SHORT COMMUNICATION

J For Res (2006) 11:461–465 © The Japanese Forest Society and Springer 2006DOI 10.1007/s10310-006-0237-4

Gerald Martin · S. P. Geetha · Sudhakar S. RajaA. V. Raghu · Indira Balachandran · P. N. Ravindran

An effi cient micropropagation system for Celastrus paniculatus Willd.: a vulnerable medicinal plant

Received: March 18, 2006 / Accepted: July 24, 2006

Abstract A micropropagation protocol was developed for Celastrus paniculatus, a vulnerable medicinal plant. Cul-tures were initiated from nodal explants collected from young shoots of a 12-year-old plant in MS basal medium. An average of fi ve shoots were produced in MS medium supplemented with 1.5 mg l−1 benzyl adenine (BA) and 0.1 mg l−1 naphthalene acetic acid (NAA) after two subcul-ture cycles with a 30-day interval. Continuous subculture in the same medium for three more cycles resulted in re-duction of the number of multiple shoots (2 or 3 shoots), vitrifi cation of the shoots, and callus formation. Vitrifi ca-tion of cultures could be overcome by the use of MS medium supplemented with lower concentrations of BA (0.05 mg l−1) and NAA (0.01 mg l−1). Among the various rooting trials, ex vitro rooting of shoots with simultaneous hardening was most effi cient. The method standardized in the present study is simple, as it eliminated separate steps for in vitro rooting and hardening. Qualitative chemical similarity of the tissue culture regenerants with the mother plant was confi rmed using high performance thin-layer chromatographic (HPTLC) profi ling.

Key words Chemical fi delity · Clonal multiplication · HPTLC · In vitro culture · Large-scale propagation

Introduction

Celastrus paniculatus Willd. (Jyotismati), commonly called the climbing staff tree, the black oil plant, or the intellect tree, is an important medicinal plant belonging to the fam-ily Celastraceae. It is a large, woody, unarmed climbing shrub reaching up to a height of 10 m, and is distributed

G. Martin · S.P. Geetha (*)· S.S. Raja · A.V. Raghu · I. Balachandran · P.N. RavindranTissue Culture Facility, Centre for Medicinal Plants Research, Arya Vaidya Sala, Kottakkal 676 503, Malappuram, Kerala, IndiaTel. +91-483-2743430; Fax +91-483-2742572e-mail: spgeetha@yahoo.co.in

throughout India up to an altitude of 1200 m, mainly in deciduous forests. The species is vulnerable in the Western Ghats of South India (Rajesekharan and Ganeshan 2002). Its bark is an abortifacient, a depurative, and a brain tonic. Root bark extract also shows antimalarial activity (Rastogi and Mehrotra 1998). Leaf sap is a good antidote for opium poisoning. Seeds are useful in abdominal disorders, lepro-sy, skin diseases, fever, and for stimulating the intellect (Prajapati et al. 2003). Among the Gonds tribe of Uttar Pradesh, India, the powdered root is considered useful for the treatment of cancerous tumors (Parotta 2001). The seed oil is bitter, thermogenic, intellect promoting, and is useful for treating abdominal disorders, beriberi, and sores (Warrier et al. 1994). Chemical constituents as revealed by phytochemical analysis were sesquiterpene alkaloids like celapagine, celapanigine, and celapanine (CSIR 1992).

Indiscriminate collection of this plant from the wild has posed a serious threat to its existence in the wild, especially when the plants are harvested well before seed set. Moreo-ver, propagation either by seed or vegetatively is rather diffi cult. Rekha et al. (2005) reported seed germination as low as 11.5%. Propagation through tissue culture is a viable alternative in this species because it could also be used as a complimentary strategy for conservation and utilization of genetic resources. Some reports (Nair and Seeni 2001; Arya et al. 2002) on the micropropagation of C. panicula-tus are available, but these protocols could not be repro-duced in our laboratory for the rapid clonal propagation of this species.

This article reports the development of an effi cient tech-nology for rapid clonal multiplication of C. paniculatus and also the comparative chemical analysis of tissue culture regenerants with the mother plants to confi rm their true-to-type nature.

