review of literature - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/11602/14... · 2015-12-04 · Review of literature 20 with activity against several kinds of cancer (Schmeller

Review of literature

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2.1 Plant as a source of alkaloids

Medicinal plants are vital source of compounds for the pharmaceutical industry and for

traditional medicine. About 80% of the population living in developing countries still use

traditional medicines derived from plants for their primary health care (De Silva 1997). The

success of health also depends on the availability of suitable drugs on a sustainable basis.

Although synthetic drugs and antibiotics are essential these days for current medical practice

plants too provide a major contribution to the pharmaceutical industry (Sahoo et al. 1997).

Alkaloids are one of the largest classes of secondary metabolites. These compounds contain

heterocyclic nitrogen usually with basic properties that makes them particularly

pharmacologically active. Alkaloids have been traditionally isolated from plants as around

20% of the plant kingdoms contain these compounds (De Luca and St Pierre 2000). A large

number of alkaloids have been used in medicine and many of them are basic components of

modern drugs (Morgan and shank 2000). The terpenoid indole-alkaloids (TIAs) form a

family of more than 3,000 members of which only a few have known physiological effects in

mammals (Geerlings et al. 2000). These types of alkaloids have been found in several

families, but are more prevalent in families like Apocynaceae, Loganiaceae, Nissaceae and

Rubiaceae (Verpoorte et al 1998), all are under Gentiales order. Among the better known and

studied plants, which produce TIAs are Catharanthus roseus, Tabernaemontana divaricata

and Rauvolfia serpentina (Cordell 1999). Due to economical interest of TIA in C. roseus, the

physiological, biochemical, cellular and molecular aspects of their biosynthesis have been

studied extensively. With this aim, the whole plant, plant parts, callus, in vitro cell

suspensions, hairy root cultures have been used as model source of materials.

Micropropagation by tissue culture offers an alternative way of plant propagation and has the

potential to provide high multiplication rates (Beck and Dunlop 2001). Some important

plants/trees can now be selected, grafted, rejuvenated, cloned through somatic embryogenesis

(micropropagated) and polyembryogenesis techniques (Beck and Dunlop 2001). The recent

large-scale cloning of spruces and eucalypts has validated the importance of

micropropagation. Thus, clonal propagation through tissue culture is receiving increased

recognition as an alternative to conventional vegetative practices (Han et al. 1997).

Micropropagation of mature tissues through tissue culture also allows for the improved

quality of selected traits such as high yield and superior pulping properties (Jones and Van

Staden 1997). Planting genetically superior clones instead of seedlings, which vary both


19

genotypically and phenotypically, may increase productivity (Beck and Dunlop 2001). The

advantage of micropropagated plants is that the regenerated plants show juvenile

characteristics such as rapid growth.

In vitro techniques have also been used in the propagation of a large number of valuable and

endangered medicinal plants (Sarasan et al. 2006). This technique is more advantageous as it

demonstrates rapid clonal multiplication as well as for germplasm conservation (Saritha and

Naidu 2007). The induction of multiple shoots through axillary branching is now marked as

an efficient technique for micropropagation and in vitro conservation of threatened plants

(Constable 1990). Micropropagation has several other advantages over conventional methods

of vegetative propagation, which suffer from several limitations. Indiscriminate, ruthless

collection of medicinal plants for their medicinal purposes is causing rapid depletion of flora,

leading to the extinction of many species. It is possible to save local flora if proper

propagation and conservation measures are taken in time (Gilani et al. 2009). Thus, there is

an increasing interest in using these techniques for rapid and large-scale propagation of

medicinal and aromatic plants (Otroshy and Moradi 2011).

2.2 Taxonomy, habit and habitat of Catharanthus roseus

Medicinal plants are the traditional source of drugs (Jha et al. 2011). Catharanthus roseus

(L.) G. Don of the family Apocynaceae is one of the most widely investigated medicinal

plants. It is a perennial, evergreen herb, 30-100 cm height that was originally native to the

island of Madagascar. It has been widely cultivated for hundreds of year and can now be

found growing wild in most warm regions of the world. The leaves are glossy, dark green (1-

2 inch long), oblong – elliptic, acute, rounded apex; flowers fragrant, white to pinkish purple

in terminal or axillary cymose clusters; follicle hairy, many seeded, 2-3 cm long; seeds

oblong, minute, black. The plant is commonly grown in gardens for beddings, borders and for

mass effect. It blooms through out the year and is propagated by seeds or cuttings. The bloom

of natural wild plants are pale pink with a purple eye in the centre, but horticulturist has

developed varieties (more than 100) with colour ranging from white to pink to purple (Junaid

et al. 2010).

2.2.1 C. roseus and its medicinal importance

It has been used in traditional medicine as a hypoglycemic agent (Singh et al. 2001). The

present interest in this plant is due to the fact that it is a source of chemotherapeutic agents


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with activity against several kinds of cancer (Schmeller and Wink 1998) and also because it

produces a great variety of terpenoid indole alkaloids (TIAs), most of them with

pharmacological activity (Van der Heijden et al. 2004). Vinblastine (VB) and vincristine

(VC) are perhaps the most important alkaloids with anti-cancerous property (Mukherjee et al.

2001). VB is used against several forms of cancer like Hodgkin’s disease (Schmeller and

Wink 1998) while VC is used in the treatment of leukemias (Schmeller and Wink 1998). This

plant also produces antihypertensive agents such as ajmalicine and serpentine, which are used

to overcome heart arrhythmias (Shanks et al. 1998). These agents improve blood circulation

in brain (Moreno et al. 1995). Some of the TIAs are used in the treatment of anxiety

(serpentine), arterial hypertension (ajmalicine) (Kruczynsky and Hill 2001) and similar other

disorders.

