Chapter 1 Lawsonia inermis L. (Syn. L.alba) family Lythraceae (2n=30) is a heavily branched glabrous perennial shrub (Aweke et al. 2005). Lawsonia inermis is a monotypic genus (Sastri 1962) which grows up to 4 to 5 feet high in hot and dry climates (Grieve 2005). The plant is commonly known as Mehndi in Hindi, Mendika, Rakigarbha in Sanskrit, Mailanchi in Malayalam, Maruthani in Tamil, Benjati in Oriya, Mayilanchi in Kannada, and Mehedi in Bengali (Kirtikar and Basu 2005). It thrives well in climate with annual rainfall of 200-4200 mm and an extended dry season, even with poor semi dry soil in range of ph 4.3 to 8 (Orwa et al. 2009). TAXONOMICAL HIERARCHY OF LAWSONIA INERMIS Kingdom: Plantae-Plants Subkingdom: Viridaeplantae Division: Angiospermae Subdivision: Spermatophytina Class: Dicotyledoneae Order: Myrtales Family: Lythraceae Genus: Lawsonia Species: inermis Fig 1.1 Lawsonia inermis plant Lawsonia inermis is commercially cultivated in Afghanistan, Bangladesh, Egypt, Iran, Libya, Morocco, Pakistan, Saudi Arabia, Somalia, Sudan, Tunisia, UAE, Western India and Yemen as shown in Fig 1.2. In India plantation of Lawsonia inermis is mainly confined to Gujarat, Haryana, Punjab, Madhya Pradesh and Rajasthan (Duke et al. 1993). Rajasthan State has 41,450 ha (2010-2011) area under henna cultivation in which Pali district of Rajasthan alone accounted for 39,400 ha (95%) largest cultivated Micropropagation of Lawsonia inermis L. and in vitro manipulations 1
area (Sukla et al. 2012). As per the FAO report, India exported 45,000 tones to 76,00 tones of henna per annum. The estimated export of henna, in all forms, was around 10,500 tones with a value of around Rs 91 crores. Map of India showing centre for Lawsonia inermis growing, processing and exporting is shown in Fig 1. 3 Fig 1.2 World map showing centre of native and exotic range of Lawsonia inermis (Source: http://www.worldagroforestry.org/sites/treedbs/treedatabases.asp) Fig 1.3 Map of India showing centree for Lawsonia inermis growing, processing and exporting (Source: http://www.heenapage. com/heena/encyclopedia/geography/indiabigma html) Micropropagation of Lawsonia inermis L. and in vitro manipulations 2
Optimum temperature range for germination is 25-30 C and it produces highest dye content in temperatures ranging from 35 C -45 C (Makhija et al. 2011). The leaves of henna are greenish brown to dull green, sub sessile 1.5-5cm long, 0.5-2 cm wide with an entire margin. Henna is commercially used for fodder, timber, firewood and dye for staining clothes, wood, leather, wool and hair (Orwa et al. 2009). Lawsonia inermis serves as an immense source of various pharmacologically important secondary metabolites such as aliphatic components, coumarins, naphthoquinone, flavonoids, derivatives, phenolic derivatives, sterols, triterpene and xanthones. Lawsone being an important secondary metabolite gets accumulated in aerial part of plant (Bakkali et al. 1997) with highest amount of 1.0-1.4% in young leaf petiole (Jones 2006; Kirkland and Marzin 2003). Lawsonia inermis tree has been extensively used in Ayurveda, Unani and Homeopathic medicine as a household remedy against various human ailments from antiquity making it a cynosure of modern medicine (Biswa et al. 2002). As the global scenario is now changing towards the use of safer non-toxic plant products having traditional medicinal applications, development of modern drugs from henna should be emphasized for the control of various diseases. Lawsonia inermis indeed is a Multipurpose Crop or Living Pharmacy due to its multivariate pharmacological properties like antiviral, immunomodulatory, antibacterial, antifungal, antifertility, hepatoprotective. It also possesses antimitotic, analgesic and antiinflammatory properties (Mikhaeil et al. 2004; Syamsudim et al. 2008; Chaudhary et al. 2010; Borade et al. 2011). It is also used for skin ailments to treat burns and wounds. A decoction is used as gargle for relax action of sore throat. Leaves of Lawsonia inermis are used as popular dye to color hands, hair and nails (Rosenberg 1999). As a medicinal plant, henna has been used for anti hemorrhagic, astringent, antineoplastic, cardio-inhibitory, hypertensive along with sedative effects and used as a folk remedy against amoebiasis, headache, jaundice and leprosy. The available data of Lawsonia inermis suggests that the use of henna or lawsone for hair dying presents no or negligible risk of genotoxicity to the consumers (Kirkland and Marzin 2003). Plant tissue culture offers tremendous potential for in vitro culture and micropropagation of several plant species. Plant tissue culture techniques, are being used for organogenesis as well as somatic embryogenesis and serve as good method for rapid multiplication. It would lead to the identification of embryogenic potential Micropropagation of Lawsonia inermis L. and in vitro manipulations 3
serving as an indicator of the loss of regeneration potential with culture age. Traditionally the plant has been propagated through seeds or by cuttings. Owing to poor viability of seeds and low multiplication rates, sufficient planting material cannot be made available during the growing season. In order to cope up with the increasing demand, there is an urgent need to develop micropropagation methodology which should not only be able to provide disease-free and true-to-type planting material but also be useful for mass multiplication. Biotechnological approaches help in increasing application of medicinal plants for secondary metabolite production. In vitro culture allows the production of secondary metabolites under controlled culture conditions. Two types of in vitro cultures are currently used for secondary metabolite production: dedifferentiated cultures (callus and suspension cultures) and differentiated cultures (transformed roots and shoots). Various approaches used for increasing the production of secondary metabolites include optimization of growth and production culture media, as well as elicitation and metabolic engineering (Giulietti and Ertola 1999). In recent years, in vitro culture of cell, tissue and organ has been used as an efficient tool for the large scale propagation of many commercially important medicinal plants viz. Arnebia hispidissima, Azadirachta indica, Cathranthus roseus, Glycyrrhiza glabra, Ocimum sacnctum, Rauwolfia serpentine, Withaina somnifera and many other sp. Plant tissue culture technology not only fulfills the demand but also aid in crop improvement programs and production of secondary metabolites at commercial level. Present investigation deals with the induction of lawsone in Lawsonia inermis by plant tissue culture obtained through biotechnologies means. Variety of Colorimetric and Spectroscopy method viz. Mass Spectroscopy, Nuclear Magnetic Resonance Spectroscopy, Infra Red Spectroscopy, FT- IR Spectroscopy, UV Spectroscopy, Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC) and High Performance Thin Layer Chromatography (HPTLC) methods are usually employed for quantitative analysis of secondary metabolites production of Gallic acid, Lawsone, Betulinic acid and Lupeol in Lawsonia inermis. Keeping in view the above facts present study was undertaken to develop an efficient protocol for micropropagation. Subsequently, the most effective hormonal concentration for in vitro establishment, multiplication and root induction and Micropropagation of Lawsonia inermis L. and in vitro manipulations 4
hardening was determined. The genetic fidelity of the in vitro regenerated plants was verified using molecular marker Inter Simple Sequence Repeat (ISSR) analysis. Therefore, the present investigation has been undertaken with following objectives: Optimization of efficient protocol for microprapogation of L. inermis Induction of the lawsone content in Lawsonia spp. Quantification of lawsone content in Lawsonia spp. Micropropagation of Lawsonia inermis L. and in vitro manipulations 5