Materials and methods

A 12-year-old plant grown in the herb garden, Arya Vaidya Sala, Kottakkal, Kerala, India, was used as the explant

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source. Two node cuttings collected from the fresh sprouts were used as explants. Leaves were removed from them leaving behind a portion of the petiole; they were washed in running tapwater for 30 min, then treated with 0.1% (w/v) mercuric chloride (HgCl2) and Tween 20 (two drops of per 100 ml) for 5 min, and washed with distilled water four or fi ve times. Explants were then taken to the laminar air fl ow chamber and treated with 0.1% (w/v) HgCl2 for 2 min and washed with sterile double distilled water four or fi ve times. The two node pieces were cut into single node pieces, and the petiole and cut ends were trimmed off and inoculated into MS (Murashige and Skoog 1962) basal medium.

The aseptic cultures with sprouting axillary buds estab-lished in the growth regulator-free medium were then transferred to MS medium supplemented with 3% sucrose, 0.7% agar, and various combinations of growth regulators to determine media requirements for optimum shoot multiplication. MS medium with varying levels of benzyl adenine (BA) and naphthalene acetic acid (NAA) with or without other growth factors, like adenine sulfate (5 mg l−1) and arginine (25 mg l−1), was used. A mixture of antioxi-dants, including ascorbic acid (50 mg l−1) and citric acid (25 mg l−1), was also used to prevent browning due to phe-nolic exudation.

Shoots (4–5 cm) excised from the multiclumps during subculturing were subjected to various rooting trials:

1. Pulse treatment with 100 mg l−1 each of napthoxy acetic acid (NOA) and indole-3-butyric acid (IBA) for 1–48 h and then in 10 mg l−1 chlorogenic acid for 1–10 min and further planting in sterilized river sand.

2. Pulse treatment and further planting in liquid MS basal medium containing washed, dried, unspun coir as matrix.

3. Agar solidifi ed half strength MS medium with 1 mg l−1 indole-3-acetic acid (IAA).

4. In vitro derived shoots directly planted in sand without pulse treatment.

5. In vitro derived shoots directly planted in a mixture of sand and coir (coconut fi ber) pith (1 : 1).

After 5 weeks, the rooted shoot tips from various treat-ments, except treatments 4 and 5, were inspected for root-ing and the rooted ones were transferred to polythene bags fi lled with a mixture of sand and soil and kept in a humid chamber until new leaves sprouted. In treatments 4 and 5, shoots were kept in a humid chamber in the greenhouse and observed for new growth as an indication of root for-mation, which was confi rmed by uprooting.

For all in vitro studies, the pH of the medium was ad-justed to 5.8 prior to the addition of agar. The culture me-dium was autoclaved at 120°C and 1.5 kg cm−2 for 20 min. The cultures were maintained at 24° ± 2°C under a 12-h photoperiod of 35–40 µmolm−2 s−1 irradiance provided by cool white fl uorescent tubes (Philips India, Mumbai).

Each treatment was carried out with 12 culture tubes with one explant per tube and each experiment repeated three times. The response in various media was expressed

Fig. 1A–H. Micropropagation of Celastrus paniculatus. A Culture initiation from mature explants, B multiple shoot induction, C pulse-treated shoots rooted in sand, D pulse-treated shoots rooted in coir as matrix with liquid Murashige and Skoog medium, E ex vitro root-ing in sand, F hardened tissue culture plants, G tissue culture plant growing in the fi eld after 1 month, H tissue culture plant growing in the fi eld after 1 year. Bars A–E 1 cm; H 1 m

in terms of percentage response, mean number of shoots or roots per explant, length of roots, etc.

For chemical analysis, air-dried leaves (50 g) of 8-month-old fi eld-grown tissue culture-raised plants and the mother plant of Celastrus paniculatus were powdered and 5 g of the powdered plant material was refl uxed with methanol at 60°C for 4 h over a water bath. The extracts were fi ltered and concentrated under reduced pressure in a rotary evap-orator below 60°C. The concentrated extracts were dis-solved in methanol and used for high performance thin-layer chromatographic (HPTLC) analysis.

