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CHAPTER - 3

To identify the secondary metabolites

from callus suspension.

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INTRODUCTION:

Many higher plants are major sources of natural products used as

pharmaceuticals, agrochemicals, flavor and fragrance ingredients, food additives, and

pesticides (Balandrin and Klocke, 1988). The search for new plant derived chemicals

should thus be a priority in current and future efforts toward sustainable conservation

and rational utilization of biodiversity (Phillipson, 1990). In the search for alternatives

to production of desirable medicinal compounds from plants, biotechnological

approaches, specifically, plant tissue cultures, are found to have potential as a

supplement to traditional agriculture in the industrial production of bioactive plant

metabolites (Ramachandra Rao and Ravishankar, 2002). Since time immemorial,

humans have been depending on plants mainly for food and medicine, apart from

various other uses (Endress 1994). Whole plants or their crude extracts have been

used as medicine without knowledge of their active constituents. It is now known that

these active principles are secondary metabolites, which in the present day, are

exploited for their use, not only in the preparation of medicines, but also as food

additives, cosmetics and agrochemicals etc. (Stafford et al. 1986; Fontanel and Tabata

1987; Fowler and Scragg 1988).

In plants, these compounds have distinct biological functions, with metabolites

like alkaloids defending against herbivores and microorganisms, or are produced as an

adaptive response to a changed environment. On the other hand, compounds which

impart colour, flavour or fragrance help in pollination or fruit dispersal by animals

(Chrispeels and Sadava 1994). Productions of secondary metabolites have also been

viewed as an expression of cell specialization triggered by cell differentiation or as an

aspect of plant development (Luckner and Nover 1977; Yeoman et al. 1982).

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Production of secondary metabolites is another important area of plant

biotechnology. Even with the advancement in synthetic chemistry, plants provide a

wide range of phytochemicals – pharmaceuticals, insecticides, flavours, fragrances,

colours etc. (Balandrin and Klocke 1988).

Plant tissue/cell culture is a frontier area of plant biotechnology with its

application in agriculture, horticulture, food, forestry and pharmaceuticals. Tissue

culture methods such as somatic embryogenesis, or micro propagation which has been

commercialized to a great extent, are instrumental in efficient multiplication of

superior clones of thousands of economically important plants. Production of

secondary metabolites from cell culture has been well established in many plant

species with commercialization in a few. Cell culture systems have also used for

production of novel compounds, which are not known to be present in the source

plant.

The capacity for plant cell, tissue, and organ cultures to produce and

accumulate many of the same valuable chemical compounds as the parent plant in

nature has been recognized almost since the inception of in vitro technology. The

strong and growing demand in today’s market place for natural, renewable products

has refocused attention on in vitro plant materials as potential factories for secondary

phytochemical products, and has paved the way for new research exploring secondary

product expression in vitro. However, it is not only commercial significance that

drives the research initiatives.The deliberate stimulation of defined chemical products

within carefully regulated in vitro cultures provides an excellent forum for in-depth

investigation of biochemical and metabolic pathways, under highly controlled

microenvironmental regimes (Karuppusamy, 2009).

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Cell suspension culture systems could be used for large scale culturing of plant

cells from which secondary metabolites could be extracted. The advantage of this

method is that it can ultimately provide a continuous, reliable source of natural

products (Mulabagal et al. 2004). Commercial production of secondary metabolites

has been achieved only in a few species like Lithospermum erythrorhyzon for

Shikonin, Coptis japonica for berberine, coleus blumei for for rosmarinic acid (Curtin

1983; Fontanel and Tabata 1987; Fujita and Tabata 1987, Chrispeels and Sadava

1994). Large scale culture has been worked out for biomass production of Panax

ginseng (Furuya et al. 1984). In addition to these, cell culture systems have also been

used for biotransformation (Reinhard and Alferman 1980; Sudhakar Johnson and

Ravishankar 1996) and production of specific enzymes (Esquivel et al. 1988).

Plants still remained, however, a great source of therapeutic agents until the

beginning of the 20th

century. The development of chemistry in the last century, plants

have been looked at the sources of new therapeutic and newer drugs of plant origin

are being discovered every year. The importance of plant derived drugs or natural

products in modern medicine are usually not fully recognized. Some 75% of the

world’s populations rely for health care on traditional medicines, which are derived

directly from natural sources (UNDP, UNEP, World Bank and WRI, 2000). Many

chemical compounds of medicinal value are still obtained from plants for one or the

other reason. These compounds provide new pharmacological properties and may

serve as a starting material for more complex biologically active compounds. Many

medicinal plants have yet to be evaluated chemically and pharmacologically

(Hostettmann et al., 1998).

