as Cassava meristem did not grow for a prolonged period on a

medium of pH 4.8 (Kartha, 1981). When plant cell cultures were incubated

in a medium containing an auxin, efflux of H+ ions through the cell wall

occurred resulting in an acidic medium. The pH of cell sap was raised and

became proportional to auxin, 2, 4 -D concentration (Krikorian, 1990).

A number of reports regarding somatic embryogenesis and plant

regeneration have been reported since the first observation of somatic

embryo formation in cell suspension cultures of Dacus carota (Reinert and

Backs, 1968; Steward et al., 1958). Two primary modes of regeneration

from plant cell and tissue cultures are organogenesis and somatic

embryogenesis. The process of embryogenesis has four stages namely

induction, proliferation, development or maturation and germination. In

Dactylis glomerata, embryos were produced directly from cells of the explant

in cultured leaves (Hanning and Conge, 1982). Plant regeneration via

somatic embryogenesis have been reported in numerous cereals and

grasses (Conger et al., 1983; Vasil, 1985).

Callus capable of producing somatic embryos (embryogenic callus)

is most reliably obtained from an explant during the initial phase of culture

and is frequently produced in conjunction with non morphogenic tissue.

During the second stage of culture, somatic embryogenesis is usually

58 Chapter II

initiated on a media with relatively high concentrations of auxin. Somatic

embryos arose from a single cell and had no vascular connection with

maternal tissues. In Dactylus glomerata the single cell developed became

densely cytoplasmic within one week. 90% of divisions of cells were

periclinal and produced uniseriate suspension cells. From these cells,

multiseriate proembryo were developed (Haccius.1978). According to

Sharp et al., (1980) although competent and non competent cells could

produce callus only that which grew from competent cells gave rise to

somatic embryos. The expression of competence depends on the use of a

suitable medium for culturing with requisite growth regulators and required

concentration. In Lolinum multiforum primary callus arising from an explant

showed no morphogenic capacity but could be induced to give rise to new

embryonic tissues during later stages (secondary stage). Ahee et al.,

(1981) used this method to propagate oil palms. Such structures grew

much faster than other cells and these were induced to produce structures

resembling embryoids which upon subculture produced plantlets.

Developmental pathways of somatic embryogenesis were chalked

out by various physical and chemical treatments. This was done to enhance

an efficient regulation for formation of plants via somatic embryogenesis

(Arnold et al., 2002). Three culture methods were compared for developing

an efficient method for carrot somatic embryos. They were using

conventional shake flask cultures, static suspension culture using

Erlenmeyer flasks and with static suspension culture using pertidishes (Li

and Kurata, 2005). Of the three methods experimented, static suspension

cultures of Dacus carota were found to be very efficient for producing

somatic embryos. Similar reports of somatic embryogenesis and plant

regeneration were obtained in Eucalyptus tereticorms (Prakash and

Gurumurthi, 2005) and in Cicer arietinum (Kiran et al., 2005). MS medium

supplemented with TDZ and NAA, 2, 4 - D and CPPU produced direct


somatic embryogenesis in Syngonium podophyllum (Zhang et al., 2006).

Somatic embryo proliferation, maturation, and germination in Catheranthus

roseus was reported in medium of MS supplemented with 2,4 - D and NAA

at 1.0 mg/l (Junaid et al., 2006).

Abscisic acid had an influence on morphogenesis in a number of

plants. Low concentrations stimulated embryogenesis in Citrus sinensis at

0.01-1 mg/l (Kochba et al., 1978). By adding ABA to the medium at a

concentration of 0.5-2mg/l morphogenic changes were more rapidly seen

in potato culture cells (Sharp et al., 1980).

Once an embryogenic callus was formed in the medium, it

continued to give rise to somatic embryos. These somatic embryos can

be subcultured over larger periods which depended on either proliferation

of proembryo or denovo formation of embryogenic tissue from young

somatic embryo during each subculture. Embryogenesis in liquid

suspension culture was reported in Pearlmillet and Guinea grass (Vasil

and Vasil, 1981) where the suspensions were initiated by supplementing

MS liquid media with 1-2.5 µg/l 2,4 - D and 2.5- 5% coconut milk. The

medium possessed a mixture of embryogenic cells which were small,

highly vacuolated and contained starch. New embryonic cells formed

were large and vacuolated. When these were plated on agar medium

without growth regulators or with very low level auxin, embryoids were

produced (Dixon, 1985).

60 Chapter II

2.3 MATERIALS AND METHODS 2.3.1 Establishment of Suspension Culture

Friable callus initiated by culturing leaf explants on MS medium

supplemented with 2, 4 - D 1mgl-1 and Kinetin 4 mgl-1 after 30 days were

chosen for initiating actively dividing cells. 250 mg of loose brown callus

were cut into fine pieces under sterile conditions and transferred to MS

medium containing 2, 4 – D and kinetin (1mgl-1 and 4mgl-1). They were kept

under 24 ± 2 0C for 14 days for establishing a suspension of cells on a

rotary shaker at 90rpm.The inoculum concentration was also standardised

using the same medium. The protocol for establishing suspension culture

in MS medium was based on works by Horn et al., (1988) on Oryza sativa,

and by Dixon and Gonzales (1994) in Zea mays and Glycine max.

2.3.1.1 Effect of various inoculum concentration

In order to study the influence of inoculum’s size on cell growth and

sub culture yield, inoculum of 4ml, 6ml, 8ml and 10ml containing single

cells were added from the mother suspension culture using sterile vials

and made up to 90 ml with MS medium. The packed cell volume (PCV),

fresh weight and dry weight of the cell suspensions were recorded at 0-30

days with 5 days interval (Franklin and Dixon, 1994). The experiment was

repeated thrice

2.3.1.2 Growth, behaviour and SDB analysis in the suspension cultures with 2,4-D (1mg/l) and Kinetin (4mg/l)

The growth pattern and their behaviour of cells in suspension

cultures in MS medium supplemented with 2,4-D (1mg/l) and Kinetin

(4mg/l) were studied. The SDB content was also analysed.

