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In vitro owering of Withania somnifera Dunal.An important antitumor medicinal plant

K.V. Saritha *, C.V. Naidu **
Plant Biotechnology Division, Department of Biotechnology, Sri Venkateswara University, Tirupati 517502, AP, India Received 8 June 2006; received in revised form 9 December 2006; accepted 14 December 2006 Available online 12 January 2007

Abstract In vitro owering, in vitro fruiting and effective micropropagation protocol were studied in Withania somnifera, an antitumor medicinal plant using axillary bud explants. The Murashige and Skoogs medium (MS) supplemented with N6-benzyl adenine (BA) 2.0 mg l1 and a-naphthalene acetic acid 0.1 mg l1 was found optimum for production of multiple shoots. The regenerated plantlets were found to form tiny green oral buds after 46 weeks of culture in MS medium supplemented with Kinetin (0.54.0 mg l1) and indole-3-acetic acid (0.1 mg l1). In vitro fruiting was observed in the presence of Kn (2.0 mg l1) and IAA (0.1 mg l1). This paper describes in vitro owering system to overcome problems associated with ower growth and development as well as fruit and seed production in vitro. # 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: In vitro owering; In vitro fruiting; Seed set; Withania somnifera

1. Introduction Withania somnifera Dunal. (Ashwagandha) belongs to the family of Solanaceae, growing to a height of 30150 cm. It is one of the important medicinal cash crops in many states of India. It has antibiotic, antiviral, antiamoebic, antiarthritic and anti-inammatory properties [1]. Withaferin A found in this plant shows marked tumour-inhibitory activity [2]. Its fruits and seeds are diuretic, hypnotic, maticatory and employed in curdling plant milk to prepare vegetarian cheese. They are also rich in saponins and can be used as substitutes of soap [35]. Multiple shoot formation in in vitro culture is more advantageous over a single shoot formation for rapid clonal multiplication as well as for its conservation. The induction of multiple shoots through axillary branching is now recognized as a useful technique for propagation and in vitro conservation of threatened plants [6]. Many authors have analyzed the resident meristem of juvenile origin for rapid clonal propaga-

Abbreviations: BA, 6-benzyladenine; Kn, kinetin; NAA, a-naphthalene acetic acid; IAA, indole-3-acetic acid * Corresponding author. Tel.: +91 877 2249496; fax: +91 877 2249611. ** Corresponding author. Tel.: +91 877 2260386; fax: +91 877 2249611. E-mail addresses: sarikv@yahoo.com (K.V. Saritha), challagundlav@yahoo.co.in (C.V. Naidu). 0168-9452/$ see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2006.12.016

tion of medicinal plant taxa such as Gymnema sylvestres [7] and Gomophrena ofcinalis [8]. Although in vitro owering has been reported for many plant species, for example, Oscimum basilicum [9] and Panax ginseng [10], reports on in vitro fruiting and seed set are limited [11,12]. In vitro owering was observed on MS medium with BA in Dendrocalamus hamiltonii [13]. Floral induction in Date palm was observed in seedlings grown in vitro [14]. In Arabidopsis thaliana isopentenyl adenosine induced oral bud formation [15]. In roses, the combination of Thidiazuron (TDZ) or Zeatin (Zn) and NAA induced owering in vitro [16]. In tobacco in vitro owering was observed after the addition of growth regulators such as BA and IAA [17]. In vitro owering of Capsicum fruitescens was observed in liquid MS medium without growth regulators [18]. In vitro owering and pod formation were observed in Arachis hypogea on MS medium with Kinetin or BA and IAA/NAA [19]. These ndings were similar to the results observed by us in W. somnifera. Knowledge on in vitro owered plantlets for the formation of fruits and seeds is highly valuable. Further knowledge on the in vitro fruiting and seed set might aid studies seeking a better understanding of fruiting and seed set or for obtaining improved strategies to overcome problems associated with premature fruit drop or poor seed set in many greenhouse or eld grown fruit bearing plants. There have been reports on in vitro

