Advances in Biological Research 4 (4): 211-216, 2010 ISSN 1992-0067 IDOSI Publications, 2010

Rapid in vitro Multiplication and Plant Regeneration from Nodal Explants of Andrographis neesiana: A Valuable Endemic Medicinal Plant
1

S. Karuppusamy and 2K. Kalimuthu

Department of Botany, The Madura College, Madurai-625 011, Tamil Nadu, India 2 Department of Microbial Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai-625 021, Tamil Nadu, India

Abstract: A rapid and efficient method for the large-scale propagation of an endemic medicinal plant, Andrographis neesiana Wight, through in vitro culture of nodal explants obtained from 30-d-old aseptic seedling has been developed. High frequency direct shoot proliferation was induced in nodal explants cultured on Murashige and Skoogs medium supplemented with thidiazuron. Amongst the various cytokinins tested (BAP, kinetin, thidiazuron and 2-isopentyl adenine), thidiazuron proved to be the most effective. The shoot forming capacity of the nodal explants was influenced by the thidiazuron concentration (1-12.5 M) and the optimal response was observed at 10 M thidiazuron, which induced an average of 34 shoots in 94% of the cultures within 4 wk. Significant differences were recorded in terms of average number of shoots per explant (8.6-34.1) among the different concentrations of thidiazuron investigated. Concentrations of all cytokinins tested reach a level that can be considered above the optimum level, as marked by a reduced frequency of shoot proliferation. The multiple shoots obtained on various concentrations of thidiazuron failed to elongate even after transfer to hormone-free MS medium. Elongation of the induced shoots was achieved on MS basal medium supplemented with 1.0 M GA3 within 2 wk. A proliferating shoot culture was established by repeatedly subculturing the original nodal explants on shoot multiplication medium after each harvest of the newly formed shoots. The explants retained their morphogenic potential even after three harvests. Therefore, in 90 d, about 60-70 shoots were obtained from a single nodal explant and the nodal explants from primary shoots further regenerated equivalent number of shoots, depicting their high frequency regeneration potential in A. neesiana. Rooting was best induced in 94% of shoots cultured on MS medium supplemented with 2.5 M indole-acetic acid (IAA), within a wk. The plantlets were successfully transferred to soil after hardening with a 92% survival rate. The system is rapid: the initiation of shoot buds to the transplanting of regenerants to soil is completed in 8-9 wk. Key words: Andrographis neesiana % Axillary proliferation % Micropropagation % Medicinal plant % Nodal explants INTRODUCTION Andrographis neesiana Wight (Acanthaceae), is an endemic to Peninsular India, used as a herbal medicine by local communities. It is laxative, bitter and overcomes difficulty in breathing, burning sensation, cough, edema, thirst, skin diseases, syphilitic ulcers, worms, acidity and liver complaints [1]. Two new flavonoids, 2',4',6',2,3,4hexamethoxychalcone and 5-hydroxy-7,2',5'trimethoxyflavone together with a known flavone glycoside, echioidinin 5-O-beta-D-glucopyranoside were isolated from the whole plant of Andrographis neesiana [2]. The high demand for its flavone glycoside andrographolide which is originally extracted from A. paniculata by the pharmaceutical industries is largely met by extraction of the compound from wild populations; however, the commercial exploitation of this compound is hampered due to its limited availability. Andrographis neesiana is a potential alternative source of andrographalide. The heavy demand of andrographolide in Indian as well as international markets has motivated Indian farmers to start commercial cultivation of this medicinal plant [3]. Conventional propagation of this species is limited to vegetative means,

Corresponding Author: S. Karuppusamy, Department of Botany, The Madura College, Madurai-625 011, Tamil Nadu, India

