European Journal of Biotechnology and Bioscience

Online ISSN: 2321-9122

www.biosciencejournals.com
Volume 3; Issue 11; November 2015; Page No. 62-66

In vitro regeneration of an endangered tree - Elaeocarpus blascoi Weibel. (Rudraksha) from Southern
Western Ghats, Tamil Nadu, India
1
SM Siva, T Amutha Priya, 3 P Balasubramanian, 4 V Manimekalai, 5 P Ravichandran
2

1, 2, 3, 5
Lab of Developmental Biology and Plant Biotechnology, Department of Plant Science, Manonmaniam Sundaranar
University, Abishekapatti - 627 012, Tamil Nadu, India.
4
Department of Botany, Sri Parasakthi College for Women, Courtallam 627 802, Tamil Nadu, India.

Abstract
Elaeocarpus blascoi Weibel. A relative species of Rudraksha, of Elaeocarpaceae is an endemic and endangered evergreen forest tree
from Palni hills, Southern Western Ghats. In vitro regeneration protocol from nodal segments of E. blascoi for clonal propagation
was developed by employing different tissue culture media. Woody plant medium (WPM) was found to be suitable for E. blascoi
shoot multiplication and regenerations. Phenolic secretion makes the explants recalcitrant and was overcome by supplement of 0.
3% charcoal / 0.5% polyvinylpyrrolidone (PVP). Nodal segments were cultured in WPM medium supplemented with cytokinins
including Thidiazuron (TDZ), 6-Benzylaminopurine (BAP), and Kinetin (KIN). Among the three plant growth regulators, TDZ
induced multiple shoots. Shoot elongation was achieved by addition of 10 mg/l silver nitrate in the shoot elongation medium, and an
average of 6 ± 0.47 shoots are produced per explants. The regenerated micro shoots were subsequently rooted in half-strength WPM
supplemented with 2 mg/l Indole -3 Butyric Acid (IBA). Thus the protocol developed is utilised for successful conservation of the
IUCN red listed species E. blascoi.

Keywords: Elaeocarpus blascoi, In vitro culture, WPM, nodal segments, Regeneration.

1. Introduction to the Western Ghats including four steno-endemics viz., E.

The richness of flora makes India one of the mega diversity
countries in the world with four biodiversity hotspots and three
mega centres of endemism. Nayar and Daniel [1] have reported
that there are 4000 species of flowering plants recorded from
the Western Ghats and 1,500 (38%) of these are endemic. The
Western Ghats of India is recognized as one of the 34 global
biodiversity hotspots of the world with over one-third of its
angiosperms being endemic [2]. The United Nations
Educational, Scientific and Cultural Organization (UNESCO)
have recognised the Western Ghats as one of the Natural World
Heritage sites due to its importance of rich endemism [3]. The
Palni hills are a part of Western Ghats which are one of the
internationally recognized hotspots of the planet and is also
known as an environmental marker zones [4]. There is an
alarming threat for extinction of species which often increases
in the wild. The reasons may be due to natural habitat
degradation, followed by anthropogenic pressure and
reproductive limitation. The conservation of red listed species
is a critical issue for any attempt to mange and utilise in a
sustainable manner.
The family Elaeocarpaceae has 12 genera and 605 species in
tropical and temperate southern regions [5]. The genus
Elaeocarpus has 350 species in the temperate, subtropical and
tropical zones throughout South- East Asia, Australia, Chile,
New Zealand and the West Indies [6-10]. Khan et al., [11] reported
that 360 species belong to world wide and 120 species in Asia.
In India there are about 29 species of which ten are endemic.
Eleven Elaeocarpus species have been reported from Tamil
Nadu. There are eight Elaeocarpus species which are confined
blascoi Weibel, E. gaussenii Weibel, E. recurvatus Corner and
E. venustus Bedd [12].
Elaeocarpus blascoi is a woody, evergreen tree, first discovered
in Kodaikanal, Palni hills, Western Ghats, India [13-15] (Fig.1).
E. blascoi is an endemic and also red listed as endangered
species by IUCN [16]. Mathew, who could not collect any
specimens, then presumed the plant to be extinct in the wild;
however the single tree was rediscovered in Kodaikanal forest
area during the year 2000 by local conservation trust team [17].
Besides, a couple of individuals were relocated around the
Vattakanal forests of Kodaikanal, Southern Western Ghats [18].
In general Elaeocarpus fruits are endowed with a hard and
highly ornamental stony endocarp and germination of the seeds
is very low and erratic since the nuts are unable to imbibe water
[19]
. As on now there is no successful report on the regeneration
of Elaeocarpus blascoi hence, an attempt was made to establish
an efficient and reproducible method for clonal propagation of
E. blascoi from mature tree explants.
2. Materials and Methods
Plant Collection- Plant specimens of Elaeocarpus blascoi were
collected from Vattakanal forest (10°12.59.6’’ N and
77°29.06.9’’E) at an altitude of 2031m, Kodaikanal, Palni hills
India. The voucher specimens were processed, mounted and
identified with the help of Flora of Palni Hills and were also
authenticated by Dr. K. Ravikumar, Deputy Director, FRLHT,
Bangalore. The labelled voucher specimens (No. 896) have
been deposited at the Herbarium, SPKCES, MSU,
Alwarkurichi, Tirunelveli 627 412.

