Large scale in vitro propagation of Stevia rebaudiana (bert) for commercial application: Pharmaceutically important and antidiabetic medicinal herb

Industrial Crops and Products 37 (2012) 111–117

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Large scale in vitro propagation of Stevia rebaudiana (bert) for commercial application: Pharmaceutically important and antidiabetic medicinal herb M. Thiyagarajan, P. Venkatachalam ∗ Plant Genetic Engineering and Molecular Biology Lab, Department of Biotechnology, Periyar University, Salem-11, Tamil Nadu, India

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Article history: Received 19 June 2011 Received in revised form 22 October 2011 Accepted 24 October 2011 Available online 7 January 2012 Keywords: Stevia rebaudiana In vitro propagation Nodal explant MS medium Large scale production Plant growth regulators

a b s t r a c t Stevia rebaudiana is a valuable medicinal plant species and it is being used for the treatment of diabetes. Currently, there is a high demand for raw material of this medicinal herb due to ever increasing diabetes disorder among the population. In order to meet the increased demand an efficient in vitro propagation of S. rebaudiana was established. Nodal explants collected from the field were cultured on MS basal medium fortified with different concentrations of BAP (0.5–3.0 mg/l) and KIN (0.5–3.0 mg/l) individually for shoot bud induction. In vitro derived nodal buds were cultured on MS medium supplemented with different concentrations (0.5–3.0 mg/l) of BAP and KIN for multiple shoot bud regeneration. In the second experiment, in vitro derived buds were placed on MS medium supplemented with different concentrations of BAP (0.5–3.0 mg/l) in combination with 0.5 mg/l IAA or IBA or NAA for shoot bud multiplication. The highest frequency (94.50%) of multiple shoot regeneration with maximum number of shoots (15.69 shoots/explant) was noticed on MS medium supplemented with 1.0 mg/l BAP. For large scale plant production, in vitro derived nodal bud explants were cultured on MS medium fortified with 1.0 mg/l BAP, in which about 123 shoots/explant were obtained after three subcultures on the same media composition. Elongated shoots (>2 cm) dissected out from the in vitro proliferated shoot clumps were cultured on half-strength MS medium containing different concentrations of NAA (0.1–0.5 mg/l) and/or MS medium fortified with various concentrations (0.5–2.0 mg/l) of auxins (NAA, IAA and IBA) for root induction. Highest frequency of rooting (96%) was noticed on half-strength MS medium augmented with 0.4 mg/l NAA. The rooted plantlets were successfully transferred into plastic cups containing sand and soil in the ratio of 1:2 and subsequently established in the greenhouse. The present in vitro propagation protocol would facilitate an alternative method for rapid and large-scale production of this important antidiabetic medicinal plant. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Medicinal plants are of great interest to researchers in the field of biotechnology as most of the drug industries depend, in part, on plants for the production of pharmaceutical compounds (Chand et al., 1997). Diabetes mellitus is a complex and a multifarious group of disorders that disturbs the metabolism of carbohydrates, fat and protein. It results from shortage or lack of insulin secretion or reduced sensitivity of the tissue to insulin. Diabetes mellitus is a common disorder among the Indian population. It is estimated that diabetes would affect approximately 57 million people by the year 2025. The management of diabetes is a global problem until now and successful treatment is not yet discovered. There are many synthetic medicines/drugs developed for patients, but it is the fact that it has never been reported that someone had

∗ Corresponding author. Tel.: +91 9952609915. E-mail address: [email protected] (P. Venkatachalam). 0926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2011.10.037

recovered completely from diabetes (Li et al., 2004). The modern oral hypoglycemic agents produce undesirable and side effects. Thus, alternative therapy is required to shift towards the different indigenous plant and herbal formulations (Satyanarayana et al., 2007). Plant drugs are frequently considered to be less toxic and free from side effects than synthetic one. With the worldwide increasing demand for plant derived medicines, there has been a concomitant increase in the demand for raw material. However, the increasing human and livestock populations affected the status of wild plants, particularly those used in herbal medicine. Stevia rebaudiana (Bert.) is a perennial sweet herb, belonging to the family Asteraceae. It is a natural, non-caloric sweet-tasting plant used around the world for its intense sweet taste. Stevia plant produces zero-calorie diterpene glycoside (Stevioside and Rebaudiooside) in its leaves as natural non-nutritive sweetener which is being used as substitute to sucrose (Chalapathi and Thimmegowda, 1997). The eight types of glycosides viz. rebaudioside (A–F), steviolbioside A and dulcoside A were identified (Starratt and Gijzen, 2004). Stevia making strides has the best alternative of the table

