chapter - 2 review 2.1. in vitro...
TRANSCRIPT
Chapter - 2
REVIEW
Huge volume of works have been completed world wide about the application
of tissue culture technology in in vitro regeneration of many plant species which are
difficult to regenerate by conventional methods and save them from endangerment and
extinction. Some of the representative works are presented here to justify the present
study.
2.1. In vitro regeneration
Tissue culture technology is employed in three occasions for plants such as i) to
make reproduction in the plant species of lower propagation efficiency and threatened
category ii) to protect the wild by getting benefits from the in vitro developed callus and
iii) to get the clones of desired characters by making gene manipulation at callus level.
In addition, with micropropagation, the multiplication rate is greatly increased and it
also permits the production of pathogen-free material (Nehra and Kartha, 1994).
In vitro regeneration attempts and standardization of basal medium for the
propagation of plant species with respect to red listed species and economically
important species are under practice world wide (Cuenca et al., 1999; Naomita and Rai,
2000). Mascarenhas and Muralidharan (1989) reviewed the tissue culture studies carried
out in India for the manipulation of important forest species. de Oliveira et al. (2003)
successfully employed the tissue culture techniques for an economically important plant
species, Tabernaemontana fuchsiaefolia to in vitro regeneration in Brazil. Earlierly,
Begum et al. (2002) standardized the basal medium for in vitro regeneration of
ethnobotanically important pan tropical herbal species, Ocimum basilicum and the
plantlets produced by this method were found to have 75% survivability in fields.
Rahman et al. (2004) successfully developed callogenesis and organogenesis in
Curcuma longa (turmeric) and the results of the hardening experiments revealed that
over 70% of transplanted plantlets of this species was survived in the filed. The species,
Spilanthes mauritiana is a native of Eastern Africa has wide medicinal uses with less
population size in its homeland, the Nigeria and for in vitro regeneration of this species,
the attempts of Bais et al. (2002) by standardizing the basal medium produced elite
plantlets effectively. Similarly, for another species of the same genus, Spilanthes
5
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
acmella, a herbal pesticidal plant of north-east India, Purabi Deka and Kalita (2005)
standardized MS medium for in vitro clonal multiplication and organogenesis.
In vitro propagation has many advantages over conventional methods of
vegetative propagation which suffer from several limitations (Murch et al., 2000). With
in vitro propagation, the multiplication rate is greatly increased and it also permits the
production of pathogen free propagules. Micropropagation from existing meristems
yields plants that are genetically identical with the donor plants (Roy et al., 1994). Plant
regeneration from shoot and stem meristems has yielded encouraging results in
medicinal plants like Cathranthus roseus, Digitalis purpurea, Dioscorea deltoidea and
Rauwolfia serpentina. For the biodiesel plant, Jatropha curcus, Kalimuthu et al. (2007)
developed micropropagation strategies. The nodal explants of this species
micropropagated successfully when cultured onto the MS medium contained BAP, Kn
and IAA at 1.5, 0.5 and 0.1mg/l respectively. Somatic embryos were induced directly
from green cotyledon explants of this species on MS medium fortified with 2 mg/l of
BAP. Subsequently, the rooting was effectively achieved on MS medium supplemented
with IAA at 1.0 mg/l.
Several factors are reported to influence the success of in vitro propagation of
different medicinal plants. The effect of auxins and cytokinins on shoot multiplication
of various medicinal plants has been reported. Maragatham and Panneerselvam (2010)
reported that the combination of auxin, NAA with kinetin at 1.0 and 2.0mg/l
respectively was effective for callus induction in the medicinal plant, Sida cordifolia.
Benjamin et al. (1987) reported that 6- benzylaminopurine (BAP) at high concentration
stimulated the development of axillary meristems and shoot tips in Atropa belladonna.
Lal et al. (1988) observed a rapid shoot proliferation rate in Picrorrhiza kurroa using
kinetin at 1.0-1.5mg/l. Direct plantlet regeneration from male inflorescences of
medicinal yam on MS medium supplemented with 13.94µM kinetin has also been
reported (Borthakur and Singh, 2002). The highest shoot multiplication of Nothapodytes
foetida is achieved in medium containing, thidiazuron (TDZ) at the concentration of 2.2
µM (Ravi, 2002). Similarly, it has been observed that cytokinin is required in optimal
quantity for shoot proliferation in many genotypes but inclusion of low concentration of
auxins along with cytokinin triggers the rate of shoot proliferation (Tsay et al., 1989;
Shasany et al., 1998; Rout et al., 1999a). Wakhlu and Barna (1989) explained that the
6
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
production of multiple shoots was higher in Plantago ovata on a medium with 4-6mg/l
kinetin along with 0.01mg/l NAA. Thidiazuron induced high frequency shoot
proliferation in Cinerraria marittima reported by Banerjee et al. (2004).
Xiangquian et al. (2002) reported that high frequency of callus induced by
11.3µM/l 2, 4-D in rose plant. It has been reported previously that GA3 has induced
somatic embryogenesis in several rose cultivators (Rout et al., 1991; Marchant et al.,
1996; Kintzois et al., 1999). Gatica Arias et al. (2010) demonstrated that the effect of
growth regulators on in vitro regeneration of five cultivars of commercially important
common bean, Phaseolus vulgaris is significantly varied. They investigated that the
basal medium with N6 – benzylaminopurine at 5 mg/l and adenine sulphate at 20 or 40
mg/l resulted in higher average of shoot formation. Trigridia pavonica, an important and
ornamental medicinal plant of Mexico was successfully regenerated by using tissue
culture technique by Jose Luis et al. (2010) to meet the demand. In this plant shoot
formation was determined to be most significant when the twin-scaling explants are
cultured on MS medium supplemented with 4.5µM 2, 4-dichlorophenoxyacetic acid in
combination with 2.2 µM BAP. Mir et al. (2010) explained that the shoot multiplication
was most effective in an endangered medicinal plant, Gardenia gummifera while
cultured onto the MS medium fortified with BAP at 2.0mg/l. They also reported that the
combination of BAP and GA3 in the MS medium was effective in shoot proliferation.
