Micropropagation of Vitex spp. through in vitro manipulation: Current status and future prospectives

G Model

ARTICLE IN PRESS

JARMAP-48; No. of Pages 10

Journal of Applied Research on Medicinal and Aromatic Plants xxx (2015) xxx–xxx

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Review article

Micropropagation of Vitex spp. through in vitro manipulation: Current status and future prospectives Naseem Ahmad ∗ , Afsheen Shahid, Saad Bin Javed, Md. Imran Khan, Mohammad Anis Plant Biotechnology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh, 202 002 Uttar Pradesh, India

a r t i c l e

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Article history: Received 11 April 2015 Received in revised form 27 August 2015 Accepted 11 September 2015 Available online xxx Keywords: Vitex trifolia Vitex negundo Vitex agnus-castus Cytokinins Auxins Plant growth regulators

a b s t r a c t The genus Vitex belonging to family Verbenaceae possesses diverse medicinal properties ranging from anti-inflammatory, anti-allergic, anti-microbial to anti-cancer and anti-HIV. Many potential bioactive molecules form an integral part, of which some are highly valued by multinational pharmaceutical companies. Unrestricted harvesting and high market demand of many medicinal plants including Vitex had drastically reduced the existing wild stocks, a concern that needs attention. Such practices necessitate the intervention of biotechnological techniques such as plant tissue culture for mass propagation and conservation of this valuable genus. Micropropagation, a key tissue culture technique provides new vistas for the conservation of many important plant species. In recent years, various researches dealing with micropropagation of medicinal plants have come forth to take up the challenge. A careful and judicious approach in selecting the right combinations of various factors plays an important role in determining the fate of the regeneration pathway. Present day knowledge on different strategies dealing with micropropagation and in vitro conservation of various species of Vitex are a subject of discussion in the present review. Moreover, role of different factors like type of plant growth regulators and additives, their dosages, explant types, etc., in developing an efficient regeneration system for the genus is discussed. © 2015 Elsevier GmbH. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Micropropagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.1. Explant type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2. Growth regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2.1. Effect of cytokinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2.2. Effect of cytokinins in combination with auxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2.3. Effect of TDZ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3. Effect of additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3.1. Coconut water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3.2. Sodium sulphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3.3. Silver nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3.4. PVP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3.5. Adenine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3.6. Phloroglucinol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.4. Callus induction and regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.5. Cyclic production of shoots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Abbreviations: 2,4-D, 2,4-dichlorophenoxy acetic acid; 2iP, isopentenyl adenine; AgNO3 , silver nitrate; BA, 6-benzyladenine; GA3 , gibberellic acid; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; Kin, kinetin; Na2 SO4 , sodium sulphate; NAA, ␣-naphthalene acetic acid; PGRs, plant growth regulators; PVP, polyvinyl pyrolidone; TDZ, thidiazuron; MS, Murashige and Skoog’s medium. ∗ Corresponding author. E-mail addresses: [email protected], [email protected] (N. Ahmad). http://dx.doi.org/10.1016/j.jarmap.2015.09.001 2214-7861/© 2015 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Ahmad, N., et al., Micropropagation of Vitex spp. through in vitro manipulation: Current status and future prospectives. J. Appl. Res. Med. Aromat. Plants (2015), http://dx.doi.org/10.1016/j.jarmap.2015.09.001

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2

2.6.

3. 4. 5. 6.

Rooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.6.1. In vitro rooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.6.2. Ex vitro rooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.7. Acclimatization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Ex situ conservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Synthetic seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Test for genetic homogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction Plants are an excellent source of biologically active compounds and thus, extremely important in pharmaceutical industry and human health. Approximately 85% of traditional medicine preparations and 40% or more of mordern pharmaceuticals are derived or at least partially derived from natural sources (Rout et al., 2000; Vieira and Skorupa, 1993). In recent years there has been renewed interest in natural medicines that are obtained from plant parts or plant extracts. Since the time immemorial, Indian subcontinent has been reputed as the treasure box of valuable medicinal plants of the world on account of vast diversity of climatic conditions. Additionally, India is one of the 12-mega biodiversity centers having about 10% of the world’s biodiversity wealth, which is distributed across 16 agro-climatic zones. Out of 17,000 species of higher plants reported to occur within India, 7500 are known to have medicinal uses. This proportion of medicinal plants is the highest known in any other country against the existing flora of the country. The use of various parts of certain wildly growing plants to cure specific ailments has been in vogue in our indigenous medicine from ancient times. Plant based products have been in use for medicinal, therapeutic or other purposes right from the dawn of history (Sharma, 2004) Increasing use of herbs for healthcare and the herbal medicine boom in recent years has imposed a great threat to the natural resources and endangered plant species. Many pharmaceutical companies depend on wild medicinal plants as raw materials for extraction of medically important compounds. The genetic diversity of medicinal plants in the world is depleting at an alarming rate due to over-harvesting and ruinous harvesting practices for production of medicines, with little or no regard to the future. Destruction of the plant-rich habitat as a result of forest degradation, agriculture encroachments, urbanization, etc., are other factors. Hence there is a need to conserve, cultivate and sustain the use of important medicinal plants for future. In this era of technology, various biotechnological tools and techniques are currently being used for the conservation of different crops, endangered or rare plants. These tools are also important for multiplication and genetic improvement of medicinal plants through in vitro regeneration, genetic transformation and production of secondary metabolites using plants as bioreactors. Plant tissue culture is an alternative technology for commercial propagation (George and Sherrington, 1984) and is being used widely for large scale propagation of a number of plant species viz., Tylophora indica (Faisal and Anis, 2003), Ruta graveolens (Faisal et al., 2005a), Rauvolfia tetraphylla (Faisal et al., 2005b), Psoralea corylifolia (Faisal and Anis, 2006a), Pterocarpus marsupium (Husain et al., 2007), Mucuna pruriens (Faisal et al., 2006), Balanites aegyptiaca (Siddique and Anis, 2009), Salix tetrasperma (Khan et al., 2011). The genus Vitex of family Verbenaceae, consists of over 270 species, predominantly trees and shrubs, and is restricted to

