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Coleus Leaves
Coleus Leaves
Micropropagation
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.
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.
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.
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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).
Cytological Analysis
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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).
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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).
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).
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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).
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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).
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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).
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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.
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
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level which result from evolutionary mechanisms such as DNA deletions, additions,
substitutions, repetitions and translocations.
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
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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.
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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.
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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.
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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).
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distinctness shown by the accessions was found to be highly correlated to volatile oil
constituents.
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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).
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.
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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).
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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.
Antioxidant Activity
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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).
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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).
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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).
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