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During the screening of plants for secondary metabolites and for their

activities only one activity will be considered. Thus it appears that the plant kingdom

has received little attention as a resource of potentially useful bioactive compounds.

Many secondary metabolites are genus or species specific, the chances are good to

excellent that many plant constitutents with potentially useful biological properties

remain undiscovered, undeveloped and unused (Balandrin et al., 1985).

The production of secondary metabolites in plant cell suspension cultures has

been reported from various medicinal plants. The production of solasodine from calli

of Solanum eleagnifolium, and pyrrolizidine alkaloids from root cultures of Senecio

sp. are examples (Nigra et al 1987; Toppel et al.1987). Cephaelin and emetine were

isolated from callus cultures of Cephaelis ipecacuanha (Jha et al. 1988). Scragg et al.

(1992) isolated quinoline alkaloids in significant quantities from globular cell

suspension cultures of Cinchona ledgeriana. Enhanced indole alkaloid biosynthesis in

the suspension culture of Catharanthus roseus has also been reported (Zhao et al.

2001).

Suspension cultures offer several distinct advantages over stationary cultures.

When grown in liquid medium the cells are very evenly exposed to nutrients vitamins

and growth regulators. This allows a more precious manipulation of media

components and consequently a better control of growth and development (Ammirato,

1984). Suspension culture is the most suitable culture mode for investigating the

physiological, biochemical and molecular basis of organ differentiation. More over

these cultures can be used effectively for the isolation of somaclonal variants or

mutant cell lines in addition they are often used as a renewable source of protoplasts

because the presence of cytoplasmic strands and absence of mature chloroplasts of

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could serve as a morphological markers for heterokaryon identification following

their fusion with mesophyll protoplast (Chand et al.1988).

Callus tissue is an essential material in plant cell culture systems. When it is

introduced into a liquid medium and agitated, the cells disperse throughout the liquid

to form a cell suspension culture. Such cells are, in theory, totipotent and should also

have a potential to synthesize any of the compounds normally associated with the

intact plant (Allan, 1996). As new cells are formed they are dispersed into the liquid

medium and become clusters and aggregates. Cells in suspension can exhibit much

higher rates of cell division than do cells in callus culture. Thus, cell suspension offers

advantages when rapid cell division or many cell generations are desired, or when a

more uniform treatment application is required (Philips et al., 1995). Cell suspensions

have also proven to be excellent starting materials for the isolation of protoplasts to be

used in a wide range of applications including cell fusion and genetic manipulation

(Hall, 1991).

Induction of somatic embryos by suspension culture may constitute a viable

means of rapid clonal propagation. The use of somatic embryos for clonal propagation

and artificial seed production and their cryopreservation in germplasm banks would

be beneficial for medicinally important species.

REVIEW O F LITERATURE:

The cell suspension culture systems have been studied to increase metabolite

biosynthesis in some species and can potentially be used for large-scale production by

introduction of elicitors or precursors to the medium because they were easily applied

under controlled conditions as compared to whole plants, organs or tissues (Rout et al.

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2000). It has been proved by many workers like, Zhong et al. (1996) increased the

synthesis of ginsenoside saponin in cell cultures of Panax quinquefolium and Rolfs et

al. (1987) reported increased production of the resveratrol-forming enzyme stilbene

synthase in Arachis hypogaea. Similarly anthocyanin pigments were produced in

Perilla frutescens (Zhong et al., 1994) and berberine in Thalictrum minus cultures

(Kobayashi et al., 1989). In the Phytolaccacea family the accumulation of betacyanin

was determined in Phytolacca americana (Sakuta et al. 1987) and two cell lines of

Phytolacca dodecandra produced dodecandrin, a ribosome inhibitor (Thomsen et al.,

1991; Bonnes and Mabry, 1992).

Mulabagal et al. (2004) in their review focus the application of tissue culture

technology for the production of some important plant pharmaceuticals and also they

emphasis the In vitro propagation of medicinal plants with enriched bioactive

principles and cell culture methodologies for selective metabolite production is found

to be highly useful for commercial production of medicinally important compounds.

The authors noticed the increased use of plant cell culture systems in recent years is

perhaps due to an improved understanding of the secondary metabolite pathway in

economically important plants.

According to Karupppusamy (2009) the main potential source for commercial

production of secondary metabolites is in vitro plant cell cultures and the introduction

of newer techniques of molecular biology in the in vitro cultures, so as to produce

transgenic cultures and to effect the expression and regulation of biosynthetic

pathways, is also likely to be a significant step towards making cell cultures more

generally applicable to the commercial production of secondary metabolites.