The cost of 1 kg of VB in the market is around a million dollars and the world annual

production is near 12 kg. On the other hand, VC has reached the price of 3.5 million dollars

for 1 kg and its annual production is 1 kg. The high cost of these alkaloids is due to the fact

that these two compounds are present in minute amounts in C. roseus leaves (around

0.0005% DW) and their extraction is carried out in the presence of many other compounds

with very similar properties (De Luca and Laflamme 2001).

2.2.2 C. roseus cultivation

The genus comes from Madagascar and has been cultivated with ornamental aim because it

produces flowers of pink or white colour for most of the year (Loyola-Vargas et al. 2007). As

an ornamental and medicinal plant, C. roseus is cultivated in tropical and subtropical regions

of the world (Yuan et al. 2011). The climatic conditions and the soil properties of some

European countries are, however unfavourable for the cultivation of C. roseus. It may be

grown only as an annual plant in greenhouses and in plastic tunnels but in that cases the

content of dimeric indole alkaloids was observed to be very low (Pietrosiuk et al. 2007). In

Poland, hydroponics technique is also used for C. roseus cultivation (Lata et al. 2007).

2.2.3 Plant tissue culture and regeneration in C. roseus

The cultivation of plant parts, i.e. shoot and root has been practised for rapid biomass

production in C. roseus and for in vitro biosynthesis of secondary metabolites (Pietrosiuk et

al. 2007). The first observations related to the formation of roots from the callus tissue were

reported by Dhruva et al. (1977). Ramavat et al. (1978) described the formation of shoots of


21

C. roseus from the callus. Plant regeneration from haploidal and diploidal callus cells of

using different combinations of the plant growth regulators (PGRs) like kinetin and IAA, was

carried out by Abou-Mandour et al. (1979). Krueger et al. (1982) established plant and leaf-

organ cultures from seeds of C. roseus, germinated aseptically on the Murashige and Skoogs

Revised Tobacco medium (MS RT) supplemented with BA. The process for conducting plant

organ cultures of C. roseus capable of producing significant amounts of indole alkaloids

including VB, VC, vindoline, catharanthine and ajmalicine was patented earlier (Miura and

Hirata 1986). Endo et al. (1987) induced root and shoot cultures from the seedlings of C.

roseus.

Plant regeneration from existing meristems is also attempted for quick biomass production in

pharmaceutical industries as it helps in obtaining regenerants of a stable invariable genotype

(Pietrosiuk et al. 2007). Furmanowa et al. (1994) successfully regenerated C. roseus plantlets

when the shoot tips were excised from 7-day-old seedlings and incubated in solid Nitsch and

Nitsch (NN) medium (Nitsch and Nitsch 1969), supplemented with kinetin, BA, IBA and

IAA in various combinations.

2.2.4 Somatic embryogenesis in plants and in C. roseus

In plant tissue culture, somatic embryogenesis method has been employed for various

purposes including elite plant propagation. It is a process by which plant’s somatic cells are

transformed into embryos in culture. It is a period of transition from the reproductive single-

cell state to the multicellular organization of the mature embryo or seedling. Although the

embryos of many other plant species display less regular division patterns, the essential

features of division pattern formation are likely to be similar because of the close

evolutionary relationship among flowering plant species (Johri et al. 1992). The formation of

somatic embryos is now recognized as a useful method of clonal propagation, but the

technique can also be used for plant regeneration from transformed cells, artificial seed

production, and for the study of plant embryogenesis (Von-Arnold et al. 2002).

Although somatic embryogenesis (SE) has been reported in a wide variety of plant genera of

angiosperms and gymnosperms (Thorpe 1995; Thorpe and Stasolla 2001; Mujib and Samaj

2006) the report of in vitro embryogenesis was rather new in C. roseus (Junaid et al. 2006).

Earlier, a preliminarily study on plant regeneration from immature zygotic embryo was

reported, in which the system for high frequency plant regeneration through somatic

embryogenesis had described in Catharanthus (Kim et al. 2004). The advantage of SE is that


22

the initial cell populations can be used as a single cellular system and their genetic

manipulation are easy and are similar to microorganisms (Junaid et al. 2006). There are

several factors that control embryogenesis in culture some of them are like the involvement

of PGRS, carbohydrates, pH, light, amino acids etc. (Junaid et al. 2008). It has been noted

that SE can also be induced by various stresses in addition to the use of specific hormonal

treatments and over expression of specific genes (Ikeda-Iwai et al. 2003). This has been

clearly observed in plants like carrot, Arabidopsis and several other genera (Kamada et al.

1993; Touraev et al. 1997; Ikeda-Iwai et al. 2003).

2.2.5 Suspension culture in C. roseus

Suspension culture studies were performed in bioreactors of different volumes and types for

large scale production of catharanthine and ajmalicine in C. roseus (Fulzele and Heble 1994).

Ten Hoopen et al. (2002) showed that the temperature has an important influence on growth

and ajmalicine production in C. roseus suspension cultures. The optimal temperature for

biomass growth and subsequent secondary metabolite production was noted to be at 27.50C.

Bhadra et al. (1993) evaluated alkaloids production in selected hairy root cultures of C.

roseus as the use of hairy roots has many advantages including their genetic and biochemical

stability compared to other cultures (Khan et al.2009).