2.3.1.3 Effect of various hormonal combinations. The effect of various hormonal combination in the production of

SDB was carried out in the suspension culture. The cells from the mother


suspension medium [2,4-D (1mg/l) and Kinetin (4mg/l)]were subcultured

into NAA (5mg/l) and BA (1mg/l). The actively dividing green cells

(Embryogenic like) obtained were again subcultured into media with

different hormonal combinations. Aliquots of 10ml of the media were

transferred to 90ml MS liquid medium supplemented with NAA and BA,

2,4 - D and BA, 2,4 - D and kinetin combinations. The suspension cultures

were maintained for 30 days. In all the cases, the growth, SDB content and

the biochemical changes were analysed at 5 day interval as mentioned

below.

20 µl of embryogenic like suspension was transferred to MS

medium with abscisic acid and MS medium with IAA, BA, and 2, 4 - D for

2-3 weeks in darkness. Later they were transferred to low light intensity at

25oC to asses the regeneration capacity of cells.

2.3. 2 Growth analysis in suspension culture 2.3.2.1 Determination of cell viability

Cell viability was determined by using 10% Evans blue (Dixon and

Gonzales, 1994). Non viable cells in both aggregates and single cells

became blue in colour; while viable cells did not take up stain. The

percentage of viability was recorded by taking 10ml of sample.

2.3.2.2 Determination of Packed Cell Volume (PCV) Packed Cell Volume was determined by transferring the whole

contents into sterile centrifuge tubes and centrifuged at 200g for 10 minutes.

Packed cell volume was expressed as a percentage of the volume of the

pellet to the entire culture volume (Dixon and Gonzales, 1994).

2.3.2.3 Determination of fresh weight and dry weight. Cells were separated from suspension by centrifuging 100ml of

suspension. Pre weighed filter paper pieces were used for transferring

pellets for fresh weight determination. Fresh weight was determined by

noting the difference in final and initial weight of the filter paper. The cells

62 Chapter II

were oven dried for 60oC of 6 hrs. till no change was observed. The

difference in the weight was noted as before and was taken as dry weight

The experiment was repeated three times (Yeoman and Evans, 1967).

2.3.3 Detection and estimation of Scopadulcic acid B 2.3.3.1 Preparation of standard compound A primary standard of scopadulcic acid B (1mg/ml) was dissolved in

methyl alcohol and was used as the working standard for spectroscopic,

chromatographic as well as spectrophotometric analysis.

2.3.3.2 Thin layer chromatography TLC Preparative thin layer chromatography was done on silica gel

plates of 20x20cm dimension using hexane and ethyl acetate as solvents

in the ratio of 95:5. Detection was done using UV Trans illuminator and the

spots were observed as blue fluorescent spots at 240nm. A standard of

Scopadulcic acid B was also used as reference. The detected spots was

marked and scratched off, dissolved in chloroform centrifuged and used for

further analysis by UV spectrometer, HPLC and IR spectroscopy.

2.3.3.3 UV analysis

UV analysis was done using UV Visible spectrophotometer

(Hitachi) at the absorption maxima of 282 nm.

2.3.3.4 HPLC analysis High performance liquid chromatography (HPLC) was done to

confirm the presence of the secondary metabolite. Analysis was done at

Sree Chitra Thirunal Institue for Medical Sciences and Technology,

Poojappura, Thiruvanathapuram.

The column conditions were HPLC column of Waters C 18 symmetry,

Column (4.6x 250mm), and Waters 600 pump 7725 rheodyne 7725 injector,

Waters 2487 dual wave length UV absorbance Detector 230nm. Mobile

phase Methanol / 0.01 M H3 PO4 (3:1), Flow rate 1.2ml /minute


2.3.3.5 FTIR analysis FTIR was performed to confirm the functional groups in the

samples and was done with Fourier Transform Infra Red Spectrometer at

Sophisticated Test and Instrumentation Centre (STIC), Cochin University,

2.3.4 Sources used for SDB extraction 2.3.4.1. Type I and Type II callus

250mg of callus (green hard and brown friable callus) were

sonicated with 5ml of chloroform for 20 minutes, centrifuged and the

supernatant was concentrated to small quantity under reduced pressure. It

was passed through silica gel column (30cmx 1 cm) and eluted with 5ml of

chloroform. The column was further eluted with 5ml of methyl alcohol. The

second elute was evaporated to dryness. The residue was dissolved in

0.1ml of methyl alcohol. The SDB content was analysed by TLC, UV

spectrophotometer, HPLC and FTIR as mentioned above.

2.3.4.2 Multiple shoots 250mg of multiple shoots obtained from indirect organogenesis

were sonicated with 5ml chloroform for 10 minutes, centrifuged; the

supernatant was concentrated to small quantity under reduced pressure,

passed through silica gel column and eluted with 5ml of chloroform. The

column was further eluted with 5ml of methyl alcohol. The second elute

was evaporated to dryness. The residue was dissolved in 0.1ml of MeOH.

The SDB content was analysed by TLC, UV spectrophotometer, HPLC and

FTIR as mentioned above.

2.3.4.3 Plants in vivo 1Kg of air dried aerial parts of S. dulcis was boiled in 70% ethyl

alcohol cooled and the residue was treated with n-hexane. (Hayashi et al.,

1987). The fractions were cooled and separated with chloroform. The

residue was dissolved in 0.1ml of MeOH. The SDB content was analysed

by TLC, UV spectrophotometer, HPLC and FTIR as mentioned above.