tropical and subtropical regions like India, China, Sri-Lanka, Indonesia, North Australia, New Caledonia and French Polynesia. 14 species of Vitex have been reported in India (Anonymous, 2003) chiefly occurring in parts of Deccan peninsula and north east India, out of which Vitex negundo and Vitex trifolia are prescribed to be major medicinal plants (Kannathasan et al., 2007). V. negundo commonly called as ‘five leaved chaste’ tree or ‘Monks pepper’ (Tandon, 2005) is a small tree (4–5 m) found mostly at warmer zones at an altitude of 1500 m in outer Western Himalayas (Anonymous, 2003). The plant improves receptive and retentive power of mind, skin complexion and hair growth (Gupta et al., 1999; Sivarajan and Balachandran, 2002). The juice made from fresh leaves is poured into the nostrils in stupor and coma (Kirtikar and Basu, 1991). The decoction of leaves is used in bath during puerperal state of women in India. Apart from this, it is widely used to treat rheumatism (Jayaweera, 1980; Anonymous, 2003), relieve pain (Nadkarni, 1976), chronic bronchitis and cold (Mahmud et al., 2009). The leaf extract is astringent, febrifuge, sedative, tonic and vermifuge (Horowitz and Gentili, 1966). Chloroform extract of seeds show anti-inflammatory activity (Telang et al., 1999; Dharmasiri et al., 2003). In Tamil Nadu (India), dried aerial parts are also used in paddy storage (Kannathasan et al., 2007). The plant possesses potent mosquito repelling activity (NguyenPouplin et al., 2007) against Aedes aegypti (Hebbalkar et al., 1992), Culex quiquefasciatus (Rahuman et al., 2009) and Culex tritaenirrhynchus (Karunamoorthi et al., 2008) and possesses anti-filarial activity against Brugia malayi (Sahare et al., 2008). It also shows anti-bacterial activity against Escherichia coli, Klebsiella aerogenes, Proteus vulgaris and Pseudomonas aerogenes (Samy et al., 1998), anti-feedant activity against Spodoptera litura and Achoea janata (Sahayaraj, 1998; Chandramu et al., 2003a; Aswar et al., 2009). V. trifolia is a shrub, 3–6 m high and commonly seen on the banks of rivers, channels and ponds. It improves memory, relieve pain, removes bad taste in mouth and cure fever (Kirtikar and Basu, 1991). Flowers and fruits are useful in fevers (Bhattacharjee and De, 2005) and amenorrhoea, leaves are used to treat hair loss (Varier, 2003). The leaf extract is anti cancerous. Aerial parts are useful in diabetes (Pullaiah and Naidu, 2003) while its stem is used for treating dysentery (Holdsworth, 1997) and headaches (Rageau, 1973; Mc Clatchey, 1996; Kok (de), 2007). In Tonga, it is employed to treat mouth infections and inflammations (Whistler, 1992; Limousin and Bessieres, 2006). Anti-inflammatory potential of V. trifolia has also been reported recently (Matsui et al., 2009). Anti-feeding activity of dichloromethane leaf extract against a pest, Spodoptera frugiperda, has been reported (Hernandez et al., 1999) while methanol extract of leaves have larvicidal activities against C. quinquefasciatus (Kannathasan et al., 2008). Petroleum ether and ethanolic extracts exhibit antibacterial activity against both gram positive and gram negative bacteria (Hossain et al., 2001). Vitex agnus-castus is a strongly aromatic, woolly-tomentose shrub or a small tree, found from the Mediterranean region, through South-West Asian countries up to Baluchistan in Pakistan

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(Anonymous, 2003). It is used in treatment of female reproductive disorders including menopausal symptoms and insufficient milk production (Balaraju et al., 2008). It is also used to treat fibroid cystosis and prevent miscarriages (Christie and Walker, 1997; Mc Guffin et al., 1997; Pierce, 1999).

2. Micropropagation In vitro cultivation of plants by exploiting the totipotent nature of plant cells is of paramount importance to ensure continuous supply of plants irrespective of season and also for effective conservation of medicinally important plant species. Micropropagation, a key tissue culture technology has been extensively utilized for conservation of valuable medicinal plants like R. tetraphylla (Faisal et al., 2005b), P. corylifolia (Faisal and Anis 2006) and B. aegyptiaca L. (Siddique and Anis, 2009) etc. Micropropagation also helps in rapidly multiplying new cultivars of important medicinal plants that would otherwise take many years by conventional methods. Likewise, micropropagation has also been successfully employed in Vitex species using various types of explants, culture regimes etc (Table 1).