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sugar in the years to come. It is being commercially cultivated in China, Taiwan, Thailand, Korea, Japan, India and Malaysia. In addition, S. rebaudiana possesses hypoglycemic, hypotensive, vasodilating, taste improving, sweetening, antimicrobial properties and increases urination function of the body. It has been found to be non-toxic, non-carcinogenic, non-mutagenic and is devoid of genotoxic effect. It does not affect blood sugar level hence safe for diabetics. The key benefit of Stevia is it stimulates the release of insulin and normalizes blood glucose levels. It is recommended for diabetes and has been extensively tested on animals and has been used by humans with no side effects. Stevia extract and stevioside are officially approved as food additives in Brazil, Korea and Japan. Stevia could be used as a major source of high potency sweetener (alternate to sucrose) in the near future (Starratt and Gijzen, 2004). Currently, S. rebaudiana is being propagated by stem cuttings. Low seed germination percentage is a major limiting factor for large scale cultivation of Stevia plant species for commercial usage. Further vegetative propagation is also limited by the less number of individuals obtained from single plant. Therefore, a suitable alternative method for large scale plant production within a short period is the use of in vitro culture technology. The micropropagation of plants through shoot tip or axillary bud culture allows recovery of genetically stable and true to type progeny. There are few reports on in vitro clonal propagation of Stevia plants using leaf, nodal, internodal segment and shoot tip explants. Bespalhok and Hattori (1997) obtained only embryogenic callus from floret explants of S. rebaudiana. In vitro plant regeneration from shoot tip explants of S. rebaudiana was reported by Patil et al. (1996), Uddin et al. (2006) and Debnath (2008). Sivaram and Mukundan (2003) obtained maximum number of shoot buds (7.9 shoots/explant) from nodal explants on MS medium supplemented with 8.87 ␮M BAP and 5.71 ␮M IAA. Although few tissue culture protocols have been reported in the recent past, there is no efficient regeneration protocol available for commercial scale production of this important medicinal plant. The major goal of this project was to develop an efficient protocol for large-scale production of Stevia plants from an elite germplasm.

2. Materials and methods 2.1. Preparation of explants S. rebaudiana plants were collected from Horticulture Research Station, Tamil Nadu Agricultural University (TNAU), Yercaud, Tamil Nadu and maintained in the greenhouse, Department of Biotechnology, Periyar University, Salem-11. For shoot bud induction, nodal explants were collected from 3 months old plants and were washed in running tap water. Explants were washed with few drops of Tween-20 to remove the superficial dust particles including microbes. Then, they were surface sterilized with 0.1% (w/v) mercuric chloride for 8 min followed by rinsing them for five times with sterile distilled water. Sterilized nodal explants were used for in vitro studies as described below.

2.2. Culture media and growth conditions The culture medium consisted of MS (Murashige and Skoog’s, 1962) salts, vitamins, 3% (w/v) sucrose and the pH of the media was adjusted to 5.6 with 0.1 N NaOH or HCl before adding of 0.7% (w/v) agar. Media (15 ml) were poured into 25 mm × 150 mm culture tubes (Borosil, Mumbai) and autoclaved at 121 ◦ C for 15 min. The cultures were incubated at 24 ± 2 ◦ C under 16/8 h (light/dark cycle) photoperiod (60 ␮E m−2 s−1 ) and irradiance provided by cool-white fluorescent tubes (Philips, India).