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Table 1. In vitro response of Celastrus paniculatus in different media combinations

Media composition (MS+) Response (%) Mean no. of shootsb

NAA BA Additivesa

(mg l−1) (mg l−1)

0.1 0.1 No 80 1.0 ± 0.2cd

0.1 0.1 Yes 40 0.3 ± 0.42c

0.1 0.25 No 100 1.8 ± 0.6d

0.1 0.25 Yes 80 1.0 ± 0.4cd

0.1 0.5 No 100 2.3 ± 0.8e

0.1 0.5 Yes 80 2.8 ± 1.1e

0.1 0.8 No 90 2.5 ± 0.75e

0.1 0.8 Yes 100 2.9 ± 1.1e

0.1 1.0 No 100 4.1 ± 1.32f

0.1 1.0 Yes 100 3.9 ± 0.9f

0.1 1.5 No 100 5.0 ± 1.2g

0.1 1.5 Yes 90 3.0 ± 1.4e

0.1 2.0 No 100 4.5 ± 1.6fg

0.1 2.0 Yes 100 4.6 ± l.48fg

0.2 0.1 No 80 1.8 ± 1.52d

0.2 0.1 Yes 50 0.5 ± 0.5c

MS, Murashige and Skoog medium (Murashige and Skoog 1962); NAA, naphthalene acetic acid; BA, benzyl adeninea Ascorbic acid (50 mg l−1) + citric acid (25 mg l−1) + arginine (25 mg l−1) + adenine sulfate (5 mg l−1)b Values are mean ± SE of three independent experiments each with 12 replicates. Means fol-lowed by the same letters are not signifi cantly different at P = 0.05 by the least signifi cant difference test

The extracts were spotted as sharp bands on precoated silica gel plate 60F254 (0.25 mm thickness) (Merck, India) using a Camag (Germany) Linomat V sample applicator. The plate was dried and the chromatogram was developed using toluene/ethyl acetate (7 : 3) as the mobile phase up to a solvent front distance of 80 mm. Then the plate was dried and scanned at 220 nm using a Camag Scanner III.

Results and discussion

A maximum of 50% of cultures could be established ini-tially. The nodal explants exhibited bud break within 7 days in MS growth regulator-free initiation medium and elongation of axillary buds were achieved in 2 weeks with-out any callus formation at the proximal end (Fig. 1A).

These cultures on subculture to MS medium supple-mented with different combinations of BA and NAA gave varying responses (Table 1). Maximum shoot induction (fi ve shoots per explant) was achieved in MS medium sup-plemented with 1.5 mg l−1 BA and 0.1 mg l−1 NAA (Fig. 1B). Continuous subculturing in the same medium led to vitri-fi cation of the cultures, increased callus formation, and de-creased culture vigor followed by death. Vitrifi cation could be overcome by transferring to lower concentrations of BA and NAA. MS medium supplemented with 0.05 mg l−1 BA and 0.01 mg l−1 NAA yielded a constant multiplication rate of 1 : 5 for four or fi ve subculture cycles after which the rate began to come down. This could be overcome by transfer-ring the cultures back to medium that gave the maximum number of shoots. Arya et al. (2002) reported multiplica-tion in a medium containing NAA and BA that was further

enriched with additives such as adenine sulfate, arginine, ascorbic acid, and citric acid. In our studies, no enhancing effect of additives was noticed. Addition of antioxidants such as ascorbic acid and citric acid was effective in reduc-ing browning of cultures due to phenolic exudation. Cul-tures retained for more than 6 weeks showed necrosis of shoot tips that may have been caused by nutritional defi -ciencies (Nair and Seeni 2001), and this can be overcome by reducing the subculturing cycle duration (Patnaik and Debata 1996) to 4 weeks.