2.1. Explant type Various explant types ranging from nodal to leaf segments and collected in different seasons have been screened for their in vitro regeneration potential by several workers (Hiregoudar et al., 2006; Vadawale et al., 2006; Ahmad and Anis, 2007; Balaraju et al., 2008; Jawahar et al., 2008). Nodal segments have been the most widely investigated explants but in addition, shoot tips and leaf segments were also used by some workers (Jawahar et al., 2008; Balaraju et al., 2008). Jawahar et al. (2008) used young healthy leaf explants for inducing indirect organogenesis in V. negundo. Nodal segments were used for in vitro production of true-to-type plants and for high frequency induction of multiple shoots of V. negundo L. (Ahmad et al., 2008; Noman et al., 2008; Steephen et al., 2010; Rathore and Shekhawat, 2011). Balaraju et al. (2008) used apical meristems consisting of the apex and nodal explants collected during the months of June and July from a healthy and mature V. agnuscastus plant for in vitro propagation. Shoot tips and nodal explants were used in V. negundo for high frequency induction of multiple shoot and rapid clonal multiplication (Sahoo and Chand, 1998; Thiruvengadam and Jayabalan, 2000; Chandramu et al., 2003b; Vadawale et al., 2006; Sharma et al., 2006; Ahmad and Anis, 2007; Afroz et al., 2008; Ahmad et al., 2008; Islam et al., 2009; Ahmad and Anis, 2011; Ahmad et al., 2013a,b). Rani and Nair (2006) used shoot tip and nodal segment (1–1.5 cm) for high frequency shoot multiplication and callus regeneration in V. negundo. Of the three explants, nodal segments produced maximum number of shoots per explant (Ahmad et al., 2008; Afroz et al., 2008; Noman et al., 2008; Balaraju et al., 2008). In V. trifolia, multiple shoot emergenceand shoot proliferation was also documented in various types of explants containing meristem like nodal and shoot tip segements (Hiregoudar et al., 2006; Ahmed and Anis, 2012; Ahmad et al., 2013b; Ahmed and Anis, 2014a,b) and shoot tip explants. Various strategies adopted for in vitro propagation of Vitex species have been summarized in Fig. 1. Still some explants types that need to be explored in Vitex species include root segments, inflorescence etc., which could broaden our knowledge on regeneration studies of this important medicinal plant. In general it is observed that an explant with a pre-existing meristem is a better choice for explant, moreover other explants that have not been used, needs to be explored for exploiting their regeneration potential.

3

2.2. Growth regulators The manipulation of cells, tissues and organs in culture for applications in micropropagation and genetic modifications of plants is highly dependent on the use of appropriate growth regulators (Murashige, 1974). Conversely, tissue culture systems are useful as bioassays to define the growth regulating activity of many compounds (Mok et al., 1987). A range of auxins and cytokinins play a central role in the regeneration of multiple shoot in many plant species (Murashige, 1974). 2.2.1. Effect of cytokinin Cytokinins are N6 - substituted adenine derivatives (Nandi et al., 1989) regulating many plant’s developmental processes like cell division, cell differentiation, leaf senescence, apical dominance (Mok and Mok, 2001), apart from influencing shoot morphogenesis, embryogenesis, germination, de-etiolation, flowering and fruit development (Binns, 1994). These are produced by actively growing tissues, particularly roots, embryos, fruits etc., and reported to promote cell division and cell expansion in plant tissue culture (Kadota and Niimi, 2003). The ability of cytokinins to trigger cell division and influence differentiation is related to the fact that they stimulate RNA and protein synthesis, which may be involved in cell division (Srinivasan et al., 2006). Different cytokinins generally express different activities in affecting axillary shoot formation in vitro (Preece and Shutter, 1991). Many studies on micropropagation have reported suitable cytokinins types and their concentration for each species (Hutchinson and Zimmerman, 1987). They are thus crucial for shoot proliferation of many medicinal plants (Chen et al., 1995; Rout et al., 2000; Martin et al., 2005). Multiple shoots were induced in V. negundo on MS medium supplemented with varying concentration of BA alone. Sahoo and Chand (1998) reported that BA at the concentration of 8.88 ␮M was the most effective in inducing bud break. Sharma et al. (2006) reported that maximum number of shoots developed on MS medium fortified with 26.63 ␮M, BA. However, BA at lower concentrations (1.0 ␮M and 5.0 ␮M) in the MS medium was used in V. negundo for shoot induction and multiplication as reported by several workers (Ahmad et al., 2008; Afroz et al., 2008; Rathore and Shekhawat, 2011), Islam et al. (2009) reported that BA (8.8 ␮M) was found to be most effective for multiple shoot induction from shoot tip explants of V. negundo. Hiregoudar et al. (2006) reported that maximum number of shoots developed on MS medium supplemeted with BA 5.0 ␮M in V. trifolia (Ahmad et al., 2013a,b; Ahmed and Anis, 2014b). BA proved to be most effective in all the investigated Vitex species and the order of effectiveness is BA > Kn > 2iP as reported by several workers (Sahoo and Chand, 1998; Sharma et al., 2006; Hiregoudar et al., 2006; Afroz et al., 2008; Ahmad et al., 2008, 2013a,b). BA along with another cytokinin such as Kn have also been used to induce multiple shoots and maximum number of shoots per explant were developed on a combination of BA and Kn in nodal segments of V. agnus-castus (Balaraju et al., 2008). Cytokinin seems to be an essential requirement for inducing regeneration in somatic tissues. However, the type and concentration of the exogenous cytokinin may vary with species and explant type. 2.2.2. Effect of cytokinins in combination with auxins The synergistic influence of cytokinins along with an auxin in shoot induction and proliferation has been reported in many medicinal plants (Faisal and Anis, 2002; Faisal et al., 2005a; Hemborm et al., 2006). Addition of auxin at lower concentration of the order 0.5 ␮M to the optimal cytokinin concentration led to the promotion of shoot number and shoot length in V. negundo (Thiruvengadam and Jayabalan, 2000; Usha et al., 2007; Ahmad et al., 2008; Islam et al., 2009). Similar response of nodal explants

