Advances in Micropropagation of a Highly Important Cassia species- A Review

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Advances in Micropropagation of a
Highly Important Cassia species- A Review
M. Anis1,2*, Iram Siddique3, Ruphi Naz1,
M. Rafique Ahmed1 and Ibrahim M. Aref2
1Plant Biotechnology Laboratory, Department of Botany,
Aligarh Muslim University, Aligarh,
2Department of Plant Production, College of Food & Agricultural Sciences,
King Saud University, Riyadh,
3Department of Botany and Microbiology, College of Science,
King Saud University, Riyadh,
1India
2,3Saudi Arabia
1. Introduction
The medicinal properties of plant species have made an outstanding contribution in the
origin and evolution of many traditional herbal therapies. Over the past few years, however,
the medicinal plants have regained a wide recognition due to an escalating faith in herbal
medicine in view of its lesser side effects compared to allopathic medicine in addition the
necessity of meeting the requirements of medicine for an increasing human population.
With an ever increasing global inclination towards herbal medicine for healthcare and their
boom in recent years has imposed a great threat to the conservation of natural resources and
endangered plant species. Currently 4,000-10,000 medicinal plants are on the endangered
species list and this number is expected to increase (Canter et al., 2005). Most of the
pharmaceutical industry is highly dependent on wild population for the supply of raw
material for extraction of medicinally important compounds. The genetic diversity of
medicinal plants in the world are getting endangered at an alarming rate because of ruinous
harvesting practice and over-harvesting for production of medicines, with little or no regard
to the future. Also, extensive destruction of the plant-rich habitat as a result of forest
degradation, agriculture encroachments, urbanization, etc. is other factors.
In modern medicine, plants are used as sources of direct therapeutic agents, as model for
new synthetic compounds and as a taxonomic marker for the elaboration of more complex
semisynthetic chemical compounds (Akerele, 1992). Wide variations in medicinal quality
and content in phytopharmaceutical preparations have been observed. These are influenced
mainly by cultivation period, season of collection, plant- to- plant variability in the
medicinal content, adulterants of medicinal preparations with misidentified plant species, a
lack of adequate methods for the production and standardization of the plants, a lack of
* Corresponding Author

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understanding of the unique plant physiology or efficacy with human consumption and
consumer fraud. Generally, herbal preparations are produced from field- grown plants and
are susceptible to infestation by bacteria, fungi and insects that can alter the medicinal
content of the preparations (Murch et al., 2000). Also there is significant evidence to show
that the supply of plants for traditional medicines is failing to satisfy the demand
(Cunningham 1993). An efficient and most suited alternative solution to the problems by the
phytopharmaceutical industry is development of in vitro systems for the production of
medicinal plants and their extracts.
2. Role of biotechnology
Biotechnology involves modern tissue culture, cell biology and molecular biology offers the
opportunity to develop new germplasms that are well adapted to changing demands.
Biotechnological tools are also equally important for multiplication and genetic
enhancement of the medicinal plants by adopting various techniques such as in vitro
regeneration and genetic transformation. It can also be harnessed for the production of
secondary metabolites using plant as bioreactors (Tripathi and Tripathi, 2003). In addition,
modern biotechnology is being increasingly applied for plant diversity characterization and
they have a major role in assisting plant conservation programmes.
3. Plant tissue culture
In recent years, tissue culture has emerged as a promising technique for culturing and
studying the physiological behaviour of isolated plant organs, tissues, cells, protoplasts and
even cell organelles under precisely controlled physical and chemical conditions. Tissue
culture can be divided into three broad categories. The most common approach is to isolate
organised meristems like shoot tips or axillary buds and induce them to grow into complete
plants (Fig1A-F). This system of propagation is commonly referred to as micropropagation. In
the second approach, adventitious shoots are initiated on leaf, root and stem segments or on
callus derived from those organs. The third system of propagation involves induction of
somatic embryogenesis in cell and callus cultures. The commercial technology is primarily
based on micropropagation, in which rapid proliferation is achieved from tiny stem cutting,
axillary buds and to a limited extent from somatic embryos, cell clumps in suspension cultures
and bioreactors. This technique is being used for large scale propagation of a number of plant
species viz. Rauvolfia tetraphylla (Faisal et al., 2005), Tylophora indica (Faisal and Anis, 2003),
Vitex negundo (Ahmad and Anis, 2007), Pterocarpus marsupium (Husain et al., 2007), Mucuna
pruriens (Faisal et al., 2006), Balanites aegyptiaca (Siddique and Anis, 2009). Although there are a
number of reviews published on micropropagation of medicinal plants, they do not provide
the comprehensive micropropagation reports on Cassia species. In this way, the present review
highlighted in vitro regeneration of medicinally important Cassia species, their significance and
the wide scope existing for investigations on mass cloning of these plants.
3.1 Cassia angustifolia Vahl. (Fabaceae)
Cassia angustifolia Vahl. is a small medicinal shrub commonly known as senna, a valuable
drought resistant plant. It is mainly grown as a cash crop in various parts of the world
(Anonymous, 1992).The leaves and pods of senna are chief source of anthraquinone,
glycoside known as sennosides, which are extensively used as a laxative. It is also used as a

Advances in Micropropagation of a Highly Important Cassia species- A Review
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febrifuge in splenic enlargements, anaemia, typhoid, cholera, biliousness, jaundice, gout,
rheumatism, tumours, foul breath and bronchitis and in leprosy (Pulliah, 2002). It is
employed in the treatment of amoebic dysentery as an anthelmintic and as a mild liver
stimulant. Poor seeds viability and low germination frequency restricts its propagation on a
large scale. Therefore, micropropagation appears to be an alternative method in order to
meet the demand for commercial production of this medicinal plant.
Fig. 1. (A-F). A. Shoots induction in Cassia occidentalis on MS medium containing BA. B.
Shoot induction in C. alata. C. Multiple shoot induction from cotyledonary node explant of
C. angustifolia D. Multiplication and elongation of shoots from cotyledonary node explant of
C. occidentalis E& F. Shoot proliferation in C. angustifolia and C. alata.