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Suspension cultures of plant cells are becoming increasingly important as

experimental material for plant growth and metabolism. The development of a system

of embryogenic suspension cultures has been a focal point for improving the

regeneration frequency in plant. In this view many researcher established

embryogenic cultures and achieved regeneration through embryogenesis. Gurel et al.

(2002) established cell suspension cultures and achieved plant regeneration in Beta

vulgaris. Chee and Tricoli (1988) reported the regeneration of Cucumis sativus from

cell cultures. Finer (1988) observed the regeneration through somatic embryogenic

suspension in Gossypium hirsutum. Sushama Kumari et al. (2000) reported

regeneration in rubber through protoplast derived suspensions and also in Hordeum

vulgare (Singh et al., 1997). In Spinacia olaracea regeneration was reported by Xiao

et al., (1997).

Many workers were also successfully achieved regeneration thorugh cell

suspension cultures. Ferreria and Handro (1988) reported in Stevia rebaudiana. Kim

et al., (2003) reported high frequency of somatic embryogenesis and plant

regeneration in cell cultures of Hylomecon vermalis. Saito and Nakrho (2002)

achieved plant regeneration from suspension cultures of Hosta sieboldoans.

Regeneration of some leguminous tree species through cell suspension culture was

achieved by Pradhana et al., (1998) in Dalbergia latifolia, Suezewa et al., (1988) in

Actinidia chinesis, Kumar et al., (1991) in Dalbergia sisso and Park and Son (1988)

in Populus alba, Jhankare et al., (2011) established embryogenic cell suspension

culture and plantlet regeneration in Withania somnifera. Tiwari et al., (2007) achieved

regeneration in Allium cepa. Roy et al., (2000) establish a protocol establish

embryogenic suspension culture and regeneration of latex producing plant Calotropis

gigantea. Ara et al., (2000) produced somatic embryos of Mangifera indica Var

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Amrapali from the proembryogenic calli from large scale production. Soomra and

Memon (2007) established suspension culture for regeneration of a biodiesel plant

Jatropha carcas.

Suzuki et al (1988) isolated an isoquinoline alkaloid, berberine in cell

suspension cultures of Thalictrum flavum and T. dipterocarpum. Seo et al., (1995)

isolated flavonoiids from cell culture of Scutellaria baccalensis. Kim et al., (2001)

reported the production of volatile flavour compounds from cell suspension culture of

Agastache rugosa. Rahman et al., (2002) produced an important saponine bacoside in

culture of Bacopa monnieri. Yamamoto et al., (1981) isolated different chemical

constituents like sitosterol, stigma sterol, camposterol, palmetic acid, linoleic acid in

cell culture of Euphorbia tirucalli and E.millii. Indole alkaloids were isolated from

Rauwolfia serpentina (Stockigt et al., (1981). Skrzypek and wysokinska (2003)

noticed the presence of two sterols, β- sitosterol and stigmasterol in the culture of

Hyssopus officinalis. Elfahmi et al., 2006 isolated lignans from the suspension culture

of Phyllanthus niruri. Lignan accumulation was also observed in Linum strictum by

Vasilev and Ionkova (2004). Hayashi et al., (1988) reported the presence of

triterpenoids in callus and suspension culture of Glycyrohiza glabra. Villarreal et al.,

(1997) dedicated an antifungal spirostanol saponin in cell culture of Solanum

chrysotrichum. Jang et al., (1998) produced a hepatoprotective cerebroside from cell

culture of Lycium chinense. Danelute et al., (2005) reported the production of phenyl

ethylamines, dopamines and tyramine in Piper cernuum and alkaloids in P.

crassivervium. Kanwar et al. (2008) reported plant regeneration in Robina

pseudoacacia from cell suspension culture. Sujanya et al., (2008) elucidate the effect

of nutritional alteration on biomass content and azadirachtine production in cell

suspensions of Azadirachta indica.

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Only few reports on suspension culture of latex producing plants have been

documented including Hevea (Wilson and Street, 1975), Papaver (Nessler, 1982),

Asclepias (Dunbar et al.1986) Calotropis (Roy et al.2000) and in Cynanchum acutum

(Ahmed, 2011). The present study aims to establish the suspension culture form the

callus of two important latex producing medicinal plants such as Oxystelma secamone

and Tragia involucrata and for regeneration and phytochemical analysis.

MATERIALS AND METHODS:

Plant material:

Suspension culture was carried out in two selected medicinal plants Tragia

involucrata and Oxystelma secamone.