Please cite this article in press as: Ahmad, N., et al., Micropropagation of Vitex spp. through in vitro manipulation: Current status and future prospectives. J. Appl. Res. Med. Aromat. Plants (2015), http://dx.doi.org/10.1016/j.jarmap.2015.09.001

Vitex negundo

Nodal segments

V. negundo

Shoot tips/nodal segments Nodal segments

BA, Kin, TDZ, IAA, IBA, GA3 BA, Kin, NAA, IAA, IBA BA, NAA, IAA, IBA, Kin BA, Kin, GA3 , IBA TDZ, BA, Kin, IAA, GA3 BA, NAA, IBA BA, Kin, TDZ, 2-iP, NAA TDZ, BA, NAA, IBA BA, Kin, NAA, IBA BA, Kin, NAA BA, Kin, GA3 , NAA

V.negundo V. negundo V. negundo V. negundo V. trifolia V. negundo V. negundo V. negundo V. negundo V. negundo V. negundo

Nodal segments Shoot tips/nodal segments Nodal segments Nodal segments Nodal segments Shoot tips Nodal segments Shoot tips and nodal segments Leaf segments Nodal segments

V. negundo

Nodal segments and shoot tip Nodal segments micro-node Nodal segments

V. negundo

Shoot tip

V. trifolia V. trifolia

Nodal Nodal

V. negundo

V. agnus-castus V. negundo

BA, 2,4-D, IAA BA, IBA, IAA, Kin, NAA BA, Kin, NAA, GA3

Adjuvant

Response (shoot formation)

No. of shoots

Optimal response

References

Axillary

04.42

BA (8.8 ␮M)

Sahoo and Chand (1998)

Axillary/ apical

BA (6.6 ␮M) BA (17.80 ␮M) + NAA (2.15 ␮M)

Thiruvengadam and Jayabalan (2000) Chandramu et al. (2003b)

Na2 SO4

Axillary

32.00 49.00 20.68

Coconut water, PVP

Axillary Axillary/apical

08.50 14.62

BA (26.63 ␮M) TDZ (1.8 ␮M)

Sharma et al. (2006) Rani and Nair (2006)

Axillary Axillary

05.00 09.00

BA (4.4 ␮M) + NAA (0.5 ␮M) BA (5.0 ␮M)

Vadawale et al. (2006) Hiregoudar et al. (2006)

Coconut water

Axillary Apical Axillary Axillary/apical

25.00 06.30 16.40 21.83

TDZ (1.0 ␮M) BA (8.87 ␮M) + NAA (2.69 ␮M) BA (5.0 ␮M) + NAA (0.5 ␮M) BA (4.44 ␮M)

Ahmad and Anis (2007) Usha et al. (2007) Ahmad et al. (2008) Afroz et al. (2008)

AgNO3

Adventitious Axillary

17.39 22.45

Jawahar et al. (2008) Noman et al. (2008)

Axillary/apical

07.70

IAA (1.71 ␮M) + BA (1.33 ␮M) BA (16.80 ␮M) + IBA (2.25 ␮M) + AgNO3 (100 mg l−1 ) BA (8.8 ␮M) + Kin (0.46 ␮M)

Axillary

16.40

BA (5.0 ␮M) + NAA (0.5 ␮M)

Ahmad and Anis (2011)

Synthetic seed

Kin (2.5 ␮M) + NAA (1.0 ␮M)

Ahmad and Anis (2010)

Apical

Syn-seed germinated 04.80

BA (5.0 ␮M) + NAA (0.5 ␮M)

Ahmad et al. (2013a,b)

Axillary Axillary

22.30 16.80

TDZ (5.0 ␮M) BA (5.0 ␮M) + NAA (0.5 ␮M)

Ahmed and Anis (2012) Ahmad et al. (2013a,b) Steephen et al. (2010)

Arulanandam and Ghantikumar (2011) Islam et al. (2009) Ahmed and Anis (2014a) Ahmed and Anis (2014b) Ahmed et al. (2015)

BA, Kin, 2iP, IAA, IBA, NAA Kin, NAA

Nodal

BA, Kin, 2iP, IAA, IBA, NAA TDZ, BA, NAA, IBA BA, Kin, 2iP, IAA, IBA, NAA BAP

PG, AgNO3

Axillary

15.12

V. negundo

Nodal segments

BAP, Kin, IAA

AA, CA

Axillary

29.00

V. negundo

BAP, Kin, IAA, NAA

ABA

Apical/axillary

03.60

Vitex trifolia

Shoot apex/nodal and leaf Leaf/internodal

BAP (4.4 ␮M) + PG (793.02 ␮M) + AgNO3 (20 mg/l) BAP (4.4 ␮M) + Kin (1.16 ␮M) + IAA (0.57 ␮M) BAP (8.8 ␮M) + NAA (2.69 ␮M)

BA, Kin, IAA, NAA

Adventitious

07.90

BAP (8.8 ␮M) + NAA (1.61 ␮M)

V. negundo V. trifolia V. trifolia V. trifolia

Shoot apex Shoot apex Shoot tip Nodal segments

BA, Kin, 2ip NAA BA, Kin, 2ip NAA TDZ, BA, NAA BA, NAA

Apical Apical Apical Synthetic seed

07.20 19.20 22.30 Syn-seed germinated

BA (8.8 ␮M) BA (5.0 ␮M) + NAA (0.5 ␮M) TDZ (5.0 ␮M) BA (2.5 ␮M) + NAA (1.0 ␮M)

Balaraju et al. (2008)

Rathore and Shekhawat (2011) Rahman and Bhadra (2011)

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PGRs used

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Table 1 Effect of different explants, PGRs, additives on in vitro morphogenesis in Vitex species.

