REVIEW OF LITERATURE

Micropropagation

Plant improvement programmes primarily depend on the availability and

efficient induction of genetic variability. The technique of plant cell and tissue
culture offers an ample scope for in vitro rapid clonal multiplication (Murashige,
1977) and somaclonal variations for the recovery of novel genotypes (Scowcroft,
1984; Evans and Sharp, 1988). These variations may involve phenotypic alterations,
chromosomal rearrangements, biochemical and molecular changes (Reddy, 1989).
The heritable variations thus emerged, forms an essential ground for selection in
quality improvement of plants.

Cell culture technology has been applied to a number of medicinal plants to

obtain pharmaceutically important drugs. Schieder (1985) has suggested that plant
cell and tissue culture, may be an alternative to conventional methods for the
improvement of medicinal plants.

Although more than 50 different devised media formulations have been used
for the in vitro culture of tissues of various plant species, the formulation described
by Murashige and Skoog (MS medium, 1962) is the most commonly used. The level
and kind of plant growth regulators included in the culture medium largely
determine the success of tissue culture. Root and shoot initiation and the process of
differentiation from unorganised callus tissue are closely regulated by the relative
concentrations of auxins and cytokinins in the medium (Skoog and Miller, 1957;
Ammirato, 1983; Bajaj et al., 1988; Rout and Das, 1997a, 2004). Auxin : cytokinin
ratios of 10 yield rapid growth of undifferentiated callus, a ratio of 100 favours
root development and a ratio of 4 favours the development of shoot morphogenesis
(Murashige, 1980). Cytokinin levels were shown to be most critical for
multiplication of many medicinal plants. The inclusion of low concentration of

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auxins along with cytokinin triggered the rate of shoot proliferation in some
genotypes (Roja et al., 1987; Ravishankar and Venkataraman, 1988; Barna and
Wakhlu, 1988; Satheesh and Bhavanandan, 1988; Upadhyay et al., 1989; Jha and
Jha, 1989; Tsay et al., 1989; Arora and Bhojwani, 1989; Sharma et al., 1993; Mao et
al., 1995; Chen et al., 1995; Sharma and Singh, 1997; Rout and Das, 1997a, 1997b;
Shasany et al., 1998; Saxena et al., 1998; Rout et al., 1999).

The tropical genus Coleus, differs from the related Plectranthus by having
the stamens united, but this difference is not quite consistent, thus Coleus has been
reduced to a synonym (Ryding, 1994). Much of the micropropagation research on
the genus Coleus was concentrated on Coleus forskohlii and Coleus blumei. The
establishment of in vitro cultures of Coleus blumei was published in 1977 by two
different groups, Razzaque and Ellis (1977) and Zenk et al. (1977). Both groups
used B5 medium (Gamborg and Eveleigh, 1968; Gamborg et al., 1968) for callus
and suspension cultures. Razzaque and Ellis (1977) supplemented this basal medium
with 1 mg/l 2,4-D and 0.1 mg/l kinetin. Zenk et al. (1977) tested 35 differently
substituted phenoxyacetic acids (10 -5 M) as sole addition and found that 2, 4-
dimethyl phenoxyacetic acid had the highest effect. Successful plantlet regeneration
in the callus and shoot tip cultures (Hervey and Robbins, 1978; Smith and
Murashige, 1982) and large scale production of rosmarinic acid in cell cultures
(Ulbrich et al., 1985) was reported in C. blumei.

Attempts to obtain high yielding plants of C. forskohlii by somaclonal

variation or UV mutation were made by Mandler-Henger (1988). The cultures were
established on B5 medium (Gamborg et al., 1968) with growth regulators 2, 4-D and
kinetin for callusing and BAP and IAA for shooting and rooting respectively. A
totally different method was used by Mersinger et al. (1988) who produced forskolin
from suspension cultures of C. forskohlii.

Bayliss (1980) produced genetic variability through cell cultures. Since then
the application of plant tissue culture for the induction of stable and heritable
variations had been demonstrated in a range of economically important plant species
(Evans and Sharp, 1985; Bajaj, 1986; Mathur et al., 1988).

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 Micropropagation from shoot tips of 20 to 30 days old aseptically
germinated seedlings of C. forskohlii was reported by Sen and Sharma (1991).
Multiple shoots were induced on MS medium supplemented with BAP. On testing
auxins for their effect on shoot proliferation, only IAA together with BAP showed
an effect. Another approach for micropropagation of C. forskohlii using nodal
segments of mature plants was reported by Sharma et al. (1991). Callus induction
and shoot proliferation was effected in phytohormone combinations of BAP and
NAA as well as kinetin and IAA respectively. The in vitro selection method
developed by Ibrahim et al. (1992) for C. blumei involved the initiation of the callus
and regeneration of plantlets on MS medium with BA and NAA.

The interest in root cultures of C. forskohlii was based on the occurrence of

forskolin in root tubers and the close correlation between root differentiation and
production of forskolin. Krombholz et al. (1992) established root cultures of
C. forskohlii from primary callus and suspension cultures and also by transformation
of young leaves of C. forskohlii with Agrobacterium rhizogenes.

Shoot tip cultures, callus cultures and excised root tip cultures from rooted in
vitro micropropagated shoots were established and investigated for their forskolin
content by Sen et al. (1992, 1993). Multiple shooting from the shoot tip explants
was promoted by BAP (0.5 - 2.5 mg/l). Callus cultures were established on MS
media and further cultivated on White's medium (White, 1963) with 1 mg/l BAP and
1 mg/l NAA where they became friable, whitish and rhizogenic. Excised root tips
from in vitro propagated rooted shoots grew to an entangled mass of roots with
primary and secondary laterals on ¼ MS basal medium with 0.5 mg/l IBA and 1%
sucrose.

