Vol 9 No1 - Journal of Cell and Molecular Biology - Haliç Üniversitesi

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Journal of Cell and Molecular Biology 9(1): 37-44, 2011 Research Article 37 Haliç University, Printed in Turkey. http://jcmb.halic.edu.tr In vitro regeneration of Cleome viscosa – an important medicinal herb Jeyaraj ANBURAJ *,1 , Chinnappan Ravinder SINGH 1 , Shenbagamoorthy SUNDARRAJ 2 Soundarapandian KANNAN 2 1 Ayya Nadar Janaki Ammal College, Department of Biotechnology, Sivakasi, India. 2 Bharathiar University, Proteomics and Molecular Cell Physiology Lab, Department of Zoology, Coimbatore, India. (* author for correspondence; geneanbu@yahoo.co.in) Received: 30 August 2010; Accepted: 26 May 2011 Abstract Cleome viscosa is an important medicinal herb. Mass collection of the plant from natural habitats has led to its depletion. We have established a protocol for its mass propagation through in vitro organogenesis using leaf explants. For callus induction it was found that Indole-3acetic acid (2mg/l) has a significant effect on the callus induction, callus index and callus physical appearance. The moderate response was observed with Indole-3butyric acid (2mg/l), and a low response was observed with 2-Napthalene acetic acid (2mg/l). 2mg/l concentration of Benzyl aminopurine significantly influenced shoot proliferation. Optimum response was observed with Benzyl aminopurine + Kinetin (5+2.5mg/l), and low response was observed with Kinetin (1mg/l). About 91% of rooting response was observed with NAA (0.1mg/l). Moderate response was observed from Indole-3butyric acid (0.1mg/l) and low response observed from IAA (0.1mg/l). After two weeks of acclimatization 90% of survival rate have been observed. The present study describes a well-documented and reliable protocol for Cleome viscosa from leaf explants with a high rate of multiplication. This protocol could be used as a basic tool for commercial cultivation of this medicinally important plant Cleome viscosa. Keywords: Cleome viscosa, organogenesis, medicinal plants, in vitro regeneration, auxins Önemli bir tıbbi bitki olan Cleome viscosa’nın in vitro rejenerasyonu Özet Cleome viscosa önemli bir tıbbi bitkidir. Bitkinin doğal ortamından toplu halde toplanması tükenmesine sebep olmuştur. Yaprak eksplantları kullanarak in vitro organogenez ile toplu üretimini sağlayacak bir protokol oluşturduk. İndol- 3-asetik asitin (2mg/l) kallus oluşumunda, kallus indeksinde ve kallus fiziksel görünümünde önemli derecede etkili olduğu belirlenmiştir. İndol-3-butirik asitin (2mg/l) orta derecede, 2naptalen asetik asitin de (2mg/ml) düşük derecede etki gösterdiği belirlenmiştir. 2mg/ml konsantrasyonda benzil aminopürin ise filiz proliferasyonunu önemli derecede arttımıştır. Benzil aminopürin + Kinetin (5+2 mg/l) ile optimum cevap, Kinetin (1mg/ml) ile de düşük dereceli cevap gözlemlenmiştir. İndol-3-bütirik asitin (0,1mg/l) orta derecede, İAA' nın ise düşük derecede cevaba sebep olduğu gözlemlenmiştir. İklimlendirmenin iki hafta sonrasında %90 hayatta kalma oranı gözlemlenmiştir. Bu çalışmada Cleome viscosa yaprak eksplantlarından yüksek oranda çoğaltım sağlayan güvenilir ve iyi belgelenmiş bir protokol tarif edilmektedir. Bu protokol tıbbi önemi olan bir bitki olan Cleome viscosa'nın ticari amaçlı kültürünün yapılabilmesi için temel bir araç olarak kullanılabilir. Anahtar Sözcükler: Cleome viscosa ,organogenez, tıbbi bitkiler, in vitro rejenerasyon, oksinler.