Faria and Illg (1995) reported that the addition of 10µm BA along with 5µm
IAA or 5µm NAA induces a high rate of shoot proliferation in Zingiber spectabile. They
have also demonstrated that the number of shoots/explant depends on concentration of
the growth regulators and the type of genotype used. Nature and condition of explants
have also been shown to have a significant influence on the multiplication rate of the
medicinal plant, Clerodendrum colebrookianum (Mao et al.,1995). A simple and
efficient micropropagation protocol for Vanilla planifolia using shoot tip and nodal
segments cultured on MS medium containing BA at 1mg/l was reported by Geetha and
Shetty (2000). As an alternative to the conventional methods of propagation, the plant
species, Crinum variable was successfully propagated in vitro using twin-scale explants
(Fennell et al., 2001). They also noted that plant growth regulators were not required for
the induction of shoots and inclusion of activated charcoal increasing the bulblet size
and the frequency. High frequency shoot multiplication of Rauwolfia serpentina was
7
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
achieved by using nodal explants culturing on MS medium containing 2.5mg/l BA and
0.1mg/l NAA (Ahmad et al., 2002). Naushaba Baig et al. (2004) have studied the single
hormonal treatments to understand the requirement of specific type of auxin or
cytokinin for the propagation of Boerhaavia diffusa through nodal segments and
explained that it depends upon the endogenous level of the hormones. They
demonstrated that the nodal explants might be having some cytokinin endogenously in
higher concentrations which act synergistically with the exogenous application of auxin
in the medium for in vitro organogenesis. An efficient protocol was developed for high
frequency plant regeneration from leaf explants of Withania somnifera on MS medium
supplemented with different concentrations of auxins and cytokinins by Sivanesan and
Murugesan (2005). In this it was reported that frequency of shoot bud regeneration
varied with the doses of plant growth regulators in the medium. It was also observed
that lower concentration of growth hormones did not produce any shoots while higher
concentrations induced callus formation. For the anticancer plant, Plumbago zeylanica,
Mallikadevi et al. (2008) developed in vitro regeneration strategies. They explained that
high amount of 90% callus was induced in the MS medium fortified with 2, 4-D at
2.0mg/l. In the subculture, the shoot formation was prominently higher (83%) in the MS
medium containing BAP and NAA at 3.5 and 0.3 mg/l respectively. The growth
hormone, IAA at 1.0 mg/l in MS medium produced roots in 90% of secondary explants
while subculturing.
The induction of callus growth and subsequent differentiation and organogenesis
are accomplished by the differential application of plant growth regulators and control
of condition in the culture medium. Cell division, cell growth and tissue differentiation
are induced by the stimulus of endogenous growth substances or by addition of
exogenous growth regulators in the culture medium. There are many reports on the
regeneration of various medicinal plants via callus culture. Kumar and Bhavanandan
(1988) reported the rapid regeneration of shoots of Plumbago rosea from the hypocotyl
derived callus when cultured on MS medium enriched with 6-BAP at 2mg/l and NAA at
0.2mg/l. Binoy Jose and Satheeshkumar (2004) established micropropagation system for
the anticancerous plant, Ophiorrhiza mungo by using seedlings germinated in the
persistent calyx cup. Chung-Chuan Chen et al. (2001) found that stem internodes have
served as better explants when inoculated in appropriate standardized MS medium for
the effective callusing and organogenesis. Plant regeneration was achieved from leaf
8
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
callus of the medicinal plant, Cepahaelis ipecacuanha on MS medium supplemented
with 4.5 mg/l NAA by Rout et al. (1992). Saxena et al. (1997) reported plant
regeneration via organogenesis from callus culture derived from matured leaves, stems,
petioles and roots of young seedling of the critically endangered medicinal plant,
Psoralea corylifolia. Impotance of liquid static culture for shoot proliferation and root
induction for this species has been demonstrated by Uikey et al. (2010) to improve the
technology to achieve 100% success to meet the growing demand of pharmaceutical
industries.
Rapid clonal multiplication and restoration of Celastrus paniculatus were
reported by using internode and leaf callus culture (Nair and Seeni, 2001). Patil and
Chavan (2004) described callus induction from leaf and stem explants of an endemic
wild species, Cucumis setosus on MS medium supplemented with BA, NAA, IBA and
2, 4-D. Callus mediated organogenesis of an important medicinal plant, Withania
somnifera by using leaves, hypocotyl, roots and cotyledonary leaf segments as explants
on MS medium supplemented with various concentrations and combinations of 2, 4-D
and kinetin (Rani et al., 2003). For the same species, Siddique et al. (2004) reported
efficient and easy to handle protocol for the micropropagation by using nodal segments
on MS medium supplemented with 2, 4-D, BAP and Kn. This protocol provides a
successful and rapid technique that can be used for ex-situ conservation and to minimize
the pressure on wild populations. Nandagopal and Ranjithakumari (2006) reported that
the organogenesis and subsequent plantlet production, in vitro flowering via callus were
found to be better for ex situ conservation of wild medicinal plant, Cichorium intybus.
Several reports on callus mediated morphogenesis and subsequent micropropagation for
certain medicinal plants like Curuligo orchioides (Vidya et al., 2005), Eclipta alba
(Baskaran and Jeyabalan, 2005a), Embelia ribes (Shankarmurthy et al., 2004), Gloriosa
superba (Sivakumar and Krishnamurthy, 2000), Rauvolfia teteraphylla (Anitha and
Kumari, 2006), Solanum nigrum (Jabeen et al., 2005), Tylophora indica (Faisal et al.,
2005; Thomas and Philip, 2005), and Withania somnifera (Manickam et al., 2000) have
been published.
Successful multiple shoot production of Clitoria ternatea was reported from
nodal explants cultured on MS medium supplemented with 2.0mg/l BA and 0.25mg/l
NAA (Rout, 2004). In the same species, Krishna Pandeya et al. (2010) also
9
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
demonstrated that in addition to nodal explants, shoot tip and cotyledonary nodal
explants also responded well for multiple shoot formation to the MS medium fortified
with BAP at 2.0 mg/l. Beena et al. (2003) reported the rapid shoot proliferation of
medicinal herb, Ceropegia candelabrum on MS medium supplemented with BA and
kinetin. In the similar fashion, John Britto et al. (2003) explained a rapid
micropropagation system for in vitro flowering and shoot multiplication from nodal
explants of a rare species, Ceropegia bulbosa when cultured on B5 medium containing
1mg/l GA3 and 0.5mg/l BA. Sinha et al. (2005) observed shoot regeneration of
Hydnocarpus kurzi on the basal medium containing 2.5mg/l BA and 0.5mg/l NAA. An
efficient protocol was developed for mass multiplication of Psoralea corylifolia from
shoot tip and nodal explants by using BA and NAA at 0.5µM (Anis and Faisal, 2005).