2.2.6 Alkaloids of C. roseus in cultured tissue

C. roseus produces numerous alkaloids most of them are of high pharmaceutical importance.

Among those alkaloids, VB and VC are extremely valuable antineoplastic medicines (Magdi

and Verpoorte 2002). Ajmalicine and serpentine are also medicinally valuable alkaloids that

have use as anti-hypertension agents. The amounts of these alkaloids, particularly VB and

VC in plant are, however, extremely low, thus, several laboratories worldwide employ plant

cell and tissue cultures as alternative means of production of alkaloids (Min et al. 2004) with

intended purpose to enhance production of these valuable alkaloids (Ataei-Azimi et al. 2008).

It was noted that the tissue differentiation plays a significant role for alkaloid yield and in the

types of alkaloids produced (Morgan and Shanks 2000). For example, the synthesis of

vindoline is restricted to the leaves of the plant (Morgan and Shanks 2000), while ajmalicine

and serpentine are the major alkaloids found in roots of the plant as well as cell suspension

cultures (Li et al. 2011). In hairy roots of Catharanthus species, tabersonine, lochnericine,

and horhammericine are the major products in addition to ajmalicine and serpentine (Lee-

Parsons and Royce 2006). Near ultraviolet light (NUV) with the peak at 370 nm stimulated


23

the production of leurosine, one of the major dimeric indole alkaloids, in multiple-shoot

cultures of C. roseus (Pietrosiuk et al. 2007). In contrast, the contents of vindoline and

catharanthine were decreased greatly by this light treatment. The results suggested that NUV

light might specifically stimulate the synthesis of leurosine and to a lesser extent, VB from

vindoline and catharanthine. Pietrosiuk and Furmanowa (2001) earlier investigated indole

alkaloid production in roots of C. roseus, cultured in vitro.

2.2.7 Use of PGRs in C. roseus

A number of PGRs are used in culture for various in vitro purposes including embryogenesis

and in most cases the right balance or the ratio of the compounds are the primiary basis for

optimization of embryogenesis at different stages of embryo development (Junaid et al.

2008). PGRs are also noted to be responsible for the diversification of alkaloids and even

these agents enrich the yield of alkaloids in several studied plants (Bush et al. 1997). In C.

roseus, Hirata et al. (1994) studied the importance of PGRs in growth and morphological

differentiation of tissues, leading to the formation and development of shoots. Yuan and Hu

(1994) investigated the influence of different combinations of auxins, cytokinins and light

intensity on the formation of multiple shoots of C. roseus in in vitro cultures. Satdive et al.

(2003) studied the effect of different concentrations of IAA and BA on the production of

ajmalicine in flasks using multiple shoot cultures of C. roseus. The roots obtained on NN

medium with addition of BA were white, thin, long and very branched, whereas roots

produced on medium with kinetin were yellow, thick and had shorter branches (Pietrosiuk

1997). An auxin–cytokinin combination was noted to promote regeneration from protoplast-

derived callus in several studied plant systems (Borgato et al. 2007). The accumulation of

PGRs, long callus phases and the use of 2, 4-D reduced the formation of somatic embryos

and caused genetic and epigenetic variations in cultured tissues (George et al. 2008a).

The effects of PGRs on the contents of C. roseus TIAs has been extensively studied (El-

Sayed and Verpoorte 2007; Zhao and Verpoorte 2007; Pan et al. 2010). PGRs such as methyl

jasmonate (MeJA), jasmonate (Lee-Parsons et al. 2004; El-Sayed and Verpoorte 2005; Ruiz-

May et al. 2008; Peebles et al. 2009), abscisic acid (ABA), salicylic acid (SA) (Bulgakov et

al. 2002; Mustafa et al. 2009) and gibberellic acid (GA3) (Srivastava and Srivastava 2007;

Amini et al. 2009) showed significant influence on TIAs production and enzymes activities of

the biosynthesis pathways in C. roseus cell suspensions cultures, hairy roots and seedlings

(El-Sayed and Verpoorte 2004; Ruiz-May et al. 2008). The addition of PGR in media affects


24

both culture growth and secondary metabolite production. Cytokinins, another group of PGR

are also important, which regulate many aspects of plant growth and differentiation (Sakai et

al. 2001). Addition of zeatin to an auxin-free medium resulted in an increase in alkaloid

accumulation in C. roseus cell cultures (Taha et al. 2009).

Previous research about the effects of PGRs on TIAs in C. roseus was mostly focused on the

production of ajmalicine, serpentine, tabersonine, ajmaline, vindoline and catharanthine (Lee-

Parsons et al. 2004; El-Sayed and Verpoorte 2005; Srivastava and Srivastava 2007; Zhao and

Verpoorte 2007; Ruiz-May et al. 2008; Amini et al. 2009; Mustafa et al. 2009; Peebles et al.

2009). There are few reports that also described the effect of PGRs on production of VB (Pan

et al. 2010).

2.3 Protoplast technology

Protoplast isolation and subsequent protoplast fusion has become an important tool for raising

new genotypes/ cell lines in plant improvement programme. The development of protoplast

technology became possible following the first successful isolation of plant protoplasts by

Cocking (1960). The first achievement on somatic hybridization was, however reported little

later in tobacco (Carlson et al. 1972). Over the years, several reviews on protoplast

technology were appeared in literature (Waara and Glimelius 1995; Johnson and Veilleux

2001). Since then a good number of reports on protoplast isolation and fusion have been

available in a wide variety of plant genera including model and important economic crop

plants like tobacco, tomato, potato, rice, citrus, etc. (Orczyk et al. 2003; Davey et al. 2005;

Grosser and Gmitter 2005). The advantages of protoplast isolation and hybridization is that it

produces hybrids even of wide intergeneric nature, which is otherwise not possible in

conventional breeding technique owing to incompatibility at different sexual stages.