An attempt was made with the help of high producing cell suspension
cultures of C. blumei to elucidate the biosynthetic pathway of rosmarinic acid and to
isolate the enzymes involved in the biosynthesis (Petersen and Alfermann, 1988;
Petersen et al., 1993, 1994, 1995). The highest production of coleonol (forskolin)
was described by Tripathi et al. (1995) in callus cultures of C. forskohlii.

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 Mukherjee et al. (1996, 2000a) demonstrated the production of forskolin in
in vitro cultures of C. forskohlii transformed with Agrobacterium tumefaciens.
Increased forskolin yield was obtained in transformed root, rhizogenic calli and cell
suspension cultures of C. forskohlii when treated with various concentrations of
auxins, auxin conjugates, cytokinins and giberellic acid (Mukherjee et al., 2003).
The induction of hairy roots by infection with Agrobacterium rhizogenes and
subsequent much higher forskolin production was described in other scientific
experimentations done in C. forskohlii (Zhou et al., 1996; Sasaki et al., 1998).
Zagrajski et al. (1997) pointed out the effectiveness of nodal explant as the best
regeneration system in C. blumei.

A better alternative for micropropagation using flowers of C. forskohlii was

suggested by Suryanarayanan and Pai (1998). Substantial callus formation was
initiated from florets, stem and shoot tips on MS medium fortified with NAA and
BA. Subculturing of the callus into another growth regulator supplemented medium
was required for organogenesis in shoot tip and stem cultures but not required in
floral culture.

An experiment was conducted to standardize the type of propagation

material (terminal, middle or basal cuttings) and growth regulators for in vitro
propagation of C. forskohlii (Sundharaiya et al., 2000). The terminal cuttings
supplemented with IBA (500ppm) recorded the highest survival percentage. A high
frequency shoot organogenesis and plant establishment protocol has been developed
for C. forskohlii from leaf derived callus cultures (Malathy and Pai, 1999; Reddy et
al., 2001). They were successfully raised somaclones exceeding the parent plants in
forskolin content.

Multiple shoot emergences from shoot tip explants was observed in

C. forskohlii by Bhattacharyya and Bhattacharya (2001) on MS medium augmented
with kinetin and IAA. Asamenew and Narayanaswamy (2001) proposed another
protocol for callus and plant regeneration in C. forskohlii with growth regulators
IAA and BAP.

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 Significant enhancement in forskolin content was observed in genetically
transformed callus cultures of C. forskohlii in presence of casein hydrolysate
(Mukherjee et al., 2000b). The efficiency of genetic transformation in callus cultures
of C. blumei using Agrobacterium was evaluated and appreciable improvement was
noticed in rosmarinic acid production (Bauer et al., 2002, 2004). Micropropagation
of Plectranthus vetiveroides was obtained on MS medium supplemented with BA
(Sivasubramanian et al., 2002). Biotechnological production of rosmarinic acid with
plant cell cultures from C. blumei has been proposed by Petersen and Simmonds
(2003).

The growth and rosmarinic acid production by C. forskohlii hairy root

cultures were examined by Li et al. (2005) in various liquid media such as MS and
Gamborg B5 medium. Their inventions recommended methyl jasmonic acid as an
effective elicitor for rosmarinic acid production. Anbazhagan et al. (2005) achieved
callus induction and shoot emergence from the leaf explants of C. forskohlii on MS
medium with 1mg BA/l and 2mg NAA/l. In vitro multiplication with same growth
regulators was tried by Rajasekharan et al. (2005).

A rapid and higher effective method of micropropagation using terminal and

axillary buds with MS medium having growth regulators BA and NAA had been
reported in C. blumei (Rani et al., 2006).

Variations shown by plants regenerated in vitro had been the subject of

numerous scientific inquiries (D' Amato, 1977; Skirvin, 1978; Earle and Demarly,
1978; Larkin and Scowcroft, 1981; Benzion et al., 1986; Sun and Zheng, 1990;
Ahmed and Sagi, 1993; Kaeppler and Phillips, 1993; Phillips et al., 1994). There
are reports of isolation of secondary metabolites in remarkable yield from tissue and
cell suspension culture of higher plants (Ellis, 1988) either in levels equivalent to or
higher than the parent plant (Basu and Chand, 1998).

Cytological Analysis

The passage of many plant tissues through an in vitro culture phase

frequently causes chromosomal instability (Constantin, 1981). When plant cells are

16
allowed to proliferate in callus or suspension culture, there is a high probability of
chromosomal variation between cells and of the emergence of cell lines with
chromosome complements differing significantly from those of cells in the parent
plant tissue. The occurrence of chromosomal changes in undifferentiated callus and
suspension cultures has been reviewed by Bayliss (1980). Chromosomally variant
cells in cultures of at least 55 plant species are mentioned in this review. Nearly all
plants studied showed cells in culture with chromosome numbers and karyotypes
different from those characteristic of cells of the intact plant. The changes which
occur as a result of the instability are characterised as both structural rearrangements
and numerical variations in the chromosomes. Mutations of chromosome
complements in tissue culture are known to be common phenomena (D' Amato,
1975; Partanen, 1965; Sunderland, 1977). The chromosome complement changes in
somatic cells of plants may be enhanced in tissue culture and plants of different
ploidy may be produced (Mitra et al., 1960; Muir, 1965; Murashige and Nakano,
1965; Torrey, 1967; Wu and Jampates, 1986).

The studies on morphogenesis in long term plant tissue cultures

demonstrated that during the prolonged period of subculture there was a progressive
loss of organ-forming capacity in all tissue strains. This loss was paralleled by
increasing abnormalities in the chromosomal constitution, including higher
chromosome numbers and greater frequency of aneuploidy (Torrey, 1967).
McClintock (1984) predicted tissue culture as one of the stress factor that could
cause widespread genomic restructuring facilitated by transcriptional transposon
activation, transposition of mobile elements and chromosome-breakage-fusion-
bridges.