Callus Induction:

Callus was induced from leaf explants of both species were used for

suspension culture. From in vitro studies it was revealed that MS basal medium

supplemented with 2.0 mg/ l exhibited a better response in inducing the friable callus.

The same callus was maintained in the aseptic condition through subculture to induce

cell culture. Initiaton of cell suspension culture.

Subculture:

In order to initiate suspension cultures of both species, four weeks old friable

calluses obtained from leaf culture were selected. The friable callus massed (2g fresh

weight) were transferred to 250 ml Erlenmeyer flasks containing 50ml of MS liquid

medium supplemented with 2,4-D( 2.0mg/l). The flasks were incubating on a rotary

shaker at 100rpm at 25⁰C with 60-70% relative humidity and a 16 hr photoperiod by

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cool white fluroscent lamps). Such suspensions were subcultured usually every 16

days by transferring 10 ml of old suspension filtrate to 40 ml of fresh medium and

incubated as indicated above.

Large clumps of embryogenic tissue, which were first seen 4-8 weeks

following initiation of suspension cultures, were transferred to the embryo

proliferation medium. The embryo proliferation medium was the as that used as the

auxin. Proliferating suspension cultures were subcultured weekly to monthly

depending on subculture inoculums density.

Embryogenic potential

In both the plants embryoid formation is brought about by transfer of

suspension from 2,4-D containing to 2,4-D omitted medium. The embryogenic

potential was based upon the number of embryoids in suspension cultures grown in a

medium for 15-20 days. The number of visible embryoids was counted by placing the

petridish. For suspension cultures 10 ml suspension was transferred to 40 ml liquid

medium in 250 ml Erlenmeyer flasks and incubated on the shaker under standard

conditions and then in a examined in a thin layer in a petridish as described above.

Size of the embryoid ranges from 0.4mm to 2.0mm in length. After two or three

culture passages in 2, 4-D containing medium, the cultures do not containing medium,

the cultures do not contain any microscopically identifiable stages and it is only on

transfer of such lacking 2, 4-D medium i.e. BAP(2.0mg/l)+ NAA(1.0mg/l) cultures

exhibit various types of embryoids such as globular, cordate and torpedo stages. There

is decline in embryogenic potential on continuing subculture and represents a true loss

of totipotency.

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Measurement of Growth

Increase in fresh and dry weights and settled cell volume of the cells in culture

was followed at 4 day intervals for a period of two weeks using freshly subculture cell

suspension cultures which consisted of 1ml settled cells from the previously

established suspension culture. Settled cell volume was determined transferring the

suspension to a graduated tube and allowing the cells to settle down by letting the

tube stand for an hour. The fresh weight and dry weight of suspension cultured cells

was determined at 4 days intervals throughout the one batch culture period (32days,

2gm fresh inoculums; Jang et al. 1998). To determine the fresh mass, cell clusters

were collected on a filter paper, drained and transferred onto reweighed aluminum foil

and immediately weighed again. Dry weight by placing the cells in until constant

weight was attained (Kwa et al.1997).

Viability of the cells:

One of the requirements for the establishment of cell culture is to count

on a reliable and efficient method to estimate cell viability. The cell viability can be

evaluated by staining the dead or living cells, because the colour is a product of cell

metabolic activity. The most used stain from dead cells is Evans blue or methylene

blue. The Evans blue is reduced by the living cells turning colourless. While the dead

cells remain blue. In the present study, Evans blue was used as a staining method

because of cheap, reliable and can be observed easily under light microscope.

Histological examination

For histological examination, the larger clumps and embryos’ from the

suspension culture were formalin: acetic acid: alcohol (FAA) dehydrated through a

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tertiary butyl alcohol: xylene series and embedded in paraffin wax (Johansen 1940).

Serial sections (10µ) were cut with a rotary microtome and stained with haematoxylin

(2%). Mounted in water mounting medium and mounted in DPX mountant.

Cytology

For cytological variations to study, tissue or suspension was pretreated with

0.01% Mohr’s salt solution for 3.30 Hrs, washed in eater, fixed in 3:1 absolute

alcohol: glacial acetic acid for 24-48 hrs, washed with water, stored in 70% alcohol

and the material is treated with 0.5% mordant for 5mins. Stained by the acetoorcein

ad observed under light microscope.

Preparation of extract:

Dried cells mentioned above were successfully extracted with methanol

using soxhlet apparatus for 18h and after recovery off the solvents, the extracts were

concentrated and aqueous extract also prepared and stored at 10°C in refrigerator for

further phytochemical analysis.