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3.1.1 Explant type
Multiple shoots were developed successfully from different explants (Cotyledonary node,
nodal and shoot tips) excised from in vitro grown seedlings of C. angustifolia (Agrawal and
Sardar 2003; Siddique and Anis 2007 a, b). Among the various explants, cotyledonary node
gave the best response (Agrawal and Sardar, 2003), (Siddique and Anis, 2007a, b). In
addition, plant regeneration has been successfully developed by using root explants
(Parveen and Shahzad 2011). Root explants are advantageous over other explants in terms of
their easy manipulation, higher regeneration potential and excellent susceptibility for
Agrobacterium transformation (Knoll et al., 1997).
In the second approach a much faster rate of multiplication has been obtained through
indirect organogenesis. Different explants (Cotyledons, leaflets and petiole) excised from
axenic seedlings were used for inducing organogenic callus. Agrawal and Sardar (2006)
used cotyledons and leaflets for in vitro propagation. Cotyledons were more responsive
where 91% cultures produced about 12 shoots per explant. Siddique et al. (2010) used
petiole for indirect shoot organogenesis in C. angustifolia.
3.1.2 Growth regulators
Plant growth regulators play an important role in growth and development (Little and
Savidage, 1987). A range of auxins and cytokinins played a vital role in multiple shoot
regeneration in many Cassia species. MS medium with optimal quantity of cytokinins (BA,
Kn, 2-iP or TDZ) is required for shoot proliferation in many genotypes but inclusion of low
concentration of auxins along with cytokinin triggers the rate of shoot multiplication (Tsay
et al., 1989). BA individually within the range of 0.5-10.0 µM was common in most of the in
vitro micropropagated plants. According to Agrawal and Sardar (2003) 1.0 µM 6-
benzyladenine (BA) was found best to induce multiple shoots in CN. However, at higher
concentration of BA (10.0 µM), a decreasing trend in response in terms of percentage
responding explants, average shoot number per explants as well as average shoot length
was seen. Also, CN gave the best response in MS medium fortified with 1.0 µM thidiazuron
(TDZ). In case of nodal explants best result was obtained with 5.0 µM TDZ and 1.0 µM
Indole-3-acetic acid (IAA). Further, transfer of shoot clusters in hormone free MS medium
considered to increase the rate of multiplication (Siddique and Anis, 2007a, b). TDZ has also
been successfully used to induce shoot bud in root explants. To avoid adverse effect of TDZ,
culture were transferred to shoot regeneration medium, where 2.5 µM BA + 0.6 µM NAA
gave the maximum response (Parveen and Shahzad, 2011).
In cotyledons and leaflets explants, multiple shoots were observed when green,
morphogenic callus (1.0 µM 2.4-D + 1.0 µM BA) was transferred to BA + NAA (Agrawal and
Sardar 2006). In case of petiole, highest number of shoots and shoot length was recorded on
MS medium along with 5.0 µM TDZ and 1.5 µM IAA (Siddique et al., 2010).
3.1.3 Somatic embryogenesis
Somatic embryogenesis is the most striking confirmation of totipotency, it 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 grow into seedlings on suitable medium. Types of

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auxins and its interaction with cytokinins significantly influenced on somatic
embryogenesis. Plant regeneration via somatic embryogenesis from single cells, that can be
induced to produce an embryo and then complete plant, has been demonstrated in many
plant species (Wann et al. 1987). In Cassia angustifolia, direct and indirect somatic embryos
were observed on MS medium supplemented with auxin alone or in combination with
cytokinins (Agrawal and Sardar, 2007). Efficient development and germination of somatic
embryos are prerequisites for commercial plantlet production.
Besides this, antimutagenic and genotoxic potential of senna have been reported by Silva et
al., (2007). Hence it is a commercially important medicinal plant which has diverse
medicinal applications, there is pressing need to conserve the plant by in situ and ex situ
multiplication in general and micropropagation.
3.2 Cassia siamea Lam
Cassia siamea Lam. (Caesalpiniaceae), is an evergreen tree, commonly planted as an avenue
and shade tree in tea estates and found useful for afforestation of degraded and wastelands
where organic manure is deficient. It decreases soil erosion, while improving soil fertility in
the plantation site, well adapted to a variety of climatic conditions within the tropics and
highly resistant to drought. The anthraquinones and cassiamin B present in the plant is an
antitumour promoting and chemopreventing agent (Sastry et al., 2003). The root and bark is
used in folklore medicine to treat stomach complaints and as a mild purgative. Therefore, a
systematic propagation of this valuable tree is important.
3.2.1 Explant type
Various explants, shoot tip, cotyledonary node and nodal segments excised from in vitro
raised seedlings were used for multiplication. Maximum response was observed with nodal
segments in MS macro salts + B5 micro salts (Sreelatha et al., 2007). In contrast, according to
Parveen et al., (2010), cotyledonary node gave the best response in MS medium
supplemented with BA and NAA. Gharyal and Maheshwari (1990) used stem and petiole
for in vitro propagation. Gharyal et al., (1983) used C. siamea for androgenesis also.
3.2.2 Growth regulators
Sreelatha et al., (2007) used the different medium MS, B5 and MS macro salts + B5 micro
salts with various hormones alone or in combinations. 0.1mg/l Kn + 0.1 mg/l TDZ + 2.0
mg/l 2-iP gave the best response on MS macro salts + B5 microsalts in nodal explants.
Parveen et al., (2010) found that MS medium augmented with 1.0 µM BA + 0.5 µM NAA
was the best medium for multiple shoot regeneration in CN explants. B5 basal medium
supplemented with 0.5 mg/l IAA + 1 mg/l BA gave the best response in stem segments
(Gharyal and Maheshwari 1990).
3.3 Cassia sophera Linn
Cassia sophera Linn (Fabaceae) is an important medicinal plant. The whole plant extracts and
leaves have expectorant properties, cures cough, asthma and acute bronchitis, anticancer
and anti-inflammatory properties. They are specific to eliminate ring worms and also useful