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regeneration of shoots and roots for W. somnifera but in vitro owering has not been known [20,21]. Hence the present investigation was undertaken to study in vitro owering and fruiting using nodal explants. 2. Materials and methods 2.1. Plant material Seeds were collected from Sri Venkateswara Ayurvedic Pharmacy, Narasingapuram, Tirupati, India. 2.2. Surface sterilization Seeds were washed in running tap water for 2 min and then with tween 20 (10%) for 5 min. Then the seeds were sterilized in 70% alcohol for 1 min and 0.01% HgCl2 for 2 min followed by rinsing them twice with sterile double distilled water. 2.3. Seed germination Seeds were surface sterilized and aseptically germinated in the Murashige and Skoog medium [22] with 30% sucrose and solidied with 0.8% agar. 2.4. Sub-culturing From 4 to 6 weeks old seedling, axillary buds were isolated and cultured on various nutrient media for multiple shoot production. For this purpose the MS basal medium supplemented with BA/Kn (1.04.0 mg l1) along with NAA (0.1 mg l1) was used and for owering, and fruiting the MS medium with BA/Kn (0.54.0 mg l1) and IAA (0.1 mg l1) was used. Hormones were added to medium, adjusted to pH 5.8 gelled with 0.8% (w/v) Bactoagar (Qualigens, India) and autoclaved for 20 min at 121 8C and 15 lb. 2.5. Culture conditions The growth room conditions maintained for in vitro cultures were 26 2 8C and 6070% relative humidity. Light intensity was 2000 lux with 20 h light and 4 h dark conditions. For each hormone treatment 10 experiments were conducted with 20 replicates. 3. Results and discussion 3.1. Seed germination and multiple shoot production Seeds showed germination in vitro 1 week after inoculation. The seedlings showed growth of 8 cm long within a period of 36 weeks. In the MS medium supplemented with BA (2.0 mg l1) and NAA (0.1 mg l1) a large number of multiple shoots (12) developed within 4 weeks (Fig. 1A) (Table 1). Considerable shoot elongation (4.2 0.1 cm) was observed in the presence of Kinetin (2.0 mg l1) and NAA (0.1 mg l1) (Fig. 1B). Kn was ineffective in inducing

proliferation. For every 4 weeks shoots were transferred on to the new medium. 3.2. In vitro owering and in vitro fruiting Sub-culture of plants regenerated in MS medium with BA and NAA or Kn and NAA on a medium of the same composition showed continuous growth without any sign of further morphogenetic differentiation. However, plants transferred on to the Kn (0.54.0 mg l1) and IAA (0.1 mg l1) medium showed owering after four successive subcultures (each lasting 4 weeks) which were grown on medium containing BA (2.0 mg l1) and NAA (0.1 mg l1) for 4 weeks. The rst ower appeared after 4 weeks of transfer of plants to the new medium and later ve to ten owers per plant were developed within the next 20 days. All of the adventitious shoots produced ower buds. Regenerants subcultured on the BA and IAA, Kn, IAA medium failed to ower in vitro. The regenerated plantlets were found to form tiny green oral buds after about 46 weeks of culture in culture bottles in the MS medium supplemented with Kn (0.54.0 mg l1) and IAA (0.1 mg l1). Within 1015 days of their formation, most of the oral buds either turned brown or dropped during their development. However, some exhibited growth and developed into white owers, which matured. Similar results were reported in Oscimum basilicum where BAP (4.0 mg l1) and IAA (3.0 mg l1) were used [9]. Empherical guidences show that exogenous auxin could act as a principal oral inhibitor [23], but in the present study IAA in the medium did not inhibit in vitro ower formation. The owers produced in vitro appeared morphologically normal, greenish yellow in colour, umbellate cymes, born in axillary clusters and the owers were hermaphrodite. Two to ten ower buds were produced for each in vitro cultured plant (Table 2) of which only one developed into a berry while others abscised (Fig. 1C and D). In vitro fruiting was observed in the presence of Kn (2.0 mg l1) and IAA (0.1 mg l1). Data on percentage of plantlets that showed owering and fruiting are presented in Table 2. Fruit formation was observed after 8 weeks of owering. The fruits were berries, small, globose, smooth, orange red when ripe, enclosed in the inated membranous calyx (Fig. 1E). Seeds were yellow in colour and reniform (Fig. 1F). It takes 7 months period for owering when grown out of culture.
Table 1 Multiplication of shoots in axillary explants of Withania somnifera Dunal. in various media after 4 weeks Treatments (mg l1) Mean number of shoots generated per explant S.E. 8 0.04 12 0.1 7 0.2 6 0.3 5 0.2 4 0.4 Mean shoot length (cm) 2.5 0.5 1.2 0.4 0.7 0.2 2.3 0.2 4.2 0.1 3.1 0.4

MS + BA (1.0) + NAA (0.1) MS + BA (2.0) + NAA (0.1) MS + BA (4.0) + NAA (0.1) MS + Kn (1.0) + NAA (0.1) MS + Kn (2.0) + NAA (0.1) MS + Kn (4.0) + NAA (0.1)

The results are the mean S.E. of 20 replicates.