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which is difficult and slow in meeting the commercial quantities required [4]. Variability among the seed-derived progenies and scanty and delayed rooting of seedlings curbs its propagation via seeds. Micropropagation is the proven method for efficient in vitro propagation of medicinal and aromatic plants and for commercial exploitation of valuable plant-derived pharmaceuticals [5, 6]. Excepting a preliminary report on plant regeneration via somatic embryogenesis (Martin 2004), no comprehensive in vitro studies have been undertaken in A. neesiana. The published protocol for its related species utilizes lengthy culture period (105 days) for initiation of somatic embryos from callus and is limited by poor conversion of embryos to plantlets, which genuinely limits its possible use in rapid generation of commercial scale planting material. Considering the medicinal importance of A. neesiana, the present investigation was undertaken to develop a rapid and efficient in vitro multiplication and regeneration system of this species using in vitro nodal explants. MATERIALS AND METHODS Mature capsules of A. neesiana were collected from the natural population of Palni hills, Western Ghats, Tamil Nadu, India during the month of November 2008. Capsules were washed thoroughly in running tap water for 30 min to remove any adherent particles, followed by 30 min of washing with a 0.1% (v/v) aqueous solution of Tween 20 (Qualigens, Mumbai, India). The fruits were then rinsed in 70% ethanol for 2 min, surface sterilized with 0.2% (w/v) aqueous HgCl2 solution for 5 min and subsequently rinsed five times with sterile distilled water under sterile conditions. The capsules were blotted dry on sterile filter paper and then carefully dissected to expose the seeds. The seeds were inoculated onto Murashige and Skoog (MS) [7] basal medium for germination. Three nodal segments (1.0 cm in length) were obtained from 30-d-old aseptic seedlings, the middle and bottom segments of which contained a single node with a dormant a xillary bud. The scale leaves were removed from the axillary buds of the nodal segments. All three of the nodal segments, upper, middle and top, were used as explants. The explants were inoculated in a vertical upright position in 150 ml x 1.5 cm wide glass tubes, with one explants per tube, each containing 15 ml of MS basal medium augmented with various cytokinins: thidiazuron (TDZ), 6-benzylaminopurine (BAP), kinetin and 2isopentenyl adenine (2-iP) at different concentrations

(1.0, 2.5, 5.0, 7.5, 10.0 and 12.5 M) in order to induce multiple shoots MS medium without cytokinin served as control. All culture media were supplemented with 3% (w/v) sucrose and 0.7% (w/v) agar agar (Hi-Media, Mumbai, India). The pH of the medium was adjusted to 5.8 prior to autoclaving at 15 psi and 121C for 20 min. All the cultures were maintained at 252C under a 16-h photoperiod with a photosynthetic photon flux density of 35 mol m?2 sG1 provided by cool white fluorescent tubes. After 4 wk in culture, the efficacy of each medium variant on shoot proliferation and growth was determined by recording the frequency of primary explants developing shoots, the number of shoots per explant and the length of the shoots. The explants with shoot clusters after 4 wk of culture on growth hormone-supplemented medium were transferred to MS basal medium for shoot elongation. The explants with shoot clusters, produced after 4 wk of culture on MS medium containing 10.0 M thidiazuron, were transferred to MS medium supplemented with varied concentrations of gibberellic acid (0.1, 0.5, 1.0, 2.5 and 5.0 M) for 2 wk to allow for the elongation of shoots. The mother explants were repeatedly subcultured on shoot multiplication medium after each harvest of the elongated shoots for further induction and harvest of shoots till the third subculture. The elongated shoots (3-4 cm) with two to three pairs of leaves were excised and transferred to MS medium supplemented with various auxins: indole-acetic acid (IAA), indole-3-butyric acid (IBA) and -naphthalene acetic acid (NAA) at different concentrations (0.5, 1.0, 2.5 and 5.0 M) for rooting. Data on percentage of rooting, mean number of roots, mean root length and duration of root initiation were recorded. Plantlets with well-developed roots were removed from the culture medium, washed gently under running tap water and transferred to plastic pots containing soil, vermiculite and vermicompost (1:1:1). Plants were covered with transparent polyethylene bags to maintain adequate moisture for a week and transferred to the greenhouse (28C day, 20C night, 16-h day-length, 70% relative humidity). After a week, the plastic covering was gradually removed and the plantlets were maintained in the greenhouse in earthen pots containing normal garden soil until they were transplanted to the nursery. Experiments were set up in a completely randomized design and each treatment had three replicates of 20 explants each. All data are statistically analyzed by ANOVA followed by Newman-Keuls multiple range test for mean comparison.