The efficient protoplast isolation and plant regeneration have been achieved in a wide variety

of species (Grosser and Gmitter 2005) (Table f). Various starting materials for protoplast

isolation have been tested in many studies, such as callus (Yang et al. 2007), cotyledons

(Dovzhenko et al. 2003) and embryogenic suspensions (Mahanom et al. 2003). Different

enzymes of different origin have been used in this process (Table d). Suspension culture is

the appropriate donor material for efficient protoplast isolation as it resulted into higher yield

of viable protoplasts than obtained from callus or mesophylls (Khatri et al. 2010). However,

isolated mesophyll cells have also been used as a source of material for protoplast isolation,

culture and subsequent regeneration in many plant species, such as Lactuca (Webb et al.


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1994), Eustoma (Kunitake et al. 1995), Echinacea (Pan et al. 2004), Morus (Umate et al.

2005), Ipomoea (Guo et al. 2006) and Solanum (Borgato et al. 2007). Viability of protoplasts

can be analysed by using different stains given in Table e. In other monocotyledons, such as

maize (Kao et al. 1975), wheat (Vasil et al. 1990) and barley (Funatsuki et al. 1992), rice

(Jain et al. 1995), cell suspension cultures have been used as primary source of material.

Suspension cultures as well as somatic embryos (especially cotyledons) were exploited as

suitable sources for protoplast isolation study in C. coum (Anika et. al 2010). However, an

efficient and successful protoplast-to-plant regeneration system is still not made in many

important crop species (Sharma et al. 2005).

Direct gene transfer via protoplasts would also be a better option for the integration of

agronomically useful genes, such as sterility and disease or insect resistance (Li et al. 2002).

The development of a ‘protoplast-to-plant’ in vitro regeneration system could also enable

somaclonal variations to be utilised in plant improvement efforts. The establishment of a

suspension culture for efficient protoplast to plant regeneration is a pre-requisite technique

such as direct gene transfer and somatic hybridization, mediated by protoplast fusion

(Thomas 2009). It provides opportunities for combining the genomes together of

taxonomically different species that cannot be combined sexually due to incompatibility

barriers. It has been considered as an efficient tool for the transfer of valuable polygenic

agronomical traits like resistances from wild to cultivated species (Liu et al. 2007).

Table d: Commercially available enzyme preparations used for protoplast isolation and

their sources:

Enzyme Source

Cellulase R-10

Meicelase-P

Hemicellulase H-2125

Macerozyme R-10

Pectinase

Pectolyase Y-23

Pectinol

Zymolase

Driselase

Trichoderma viride

Trichoderma viride

Rhizopus sp.

Rhizopus sp.

Aspergillus niger

Aspergillus japonicus

Aspergillus sp.

Arthrobacter luteus

Irpex lactes


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Table e: Stains used to check the viability and yield of isolated protoplasts (Endress

1994).

Staining agent Dead protoplasts Living protoplasts

Evan’s blue Coloured (blue) Colourless

Flourecein diacetate (FDA) Colourless FDA is cleaved by estearses forming fluorecien; fluorecien cannot pass membrane of living class; after UV excitation: fluorescence (≥ 470 nm)

Methylene blue Coloured (blue) Reduced: yellow

Neutral red (=toluylene red) Clolourless Coloured (red)

Phenosafranine Coloured (red) Colourless

2,3,5-Triphenyltetrazolium-chloride (TTC) (water solubleand colourless)

---------- Formation of formazan by dehydrogenases (water-insoluble, red)

Table f: Some plants from which protoplast has been isolated

Plant Part/tissue used Workers

Nicotiana tabacum Mesophyll cells Nagata and Takebe et al. 1970

Petunia hybrid Leaf Frearson et al. 1973

Triticum aestivum L. Embryogenic suspension culture Vasil et al. 1990

Indica rice Mesophyll cells Gupta et al. 1993

Phalaeanopsis spp. Non embryogenic callus Kobayashi et al. 1993

Litchi chinensis Embryogenic suspensions. Yu et al. 2000

Primula malacoide Embryogenic suspensions. Mizuhiro et al. 2001

Artemisia judaica L Mesophyll cells Pan et al. 2003

Echinacea angustifolia Non embryogenic callus Zhu et al. 2005

Cyclamen persicum embryogenic suspension cultures Winkelmann et al. 2006

Vitis vinifera Embryogenic suspension culture Xu et al. 2007

Musa paradisiacal ABB. Linn Embryogenic suspensions. Xue et al. 2010

Maesa lanceolata in vitro cultures and hairy roots Lambert et al. 2010


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2.4 Synthesis of secondary metabolites

2.4.1 Biosynthesis of amino acid (tryptophan) required for VB and VC synthesis

Anthranilate is formed from chorismate through shikimate pathway, from which tryptophan

is formed, which is an aromatic amino acid. Biosynthetic pathway of L-Tryptophan consists

of five enzymatically controlled steps. The first step involves the formation of anthranilate

from chorismate in the presence of an enzyme called anthranilate synthase. Anthranilate

synthase was first isolated and purified to apparent homogeneity from C. roseus (Poulsen et

al. 1993). The second step is the formation of N-(5-phosphoribosyl) anthranilate and this step

is catalysed by the enzyme phosphoribosyl diphosphate- anthranilate transferase. The third

step is the formation of 1-(O-carboxyphenylamino)-1-deoxyribulose phosphate and that is

catalysed by the enzyme phosphoribosyl diphosphate-anthranilate isomerase. The following

step is formation of indole-3-glycerol phosphate and the reaction is catalysed by the enzyme

indole-3-glycerol phosphate synthase. The next step is formation of indole, which is catalysed

by the enzyme tryptophan synthase α. The fifth step involves the formation of L-tryptophan,

which is catalysed by the enzyme tryptophan synthase β.