2.3. Shoot bud initiation Surface sterilized nodal explants were cultured on MS medium supplemented with different concentrations of BAP (0.5–3.0 mg/l) and/or KIN (0.5–3.0 mg/l) for shoot bud induction. After two weeks of culture, direct shoot bud initiation from the nodal explants was noticed. 2.4. Induction of multiple shoots In order to achieve multiple shoot bud regeneration, two different experiments were performed as described below. 2.4.1. Experiment: I In the first experiment, the effect of different concentrations of two cytokinins on multiple shoot regeneration was examined. Nodal explant derived in vitro regenerated shoot buds as explant source were cultured on MS medium fortified with different concentrations (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) of BAP and KIN individually for multiple shoot bud development. 2.4.2. Experiment: II In the second experiment, the influence of different concentrations of BAP in combination with three auxins on induction of multiple shoots was evaluated. To identify the best auxin for shoot bud multiplication, nodal explant derived in vitro developed shoot buds as explant source were cultured on MS medium containing different concentrations of BAP (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) in combination with 0.5 mg/l IAA or IBA or NAA. 2.5. Large scale production of shoot buds For large scale plant production, in vitro regenerated shoot buds were cultured on MS medium supplemented with 1.0 mg/l BAP using 250 ml flasks. The cultures were subcultured onto the fresh same media composition once in 3 weeks interval. This process was repeated for another three subcultures (each 21 days) to examine the effect of subculture on production of large scale shoot buds. After 65 days of culture multiple shoots were counted for analysis of total number of regenerated shoot buds. 2.6. Rooting of elongated shoots and acclimatization The elongated shoots (>2.0 cm height) were transferred onto half-strength MS medium fortified with different concentrations of NAA (0.1–0.5 mg/l) and full-strength MS medium with various concentrations of IAA or IBA or NAA (0.5–2.0 mg/l) for root induction. Plantlets with well-developed roots were removed from the culture tubes and gently washed under running tap water to remove adhering medium. Subsequently, they were transferred to plastic cups containing sterile sand and soil mixture in 1:2 ratio. The potted plantlets were initially maintained in the controlled environment for two weeks and subsequently they were shifted to the greenhouse. After twenty days, the plantlets were successfully established in the field. 2.7. Statistical analysis Experiments were set up in a completely randomized block (CRB) design and each experiment had three replicates. The cultures were observed periodically and percent of response for shoot bud regeneration, multiple shoots development and rooting. A total number of shoots as well as roots were also recorded by visual observations. The analysis of variance (ANOVA) was performed

M. Thiyagarajan, P. Venkatachalam / Industrial Crops and Products 37 (2012) 111–117 Table 1 Effect of different concentrations of two cytokinins on shoot bud induction from nodal explants of S. rebaudiana. Cytokinin conc. (mg/l)

BAP

KIN

0.5 1.0 1.5 2.0 2.5 3.0 – – – – – –

– – – – – – 0.5 1.0 1.5 2.0 2.5 3.0

Percent of shoot induction (Mean ± SE)

71.42 85.71 75.42 65.42 55.45 50.14 75.00 75.00 50.00 45.00 42.25 37.75

± ± ± ± ± ± ± ± ± ± ± ±

2.02c * 2.88a 3.46c 1.15c 1.73c 1.73d 1.15b 4.04b 1.55d 1.30d 1.25d 1.15e

No. of shoots/explant (Mean ± SE)

1.40 2.00 1.80 1.60 1.20 1.25 1.66 1.33 1.00 1.00 1.00 1.00

± ± ± ± ± ± ± ± ± ± ± ±

0.24 0.18 0.24 0.24 0.20 0.25 0.66 0.33 0.57 0.57 0.32 0.15

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Table 2 Effect of different concentrations BAP and KIN on shoot bud multiplication from in vitro derived shoots buds of Stevia rebaudiana. Cytokinin conc. (mg/l)

BAP

KIN

0.5 1.0 1.5 2.0 2.5 3.0 – – – – – –

– – – – – – 0.5 1.0 1.5 2.0 2.5 3.0

Percent of multiple shoot bud induction (Mean ± SE)

80.00 94.50 90.00 70.00 60.00 66.66 80.00 60.00 66.59 60.00 57.55 48.15

± ± ± ± ± ± ± ± ± ± ± ±

2.80b * 1.32a 1.15a 1.73c 1.34d 2.38d 2.30b 2.43d 2.30d 1.73d 1.21e 0.95f

No. of shoots/explants (Mean ± SE)

8.80 15.69 11.22 8.85 6.80 4.75 3.25 2.66 2.50 2.25 2.05 1.75

± ± ± ± ± ± ± ± ± ± ± ±

3.77b * 3.45a 2.91a 3.29b 3.11c 1.71d 0.96d 0.58e 0.58e 0.96e 0.86e 0.75f

* Mean values within the column followed by the same letter in superscript are not significantly different at P < 0.05 level.

* Mean values within the column followed by the same letter in superscript are not significantly different at P < 0.05 level.

using SAS programme. The differences among means were determined by Student–Newman–Keuls Test at 5% significance level.

