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SHORT COMMUNICATION
J For Res (2006) 11:57–60 © The Japanese Forest Society and Springer-Verlag Tokyo 2006
DOI 10.1007/s10310-005-0188-1
A.V. Raghu · S.P. Geetha · Gerald Martin
Indira Balachandran · P.N. Ravindran
Direct shoot organogenesis from leaf explants of
Embelia ribes
Burm. f.:
a vulnerable medicinal plant
Received: April 14, 2005 / Accepted: July 11, 2005
Western Ghats of Tamil Nadu and Karnataka states of
India, and at lower risk in Kerala state (Ravikumar and Ved
2000). Dried fruits of the plant are the source of an
ayurvedic drug, vidanga. It is a highly esteemed medicinal
plant having powerful anthelmintic, carminative, antibacte-
rial, antibiotic, and hypoglycemic properties and is an ingre-
dient in about 75 traditional ayurvedic drug formulations.
The drug gained particular importance in view of the wide
experimental and clinical trials on its contraceptive
potential (Anon. 1966, 2002; Sivarajan and Balachandran
1994). The main active component, embelin, isolated from
the berries has antifertility activity, which is reversible.
The pharmacological and clinical investigations by various
workers gave promising results about its antifertility activity
without any side effects (Mitra 1995; Anon. 2002).
Propagation of the species is possible through seeds as
well as vegetative means, although both methods are unre-
liable and difficult. The embryos are small when present and
most of the seeds are abortive (Anon. 1990). Based on
the preliminary studies conducted on seed and vegetative
propagation, specific habitat conditions are required for its
survival and growth. Regeneration is also very slow. All
such factors, coupled with unsustainable and indiscriminate
harvesting from the wild, have posed threats to this species.
Thus, conventional propagation through seeds and vegeta-
tive cuttings is not sufficiently reliable or adequate to meet
the demand for planting material. The development of an in
vitro propagation method will be of great importance for
the production of planting material to build up the resource
base of this threatened species. This is the aim of the
present study. No previous reports are available on in vitro
propagation of this threatened medicinal plant.
Materials and methods
Establishment of in vitro shoot cultures
Mature fruit were collected from the wild populations of
Embelia ribes in the Western Ghat forests of Kannur Dis-
Abstract An efficient system was developed for direct plant
regeneration from in vitro-derived leaf explants of Embelia
ribes Burm. f., a vulnerable medicinal woody climber of the
Western Ghats of India. The in vitro procedure involved
three steps that included induction of shoot initials from
leaf tissue, regeneration and elongation of shoots from the
shoot initials, and rooting of shoots. The induction of shoot
initials was achieved on Murashige and Skoog (MS) solid
medium supplemented with different concentrations of
thidiazuron (TDZ). The best medium for shoot induction
was MS with 0.272mM TDZ. Numerous shoot primordia
developed within 2–3 weeks on the leaf margin as well as on
the midrib region, without any callus phase. In the second
step, the shoot clumps separated from the leaf explant on
transfer to MS basal medium, resulting in the differentia-
tion of 90% of the shoot initials into well-developed shoots.
The 2- to 3-cm-long shoots rooted on half-strength MS basal
medium supplemented with 4.90mM indole-3-butyric acid
(IBA) and 3% (w/v) sucrose in the third stage. The rooted
plants could be established in soil with 70% success. This
protocol could be utilized for in vitro propagation and con-
servation of this important threatened medicinal plant.
Key words Embelia ribes · Shoot organogenesis · Medicinal
woody climber · Vulnerable · Conservation
Introduction
Embelia ribes Burm. f. (Myrsinaceae), an important vulner-
able medicinal plant, is a climbing shrub found in the
semievergreen to evergreen forests of India, Sri Lanka,
Malaysia, and China at altitudes above 400m and up to
1200m. The species is reported to be vulnerable in the
A.V. Raghu · S.P. Geetha (*) · G. Martin · I. Balachandran ·
P.N. Ravindran
Tissue Culture Facility, Centre for Medicinal Plants Research, Arya
Vaidya Sala, Kottakkal 676 503, Malappuram, Kerala, India
Tel. +91-48-3274-3430; Fax +91-48-3274-2572
e-mail: spgeetha@yahoo.co.in
58
trict (1200m a.s.l.), Kerala, India, during April 2003. Fruit
were shade-dried and used immediately. They were soaked
in water for 3h and then treated with 0.1% (w/v) mercuric
chloride solution and Tween 20 (2 drops in 100ml) for
10min. After rinsing four to five times with sterile distilled
water, the surface-disinfected fruits were depulped and in-
oculated aseptically on Murashige and Skoog (MS) basal
medium (Murashige and Skoog 1962) containing 0.7% agar
and 3% sucrose.