62 
 
Culture Media Conditions -The nodal explants were collected
from the healthy disease-free mature branches of the existing
plants in the field and brought to the laboratory in sealed
polythene covers. Expanded leaves from the shoot apex were
first trimmed, and the shoot tip explants (3.0 – 3.5 cm) were
washed thoroughly under running tap water for 30 minutes,
then the explants were immersed for 5 – 8 min in 10% sodium
hypochlorite (Merck, Mumbai) with two or three drops of
Teepol. After three consequent washes in sterile distilled water,
explants were treated with 70% ethanol for 30 seconds and
thoroughly rinsed in sterile distilled water. The explants were
then surface sterilized with 0.05% mercuric chloride (Hi Media,
Mumbai) which was followed by two or three washes with
autoclaved distilled water, followed by immersion in
antimycotic solution (Hi Media, Mumbai) for 5 min. Explants
were finally trimmed to smaller pieces (2 – 2.5 cm) and were
transplanted on Woody Plant Medium (WPM) of Lloyd and
McCown [20] to initiate multiplication.
The basal medium of WPM consisted of the mineral salts and
nutrients with 2% sucrose and 0.8% agar. The basal medium of
the in vitro cultures experiments was variously supplemented
with factorial combination of different plant growth regulators
such as TDZ, BAP, Kin, IBA, IAA and NAA at different
concentrations ranging from 0. 22 µM to 4.5 µM for shoot
induction, 2.4 µM to 17 µM for rooting. All the supplements
were added to the molten agar at around 50 °C and the pH of
the medium was adjusted to 5.8 before autoclaving at 121 °C
for 15 min (15 lb). The cultures were maintained at 24 ± 2°
under 16 h photo-period with light intensity of 60 – 70 µ Em-2
s-1 from Crompton cool white fluorescent lamp.
3. Results and Discussion
Explant size, surface contamination, phenolic exudation
and regeneration capacity
Fungal contaminants cause a major threat at every stage of plant
cultures and it is difficult to eradicate 100% from the in vitro
cultures. Shoot induction and multiplication in E. blascoi was
very difficult initially due to heavy fungal and bacterial growth.
This condition may be due to its natural habitat characterized
by low temperature (8°- 23 °C) and high humidity (85 – 99%)
throughout the year which favours microbial growth. It has
been reported that the leaves of 27 endemic endangered rare
taxa of the Southern Western Ghats harbour Asterinaceous
fungi. The black colonies of these fungi increase the
temperature of the infected parts and cause physiological
imbalance in the entire leaves and they decrease the
photosynthetic efficiency of the plants, affecting the hormonal
and phenolic compound level thus, the efficiency of the plants
in total [21]. Elaeocarpus species are also found to be associated
with Asterina fungus persisting even after sterilization with
Mercuric Chloride. As many as 1172 fungal colonies were
reported from two Elaeocarpus species [22] and 200 fungal
colonies were associated in a single Elaeocarpus tree [23].
Hosagoudar [24] reported that four Elaeocarpus species were
associated with exogenous Asterina which are E. munronii, E.
serratus, E. tectorius and E. tuberculatus. The leaves of E.
blascoi in the field were also found with exogenous fungi
(Fig.2). However, the contamination on the explants of E.
blascoi was reduced to 10% – 20% in the present study after
Mercuric Chloride treatment followed by antimycotic solution
treatment for 5 min which helped to achieve 80% – 90% sterile
cultures. Besides fungal and bacterial contamination another
63 
major problem with in vitro cultures of E. blascoi was phenolic
exudation from explants and browning of the culture which
often resulted in the necrosis. This result corroborates with the
findings of micropropagation studies in other woody plants [25,
26]
. The exudation of phenols and browning of the medium was
overcome by addition of 0.3% charcoal and 0.5% polyvinyl
pyrrolidone (PVP) in the medium, decreasing the passage time
by frequent subcultures and placing the cultures in dark
condition for few days. The successful in vitro propagation
method requires the correct size and physiological status of
explants which play a crucial role during regeneration. Healthy
and younger nodes with 2-2.5 cm long (Fig. 3) respond very
well in woody plant medium. The explants response recorded
maximum (80%) among 2 – 2.5 cm long shoot explants. Among
the different size of explants tested 2 – 2.5 cm gave a maximum
survival rate. While the smaller sized explants lost their
regeneration potential which lead to tissue blackening and death
after two weeks.
Induction of shoot growth and multiplication
The node was initially cultured on woody plant basal medium
with different concentrations of plant growth regulators such as
TDZ, BAP and KIN. In the absence of growth regulators shoot
multiplication was not observed from the nodal explant rather