(0.5–3.0 mg/l). Among the concentrations tested, BAP at 1.0 mg/l was found to be the best concentration for induction of highest percent of shoot bud regeneration (94.5%) with 15.69 shoots/culture (Fig. 1B) and the data were statistically significant at 5% level (Table 2) (Fig. 2). Of the two cytokinins used, BAP was proved to be the most efficient cytokinin for multiple shoot bud regeneration compared to KIN. Both the plant regeneration frequency and the number of shoot buds per culture increased with increasing the BAP concentration up to the 1.0 mg/l. However, shoot bud regeneration frequency as well as the number of shoot buds were declined when the BAP concentration was increased beyond 1.0 mg/l in the medium. Similar results were also reported earlier by Faisal and Anis (2003). The effect of BAP on multiple shoot formation has also been studied in various medicinal plant species such as Chlorophytum borivilianum (Purohit et al., 1994), E. alba (Franca et al., 1995), Ocimum (Patnaik and Chand, 1996), Ceropegia (Patil, 1998) and Gymnema sylvestre (Komalavalli and Rao, 2000).

3. Results and discussion 3.1. Shoot bud initiation In the present study, nodal explants from mature S. rebaudiana plants were placed on MS medium supplemented with different concentrations of BAP (0.5–3.0 mg/l) and KIN (0.5–3.0 mg/l) for shoot bud initiation. Among the cytokinin concentrations tested highest percent of shoot bud regeneration (85.7%) was noticed on MS medium containing 1.0 mg/l BAP while 75% shoot bud regenerations was observed on MS medium fortified with 0.5 mg/l KIN. However the shoot bud induction was suppressed at higher concentrations of cytokinins (Table 1). The nodal explants produced minimum of two shoots on MS medium containing 1.0 mg/l BAP within two weeks of culture (Fig. 1A). The presence of cytokinins in the medium was essential to induce bud break and shoot proliferation from nodal explants. Of the two cytokinins used, BAP was found to be more effective than KIN for shoot bud development from nodal explants (Table 1). The percent of shoot bud proliferation increased with increasing the concentrations of BAP up to 1.0 mg/l, thereafter, shoot bud induction was gradually decreased with further increase the concentration of BAP. In contrast, when KIN concentration was increased beyond 0.5 mg/l, shoot bud regeneration was gradually suppressed. An inhibitory effect of higher concentrations of BAP on shoot formation has also been reported earlier in Pterocarpus marsupium (Anis et al., 2005). The superiority of BAP over other cytokinins in shoot bud regeneration has been well documented in Syzygium alternifolium (Sha Valli Khan et al., 1997) and in P. marsupium (Chand and Singh, 2004). Similarly the influence of BAP on induction of multiple shoot buds from nodal explants has been reported in various plant species, including Quercus euboica (Kartsonas and Papafotiou, 2007), Eclipta alba (Dhaka and Kothari, 2005), Ulmus parvifolia (Thakur and Karnosky, 2007) and Sarcostemma brevistigma (Thomas and Shankar, 2009). 3.2. Multiple shoot bud induction 3.2.1. Experiment I. Effect of different concentrations of two cytokinins on multiple shoot bud development In order to induce multiple shoots, in vitro regenerated shoot buds from nodal explants were cultured on MS medium supplemented with different concentrations of BAP (0.5–3.0 mg/l) and KIN

3.2.2. Experiment II. Effect of different concentrations of BAP in combinations with various auxins on multiple shoot bud regeneration In the second experiment, in vitro developed shoot buds from nodal explants were placed on MS medium supplemented with different concentrations of BAP (0.5–3.0 mg/l) in combination with 0.5 mg/l IAA/IBA/NAA for shoot bud multiplication. Among the combinations used, BAP (1.0 mg/l) + IAA (0.5 mg/l) combination was found to be best for multiple shoot bud induction (8.5 shoots/explants). The highest percent (92%) of multiple shoot bud formation was noticed on MS medium fortified with 1.0 mg/l BAP and 0.5 mg/l IAA combination which was statistically significant at 5% level (Table 3) (Fig. 3). Of the three auxin combinations tested, BAP + IAA combination was found to be superior for induction of highest percent (92%) of multiple shoot bud development, followed by BAP + NAA (83%) and BAP + IBA (75%) combinations. However, both the BAP + NAA and BAP + IBA combinations produced more callus with low percent (50%) of multiple shoot bud regenerations. The lower percent of multiple shoot bud regenerations noticed on a medium containing BAP + NAA and BAP + IBA combinations may be due to the profuse callusing at the basal part of differentiated shoot buds. Similar observation was reported in Jatropha curcas (Kumar et al., 2008). The present study clearly suggests that BAP and IAA combination was found to be best for shoot bud multiplication without producing any callus in the culture as compared to BAP + NAA and BAP + IBA combinations. Nunes et al. (2002) also described that BAP in combination with low concentration of auxin

