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 Indian Journal of Tropical Biodiversity
© Society for Promotion of Tropical Biodiversity, Jabalpur

IN VITRO PROPAGATION OF GARCINIA TRAVANCORICA – AN ENDEMIC AND

ENDANGERED TREE SPECIES OF WESTERN GHATS, INDIA

RAMASUBBU RAJU*, MANIKANDAN GURUSAMY AND SASIKALA NAMBI

Department of Biology
The Gandhigram Rural Institute – Deemed University,
Gandhigram, Dindigul, Tamil Nadu, India.
*Corresponding author: racprabha@gmail.com

ABSTRACT: The current study focused on conservation through in vitro propagation of Garcinia travancorica Bedd., an
endemic and endangered tree species of Western Ghats India. Shoots were initiated from nodal explants on Murashige
and Skoog (MS) medium supplemented with 4.0 mg/l BAP with major combination of activated charcoal. Multiple shoots
and shoot elongation were achieved in MS medium with the combination of 4.0mg/l BAP and 1.0 mg/l of α-Naphthalene
acetic acid (NAA). Individual shoots with a minimum of one node were excised and rooted in vitro on half strength basal
medium supplemented with 0.5-2.5 mg/l of IAA and IBA transferred to the mist house for acclimatization.
Key words: Garcinia; Agasthyamalai; BAP; hydroxy citric Acid; conservation
Abbreviations: IAA - Indole acetic acid; Kn - Kinetin; BAP - Benzylaminopurine; IBA -Indole buteric acid; NAA - α - Naphthalene
acetic acid.
Citation: Ramasubbu R, Manikandan G, Sasikala N (2016) In vitro propagation of Garcinia travancorica – an endemic and
endangered tree species of Western Ghats, India. Indian J Trop Biodiv 24(1): 64-69

Received on : 26 Oct. 2015

Accepted on : 07 Mar. 2016
Published on : 30 Jun. 2016

The genus Garcinia is represented by 35 species in
India, many of which are endemic and economically
important with immense medicinal properties. Garcinia
travancorica Bedd. is an endangered tree species
locally known as Malampongu belongs to the family
Clusiaceae (Guttiferae). This little known tree has been
distributed in the restricted patches of high altitude
forest areas (2000-2500m asl) of Agasthyamalai
Biosphere Reserve of Southern Western Ghats of India.
The habitat of the tree is reported as one among the 25
globally identified biodiversity hotspots, recognized as
one of the five important centers of plant diversity in
India and also as one of the 24 microcenters of
endemism in India. In the forest, the tree appears as
medium-sized, straight stemmed with horizontal
branches. The trees were overexploited for several
commercial (gum resin) or medicinal products and very
meager number of individuals alone existing in the
natural forest areas. Yellow gamboge, a gum-resin
obtained from this plant is used as an ointment, as a
yellow dye, as an illuminant and in varnishes, water
colour preparation etc. (Uphof, 1959). Garcinia is also a
major source for a natural diet ingredient Hydroxy Citric
Acid (HCA) having anti obesity properties. Gracinia
atroviridis, G. dulcis and G. cowa have reported with high
concentration of HCA, particularly in the fruits and leaves
which has been shown to inhibit ATP dependent citrate
lyase, a key enzyme in diverting carbohydrate to fatty
acid and for the synthesis of cholesterol (Lewis and
Neelakantan, 1965). Other than these compounds the
essential oil of leaves of G. travancorica contain larger
quantities of bioactive compounds like 5,9,13
Pentadecatrien 2 one, 6,10,14 trimethyl, (E,E) and
5,9,13Pentadecatrien2one, and minimal amount of
6,10,14 trimethyl Nerolidyl acetate 1, 6, 10
D o d e c a t r i e n 3 o l , 3 , 7 , 11 t r i m e t h y l , ( E ) 2 , 6 , 1 0
Dodecatrien1ol, 3,7,11trimethyl in the essential oils
(Ramasubbu, 2015- unpublished data).
Since, the trees have polygamo dioecious habit in
which male and female flowers developed in two
different branches of trees at different time intervals.
Therefore, the sexual life between male bisexual and
female flowers is not possible in all times and the
distribution is also restricted to few square kilometers.
The distributional range and number of mature individual
particularly trees with female flowers were very low. The