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in the treatment of gonorrhoea and syphilis. The bark is used in the treatment of diabetes;
wounds and ascites (Anonymous, 1992). The root is administered internally with black
pepper for snake bite. The seed extract exhibited important pharmacological effects like
analgesic, hypnotic, sedative and antiepileptic effects (Bilal et al., 2005). Nature has provided
us a rich store house of herbal remedies to cure most diseases. In vitro regeneration is the
best alternative to overcome these hurdles. Conventionally C. sophera can be propagated
through seeds. The seeds however remain viable for a short period and germinated poorly.
Because of wide spectrum of its medicinal properties, Parveen and Shahzad (2010) has
developed a protocol for rapid multiplication of this valuable plant through cotyledonary
node, excised from in vitro raised seedlings.
3.3.1 Explant types and growth regulators
Cotyledonary node explants were cultured on Murashige and Skoog medium (MS)
supplemented with thidiazuron (TDZ, 0.1 -10.0 µM). 2.5 µM TDZ proved to be optimal for the
production of maximum number of shoots. For further multiplication and elongation, shoot
clusters were transferred to various concentrations of BA. 1.0 µM BA showed better response.
3.4 Cassia fistula Linn
Cassia fistula Linn. (Caesalpiniaceae) commonly known as ‘Indian Laburnum’ has been
extensively used in Ayurvedic system of medicine for various ailments. The whole plant
possesses medicinal properties useful in the treatment of skin diseases, rheumatism,
anorexia, jaundice, antitumour, antiseptic and antimicrobial (Kirtikar and Basu 1991) and
antifungal activity (Gupta, 2010). It possesses hepatoprotective, anti-inflammatory and
antioxidant activities (Ilavarasan et al., 2005). Both the leaves and pods were widely used in
traditional medicine as strong purgative and laxatives (Kirtikar and Basu 1975, Elujoba et
al., 1999) due to presence of sennoside (Van, 1976). In Ayurvedic medicinal system, it was
used against various disorders such as pruritus, leucoderma, diabetes and other ailments
(Satyavati and Sharma 1989, Alam et al.,1990, Asolkar et al.,1992). Leaves were also found
effective against cough and ring worm infections (Chopra et al., 1956, Biswas and Ghose
1973). It is also used in treating bone fracture (Ekanayak 1980). Kuo et al., (2002) have
isolated and identified oxyanthraquinones, chrysophenol and chrysophanein from the
seeds. Extensive studies have been carried out on its medicinal values and the synergistic
actions. Patel et al., (1965) reported analgesic and antipyretic action. Mazumdar et al., (1998)
reported sedative and analgesic action of C. fistula seeds. Gupta and Jain (2009) have
reported hypolipidemic activity of this important legume. It has also been reported for anti-
inflammatory (Suwal, 1993), hypoglycaemic activity (Alam et al., 1990; Esposito et al., 1991),
antiperiodic (Kashiwada et al., 1990), anti rheumatic (Suwal, 1993), anti-tumor (Bodding,
1983; Gupta et al., 2000), hepato-protective (Bhakta et al., 1999), antioxidant (Luximon
Ramma et al., 2002; Sidduraju et al., 2002), anti fungal and anti bacterial activities (Patel and
Patel, 1956; Ramakrishna and Indragupta, 1997; Dhar and Qasba, 1984; Perumal et al., 1998).
3.4.1 Explant type and growth regulators
There are only few reports available for in vitro regeneration of C. fistula. Gharyal and
Maheshwari (1990) used the stem and petiole for shoot regeneration. Stem and petiole were

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cultured on B5 basal medium supplemented with 2 mg/l NAA + 0.5 mg/l BA (medium a)
or 0.5 mg/l IAA + 1.0 mg/l BA (medium b). Medium b gave the best response where well
differentiated shoots were developed.
3.5 Cassia obtusifolia L Syn Cassia tora
It is also an important medicinal plant. The seeds are effective for insomnia, headache,
constipation, oliguria, cough, opthalmia, dacryoliths, omblyopia and hypertension (Purohit
and Vyas, 2005). The roots extract contain tannins, flovonoids, alkaloids (Olabiyi et al.,
2008), betulinic acid, chrysophanol, physcion, stigmasterol and aloe-emodin (Yang et al.,
2006). Doughari et al., (2008) reported that leaf extracts contain the activity against both
gram positive and gram negative bacteria and fungi that can therefore be employed in the
formulation of antimicrobial agents for the treatment of various bacterial and fungal
infections including gonorrhea, pneumonia, eye infections and mycotic infections. Also
Joshi (2000) reported that the plant extract is antiviral, spasmolytic and diuretic used against
epilepsy, scabies and sores.
3.5.1 Explants and growth regulators
Hasan et al., (2008) used shoot tips for callus induction and shoot regeneration. Shoot tips
were cultured in MS medium supplemented with different concentrations and combinations
of 2,4-D and Kn. 2.0 mgl-1 2,4-D + 0.2mgl-1 Kn were found best for shoot induction as well as
elongation.
3.6 Cassia occidentalis Linn
Cassia occidentalis (Linn) (Caesalpiniaceae) commonly known as Coffee Senna. It is an
ayurvedic plant with huge medicinal importance. It is used for fever, menstrual problems,
tuberculosis, diuretic, anemic, liver complaints ( Kritikar and Basu 1999). This weed has
been known to possess antifungal and anti-inflammatory activity. An infusion of the bark is
given in diabetes (Anonymous 1998). Leaf extracts have antibacterial (Jain et al., 1998 and
Saganuwan and Gulumbe 2006), antimalarial (Arya et al., 2010), antimutagenic (Tona et al.,
1999 and Jafri et al., 1999), antiplasmodial (Sharma et al., 2000), anticarcinogenic (Tona et al.,
2004) and hepatoprotective (Sharma et al., 2000) and analgesic and antipyretic (Sini et al.,
2010) activity. A wide range of chemical compounds including achrosin, aloe-emodin,
emodin, anthraquinones, anthrones, apigenin, aurantiobtusin, campesterol, cassiollin,
chryso-obtusin, chrysophanic acid, chrysarobin, chrysophanol, chrysoeriol etc. have been
isolated from this plant. Further, micropropagation of C. occidentalis Linn is being conducted
in tissue culture laboratory of Botany department at AMU Aligarh.
3.7 Cassia alata L.
Cassia alata L. (Fabaceae) commonly known as Ringworm Bush is an erect medicinal shrub
or small tree distributed mainly in the tropics and subtropics. The plant is a source of
chrysoeriol, kaempferol, quercetin, 5,7,4'-trihydroflavanone, kaempferol-3-O-β-D-
glucopyranoside, kaempferol-3-O-β-D-glucopyranosyl-(1->6)-β-D-glucopyranoside, 17-
hydrotetratriacontane, n-dotriacontanol, n-triacontanol, palmitic acid ceryl ester, stearic