2.4.2 Biosynthesis of bisindole alkaloids

VB and VC are bisindole alkaloids which are of great interest. These two compounds are

synthesized from the coupling of the monomeric alkaloids catharanthine and vindoline. The

information on catharanthine biosynthesis is very limited. Geissoschizine fed to C. roseus

plants was incorporated into catharanthine (El-Sayed and Verpoorte 2007). Brown et al.

(1971) suggested that geissoschizine could be converted into stemmadenine or akuammicine.

Feeding stemmadenine to C. roseus cell suspension cultures resulted in the formation of

catharanthine and tabersonine in a few hours (El-Sayed et al. 2004). It has been established

that tabersonine is transformed into vindoline by a sequence of six steps and these steps

include: aromatic hydroxylation, O-methylation, hydration of the 2, 3-double bonds, N (1)-

methylation, hydroxylation at position 4 and 4-O-acetylation (Balsevich et al. 1986). The

product resulting from the coupling is α-3'4'-anhydrovinblastine which is converted into VB

that converted into VC later (Verpoorte et al. 1997) (Figure b). The coupling process is

catalysed by the enzyme anhydrovinblastine synthase (Table g). These dimeric alkaloids are

used as antitumour agents and produced in trace amounts (0.0005% dry weight). The natural

high abundance of vindoline and catharanthine in C. roseus plants led to the establishment of


28

a semisynthetic process for coupling the monomers either chemically (Kutney et al. 1976) or

enzymatically using horseradish peroxidase (Goodbody et al. 1988).

2.4.3 Enzyme catalyzing the dimerization process – Basic Peroxidase

Coupling vindoline with catharanthine by a peroxidase into anhydrovinblastine which is a

reduction product from a highly instable dihydropyridinium, an iminium, is the true precursor

to the other bisindole alkaloids vinblastine, vincristine and leurosine. The localization of the

enzyme has been reported to occur in the vacuole associated to specific spots of the internal

face of the tonoplast (Sottomayor et al. 1996).

2.4.4 Regulation of terpene indole alkaloids biosynthesis (TIA)

The biosynthesis of VB and VC in C. roseus is quite complex. The process of synthesis

starts with the amino acid tryptophan. The schematic pathway presented in Figure b clearly

suggests the participation of several enzymes, proteins, genes including regulatory genes and

compartments. At each step regulation is possible and this regulation of TIAs synthesis in

fact, can be controlled either by developmental or exogenous signals. The exogenous signal/

chemicals improve the yield in C. roseus in many cases for example, betaine, malic acid,

tetramethyl ammonium bromide and rare elements increased the yields of ajmalicine and

catharanthine in cell cultures about five to six fold (Zhao et al. 2000a). Increasing the

substrate supply via precursor feeding overcomes the rate-limiting steps in the production of

alkaloids. Particularly the terpenoid pathway seems rate limiting for alkaloid production and

feeding with secologanin or loganin was proved to be an efficient way to improve

accumulation of alkaloids (Moreno et al. 1993).

Genetic and environmental factors are involved to influence the secondary metabolite

synthesis in C. roseus. The precursors and enzyme complex necessary for biosynthetic

pathway are known. Tryptophan decarboxylase (TDC) helps in trypamine synthesis and

strictosidine synthetase (SSS) catalyses the coupling of trypamine and secologanin to form

strictosidine. Other enzymes like geranoil 10-hydroxylase (g10H), NADPH: cytochrome P-

450 reductase, anthranilate synthetase (AS) are the enzymes with activity same as TDC and is

involved in alkaloid biosynthesis (Poulsen et al. 1993). Pennings (1989a) purified the TDC

activity from cell suspension culture and subsequently isolation of cDNAwas done by

Pasquali (1992). In C. roseus, mevalonate biosynthesis is considered as integrated part of

indole alkaloid and the enzymes involved at different stages were investigated. Enzyme 3-


29

hydroxy 3-methylglutaryl coenzyme A reductase and the gene encoding this enzyme was

investigated extensively (Van der Heijden et al. 1994). Alkaloid metabolism is under

developmental regulation as differential mRNA level for TDC, SSS and cytochrome P-15

were noticed at various stages of development, maximum was found in root. The content of

vindoline, catharanthine, 3, 4-anhydrovinblastine varied at different developmental stages

(Pasquali et al. 1992).

It has been reported that that the precursors for alkaloid (tryptophan to trypamide) are located

in the cytosol whereas the enzyme SSS is located in vacuole (Stevens et al. 1993). Usually, it

seems that three cellular components namely: vacuole, cytosol and plastid are the part in

alkaloid synthesis. Moreno et al. (1995) described that subcellular compartmentation is an

important factor in the regulation of secondary metabolism and it enables to separate the

enzymes from substrate and their end products.

Figure b: Different Catharanthus

formation of VB and VC:

El-Sayed and Verpoorte (2007)

Review of

Catharanthus indole alkaloids biosynthetic pathways leading

of literature

30

indole alkaloids biosynthetic pathways leading to the

Table g: Enzymes involved in biosynthesis of indole alkaloids of

El-Sayed and Verpoorte (2007)

2.5 Elicitation in C. roseus

An elicitor is defined as a substance that induces the synthesis of compounds, used in defense

responses (Koga et al. 2006) (Table h

been enhanced by compounds, which are biotic and abiotic in nature.

studied the effect of elicitation on different met

metabolism in C. roseus. Zhao et al. (2001a) tested various fungal elicitors derived from 12

fungi and their effect on improving indole alkaloid production in

culture. These authors observed

Review of

Enzymes involved in biosynthesis of indole alkaloids of C. roseus

elicitor is defined as a substance that induces the synthesis of compounds, used in defense