64 Chapter II

2.3.4.4 Suspension culture

SDB content was analyzed from zero to 30 days at 5 days interval

using spectrophotometer. Total SDB content was noted after 30 days in

each set of experiment. 10ml of culture medium was removed and

centrifuged. The supernatant was extracted with 5ml of CHCl3. The organic

layer was collected, evaporated to dryness dissolved in 5ml methyl alcohol.

The SDB content was analysed by TLC, UV spectrophotometer, HPLC and

FTIR as mentioned above.

2.3.5 Biochemical Analysis 2.3.5.1 Estimation of Protein Protein content was estimated by Lowry’s Method (Lowry et al., 1957).

10ml of suspension were centrifuged for 5 minutes at 2000g. The supernatant

and pellet were separated. Protein estimation was done for the supernatant

(extracellular) and cells (intracellular) at an interval of 5 days for 30 days. For

determining protein content of pellet, they were treated with 10% TCA to

remove all other interfering constituents (Khanna et al., 1963). Bovine serum

albumin was used as standard. The experiment was repeated three times and

the intracellular protein value was expressed as µg/g of pellet and

extracellular protein was expressed as µg /ml.

2.3.5.2 Estimation of carbohydrate content in the medium Carbohydrate content in the medium during the cell growth from 0-

30 days was estimated by Anthrone method. The results were plotted on

standard graph and was expressed in g/l (Hedge and Hofreiter, 1962).

2.3.5.3 Estimation of Phenol

Estimation of phenol was determined by Folin Ciocalteau method.

Standard graph was plotted using catechol as internal standard and

readings were taken at an interval of 5 days (Malik and Singh, 1980).


2.4 RESULTS 2.4.1 Establishment of medium for suspension culture

a b

c

Plate 2.1 Cells of Scoparia dulcis in suspension culture

2.1 a Single cell in suspension culture 2,4 – D (1mg/l) and Kinetin (4mg/l) X 400 2.1 b Cell aggregates in suspension culture 2, 4 – D (1mg/l ) and Kinetin (4mg/l) X 400 2.1 c Cell aggregates in suspension culture 2,4-D (1mg/l) and Kinetin (4mg/l) X 400

66 Chapter II

When friable callus was added to MS medium supplemented with

2, 4 - D (1mg/l) and kinetin 4(mg/l) it was found to induce single cells. But

at this combination the number of cells was less and the production rate

was low. Also, two types of loose cells were observed. Cells with dense

cytoplasm containing starch grains and large vacuoles (Plate 2.1a) and

another type with dense cytoplasm in small groups. The large cells were

ellipsoidal and differently shaped (Plate2.1b). The proportion of the two

types of cells in the medium appeared to be dependent on cell density of

the inoculum, composition of the media and duration of cell culture. Cells

were large, spherical, and some were formed in aggregates (Plate 2.1c).

2.4.2 Standardisation of inoculum

Table 2.1 Effect of 4% inoculum in MS medium with 2,4- D (1mg/l) and Kinetin (4mg/l) for the suspension culture in Scoparia dulcis

Days PCV (%) F.wt.(g) D.wt. (g)

0 0.98 0.633±.030 0.022±.001

5 1.30 0.698±.020 0.023±.004

10 1.70 0.807±.030 0.032±.002

15 1.80 0.820±.020 0.038±.003

20 1.80 0.840±.010 0.039±.007

25 1.80 0.829±.020 0.038±.008

30 1.80 0.825±.040 0.038±.001



Days PCV (%) F.wt. (g) D.wt. (g)

0 0.97 0.625±.020 0.023±.004

5 1.30 0.638±.010 0.026±.002

10 1.50 0.789±.080 0.024±.002

15 1.50 0.796±.090 0.025±.006

20 1.80 0.821±.040 0.038±.001

25 1.80 0.811±.010 0.034±.006

30 1.80 0.810±.060 0.034±.003



0 0.97 0.650±.040 0.023±.004

5 1.60 0.710±.010 0.026±.005

10 1.80 0.800±.050 0.024±.002.

15 1.87 0.835±.026 0.025±.006

20 1.85 0.828±.035 0.038±.003

25 1.85 0.826±.040 0.034±.023

30 1.85 0.800±.045 0.033±.004

68 Chapter II



0 0.98 0.620 ±.040 0.021±.001

5 1.75 0.798 ±.020 0.025±.004

10 1.80 0.854 ±.030 0.034±.003

15 1.85 0.878 ±.040 0.035±.002

20 1.90 0.906±.050 0.035±.001

25 1.98 0.995±.080 0.029±.005

30 1.95 0.990 ±.010 0.028±.003

0

0.2

0.4

0.6

0.8

1

1.2

5 10 15 20 25 30

Time (Days)

F.w

t.(g)

4ml6ml8ml10ml

Fig. 2.1 Growth pattern of cells in MS medium with 2, 4 –D (1mg/l) and Kinetin (4mg/l) at various inoculum concentrations of mother suspension cells in suspension cultures of Scoparia dulcis


Standardization of inoculum for producing fine single cells in MS

medium supplemented with different combinations of hormones were

done by adding 4ml, 6ml, 8ml and 10 ml of inoculum into the medium and

the final volume was adjusted to 100 ml. By analyzing the fresh weight

and pcv it was found that when 4 ml inoculum was added maximum fresh

weight was 840 mg with a packed cell volume of 1.80 % on the 20th day.

(Table 2.1). Whereas, when 6ml inoculum was added, pcv was 1.8% and

the fresh weight was 821mg on 20th day (Table 2.2). When 8ml was used

as inoculum on 15th day pcv was 1.87 with fresh weight 825mg.

(Table2.3). Addition of 10ml inoculum to the medium showed a maximum

fresh weight of 995mg with a pcv of 1.98 on 25th day. Since maximum

growth was observed between 20-25 days when 10 ml was used as

inoculum it was further used in all the experiments (Fig 2. 1).