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acid, palmitic acid (Liu et al., 2009). C. alata leaf is also credited for the treatment of
haemorrhoids, constipation, inguinal hernia, intestinal parasitosis, blennorrhagia, syphilis
and diabetes (Abo et al., 1998; Adjanahoun et al., 1991). The flowers and leaves are used for
the treatment of ringworms and eczema. The other uses of C. alata are as an antihelminthic,
antibacterial, laxative, diuretic, for treatment of snakebites and uterine disorders (Kirtikar
and Basu, 1975). Besides the leaf extract of this species has shown several pharmacological
properties such as antimicrobial, antifungal (Khan et al., 2001), antiseptic (Esimone et al.,
2008), anti-inflammatory, analgesic (Palanichamy and Nagarajan, 1990) and anti-
hyperglycemic activities (Palanichamy et al., 1988). It contains therapeutic (Damodaran and
Venkataraman, 1994) and anti-ageing activities (Pauly et al., 2002) also.
3.7.1 Explant and growth regulators
FettNeto et al., (2000) used cotyledonary node along with one third of the hypocotyl and
cotyledons. The best result was obtained on 0.5 micro MS salts + 0.38 mgl-1 of BA and 0.005
mgl-1 IBA.
3.8 Root development and acclimatization
The induction of roots in vitro is an important step in plant micropropagation and genetic
transformation. In vitro root induction from growing shoots has been achieved in standard
media containing auxin and in media in the absence of auxin depending on plant genotype
(Rout et al., 1989) (Fig. 2 A-C). There is marked variation in the rooting potential of different
plant species and systematic trials are often needed to define the conditions required for
root induction. Agrawal and Sardar (2006) examined the effectiveness of various auxins on
rooting of C. angustifolia microshoots and found that 10.0 µM IBA was superior to IAA or
NAA. However 200 µM IBA was best for ex vitro rooting in C. angustifolia (Parveen and
Shahzad 2011). According to Parveen et al., (2010) 2.5µM IBA gave the maximum roots in C.
siamea. Sreelatha et al., (2007) reported that NAA (1.0 mg/l) + IBA (0.25 mg/l) produced
long and well developed roots in C. siamea. 0.1 mg/l IAA exhibited the positive effect on
root induction in C. fistula (Gharyal and Maheshwari 1990).
Prolific rooting on in vitro grown microshoots is critical for the successful establishment of
these shoots in the greenhouse or field. Plantlets were developed within the culture vessels
under low level of light, aseptic conditions, on a medium containing sugar and nutrients to
allow for heterotrophic growth and in an atmosphere with high level of humidity. These
contribute a culture- induced phenotype that cannot survive the environmental conditions
when directly placed in a greenhouse or field. The physiological and anatomical
characteristics of micropropagated plantlets necessitate that they should be gradually
acclimatized to the environment of the greenhouse or field (Fig 2 D, E). In C. siamea,
Sreelatha et al., (2007) reported that when micropropagated plantlets were transferred to
pots containing (3:1) vermiculite: sand under greenhouse conditions, about 40 % of the
plants survived. A high survival 85 % was recorded when plantlets of C. siamea were
transplanted into 1:1 sterilized garden soil and garden manure (Parveen et. al., 2010).
Siddique and Anis (2007) noted the highest survival of C. angustifolia when the plants were
maintained inside the growth room in sterile soilrite for 4 weeks and eventually transferred
to natural soil. Approximately 70 % of rooted plants of C. obtusifolia survived in pots
containing a 1:1:1 mixture of sterile sand, soil and farmyard manure (Hasan et al., 2008).

Advances in Micropropagation of a Highly Important Cassia species- A Review
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Plant name Explant Media/
Adjuvant
PGRs
used
Response Optimal
response
No of
shoots
References
Cassia
angustifolia
CN, N,
St
MS BA, Kn Direct and
Indirect
1.0µM BA 2 Agrawal and
Sardar, (2003)
C.an
g
usti
f
olia L, C MS 2,4
-
D,BA,
Kn,NAA
Indirect 5.0 µM BA+
0.5µM NAA
12 Agrawal and
Sardar, (2006)
C. an
g
usti
f
olia N MS BA, TDZ,
IAA,
NAA
Direct 5.0 µM TDZ+
1.0 µM IAA
12 Siddique and
Anis, (2007a)
C. an
g
usti
f
olia CN MS TDZ Direct 1.0 µM TDZ 17 Siddique and
Anis, (2007b)
C. an
g
usti
f
olia C MS 2,4-D,
NAA
,BA,Kn,
2-iP
Zeatin
Somatic
embryo
10.0 µM 2,4-D+
2.5 µM BA or
5.0 µM BA
19 Agrawal and
Sardar
(2007)
C. an
g
ustiolia P MS 2,4-
D,TDZ,
BA, Kn
Indirect 5.0 µM TDZ +
1.5 µM IAA
12 Siddique et al.,
(2010)
C. an
g
usti
f
olia R MS BA, Kn,
TDZ
IAA,
NA
A
Indirect 2.5 µMBA+ 0.6
µM NAA
24 Parveen and
Shahzad
(2011)
Cassia siamea A B5/Cocon
ut
milk
2,4-D,
Kn
Indirect 2mg/l 2,4-D,
0.5mg/l Kn,
15%
coconut milk
Polle
n
Embry-
oids
Gharyal et al.,
(1983)
C. siamea S, P B5/PVP,
PVPP
BA,IAA,
NAA
Indirect 0.5 mg/l IAA
+ 1mg/l BA
Only
green
Meriste
moid
Observe
d
Gharyal and
Maheshwari
(1990)
C. siamea St, CN,
N
MS macro
salt
+ B5 micro
salt
BA,Kn,2-
iP,
TDZ,
NAA,
IBA, IAA
Direct 0.1 mg/l Kn+
0.1mg/l TDZ+
2mg/l 2-iP
20 Sreelatha et al.,
(2007)
C. siamea CN MS BA,Kn,
TDZ
Direct 1.0 µM BA
+0.5 µM NAA
12 Parveen et al.,
(2010)
Cassia so
p
hera CN MS BA, TDZ Direct 1.0 µM BA 14 Parveen and
Shahzad, (2010)
Cassia
f
istula S, P B5/PVP,P
VPP
BA,
NAA,
IAA
Indirect 0.5 mg/l IAA
1.0 mg/l BA
_
Gharyal and
Maheshwari
(1990)
Cassia alata CN
with
H, C
MS macro
+ B5
micro salt
BA,
NAA,
IBA
Indirect 0.38mg/l BA,
0.05 mg/l IBA
Fett Neto et al.,
(2000)
Cassia
obtusifolia
St MS Kn, 2,4-
D
Indirect 2 mg/l 2,4
-
D+
0.2 m
g
/l Kn
5 Hasan et al.,
(2008)
Abbreviations: CN- Cotyledonary node, N- Nodal, St- Shoot tip, L- Leaflet, C- Cotyledon, P- Petiole, R-
Root, A- Anther, S- Stem, H- Hypocotyl
Table 1. In vitro multiplication of different Cassia species