Isolation and identification of phenols form T. involucrata:

Methanol extract of both aqueous and dried cell suspension samples were

dissolved in methanol and were subjected to HPLC (model LC-6A, Shimadzu)

analysis on a SphereClone 5µ ODS 2 column (4.6 mm x 150 mm, Phenomenox) using

UV-detection system. Chromatographic run was done with mobile phase consisting of

an isocratic solvent mixture of water: acetic acid: methanol (80: 5: 15 v/v/v) with a

flow rate of 1 mL/min the chromatograms were monitored at 280 nm (Sureshkumar et

al. 2006). The standard phenolic acids dissolved in methanol (1 mg/mL) were also run

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on the same HPLC column under similar conditions. The retention time of the

samples were compared with that of standard phenolic acids to identify the respective

phenolics.

RESULTS

Yellowish, friable calli began to form on the cut edges of leaf explants after

two weeks of culture on medium with 2, 4-D and on medium with combinations of

BAP or Kn and NAA or IAA. After four weeks of culture, some of the primary

yellowish, friable calli produced secondary white, friable and nodular calli. These

nodular structures were separated using sterilezed forceps and subcultured on the

liquid medium of the same composition as used in the original culture. These calli

masses proliferated and produced somatic embryos of various stages in the leaf calli

of both the species, indicating that the white, friable calli were embryogenic.

Evans blue is an indicator of non-viable cells, in the present study the viable

cells were non stained (Fig.3.4), whereas the viable cells did not stain the evans blue

method yielded a 80% of viablility and it was low at room temperature (20%) and

increased as temperature is increased.

At the out set, it was important to study the growth of the callus tissues in

liquid medium of complex constitution and to use the behaviout here as a basis for

comparison with growth in synthetic medium. For this purpose, liquid medium was

prepared of the constitution MS with 2.4-D. on the basis of their growth performance

4-5 fold increase in fresh weight and dry weight during the 16 days of culture

(Table3.1 and 3.2). the percent dry matter of both these plants remained with in 3-4%

of the frersh weight during the optimum growth period.

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On MS medium with 2 mg/l 2,4-D and 2.0 mg/l BAP spontaneously produced

proemryogenic clusters developing into globular stage embryos. These embryos were

observed when the cultures were of four weeks old. The embryogenic capacity of the

suspension cultures has been retained for eight weeks. The cultures turned green and

produced embryos in different stages of development. These embryos were isolated

and placed in the high intensity of white light for 18 hrs and these embryos developed

through the different stages of embryogenesis such as one celled, bi or tricelled ,

heart, torpedo and cotyledon stage into small plantlets with leaf promordia. Within

five weeks the plantlets developed further. Besides the embryos, adventitious roots

appeared from the callus cultures.

Morphologically the callus in suspension in both the selected species, mainly

consisted of cell aggregates of round to oval shaped cells. In order to maintain the

suspension culture, a portion (10 ml) of liquid suspension cells were transferred to

fresh MS liquid medium at 3-4 week intervals.

Sequential filtration through 500-100 µm sieve was performed every 2-3

subcultures. In this the cultures were composed of isolated cells (15-30%) and small

cellular aggregates (10-60 cells) were obtained in three weeks. Some mitotic

abnormalities such as papillate projections , multinucleate, multinucleolate cells with

differeing nucleoli number in each nucleus and cell with gigantic nucleus and nuclear

material migration from cell to cell observed.

Variations in both the species after staining with acetoorcein also showed

clump of three cells (Fig.3.1), some cellls actively divided to produce the filamentous

embryo (Fig.3.4 and 3.12), papillate projections from the isolated cells were evident

with nucleus entering into the papillate structure (Fig.3.3 and 3.3a), some cells

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(polyploidy) possess gigantic nucleus or with tetranucleolate nucleus (Fig.3.7 and

3.9). Binucleated cells were also observed (Fig.3.11 and 3.14).

Mass of embryogenic callus with peripheral developmental stages (Fig.3.16

and 3.26), two celled or three celled embryo (Fig.3.17 and 3.25), cordate shaped two

celled embryo with a stalk (Fig.3.21), single celled pro embryo with suspensor

(Fig.3.27), torpedo shaped embryoid (Fig.3.29).

Besides these abnormalities in both the species vascularised cells with various

types of thickenings (Fig.3.8 and 3.20) has been observed. These cytodifferentiated

cells differ in their percentage and ranges from 5-10% when compared to normal

isolated cells.