only a single shoot grew from it. However, in the presence of
growth regulators the mean number of shoots varied
significantly with the type of cytokinin in the medium. Results
showed that TDZ induced 2 - 4 shoots per node (Tab. Figs. 9-
10) when compared to BAP and KIN. Among the different
concentrations 0.22 µM TDZ induced 2 - 4 shoots followed
BAP at a concentration of 0.44 µM (1.83±0.76) and KIN at 1.16
µM (1.53±0.51) per node after 5 weeks. In TDZ (0.22 µM)
medium showed a maximum number of 7 shoots per node in
three subcultures over a period of 150 days. High
concentrations above 3 µM induced profuse basal callusing in
all the three plant growth regulator combinations treated. When
compared to BAP and KIN, TDZ was very active in inducing
basal callus even at low concentration (Figs. 6-7). Basal callus
was compact type which did not regenerate into shoots when
transferred to medium lacking plant growth regulators. During
subculture newly formed shoots were transferred to fresh
medium of same composition. The shoots continued to
proliferate through several subcultures with an average of four
new shoots per transfer. Even though, multiple shoots were
produced on TDZ medium the shoots failed to elongate
therefore, they were transferred to medium containing
gibberellic acid (5- 20 µM) and silver nitrate 10 mg/l for
elongation. The formation of stunted shoots on TDZ
supplemented medium has been reported for several tree
species such as Miconia sp. [27] Malus [28] and Rhododendron [29,
30]
. Inhibition of shoot elongation may be due to the high
cytokinin activity of TDZ [31]. Compared to the adenine-type
cytokinins (BAP, KIN), the synthetic TDZ was found more
effective at lower concentrations; the dominance of TDZ has
been reported in some trees [32, 33]. The potential effect of TDZ
may be due to its resistance of cytokinin oxidase [34]. The shoot
elongation was accomplished on a medium containing 10 µM
GA3 and 10 mg/l AgNo3. Maximum of 6 cm long shoot was
produced after 5 weeks of culture. The exact role of AgNo3 in
shoot elongation is not known however, the results showed that
silver nitrate has the ability for shoot elongation at a
concentration of 10 mg/L in E. blascoi micro shoots after 5
weeks either alone or along with GA3. Bais et al., have reported Root induction
that AgNo3 at 40 µM influenced root induction in Decalepis The elongated shoots were transferred to half strength woody
hamiltonii shoots in vitro by accelerating endogenous plant medium containing either one of different concentrations
polyamines [35]. ranging from 2.4 µM to 17.12 µM of auxin type PGR’s such as
IBA, NAA, IAA and to a basal medium, treated as control. IBA
Table 1. Effect of plant growth regulators on shoot induction and 9.84 µM was found to be the ideal concentration for promoting
multiplication from nodal segments of E. blascoi rooting in E. blascoi. Out of three auxin type PGR’s, IAA did
Concentration % of Shoot Number of shoots not induce rooting at any concentration as it is well known that
PGR supplements of direct auxin in the culture medium do not
(µM) Induction (%)r per explants r
(0.22 µM) 80.00±1.0 3.86±0.61 necessarily induce roots [36]. However, IBA and NAA were
(0.45 µM) 46.66±2.3 2.06±0.40 effective in inducing roots. A maximum 80% of shoots were
TDZ (1.13 µM) 30.00±2.0 1.37±0.15 rooted on half strength WPM+ IBA 9.84 µM with 3 to 5 weeks
(2.27 µM) 16.66±0.57 1.50±0.50 whereas 55% of shoots were rooted medium containing half
(4.50 µM) 13.33±1.52 1.26±0.28 strength WPM+8.05 µM NAA. The basal medium failed to
(0.22 µM) 28.33±2.08 1.50±0.50
induce any roots. The collection of time of explants play a vital
(0.44 µM) 38.33±3.70 1.83±0.76
role in inducing in vitro multiplication and rooting of E. blascoi.
BAP (1.10 µM) 45.00±1.00 1.39±0.22
The explants collected during April to May induced shoots and
(2.21 µM) 23.33±3.05 1.57±0.37 roots on auxin containing medium. However, explants collected
(4.43 µM) 18.33±2.00 1.30±0.26 during other months and seasons showed a poor shoot
multiplication and rooting response. Singh et al., [37] and
(0.24 µM) 10.00±1.73 1.16±0.15
Choudhary et al., [38] have reported that IBA alone can induce
(0.46 µM) 20.00±2.00 1.58±0.36
highest percentage of rooting in woody trees - Shorea robusta
KIN (1.16 µM) 28.33±2.51 1.53±0.51
and Prunus dulcis. The findings of the present study also show
(2.32 µM) 11.66±0.57 2.26±0.95
that IBA alone induces maximum roots in E. Blascoi (Fig.13).
(4.64 µM) 6.00±1.15 1.30±0.30
r The value represents the mean (± SE) of three independent For hardening rooted plantlets were transferred to potting
experiments. Twenty explants were used for each experiment. mixture which has a combination of farmyard manure, garden
soil and river sand in the ration of 2:1:1. In vitro derived
plantlets were successfully acclimated and trained to withstand
the outer environmental conditions and are ready to be
reintroduced into natural habitats.