The relative importance of genotype, explant and their interactions for in vitro plant
regeneration via organogenesis in Solanum melongea has been investigated by Sharma
and Rajam (1995). Sathees Kannan et al. (2006) developed a high frequency and rapid
in vitro regeneration system for Solanum nigrum by using meristematic explants on MS-
B5 medium with various growth hormones. Raman and Senthilkumar (2001) described
a technique for high frequency plant regeneration in Solanum trilobatum, a medicinal
plant by internodal explants cultured on MS medium supplemented with BA at 2.0mg/l.
This technique should be beneficial for the sustainable utilization of this important
medicinal plant and it is an alternative method for the production of bioactive
compounds without destroying whole plant that are usually not under cultivation. For
this species, Jawahar et al. (2004) devised another efficient protocol for rapid
propagation by using shoot tip and nodal explants.
For germplasm conservation and the mass multiplication of an endangered
medicinal plant, Hemidesmus indicus, Siddique et al. (2003) formulated an efficient
protocol by using nodal segments derived callus on MS medium supplemented with 2,
4-D at 2.0mg/l, NAA at 1.0mg/l and Kn at 2.0mg/l. This protocol provides a successful
and rapid technique that can be used for ex situ conservation. A successful system of
direct organogenesis is described for an endangered species of South-East Asia, Citrus
halimii cultured on MS medium supplemented with 0.4 - 11.1 µm 6-benzyladenine
(Normah et al., 1997). McCartan and Crouch (1998) developed a micropropagation
strategy for conserving an endemic plant species, Mondia whitei using single-node
explants from in vitro grown seedlings. They also indicated that addition of charcoal to
10
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
the medium found to significantly reduce vitrification and this protocol is very suitable
for producing large quantities of the elite plantlets. An efficient protocol for in vitro
multiplication through the callus and reintroduction of regenerants to the natural
condition have been standardized for the rare medicinal species, Celastrus paniculatus
on MS medium enriched with 2% fructose, 6µM Kn and 1µM IBA (Maruthi et al.,
2004). Shashikala et al. (2005) developed a micropropagation strategy through
employing tissue culture techniques for an endangered plant species, Centella asiatica.
For sustainable utilization and conservation of endangered medicinal plant, Pimpinella
tirupatiensis, Prakash et al. (2001) developed an efficient protocol by inducing somatic
embryogenesis using hypocotyl explants. Thoyajaksha and Rai (2001) developed
micropropagation strategy through employing tissue culture techniques for an
endangered plant species endemic to the Western Ghats region of Karnataka,
Dictyospermum ovalifolium as a conservation measure. Abrie and van Staden (2001)
formulated micropropagation protocol for the highly endangered medicinal plant, Aloe
payphylla using MS medium supplemented with 1mg/l BA. For this same species,
Chukwujekwu et al. (2002) also demonstrated that BA at 1-2mg/l and other growth
regulators such as zeatin, IBA, NAA and kinetin used either singly or in combination
gave better results for in vitro regeneration. For the medicinal plant, Aloe vera,
Kalimuthu et al. (2010) standardized the MS medium by establishing simple two steps
protocol by enhancing axillary branching. The study revealed that MS medium
containing BAP at 1.5 mg/l and AS (adenine sulphate) 50 mg/l gave effective
multiplication.
Rhizome explants were used to develop a protocol for in vitro plantlet
regeneration of Podophyllum hexandrum, a crtically endangered medicinal plant
through direct organogenesis (Chakraborthy et al., 2010). Highest rate of multiple shoot
formation was noted in MS medium supplemented with 11.42 µM IAA within three
months. In other plant, Bacopa monneiri, Neethu Sharma (2005) reported that best
medium for initiation and development in terms of bud breakage percentage, cluster
formation and maximum shoot length were with the growth regulators, BAP 0.5 mg/l
plus NAA 0.5 mg/l, BAP 4.0 mg/l plus NAA 0.4 mg/l and BAP 1.0 mg/l plus Kn 1.0
mg/l respectively.
Formation of healthy shoots and its higher multiplication are the prerequisites of
an economically viable micropropagation protocol. Karuppusamy and Pullaiah (2007)
11
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
determined the effect of different growth regulators on shoot formation and
multiplication of genetically stable multiple shoots from shoot tip and nodal explants of
Bupleurum distichophyllum using MS medium. Nithiya and Arockiasamy (2007)
reported a simple reproducible protocol for in vitro micropropagation of the medicinal
plant, Datura metal through somatic embryos cultured on MS medium supplemented
with BAP at 4.0mg/l, GA3 1.0mg/l and IBA 1.0mg/l and they further observed that 90 %
of the plantlets have successfully survived under field conditions. For the same species,
Madhavan and Joseph (2001) reported that the organogenesis and subsequent plantlet
production via callus were found to be better. For a Chinese medicinal herb, Gentiana
straminea which is rich in secoiridoids, Yunfei Coi et al. (2009) have developed
efficient tissue culture system for high frequency plant regeneration. They have
demonstrated that 2, 4- dichlorophenoxyacetic acid (2, 4-D) is efficient for both callus
induction and embryogenesis, indole-3-acetic acid is suitable for embryogenic callus
proliferation, and N6- (benzyl) – adenine promotes both embryo development and the
accumulation of gentiopicroside in the cultures. The sub-temperate Himalayan
medicinal plant, Hedychium spicatum has been reported to micropropagate well in
various combinations of certain growth regulators (Badoni et al., 2010). The in vitro
shoot tip of this plant showed pronounced shoot elongation and root formation while
subcultured onto the MS medium contained 5.0 µM/l Kn and 1.0 µM/l IAA.
Increasing concern for conservation and systematic propagation of medicinally
important plant species, Pergularia daemia, Kiranmai et al. (2008) established a
protocol for callus culture and plant regeneration from in vitro grown seedling explants
on MS medium supplemented with different concentrations of auxins, and shoots were
rooted considerably on half strength MS medium supplemented with 0.1 mg/l IBA.