Table h). The production of many secondary metabolites

compounds, which are biotic and abiotic in nature. Moreno

studied the effect of elicitation on different metabolic pathways involved in

Zhao et al. (2001a) tested various fungal elicitors derived from 12

fungi and their effect on improving indole alkaloid production in C. roseus cell suspension

ors observed enhanced catharanthine production on combined elicitor

of literature

31

elicitor is defined as a substance that induces the synthesis of compounds, used in defense

secondary metabolites has

Moreno et al. (1996)

abolic pathways involved in secondary

Zhao et al. (2001a) tested various fungal elicitors derived from 12

cell suspension

combined elicitor


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treatment in shake flasks and in bioreactors. The introduction of different elicitors alone, e.g.,

homogenates of fungal mycelium (Eilert et al. 1986), non-biotic elicitors such as vanadium

(Tallevi and DiCosmo 1988) was also found very effective in enriching yield. Various

research groups noticed that the catharanthine production by cell cultures in C. roseus may be

enhanced by the improvement of cultivation conditions (Smith et al. 1987a), and by using

immobilization techniques (Facchini and DiCosmo 1991). Smith et al. (1987b) reported that

the increase in sucrose concentrations from 4-10 % (w/v) stimulated alkaloid content in

cultured cells of C. roseus. DiCosmo and Towers (1984) noted that the addition of 200 mM

sorbitol resulted in 63% increase in catharanthine content. Ajmalicine and catharanthine were

also similarly induced by the addition of tetramethyl ammonium bromide and Aspergillus

niger homogenate, which proved that the combination of abiotic and biotic elicitors added to

C. roseus cell suspension cultures, improved TIAs production (Zhao et al. 2001). The fungal

elicitor Penicillum sp. exhibited dual effect i.e, the production and release of indole alkaloids

in culture (Sim et al. 1994).

2.5.1 Yeast Extract (YE) as biotic elicitor in relation to alkaloids

Currently, YE is employed as a biotic elicitor for the induction and enhancement of

secondary metabolites production (Abrahim et al. 2011). YE is used as a supplement in order

to promote cultural growth as it contains high amino acid content (George et al. 2008b). In

suspension cells, the perception of YE leads to the induction of TIA biosynthetic genes

including those encoding strictosidine synthase (STR) and tryptophan decarboxylase (TDC)

(Pauw et al. 2004). The only detectable YE component inducing TIA biosynthetic gene

expression in Catharanthus is a water-soluble, low molecular weight fraction, which is

probably a small peptide (Menke et al. 1999a).

YE was noted to be responsible for both the activation of ROS generation and for the

induction of TIA biosynthetic gene expression (Simone 2010). The generation of ROS

through oxidative burst was induced by a variety of elicitors, such as chitin oligosaccharides

in tomato (Felix et al. 1993), fungal oligosaccharides in chickpea cell cultures (Otte and Barz

1996), YE in tobacco (Baier et al. 1999). Fungal elicitors were noted in several studied plants

like in spruce (Schwacke and Hager 1992) and in parsley cell suspensions (Jabs et al. 1997),

produce ROS via oxidative stress. In Curcuma mangga cultures, YE was used as supplement

in medium but it failed to promote shoot proliferation; the in vitro raised plantlets, grown in

medium with over 3.5 mg L-1 YE also showed sign of morphological abnormalities and these


33

alteration was thought to be due to accumulation of secondary metabolites in response to YE

elicitation (Abrahim et al. 2011). During elicitation, different species respond differently i.e,

in Taxus baccata addition of high concentration of YE into the medium inhibited growth

while lower concentrations of YE promoted in vitro cultural growth (Bonfill et al. 2006). YE

also enhanced the accumulation of alkaloids like 6-methoxymellein in carrot cells (Guo and

Ohta 1994) and polyphosphoinositol in Cupressus lusitanica cell cultures (Zhao et al. 2004).

The addition of YE increased both the growth and alkaloid yield in callus of Hyoscyamus

muticus L. (Ibrahim et al. 2009). Rosmarinic acid accumulation was also induced by addition

of YE in Lithospermum erythrorhizon (Mizukami et al. 1992) and in Orthosiphon aristatus

suspension cultures (Sumaryono et al. 1991). YE enhanced the level of shikimic acid, a

precursor of phenylpropanoid pathway, thus enhanced the end product of the same pathway

in cell cultures of Medicago truncatula (Goyal and Ramawat 2008). YE was shown to induce

a transient increase in cytosolic calcium levels in C. roseus cells, which was necessary for the

induction of JA accumulation, STR and TDC gene expression (Memelink et al. 2001).

Rodriguez et al. (2003) showed that methyl jasmonate, a chemical inducer of secondary

metabolism, promoted tabersonine biosynthesis in hairy root cultures of C. roseus.


34

Table h: Elicitor defence and and defence like responses in plants. I=oligosaccharides, II-peptides and proteins, III-glycopeptides and proteins, IV=glycolipids, V=lipophilic elicitors.

(Montesano et al. 2003)


35

2.6 Biochemical attributes in relation to elicitors

YE is an autolysate of baker’s yeast (Saccharomyces cerevisiae) cell walls, activated the

generation of ROS in C. roseus cells (Pauw et al. 2004). The most likely source of YE

induced ROS production in Catharanthus is a membrane-bound NADPH oxidase complex,

which used molecular oxygen to make superoxide (Torres et al. 2002). Cell and tissue culture

has been used to study various physiological and biochemical processes affected by induced

stress (Ikeda-Iwai 2003). Salt stress induces various biochemical and physiological responses

in plants and affects almost all plant processes (Jaleel et al. 2007).