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Fig. 1. Schematic representation of methodology used in propagation of Vitex species.

was recorded by Chandramu et al. (2003b) where combination of cytokinins and auxins proved effective for shoot induction and multiplication. Variation in shoot bud growth response was very much obvious even when minor differences were made in the respective concentrations in the studies of V. agnus-castus using apical meristem and nodal explants (Balaraju et al., 2008) and reported that a relatively high caulogenic response was observed when the medium was supplemented with BA and NAA. Of a range of combinations inves-

tigated using nodal segments, BA(17.80 ␮M) along with 2.15 ␮M NAA was found to be the best resulting in 20.6 ± 0.56 shoots per explant with maximum elongation (4.54 ± 0.14 cm). Other combinations investigated include BA + IAA, BA + IBA using various concentrations (Chandramu et al., 2003b; Vadawale et al., 2006; Usha et al., 2007; Ahmad et al., 2008; Rahman and Bhadra, 2011). In V. trifolia the combination of BA and NAA was also very effective as described by Ahmad et al. (2013a,b) and Ahmed and Anis (2014b). It was concluded that the combination of either

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BA or Kn with IBA was least productive as compared to those with IAA or NAA and the combination of BA and NAA was most effective in all the treatments using nodal explants tried by various workers. Addition of low concentration of auxin seems to augment shoot regeneration in Vitex, however, higher concentrations inhibits cytokinin action. Although auxins have been reported to show antagonistic effect to cytokinins, but synergistic effect has been frequently reported in plant tissue culture. 2.2.3. Effect of TDZ Number of Urea-derived herbicides including few synthetic compounds such as N-(2-chloro-4-pyridyl)-N-phenyl urea and diphenyl urea (DPU, N, N -diphenylurea) were reported to have cytokinin-like activity (Woo and Wick, 1995). Thidiazuron (Nphenyl-N -1,2,3-thidiazol-5-yl urea), a derivative of phenyl urea initially developed as a Cotton defoliant (Arndt et al., 1976), later detected to have high cytokinin activity (Mok et al., 1982); has been frequently used as an effective growth regulator in many crops (Thomas and Katterman, 1986; Lu, 1993) and woody plants (Huetteman and Preece, 1993; Khurana et al., 2005). Use of TDZ for in vitro morphogenesis is quite common in many important medicinal plants (Faisal et al., 2005b in R. tetraphylla; Husain et al., 2007 in P. marsupium; Siddique and Anis, 2007 in Cassia angustifolia) and V. negundo is no way an exception. TDZ is known to bud break dormancy, stimulate growth and induce multiple shoots in various explants. It is very effective in amounts as low as 0.0045–0.09 ␮M which alone is more efficient than various combinations of other cytokinins and auxins. Going by various reports in Vitex species, one could clearly conclude that TDZ was very much beneficial in shoot proliferation amounting to about 25 shoots per nodal explants of V. negundo (Ahmad and Anis, 2007). However, prolonged exposure to TDZ for more than 4 weeks led to distortion, hyperhydricity and fasciation of induced shoots and was found to be deleterious on growth and multiplication of induced shoots. TDZ exposed nodal segments of V. negundo continued to proliferate in MS medium devoid of any growth regulators (Ahmad and Anis, 2007). Ahmed and Anis (2012) reported similar effects of TDZ on V. trifolia and obtained optimum shoot multiplication and elongation with 5.0 ␮M TDZ and later to avoid the adverse effects of prolonged TDZ exposure they transferred the cultures to TDZ free MS medium or MS medium fortified with various combinations of BA alone or in combination with NAA. Likewise, Rani and Nair (2006) obtained optimum response with 1.8 ␮M TDZ. In contrast, Sahoo and Chand (1998) obtained lower morphogenic response with 2.27 ␮M TDZ supplemented MS medium which could possibly be due to the exposure duration. TDZ is a potent growth regulator that mimics the action of both auxin and cytokinin, however, it also induces various physiological disorders on prolonged exposure or over dosage. TDZ has also been found to cause genetic changes inducing somaclonal variations. Therefore, the use of TDZ is not preferred for species in which other hormones are effective in inducing regeneration. The use of TDZ should be avoided in Vitex except for woody species showing high level of recalcitrance. 2.3. Effect of additives Addition of organic and inorganic supplements is a common phenomenon in plant tissue culture. In Vitex species, effect of various additives like coconut water, polyvinyl-pyrrolidone, Sodium sulphate and silver nitrate have been reported (Chandramu et al., 2003b; Rani and Nair, 2006; Afroz et al., 2008; Noman et al., 2008). 2.3.1. Coconut water Addition of coconut water (CW) to the medium increased the number of shoots in Elaeocarpus robustus (Roy et al., 1998) and Gloriosa superba (Hassan and Roy, 2005). But the addition of 5% CW