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 Indian Journal of Tropical Biodiversity, 24(1) 2016
previous studies on population analysis on G.
travancorica indicated that, the extent of occurrence
was estimated to about less than 50 km2 and the area of
occupancy was restricted to less than 10 km2. The
populations are severely fragmented and exist in less
than 10 locations (Manikandan and Ramasubbu, 2013).
The habitat of the species is being altered due to
extension of tea estates by private companies and also
by raising commercial plantations by the forest
department. It was also observed that there were no
sub-populations in the study area. The number of
(mature individuals) individuals which produce new
recruits and individuals having reproducing units within
the populations was reported as ±227 in the entire
distributional areas. There is an extreme fluctuation
observed in every year in case of populations and also
in the number of individuals due to the disturbance in the
forest ecosystem (Manikandan and Ramasubbu,
2013). Garcinia travancorica is a strict endemic species
to Agasthyamalai region and categorized under
Endangered (IUCN Red List of Threatened species,
Version 2012.2). Due to lack of awareness, coupled with
habitat destruction is leading to genetic erosion of this
forest resource. By considering this situation, to
increase the number of trees among the population of
G. travancorica, the present study has been attempted
to develop a simple and efficient method for plant
regeneration from the shoot tip, nodal segments, flower
buds and leaf explants through tissue culture.
Therefore, the protocol developed offers a scope for
large scale production and conservation of the tree
species.
MATERIALS AND METHODS
Collection of Explants
Seedlings of G. travancorica Bedd. were collected from
the Upper Kodayar and Muthukzivayal forest areas of
Western Ghats and maintained in the mist house which
has served as a source of explants. For the current
study, non-woody branches from wild trees of G.
travancorica and from young seedlings maintained in
the mist chamber were used as explants. Nodal and
shoot tip explants were excised free from leaves,
trimmed into (2-3 cm) segments comprising a single
node and washed thoroughly with running tap water for
15 min. Then, explants were rinsed thoroughly with
liquid detergent for several times and again rinsed with
running tap water. Explants were also sterilized by
shaking with 1% (v/v) Clorox for 15min. After several
washes in sterile distilled water and the segments were
surface sterilized by using 0.01% (w/v) HgCl2 for 20 min
with continuous shaking and subsequently washed with
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sterile distilled water. Finally, explants were surface
sterilized by using 70% (v/v) ethanol for 10 min and
thoroughly washed (three times, 5 min each) with sterile
distilled water.
Surface sterilized segments were inoculated on the
MS medium supplemented with 3% (w/v) sucrose and
solidified with 0.7% (w/v) agar. The pH of the medium
was adjusted to 5.6-5.8 with 0.1N NaOH or 1% HCl and
autoclaved for 20 min at 121°C under 16h photoperiod of
light intensity at 2000-3000 lux with cool white-
fluorescent lamp. Explants were cultured on MS medium
fortified with 1-10mg/l of BAP or Kn alone and in
combination with 1-5 mg/l of NAA. In addition, activated
charcoal was incorporated to the medium to control the
phenolic exudation and blackening of the medium
explants. Explants were sub-cultured after 21 days
regularly in the fresh media with the same
concentrations of plant growth regulators for further
multiplication and elongation of the shoots. Healthy
elongated shoots were excised and transferred to half
strength MS medium supplemented with various
concentrations of IAA and IBA (0.5 -2.5mg/l) for root
induction. Plantlets with well developed shoot and root
systems were removed from the culture, washed
carefully with tap water and transplanted into plastic pots
(80 mm diameter) containing sterile soil and covered
with transparent polyethylene bags to ensure high
humidity and kept in greenhouse for further
development.
RESULT AND DISCUSSION
In vitro propagation
The present study focused to derive plantlets by using
suitable explants, surface sterilizing agents and growth
regulators. Both wild and mist chamber grown explants
were used for the development of plantlets through in
vitro propagation. The explants attempted from mist
chamber exhibited better results through axillary bud
breakup within fortnight. While, the wild grown explants
showed contamination and necrosis during the phase of
establishment and even the field grown plants which are
always at high risk of internal and external
contamination. At the same time age of explants also
showed a significant effect on bud sprouting. However,
in vitro establishment and multiplication of plantlets
using mature vegetative tissues is difficult in many
tropical tree species (Rajwant et al. 2012). About 65% of
explants were sprouted from seedlings grown in the mist
chamber. Whereas, no response was observed and also
exhibited the fungal contamination after the axillary bud
breakup on explants collected from the wild.
 Indian Journal of Tropical Biodiversity, 24(1) 2016
Nodal explants were exhibited more response
towards the rapid bud break. The frequency of axillary
bud breakup and the rate of multiplication depended on
the type of explant, and the concentration of cytokinin
either alone or in combination. Although Table. 1
elucidates that, Cytokinins and auxin combination
showed the better response in axillary bud breakup.
Exudation of phenolic compounds (Yellow Gamboge)
from the explants also one of the major constrain to
Table 1. Effect of various concentrations of BAP and NAA on axillary bud breakup of Garcinia travancorica Bedd.
(Data scored within 21days, 12 replicates for each treatment, repeated twice)
Hormonal supplements (mg/l)
BAP NAA
4.0 1.0
4.0 1.5
4.0 2.0
4.0 2.5
4.0 3.0
4.0 3.5
4.0 4.0
4.0 4.5
4.0 5.0
Among the different growth hormones tested, no
significance number of shoot-lets obtained and average
response was noted when NAA was combined with
BAP. The maximum number of shoots (5.6±0.3) was
recorded in treatments with 4.0 mg/l of BAP and 1.5 mg/l
of NAA and above 2.5 mg/l of NAA produced minimum
number of shoots per explants. After fortnight of culture
the explants undergone relatively different stages such
as shoot bud breakup and initiation of bud proliferation.
This observation is in accordance with Rahman et al.
(1993) and confirmed that presence of auxin at lower
concentration is favorable for optimal shoot
multiplication in woody plants. Explants formed first
with two shoots per node were sub-cultured regularly
with the same concentration for further shoot
proliferation. Finally, the maximum numbers of shoots
(5.6±0.3) were obtained at the stage of fifth subculture.
On the accordance, Kulkarni and Deodhar (2002)
reported a positive effect of NAA in morphogenetic
responses of seeds of G. indica and leaf explants of G.
quaestia (Farzana et al. 2010). The variable response of
different genotypes and species to auxin supplemented
media may be due to different endogenous levels of
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obtain the multiple shoots from G. travancorica and
phenolic exudation has controlled by adding charcoal
(0.5%) into the medium. The culture was largely
affected by the exudation of yellow gamboge during the
development. Fridborg et al. (1978) also proved that
better growth responses of plant tissues have been
associated with addition of activated charcoal as it
removes inhibitory substances from the growing
tissues.
Response of Days taken for bud
explants (%) break
86.66 08-11
80.00 08-12
80.00 09-13
76.66 11-14
76.66 13-16
73.33 09-12
70.00 15-18
73.33 14-16
70.00 16-19
auxins. The inhibition of shoot formation is due to action
of auxins accumulated at the basal end of the explants.
The plant growth regulators are rarely specific in their
ultimate influence on growth and development. The
responses of cells, tissues and organs in vitro can vary
with cultural conditions, the type of explants and the
genotype. This is because of the considerable variability
existing among genera, species and even cultivars in the
type and amount of hormones required for induction of
morphogenesis (Rajwant et al. 2012) and also auxin
(NAA) decreasing the browning of explants on the
medium (Preece and Compton, 1991).
In addition, different dilutions of cytokinins with basal
medium were assessed for shoot multiplication. Better
response was obtained in the concentration of 4.0 mg/l
BAP which induced a mean value of 2.8±0.7 shoots per
node and almost equal number of shoots (1.6±0.5) were
noted at the same concentration replaced with Kn.
Exposure of explants to higher BAP concentrations
during induction phase may lead to accumulation of
cytokinins which severely inhibits further cell
development and shoot growth (Fridborg et al. 1978).
The effectiveness of BAP in shoot induction and multiple
 Indian Journal of Tropical Biodiversity, 24(1) 2016