Shoots that were pulse-treated in a solution of 100 mg l−1 each of IBA and NOA for 2 h and then for 3 min in 10 mg l−1 chlorogenic acid gave 90% rooting when planted in culture bottles containing a mixture of soil and sand (Table 2). Two to three roots of 2–4 cm in length were produced within a period of 5 weeks (Fig. 1C). These were then transplanted to polythene bags, although the establishment rate was only 30% due to disturbance of the root system while transplanting. This transplantation shock could be over-come by using coir as a matrix for rooting. When coir was used as a matrix, 95% of the pulse-treated shoots rooted (Table 2). Three to fi ve roots of 2–5 cm in length were produced within 5 weeks (Fig. 1D). The produced roots were profusely branched in contrast with the unbranched roots in the sand medium. This may be due to the effect of the liquid medium in which the coir was immersed. These plantlets were transferred along with the coir to polythene bags fi lled with sand and soil mixture, resulting in a high establishment rate of 80%. Nair and Seeni (2001) reported that MS solid medium supplemented with IAA, IBA, NAA, and 7 gl−1 agar, for which IAA under initial dark conditions gave the maximum rooting within a period of 5

464

weeks in Celastrus paniculatus. However, in our trials, me-dium with IAA produced roots that were hard and brittle, black in color, and diffi cult to establish.

In order to reduce the cost and time involved, attempts were made to directly plant the in vitro grown shoot tips in polythene bags fi lled with river sand and a mixture of river

sand and coir pith compost (1 : 1). These were then kept in humid chambers in greenhouses and shoots were success-fully rooted (98%–99%) within 9 days, hence reducing the number of separate steps and the time for root induction and hardening (Table 2). Rooting could be obtained with well-developed root systems (Fig. 1E). River sand and coir pith mixture was found to be more suitable than river sand alone. A tuft of well-branched roots was produced with an average of four roots per shoot. A separate hardening step was not required because 100% of the rooted plants accli-matized with rooting (Fig. 1F). These plantlets could be transferred to the fi eld within 15 days after removal from in vitro conditions. This method has been found to be suc-cessful in other species also (Martin et al. 2005; Geetha et al. 2005). The fi eld-transferred plants performed as nor-mally as the conventionally raised plants (Fig. 1G, H). HPTLC fi ngerprint profi les of the regenerated and mother plants were identical, indicating their qualitative chemical similarity (Fig. 2).

Conclusions

The described method can be successfully employed for the large-scale multiplication and establishment of Celas-trus paniculatus, an endangered medicinal plant. The use of a simple multiplication medium and a single-step ex vitro rooting with simultaneous hardening is a marked achievement and can be used for low-cost large-scale micropropagation and restoration for this important threat-ened medicinal plant within a short period of time.

Acknowledgments The authors are thankful to Sir Dorabji TATA Trust, Mumbai, for fi nancial assistance, and to Arya Vaidya Sala for providing facilities and support.

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Arya V, Singh RP, Shekhawat NS (2002) A micropropagation proto-col for mass multiplication and off-site conservation of Celastrus paniculatus – a vulnerable medicinal plant of India. J Sustain For-est 14:107–120

Table 2. Rooting response of Celastrus paniculatus to various treatments

Treatment Planting Time for Mean no. of Mean Type of Rooting (%) Establishment(%) Hardening media rooting roots length of roots roots (cm)

Pulse Sand 5 weeks 2 ± 0.66 0.8–2.0 Unbranched 90 30 Required treatmentPulse Coir in 5 weeks 3 ± 0.66 3–5 Highly 95 80 Required treatment liquid media branchedMS solid Sand 5 weeks 2.2 ± 1.03 1.0–1.5 Unbranched, 50 10 Required media with hard, blackIAANil River sand 9 days 2.8 ± 0.63 1–2 Unbranched 98 100 Not requiredNil River sand 9 days 9.5 ± 1.08 1.0–2.5 Tuft of roots 99 100 Not required and coir pith

IAA, indole-3-acetic acid

A

B

Fig. 2. High performance thin-layer chromatographic scans (220 nm) of leaves of A Celastrus paniculatus mother plant and B Celastrus paniculatus tissue-cultured plants

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