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Fig. 1. (A) Shoot bud induction; (B) proliferation of multiple shoots from in vitro derived nodal explants; (C) large scale production of Stevia rebaudiana; (D) and (E) rooting of in vitro regenerated shoots; and (F) plantlets growing in plastic cups.

was found to be most effective for multiple shoot bud induction while shoot bud regeneration was inhibited at higher concentrations of auxin in Cedrela fissilis. 3.3. Large scale production of shoots The nodal explants derived in vitro regenerated shoot buds were cultured on MS medium supplemented with 1.0 mg/l BAP and the cultures were repeatedly subcultured for multiple shoot bud regeneration. This process was performed for increasing the number of multiple shoots per culture. After three subcultures maximum number of multiple shoots obtained was 123 shoots/nodal explant (Table 4) (Fig. 1C). Repeated subculture of nodal explants derived shoots through the first three passages could enabled in

continuous production of healthy callus-free shoots without any sign of decline so far. Similar results were also reported earlier in E. alba by Borthakur et al. (2000) and Husain and Anis (2006). Repeated subculture is usually applied for increasing the shoot bud multiplication rate. Prakash et al. (2006) had successfully used this technique to increase the number of shoot buds in Ptertocarpus santalinus. They observed an increase in shoot bud multiplication rate up to the six subculture stage. In contrast, the number of shoots per culture showed a continuous increase in each subculture up to the third subculture stage and thereafter in decreased in Cyclea peltata (Abraham et al., 2010). In the present study, each explant produced on an average of 123 shoots/culture on shoot bud multiplication medium within three subcultures and the number of multiple shoots increased almost six fold (Table 4).

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Fig. 2. Effect of different concentrations BAP and KIN on shoot bud multiplication from in vitro derived shoot buds of Stevia rebaudiana.

Table 3 Effect of different concentrations of BAP in combination with 0.5 mg/l IAA or IBA or NAA on shoot bud multiplication from in vitro derived shoot buds of Stevia rebaudiana. Hormone conc. (mg/l)

Percent shoot regeneration (Mean ± SE)

BAP

IAA

IBA

NAA

0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0

0.5 0.5 0.5 0.5 0.5 0.5 – – – – – – – – – – – –

– – – – – – 0.5 0.5 0.5 0.5 0.5 0.5 – – – – – –

– – – – – – – – – – – – 0.5 0.5 0.5 0.5 0.5 0.5

*

86.0 92.0 83.0 75.0 68.0 63.5 75.0 50.0 75.0 75.0 50.0 47.5 75.0 83.0 75.0 60.0 20.0 17.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.30b * 1.73a 3.30b 2.30c 1.70d 1.25d 2.80c 1.70e 1.15c 2.04c 1.73e 1.43e 3.15c 2.04b 1.73c 1.15d 2.75f 1.56f

No. of shoots/explant (Mean ± SE)

6.00 8.50 5.40 3.30 2.75 1.85 3.75 4.25 4.00 4.50 2.75 2.21 2.25 2.75 3.50 2.00 1.50 1.25

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.22b * 2.64a 1.47b 0.33c 0.62d 0.32e 0.47c 1.47b 0.70b 2.64b 0.28d 1.32d 1.47d 0.47d 0.28c 0.40d 0.28e 0.57e

Length of shoots (cm) (Mean ± SE)

3.83 6.33 4.03 3.40 3.50 3.20 3.75 5.02 2.50 3.35 3.50 3.10 2.66 4.20 3.60 4.50 2.30 2.10

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.64d * 2.44a 1.17c 1.34d 1.17d 1.06d 3.43d 1.22b 2.26e 2.17d 0.40d 1.02d 0.44e 3.43c 0.75d 2.62c 1.42e 1.05e

Mean values within the column followed by the same letter in superscript are not significantly different at P < 0.05 level.

Fig. 3. Effect of different concentrations of BAP in combination with 0.5 mg/l IAA or IBA or NAA on shoot bud multiplication from in vitro derived shoot buds of Stevia rebaudiana.