Histologically in both the species four week old fast growing suspension

culture revealed embryogenic cluster of cells, peripherally showing one or two celled

proembryos with single celled suspensor. Further more embryos of all stages

(filamentous, globular, cordate and torpedo shaped) were observed. The differentiated

embryos matured in the same liquid medium in 5-6 weeks. The number of

differntiated embryos varied in the subsequent passages. However, after the fourth

passage, the number of embryos were reduced and in the fifth subculture, the

suspension culture lost its embryogenic potential. The embryos remained dormant in

both the species and did not germinate into plantlets.

Phenolic compounds were isolated besides carbohydrates, alkaloids, steroids,

flavonoids, tannins and triterpenoids from the cultured cells of suspension in 250ml

flasks (Table.3.3 and 3.4). This was identified by various analytical methods

including HPLC technique subjecting methanol extract of T. involucrata leaf callus

suspension. HPLC analysis revealed the presence of various types of phenolic

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compounds based on retention time such as gallic acid, vanillic acid, chlorogenic acid,

protocatechuic acid and ferulic acid.

Ferulic acid could be detected besides gallic acid, vanillic acid, chlorogenic

acid and protocatechuic acid. It was found be de novo prduct of cell suspension

culture which was found in trace amount in the in vivo extracts of T. involucrata leaf

extracts (Graph.3.1 and 3.2).

DISCUSSION

The effectiveness of a sieve has a means of initiating suspension culture of O.

secamone and T. involucrata probably was due to the fractionation of the cell

population and the better acess to media components. However, our results are not in

accordance with those of Nomura and Komamine (1985), who found that a small

mesh size (16µm) followed by centrifugation through a percoll radiant gave best

regeneration from Daucus carota. The centifugation may be the reason for their

success with small cell agreegates.

The bulk of material in embryogenic suspension cultures of T. involucrata and

O. secamone was composed of large cellular units, the embryogenic fraction being

essentially restricted to calli and cell aggregates. Similar observation made for long

term suspension cultures of Daucus carota propagated by subculturing nodules 1mm

in diameted every month (Sussex, 1972).

The embryo proper consisted of a small cluster of dense cytoplasmic cells that

appeared yellow-brown when viewed on an inverted microscope. The suspensors

were single celled (stalk cell). Similarly in other studies (Hakman and Fowke, 1987),

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the suspensors were attached to small embryos and were observed to occur as single

strands.

Transfer of embryogenic cell suspensions to MS medium with 2,4-D resulted

in recallusing of embryos. The traser of embryogenic mass to a medium containing

BAP (2.0mg/l) + NAA (1.0mg/l) was essential for further development of

embryogenic mass and after four to five weeks growth led to greening and

development of embryoids in the species of Gramineae (Vasil, 1985). Somatic

embryogenesis has been reported in a number of plant species in vitro and there is a

clear evidence of the origin of somatic embryos from single cells as reported by Mc

William et al.(1974) in Daucus carota and Konar et al (1972) in Ranaculus scleratus.

According to Haccius (1978), somatic embryos in vitro araise from single cells. In

support to this in our present investigation, the suspension cultured cells of both the

species have developed embryoids from single cells. Variations observed in the

development of embryoids in these two species are also common in many other

species, for eg in carrot the proembryoids develop into structures which show varying

degrees of root and shoot development with little resemblence to normal embryos

(Halperin, 1964).

Evans blue stain only the dead cells because in the living cells the dye is

reduced to a colourless form, but it is difficult to distinguish the dead cells in a blue

background due to the low extinction co efficent of this dye (Huang et al. 1986). In

agreement with these authors, we also observed that it is difficult to discriminate

between living and dead cells. Previous research has also showed that the time

required to observed the colours in the cells varies with the compound. For eg, evans

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blue stains the cells raphidly (10 mins) simillar to fluorescin di acetate (3mins)

(Widholm, 1972).

It includes the deposition of a specifically patterned secondary cell wall, its

subsequent lignification, then loss of the nucleus, cell contents and death of the cell.

In T. involucrata and O. secamone cytodifferntiation occurred on the medium

containing both auxin and cytokinin. Similar type of behavior has to the formation has

been observed in Allium sativum by Havranck and Novak (1973) cultured on a

medium supplemented with auxin and cytolinin. They emphasisied that xylogenesis is

favoured by auxin-cytokinin balance in the medium. In O. secamone xylogenesis is

also observed on the medium supplemented with 2,4-D which is quite similar to the

work of Fosket and Torrey (1969) who cultured the soyabean on a medium

containning 2,4-D even in the absence of the cytokinin. The presence of vascular

elemetnts in the cultures indicates the potentiality to regeneratre the plant lets from

the callus cultures.