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Fig. 2. (A-E) A In vitro rooting of C. alata B&C. Ex vitro and In vitro rooting in C. occidentalis
D&E. Acclimatized plants of C. alata and C. occidentalis.
4. Conservation strategies used for the propagation
Due to growing demand, the availability of medicinal plants to the pharmaceutical
companies is not enough to manufacture herbal medicines. There is need to conserve the
economically important plants. Tissue culture techniques have been used as tools for
germplasm conservation of rare or threatened as well as medicinal plants (Zornig, 1996).
The utilization of in vitro techniques for germplasm conservation is of great interest in plant
species (Costa Nunes et al., 2003). The in vitro conservation can be for medium and long

Advances in Micropropagation of a Highly Important Cassia species- A Review
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periods. The conservation for a medium period is done by decreasing the growth of cultures.
The long period conservation is done by cryopreservation techniques (Engelmann, 1998).
The establishment of in vitro germplasm banks in developing countries has great
importance, but these techniques must be associated with other plant genetic resources
conservation practices (Engelmann, 1997). The in vitro conservation techniques allow
material exchanges among germplasm banks and the germplasm keeps its sanitary
conditions and viability during the transport (Ashmore, 1998). The powerful techniques of
plant cell and tissue culture, recombinant DNA and bioprocessing technologies have offered
mankind a great opportunity to exploit the medicinal plants under in vitro conditions.
5. Conclusion and future prospects
The biotechnological strategies have opened up new vistas in all aspects of plant germplasm
characterization, acquisition, conservation, exchange and genetic resource management.
Future prospects are highly encouraging in terms of the development and application of
new techniques and protocols within the context of germplasm conservation. It is useful for
multiplying the species which are difficult to regenerate by conventional methods and save
them from extinction. Further, the technology delivery with effective dissemination
channels has to play a major role in the commercial production of micropropagated plants,
it surely needs to be revived and utilized in a broader spectrum rather than confined to
publications. For instance, adoption of tissue culture technology will have to facilitate the
use of genetically engineered plants as soon as they become available in near future.
Furthermore, technology has always to be understood in a dynamic way. Recent
developments in transgenic plants can have multidirectional benefits. The benefits range
from manipulating generation time, plant protection, wood quality, production of
compounds of pharmaceutical value and improvements to polluted soil.
6. Acknowledgement
Authors are grateful to the University Grants Commission, Govt of India, New Delhi for
providing research support under UGC- SAP (2009) program. The award of a Research
Associateship to I. S. by the Council of Scientific and Industrial Research (CSIR), is gratefully
acknowledged.
7. References
Abo KA, Adediwura AA, Ibikunle AJ (1998) 1st International Workshop on Herbal
Medicinal Products, University of Ibadan, Ibadan, Nigeria.pp. 22 - 24.
Adjanahoun E, Ahyi MRA, Ake-Assi L, Elewude JA, Fadoju SO, Gbile ZO, Goudole E,
Johnson CLA, Keita A, Morakinyo O, Ojewole JAO, Olatunji AO, Sofowora EA
(1991). Contribution to ethnobotanical floristic studies in Western Nig. Pub.
Organization of African Unity: Lagos, Nigeria p.420.
Agrawal V, Sardar PR (2003) In vitro organogenesis and histomorphological investigation in
senna (Cassia angustifolia)- a medicinally valuable shrub.Physiol Mol Biol Plants
9:131-140.
Agrawal V, Sardar PR (2006) In vitro propagation of Cassia angustifolia (Vahl) through leaflet
and cotylerdon derived calli. Biol Plant 1:118-122.