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Table 4 In vitro derived shoot buds from nodal explants were cultured on MS medium enriched by 1.0 mg/l BAP and subcultured onto the same medium for large scale propagation. BAP (1.0 mg/l)

No. of shoots/explants (Mean ± SE)

1st subculture 2nd subculture 3rd subculture

49.0 ± 9.80c * 74.0 ± 4.60b 123.0 ± 10.53a

3.5. Acclimatization

* Mean values within the column followed by the same letter in superscript are not significantly different at P < 0.05 level.

Table 5 Effect of different concentrations of auxins on in vitro rooting of elongated shoots of Stevia rebaudiana. No. of roots/shoot (Mean ± SE)

Root length (cm) (Mean ± SE)

Half strength MS medium + NAA 80 ± 2.8b * 0.1 0.2 80 ± 1.7b 0.3 80 ± 0.5b 0.4 96 ± 1.2a 0.5 94 ± 1.7a

4.0 ± 1.79c * 6.2 ± 1.43b 8.3 ± 1.34b 14.4 ± 3.41a 11.6 ± 2.51b

4.75 ± 0.92c * 5.32 ± 0.95c 5.70 ± 0.68c 11.5 ± 1.33a 8.87 ± 1.09b

MS medium + NAA 0.5 1.0 1.5 2.0

86 ± 2.3b 60 ± 5.1d 50 ± 4.04d 0.00 (callusing)

8.3 ± 1.28a 3.4 ± 0.35d 2.5 ± 1.06b 0.00 (callusing)

6.13 ± 1.10b 4.43 ± 0.84c 2.54 ± 0.64d 0.00 (callusing)

MS medium + IAA 0.5 1.0 1.5 2.0

73 ± 2.88c 80 ± 2.30b 45 ± 1.10d 0.00 (callusing)

3.8 ± 1.32d 4.6 ± 2.06c 4.2 ± 0.09c 0.00 (callusing)

4.40 ± 0.49c 5.40 ± 0.49c 3.50 ± 0.20d 0.00 (callusing)

MS medium + IBA 0.5 1.0 1.5 2.0

68 ± 2.30d 42 ± 1.73e 38 ± 0.57f 0.00 (callusing)

4.8 ± 0.89c 3.1 ± 0.78d 2.8 ± 0.63d 0.00 (callusing)

5.03 ± 0.90c 3.43 ± 0.73d 2.00 ± 0.11c 0.00 (callusing)

Auxins conc. (mg/l)

Percent of rooting (Mean ± SE)

roots induced on half-strength MS medium fortified with different concentrations of NAA were found to be thick and long (11.5 cm length) with fine roots whereas the roots were thin and short (2.0 cm length) on MS medium augmented with different concentrations of NAA or IAA or IBA.

* Mean values within the column followed by the same letter in superscript are not significantly different at P < 0.05 level.

3.4. Rooting of shoots For rooting, elongated shoots were transferred to either halfstrength MS medium supplemented with different concentrations of NAA (0.1, 0.2, 0.3, 0.4 and 0.5 mg/l) or MS medium fortified with various concentrations of IAA or IBA or NAA (0.5, 1.0, 1.5 and 2.0 mg/l). Shoots produced roots within two weeks of culture and the data were recorded. The first roots emerged directly from the basal part of the shoots 7 days after culture with no intervening callus. The highest percent (96%) of root induction was observed on half-strength MS medium fortified with 0.4 mg/l of NAA and maximum number of roots obtained was 14.4 roots/shoot and it was statistically significant at 5% level (Fig. 1D and E) (Table 5). The percent of rooting was increased with increasing the concentration up to 0.4 mg/l while the rooting was declined beyond 0.4 mg/l NAA used in the half-strength MS medium. Similar observation was made by Thiruvengadam and Jayabalan (2000), Arockiasamy et al. (2002), Raman and Jaiwal (2000) and Jeyakumar and Jayabalan (2002). It is interesting to note that the rooting response was decreased when the auxin (NAA, IBA and IAA) concentration was increased from 0.5 to 1.5 mg/l and root initiation was inhibited at higher concentration (2.0 mg/l) (Table 5). Results of this study are corroborated by the findings of a previous reports (Anitha and Pullaiah, 2002) and (Latha et al., 1998). Among the three auxins used, maximum percent of rooting was obtained on MS medium fortified with NAA which was found to be superior for rooting over other two auxins (IAA and IBA). It is interesting to note that, the

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