The present study has demonstrated that both these plants are capable of

producing different kinds of metabolites, particularly phenols. Phenol production in T.

involucrata and O. secamone was greatly promoted by simultaneous administration of

high concentrations of 2, 4-D and subsequently by the addition of BAP contrarily in

the Thalictrum minus (Nakagawa et al. 1986) high concentration of NAA and BAP

are supplemented for the production of phenols. Schulte et al (1984), who investigated

optimal hormonal conditions ffor anthraquinone formation in cell cultures of different

species belonging to Rubiaceae. They have indicated that there is no general rule in

optimizing the anthraquinone production among cell cultures derived from different

cell cultures of the family or even of the same genus.

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The production of phenol in cell cultures of these two species is maximum at

the senescence period off growth phase. The secretory activity of these species was

evidenced in releasing most of the phenols into the medium. By contrast, in

Thalictrum cultures proved to accumulate berberine exclusively in the cells, never

secreting it under any hormonal condition tested.

Among phenols of varous produced in cultures of T. involucrata ferulic acid is

in greater amounts when compared to the amount of ferulic acid produced in the leaf

extract. This result indicates the advantages and abilities of suspension cultures over

the natural source with respect to obtaining substantial amounts of ferulic acid

contrarily secondary metabolites exhibit non growth associated production kinetics in

cell culture (Sahai and Knuth, 1985).

In accordane to an earlier report (Pal and Sarin, 1992), as well as the present

observation with respect to the leaff extract, the phenol content always predominated

in both the species throughout growth. To our knowledge, isolation of phenols in

general ferulic acid in particular in higher amount, this is the first isolation from

cultureed cells of T. involucrata. Ferulic acid (Hydroxy cinnamic acid)is a component

of lignin of the cell wall. This may serve as an important antioxidant function in

preserving physiological integrity of cells exposed to both air and impinging UV

radiations, it also gives protection against various inflammatory diseases. Efffect of

ferulic acid on gastroinstestinal tract both in vivo and in vitro has been studied besides

protective effect of ferulic acid on hyperlipidemic diabetic rats.

The present work is a necessary step in the direction of obtaining such cellular

material for study. The fact that cell suspensions of higher plant tissues can be

cultivated in c completely defined synthetic medium under relatively simple

conditions means that biochemical studies can now be pursued with these tissues. The

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146

evidence is already at hand that cell suspensions represent ideal material for studying

especially problems of cellular diffrentiation. Considerable improvement can be made

to increase tissue growth and seperation and to simplify the medium itself to meet

more precisely the specific requirements of the tissue studied.

With a synthetic medium now available the problem is open to study. Other

factor in the medium may also be important in tissue dissociation, notably ascorbic

acid. The present experiments suggest further that the nutrient requirements for

continued proliferation of cell suspensions derived from higher plant tissues may be

different and more complex than those for the same tissues grown as a multi cellular

mass on a solidified medium.

It has been well known that the cells are totipotent and can produce all the

compounds originally found in the plants. Cells of T. involucarata and O. secamone

retain the ability to synthesize chlorogenic acid, gallic acid, protocatechuic acid,

vanillic acid and ferulic acid which are antioxidants. Of these the content of ferulic

acid was more in the suspension culture cells than leaf extract. If cell medium or

suspension environment were improved the content of these phenolics could be

increased to a greater extent.

The presence of phenols in these medicinal plants indicates that they could act

as antioxidants, immune enhancers and hormone modulators. Phenols have been the

subjects of extensive research as bioactive compounds used in disease prevention.

Phenols have been responsible in having the ability to block specific enzymes that

causes inflammatory disorders. They also modify the prostaglandin pathways and

thereby protect platelets from clumping (Duke, 1992). These findings justify the

traditional use of these plants as antioxidants in traditional medicinal practice.

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147

Fig 3.1: Clump of three cells in O. secamone callus suspension

Fig 3.2: Embryogenic mass of cells in O. secamone callus suspension

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148

Fig 3.3: A single isolated cell in O. secamone callus suspension

Fig 3.3a: Cell in O. secamone callus suspension showing papillate projection

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149

Fig 3.4: Actively dividing cell forming filamentous nature in O. secamone callus

suspension

Fig 3.5: Isolated cells in a microscopic field in cell suspension of O. secamone

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150

Fig 3.6: Clump of dividing cells and nuclear materiaI migration between

adjacent cells in suspension of O. secamone

Fig 3.7: An isolated cell with gigantic nucleus in suspension of O. secamone

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151

Fig 3.8: Cytodifferentian and a single cell embryoid with a single celled stalk in

cell suspension of O. secamone

Fig 3.9: A polyploid cell with tetranucleate nucleus in cell suspension of O.

secamone

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152

Fig 3.10: An isolated cell in dividing mitotic metaphase in O. secamone

suspension

Fig 3.11: Binucleated cell of O. secamone showing variation in the number of

nucleoli in two nuclei.