Fig 1-4: 1. Habit of more than 50 years old Elaeocarpus blascoi from Kodaikanal forest (Palni Hills); 2. Flowering twig with exogenous fungal
infection in leaf; 3. Uninfected young leaf of E. blascoi 4. Mature fruits of E. blascoi with hard endocarp
64 
 
 Fig 5-13: In vitro clonal propagation of Elaeocarpus blascoi from nodal explants. 5. Shoot initiation from nodal explant in WPM+TDZ (0.227
µM); 6. Shoot and basal callus formation from nodal explant on WPM+TDZ (0.45 µM); 7. Compact callus induced from nodal segment on TDZ
containing medium (1.13 µM); 8. Shoot and root induction on WPM medium; 9-10 Multiple shoot induction on WPM+TDZ (0.227 µM); 11-12.
Shoot elongation on medium containing GA3 + Ag NO3 (10mg/l); 13. Well grown shoot on root induction medium WPM+ IBA

4. Conclusion 2. Myers N, Mittermeier RA, da Fonseca GAB, Kent J.

The clonal propagation through nodal culture of E. blascoi was
successful using WPM supplemented with cytokinin type
PGR’s. This is the first report on in vitro culture conservation
attempt which was made to establish shoot multiplication and
whole plant regeneration of E. blascoi. These results show that
it is possible to propagate this E. blascoi endangered tree
species of Western Ghats through in vitro clonal propagation in
order to overcome the threat of the extinction.
5. Acknowledgment
The authors are thankful to the Department of Biotechnology
(DBT), Ministry of Science and Technology, New Delhi, India
for providing financial support. Project No. BT/PR
9365/BCE/08/562/2007 and the Tamil Nadu Forest department
for providing permission (Proc. No. WL5/4799/08 dt. 30.01.
2008; C. No. 2935/2009/M dt. 18. 08. 2009) to collect explants
and carry out field studies.
6. References
1. Nair NC, Daniel P. The floristic diversity of the Western
Ghats and its conservation: A review. Proceedings of the
Indian Academy of Sciences (Animal Science/Plant
Science) Supplement 1986, 127-163.
Biodiversity hotspots for conservation priorities. Nature.
2000; 403:853-857.
3. UNESCO Western Ghats. http://whc.unesco.org/en/
list/1342., 2012.
4. Matthew KM. Part 1 – Polypetalae; The Flora of Palni
Hills, South India. (The Rapinat Herbarium, Tiruchirapalli,
1999, 45-146.
5. Gray AM. 71 Elaeocarpaceae, version 2011:1. In MF
Duretto (Ed.) Flora of Tasmania Online. (Tasmanian
Herbarium, Tasmanian Museum & Art Gallery: Hobart),
www.tmag.tas.gov.au/floratasmania., 2011.
6. Coode MJE. Elaeocarpaceae. In Henty EE (ed.),
Handbooks of the Flora of Papua New Guinea. (Melbourne
University Press: Carlton). 1981, 38-185.
7. Hyland BPM, Whiffin T, Christophel DC, Gray B, Elick
RW. Australian Tropical Rainforest Plants. (CDROM).
(CSIRO Publishing: Melbourne), 2003, 2.
8. Coode MJE. Elaeocarpaceae. In Kubitzki, K. (ed.), the
Families and Genera of Vascular Plants (Springer-Verlag:
Berlin) 2004; 6:135-144.
9. Coode MJE. Elaeocarpaceae for flora Malesiana: New
information on Elaeocarpus from Borneo and Sulawesi.
Kew Bull 2007; 62:329-332.