Karthikeyan et al. (2008) standardized the basal medium for in vitro propagation of
Phyllanthus niruri, a medicinal plant from leaf, shoot tip and nodal explants. They
observed that rooting in the medium is successful by supplementing NAA and BAP at
1mg/l each. For the same species, Chitra et al. (2009) outlined a procedure for indirect
organogenesis by using leaf discs and internodes. They found that lower amount of
auxin and cytokinin is desired for obtaining complete plantlets. Ioan Bacila et al. (2010)
established an effective in vitro propagation protocol for the important medicinal plant,
Hypericum maculatum using nodal segments. The nodal pieces inoculated individually
in MS basal medium supplemented with 0.5 mg/l 2iP plus 0.2 mg/l BA plus 0.1 mg/l K
plus 0.5 mg/l NAA produced multiple shoots.
12
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
An efficient plant regeneration protocol was described for Ricinus communis
(Alam et al., 2010) using cotyledonary nodes as explants. For this explants, use of BAP
at 3.0 mg/l induced the highest frequency of shoot induction as well as maximum
number of shoots per explants. Alka Jajoo (2010) developed an efficient and highly
reproduciable plant regeneration protocol from nucellar embryos for the medicinal
plant, Citrus limonia. It was noted that 6- benzylaminopurine at a concentration of 2.22
µM induced highest number of multiple shoots. Further, IBA at 2.46 µM and 13 AP at
1.11 µM proved to be the best combination for rooting of shoots. For the species,
Bauhinia cheilantha, an important medicinal plant of Brazil, Gutierrez et al. (2011)
standardized MS medium for in vitro regeneration and acclimatization. For the
economically important medicinal plant, Rorippa indica, Ananthi et al. (2011)
developed micropropagation strategies. The nodal explants of this species
micropropagated successfully when cultured onto the MS medium contained BAP and
Kn at 3.0 mg/l each.
Das et al. (2008) have been established callus cultures from needles of
aseptically germinated seeds of Taxus wallichina and maintained on different media
with regulators. Furthermore, they found that callus contains an important secondary
metabolite, taxol. For the in vitro clonal propagation of Saraca asoca, Rama Subbu et
al. (2008) reported the regeneration of shoots from the shoot tip, nodal and internodal
explants when cultured on MS medium enriched with different growth regulators like 2,
4-D, BAP and Kn. Influence of different growth regulators like 2, 4-D, BAP, Kn and
IAA on growth of calli was reported by Roy et al. (2008). They found that the highest
efficiency of callus formation was observed in the medium containing different
concentrations of 2, 4-D and Kinetin. Abdeliatef and Khalafallah (2008) explained the
influence of growth regulators on callus induction from hypcotyls of Gossypium
hirsutum. They found that B5 medium without growth hormones produced no callus,
while B5 with 2, 4-D induced brown coloured low quantity of calli than B5 with NAA.
Somatic embryogenesis is a process where groups of somatic cells/tissues lead to
the formation of somatic embryos which resemble the zygotic embryos of intact seeds
and can grow into seedlings on suitable medium. Plant regeneration via somatic
embryogenesis from single cells that can be induced to produce an embryo and then a
complete plant has been demonstrated in many medicinal plant species. Arumugam and
13
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
Bhojwani (1990) and Basu and Chand (1996) noted the development of somatic
embryos from zygotic embryos of Podophyllum hexandrum on MS medium containing
2 µM BA and 0.5 µM IAA. Ghosh and Sen (1991, 1994) reported regeneration and
somatic embryogenesis in Asparagus cooperi on MS medium having 1.0 mg/l NAA and
1.0 mg/l kinetin. Embryogenic calli and germination of somatic embryos in nine
varieties of Medicago sativa have been achieved (Fuentes et al., 1993). Using a medium
containing 2, 4-D and TDZ, Zhou et al. (1994) have achieved the induction of somatic
embryogenesis in cells from Cayratia japonica. Somatic embryogenesis and subsequent
plant regeneration from callus derived from immature cotyledons of Acacia catechu
have also been achieved on medium supplemented with 13.9 µM kinetin and 2.7µM
NAA (Rout and Samantaray, 1995). Gastaldo and Caviglia (1996) induced somatic
embryos from bark derived callus of Aesculus hippocastanum on MS medium
supplemented with 2.0 mg/l kinetin, 2.0 mg/l 2,4-D and 2.0 mg/l NAA. High frequency
somatic embryogenesis and plant regeneration from suspension cultures of
Acanthopanax koreanum have been reported on a medium containing 4.5 µM 2, 4-D
(Choi et al., 1997). Das et al. (1999) reported high frequency somatic embryogenesis in
Typhonium trilobatum on medium containing 1.0 mg/l kinetin and 0.25 mg/l NAA. The
suspension culture of Catharanthus roseus from stem and leaf explants on medium
containing NAA and kinetin has been established by Zhao et al. (2001). Chand and
Sahrawat (2002) have reported the somatic embryogenesis of Psoralea corylifolia from
root explants on medium supplemented with NAA and BA.
Efficient development and germination of somatic embryos are the prerequisites
of commercial plantlet production. Lowering of growth regulator concentrations in
culture media has improved embryo development and germination of many medicinal
plants (Arumugam and Bhojwani, 1990; Wakhlu et al., 1990; Kumar, 1992).
Germination of the somatic embryos is achievable on MS medium without the growth
regulator (Zhou et al., 1994; Choi et al., 1997). However, Arumugam and Bhojwani
(1990) noted that the inclusion of BA (2 µmL) and gibberellic acid (GA3, 2.8 µmL) in
the medium stimulated embryo development of Podophyllum hexandrum, although 75%
of the embryos germinated on MS medium devoid of growth regulator. Similar results
were reported on the germination of embryos of Psoralea corylifolia (Chand and
Sahrawat, 2002). Wakhlu et al. (1990) have reported that the somatic embryos of
Bunium persicum matured and germinated on the MS medium supplemented with 1.0
14
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
mg/l kinetin. Further, Kunitake and Mii (1997) reported that 30– 40% of somatic
embryos of Asparagus officinalis germinated after being treated with distilled water for
a week; they were subsequently transferred to half-strength MS medium supplemented
with 1.0 mg/l IAA, 1.0 mg/l GA3 and 1% sucrose. However, the somatic embryos of
Typhonium trilobatum have been germinated on MS medium supplemented with 0.01
mg/l NAA and 2% (w/v) sucrose after 2 weeks of culture (Das et al., 1999).