New Perspectives in Plant Protection
202
Agrawal V, Sardar PR (2007) In vitro regeneration through somatic embryogenesis and
organogenesis using cotyledons of Cassia angustifolia (Vahl). In Vitro Cell Dev Biol
Plants 43: 585-592.
Ahmad N, Anis M (2007) Rapid clonal multiplication of a woody tree, Vitex negundo L.
through axillary shoots proliferation. Agroforestry Syst 7:195–200.
Ahmad Z, Ghafoor A (2002) Resource Base and Conservation Strategies of MAPs in
Pakistan. In Sharing Local and National Experience in Conservation of Medicinal
and Aromatic Plants in South Asia. Edited by: Bhattarai N. and Karki M. HMGN,
IDRC and MAPPA. pp: 105-109.
Akerele O (1992) WHO guidelines for the assessment of herbal medicine . Fitoterapia 62:99-
110
Alam MM, Siddique MB, Hussain W (1990) Treatment of diabetes through herbal drugs in
rural India. Fitoterapia 61:240-242.
Anonymous (1992) The Wealth of India: A Dictionary of Indian Raw Materials and
Industrial Products (Vol III). CSIR New Delhi, 366-367
Anonymous (1992) The wealth of India: a dictionary of Indian raw materials and industrials
products, vol 3.CSIR, New Delhi 354-363.
Anonymous (1998), The Wealth of India (1998) A dictionary of Indian raw material and
industrial products. Council of Scientific and Industrial Research New Delhi, pp
350.
Arya V,Yadav S, Kumar S, Yadav JP (2010) Antimicrobial activity of Cassia occidentalis L
(leaf) against various human pathogenic microbes. Life Sci Med Res 1-11.
Ashmore SE (1998) The status of in vitro conservation of tropical and subtropical species.
Proceedings of the Internatonal Symposium on Biotechnology Tropical &
Subtropical Species. Acta Hort 461:477-483.
Asolkar LV, Kakkar KK, Chakre OJ (1992) Second supplement to glossary of India medicinal
plant with active principles. In: Publication and Information Directorate, New
Delhi. CSIR 1:177.
Bhakta T, Mukherjee PK, Mukherjee K, Banerjee S, Mandal SC, Maity TK, Pal M, Saha BP
(1999) Evaluation of hepatoprotective activity of Cassia fistula leaf extract. J
Ethnopharm 66: 277-282.
Bilal A, Khan NA, Ghufran A, Inamuddin (2005) Pharmacological investigation of Cassia
sophera Linn var Pupurea Roxb. Med J Islamic World Ac Sci 15:105-109.
Biswas K,Ghose AB (1973) In Bharatia Banawasasadhi, Calcutta University, Advertisement
of learning, Calcutta 2:336.
Bodding PO (1983) Santhal medicine, Laxmi Janardhan Press, Calcutta. 21.
Canter PH, Thomas H, Ernst E (2005) Bringing medicinal plants into cultivation:
opportunities and challenges for biotechnology. Trends Biotechnol 23:180–185.
Chopra RN, Nayer SL, Chopra IC (1956) Glossary of India medicinal plants. CSIR, New
Delhi 54.
Constabel F (1990) Medicinal Plant biotechnology Planta Med. 56: 421-425.
Costa Nunes EC, Benson EE, Oltramari AC, Araujo PS, Moser JR, Viana AM (2003) In vitro
conservation of Cedrella fissilis Vellozo (Meliacea), a native tree of the Brazilian
Atlantic Forest. Biodiversity and Conservation 12:837-848.
Crockett Co, Guede-Guina F, Pugh D, Vangah-Manda M, Robinson TJ, Olubadewo JO,
Ochillo RF (1992) Cassia alata and the preclinical search for therapeutic agents for
the treatment of opportunistic infections in AIDS patients. Cell Mol Biol 38: 799-
802.

Advances in Micropropagation of a Highly Important Cassia species- A Review
203
Cunningham AB (1993) African medicinal plants: setting priorities at the interface between
conservation and primary health care. People and plant initiative working paper1,
Nairobi: UNESCO.
Damodaran S, Venkataraman S (1994) A study on the therapeutic efficacy of Cassia alata,
Linn. leaf extract against Pityriasis versicolor. J. Ethnopharmacol 42: 19-23.
Dhar DN, Qasba GN (1984) Screening of some plant extract for antifunlal activity against
venturia inaequalis. Sci Cult 50: 209.
Doughari JG, El-mahmood AM, Tyoyina I (2008) Antimicrobial activity of leaf extracts of
Senna obtusifolia L. J Pharm Pharmacol. 2: 7-13.
Ekanayak DT (1980) Plant use in the treatment of skeletal fracture in the indigenous system
of medicine in Srilanka. The Srilanka Forester 14: 145-152.
Elujoba AA, Abere AT, Adelusi SA (1999) Laxative activities of Cassia pods sourced from
Nigeria. Nig J Nat Prod Med. 3: 51-53.
Engelmann F (1997) Present development and use of in vitro culture techniques for the
conservation of plant genetic resources. Acta Hort 447: 471-475.
Engelmann F (1998) In vitro germplasm conservation. Acta Hort 461: 41-47.
Englemann F (2011) Use of biotechnologies for conservation of plant biodiversity, In Vitro
Cell Dev Biol Plant 47: 5-16.
Esimone CO, Nworu CS, Ekong US, Okereke B (2008) Evaluation of the antiseptic properties
of Cassia alata-based herbal soap, The Intern J Alternat Med 6: 23-32.
Esposito AM, Diaz A, De- Gracia I, De- Tello R, Gupta MP (1991) Evaluation of traditional
medicine: effects of Cajanus cajan L and Cassia fistula L on carbohydrates
metabolism in mice Rev Med Panama 16: 39-45.
Faisal M, Ahmad N, Anis M (2005) Shoot multiplication in Rauvolfia tetraphylla using
thidiazuron. Plant Cell Tiss Org Cult 80:187–190.
Faisal M, Anis M (2003) Rapid mass propagation of Tylophora indica Merrill via leaf callus
culture. Plant Cell Tiss Org Cult 75: 125-129.
Faisal M, Siddique I, Anis M (2006) In vitro rapid regeneration of plantlets from nodal
explants of Mucuna pruriens- a valuable medicinal plant. Ann Applied Biol 148: 1-6.
FettNeto AG, Fett J P, Aquila MEA, Ferreira AG (2000) In vitro propagation of Senna alata.
Planta Medica 66:195-196.
Gadgil M, Rao PRS (1998) Nurturing Biodiversity: An Indian Agenda. Center for
Environ.Edu., Ahamdabad, India.
Gharyal PK, Maheshwari SC (1990) Differentiation in explants from mature leguminous
trees. Plant Cell Rep 8: 550-553.
Gharyal PK, Rashid A, Maheshwari SC (1983) Androgenic response from cultured anthers
of a Leguminous Tree, Cassia siamea Lam. Protoplasma 118: 91-93.
Gupta M, Mazumder UK, Rath N, Mukhopadhay DK (2000) Antitumour activity of
methanolic extract of Cassia fistula L. seed against Ehrlich ascites carcinoma. J
Ethnopharm 72: 151-156.
Gupta RK (2010) Medicinal and Aromatic plants, CBS publishers and distributors, Ist edition
116-117.
Gupta UC, Jain GC (2009) Study on hypolipidemic activity of Cassia fistula legume in rats.
Asian J Exp. Sci. 23:241-248.
Hasan MF, Das R, Rahman MS, Rashid MH, Hossain MS, Rahman M (2008) Callus
induction and plant regeneration from shoot tips of Chakunda (Cassia obtusifolia L).
Int J Sustain Crop Prod. 3: 6-10.
Husain MK, Anis M, Shahzad A (2007) In vitro propagation of Indian Kino (Pterocarpus
marsupium Roxb.) using thidiazuron. In Vitro Cell Dev Biol Plants 43: 59-64.