38 Jeyaraj ANBURAJ et al. Introduction Cleome viscosa Linn. (Capparidaceae) is commonly known as Tickweed, wild mustard or Spider plant (English), Hurhur (Hindi) and Hurhuria (Bengali). It occurs in woodland and grassland, and is a weed of fallow land, fields, roadsides and wasteland, often occurring on sandy soils, but sometimes on calcareous and rocky soils. It is a widely distributed herb with yellow flowers and long slender pods containing seeds. The whole plant is sticky in nature and has a strong odour resembling asafoetida. It is found in both seasonal dry and humid conditions, from mean sea level up to an altitude of 1000 m. The seeds have no dormancy and germinate readily after shedding. Plants start flowering 3–4 weeks after germination and the lifecycle is about 3 months. Cleome viscosa Linn (Capparidaceae), also called “Dog mustard”, is a herb that grow up to 1m height in India (Parimala et al., 2004). Cleome is a large genus included in the Capparaceae family, which comprises 427 species occurring in tropical and subtropical regions of the world (Brummit, 1992). In Asia and Africa the leaves and seeds are used as a rubefacient and vesicant and to treat infections, fever, rheumatism and headache. The whole herb is used in the treatment of ringworm, flatulence, colic, dyspepsia, constipation, cough, bronchitis, cardiac disorders (Kirtikar and Basu, 1975; Saxena et al., 2000). Leaves are used as the external application to inflammation of the middle ear and applied on wounds and ulcers (Chopra et al., 1986), hepatoprotective activity (Gupta and Dixit, 2009). Traditionally, this plant is used in various disorders such as diarrhoea, fever, inflammation, liver diseases, bronchitis, skin diseases, and malarial fever. Its juice is useful in piles, lumbago and earache (Rukmini, 1978). The analgesic, antipyretic and anti-diarrhoeal activities of the extract have been reported by researchers. A decoction is used as an expectorant and digestive stimulant and the vapour from a steaming decoction of the whole plant is inhaled to treat headache (CSIR, 1950). The seeds and its oil have anti-helminthic properties but they are ineffective in treating roundworm infections (Saxena et al., 2000). They also have anti-convulsant effect in Swiss albino mice (Mishra et al., 2010). The root is a remedy for scurvy and rheumatism (Rukmini, 1978). An aqueous seed extract displayed significant analgesic activity in mice and local anaesthetic activity in guinea pigs (Singh and West, 1991; Parimaladevi et al., 2003). In tests with rats, the anti-diarrhoeal (Devi et al., 2002) and anti-pyretic (Devi et al., 2003) activities of the extracts have been confirmed. Leaves and young shoots are cooked as a vegetable (Manandhar, 2002). It has a sharp mustard like flavor (Facciola, 1990). The pungent seed can be pickled or used as a mustard substitute in curries (Manandhar, 2002; Facciola, 1990). The juice of the plant is used as a condiment (Facciola, 1990). Oil obtained from the seeds is used for cooking (Manandhar, 2002). All parts of the plant are used in liver diseases, chronic painful joints and mental disorders (Chatterjee and Pakrashi, 1991). The induction of callus growth and subsequent differentiation and organogenesis is accomplished by the differential application of growth regulators and the control conditions in the culture medium. With the stimulus of endogenous growth substances or by addition of exogenous growth regulators to the nutrient medium cell division, cell growth and tissue differentiation are induced (Tripathi and Tripathi 2003). Organogenesis permits the manipulation of large numbers of high quality plant material within a short period of time. The vast usage of the Cleome viscosa in medicinal purpose, based on the above reason, it is increasing commercial demand of Cleome viscosa. The present study was undertaken to develop a more efficient protocol for rapid in vitro multiplication of Cleome viscosa using leaf explants as an initial plant material. Materials and methods The plants were collected from in and around Virudhunagar district, Tamil Nadu, India (Figure 1). Small young leaves were collected from healthy plants of Cleome viscosa L defoliated and sectioned into many segments. They were washed under continuous flashing of running tap water for 30 minutes and then with distilled water three times. Leaves were treated with 0.1% mercury chloride for 1.5 minutes and washed with sterilized double distilled water for 3 times. The small washed explants were treated with 3% hydrogen peroxide for 1.5 minutes and then washed with sterilized double distilled water for 3 times. At final the explants were treated with 70% ethanol for 0.5 minutes and then washed with sterilized double distilled water for 3 times in laminar air flow cabinet.
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