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RESULTS AND DISCUSSION Establishment of Aseptic Plantlets: Ninety-six percent of A. neesiana seeds germinated within 5 d of inoculation on a growth regulator-free MS basal medium. The seeds developed into plantlets (3-4 cm) consisting of 2-3 pair of leaves within 15 d of germination. Nodal explants from these plantlets were subsequently used for all experiments. Shoot Multiplication and Proliferation: The morphogenetic responses of nodal explants to various cytokinins (TDZ, BAP, kinetin and 2-iP) were evaluated (Table 1). Nodal explants cultured on growth regulatorfree MS medium showed no sign of bud break even after 30 d. Addition of a cytokinin was essential to induce bud break and multiple shoot formation from the explants. Of the four cytokinins tested, TDZ was more effective than others in inducing shoot development and multiple shoot induction. The difference in regeneration response between the middle and bottom nodal explants was insignificant (data not shown). The upper nodal segment showed no regeneration response in any of the cytokinincontaining medium, suggesting that the presence of an axillary bud was essential for development and induction of multiple shoots in A. neesiana. The nodal segments responded by an initial enlargement of the dormant axillary buds followed by bud break within a week and multiple shoot induction and

proliferation (Fig. 1a) within 4 wk of culture on TDZ containing media. The new shoots developed adjacent to the axillary shoot. The frequency of axillary shoot proliferation and the number of shoots per explant increased with increasing concentration of TDZ, up to the optimal level (Table 1) such that TDZ at 10.0 M showed the highest shoot regeneration frequency (94%) and number of regenerated shoots (34.1) (Fig. 1b). The frequency of shoot proliferation was relatively low and there were fewer shoots per explant when medium was supplemented with kinetin, BAP or 2-iP instead of with TDZ (Table 1) (Fig. 1c). The shoot buds that developed on medium containing BAP and 2-iP were stunted and often fascinated and subsequently failed to elongate. The formation of stunted shoots, or the fasciation of the shoots formed on BAP-supplemented medium, has been reported for several plant species such as Rhododendron spp. [8] and Dalbergia sisoo [9]. Thus, TDZ when added singly in the medium was the most effective plant growth regulator indicating the cytokinin specificity of nodal explants of A. neesiana for multiple shoot regeneration. The stimulating effect of TDZ on multiple shoot formation has been reported earlier for several medicinal and aromatic plant species [10]. Shoot Elongation: In the present study, TDZ alone induced both multiple shoot bud induction and proliferation, however, the multiple shoots obtained on 5.0, 7.5, 10.0 and 12.5 M of TDZ failed to elongate

Table 1: Effects of phytohormoes on the induction of axillary shoots, callus and root formation in the nodal explants of A. neesiana after 4 weeks of culture on MS medium. Values represent mean SE (n = 15); means followed by different letters within a column are significantly different at P < 0.05 using Tukey-Kramer multiple comparisons test Phytohormones Hormone free BAP Concentrations [M] 00 0.1/0.0 0.5/0.5 1.0/0.5 2.0/0.5 3.0/0.5 0.1/0.0 1.0/0.5 2.0/0.5 3.0/0.5 4.0/0.5 5.0/0.5 0.5/0.0 1.0/0.0 2.0/0.5 1.0/0.5 2.0/0.5 3.0/0.5 Axillary shoots number 1.9 0.17a 2.5 0.22a 2.7 0.25ab 3.7 0.36bc 3.9 0.35c 2.1 0.17a 2.8 0.24a 2.6 0.23a 5.7 0.48b 6.2 0.5b 7.0 1.07b 2.1 0.23a 6.2 0.92d 5.1 0.42bc 3.0 0.22ab 6.7 0.68cd 8.1 0.97d Length [cm] 3.31 0.23a 3.65 0.30ab 3.67 0.31ab 3.97 0.28ab 4.63 0.37b 3.38 0.27a 4.54 0.45ab 4.53 0.42ab 5.21 0.45b 5.10 0.42b 5.72 0.38b 3.5 0.22a 4.5 0.25b 4.7 0.25b 5.0 0.32b 5.2 0.35b 5.7 0.36b Callus formation [%] 50 60 80 100 30 Rooting [%] 70 90 50 50 40