It was recently noted that the total soluble protein content increased in response to biotic

elicitor application in Euphorbia pekinensis (Gao et al. 2011). Changes in protein expression,

accumulation and synthesis have been observed in many plant species as a result of plant

exposure to drought stress during growth (Cheng et al. 1993). Both quantitative and

qualitative changes to proteins were detected during drought stress (Riccardi et al. 1998).

Accumulation of proline may occur through an increase in its synthesis constantly with

inhibition of its catabolism (Jaleel et al. 2007) and may be a mechanism for stress tolerance.

Plant species usually have low amounts of proline when grown in well-watered and non-

saline soils, accumulate the level upon imposition of drought or salt stresses (Sakamoto and

Murata 1998). Accumulation of proline in plants under stress is a result of reciprocal

regulation of two pathways: [(• -1-pyrroline-5-carboxylate synthetase (P5CS) and P5CR] and

repressed activity of proline degradation (Kavikishore et al. 2005). An increase in proline

content was observed in both cultivars i.e., Catharanthus roseus and Catharanthus alba with

increasing NaCl in the growth medium (Garg 2010). Reports on proline accumulation under

stress conditions were also observed in seedlings, as well as in fully grown plants (Ghoulam

et al. 2002). Proline acts as an ampiphilic osmolyte, binds onto hydrophobic surfaces via its

hydrophobic moiety and converts them into hydrophilic surfaces (Szabados and Savoure

2009). The conversion enables the cell to preserve the structural integrity of cytoplasmic

proteins under conditions of cell dehydration, which develops in plants during frost treatment

(Papageorgiou and Murata 1995).

Ghoulam et al. (2002) reported that the total sugar content in the shoots declined after an

initial increase in the tolerant cultivar. This increase in sugar content in the tolerant cultivar

may facilitate osmotic adjustment in the plant. The increase in soluble sugar content in the

cold-acclimatized stage, has been proposed to be a part of cold acclimatization mechanisms


36

in olive plants (Eris et al. 2007). In many other observations, increment in total sugar content

in response to cold stress was observed in plants like peach cultivars (Burak and Eris 1992),

winter rye leaves (Antikainen and Pihakaski 1994) and in cabbage genotypes (Sasaki et al.

2001).

2.6.1 Influence of elicitors on enzyme activities

An efficient and highly redundant plant ROS system composed of antioxidative enzymes are

responsible for maintaining the levels of ROS under tight control (Gechev et al. 2006). Cell

and tissue culture may act as a useful way for the assessment of salt tolerance competence in

plants since it allows relatively fast responses such as short generation time in controlled

environment (Wang et al. 2011). Phenylalanine ammonia lyase (PAL) is the first enzyme in

the general phenylpropanoid pathway and was reported to be regulated in C. roseus cell

cultures induced by Aspergillus niger elicitor (Xu and Dong 2005a). Peroxidase participates

in a variety of plant defense mechanisms, and is involved in plant resistance to diseases (Silva

et al. 2008; Dutsadee and Nunta 2008). CAT and SOD, which play important role in the

metabolism of ROS, could be induced by environmental stresses including fungal elicitor

(Tanabe et al. 2008).

It is widely accepted that secondary metabolites are produced by plants to protect themselves

against the attacks from insects, herbivores and pathogens, or to survive under other biotic

and abiotic stresses (Zhao et al. 2005). The activities of PAL, POD, SOD, and CAT are

usually used to evaluate physiological and biochemical responses of plants to biotic and

abiotic stresses and the plant systemic acquired resistance (SAR) (Gechev et al. 2003).

Mannitol improves tolerance to stress through scavenging of hydroxyl radicals (OH-) and

stabilization of macromolecular structures (Shen et al. 1997). The importance of mannitol as

a scavenger of the hydroxyl radical has been demonstrated in vitro (Smirnoff and Cumbes

1989) and in vivo using transgenic tobacco (Shen et al. 1997). The exposure of cells to

oxidative stress has multiple effects on redox-regulated activities of the cell (Powis et al.

1995). Antioxidants as carotenoids, ascorbate, α-tocopherol, glutathione and flavonoids, as

well as antioxidant enzymes such as peroxidases, superoxide dismutase and catalase can be

synthesized in order to protect the plant cells (Tanaka et al. 1990).

Willekens et al. (1997) reported that the CAT activity belongs to the normal operation of the

photosynthetic apparatus in tobacco plants and is essential for the antioxidant defense in plant

cells under stress conditions. Thermal stress was associated with more activation of CAT


37

activity in adapted clones of Gladiolus (Bettaieb et al. 2007) and transgenic rice (Matsumura

et al. 2002) as compared to their controls. CAT reduces H2O2 into H2O and O2, whereas POX

decomposes H2O2 by oxidation of co-substrate such as phenolic compounds (Gadalla 2009).

The equilibrium between the production and scavenging of ROS may be perturbed under

adverse abiotic stresses thereby results into reduction in crop yield (Hilaly and El-Hosieny

2011).

Ascorbate peroxidase is an antioxidant enzyme that participates in the ascorbate-glutathione

cycle, acts in chloroplasts and cytosol. It reduces H2O2 to H2O by using ascorbate as reducing

agent, protecting thus the plant (Meloni et al. 2003).

SOD is an enzyme that catalyzes the dismutation of superoxide into hydrogen peroxide and

molecular oxygen which is less harmful than H2O2 (Hilaly and El-Hosieny 2011). Under low-

temperature stress, high activity of SOD is very important for the plants to enhance cold

resistance (Bowler et al. 1992). In transgenic alfalfa, SOD enhanced the tolerance to freezing

stress (Mckersie et al. 1993). Enzymatic activity generally becomes stronger as the stress

increases, or increases at the beginning and then decreases by the end of stress treatment (Ren

et al. 2002).