Shoot regeneration from leaf tissue
The in vitro raised seedlings served as explant source.
Mature and semimature leaves along with a portion of
the petiole were excised and were cultured on solid me-
dium with their abaxial sides touching the medium. MS
basal medium supplemented with 3% (w/v) sucrose and
different concentrations of thidiazuron (TDZ), benzyl ad-
enine (BA), and kinetin were tested for shoot regeneration
experiments. The cultures were observed constantly for any
response.
Shoot elongation and rooting of shoots
The clumps of shoots produced on the leaf tissue were
excised from the parent culture and transferred to MS basal
medium devoid of growth regulators for shoot elongation.
After 4 weeks of culture, the elongated shoots (2–3cm)
were excised and kept for rooting. Full-strength and half-
strength MS basal medium supplemented with indole-3-
butyric acid (IBA) and 3% (w/v) sucrose were used.
Acclimatization
Rooted shoots were carefully taken out of the medium and
washed thoroughly in running tap water to remove all traces
of medium attached to the roots without damaging the
roots. The plantlets were planted in 5-cm thermocol cups
(Boss–CFC free; Utsav Polymers, Aurangabad, India) con-
taining a mixture of sand and soil in the ratio 1:1. The cups
were covered with polythene bags to maintain humidity and
kept in a shade house where they were observed for further
growth and establishment. After 20–25 days, the established
plants were transplanted to polybags and then to pots con-
taining garden soil and farmyard manure.
Culture conditions and data analysis
Uniform culture conditions were applied in all experiments.
The pH of the medium was adjusted to 5.8 using 0.1N
NaOH, prior to autoclaving at 121°C for 20min. The cul-
tures were maintained at 25° ± 2°C under a 12-h photope-
riod with a light intensity of 35–40mm-2s-1 (Philips India,
Mumbai, India).
All experiments were repeated at least three times with
more than 12 replicates. Standard errors of means were
calculated, and statistically significant mean differences
were determined by the least significant difference (LSD)
test.
Results and discussion
Shoot regeneration from leaf tissue
In the present investigation it was possible to obtain high
frequency of shoot organogenesis directly from in vitro-
derived leaf explants of Embelia ribes in TDZ-containing
medium.
In the process of establishment of in vitro cultures, 20%
of the seeds germinated after 45 days of incubation. The
seedlings produced harvestable leaves within 30–40 days.
Leaf explants derived from in vitro-raised seedlings exhib-
ited differentiation of shoots in MS medium supplemented
with different concentrations of TDZ ranging from 0.009 to
0.45mM (Table 1). Semimature and expanded young leaves
gave good response in all trials. Similar to the observations
made in Plumbago species (Das and Rout 2002) and
Zizyphus jujuba (Gu and Zhang 2005), shoot organogenesis
in E. ribes is dependent on the maturity of explants.
The medium supplemented with BA or kinetin sepa-
rately or the combination of the two did not give any
response (data not shown). Within 2 weeks of incubation,
direct shoot organogenesis was observed on the surface of
leaf explants (Fig. 1a). Shoot initials developed in all parts
of the lamina, although they were more concentrated in the
midrib regions (Fig. 1b). At low concentrations of TDZ,
more shoot initials developed at the petiole portion of the
midrib and surrounding area (Fig. 1c).
The number of shoot initials increased with increasing
concentration of TDZ. The highest rate of shoot bud regen-
eration took place in the medium containing 0.272mM TDZ
(Fig. 1d). Magioli et al. (1998) have reported the superiority
of lower concentration of TDZ in Solanum melongena using
similar explant. Such a response may perhaps be due to the
increase in the levels of endogenous cytokinins by the effect
of the growth regulator used. Hare and Van Staden (1994)
reported that TDZ has a capacity to inhibit (at least partly)
Table 1. Effect of thidiazuron (TDZ) concentration on direct shoot
organogenesis from in vitro-derived leaf explants cultured on MS
medium
TDZ (mM) Relative Mean no. of
response (%) shoots/explant
0.009 22.2 (± 0.6) 3.4 (± 0.6)a
0.023 31.6 (± 0.5) 4.6 (± 0.7)a
0.045 52.4 (± 0.4) 7.4 (± 0.7)b
0.091 54.3 (± 0.4) 10.2 (± 0.4)c
0.182 71.6 (± 0.3) 12.2 (± 0.5)d
0.272 83.6 (± 0.2) 16.3 (± 0.9)e
0.363 81.3 (± 0.2) 14.7 (± 0.9)de
0.454 62.8 (± 0.4) 9.6 (± 0.7)c
Each value was recorded after 28 days of incubation. Data are given as
mean (± SE) of three independent experiments each with 12 replicates.