The Nilgiri Biosphere Reserve, Western Ghats, India, a repository of many plant
species, harbours a very considerable number of endemic, rare and endangered plant
species with lot of medicinal properties. It has been reported that several intrinsic and
external factors are responsible for their poor population size (Paulsamy et al., 2008). In
addition to clonal propagation strategies, for many species in vitro regeneration attempts
have been successfully completed. Padmavathy et al. (2007) reported that callus
formation was most effective for the medicinal herb used for curing ulcer, Lycianthes
bigeminata distributed in the high hills of Nilgiris, the Western Ghats, India when
cultured onto the MS medium fortified with the growth regulators, BAP and NAA at 0.5
mg / l each. Senthilkumar et al. (2007) standardized the MS medium for another
medicinal herb, Acmella calva inhabiting the understories of Nilgiri shola forests for
callus culture and organogenesis from the leaf explant. They have also investigated that
the survivability of plantlets of this species was more pronounced in the hardening
medium composed by coir pith and soil in the ratio of 1: 1 by volume. Ganesan and
Paulsamy (2011) for the high altitude medicinal plant of Nilgiris, Artemisia annua
developed a reliable protocol for callus induction and organogenesis and successful
plantlet survivability through hardening by using leaf explants. They reported that MS
medium with NAA at 0.9 mg/l and BAP and GA3 0.5 and 1.0 mg/l respectively induced
high amount of callus formation and shooting.
For the rare and threatened medicinal plant, Disporum leschenaultianum which
is distributed in the high altitudes (>1800 m above msl) of Nilgiris, Senthilkumar et al.
(2009a) standardized the protocol for in vitro multiplication. The nodal explant
produced high degree of shoot proliferations when cultured on MS medium with BAP
and NAA at 4.0 and 0.5 mg/l respectively. The shoot multiplication was noted to be
effective when cultured onto the MS medium fortified with BAP, NAA and Kn at 4.0,
0.5 and 2.5 mg/l respectively. The regenerated shoots of this species further reported to
15
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
be successfully rooted in MS medium supplemented with 2.0 mg/l of IBA. For another
red listed medicinal species, Ophiorrhiza mungos distributed in the fringes of small
streams in the shola forests of Nilgiris, Senthilkumar et al. (2009b) established in vitro
regeneration protocol. The response of nodal explant of this species was found to be
most significant during culturing on MS medium with BAP at 3.0 mg/l. Low level of
growth regulators produced high degree of shoot formation in this species.
Subsequently, NAA at 0.5 mg/l produced higher number of roots from the secondary
explant, the shoots. The endemic and threatened medicinal plant, Cayratia pedata
distributed in the high hills of Nilgiris which is used for the treatment of cough,
bronchitis, asthma, joint pain etc showed better response to certain combinations and
concentrations of growth regulators for in vitro regeneration (Senthilkumar and
Paulsamy, 2009). Micropropagation strategies are evaluated for an endangered
medicinal plant, Ceropegia spiralis distributed in the grasslands revealed that multiple
shoot induction was more successful using nodes as explants on MS medium containing
BAP, 2.22 µM (Chyuam- Yih Ng et al., 2010; Sri Rama Murthy et al., 2010).
2.2. Synthetic seeds
Production of artificial seeds has unraveled new vistas in plant biotechnology.
The synthetic seed technology is designed to combine the advantages of clonal
propagation with those of seed propagation and storage. Despite the fact that the
technology is an exciting and rapidly growing area of research in plant cell and tissue
culture, there are many limitations of practical use.
The production of synthetic seeds for the first time by Kitto and Janick (1982)
involved encapsulation of carrot somatic embryos followed by their desiccation. Of the
various compounds tested for encapsulation of celery embryos, Kitto and Janick (1982)
selected polyoxyethylene which is readily soluble in water and dries to form a thin film,
does not support the growth of micro-organisms and is non-toxic to the embryo. Janick
et al. (1993) have reported that desiccated artifical seeds were produced by coating a
mixture of carrot somatic embryos and callus in polyoxyethlene glycol. The coating
mixture was allowed to dry for several hours on a teflon surface in a sterile hood. The
dried mixture was then placed on a culture medium, allowed to rehydrate, and then
scored for embryo survival.
16
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
Redenbaugh et al. (1984) developed a technique for hydrogel encapsulation of
individual somatic embryos of alfalfa. Since then encapsulation in hydrogel remains to
be the most studied method of artificial seed production (Redenbaugh and Walker,
1990; Mckersie and Bowley, 1993). A number of substances like potassium alginate,
sodium alginate, carrageenan, agar, gelrite, sodium pectate, etc. have been tested as
hydrogels but sodium alginate gel is the most popular (Redenbaugh et. al., 1993).
Hydrated artificial seeds consist of somatic embryos individually encapsulated in a
hydrogel. To produce hydrated synthetic seeds, the somatic embryos are mixed with
sodium alginate gel (0.5-5.0% w/v) and dropped into a calcium salt solution [(CaCl2
(30-100mM) and Ca(NO3)2 (30-100mM)] where ion-exchange reaction occurs and
sodium ions are replaced by calcium ions forming calcium alginate beads or capsules
surrounding the somatic embryos. The size of the capsule is controlled by varying the
inner diameter of the pipette nozzle. Hardening of the calcium alginate is modulated
with the concentrations of sodium alginate and calcium chloride as well as the duration
of complexing. Usually 2% sodium alginate gel with a complexing solution containing
100mM Ca2+ is used and is found to be satisfactory (Redenbaugh and Walker, 1990;
Redenbaugh et al., 1993; Ara, et al., 1999). However, Molle et al. (1993) found that for
the production of synthetic seeds of carrot, 1% sodium alginate solution, 50mM Ca2+
and 20-30 min time period were satisfactory for proper hardening of calcium alginate
capsules. They have suggested the use of a dual nozzle pipette in which the embryos
flow through the inner pipette and the alginate solution through the outer pipette. As a
result, the embryos are positioned in the centre of the beads for better protection.