TDZ

2iP

Kn

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Fig. 1: In vitro multiplication of Andrographis neesiana Wight. a. Axillary bud breaking of nodal explant within a week; b. Multiple shoo developed in medium containing TDZ 10 M after 4 week; c. Multiple shoots from medium supplemented BAP 4 M in the culture medium after 4 week; d-g. Multiple shoots in the medium containing TDZ 5.0, 7.5, 10.0 and 12.5 M after 4 week of culture; h-j. In vitro rooting of microshoots on medium containing IAA in different concentrations (Figs. d-g) on the same medium. TDZ has often been reported to stimulate shoot proliferation while inhibiting shoot elongation [11]. The multiple shoots obtained on various concentrations of TDZ failed to elongate even after transfer to hormone-free MS medium (data not shown). Hence, it was necessary to develop a suitable media for proliferation and elongation of shoot clumps. GA3 at 0.1-5.0 M was used in efforts to stimulate shoot elongation. Incorporation of 1.0 M GA3 enhanced the shoot elongation by 2.25-fold in 96% of shoot cultures within 2 weeks, whereas the shoot elongation was completely absent in medium devoid of GA3. The explants continuously produced shoots during successive subculture on MS medium supplemented with 10 M TDZ. These mother explants could be subcultured twice without affecting their shoot forming potential (data not shown). It is generally desired to establish cultures that can be continuously multiplied in order to produce a regular supply of shoots for field transfer. For this the nodal explants from the primary shoots, obtained after each harvest, were repeatedly subcultured on MS medium supplemented with 10 M TDZ to produce multiple shoots. Therefore, in 90 d about 60-70 shoots were obtained from a single nodal explant and the nodal explants prepared from these primary shoots further regenerated equivalent number of shoots depicting the high frequency regeneration potential of these explants in A. neesiana. Rooting: Among the different auxins tested, IAA was found most effective in inducing roots. The maximum frequency of root formation (94%), number of roots (7.2) and root length (2.1 cm) were achieved within a wk when shoots were cultured on medium containing 2.5 M IAA (Table 2, Figs. 1h-j). The roots were induced directly from the shoot base without an intervening callus phase on media supplemented with IAA. In contrast, rooting of shoots occurred through an intervening callus phase on IBA and NAA supplemented media.

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Table 2: Effects on NAA and IBA on rooting in vitro produced shoots of A. neesiana after 4 weeks of culture on MS medium. Values represent mean SE (n = 15); means followed by different letters within a column are significantly different at P < 0.05 using Tukey - Kramer multiple comparisons test Phytohormones NAA Concentrations [M] 0/0 0.5 1.0 1.5 2.0 2.5 IAA 0.0 0.5 1.0 1.5 2.0 2.5 IBA 0.0 0.5 1.0 1.5 2.0 2.5 roots/shoots number 1.2 0.21a 9.2 0.72b 15.5 1.24c 20.5 1.13d 20.6 1.80d 22.3 0.84d 1.1 0.25a 12.0 0.48b 20.1 0.71c 25.2 0.85d 28.4 0.68e 30.2 0.62e 1.1 0.25a 5.0 0.48b 8.1 0.71b 9.2 0.85b 8.4 0.68b 7.2 0.62b Root length [cm] 0.2 0.03a 1.5 0.14b 2.0 0.15b 3.2 0.18c 3.0 0.18c 3.3 0.14c 0.23 0.05a 0.86 0.07b 1.62 0.07c 1.45 0.07c 1.63 0.08c 2.83 0.06d 0.23 0.05a 0.86 0.07b 1.62 0.07b 1.45 0.07b 1.03 0.08b 0.83 0.06a Rooting [%] 10 20 20 50 60 80 20 60 100 100 100 100 20 60 70 70 70 60 Basal callus [%] 20 40 50 80 90 100 -

Acclimatization: Plantlets with four to five pairs of fully expanded leaves and well-developed roots were successfully acclimatized in the greenhouse, in pots containing soil, vermiculite and vermicompost (1:1:1) within 2 wk and eventually established in a nursery. About 92% of the micropropagated plants survived and showed normal growth and morphological characteristics. The present study demonstrates a simple and efficient method for high frequency direct shoot regeneration from nodal explants of A. neesiana. The system is rapid: starting with the initiation of tissue culture and ending with the transplanting of regenerants to soil 8-9 wk later. Ninety-four percent of the explants proliferated, with an average 34 shoots per explant, on MS medium supplemented with 10 M TDZ. Such a high regeneration frequency would be useful for mass propagation and multiplication of this valuable medicinal plant. Use of nodal explants ensures clonal multiplication of selected species and explants prepared from in vitro raised plantlets rule out contamination associated with ex vitro explants. Commercial exploitation of this developed protocol is possible, as the nodal segments from in vitro raised shoots can be employed as propagules for further multiplication, obviating the dependence on field material. The in vitro cultures can produce a stable supply of the bioactive compound (andrographolide). Moreover, this efficient and reliable plant regeneration system via direct shoot organogenesis can be exploited for genetic manipulation experiments for enhanced biomass growth and eventual increased production of andrographolide. 215

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