2.4.3 Growth, Biochemical and SDB analysis in the suspension cultures of Scoparia dulcis at 2,4-D (1mg/l) and kinetin 4mg/l)

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30

Time (Days)

F.w

t/D.w

t (g)

F.w t(g)

D.w t (g)

Fig. 2.2 Effect of 2,4 –D (1mg/l) and Kinetin (4mg/l) on fresh weight (g)and dry weight (g) in suspension cultures of Scoparia dulcis

70 Chapter II

01020304050607080

0 5 10 15 20 25 30

Time (Days)

Con

(mg/

l)

2,4-D1(mg/l) Kinetin (4mg/l)

Fig. 2.3 Effect of 2,4 - D and kinetin in the production of SDB(mg/l) in suspension cultures of Scoparia dulcis at 10% v/v inoculum

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30

F.w

t/D.w

t (g)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

SDB

(mg/l)

Fwt(g)SDB (mg/l)

Fig. 2.4 Relationship between fresh weight (g) and SDB (mg/l)

production in suspension cultures of Scoparia dulcis at 10% v/v inoculum

102


0

100

200

300

400

500

600

700

0 5 10 15 20 25 30

Time (Days)

Con

( µ g

)

Intra Cellular m g/g

Extra Cellular m g/ml

Fig. 2.5 Variation in concentration (µg) of extra and intracellular

proteins in suspension cultures with 2,4–D (1mg/l)and Kinetin (4mg/l) at 10% v/v inoculum in MS Medium.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 5 10 15 20 25 30

Time (Days)

Con

(mg/

l)

Phenol( mg/l)

Fig. 2.6 Variation in concentration (mg/l) of phenol in suspension cultures with 2,4–D (1mg/l) and Kinetin (4mg/l) at 10% v/v inoculum in MS Medium.

(µ/g)

(µ g/ml)

72 Chapter II

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 5 10 15 20 25 30

Time (Days)

Con

(g/l)

carbohydrate (g/l)

Fig. 2.7 Variation in concentration (g/l) of carbohydrate in suspension

cultures with 2,4-D (1mg/l) and Kinetin (4mg/l) at 10% v/v inoculum in MS Medium.

Growth pattern of cells indicated that maximum fresh weight and

linear sigmoid growth curve pattern of single cells were observed in

suspension cultures inoculated with 10 ml of inoculum. Growth of cells

was measured as increase in fresh weight. On analyzing fresh weight

and dry weight of cells in suspension culture with 2, 4-D (1mg/l) and

kinetin (4 mg/l) an increase in fresh weight was observed on 5th day.

Subsequently there was only slight increase in fresh weight up to 30 days

(Fig. 2.2). Maximum SDB content (72 mg/l) was observed on 25th day

(Fig. 2.3). For studying growth pattern of cells, protein content was also

analyzed. It was also observed that as the fresh weight increased there

was a proportional increase in SDB content (Fig. 2.4). Maximum

102


intracellular protein content (250 µg/g) was observed on 10th and 25th day.

In the medium also a corresponding increase in the extracellular protein

content (640 µg/ml) was observed. (Fig.2.5). Extracellular phenolic

content was determined by Folin’s Ciocalteau method. Phenol content

gradually increased from zero day to 30th day and maximum amount

(0.29 mg/l) was recorded on 20th day (Fig. 2.6). A linear decrease of

sucrose content in the medium indicated its consumption during cell

division and growth (Fig. 2.7). As fresh weight increased, sugar

consumption also increased resulting in the complete depletion of

sucrose from medium on 30th day.

2.4.4 Sub culturing of cells in NAA (5mg/l) BA (1mg/l) When cells were sub cultured from 2,4 - D (1mg/l) and kinetin

(4mg/l) to NAA (5mg/l) and BA (1mg/l) embryogenic like aggregates were

observed. They were dense yellow to brown in colour and with relatively

smooth surface (Plate 2.2 a) and (Plate2.2b). Embryogenic like aggregates

rapidly proliferated and were yellow to brown while non embryogenic like

cells were colourless (Plate 2.2c). All the cells in the conical flasks were

dark green in colour. They were highly meristematic in active stage of

division (Plate 2.2d).

2.4.5 Effect of different hormone concentrations in the suspension cultures of Scoparia dulcis

Growth pattern of cells in different concentration of auxins and

cytokinins were experimented with combinations of 2,4 - D / Kinetin,

2,4 - D /BA and NAA / BA (Fig . 2.8 -2.11 and Table 2.5).

74 Chapter II

Table 2.5 Effect of different hormone concentrations on the pcv, fresh weight, dry weight and SDB content in the suspension cultures of Scoparia dulcis in MS medium at 5 day intervals after subculturing from NAA (5mg/l) and BA (1mg/l) at 10% v/v inoculum

PGR Days PCV (%) F.wt. (g/l) D.wt. (g/l) SDB( mg/l)

2,4 - D (1mg/l)+K(4mg/l)

0 5 10 15 20 25 30

5.5 12 8 9 8 6

7.3

0.25 8.05 7.34 6.39 6.26 7.92 7.86

0.02 0.53 0.40 0.24 0.39 0.34 0.36

16.27 50.61 71.17 55.17 58.27 71.71 55.17

2,4 – D(1mg/l)+ BA(3mg/l)

0 5 10 15 20 25 30

4.5 10 15 20 25 18 17

0.34 8.06 8.43 7.49 7.18 6.99 5.98

0.04 0.48 0. 36 0.32 0.40 0.32 0.38

17.24 55.45 72.03 56.18 59.31 72.03 56.18

NAA(5mg/l) +BA(1mg/l)

0 5 10 15 20 25 30

5 7 9

7.2 6.8 10 10

0.14 8.09 6.54 5.29 5.36 5.98 6.84

0.01 0.65 0.38 0.41 0.38 0.36 0.40

15.24 45.78 70.1

54.16 57.25 80.6

54.16

Maximum fresh weight was recorded for 2,4 - D (l mg/l) and kinetin

(4 mg/l) as 8.05 g on 5thday (Fig.2.8) and for 2,4 - D (1 mg/l) and BA

(3 mg/l) 8.43 g on 5th day (Fig 2. 9). Similarly on 5th day in a combination

of NAA (5 mg/l) and BA (1mg/l) 8.09 g fresh weight was obtained

(Fig.2.10) (Appendix 5 and 6).