Calli obtained from root explants of Zea mays exhibited chromosomal

abnormalities like inhibition of cell plate formation, chromosomal breakage,
stickiness and clumping of chromosomes, asynchronous division, chromosomal
grouping, laggards and micronuclei formation. Numerical variation in chromosomes
including hypo- and hyper-diploid cells was observed (Mohanty et al., 1986).

17
 Although the chromosomal constitution of certain plants seems to be highly
stable in vitro (Bhaskaran, 1989) much of the variability found in tissue culture can
be directly or indirectly attributed to gross chromosomal changes and chromosomal
abnormalities. These chromosomal variations shown by the cultured cells may be
due to the mixoploid nature of the source (explant) used or due to culture conditions
(Phillips et al., 1994).

Several reports indicate that plants regenerated from callus or suspension

cultures may show genetic changes ranging from increased phenotypic variability
(Nishi et al., 1968; Williams and Collin, 1976; Lester and Berbee, 1977) through
mitotic evidence of chromosomal rearrangements (Cummings et al., 1976) to
complete polyploid or aneuploid plants (Murashige and Nakano, 1965; Sacristan and
Melchers, 1969, 1977). In contrast, propagation techniques employing meristem
culture successfully regenerate large numbers of genetically uniform plants
(Murashige, 1974; Holdgate, 1977; Rao, 1977) suggesting that significant
chromosomal changes occur principally in dedifferentiated callus or suspension
cultures (Malnassy and Ellison, 1970).

It has been clear since early in the development of plant tissue culture
methodology that a consequence of growth in vitro was the appearance of dividing
cells with chromosome numbers and karyotypes not usually found within the intact
plant (Partanen, 1963; Sheridan, 1975; D' Amato, 1975, 1977).

A correlation between nuclear state and potential for morphogenic

expression has been observed. Calli derived from various explanted organs or cells
in suspension show cytological alterations with prolonged subculture, the individual
cells becoming progressively polyploid and aneuploid (D' Amato, 1977). Several
literature reviews dealing with the genetically variable cells exhibiting alterations in
chromosome number and structure in plant tissue cultures are available (Sacristan,
1971; Bayliss, 1973, 1975; D' Amato, 1978; Roy, 1980; Gupta and Ghosh, 1983;
Bajwa and Wakhlu, 1986; Nair et al., 1993).

Plant regeneration appears to be linked with chromosomal behaviour of the

source and callus culture (Navok, 1980; Jha and Roy, 1982; Henry et al., 1994;

18
Kumar and Mathur, 2004). Variation in chromosome structure and number disturbs
the physiological and genetic balance of the callus leading to a loss in the capacity to
regenerate plants (Torrey, 1967; Singh, 1986). Regenerative capacity of cell
population decreases, as karyotypic abnormalities increase (Rice and Carlson, 1975).

Of the various changes attributable to somaclonal variation in tissue-cultured

plants, cytogenetic changes are considered most prevalent. Chromosome based
variation resulted from changes in chromosome number, chromosome
rearrangements, breakage and lagging has been a common phenomenon in tissue-
cultured plants of different species (Bayliss, 1980; D' Amato, 1985; Lee and
Phillips, 1988; Phillips et al., 1994; Gupta, 1998). Chromosomal changes may also
give rise to DNA variation that result in mutation at different levels (Vazquez,
2001).

Kaeppler et al. (2000), in their review article describes evidence indicating

that epigenetic variation is an important mechanistic basis of somaclonal variation in
plants. Epigenetics occurs in clonal expansion of a single cell leading to a diversity
of cell types (Holliday, 1993). Recent epigenetic research suggests that DNA
methylation is one of the major mechanisms. DNA methylation, being reversibly
changeable over generations, inducing no base alteration and strongly affecting gene
expression through regulation of chromatin structure appears to be one of the major
mechanisms involved in epigenetic inheritance (Akimoto et al., 2007). In addition,
the genes constantly silenced due to hypermethylation can be transcriptionally
activated by demethylation, resulting in phenotypes and these changes are stably
inherited. The biological function of DNA methylation has been proposed to be
involved in gene silencing, often being associated with hypermethylation of
promoter sequences (Paszkowski and Whitham, 2001; Bird, 2002).

Variations occurring in the plant genome in response to passage of plant cells

through cycles of tissue culture and regeneration have been mentioned by Cullis and
Creissen (1987). According to him, the genomic changes mainly occur in the highly
repetitive fraction of the genome and are limited to a specific subset of these
sequences which may be localized at particular chromosomal sites.

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 The occurrence of variability in chromosome number in in vitro cultures and
the probable reasons for the variations were reviewed by Chatterjee and Prakash
(1993). Chromosomal abnormalities especially chromosome doubling is a common
feature associated with tissue culture (Morel, 1971). Chromosomal changes found in
plants regenerated from tissue culture can be induced by media components, culture
age, explant tissue and genotype. Chromosome breakage and its consequences
(deficiencies, duplications, translocations and inversions) are events quite frequently
observed in plant tissue culture. Such breakpoints are often associated with late
replicating chromosome regions (Peschke and Phillips, 1992). Rearrangements
involving chromosome breaks at heterochromatin was described by Sacristan (1971)
in Crepis capillaris cultures. Since then, breakpoints involved in chromosome
alterations associated with heterochromatic regions have been detected in
regenerated plants from several species (McCoy et al., 1982; Lapitan et al., 1984;
Johnson et al., 1987).