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Fig. 1. In vitro owering, fruiting and rapid propagation of Withania somnifera Dunal. (A) Regeneration of shoots from axillary bud on MS medium supplemented with BAP 2.0 mg l1 and NAA 0.1 mg l1 (bar 1.0 cm = 0.92). (B) Regeneration of shoots from axillary bud on MS medium supplemented with Kn 2.0 mg l1 and NAA 0.1 mg l1 (bar 1.0 cm = 0.9). (C) Explant cultured on MS medium supplemented with Kn 2.0 mg l1 and IAA 0.1 mg l1 showing fully developed owers (bar 1.0 cm = 0.85). (D) In vitro fruiting of W. somnifera shoots regenerated from axillary bud on MS medium supplemented with Kn 4.0 mg l1 and IAA 0.1 mg l1 (bar 1.0 cm = 0.78). (E) Ripened In vitro fruits of W. somnifera shoots regenerated from axillary bud on MS medium supplemented with Kn 4.0 mg l1 and IAA 0.1 mg l1 (4). (F) Cross section of in vitro fruit showing seeds (4).

An interesting feature of the present study was that the potential of seedling explants were embarked upon owering in vitro in response to Kn and IAA. The phenomenon assumes signicance considering the fact that the explants were obtained from seedlings and there was a maturation period spanning a few years before a plant bore owers. There have been reports that BA promotes owering in some plants especially in species of Lemna [24] and Bamboo [25]. The importance of in vitro

owering has been discussed by Staden and Dickens [26]. Earlier studies have indicate the benecial effect of cytokinins on the induction of owering in vitro for other plants such as Orchids [27], Fortunella hindsii [28]. Kintzios and Michaelakis [29] stated that Kn inhibited the in vitro induction of owers in Chamomile. Contrary to the above statement, in the present study in vitro owering was induced by the MS medium with Kinetin and IAA in W. somnifera. In vitro owering was

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Table 2 In vitro owering responses of nodal explants of W. somnifera Dunal. on MS medium supplemented with various hormonal combinations Treatments (mg l1) MS + BA (0.5) + IAA (0.1) MS + BA (1.0) + IAA (0.1) MS + BA (2.0) + IAA (0.1) MS + BA (4.0) + IAA (0.1) MS + Kn (0.5) + IAA 0.1) MS + Kn (1.0) + IAA 0.1) MS + Kn (2.0) MS + Kn (2.0) + IAA (0.1) MS + Kn (4.0) MS + Kn (4.0) + IAA (0.1) MS + IAA (0.1) Flowering response No owering response No owering response No owering response No owering response Flowering response Flowering response No owering response Flowering response No owering response Flowering response No owering response Plantlets owered (%) 52 67 75 93 No. of owers/plantlet 5.4 1.1 6.7 0.07 10.3 0.2 8.1 0.7 Plantlets fruited (%) 5 7.5 No. of fruits/plantlet 1 1

For each hormone treatment 10 individual experiments were conducted with 20 replicates each. The results are the mean S.E. of 20 replicates.

observed in Arachis hypogea on MS medium with Kinetin or BA and IAA/NAA and pod formation was observed in modied MS medium supplemented with Kinetin [19]. These ndings were similar to the results observed by us in W. somnifera. During the last two decades, considerable importance has been focussed on W. somnifera world wide due to its several commercial uses. In the present investigation on W. somnifera, by using axillary buds for producing of multiple shoots, owering, fruiting and seed set were observed. Work on in vitro owering, fruiting and seed set by using axillary bud has not been reported so far. Therefore, the present work is to be regarded as the rst report on owers developed in vitro, some of which developed into ripe fruits with viable seeds. Further research is required to study the seed physiology. The present in vitro fruiting could offer novel opportunities for studies into the molecular physiology of fruit ripening under controlled conditions. In the present study an attempt was made to nd out the most favourable sets of environmental and nutrition conditions for ower induction and seed formation in vitro. The observations reported here are novel, and further experiments should lead to a better understanding of the physiological and molecular events underlying the shift from the vegetative state to the ower state, the specic roles of cytokinins in inducing ower and fruit development. This protocol also can be extended to plant breeding studies for the purpose of quick owering and fruit formation under in vitro conditions. References
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