0 1 2 3 4 5 6 7 8 9

0 5 10 15 20 25 30Time

F.w

t/D.w

t (g)

F.wt(g)

D. wt (g)

Fig. 2.8 Effect of 2, 4 - D (1mg/l) and (Kinetin 4mg/l) on fresh weight

(g) and dry weight (g) of cells in suspension cultures of Scoparia dulcis at 10% v/v inoculum after subculturing from NAA (5mg/l) and BA (1mg/l)

0

1

2

3

4

5

6

7

8

9

0 5 10 15 20 25 30

Time (Days)

Fwt/D

wt (

g)

F. wt(g)

D. wt (g)

Fig. 2.9 Effect of 2,4 D (1mg/l) and (BA 3mg/l) on fresh weight (g)and

dry weight (g) of cells in suspension cultures of Scoparia dulcis at 10% v/v inoculum after subculturing from NAA (5mg/l) and BA (1mg/l)

76 Chapter II

0

1

2

3

4

5

6

7

8

9

10

0 5 10 15 20 25 30

Time (Days)

F. w

t / D

. wt (

g)

F.wt(g)

D.wt (g)

Fig. 2.10 Effect of NAA (5mg/l) and (BA 1mg/l) on fresh weight (g) and dry weight (g) of cells in suspension cultures of Scoparia dulcis at 10% v/v inoculum

On comparing the yield of SDB at different combinations of

hormones maximum yield of SDB was observed on 25th day in all the

three combinations of the hormones. The maximum yield at 2,4-D and

kinetin combination was 71.71 (mg/l) where as it was 72.03 (mg/l) at

2,4-D and BA combinations. The highest SDB yield (80.6 mg/l) was

observed at NAA and BA combinations. This combination was taken for

further studies (Table 2.5 and Appendix Table 7).


0102030405060708090

Con

(mg/

l)

0 5 10 15 20 25 30

Time (Days)

2,4-D (1mg/l)+K(4mg/l)

2,4-D (1mg/l)+BA(3mg/l)

NAA (5mg/l)+BA (1mg/l)

Fig. 2.11 Effect of different combinations of hormones in production

of SDB (mg/l) in suspension cultures of Scoparia dulcis at 10% v/v inoculum

78 Chapter II

a b

c d

Plate 2.2 Embryogenic like cells in division in NAA (5mg/l) and BA (1mg/l)

2.2 a Embryogenic like cell initiation in NAA (5mg/l) and BA (1mg/l) medium x 1000 2.2 b Embryogenic like cells in active division in chains in NAA (5mg/l) and BA (1mg/l)

medium x 1000

2.2 c Embryogenic like and non embryogenic cells x 1000 2.1 d Green cells in active division in 8 day old culture grown in NAA (5mg/l) and BA (1mg/l)

X 100


2.4.6 TLC, HPLC and FTIR analysis of the SDB content in various sources

2.4.6.1 TLC, HPLC and FTIR analysis of standard Scopadulcic acid B

.

Plate 2.3 TLC of Scopadulcic acid B (Standard)

TLC of standard compound SDB showed an Rf value at 0.15

(Plate2.3). UV analysis of SDB showed an absorption peak at 282.2.

HPLC analysis showed a peak at retention time 9.891 minutes (Fig. 2.12).

IR spectrum revealed absorption bands at 3332cm-1, 1656 cm-1 and

1449 cm-1 indicating hydroxyl, carbonyl and phenyl groups in the molecule

(Fig. 2.13).

80 Chapter II

Fig.2. 12 HPLC of Standard Scopadulcic acid B

Fig. 2.13 IR spectrum of Standard Scopadulcic acid


2.4.6.2 TLC, HPLC and FTIR analysis of type I callus derived from 2,4-D (1mg/l) and Kinetin (4mg/l)

Plate 2.4 TLC of SDB in callus derived from (1) 2,4 - D (1mg/l)and K(4mg/l) type II (2) IAA(0.3mg/l and BA (0.3mg/l) type I (3) NAA (5mg/l) and BA (1mg/l) suspension culture.

TLC of friable callus from MS medium supplemented with 2,4 D

(1mg/l) Kinetin (4mg/l) showed a Rf value at 0.15 (Plate 2.4). UV

analysis gave an absorption peak at 280.5nm. HPLC analysis showed a

peak at retention time of 9.09 minutes (Fig. 2.14). IR analysis gave bands

at 3344 cm-1, 1893 cm-1, and 1458 cm-1 indicating hydroxyl, carbonyl and

phenyl groups of the molecule (Fig. 2.15).

(1) (2) (3)

82 Chapter II

Fig. 2.14 HPLC of the extract of friable callus of Scoparia dulcis from MS medium supplemented with 2, 4 D (1mg/l) and Kinetin (4mg/l) on agar medium

Fig. 2.15 IR spectrum of the extract of callus derived from MS agar medium supplemented with friable callus with 2, 4 D (1mg/l) and Kinetin (4mg/l) on agar medium


2.4.6.3 TLC, HPLC and FTIR analysis of type II callus derived from IAA (0.3mg/l) and BA (0.3mg/l)

TLC of callus on MS medium supplemented with IAA and BA

(0.3mg/l) showed a Rf value at 0.15 (Plate 2.4). UV analysis showed an

absorption peak at 282 nm. HPLC analysis showed a peak at retention

time of 9.96 minutes (Fig. 2.16) and IR analysis showed absorption bands

at 3019 cm-1 and 1519 cm-1 (Fig.2.17).