Cytogenetic instability is a common observation in plants regenerated from

maize callus cultures (Lee and Phillips, 1987; Benzion and Phillips, 1988). Among
the screened cell population, cells having different ploidy status and structurally
altered chromosome complement have been noticed frequently in the callus and also
in plants regenerated from this callus (Edallo et al., 1981; McCoy and Phillips,
1982; Fluminhan et al., 1996). Cytological analysis of the cultured cells carried out
by Sengupta et al. (1986) revealed a heterogenous population of dividing cells with
both euploid and aneuploid chromosome numbers. Among the species reported,
polyploidy is the most frequently observed chromosomal abnormality in the
regenerants. In the cytogenetic study performed in eight callus lines, a high level of
polyploidization of all lines during callogenesis and in the subsequent culture,
irrespective of their diploid or tetraploid origin was reported (Fras and Maluszynska,
2003). Although diploid, triploid and tetraploid plants were regenerated from the
investigated callus lines, the majority of the regenerants were diploid. In the
regenerative callus culture of Dianthus, 93% of regenerated shoots were diploids,
despite their origin from a callus with higher ploidy level cells, indicating that
diploid cells have a higher ability to regenerate shoots (Nontaswatsri and Fukai,

20
2005). Strong selection of cytogenetically normal plants was observed in some
plants in the process of regeneration from calluses, which had karyological changes
(Swedlund and Vasil, 1985; Hahne and Hoffmann, 1986). On the other hand,
considerable variations in chromosome numbers among regenerants were also
reported (Bennici and D' Amato, 1978; Asakura et al., 1995; Aida and Shibata,
2002). The investigation carried out on cytological stability of callus of pearl millet
exposed different types of chromosomal abnormalities in the cell populations
including hypo-haploidy, aneuploidy and polyploidy. The regenerants were all
diploids (Mythili et al., 1995). There are many well documented examples of
numerical abnormalities with altered ploidy levels and structural rearrangements in
the chromosome complement of cell populations associated with in vitro culture and
plant regeneration (Krikorian et al., 1983; Renfroe and Berlyn, 1985).

Cytological methods have been employed to assess the somaclonal variants

within garlic callus culture (Dolezel and Novak, 1985; Novak et al., 1986) and also
within subsequent regenerants (Novak, 1980; Al-Zahim et al., 1999). Sinha et al.
(1987) reported numerical variation in chromosomes in the long term callus cultures
raised from cotyledons of Sesbania grandiflora. Shanker and Mohanram (1993)
disclosed changes in chromosome numbers both in the callus cells and in shoot buds
regenerated from the callus. Cytological analysis of fresh callus and 12 months old
callus developed in Capsicum annuum exhibited variations in chromosome structure
and number with cells showing metaphase clumping, chromosome bridges,
anueploidy and polyploidy (Nair and Kumar, 1998). Piola et al. (1999) described
the occurrence of chromosomal instability and somaclonal variation with plant
regeneration from unorganised callus producing adventitious buds rather with
cultures derived from axillary meristems. Detailed morphometrical analysis carried
out in the somaclonal variant of Ocimum basilicum revealed only slight structural
variations of individual chromosomes (Tajo and Thoppil, 2003). Chromosomal
instability, both structural and numerical was found in callus culture of Pisum
sativum (Kumar and Mathur, 2004). The frequency of altered chromosome
constitution increased with increasing concentrations of plant growth regulators.

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 Karyological examination of in vivo and in vitro plants that helps to identify
the chromosome associated somaclonal variation is an area of immense interest
among researchers. Some workers have reported the karyological data of the genus
Plectranthus earlier. Thoppil (1993) studied the karyomorphology of Plectranthus
zeylanicus, in which he identified the diploid chromosome number as 2n = 28.

Random Amplified Polymorphic DNA (RAPD) Analysis

The polymerase chain reaction (Saiki et al., 1988) has been the basis of a
growing range of newer techniques in plant identification based on detection of
DNA sequences. The amplification of DNA using arbitrary 10-base oligonucleotide
primers has been described as a strategy to detect RAPD in many eukaryotic
organisms (Williams et al., 1990). Polymorphisms generated by random amplified
polymorphic DNA (RAPD) analysis have been used for fingerprinting (Connolly et
al., 1994) and evaluating genetic relationships among cultivars (Stiles et al., 1993).
RAPD - PCR has entered plant research in a revolutionary way due to its technical
simplicity and genetic informativeness. This molecular marker using PCR in
association with short primers of arbitrary sequence has been demonstrated to be
sensitive in detecting variation among individuals (Xena de Enrech, 2000).

Among the main concerns of the in vitro cloning are the occurrence of
somaclonal variations and their methods of detection. RAPD or any other
polymerase chain reaction (PCR) based analysis would be an attractive method for
the detection of somaclonal variations. Polymorphism at the DNA level revealed by
PCR based approaches such as RAPD have found extensive applications in plant
genetics (Wilkie et al., 1993; Yang and Quiros, 1993; Yu and Pauls, 1993;
Lashermes et al., 1993). DNA based marker will be more accurate in determination
of relationships between accessions that are too close to be accurately differentiated
by other variables. RAPDs have been used to analyze genetic variation in several
species (Hu and Quiros, 1991; Chalmers et al., 1992; Demeke et al., 1992; Halward
et al., 1992; Lanham et al., 1992; Vierling and Nguyen, 1992). RAPD method for
generating DNA fingerprints elucidates genetic differences at the DNA sequence

22
level which result from evolutionary mechanisms such as DNA deletions, additions,
substitutions, repetitions and translocations.

