Chapter - 2 REVIEW 2.1. In vitro regenerationshodhganga.inflibnet.ac.in/bitstream/10603/33765/3/...regeneration via organogenesis from callus culture derived from matured leaves, stems,

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

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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

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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

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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

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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

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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

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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)

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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.

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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

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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

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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


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.

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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


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


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


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


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


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


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


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


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.

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