in the nutrient medium was ineffective for further shoot multiplication and growth as reported in V. negundo (Afroz et al., 2008). In contrast, addition of 10% coconut water was found to improve shoot multiplication by Rani and Nair (2006). 2.3.2. Sodium sulphate Sodium sulphate along with phytohormones increases bud breaking and multiple shoot induction. Significant increase in the percentage of explants responding to shoot induction and multiple shoot formation per explant was recorded on a medium containing sodium sulphate in V. negundo. The addition of sodium sulphate did not cause much difference in the percentage of explant response for shoot induction, but a 2–3 fold increase in the multiple shoot production than in cultures without sodium sulphate was reported (Chandramu et al., 2003b). Thus, sodium sulphate at the optimum concentration (100 mg/l) with 5% (w/v) sucrose was found most effective in nodal explants of V. negundo (Chandramu et al., 2003b). 2.3.3. Silver nitrate The bud breaking and shoot induction have been reported to increase when silver nitrate was added in the medium along with the various plant growth regulators. This increase in the bud breaking and multiple shoot induction may be due to the synergistic effect of silver nitrate and the hormone. Silver nitrate at an optimum concentration (100 mg/l) with 5% (w/v) sucrose was found effective for multiple shoots induction from the nodal explants in V. negundo (Noman et al., 2008). The addition of silver nitrate did not cause much difference in the percentage of explants response for shoot induction however there was a 2–3 fold increase in the multiple shoot production than in cultures without silver nitrate (Noman et al., 2008). According to Steephen et al. (2010), silver nitrate (20 mg/l) was found to influence maximum shoot proliferation from the nodal explants in V. negundo and silver nitrate along with BA (4.44 ␮M) enhanced bud break within a week of initial culture and significantly enhanced the number of shoots per explant within 60 days of culture. 2.3.4. PVP Browning of cultures due to phenolics exudation is a common phenomenon in tissue culture of woody species. Phenolics are secondary metabolites that modulate plant development (Arnaldos et al., 2001) and protect plant against biotic and abiotic stresses (Kefeli et al., 2003; Conceicao et al., 2006; Fan et al., 2006). However many authors have observed that phenolics also generate toxic compounds in tissue culture media. These compounds negatively affect in vitro regeneration in some tree species (Laukkanen et al., 1999; Dibax et al., 2005). The phenolic compounds are oxidized by polyphenoloxidase (PPO) and peroxidase (POD) participates in blackening of culture (Down and Norton, 1995; Whitaker and Lee, 1995). Oxidized phenolic compounds inhibit enzyme activity and darken the culture medium as explants get brown or blacken and ultimately die. Several authors suggested the application of various antioxidants to minimize the lethal browning or blackening of explants caused by phenolic compounds in plant tissue culture. These include treating explants with PVP (Laine and David, 1994), Ascorbic acid (Arditii and Ernst, 1993). Similarly in V. negundo, Rani and Nair (2006) used PVP in the medium for callus regeneration in order to protect the culture from turning brown. They observed that 1% PVP was essential for callus induction. The significant effect of PVP on callus regeneration might be due to its ability to bind phenolics and some toxins thereby protecting the cultures from turning brown (Chang and Yang, 1996). 2.3.5. Adenine In V. trifolia, various concentrations (0.25–10.0 ␮M) of adenine were tested on nodal explants. The best regeneration response and

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maximum number of shoots were found on 1.0 ␮M ADE augmented MS medium (Hiregoudar et al., 2006).

in vitro regenerated shoots (2–3 cm) were used for further shoot proliferation (Balaraju et al., 2008).

2.3.6. Phloroglucinol Phloroglucinol (PG) is often used as a supplement to other plant growth regulators (Teixiera da Silva et al., 2013). PG is used to prevent hyperhydricity in micropropagation (Phan and Hegedus, 1985), increase the number of somatic embryos produced from embryogenic callus in oil palm (Hanowar and Hanowar, 1984) and increase the bud induction response in Capsicum annum by 17–18% (Kumar et al., 2005). In V. negundo, shoot proliferation form the nodal explants was enhanced on the MS medium fortified with BA 4.44 ␮M and PG 793.02 ␮M in the absence of PG explants developed callus or abscission like outgrowths followed by poor sprouting and shoot proliferation (Steephen et al., 2010). Different additives have been found to enhance the regeneration ability and quality of in vitro regenerated plants. Studies conducted in Vitex species found that addition of supplements like coconut water, sodium sulphate, silver nitrate, PVP, adenine and phloroglucinol may help to alleviate problems such as phenolic exudation and promote regeneration.