65 
 
10. Mabberley DJ. The Plant Book- A portable dictionary of 29. Preece JE, Imel MR. Plant regeneration from leaf explants
plants and their classification and uses. (Cambridge of Rhododendron P.J.M hybrids. Horti. Sci. 1991; 48:159-
University Press, United Kingdom), 2008, 1040. 170.
11. Khan ML, Bhuyan P, Tripathi RS. Regeneration of 30. Mohammad Hussain T, Chandrasekhar T, Rama Gopal G.
Rudraksha (Elaeocarpus ganitrus Roxb.)- A threatened High frequency shoot regeneration of Sterculia urens
tree species: germination Strategies. Inter j Ecol and Roxb. an endangered tree species through cotyledonary
Environ sci. 2003; 29:255-260. node cultures. African J Biotech. 2007; 61:1643-1649.
12. Murthi SK. Elaeocarpaceae. In: Flora of India, edited by 31. Hutteman CA, Preece JE. Thidiazuron: A Potent cytokinin
Sharma, B.D. &. Sanjappa, M. (Botanical survey of India, for woody plant tissue culture. Plant cell tiss. org. 1993;
Calcutta), 1993, 529-562. 33:105-119.
13. Blasco F. Elaeocarpus blascoi Vascular plant type 32. Preece JE, Hutteman CA, Puello CH, Neuman MC. The
collection. 1968. influence of thidiazuron on in vitro culture of woody
14. Weibel R. Morphologie de I’embryon et de la graine des plants. Horti sci. 1987; 22:1071.
Elaeocarpus. Condollea. 1972; 27(1):16. 33. Bates S, Preece JE, Navarrete NE, Van sambeek JW,
15. Matthew KM. The Flora of the Palni Hills, South India. Gaffney GR. Thidiazuron stimulates shoot organogenesis
The Rapinat Herbarium, India. 1999, 3. and somatic embryogenesis in white ash (Franxinus
16. World Conservation Monitoring Centre. Elaeocarpus americana L.) Plant cell tiss org. 1992; 31:21-30.
blascoi. The IUCN Red List of Threatened Species. 34. Mok MC, Mok DWS, Turner JE, Mujer CV3. Biological
Version 2015.4. <www. iucnredlist.org>, 1998. and biochemical effect of effect of cytokine-active phenyl
17. Robert Stewart, Tanya Balcar. Rare flora of Upper Palnis: urea derivatives in tissue culture systems. Horti. Sci. 1987;
in Envis: Special habitats and threatened plants of India. 22:1194-1196.
Envis bulletin 2008; 11(1):135-138. 35. Bais HP, Sudha G, Suresh B, Ravishankar GA. Silver
18. Ravichandran P, Manimekalai V. Ministry of Science and nitrate influences in vitro root formation in Decalepis
Technology, Department of Biotechnology, New Delhi, hamiltonii Wigth & Arn. Current Science. 2000; 79(6):
final report. (Unpublished), 2014. 894-898.