For the past several years other unipolar structures such as apical shoot tips and
axillary shoot buds as well as apolar protocorms or protocorm-like bodies and even
undifferentiated embryogenic calli are also being employed in synthetic seed
production. The technology of hydrogel encapsulation is also favoured for synthetic
seed productions from these micropropagules. For the production of synthetic seeds
from apical shoot tips and axillary shoot buds, these organs are usually first treated with
auxins for root induction and then their microcuttings (approximately 4 or 5 mm in
length) are encapsulated in sodium alginate gel following the method described by
Redenbaugh et al. (1984) for Alfalfa somatic embryos. However, mulberry (Bapat and
Rao, 1990) and banana (Ganapathi et al. 1992) plantlets were obtained from alginate-
encapsulated shoot buds without any specific root induction treatment. For the
17
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
endangered orchid species, Grodorum densiflorum, Datta et al. (1999) used protocorm-
like bodies for the encapsulation to produce synthetic seed successfully and the
germination was determined to be successful during culturing onto the modified
Knudson C medium contained coconut milk (15%), peptone (2g/l), BAP (2mh/l) and
NAA (1mg/l). Guerra et al. (2010) used pre-germinated torpedo shaped somatic
embryos for encapsulation and determined that treatment of beads with KNO3 for 20
min. enhanced the germination. To avoid bacterial contamination, Ganapathi et al.
(1992) added an antibiotic mixture (0.25mg/l) containing rifampicin (60mg), cefatoxime
(250mg) and tetracycline-HCl (25mg) dissolved in 5 mL dimethyl sulphoxide to the gel
matrix. Activated charcoal (0.1%) was also added to the matrix to absorb the polyphenol
exudates of the encapsulated shoots of banana (Ganapathi et.al., 1992). Pouzi et al.
(2011) established highest viability of protocorm-like bodies of Dendrobium sonia- 28
through the encapsulation –dehydration technique. Similarly, Mikula et al. (2011)
enhanced the embryogenic capacity through encapsulation method under
cryopreservation for the medicinal plant, Gentiana cruciata.
2.3. Biological studies
The studies on bioactivity in plants are carried out world wide to confirm the
therapeutic value of many medicinal plants. Further, these studies are more important
for any medicinal plant to go before commercialization by pharmacological industries.
Some important reports in the area of antimicrobial studies are presented here to know
the update information in this area.
2.3.1. Antimicrobial studies
There has been an increasing incidence of multiple resistances in human
pathogenic microorganisms in recent years, largely due to indiscriminate use of
commercial antimicrobial drugs commonly employed in the treatment of infectious
diseases. This has forced scientists to search for new antimicrobial substances from
various sources like medicinal plants. The antibacterial activity is due to different
chemical agents in the extract including essential oils, flavanoids and triterapenoids and
other natural phenolic compounds or free hydroxyl groups and these are classified as
active antimicrobial compounds (Rojas et al., 1992). It is reported that at least
12,000 such secondary metabolites have been isolated, a number estimated to be less
18
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
than 10% of the total (Schultes, 1978). In many cases, these substances serve as plant
defense mechanisms against predation by microorganisms, insects, and herbivores.
Some, such as terpenoids, give plants their odors; others (quinones and tannins) are
responsible for plant pigment. Many compounds are responsible for plant flavor (e.g.,
the terpenoid capsaicin from chili peppers), and some of them are useful medicinal
compounds also. According to World Health Organization (Santos et al., 1995),
medicinal plants would be the best source to obtain a variety of drugs. About 80% of
individuals from developed countries use traditional medicine, which has compounds
derived from plants. Therefore, such plants should be investigated for their properties,
safety and efficiency (Ellof, 1998). The use of plant extracts and phytochemicals, both
with known antimicrobial properties can be of great significance in therapeutic
treatments. In the last few years, a number of studies have been conducted in different
countries to prove such efficiency (Almagboul et al., 1985; Sousa et al., 1991; Shapoval
et al., 1994; Artizzu et al., 1995; Nanda et al., 2001; Velickvic et al., 2002;
Sureshkumar et al., 2004; Guleria and Kumar, 2006; Parekh and Chanda, 2007a). The
antimicrobial traits of the plants due to secondary metabolites are known by their active
substances, for example, the phenolic compounds which are the part of essential oils
(Jansen et al., 1987), as well as in tannin (Saxena et al., 1994). The antimicrobial
properties of plants have been investigated by a number of researchers world wide,
some of the representative studies are presented here.
The substance inhibits the growth of pathogens or kill them and have no toxicity
to host cells are considered as sources for developing new antimicrobial drugs. Olukoya
et al. (1993) enlisted ten medicinal plants which include Authocleista vogelii, Boerhavia
diffusa, Combretum bracteatum, Emilia coccinea and Phyllanthus discoideus etc., used
for the treatment of various microbial infections. Diarrhea is still the main cause of
morbidity and mortality in developing countries. Caceres et al. (1993a) investigated that
the species of Central American region, Tagetes lucida has strong antibacterial property
against the bacterial strain, Vibrio cholerae and later this finding was helpful in the
scientific validation of T. lucida as a supplementary support in the treatment of cholera.
Caceres et al. (1993b) further studied antibacterial activity for forty eight extracts from
16 plant species against three gram-positive bacteria, common causal organisms of
respiratory and skin infections in the infant population.
19
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
Medicinal plants exhibit antibacterial activity since they contain innumerable
biologically active chemical constituents. The extracts of herbal plants play a vital role
in the ethnomedicine due to their therapeutic properties. The Fabaceae member,
Indigofera suffrticosa has been used as infusion or decoction in traditional medicine
system of Brazil, and Leite et al. (2006) investigated antimicrobial activity of various
organic and aqueous leaf extracts of this plant against five human pathogenic bacteria
and seventeen fungal strains and the study exhibited that the aqueous extracts have
significant antimicrobial activity against both gram-positive and gram-negative bacteria
and fungal species. In another study, Adekunle et al. (2006) reported that the aqueous
extracts of Funtumia elastica and Mallotus oppositifolius have significant antifungal
activity against most of the fungal species tested. Traditionally, the aqueous extracts of
these two species are used orally or topically to cure skin diseases in Nigeria.