Molecular markers such as RAPD, RFLP, AFLP, etc. have proven to be

useful in identifying and estimating the genetic diversity among closely related
cultivars and wild species. They contribute much for establishing taxonomic
discrimination and phylogenetic relationships among taxa. The initial evaluation of
genetic variation in the species among zones, among populations and among
individuals can be effected by RAPD markers. For the identification of pigeonpea
cultivars and their related wild species, RAPD markers were used and the level of
polymorphism revealed the immense potential of RAPD in the genetic fingerprinting
of pigeonpea (Ratnaparkhe et al., 1995). Phylogenetic relationships in Ocimum
(Singh et al., 2004a), Salvia (Khalil et al., 2005) etc. have been determined by
analysing their genetic pattern using RAPD.

The usefulness of RAPD technique for detecting genetic variation among

cultivars and identifying germplasms is well established (Welsh and McClelland,
1990; Hu and Quirios, 1991; Halward et al., 1992; Wilde et al., 1992). Genetic
variation in rice cultivars was investigated at the DNA level using RAPD method.
The extensive polymorphism assisted selection for genetic improvement in rice
breeding (Yu and Nguyen, 1994).

During the last few years, various molecular DNA markers, which screen
nuclear and organellar genomes have been utilized for the fast and unambiguous
assessment of the genetic fidelity of micropropagated plants (Rani and Raina, 1998,
2000, 2002). Bouman et al. (1992) found intraclonal RAPD polymorphism among
micropropagated Begonia, but at a lower frequency than phenotypic variations. Rani
et al. (1995) observed RAPD variations among 23 morphologically similar
micropropagated Populus deltoides plants originating from the same clone. RAPD
technology has been used in the analysis of culture derived somaclones of two
grasses, Lolium (Wang et al., 1993) and Triticum (Brown et al., 1993) and changes
in banding pattern have been reported. Utility of RAPD as a means of molecular
analysis of in vitro regenerated plants has been amply demonstrated in a large array

23
of plants by many workers (Isabel et al., 1993; Gupta and Varshney, 1999; Latto et
al., 2005). Genetic molecular markers are considered to be reliable in monitoring
variability in the DNA sequences of the plants. Several groups have applied the
RAPD technique to detect somaclonal variations (Munthali et al., 1996; Bohm and
Zyprian, 1998; Al-Zahim et al., 1999; De Verno et al., 1999).

Godwin et al. (1997) analyzed eight rice somaclones generated from mature
seed-derived callus cultures by RAPD method and identified that all somaclonal
families differed significantly from the original material, indicating genomic
alterations in all families. The genetic fidelity of the micropropagated plants were
recently analyzed using RAPD as molecular marker for confirmation of genetic
homogeneity of the in vitro raised plantlets (Piccioni et al., 1997). Rout et al. (1998)
used 15 different decamers to assess the genetic stability of the micropropagated
plants of Zingiber officinale. The amplified products exhibited monomorphisms
among all the in vitro raised plants and were similar to those from the mother plants.

RAPDs have been used in the diversity studies in some medicinal plants.
Padmesh et al. (1999) studied the diversity of Andrographis paniculata. They
collected germplasm from different parts of India and South East Asia and a
dendrogram was produced which formed 5 clusters with most of the genotypes from
same geographical locations falling together in the same clusters. Shasany et al.
(1999) worked out the relatedness and diversity among 23 accessions of garlic from
different parts of India and Argentina and obtained high degree of polymorphism in
the RAPD banding pattern.

Molecular marker, RAPD was used to identify the level of polymorphism of

somaclonal variants in Triticum and Saccharum (Taylor et al., 1995a, 1995b). The
variation expressed in phenotypes of in vitro derived Musa acuminata was found to
be accompanied with the genetic variation as revealed by the DNA polymorphism in
the RAPD profile (Vidhya and Nair, 2002).

Molecular genome analysis using RAPD proved to be suitable for screening

genetic variation in Stachys sieboldii regenerants obtained at various phytohormone
concentrations. High DNA polymorphism was demonstrated for two types of

24
S. sieboldii callus cultures and for plants regenerated from a callus culture (Kochieva
et al., 2002). RAPD is referred as an appropriate tool for examining the clonal
identity and for certifying genetic fidelity of in vitro propagated plants (Gupta and
Rao, 2002; Carvalho et al., 2004). The results of the experiment carried out by
Feuser et al. (2003) in pineapple provided a contribution towards the abilities of
isozyme and RAPD markers to detect somaclonal variants in pineapple
micropropagated plantlets when genotypic fidelity monitoring is necessary. The
sensitivity of RAPD for revealing the genetic basis for somaclonal variation was
demonstrated by Kim et al. (2003) and detected significantly higher level of
genotypic polymorphisms between regenerants derived from a single genotype.

Genome characterization through RAPD showed distinct variation in profiles

to confirm menthol tolerance and high menthol content character of the genotype
that favoured the in vitro selection of Mentha arvensis clones (Dhawan et al., 2003).
The changes in the banding pattern obtained in basil plants regenerated in vitro
suggested the existence of genetic variation that might affect the biochemical
synthesis of phytoproducts (Rady and Nazif, 2005).

Essential Oil Analysis

The main phytochemical constituents of the genus Plectranthus are

diterpenoids, essential oils and phenolics. The major essential oil ingredients of
Plectranthus include mono- and sesquiterpenes (Abdel-Mogib et al., 2002).
Variations in the chemotypes of essential oil have been observed for several forms
of plants gathered from ecologically dissimilar regions (Grayer et al., 1996; Silva et
al., 2003). The effect of a range of nutritional and hormonal conditions on the
synthesis and accumulation of secondary compounds in vitro has been the subject of
intense investigation (Mantell and Smith, 1983). Many works on medicinal plants
regarding the production of active principles by in vitro cultures developed from any
plant part and optimization of the product yield by manipulating the medium
composition and the physical factors are described (Rideau, 1987). Extensive
investigations on secondary metabolism of plant species in culture revealed diverse
findings. In Mentha species, absence of accumulation of terpenoids (Wang and

25
Staba, 1963; Becker, 1970; Suga et al., 1980), accumulation at reduced levels
(Bricout et al., 1978) and cultures that match whole plants in their biosynthetic
capabilities (Kireeva et al., 1978; Charlwood and Charlwood, 1983) were reported.