19. Bhuyan P, Khan ML, Tripathi RS. Regeneration status and 36. Shen X, Castele WS, Gmitter FG. In vitro shoot
population structure of Rudraksha (Elaeocarpus ganitrus proliferation and root induction of shoot tip explants from
Roxb.)- In relation to cultural disturbances in tropical wet mature male plants of Casuarina cunninghamiana miq.
evergreen forest of Arunachal Pradesh. Cur. Sci. 2002; Hortisci. 2010; 45(5):797-800.
83:1391-1394. 37. Singh M, Sonkusale S, Niratker CH, Shukla P.
20. Lloyd G, McCown B. Commercially feasible Micropropagation of Shorea robusta an economically
micropropagation of mountain laurel, Kalmia latifolia by important woody plant. J forest. 2014; 60:70.
use of shoot tip culture. Proc Int plant propagators’ Soc. 38. Choudhary R, Chadhary R, Kumar Malik S, Sharma KC.
1980; 30:421-427. An efficient regeneration and rapid Micropropagation
21. Hosagoudar VB, Biju CK, Abraham TK. Diviersity of protocol for almond using dormant axillary buds as
follicolous microbionts in Munnar and Wyanad forest explants. Indian journal of experimental biology. 2015;
region of Kerala. J. Mycopatho. Res. 2002; 40:191-196. 53:462-467.
22. Akash K, Ivanov D, Kishore Dubey N. Fungal
contamination of stony endocarp (Rudraksha) of
Elaeocarpus spp. From three different countries. Indian J
nat prod resources. 2013; 5:97-100.
23. Aptroot A. Lichenized. Saprobic fungal biodiversity of a
single Elaeocarpus tree in Papua, New Guinea with the
report of 200 sp of Ascomycetes associated with one tree.
Fungal diver 2001; 6:1-11.
24. Hosagoudar VB. The genus Asterina and its anamorph on
Elaeocarpus species in Southern Western Ghats of
Peninsular India. Journal of natural and applied sciences.
2009; 1(1):27-30.
25. Anna Karkonen LK, Simola Timokoponen.
Micropropagation of several Japanese woody plants for
horticulture purposes. Ann Bot 1999; 36:21-31.
26. Iyad Musallam M, Duwayri, Shibli RA. Micropropagation
of caper (Capparis spinnosa L.) from wild plant.
Functional plant sci. and biotech 2011; 5:17-21.
27. Luis Pedro BC, Adriane CMG, Sílvia BRDC, Batista T.
Micropropagation of Miconia sp, a woody Melastomaceae
from Brazil, using thidiazuron as plant growth regulator. R.
Bras. Fisiol. Veg 1997; 9(1):21-25.
28. Van Nieuwkerk JP, Zimmerman RH, Fordham I.
Thidiazuron stimulates an apple shoot proliferation in
vitro. Horti Sci 1986; 21:516-518.
66