2.6. Rooting

2.4. Callus induction and regeneration Auxin has a property to induce cell division and formation of callus. Low concentration results in root formation whereas high concentration results in callus formation. Indirect organogenesis via callus formation is commonly induced by the manipulation of exogenous phytohormones’ level (Faisal and Anis 2003 in T. indica; Faisal et al., 2006 and Ahmad et al., 2010 in R. graveolens). Rani and Nair (2006) induced callus tissue in V. negundo using leaf and stem segments on MS medium containing a combination of TDZ (0.5–2.15 ␮M) and IAA (1.7 ␮M) with 1% PVP. They reported maximum number of shoots when subcultured onto a medium containing 2.7 ␮M TDZ. The leaf derived calli showed better regeneration as compared to that derived from stem explants. Jawahar et al. (2008) also developed a protocol for inducing indirect organogenesis using leaf explants of V. negundo on MS medium containing 2,4-D and IAA in combination with BA. They reported that high frequency of shoot bud initiation and shoot proliferation was observed on MS medium containing 1.71 ␮M IAA and 1.33 ␮M BA. Arulanandam and Ghantikumar (2011) used leaf and internodal explants for callus induction in V. trifolia using 2,4-D in combination with Kn and reported, 6.79 ␮M 2,4-D along with 1.39 ␮M Kn to produce highest percentage of callus induction. While, highest shoot induction in calli was recorded on MS medium supplemented with 8.88 ␮M BAP and 1.39 ␮M NAA. 2.5. Cyclic production of shoots The emergence of fresh shoots in Vitex spp. is season dependent and therefore, the availability of explants for the clonal propagation becomes a limitation. To overcome this problem, Ahmad and Anis (2011) used in vitro proliferated shoots of V. negundo as a source of explants for in vitro multiplication and continuous production of shootlets. They have also reported that micro-nodes excised from the microshoots exhibit better regeneration response compared to the nodal explants derived from a mature tree. Shoot regeneration frequency was also optimized by manipulating pH and using various media (B5, MS, WPM and L2). MS medium with 5.0 ␮M 6benzyladenine (BA) and 0.5 ␮M ␣-naphthalene acetic acid (NAA) and pH 5.8 were found to be the optimum for maximum regeneration in V. negundo. The protocol so developed could produce a large number of shoots in a short span from a single nodal explants derived from an adult tree at any time of the year with a low risk of generation variation. Similarly in V. agnus-castus, explants from

During micropropagation, rooting of microshoots is often problematic and losses at this stage have vast economic consequences (De Klerk, 2002). Thus, research on adventitious root formation in tissue culture raised shootlets is highly important from the practical point of view. Adventitious root production in isolated micro-cuttings of Vitex species was accomplished in the presence of various auxins (NAA, IAA, IBA). Both in vitro and ex vitro rooting has been reported in Vitex species by several workers (Sahoo and Chand, 1998; Hiregoudar et al., 2006; Ahmad and Anis, 2007; Usha et al., 2007; Balaraju et al., 2008; Ahmed and Anis, 2014a). 2.6.1. In vitro rooting Regenerated shoots from various explants such as nodal, apical meristem and leaf were excised for root induction and success has been achieved using three common auxins (IAA, NAA and IBA) at various concentrations. In general, in vitro regenerated healthy shoots failed to root in half or full strength MS medium without any growth regulators even after 30 days of culture (Balaraju et al., 2008; Usha et al., 2007; Rani and Nair, 2006; Chandramu et al., 2003b; Sahoo and Chand, 1998). IBA supplemented with half or full strength MS media produced maximum number of roots in V. negundo (Ahmad et al., 2008; Vadawale et al., 2006; Chandramu et al., 2003b; Sahoo and Chand, 1998). The relative effectiveness of different auxins for in vitro rooting revealed the order of effectiveness as IBA > IAA > NAA (Sahoo and Chand, 1998; Ahmad and Anis, 2011). However, in V. trifolia, best rooting was recorded on half strength MS medium supplemented with 0.25 ␮M NAA (Hiregoudar et al., 2006) but according to Ahmed and Anis (2014a) best rooting (88.3 ± 1.2%) was achieved on full strength MS medium supplemented with 0.5 ␮M NAA. In V. agnus-castus, the number of roots produced per shoot was significantly higher in a treatment containing 0.57 ␮M IBA in half-strength MS medium than on medium containing IAA or NAA. 2.6.2. Ex vitro rooting For ex vitro rooting, individual in vitro raised microshoots were excised from shoot clusters and their basal cut ends were dipped in various concentration of IBA for 10 min, washed with sterilized distilled water and planted in thermocol cups filled with autoclaved soilrite. Ex vitro rooting was successfully reported for the first time by Ahmad and Anis (2007) in V. negundo. Rooting with this technique combines rooting with hardening step and saves time (Balaraju et al., 2008). 2.7. Acclimatization For acclimatization, different planting substrates such as vermiculite, vermi-compost soilrite mixture and garden soil have been used by different workers. Of the different types of planting substrates tried, percentage survival of the plantlets was highest in Vermi-compost in V. negundo with a maximum survival rate of 96% followed by 93% in soilrite mixture (Sahoo and Chand, 1998; Chandramu et al., 2003b). Rooted plantlets with 4–6 fully expanded leaves and well-developed root systems were excised and transferred to pots containing soilrite moistened with half MS lacking vitamins and covered with transparent polyethylene bags to ensure high humidity. The bags were removed after 2 weeks in order to acclimatize the plants. The acclimatized plants were transferred to a green house and finally to the field under natural light (Ahmad and Anis, 2007). The acclimatized plants exhibited normal growth,