Callus cultures of Pimpinella anisum, Foeniculum vulgare and Mentha

piperita did not produce appreciable amount of monoterpenes comparable with that
of intact plants (Becker, 1970). A very low and insignificant level of monoterpene
accumulation and composition was found in callus cultures of Geranium (Brown
and Charlwood, 1986). Kulkarni et al. (1996) established intraclonal variations in
terms of oil content and composition in plants derived from leaf cuttings of rose
scented geranium. Both rooty and leafy callus cultures established in Origanum
vulgare by Isabel et al. (1998) failed to synthesize mono- and sesquiterpenoids
which are the major compounds in the parent plant. But there was increase in the
quantity of other components.

Accumulation of essential oils in plant tissue culture is the result of de novo

synthesis and in many cases the type and proportion of their components differ
greatly from those in the parent tissues. Increased or decreased essential oil
concentration was detected in different growth hormones. All the micropropagated
plantlets of Lavandula dentata showed positive correlation between oil
accumulation and the percentage of glandular hairs in the secretory stage.
Quantitative changes in the major mono- and sesquiterpene components of plantlet
oil were also noticed in response to the effect of varying growth regulator
concentration in the culture medium (Sudria et al., 1999; Bonfill et al., 2002).

Higher production of secondary metabolites in the callus cultures of various

plant species has been reported (Huang and Staden, 2002; Karam et al., 2003).
Callus cultures of Melissa officinalis ssp. altissima were established and cultured on
MS media supplemented with various phytohormones. Several new compounds
were detected in callus cultures. Changes in oil production and composition as well
as enhanced accumulation of sesquiterpenes/monoterpenes were observed depending
upon the changes in phytohormone concentration (Binder and Mandour, 2000).
Samresh et al. (2000) reported unchanging oil profile in in vitro raised plants of

26
Ocimum basilicum even after 8 subculturing passages. The effect of benzyl adenine
(BA) and IBA on ultrastructures, gland formation and essential oil accumulation
resulted in the finding that BA stimulates essential oil production and IBA decreases
oil concentration of in vitro derived plantlets in Lavendula dentata with respect to
control (Sudria et al., 2001). The variation in morphology and essential oil
components in 63 regenerated plantlets of Lavandula vera was investigated by Tsuro
et al. (2001). None of the regenerated plantlets produced as much essential oil as the
original plant. The regenerated plantlets had a different fragrance characterised by
higher levels of distinct components. Tissue culture raised plants of Mentha arvensis
showed similar essential oil profile as that of the parent plant, but at early stages of
growth there was distinct change in oil composition (Phatak and Heble, 2002).

In vitro culture of lavender plants were carried out to determine the effects of
plant growth regulators on the multiplication, oil production and chemical
composition of lavender essential oil (Badawy et al., 2003). Genetically variable
clones with high oil content of patchouli were identified in in vitro culture (Mariska
and Lestari, 2003). In vitro shoots of sage (Salvia officinalis) were established under
8 different hormonal supplementations. The respective essential oils were composed
of more than 75 compounds and the percentage compositions of the oils were found
to exhibit a narrow range of variation. However, the type and concentration of
growth regulators apparently influenced the accumulation of essential oils (Gomes
and Ferreira, 2003).

The essential oil composition and genetic variability of six commercial

cultivars of thyme (Thymus vulgaris) were analysed by GC-MS and RAPD. All
evaluated cultivars belong to the thymol chemotype with differences in the
concentrations of thymol, -terpinene, p-cymene and other minor components
(Echeverrigaray et al., 2001). Morphological, chemical and genetic differences of
Ocimum gratissimum accessions were studied to determine whether volatile oils and
flavanoids can be used as taxonomic markers and to examine the relationship
between RAPDs and these chemical markers (Vieira et al., 2001). The genetical

27
distinctness shown by the accessions was found to be highly correlated to volatile oil
constituents.

GC-MS analysis revealed carvacrol, -terpinolene and p-cymene as the

abundant components of Plectranthus cylindraceus oil. The presence of these
components was further confirmed by 13CNMR analysis (Marwah et al., 2007). The
oil of P. cylindraceus was found to be chemotypically similar to the oils of
P. coleoides (Buchbauer et al., 1993), P. tenuiflorus (Mwangi et al., 1993),
P. amboinicus (Vera et al., 1993) and P. melissoides (Mallavarapu et al., 2005) and
the major components identified were carvacrol and cymene. The phytochemical
analyses of the extracts of Plectranthus spp. have revealed the presence of abietane
diterpenoids and eudesmane sesquiterpenes (Orabi et al., 2000).

Thoppil (1993) reported the essential oil composition of Coleus zeylanicus

with -terpineol and -cadinene as the major components. Misra et al. (1994)
examined the oil from the roots of 10 genotypes of C. forskohlii and reported the
presence of 3-decanone, bornyl acetate, -sesquiphellandrene and γ-eudesmol as
major constituents. Chowdhury and Sharma (1998) identified 18 important
compounds in the oil from C. forskohlii of which 22% were hydrocarbons and 69%
oxygenated compounds with -fenchyl acetate and -pinene as the major
components. The essential oil characterization studies conducted on the genus
Coleus lead to the discovery of -ionone and -humulene as the major components
in C. laciniatus and -thujone and -farnesene as the active principles in
C. parviflorus (Thoppil and Jose, 1995). The composition of essential oil of the
leaves of C. zeylanicus was analyzed by GC-FID, GC-MS and olfactoric evaluation.
80 compounds were detected with monoterpenes especially geraniol and nerol
derivatives, hexane- and octane derivatives as the main constituents (Jirovetz et al.,
1998).