Fig. 2.16 HPLC of the extract of callus derived from MS agar medium

supplemented with IAA (0.3mg/l) and BA (0.3mg/l)

Fig 2.17 IR spectrum of the extract of the callus derived from MS

agar medium supplemented with IAA (0.3mg/l) and BA (0.3mg/l)

84 Chapter II

2.4.6.4 TLC, HPLC and FTIR analysis of cells in suspension culture in MS medium with NAA (5mg/l) and BA (1mg/l)

TLC of cells in medium supplemented with NAA (5mg/l) and BA

(1mg/l) indicated a Rf value at 0.15 (Plate 2.4). UV analysis showed an

absorption peak at 282.6. HPLC analysis showed a peak at retention time

of 9.68 minutes (Fig.2.18). IR analysis showed absorption bands at 3433

cm-1 and1639 cm-1 (Fig. 2.19).

Fig 2.18 HPLC of the extract of the cells in suspension culture in MS

medium with NAA (5mg/l) and BA (1mg/l)

Fig. 2.19 IR spectrum of the extract of the cells in suspension culture

in MS medium with NAA (5mg/l) and BA (1mg/l)


2.4.6.5 TLC, HPLC and FTIR analysis of extract of multiple shoot derived from MS medium supplemented with IAA (3mg/l) and BA (3mg/l)

Plate 2.5 TLC of SDB from extract of (1) multiple shoot derived from MS medium supplemented with IAA (3mg/l) and BA (3mg/l) and (2) leaf extract

TLC of in vitro derived multiple shoots showed a Rf value at 0.15

(Plate2.5). UV analysis showed absorption peak at 282 nm. HPLC analysis

showed the peak at retention time of 9.09 minutes (Fig. 2.20). IR gave

absorption bands at 3345 cm-1, 1641 cm-1 and 1449 cm-1 indicating hydroxyl,

carbonyl and phenyl groups of the molecule (Fig. 2.21).

1 2

86 Chapter II

Fig. 2.20 HPLC of the extract of multiple shoot of Scoparia dulcis from MS medium supplemented with IAA (3mg/l)and BA (3mg/l)

Fig. 2.21 IR spectrum of the extract of multiple shoot of Scoparia dulcis from MS medium supplemented with IAA (3mg/l)and BA (3mg/l)


2.4.6.6 TLC, HPLC and FTIR analysis of leaf extract of Scoparia dulcis in vivo

TLC of air dried leaves showed a Rf value at 0.15 (Plate 2.5). UV

analysis indicated an absorption peak at 282.2 nm. HPLC analysis showed

a peak at retention time of 9.90 minutes (Fig. 2.22). IR revealed absorption

bands at 3330 cm-1, 1658 cm-1 and 1449 indicating hydroxyl, carbonyl and

phenyl groups of the molecule (Fig.2.23).

Fig. 2.22 HPLC of leaf extract of Scoparia dulcis

Fig. 2.23 IR spectrum of the leaf extract of Scoparia dulcis

88 Chapter II

2.4.6.7 TLC, HPLC and FTIR analysis the extract of supernatant in

suspension culture of Scoparia dulcis at NAA (5mg/l) and BA (1mg/l)

Plate 2.6 TLC of the extract of supernatant in suspension culture of

Scoparia dulcis at NAA (5mg/l) and BA (1mg/l)

TLC of suspension cultures in NAA (5mg/l) and BA (1mg/l) showed

a Rf value at 0.15. (Plate 2.6). UV analysis showed absorption peak at 282

nm. HPLC analysis showed a peak at a retention time of 9.9 minutes

(Figure 2.24). IR analysis showed absorption bands at 3413 cm-1, 1631 cm-1

and 1535 cm-1 indicating hydroxyl, carbonyl and phenyl groups of the

molecule (Fig. 2.25).


Fig. 2.24 HPLC of the extract of the supernatant in the suspension culture of Scoparia dulcis at NAA (5mg/l) and BA (1mg/l)

Fig. 2.25 IR of the extract of the supernatant in the suspension culture of Scoparia dulcis at NAA (5mg/l) and BA (1mg/l)

90 Chapter II

2.5 DISCUSSION

The ability of plant cells to synthesise a vast range of compounds

exceeds the biosynthetic diversity of other kingdoms. Plant cell suspension

cultures of S.dulcis were initiated, maintained and analysed for the

secondary metabolite production. For this, both qualitative as well as

quantitative analysis were performed. The growth of an undifferentiated

callus and suspension cultures were quantified by analysing an increase in

fresh weight, dry weight and pcv. A generalised growth pattern takes the

form of a sigmoid curve, where there is a lag phase, log phase, and a

stationary phase (Lindsey and Jones, 1989).

Experiments were conducted to standardize the medium for

production of maximum amount of SDB. The ease of establishment of

suspension culture from callus was influenced by the friability of the callus

tissue. Friability of callus was increased by increasing the auxin

concentration in the medium (Lindsey and Jones, 1989). With this in view,

mother suspension was initiated using friable callus from leaf explant in

MS medium supplemented with 2, 4 D (1mg/l) and kinetin (4mg/l). The

newly established suspension cultures had free cells and aggregates as

well as clumps and hence were kept for two weeks to attain fine densely

dispersed cells.

When the suspension cultures were sub cultured on different

hormone combination and concentrations (2,4-D and Kinetin,2,4 - D and

and BA, NAA and BA) there was a marked difference in morphology of

cells. Both embryogenic like as well as non embryogenic like cells were

observed. Similar embryoid cells were reported to develop in late

stationary phase in suspension cultures of tropane alkaloids like atropine

and scopolamine (Lindsey and Yeoman, 1979). Statistical analysis showed

that MS medium supplemented with NAA and BA (5mg/l )and BA (1mg/l)


yielded more SDB than 2,4 D(1mg/l) and BA (3mg/l) or 2,4 D(1mg/l) and

kinetin(4mg/l).