Due to the emergence of drug resistant strains of many infectious
microorganisms, ethnobotany may provide new, effective pharmaceutical alternatives to
existing drugs. Hess et al. (1995) reported antibacterial activity for different extracts and
some isolated compounds derived from Vochysia divergens against different human
pathogenic bacteria causing infections and asthma. Meyer and Afolayan (1995)
investigated antibacterial activity of different extracts from Helichrysum aureonitens
against five gram-positive and gram-negative bacteria and identified dichloromethane
extract was active against all the five gram-positive bacteria and none of the extracts
inhibited the growth of the five gram-negative bacteria tested. Vijaya et al. (1995)
designed a study to prove the antibacterial effect of two plants viz., Camellia sinensis
and Euphorbia hirta against dysentery causing Shigella sp. They explained that all the
extracts inhibited the growth of this pathogen at their respective doses. Grange and Snell
(1996) reported the benzylamines, bromhexine and ambroxol, semi-synthetic derivatives
of vasicine from Indian shrub, Adhatoda vasica, which is widely used as mucolytics and
it has pH dependent growth-inhibitory effect on Mycobacterium tuberculosis. Locher et
al. (1995) directly evaluated the antimicrobial activity and anti-complement activity for
seventy three plant extracts obtained from sixteen Hawaiian medicinal plants which are
used in the traditional medicine system for the treatment of infectious diseases. The
study revealed that majority of the studied plants has curative proprieties against
infections using biological assays in vitro.
20
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
Many pharmaceutical products are of plant, bacterial or fungal origin and have
proved their value in a broad range of human infections, although synthetic organic
compounds are substantially used. Antibacterial activity of numerous drugs has been
obtained from the actinomycetes and bacillus also. Nedialkova and Naidenova (2004-
2005) reported the antimicrobial activity of forty actinomycetes strains against seven
gram-positive and gram-negative bacteria, yeast, sixteen pathogenic fungi. The study
revealed that 60% of the strains showed inhibition potential against tested
microorganisms and ten of them had a broad spectrum of antimicrobial activity.
Flavanones seem to have low toxicity because the related falvonoids are widely
distributed in edible and beverages and have been used in medicine (Havsteen, 1983 and
Pathak et al., 1991). Differently substituted flavanones were isolated from leguminosae
and their antimicrobial activity was comparatively studied against methicillin-resistant,
Staphylococcus aureus (MRSA). Among the thirteen flavanones tested,
tetrahydroxyflavanones showed intensive activity to inhibit the growth of all MRSA
strains and it would be useful in the phytotherapeutic strategy against MRSA infections
(Tsuchiya et al., 1996). Erybraedin A is a flavonoid isolated from many Erythrina
species like E. latissima, E. mey, E. zeyheri, E. senegalensis etc. is an antimicrobial
agent and has strong activity against yeast spores (Wanjala et al., 2002). Sato et al.
(2004) also reported that combination of erybraedin and vancomycin acts either
synergistically or additively against vancomycin resistant enterococci (VRE) and
multiresistant, Staphylococcus aureus (MRSA) and these antibacterial activities were
based on bacteriostatic action. The isoflavonoid 6–8-diprenylgenistein isolated from the
stem bark of E. senegalensis inhibited the growth of 36 different strains of
Staphylococcus aureus, 29 strains of Shigella sp. and 27 strains of Salmonellae sp.
(Dastidar et al., 2004). Senegalensein, a novel prenylated flavanone isolated from the
stem bark of E. senegalensis exhibited a HIV inhibitory activity (Meragelman et al.,
2001) and also has antibacterial activity against methicillin resistant Staphylococcus
aureus, and vancomycin resistant Enterococcus faecium (Salvatore et al., 1998).
Holetz et al. (2002) screened different extracts of 13 Brazilian medicinal plants
for their antimicrobial activity against bacteria and yeasts and the in vivo data obtained
may be helpful in determining the potential usefulness of these plants for the treatment
of infectious diseases. Rajakaruna et al. (2002) listed 32 plant species collected from
21
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
serpentine soils in Sri Lanka and were screened for antimicrobial proprieties against
three gram-positive and two gram-negative bacteria, a non-acid bacterium and the yeast,
Candida albicans. The study revealed that plants from serpentine environments may
have altered antimicrobial activities when compared to their relatives from non-
serpentine environments, urging the need to pay attention to substrate, habitat etc., when
collecting plants to test for antimicrobial proprieties. Janovska et al. (2003) analyzed the
antimicrobial activity of crude ethanolic extracts of 10 Chinese traditional medicinal
plants which were tested against five bacterial pathogens. Sheela and Kannan (2003)
reported the antibacterial activity of aqueous and methanol extracts of three different
medicinal plants viz., Thespesia populnea, Centella asiatica and Solanum trilobatum
against some human pathogenic bacterial strains. This study described that the
antibacterial activity of medicinal plants is varied according to species and it clearly
emphasized that the efficiency of antibacterial activity of the plant should be determined
by the physiological and biochemical synthesis of antimicrobial principles. Sashikumar
et al. (2003) evaluated the antibacterial assay of 16 crude extracts of 4 ethnomedicinal
plants viz., Aegle marmelos, Gmelina arborea, Holorrhena antidysentrica and Piper
longum used in Nilgiri Biosphere Reserve by testing against Esherchia coli, Klbesieela
pneumoniae and Streptococcus aureus and they reported that the employed extracts of
all the four traditional medicinal plants exhibited potential antibacterial activity against
the tested pathogens.
Antibacterial effect of volatile substances of medicinal plants has been studied
by several authors. The volatile substances of aromatic plants and essential oils are
extensively used in ayurveda and bio-dynamic action (Larrondo et al., 1995 and Perez
et al., 1999) Ramasamy and Manoharan (2004) assessed the antibacterial effect of
volatile components of selected medicinal plants like Anisomeles indica, A. malabrica,
Blumea lacera and Melia azadirachta against human pathogens and it was confirmed
that the volatile components of all tested plants posses good antibacterial property.
Subsequently pure chemical compounds responsible for this activity has been identified,
isolated and finally they used the compounds as prototypes. The essential oil extracted
from the aerial parts of the plant species, Baccharis trinervis during different times in
the same population has been investigated for its in vitro antimicrobial activity against
the four human pathogenic bacteria and one fungus. The volatile constituents of this
plant proved to be active against all tested microorganisms (Albuquerque et al., 2004).
22
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
Sartoratto et al. (2004) studied the antimicrobial proprieties and anti-Candida albicans
activity of essential oils from aromatic plants used in Brazil by bioautographic method
and most of the essential oils studied were effective against all tested human pathogenic
microorganisms. Efficacy of aqueous and methanol extracts of twelve medicinal plants
were studied for potential antibacterial activity against five medicinally important
bacterial strains and found that only one species showed significant antibacterial activity
and suggested the extract of that species can be used as antimicrobial agents in new
drugs for the therapy of infectious diseases caused by pathogens (Parekh et al., 2005).