Constituents of the essential oil of P. amboinicus contained carvacrol,

p-cymene and γ-terpinene as the major ones (Mallavarapu et al., 1999). The root
essential oil of C. forskohlii separated in the GC column included four classes of
compounds as monoterpenes, sesquiterpene hydrocarbons, sesquiterpene alcohols

28
and diterpenes in varying proportions (Patil and Hulamani, 1999). The essential oil
of fresh and dried leaves of P. glandulosus were analysed by GC and GC/MS. A
high percentage of oxygenated monoterpenes was obtained (Ngassoum et al., 2001).
Chemical investigations of the leaf essential oil of C. amboinicus by GC and
GC-MS techniques indicated the presence of 6 components accounting for 97% of
the total oil. The major components were thymol followed by carvacrol, 1,8-cineole,
p-cymene, spathulenol, terpinene-4-ol and an unidentified component (Singh et al.,
2002). The analysis of essential oils from leaves, stems and roots of P. barbatus
gave -pinene in the leaves, -phellandrene in the stems and -ocimene in the roots
as the major constituents (Kerntopf et al., 2002).

Trichome Observations by SEM

Labiates carry a great diversity of epidermal hairs, many of which are non-
glandular (El-Gazzar and Watson, 1970; Werker et al., 1985b). Glandular trichomes
which store volatile oils vary in morphology between species, and more than one
type can occur on a single leaf (Bruni and Modenesi, 1983; Venkatachalam et al.,
1984; Werker et al., 1985b). The number of glandular trichomes per unit area of
epidermis varies considerably amongst labiatae species. The presence of peltate and
capitate glandular trichomes is a characteristic feature of Lamiaceae species.
Different versions and combinations of these two morphological types also exist in
different species. The two types can be distinguished by head size and stalk length.
As a rule, in a capitate trichome, the length of the stalk should be more than half the
height of the head (Abu-Asab and Cantino, 1987), whereas peltate trichomes are
short with a uni- or bicellular stalk and a large secretory head with 4 to 18 cells
arranged in one or two concentric circles (Werker, 1993). Capitate trichomes are
extremely variable in stalk length, head shape and secretion process, and can be
subdivided into various types (Werker et al., 1985a). In all the Lamiaceae species,
the secretory material of peltate glands accumulate in the subcuticular space and the
rupture of the cuticle lead to the release of the exudate. Whereas in capitate hairs, the
secretory product accumulates in the apical cells and the release of secretion
probably occurs through cuticular micropores.

29
 A survey of trichome types on vegetative and reproductive organs of several
Lamiaceae species (Werker et al., 1985b) listed three kinds of capitate trichomes
according to their morphology and secretion processes. The leaves, stems and
reproductive structures of plants of Origanum vulgare carry both capitate and peltate
hairs at densities of 10-20 peltate hairs per mm2, in addition to non-glandular hairs
(Werker et al., 1985a). Trichome density also varies with ontogeny and between
different tissues. There are reports of higher trichome densities on the abaxial than
the adaxial surfaces of labiate leaves, and in the vicinity of vascular bundles (Werker
et al., 1985b). In Satureja thymbra, peltate trichomes with 12 head cells occupied
6% of the leaf surface (Bosabalidis, 1990).

The types of glandular hairs and their pattern of distribution on leaves of

Ocimum basilicum at different stages were investigated by Werker et al. (1993). The
density of the glandular hairs appeared to be very high on young meristematic leaves
and on meristematic regions of older leaves. The glandular hairs consist of small
capitate hairs and larger peltate hairs. Quantitative and qualitative variability in oil
constituents of the glandular hairs with age of the hairs was observed in Mentha
piperita (Maffei et al., 1989).

The types of glandular trichomes, their ontogeny and pattern of distribution

on the vegetative and reproductive organs of Leonotis leonurus at different stages of
development were studied by light and scanning electron microscopy (Ascensao et
al., 1995). Two morphologically distinct types of glandular trichomes - capitate and
peltate - with difference in secretion process were described. Uniseriate,
multicellular and point-shaped non-glandular trichomes were also observed.
Structural investigations of the secretory hairs of Salvia aurea leaves explained two
types of glandular trichomes - peltate glands characterized by a short stalk and a
large 6-8 celled head as well as capitate trichomes with unicellular or multicellular
stalk and unicellular or bicellular head (Valenti et al., 1997).

Ascensao et al. (1998) conducted a study to view the glandular secretory

structures of Plectranthus madagascariensis. Besides non-glandular trichomes,
peltate and capitate glandular trichomes were reported. Segmented nature of the

30
peltate trichomes was noted. Five distinct types of glandular hairs - one peltate and 4
capitate - with different localization, secretory modes and secretions were identified
in Salvia officinalis (Corsi and Bottega, 1999). The peltate hairs comprise the
secretory head composed of 12 cells arranged in a shield. The non-glandular hairs
were multicellular, unbranched and consisted of 3-4 elongated cells. Morphological
studies of epidermal hairs of Salvia blepharophylla unearthed three types of
glandular trichomes, in addition to non-glandular hairs. The glandular hairs included
peltate and two types of capitate trichomes (Bisio et al., 1999).

The types of glandular trichomes and their distribution on leaves and flowers
of Plectranthus ornatus were investigated at different stages of their development.
Five morphological types of glandular trichomes - peltate, long stalked capitate,
short stalked capitate, digitiform and conoidal trichomes on the reproductive organs
- were described (Ascensao et al., 1999). The essential oil secretory tissues of
Prostanthera ovalifolia included glandular trichomes of the peltate type with a 16-
celled secretory head (Gersbach, 2002). No capitate trichomes were noticed.