shoot regeneration has been reported in G. effectiveness of IBA at lower concentration in

mangostena (Mohammad et al. 2008; Goh et al. 1994; rhizogenesis has been reported in G. mangostena (Ika
Huang et al. 2000). et al., 2008). IAA supplemented medium produced a
Microshoots with an average length with nodes maximum of 3.8±0.4 number of roots at concentration of
were sub-cultured on half strength basal medium 2.0mg/l. The generally accepted view that, the
supplemented with 0.5-2.5 mg/l of IAA or IBA for rooting. superiority of IBA over IAA in in vitro multiplication is due
Rooting was observed within 13–18 days on MS to the relative higher stability. It is supported by Scott et
medium with IBA. The lower concentration of auxin al. (1990) in which IBA was significantly more stable than
facilitated better root formation. Higher numbers IAA at autoclave. The rooted plants were washed free of
(6.1±0.3) of roots were achieved in half strength MS agar, transferred to polybags containing sterilized sand
medium containing 2.0 mg/l of IBA (Table 2). The and soil mixture (1:1) and placed in a mist chamber at
70% relative humidity for hardening.

3.0 BAP
Kn
2.5
Mean of shoots per explant

2.0

1.5

1.0

0.5

0 2 4 6 8 10
Concentration of BAP & Kn (m g/1)

Fig. 1. Effect of BAP and Kn on mean no. of shoots per explant of G. travancorica Bedd. Data analyzed by one-way ANOVA, and
the values were compared within the column and between the columns. At P<5% level the mean values are highly
significant.
B
6.0
5.5
Mean of shoots per explant

5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5 --
4.0+1.04.0+1.54.0+2.04.0+ 2.54.0+ 3.04.0+3.54.0+4.04.0+4.54.0+ 5.0
Concentration of BAP+NAA (m g/1)

Fig. 2. Combined effect of BAP+NAA on mean no. of shoots per explant of G. travancorica Bedd.