The minimum density of the cells required for cell suspension

culture depended on the rate of growth and composition of the medium.

Previous reports indicated that 10% of initial cell density to the total volume

was necessary to initiate suspension culture (Dixon and Gonzales, 1994).

There is a minimum inoculum size at which the cell suspension does not

readily resume active growth after being transferred to a new medium. The

minimum density of cells required for cell suspension culture depended on

the rate of growth and composition of the medium. Hence the second step

was to determine the appropriate inoculum size. In this study, 4, 6, 8 and

10ml of volume of inoculum size were experimented and it was found that

10% inoculum size was suitable for experiment as it gave better biomass

(Table 4). This is in agreement with previous reports of Solanum aviculare

(Kittipongapattana, 1998) where 10% inoculum size proved successful. But

in Solanum chrysotrichum the inoculum size was 2% for efficient

production of Spirostanol saponin (Villarreal et al., 1997). Thus inoculum

size was dependant on plant species and was not specific. Unlike micro

organisms plant cells have a critical inoculation density below which

growth will not occur. Plant cells require either cell to cell contact or

substances produced by cells for their optimum growth.

Quantitative studies were determined by measuring the growth of

cells. It was confirmed by studying the pcv, fresh weight, dry weight of the

cells in culture.

From the day zero, when the cells were introduced to fresh medium

(subculture day zero) to the 3rd day, the cells were in the lag phase and

cell growth was very slow due to initiation of a series of metabolic process

which prepared the cell for mitosis. The cells were acclimatizing to fresh

medium and were beginning to uptake nutrients from the media (Table 1).

92 Chapter II

From day 3 to 20, the exponential phase, the cells which were acclimatized

to the environment rapidly grew and divided. From day 20 to 30 the

deceleration phase, the cells used up almost all the available nutrients and

the growth was very slow. Subsequent transfer to fresh medium was

necessary for further survival of the cells. This is in agreement with

previous reports of cell suspension culture studies for the production of

Sopadulciol (Hayashi et al., 1993) and Sopadulcic acid B from cultured

tissues of S. dulcis (Hayashi et al., 1997)

Presence of protein content in the cells can be accepted as an

indication of active metabolism of proliferated mass of cells. Callus is a

heterogeneous mass with high rate of division. Therefore estimation of

protein content in cell samples were undertaken with the aim of

investigating the link between growth (fresh weight) and primary

metabolism (protein) and secondary metabolism. This was found true with

production of intracellular proteins of S. dulcis suspension culture.

Extracellular proteins were detected in embryogenic suspension cultures of

Picea abies. These proteins secreted extracellularly were found to be

necessary for the development of embryogenic cell lines in somatic

embryogenesis. (Egertsdotter et al., 1993). Similarly, extracellular proteins

namely Aspiring, Karpiro, Pacifira and Tiroa were extracted from media of

actively growing embryogenic suspension cultures of Asparagus officinalis.

They were reported to play a significant role in formation of the somatic

embryos (Hollingsworth et al., 2008).

Sucrose consumption from medium is an indication of cell division

and growth. A typical 30 day culture medium of Symphytum officinale

showed decrease in sucrose content linearly (Kerner et al., 2000) . Similarly

a decrease in concentration of sucrose was observed in in vitro cultures of

cells from cereals, Saccharum officinarum and Nicotiana tabaccum. It

indicates hydrolysis of sucrose in these medium (Schmitz and Skoog, 1970).


In carrot suspension cultures, a decrease in sucrose level from 30mg/l to

zero was reported by Kanabus et al., (1986). When cells were cultured in

MS medium with 2,4 - D (1mg/l ) and Kinetin (4 mg/) , there was a linear

decrease of sucrose content from zero day to 30th day. In all the

experiments invaribly, the same observations were repeatedly seen.

Production of phenols was seen in cells when they enter into the

stationary phase (Forrest, 1969). The natural rate of formation of some

phenolic compounds has been observed to depend on rate of growth of

cultured tissues on auxin cytokinin levels within the medium (Sargent and

Skoog, 1970) and (Barz, 1977) .The phenol content of cells in MS medium

supplemented with 2,4 - D (1mg/l ) and Kinetin (4 mg/l) increased with

increase in fresh weight. There are reports on increase in secondary

metabolite production when cellular growth decreased (Pareilleux and

Vines, 1984).

Experiments were conducted to show the relation between growth

and SDB production. In all the experiments growth of cells and SDB

production was directly related (Fig. 2.4). Auxins were capable of initiating

cell division and were involved in the origin of meristematic tissues or

defined organs. Various combinations of auxins (NAA, IAA) and cytokinins

(Kinetin and BA) were tested. When 2, 4 - D and Kinetin were

supplemented to MS medium, small round cells were formed. Friable

callus under agitation produced single cells and small groups of cells.

Auxin growth regulants increased the specific activity of enzymes which

brought about dissolution of middle of plant cell walls. Cytokinins and

auxins used in combination in growth medium were more effective than

when used singly (George, 1993). Higher ratio of cytokinin and auxin were

beneficial for biomass of salidrose accumulation (Xu, 1999). But in S.dulcis it

was found that the best results were obtained when 2,4 - D and kinetin were

used at a concentration of 1mg/lm and 4mg/l respectively. Here cells were

94 Chapter II

large and non embryonic, characteristically elongated and twisted, with large

vacuoles and thin walls. The growth curve showed a sigmoid pattern.