The leaves of medicinal plants used in controlling infectious diseases were

studied for their ash values and mineral contents. Medicinal properties and Cobalt
and Manganese concentrations in the underground and aerial parts of some herbal
drugs were presented in the study carried out by Rai et al. (2001). Variation in heavy
metal concentration was observed in roots of Coleus forskohlii procured from
different geographical zones of India (Srivastava et al., 2002). The highest content
of Sodium and Potassium was reported in Coleus aromaticus (Udayakumar and
Begum, 2004).

Antioxidant Activity

Phenolic antioxidants, a specific group of secondary metabolites, play the

very important role of protecting organisms against harmful effects of oxygen
radicals and other highly reactive oxygen species. Using tissue culture techniques
several high phenolics containing clonal lines have been isolated with high
antioxidant activity.

31
 Secondary metabolites in Mentha spicata - rosmarinic acid and related
phenolics - are natural antioxidants. Tissue culture-based selection techniques was
employed to isolate high rosmarinic acid and phenolic antioxidant-producing clonal
lines from a heterogeneous bulk seed population of M. spicata (Al-Amier et al.,
1999). Tissue culture selection for phenolics and rosmarinic acid in thyme (Thymus
vulgaris) was performed by Al-Amier et al. (2001). The absence of linear
correlation between total phenolics and antioxidant activities was noticed when in
vitro studies were carried out using different vegetables, fruits and medicinal plants
(Gazzani et al., 1998a, 1998b; Velioglu et al., 1998; Al-Mamary, 2002).

Concentrations of phenolics in shoot cultures of 9 Scutellaria species were

determined and they were classified into four groups on the basis of the major
phenolics (Nishikawa et al., 2000). Influence of phytohormones in the production of
antioxidant phenolics were assessed in the in vitro proliferated shoots of Salvia
officinalis (Gomes et al., 2002). Increased accumulation of phenolic compounds was
observed in presence of Kinetin (1.5, 2 & 4 mg/ml) and 2, 4-D (0.05 mg/ml).
Accumulation of total phenolic compounds was found to be increased during the
first two weeks of callus culture of Salvia officinalis. The antioxidant activity in
vitro was not correlated with the observed accumulation of phenolics (Kintzios et
al., 2002). Highest specific accumulation of total phenolics was reported in Salvia
officinalis callus cultures (Gomes et al., 2003).

In view of an enormous data from several studies showing a clear

implication of oxidative stress in causation and progression of various diseases
globally, it appeared worth to have more antioxidant agents. Indian medicinal plants
with antioxidant and immunomodulatory activities have been identified and their
effects were reviewed by Devasagayam and Sainis (2002). The antioxidant
characteristics of plant derived materials can be attributed to their content of
polyphenols (Lugasi et al., 2003). Evaluation of antioxidant activities of the
essential oil, water soluable (polar) and water insoluable (nonpolar) subfractions of
the methanol extracts from aerial parts of Satureja hortensis plants, and methanol
extract from calluses was performed by Gulluce et al. (2003). The strongest

32
antioxidant effect was observed for the tissue culture extract, with an IC 50 value of
23.76  0.80 g/ml which could be compared with the synthetic antioxidant agent
butylated hydroxytoluene (BHT).

In vitro manipulation of plant regeneration in the Chinese medicinal species

Scutellaria baicalensis Georgi resulted in 26 chemically distinct germplasm lines.
The selective markers used to identify elite lines included antioxidant potential,
growth rate and concentration of particular components (Murch et al., 2004).
Phenolic phytochemicals isolated from grape seeds show strong antioxidant activity
in vitro and in low concentration afford significant protection against oxidative
damage in the DNA of mice spleen cells (Fan and Lou, 2004). The green leafy
vegetables provide antioxidant vitamins, minerals, phenolics and various other
phytochemicals that increase their antioxidant capacity (Simopoulos, 2004). The
antioxidative activity of essential oils from oil-rich plants including Coleus
aromaticus were analysed and was found to be effective (Singh et al., 2004b).

Estimation of total phenolics of clonal oregano (Origanum vulgare) led to

the conclusion of the highest phenolic concentration in 60% ethanol extracts of the
clones (Chun et al., 2005). They evaluated the antioxidant activity of phenolic-
enriched clonal oregano extracts and were compared to commercial oregano from
heterogeneous sources. Antioxidant activity was found to be correlated to the
amount of total phenolics. Stimulation of total phenolics in tissue cultures of Mentha
pulegium in response to Pseudomonas mucidolens was demonstrated
(Al-Amier et al., 2005).

Determination of total content of phenolic compounds and their antioxidant

activity in vegetables by spectrophotometric method confirmed correlation between
content of the phenolics and antioxidant activities (Stratil et al., 2006).
A preliminary study was undertaken to elucidate in vitro free radical scavenging
potential and inhibition of lipid peroxidation by Coleus aromaticus hydro-alcoholic
extract. The results established an efficient antioxidant, anticlastogenic and
radioprotective potential of the extract (Rao et al., 2006).

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 In a comparative study of the antioxidant activity of Plectranthus grandis
and P. ornatus essential oils, the oil of P. ornatus showed a higher antioxidant
activity than that of P. grandis, probably due to its higher yield of the phenolic
compounds eugenol and thymol (Albuquerque et al., 2006).

The extensive survey of literature confirms the added advantage of the

application of in vitro method in the quality improvement of medicinal and aromatic
plants. The beneficial aspects of variation originated in the artificial environment of
in vitro culture of many plants inspire to aim on the probability of quality
improvement in this plant too. The comparative evaluation of the in vitro plants with
their parent in different aspects such as cytological, genetical, phytochemical,
structural and biological activities tried in a wide spectrum of plant species that
helps to confirm the beneficial outcome of tissue culture could be useful to this
study also.

34