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true to type morphology with flowering. However, in V. agnuscastus, percentage of survival was highest in organic manure and sand (Balaraju et al., 2008) and in V. trifolia, the rooted plantlets were transferred to plastic vessels containing sterile soil and Vermiculite. Pots were irrigated with Hoagland solution once a week (Hiregoudar et al., 2006). Ahmed and Anis (2014a) found soilrite to be most effective for maximum (92%) planlet survival rate for V. trifolia. 3. Ex situ conservation In vitro culture techniques have also been used in ex situ conservation of many plant species (Kartha et al., 1988; Bajaj, 1988; Kondo et al., 1989) and in conjunction with cryopreservation or the use of growth retardant can be adopted for ex situ conservation of threatened and rare medicinal plants. Rahman and Bhadra (2011) used different concentrations of abscisic acid (ABA) and low nutrient levels as growth retardant for short term ex situ conservation of V. negundo, to keep the in vitro growth of seedlings stunted so that they can be kept for longer period. The growth of seedlings was lowest (0.7 ± 0.10 cm) in half strength MS medium containing 0.4 mg/l ABA. According to Rahman and Bhadra (2011) seedlings could survive normally under this stunted condition for more than 8 months without any subculture. 4. Synthetic seeds The artificial seed is an encapsulated somatic embryo, sown and germinated in the same manner as a conventional seed. Due to low success and high cost of somatic embryo production, buds, shoots, bulbs or other meristematic tissue that can produce a whole plant may also be encapsulated which are also considered as ‘artificial seeds’ (Pond and Cameron, 2003). Till date there are no reports on somatic embryogenesis of any of the Vitex species. However, encapsulation of nodal segments of V. negundo has been developed for the production of non-embryogenic synthetic seeds (Ahmad and Anis, 2010). Encapsulation was accomplished by mixing the nodal segments of V. negundo into the sodium alginate solution and dropping them into the calcium chloride solution. The droplets containing the explants were held for at least 30 min to achieve polymerization of the sodium alginate. The alginate beads were then collected, rinsed with sterile liquid MS medium and transferred to sterile filter paper in petridishes for 5 min under the laminar airflow hood to eliminate the excess of water and thereafter planted into petridishes containing MS nutrient medium with various combinations of Kn and NAA. A 3% (w/v) sodium alginate with 100 mM CaCl2 solution has been found to be optimum concentration for the production of uniform synthetic seeds. They also reported that MS medium containing 2.5 ␮M Kn in combination with 1.0 ␮M NAA gave maximum (92.6 + 3.71%) plantlet conversion frequency. Encapsulated nodal segments of V. negundo were viable (50%) even after 8 weeks of cold-dark storage while only 8% conversion frequency was observed in non-encapsulated explants. Ahmed et al. (2015) used the same methodology for encapsulation of nodal segments of V. trifolia, and recorded 84.9 ± 0.4% plantlet conversion response on MS medium supplemented with 5.0 ␮M BA and 0.5 ␮M NAA. Upto 42.5% encapsulated nodal segments remained viable after storage at 4 ◦ C for 8 weeks. Thus, axillary bud encapsulation offers possibility for germplasm conservation and exchange among laboratories. Such reports on synthetic seed production using encapsulation technique on other Vitex species have not been received so far. These strategies should be developed to effectively utilize the developments made in the field. Transportation of propagules to the field from the lab is essential which might be quite far. As it

not possible/feasible to develop a laboratory near every cultivation these strategies are essential. 5. Test for genetic homogeneity In vitro regenerated plants are usually susceptible to genetic changes due to culture stress and may exhibit somaclonal variation (Larkin and Scowcroft, 1981). The occurrence of somaclonal variation is a potential drawback during micropropagation of an elite plant, where clonal fidelity is required to maintain the advantages of the desired genotype. Several methods can be used to evaluate the genetic integrity of clones, but most have limitations. Isozyme markers provide a convenient method to assess variation, but are subjected to ontogenic variation (Rout et al., 1998). However, molecular techniques such as RAPD and ISSR DNA markers have proved to be a powerful tool to assess the genetic fidelity of in vitro raised plants (Arachak et al., 2003; Bindiya and Kanwar, 2003; Martin et al., 2004; Hemborm et al., 2006; Ray et al., 2006). Of the reports available on the in vitro propagation of Vitex species, Ahmad et al. (2008) has evaluated the genetic homogeneity of nodal segment derived micropropagated plants of V. negundo using ISSR markers. Ten randomly selected in vitro raised plants and the mother plant were subjected to ISSR fingerprinting using twenty UBC primers. Out of the 20 anchored microsatellite primers tested individually, only four (UBC-801, UBC-880, UBC-899 and UBC-900) generated well-resolved reproducible banding profiles. The ISSR profiles generated for the selected, in vitro derived progeny and the donor plant were similar in all aspects, which indicated the clonal or true-to-type nature of the progenies. RAPD has also been utilized to check out the genetic stability among the regenerants of V. negundo, out of the 10 primers tested (OPA1-10), 3 generated well-resolved and reproducible banding pattern, all the 10 micropropagated and mother plants showed monomorphic banding pattern showing the absence of any genetically varied plant (Ahmad and Anis, 2011). When the aim of developing the protocol requires complete uniformity among regenerants, these studies should be carried out to authenticate the protocol. 6. Conclusion Habitat destruction and increased demand have posed a serious threat to many medicinal plants. With the aim to conserve the wild stocks of medicinal plants like Vitex, biotechnological approaches like plant tissue culture have been successfully employed. The micropropagation protocols available in this genus will facilitate mass multiplication and hence conservation of the genus. Moreover, the protocols available for callus production and regeneration may be used for production of secondary metabolites using cell suspension cultures of calli. In short, such protocols may be exploited by pharmaceutical companies for development of bioreactors for large scale production of bioactive compounds. Such attempts would result in harnessing the pharmaceutically important compounds without disturbing the phytodiversity of the species. The incorporation of Agrobacterium rhizogens for hairy root culture would significantly enhance the yield of secondary metabolites which may be attempted in future. However, the need of the hour is to develop appropriate protocols for long term storage of germplasm for conservation and exchange between repositories. Hence, cryopreservation needs to be standardized for Vitex species in order to successfully tackle the problem of germplasm erosion. Conflict of interest The authors declare that they have no conflict of interest.

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