Means followed by the same letters are not significantly different at
P = 0.05 by the least significant difference test
59
the action of cytokinin oxidase, which in turn may increase
the levels of endogenous cytokinins. However, higher con-
centrations caused reduction in shoot length and callusing.
Multiple shoot initials induced by TDZ failed to elongate.
Similar results were observed in other species (Preece and
Imel 1991; Dalal and Rai 2004). Cytokinins commonly
stimulate shoot proliferation and inhibit their elongation.
Therefore, inhibition of shoot elongation by TDZ may be
consistent with its high cytokinin activity (Huetteman and
Preece 1993).
Shoot elongation and rooting of shoots
The length of the shoots obtained in the regeneration me-
dium containing TDZ was not suitable for persistent root-
ing. Therefore, after 28 days, the clumps of shoot initials
(0.5–1.0cm) were excised from the parent culture and trans-
ferred to growth regulator-free MS basal medium for elon-
gation. The shoot buds elongated sufficiently in this
medium within 20 days (Fig. 1e). These differentiated cul-
tures were further used as the source of leaf explant for
shoot induction in subsequent cycles. Thus, the three-step
plant regeneration protocol was completed within 132 days
initially and the subsequent cycles were completed within
87 days (Fig. 2).
Shoot tips measuring about 1.5–2cm in length with two
or three leaves were used for rooting. The auxin IBA, which
often induces rooting in tissue culture, was employed in the
present study. Results differed depending upon the concen-
tration of IBA as well as the strength of the basal medium.
However, rooting percentage was lower in full-strength MS
medium than in half-strength medium. Among the media
tried (data not shown) half-strength MS medium supple-
mented with 4.90mM IBA and 3% sucrose was the best.
Profuse root formation was observed after 1 month of in-
cubation (Fig. 1f).
Acclimatization
Rooted plantlets were successfully transferred to thermocol
cups containing sand and soil (1:1 v/v mixture) and new
growths were observed after 3 weeks (Fig. 1g). These plants
exhibited a 70% survival rate 1 month after transfer.
In conclusion, an efficient protocol was developed
for micropropagation of E. ribes, a vulnerable medicinal
plant of Western Ghats of India. Our results show that
micropropagation of this plant has a three-step protocol and
TDZ is required for adventitious shoot regeneration from
the leaf explants. Similar results have also been reported
earlier in Alstromeria species by Lin et al. (1997). This
protocol provides a successful and rapid technique that can
be used for the propagation and ex situ conservation of this
Fig. 1a–g. Direct shoot organogenesis from leaf explants of Embelia
ribes. a Initiation of shoot primordia on leaf surface within 2 weeks, b,
c shoot organogenesis concentrated on the midrib (b) and petiolar (c)
regions, d shoot bud regeneration from all over the leaf surface in
Murashige and Skoog (MS) medium with 0.272 mM thidiazuron (TDZ),
e elongation of shoots in MS basal medium, f rooting of in vitro
shoots in MS medium with 4.90mM indole-3-butyric acid, g acclima-
tized plantlet
14 days
Explant: aseptic seeds
In vitro raised seedlings 20% germination
Semi mature leaves
Shoot induction
In vitro rooting (68%)
Acclimatization 70% survival1:1-Soil + Sand
½ MS + 4.90 M IBA
45 days
Shoot elongation (90%)
Leaf explant
28 days
20 days
MS growth regulator free
MS + 0.272 Thidiazuron
25 days
1:16 shoots
MS growth regulator free
14 days
Fig. 2. Schematic representation for the micropropagation protocol of
Embelia ribes using leaf explants. IBA, indole-3-butyric acid
60
important species. Application of this protocol could help in
minimizing the pressure on wild populations and contribute
to the conservation of the valuable flora of the Western
Ghats. In addition to this, the present system could permit
genetic transformation studies in this threatened medicinal
liana in the near future.
Acknowledgment The authors are grateful to the Sir. Dorabji TATA
Trust, Mumbai, India, for providing financial assistance.
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