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153

Fig 3.12: Actively dividing cell forming filamentous nature in T. involucrata

suspension

Fig 3.13: Isolated cells in a microscopic field in cell suspension of T. involucrata

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154

Fig 3.14: Binucleated cell of T. involucrata in callus suspension

Fig 3.15: Highly vacuolated elongated suspension cells of T. involucrata

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155

Fig 3.16: Embryogenic callus of T. involucrata with peripheral developmental

stages.

Fig 3.17: Two celled embryo in cell suspension of T. involucrata

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156

Fig 3.18: Embryogenic callus of T. involucrata showing an embryoid

Fig 3.19: Cytodifferentiation in cell suspension of T. involucrata

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157

Fig 3.20: Bicelled embryos in embryogenic cluster of T. involucrata

Fig 3.21: Cordate shaped 2-celled embryo with a stalk in cell suspension of T.

involucrata

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158

Fig 3.22: Embryoids of T. involucrata (2-celled) with stalk cells

Fig 3.23: One celled embryoid with stalk cell in T. involucrata

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159

Fig 3.24: A filamentous embryo of T. involucrata

Fig 3.25: 2- celled , 3- celled embryoids with suspensor in T. involucrata

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160

Fig 3.26: Embryogenic mass in suspension of T. involucrata showing

peripherally situated enbryoids of various stages.

Fig 3.27: Proembryos with suspensor in T. involucrata

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161

Fig 3.28: Torpedo shaped embryoid with suspensor in T. involucrata

Plate 3.1: Methanol extract of leaf and suspension on TLC plate showing

presence of phenolics.

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162

Table. 3.1: Settled cell volume (SCV), Fresh weight (FW) and dry weight (DW)

of primary cell suspension culture initiated from leaf derived callus

of O.secamone

Data are mean ± SD from two independent experiments

Days in culture SCV (ml/flask) FW mg/flask DW mg/flask

0 1.2 545 ± 26 42.11

4 1.5 665 ± 54 55 ± 6

8 1.8 723 ± 58 65 ± 3

12 2.0 805 ± 45 75 ± 5

16 2.2 915 ± 111 95 ± 9

Table.3.2: Growth characteristics of T.involucrata suspension cultures as

measured by packed cell volume (PCV), Fresh weight (FW) and dry

weight (DW).

Data are mean ± SD from two independent experiments

Days in culture SCV (ml/flask) FW mg/flask DW mg/flask

0 1.9 158.4 ± 3.6 11.0 ± 0.62

4 2.5 178 ± 4.4 13.3 ± 0.40

8 4.9 250 ± 8.4 21.7 ± 0.80

12 8.3 495.8 ± 8.4 47.9 ± 1.4

16 12.6 1066.0 ± 32.0 80.6 ± 2.6

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163

Table 3.3: PRELIMINARY PHYTOCHEMICAL ANALYSIS OF

T.INVOLUCRATA LEAF AND ITS DRIED SUSPENSION

CULTURE EXTRACTS

+ = presence of compounds - = absence of compounds

Table 3.4: PRELIMINARY PHYTOCHEMICAL ANALYSIS OF

O. SECAMONE LEAF AND ITS DRIED SUSPENSION

CULTURE EXTRACTS

+ = presence of compounds - = absence of compounds

Tests for Methanol extract Aqueous extract

Leaf Suspension Leaf Suspension

Alkaloids + + + +

Glycosides + + + +

Carbohydrates + + + +

Saponins + + + +

Phytosterols + + + +

Phenolics and tannins + + + +

Sugars - - + +

Tests for Methanol extract Aqueous extract

Leaf Suspension Leaf Suspension

Alkaloids + + + +

Glycosides + + + +

Carbohydrates + + + +

Saponins - - + +

Phytosterols + + + +

Phenolics and tannins + + + +

Sugars - - + +

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164

GRAPHS 3.1: Methanol extract of T. involucrata leaf (in vivo)

Retention time Compounds

2.33 Gallic acid

2.923 Chlorogenic acid

4.357 Protocatechuic acid

10.477 Vanillic acid

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165

GRAPHS 3.2: Methanol extract of T. involucrata leaf callus (in vitro)

Retention time Compounds

2.427 Gallic acid

3.06 Chlorogenic acid

4.48 Protocatechic acid

11.1 Vanillic acid

28.3 Ferulic acid