There has been growing interest in the investigation of the natural products from
plants for the discovery of new antimicrobial and antioxidant agents. Rajeshwar et al.
(2005) investigated in vitro lipid peroxidation and antimicrobial activity of the methanol
extract of the plant, Mucuna pruriens seeds with various gram-positive and gram-
negative bacteria and it showed broad-spectrum of antibacterial activity against all the
tested microorganisms except Staphylococcus aureus and Vibrae cholera. The results
indicate that Mucuna pruriens can be a potential source for natural antibacterial
property. Bessong et al. (2004) reported the in vitro activity of three selected South
African medicinal plants viz., Bridellia micrantha, Combretum molle and Terminallia
sericea against human immunodeficiency virus type 1 reverse transcriptase and they
observed that methanol extract of Terminalia sericea found to be active against HIV-
1RT. Some of the medicinal plants are used in Colombian folk medicine to treat
infections of microbial origins. Rojas et al. (2006) analyzed antimicrobial activity of
these plants against five bacteria and one yeast and they were found to be effective
against three or more of the pathogenic microorganisms. Parekh and Chanda (2007b)
reported in vitro antimicrobial activity of some Indian medicinal plants and they
explained that Caesalpinia pulcherima extracts possess a broad spectrum of activity
against a panel of bacteria responsible for the most common bacterial disease. These
promissory extracts open the possibility of finding new clinically effective antibacterial
compounds. Akpomie and Olorungbon (2011) studied the antimicrobial activity of two
medicinal plants, Terminalia avicennoides and Acalypha wilkesiana against different
pathogens and reported that both plant extracts at high concentrations have effective
inhibition on microbial growth.
23
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
In order to give scientific authentication of the plant for its potential activities
against certain pathogenic microbes, Amerjothy et al. (2007) confirmed the
antimicrobial efficiency of the leaf extracts of Xanthium indicum against human
pathogenic bacteria and fungi and they stated that the plant is said to be a source of
many bioactive principles acting against some human ailments. Matasyoh et al. (2007)
reported antimicrobial activity of the essential oil of the plant species, Ocimum
gratissimum growing in Eastern Kenya against both gram-positive and gram-negative
bacteria and a pathogenic fungus, Candida albicans. The results showed that the oil had
pronounced antibacterial and antifungal activities on all the tested microbes.
2.3.2. Minimum Inhibition Concentration (MIC) studies
After preliminary studies on the effect of plant extracts on the inhibition of
microbial growth, further studies are generally carried out to know the specific
concentration of respective plant extract for effective control of microbial colonies by
following minimum inhibitory concentration (MIC) method. MIC is the highest dilution
of a plant extract that still retains an inhibitory effect against the growth of a
microorganism (Misra and Dixit, 1978). The sensitivity of the microorganisms to plant
extract is varied due to many factors. Gaill and Jon (1995) stated that the variations in
the sensitivity could be attributed to the differences in growth rate of the tested
organisms, nutritional requirements, temperature and inoculum size. Further, the
difference in the inhibition zones could be due to the different extraction methods used
or regional variation in the chemical constituents of the plants (Frey and Meyers, 2010).
Majorie (1999) pointed out that the MIC activity is probably due to their ability to
complex with extra cellular and soluble proteins and to complex with bacterial cell
walls. The highest sensitivity of bacterial microorganisms may be due to its cell wall
structure and outer membrane (Zaika, 1988).
The various kinds of phytochemicals found in the plant parts are the major factor
to control the growth of the microbial colonies (El-Mahmood et al., 2010). Steroids
have been reported to have antibacterial properties, the correlation between membrane
lipids and sensitivity for steroidal compounds indicate the mechanism in which steroids
specifically associate with membrane lipid and exerts its action by causing leakages
from liposomes (Epand, 2007).
24
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.
2.4. Antioxidant and anti-inflammatory activities
Plants are potent biochemical factories and have components of phytomedicine.
Plant based natural constituents can be derived from any part of plant like bark, leaves,
flowers, roots, fruits, seeds, etc i.e. any part of the plant may contain active components
(Makari et al., 2008). The beneficial medicinal effects of plant materials typically result
from the combinations of secondary products present in the plant. The medicinal actions
of plants are unique to particular plant species or groups are consistent with this
concept, as the combination of secondary products in a particular plant is taxonomically
distinct (Wink, 1999). The majority of the active antioxidant compounds are flavonoids,
isoflavones, flavones, anthocyanins, coumarins, lignans, catechins and isocatechins
(Iranshahi et al., 2009). In addition to the above compounds found in natural foods,
vitamins C and E, betacarotene and tocopherol are known to possess antioxidant and
anti-inflammatory potentials (Ara and Nur, 2009). During the last 3 decades, antioxidant
and anti-inflammatory based drug formulations are used for various diseases (Ames et
al., 1993; Prior, 2003; Hennebelle et al., 2006; Makari et al., 2008; Saha et al., 2008;
Shah et al., 2010; Warokar et al., 2010). Several herbs and spices have been reported to
exhibit antioxidant and anti-inflammatory activity, including rosemary, sage, thyme,
nutmeg, turmeric, white pepper, chili pepper and ginger (Ara and Nur, 2009).
For certain species of the families, Mimosaceae and Euphorbiaceae, the
antioxidant and anti-inflammatory activities have been reported elsewhere (Pedilanthus
tithymaloides – Abreu et al., 2006; Acacia arabica – Sundaram and Mitra, 2007;
Faidherbia albida – Tijani et al., 2008; Albizia amara and A. lebbeck – Sen et al., 2010;
Acacia nilotica – Kalaivani et al., 2011; Euphorbia nerifolia - Pracheta et al., 2011;
Ricinis communis – Kadri et al., 2011; Jatropha curcas – Oskoueian et al., 2011).
The informations furnished in this chapter related with various aspects of
regeneration and medicinal properties were highly useful for planning and execution of
the work for the two study species, Acacia caesia and Acalypha fruticosa. The
discussion on results and other observations was made satisfactorily on basis of past
work mentioned in this review part.
25
Please purchase PDF Split-Merge on www.verypdf.com to remove this watermark.