New Perspectives in Plant Protection
204
Hutchinson J, Dalziel JM (1958) Flora of West Tropical Africa, Second Edition, Vol-1, part
2,Crown Agents for oversea Governments and Administrations, London, 450-455.
Ilavarasan R, Mallika M, Venkataraman S (2005) Anti-inflammatory and antioxidant
activities of Cassia fistula Linn barks Extracts. Afr J Trad Comp Alter Med 2: 70-85.
Jafri MA, Subhani MJ, Javed K, Singh S (1999) Hepatoprotective activity of leaves of Cassia
occidentalis against paracetamol and ethyl alcohol intoxification in rats. J
Ethnopharmacol 66: 355-61.
Jafri, S.M.H. (1966) The Book Corporation, Karachi. p. 156.
Jain SC, Sharma RA, Jain R, Mittal C (1998) Antimicrobial screening of Cassia occidentalis L in
vivo and in vitro. Phytotherapy Res 12: 200-204.
Joshi SG (2000) Medicinal plants Oxford & IBH Publishing Co. pvt Ltd. New Delhi, India
122-123.
Kashiwada Y, Toshika K, Chen R, Nonaka G, Nishioka I (1990) Tannin and related
compounds. XCIII. Occurrence of enantiomeric proanthocyanidins in the
leguminosea plant, Cassia fistula L; Cassia javanica L, Chem Pharm Bull 38: 888- 893.
Khan MR, Kihira M, Omoloso AD (2001) Antimicrobial activity of Cassia alata, Fitoterapia.
75, 561-564.
Kiritikar KR, Basu BD (1975) Indian medicinal plants, Vo.l III. Reprint Ed., LN Basu
Allahabad, 856.
Kirtikar KR, Basu BA (1991) Indian medicinal plants. Vol. II. 2nd edition, periodical experts
book agency, New Delhi.277-282.
Kirtikar KR, Basu BD (1935) page 854-879, Vol.1I, published by L.M. Basu.
Knoll KA, Short KC, Curtis IS, Power JB, Davey MR (1997) Shoot regeneration from cultured
root explants of spinach (Spenacia oleracea L): a system for Agrobacterium
transformation. Plant Cell Rep 17:96-101.
Krithikar KR, Basu BD (1999) Cassia occidentalis Indian Medicinal Plants II edition. p. 860.
Kunwar RM, Nepal BK, Kshhetri HB, Rai SK, Bussmann RW (2006) Ethnomedicine in
Himalaya: a case study from Dolpa, Humla, Jumla and Mustang districts of Nepal.
J. Ethnobiol Ethnomedicine 2:27.
Kuo YH, Lee PH, Wein YS (2002) Four new compounds from the seeds of Cassia fistula. J Nat
Prod 65: 1165-1167.
Little CHA, Savidge RA (1987) The role of plant growth regulators in forest tree cambial
growth. Plant Growth Regul 6:137-169.
Liu A, Xu L, Zou Z, Yang S (2009) Studies on chemical constituents from leaves of Cassia
alata. Zhongguo Zhong Yao Za Zhi. 34, 861-863.
Luximon- Ramma A, Bahorun T, Soobrattee MA, Aruoma OI (2002) Antioxidant activities of
phenolic , proanthocyanidins and flavonoid components in extracts of Cassia fistula.
J Agric Food Chem 50:5042-5047.
Mazumdar UK, Gupta M, Rath N (1998) CNS activities of Cassia fistula in mice.
Phytotherapy Res 12: 520-522.
Murch SJ, KrishnaRaj S, Saxen PK (2000) Phyto-pharmaceuticals, mass production,
standardization and conservation. Herbal Med 4: 39-43.
Nadkarni KM (1954) page 284, 285, 290, 291. V.M. Puranik, G.R. Bhatkal. Publishers,
Bombay.
Nalawade SM, Tsay HS (2004) In vitro propagation of some important chinese medicinal
plants and their sustainable usage, In Vitro Cell Dev Biol Plants 40: 143-154.
Olabiyi TI, Oyedunmade EEA, Idikunle GJ, Ojo OA, Adesiva GO, Adelasoye KA,
Ogunniran TA (2008) Chemical composition and Bio-Nematicidal potential of some

