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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
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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
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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
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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
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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
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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
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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-
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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)
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Catharanthus indole alkaloids biosynthetic pathways leading
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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
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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
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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.
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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)
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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
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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
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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).
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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
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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).