The tripeptide glutathione (g-L-glutamyl-L-cysteinyl-glycine, GSH), which is abundantly

distributed in most living cells, is a principal antioxidant having a low-molecular weight and

non-proteinous thiol compound. GSH plays an pivotal role in maintaining the intracellular

thiol redox state and protecting cells against oxidative damage, xenobiotic organic chemicals,

and heavy metals (Meister 1989). GSH is synthesized in the cell cytosol involving two ATP-

requiring enzymatic steps: the formation of g- glutamylcysteine from L-glutamate and L-

cysteine, and the formation of GSH from g-glutamylcysteine and glycine. Reactive oxygen

intermediates and other harmful compounds are produced during the normal growth of

aerobic cells, and these may stop cell growth (Brand and Nicholis 2011). Defense systems

such as antioxidant and redox enzymes are required for the normal growth of the cells. GSH,

known as a major antioxidant, is present in high concentrations (up to 10 mM in the liver) in

most living cells, from microorganisms to humans, and is known to be involved in cellular

responses to various stresses (Penninckx 2000). Moreover, endogenous GSH concentrations

can alter cellular responses to oxidative stress, and increases in GSH have been proposed as a

potential mechanism for enhancing cellular antioxidant defense (Mollering et al. 1998).


38

Various agents modulate the transcription of the g-glutamylcysteine synthetase genes and

GSH levels in different cell types.

2.7 Synthetic seed technology

Artificial seeds are the structures obtained by encapsulating somatic embryos (Furmanowa et

al. 1991a), root segments or shoot primordia (Nakashimada et al. 1995). Synthetic seed

technology is an alternative to traditional micropropagation for the production and delivery of

cloned plantlets. Several aspects of the technique are still under developed and hinder its

commercial application (Brischia et al. 2002). This technology has also been employed for

germplasm storage and exchange purposes (Danso and Ford-Lloyd 2003). Mandal et al.

(2000) reported the conservation and propagation of four pharmaceutically important herbs

using axillary vegetative buds as source of encapsulation material. A new method to produce

encapsulatable units for synthetic seeds in Asparagus officinalis was reported by Kanji and

Yuji (2001). Shoot buds were also encapsulated in Na alginate in Adhatoda vasica (Anand

and Bansal 2002). Brischia et al. (2002) reported that synthetic seeds of M26 apple rootstock

can be produced through organogenesis. Encapsulation of somatic embryos produced in

tissue culture to make synthetic seeds was investigated in a number of plants like in carrot

(Timbert et al. 1995), Citrus reticulata Blanco (Antonietta et al. 1998), Carica papaya L.

(Castillo et al. 1998), Siberian ginseng (Choi and Jeong 2002) and others. In vitro

propagation can also be done from hairy roots using encapsulation technique (Uozomi et al.

1996). Artificial seeds using hairy roots have a further potential for mass propagation, and

modification in a bioreactor setting (Giri and Narasu 2000).

Isolated shoot buds from multiple shoot cultures of Adhatoda vasica Nees. were encapsulated

in 3% Na-alginate with different gel matrices (Anand and Bansal, 2002). In Gypsophila

paniculata L., 4% Na-alginate dissolved in MS salt produced the highest shoot number with

low shoot length (Rady and Hanafy 2004). Higher or lower concentrations of Na-alginate

used in encapsulation reduced the conversion frequency of beads (Redenbaugh et al. 1987).

The beads can potentially act like a reservoir of nutrients that may help in survival and

increase in speed of growth rate (Redenbaugh et al. 1991). The alginate matrix containing

nutrients reduced the viscosity and help in improving the ability of the gel to form solid

beads. Germination frequency of synthetic seeds also depends on condition of exposure time

in complexing gel. Soneji et al. (2002) reported that a concentration of 3% sodium alginate

was most effective for shoot encapsulation in Ananas comosus. Daud et al. (2007) also


39

reported 3% sodium alginate and 100 mM CaCl2⋅2H2O in medium effectively improved

encapsulation of micro shoots in Saintpaulia ionantha. Awal et al. (2007) reported for

90.48% germination when encapsulated (3% sodium alginate) microshoots of Begonia ×

Hiemalis Fotch were cultured on regeneration medium. Nayak et al. (1998) reported that 4%

sodium alginate and 75 mM CaCl2⋅2H2O were most suitable for formation of clear beads.

2.7.1 Storage of synseeds

Synthetic seeds can be stored at low temperature, then regenerated and cultivated when

needed (Pietrosiuk et al. 2007). Rady and Hanafy (2004) encapsulated shoot-tips (beads),

stored at 40C for 30 days that germinated and developed into shoots on MS medium. Bapat et

al. (1987) earlier evaluated that the encapsulated axillary buds of mulberry could be stored at

40C for 45 days without the loss of viability, that later regenerated into complete plantlets on

an appropriate medium. Redenbaugh et al. (1987) reported however, that in alfalfa, the

conversion frequencies of encapsulated somatic embryos was decreased after 7 days of

storage and this decline was thought to be due to inhibition of embryo respiration inside the

alginate capsules. Encapsulated somatic embryos of Pinus patula stored at 2 or 4°C for four

months showed high conversion rates (73 to 61%, resp.), but when stored at higher

temperatures (e.g., room temperature 27°C) for 40 days they showed only 6% conversion

(Malabadi and van Staden 2005). Non-encapsulated embryos did not germinate at all after

storage at 00C, 40C and 250C (Katouzi et al. 2011).

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