Advances in Micropropagation of a Highly Important Cassia species- A Review
205
weed extracts on Meliodogyne incognita under laboratory conditions. Plant Sci Res
1:30-35.
Palanichamy S, Nagarajan S (1990) Analgesic activity of Cassia alata leaf extract and
kaempferol 3-Osophoroside. J. Ethnopharmacol 29: 73-78.
Palanichamy S, Nagarajan S, Devasagayam M (1988) Effect of Cassia alata leaf extract on
hyperglycemic rats, J. Ethnopharmacol 22: 81-90.
Parveen S, Shahzad A (2010) TDZ-induced high frequency shoot regeneration in Cassia
sophera Linn. Via cotyledonary node explants.Physiol Mol Biol Plants 16: 201-206.
Parveen S, Shahzad A (2011) A micropropagation protocol for Cassia angustifolia Vahl. from
root explants. Acta Physiol Plant 33: 789-796.
Parveen S, Shahzad A, Saema S (2010) In vitro plant regeneration system for Cassia siamea
Lam., a leguminous tree of economic importance. Agroforest Syst 80: 109-116.
Patel D, Karbhari D, Gulati D, Gokhale D (1965) Antipyretic and analgesic activities of
Aconatum spicatum and Cassia fistula. Pharm Biol 157: 22-27.
Patel RP, Patel KC (1956) Antibacterial activity of Cassia fistula. Indian J Pharm 18: 107-110.
Pauly G, Danoux L, Contet-Audonneau JL (2002) Method for analysing the ageing of the
skin by inducing apoptosis in cell cultures and screening the effects of cosmetic
substances. PCT Int. Appl. 8pp, Coden:PIXXXD2WO 0221127 A2 20020314.
Perumal R, Samy S, Igganacimuthu S, Sen A (1998) Screening of 34 medicinal plants
antibacterial properties. J Ethnopharm 62: 173-182.
Pulliah T (2002) Medicinal plants in India. Regency Publ New Delhi India. 137-139.
Purohit SS, Vyas SP (2005) Medicinal plant cultivation- A Scientific Approach. Shyam
printing press, Jobhpur, India. p. 348-349.
Ramakrishna R, Indra G (1997) A note on the antifungal activity of some indigenous plants.
Indian J Anim Sci 47: 226-228.
Rout GR, Samantary S, Das P (2000) In vitro manipulation and propagation of medicinal
plants. Biotechnol Adv 18: 91-120.
Saganuwan AS, Gulumbe ML (2006) Evaluation of in vitro antimicrobial activities and
phytochemical constituents of Cassia occidentalis. Animal Research International 3:
566-569.
Sarasan V, Cripps R, Ramsay MM, Atherton C, McMichen M, Prendergast G, Rowntree JK
(2006) Conservation in vitro of threatened plants—progress in the past decade. In
Vitro Cell Dev Plants 42: 206–214.
Sastry BS, Sreelatha T, Suresh Babu K, Madhusudhana Rao J (2003) Phytochemical
investigation of Cassia siamea Lam. National seminar on biodiversity conservation
and commercial exploitation of medicinal plants, Department of Botany. Osmania
University, Hyderabad. 39-40.
Satyavati GV, Sharma M (1989) In: medicinal plants in India. ICMR New Delhi.
Sharma N. Trikha P, Athar M, Raisuddin S (2000) In vitro inhibition of carcinogen induced
mutagenicity by Cassia occidentalis and Emblica officinalis. Drug and Chemical
Toxicol. 23: 477- 84
Shrestha PM, Dhillion SS (2003) Medicinal Plant Diversity and Use in the Highlands of
Dolakha District, Nepal. J Ethnopharmacol 86: 81-96.
Siddhuraju P, Mohan PS, Becker K (2002) Studies on the antioxidant activity of Indian
Laburnum (Cassia fistula L.); a preliminary assessment of crude extracts from stem
bark, leaves, flowers and fruit pulp. J Agric Food Chem 79: 61-67
Siddique I, Anis M (2007a) High frequency multiple shoot regeneration and plantlet
formation in Cassia angustifolia (Vahl) using thidiazuron. Medicinal and Aromatic
Plant Sci Biotech 1:282-284.

New Perspectives in Plant Protection
206
Siddique I, Anis M (2007b) In vitro shoot multiplication and plant regeneration from nodal
explants of Cassia angustifolia (Vahl): a medicinal plant. Acta Physiol Plant 29: 233-
238.
Siddique I, Anis M (2009) Direct plant regeneration from nodal explants of Balanites
aegyptiaca L.(Del) ; a valuable medicinal tree. New Forests. 37: 53-62.
Siddique I, Anis M, Aref IM (2010) In vitro adventitious shoot regeneration via indirect
organogenesis from petiole explants of Cassia angustifolia Vahl. a potential
medicinal plant. Appl Biochem Biotechnol 162: 2067-2074.
Silva CR, Monterio MR, Rocha HM, Ribeiro AF, Caldeira-de- Araujo A, Leitao AC, Bezerra
RJAC and Padula AC (2007) Assessment of antimutagenic and genotoxic potential
of Senna (Cassia angustifolia Vahl) aqueous extract using in vitro assays. Toxicol. In
Vitro 20: 212-218.
Sini KR, Karpakavalli M, Sangeetha PT (2010) Analgesic and Antipyretic activity of Cassia
occidentalis Linn. World App Sci J 11: 1216-1219.
Sreelatha VR, Prasad PJN, Karuppusamy S, Pulliah T (2007) Rapid micropropagation of
Cassia siamea .Plant Cell Biotech Mole Biol 8:173-178.
Srivastava J, Lambert J, Vietmeyer N (1995) Medicinal plants; an expanding role in
development. World Bank technical paper No. 320. Washington DC. World Bank
Agriculture and Forestry Systems.
Suwal, P.N. (1993) Medicinal plants of Nepal. 4th Ed., His Majesty’s Government of Nepal,
P 30-33.
Tona L, Cimanga RK, Mesia K, Musuamba CT, Bruyne T D, Apers S, Hernans N, Miert SV,
Pieters L, Totte J Vlietinck AJ (2004) In vitro antiplasmodial activity of extracts and
fractions from seven medicinal plants used in the Democratic Rebublic of Congo. J
Ethnopharmacol. 93: 27-32.
Tona L, Ngimbi NP, Tsakala M, Mesia K, Cimanga K, Apers S, Bruyne TD, Pieters L, Totte J
Vlietinc AJ (1999) Antimicrobial activity of 20 crude extracts from nine African
medicinal plants used in Kinshasa Congo. J Ethnopharmacol 68: 193-203.
Tripathi L, Tripathi JN (2003) Role of biotechnology in medicinal plants. Tropical of Pharma
Res 2:243-253.
Tsay HS, Gau TG, Chen CC (1989) Rapid clonal propagation of Pinellia ternata by tissue
culture. Plant Cell Rep. 8: 450-454
Van OFHL (1976) Some aspects of the pharmacology of anthraquinone drugs.
Pharmacology (supl). 14:18-29.
Vieira RF, Skorupa LA (1993) Brazilian medicinal plants gene bank. Acta Hort 330: 51-58.
Wann SR, Johnson MA, Noland TL, Carlson JA (1987) Biochemical differences between
embryogenic and nonembryogenic cells of Picea abies (L) Karnst. Plant Cell Rep 6:
39-42.
Yang S, Guo H, Guo D, Zheng J (2006) Studies on chemical constituents of hairy root of
Cassia obtusifolia. J Chinese Materia Medica 31: 217-219.
Zornig R K (1996) Micropropagation of bromeliads. Revista da Sociedade Brasileira
deBromeliads 3: 3-8.