Staining with Evan’s blue indicated that the cells were viable. But rate of

division was slow. Role of kinetin in suspension cultures was emphasized in

earlier reports of S.dulcis where BA was used for sub culturing callus to yield

Scopadulciol. Similarly for production of diterpenes Scoparic acid A and

Scoparic acid B by suspension culture, Hayashi et al., (1993) used Gamborg

B5 liquid medium supplemented with NAA (5mg/l) and BA (1mg/l).

When friable callus from 2,4 - D (1mg/l) and kinetin (4mg/l) were

sub cultured in MS medium supplemented with NAA and BA at various

concentrations, cells with high meristematic activity resembling initiation of

somatic embryogenesis were observed (Table 2.5). These cells grew

much faster than other cells and were similar to embryogenic like cells.

The dividing cells were dark green in colour and grew in aggregates. From

the same mother suspension, cells were again sub cultured in to medium

with 2,4 - D (1mg/l) and kinetin (4 mg/l) and 2,4 – D (1mg/l) and BA

(3mg/l). In all these media, initiation of embryogenic like cells was observed.

The comparatively high fresh weight obtained in these experiments are

due to this reason. But when SDB content in the three different

combinations were compared, statistical analysis proved that at a

concentration of 5 mg/l of NAA and 1mg/l of BA the maximum yield was

obtained. In the experiment, embryogenic like aggregates were dense

yellow to brown in colour and were relatively with smooth surface. Some

cells had protrusions from surface. This is in true agreement with previous

reports form Dixon and Gonzales (1994) regarding suspension cell

cultures of Glycine max and Zea mays, where the embryonic cells were

more vacuolated, less dense and translucent and appeared white in colour.

The flasks with the culture medium turned white and cloudy on the 3 rd day.

From 3 – 30 days the medium became dense and dark green in colour.


The green colour was due to presence of chlorophyll. When cytokinins

were present in medium, chlorophyll induction was promoted and triggered.

Even though profused proliferation of proembryonic tissues were obtained

none of them were able to develop in to fully developed embryos. This may

be due to senescence of embryos. When the suspension culture from NAA

and BA were sub-cultured to different combination of 2,4 - D and BA , the

same type of growth were observed. But the time taken to change from

white cloudy appearance to green colour was more than three days. Here

the amount of embryogenic like cells were more in all the three medium.

The cells were all in dividing stages, single to aggregate of clumps with

and without suspensor like cells were also seen. Number of non viable

cells (cells with reduced or shrunken irregular cytoplasm) were very low in

number as evident from Evan’s blue staining.

Experiments in suspension cultures using leaf explants of S.

dulcis in the production of diterpenes were performed in continuous light

irradiation (Hayashi et al., 1996). Maximum fresh weight and SDB

content in S.dulcis was at 20-30 days when cultured in MS medium.

Where as, cell growth and diterpene content in cultured tissues were

maximum at 12-15 days for Scoparic acid B type (SDB) and scopadiol

(SDX) type. Scopadulciol (SDY) content and fresh weight of callus

increased by 10-15 days while in suspension culture in light, a maximum

of 1-12 days were taken and the amount of SDY decreased by 15th day.

In dark, the content of SDY increased by 4-6 days and the amount of

SDY formed were determined from 7th day onwards. Etiolated tissues

were found to contain significantly less amount of diterpenes in

comparison with green leaf organ (Hayashi et al., 1997). Growth of cells

in light was much better that in dark for production of Scopadulciol by

cultured tissues of S. dulcis (Hayashi et al., 1998).

96 Chapter II

An attempt was made to evaluate the potential of various in vitro

plant cell cultures of Scoparia dulcis in the production of Scopadulcic acid B.

Although it was possible to get plant metabolites out of various strategies, it

was very difficult to separate the desirable metabolite from the rest of the

contaminants. Separation and purification of the plant metabolite is a bottle

neck as it always exists in combination with other metabolites. In the present

study, a similar pattern was obtained. The results obtained in TLC, UV

absorption, HPLC and IR using the pure scopadulcic acid B standered

revealed Rf value in TLC at 0.15, UV absorption peak at 288.2, HPLC peak

at retention time of 9 minutes and IR bands at 3332 cm-1 , 1656 cm-1 and

1449 cm-1 (Plate 2.3, Fig.2.12 and 2.13). Although good spots were

obtained on TLC plate, the HPLC of the purified fraction gave many peaks in

addition to the desirable peak at 9min (Fig 2.14, 2.18, 2.20, 2.22 and 2.24).

The HPLC of the callus extract of type II callus was indicative of better purity

in SDB (Fig 2.16). But the type II callus was incapable of triggering the

suspension culture. The IR spectrum taken in each case was indicative of

Scopadulcic acid B.

The suspension culture is the ideal choice for the production of

secondary metabolite as the product isolation is possible from the extra

cellular medium. Thus the homogenization of the callus and the

extraction of the metabolite may be avoided. However the amount of

metabolite formed may be enhanced. There is ample scope for

evaluating the inducing effect of various ingredients such as precursor,

elicitor, surfactant etc. The purity of the fraction may be improved by the

application of modern strategies. Further, optimization and improvement

of the process may be required before putting the suspension culture for

large scale operation. Production of the metabolite under restricted and

finely tuned environment in a bioreactor could also be a possibility for the

enhanced production.


In conclusion, standardization of medium and initiation of

suspension culture were done using friable callus of Type I from leaf

explants of Scoparia dulcis. Production of SDB in various combinations

and concentrations of hormones like 2,4 - D and K, 2,4 - D and BA, NAA

and BA was attempted and it was found that NAA (5mg/l) and BA (1mg/l)

was the best medium for getting maximum yield of SDB. Embryogenic like

cells were observed in all the three medium after subculture from 2,4 D

and K. SDB was extracted and its presence was confirmed by TLC, UV,

HPLC and IR analysis.

In vitro secondary metabolite production in Scoparia...

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