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 Indian Journal of Tropical Biodiversity, 24(1) 2016
7.0
6.5
6.0
5.5
Mean no. of roots per node
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
Concentration of IBA & IAA (m g/1)
Fig. 3. Effect of IBA & IAA on mean no. of roots of G. travancorica Bedd. Data analyzed by one-way ANOVA, and the values were
compared within the column and between the columns. At P<5% level the mean values are highly significant.
CONCLUSION
An efficient tissue culture protocol for high frequency
shoot regeneration was developed for the first time
directly from nodal explants. The present study opens
the way to scale-up studies to enhance rapid growth
and that can be used for the propagation and ex situ
conservation of this strict endemic and endangered
species. By using this protocol we could raise clonal
plants and to conserve the germplasm.
ACKNOWLEDGEMENT
We thank University Grants Commission, New Delhi,
India for the financial support to carry out the research
work through Major Research Project (F. No. 42-
924/2013 (SR) dated 22/03/2013).
REFERENCES
Farzana AFR, Bandara RMIEK, Aluwihare PC,
Eeswara JP (2010) In vitro regeneration of shoots
from Garcinia quaestia leaf explants. J Nat. Sci.
Founda. Sri Lanka, 38 (3): 157-162.
Fridborg G, Pedersen M, Landstrom CE, Eriksson J
(1978) The effect of activated charcoal on tissue
cultures, adsorption of metabolites inhibiting
morphogenesis. Physiol. Plant. 43:104-106.
Goh CJ, Lakshmanan P, Loh CS (1994) High frequency
direct shoot bud regeneration from excised leaves
IBA
IAA
1.0 1.5 2.0 2.5
of mangosteen (Garcinia mangostana L.). Plant
Science (Limerick). 101(2): 173-180.
Huang LC, Huang BL, Wang CH, Kuo CI, Murashige T
(2000) Developing an improved in vitro propagation
system for slow-growing species using Garcinia
mangostana L. (mangosteen). In Vitro cell. Dev.
Biol. 36(6): 501-504.
Ika Rostika, Novianti Sunarlim, Ika Mariska (2008)
Micropropagation of Mangosteen. Indonesian J
Agri. 1(1): 28-33.
IUCN. 2012. www.iucn red list.org (Version 2012.2).
Kulkarni MD, Deodhar MA (2002) In vitro regeneration
and hydroxycitric acid production in tissue cultures
of Garcinia indica Choisy. Indian J. Biotech.
1:301–304.
Lewis YS, Neelakandan S (1965) Hydroxy citric acid –
the principal acid in the fruits of Garcinia cambogia.
Phytochemistry 4: 619.
Manikandan G, Ramasubbu R (2013) Population
degradation, seed predation and limited distribution
of Garcinia travancorica Bedd.: An endemic,
endangered and medicinally important tree
species. In: A Treatise on Medicinal Plants and
Herbal Products, (eds. Mehalingam, P.,
Rajarathinam, K. & Jayakumar, M.) pp.339-345.
(68)
 Indian Journal of Tropical Biodiversity, 24(1) 2016
Mohammad Hossein Torabi Sirchl, Kadir MA, Aziz MA,
Rashid AA, Arash Rafat, Javadi MB (2008)
Amelioration of mangosteen micro propagation
through leaf and seed segments (Garcinia
mangostana L.). African J Biotech.7(12): 2025-
2029.
Preece JE, Compton ME (1991) Problems with explant
exudation in micropropagation. In: Biotechnology in
agriculture and forestry, vol. 17., Bajaj YPS (ed.),
High-tech and micropropagationI. Berlin,
Heidelberg: Springer-Verlag, pp.168-186.
Rahman SM, Hossain M, Biswas BK, Joarder OI, Islam
R (1993) Micropropagation of Caesalpina
(69)
View publication stats
pulcherrima through nodal bud culture of mature
tree. Plant Cell Tiss. Org. Cult. 32: 363–365.
Rajwant K, Kalia, Malik SK, Rekha Chaudhury (2012) In
vitro morphogenetic studies on three apomictic
species of Garcinia. J. Plant Biochem. Biotechnol.
21(2):279–285.
Scott JN & Ellen GS (1990) Stability of IAA and IBA in
nutrient medium to several tissue culture
procedures, Hort. Sci. 25(7): 800-802.
Uphof JCT (1959) Dictionary of economic plants.
Lubrecht &cramer Ltd. (II edi. 1968), USA.