Department of Biotechnology, Dr. Y. S. Parmar University of Horticulture and Forestry, Nauni,

Corresponding author Email: shailjabiotech@gmail.com

Article Publishing History

Introduction

Swertia chirayita Buch.- Hams. ex Wall.belongs to family Gentianaceae. It is commonly known as “Chirata” and in sanskrit it is called as Anaryatikta, Ardhatikta, Bhunimba, Chiratika, Chiratitka, Haima, Jvarantaka, Kairata, Kandatiktaka, Kiranta, Kirataka, Kirata Tikta, Naditikta, Naipala, Nepalanimba, Nidrari, Ramasenka, Sannipatha, Sutiktaka, Trinanimba, and Viktaka (Anon, 1982 and Kritikar 1984). It is an indigenous species of temperate Himalayas. Among many species of Swertia in India chirayita is the only species which is considered most important for its medicinal properties (Joshi and Dhawan, 2005).There is no steadiness in literature that plant is either annual ( Anon, 1982 and Kritikar 1984), or biennial/pluri- annual (Edwards, 1993). The plant has long been used for its blood-purifying, antifungal and antihelmintic properties (Pant et al. 2011). S. chirayita plants contain several active constituents such as xanthones, flavonoids, iridoids and secoiridoid glycosides that are responsible for its therapeutics properties (Kumar and Chandra, 2013).

The major phytochemicals of the bitter-tasting plant include swertiamarin, amarogentin and mangiferin, a xanthone C-glucoside (Phoboo et al. 2013). Swertiamarin is reported to be effective against hepatitis (Wang et al. 2011) and shown to exhibit anti-diabetic (Vaidya et al. 2013), anticancer (Kavimani and Manisenthlkumar, 2000) activities. Amarogentin is known to be anti-diabetic(Phobooet al. 2013), anticancer (Pal et al. 2012) and anti-arthritic (Saravanan et al. 2014).

The plant has an erect, about 2-3 ft long stem and the whole plant is bitter in taste. It has lanceolate acute leaves with orange brown or purplish coloured stem, and contains large continuous yellowish pith. The roots are simple, tapering , stout, short and almost 7 cm long. The flowering & fruiting occurs between July to September. Flowers of Swertia chirayita are in the form of numerous small, axillary, opposite, lax cymes arranged as short branches small, stalked, green-yellow, tinged with purple colour, rotate and tetramerous. The corolla is twice as long as the calyx and divided near the base into four ovate–lanceolate segments. The upper surface of the petal has a pair of nectaries covered with oblong scales and ending as fringes. Fruit is a small, one-celled capsule with a transparent yellowish pericarp. It dehisces from septicidally into two valves. Seeds are numerous, minute many-sided and angular. Floral characteristics such as colourful corolla and presence of nectaries support cross-pollination in the species. Swertia chirayita contains a yellow bitter ophelic acid and two bitter glucosides chiratin (Joshi and Dhawan., 2005, Brahmchari et al., 2004 Pant et al., .2010, Chandra et al., 2012, Kumar and Staden, 2016).

Swertia chirayita is difficult to propagate on mass scale via seed owing to non-availability of seeds due to harvesting of plants before seeds mature. So instead of going for conventional approaches of, the application of alternative reproducible micropropagation strategies has become inevitable for mass propagation and sustainable utilization of this age-old medicinal plant. Due to its over exploitation for different medicinal uses and commercial purposes its availability is decreasing day by day so it’s becoming extinct. S. chirayita conservation status has been categorized as “critically endangered” (Joshi and Dhawan, 2005 and Padhan et al. 2015). Developing an in vitro regeneration protocol for Swertia chirayita is urgent to promote large-scale production for ex situ conservation and for satisfying the pharmaceutical needs. Synthetic seed technology is also an applied application of modern plant biotechnology which offers tremendous potential for easy handling, micropropagation and plant germplasm conservation through cryopreservation (Gantait et al. 2015 and Kumar and Staden, 2016).

This article briefly reviews the in vitro propagation and in vitro conservation of the plant. This is an attempt to compile and document information on micropropagation and in vitro conservation of S. chirayita and highlight the need for research.

In Vitro Propagation

Wawrosch et al. (1999) developed as protocol for micropropagation of Swertia chirayita. They found that multiplication by adventitious shoot regeneration from root explants is most suitable method for the propagation of Swertia chirayita. A two-step system consisting of an initial 3 weeks cultivation on modified MS medium supplemented with 3 µM 6-benzyladenine followed by another period of 3 weeks in plant growth factor free medium was used. The pH of all nutrient media was adjusted to 5.8± 0.1. The root explants taken from 6-to 8-week-old plants are very well suited for the multiplication of Swertia chirayita through regeneration of adventitious shoots. The explants were cultured on modified basal MS medium with 3 mMBAP for 3 weeks, followed by another 3 weeks on hormone-free basal medium. An average of 1.9 very healthy shoots per 5-mm explant were obtained. Dipping of the shoots in an aqueous solution of NAA (15 ppm) followed by 3 to 4 week cultivation period on hormone-free, half-strength MS medium proved to be the most efficient method for rooting of Swertia chirayita.

Chaudhuri et al. (2007) produced genetically uniform plants from nodal explants of Swertia chirayita Buch. Ham. ex Wall. Shoot regeneration was obtained in shoot inducing medium containing half-strength MS basal medium supplemented with 0.44 µM 6-BAP and 4.65 µM 6-furfurylaminopurine. The highest number of shoots, that is 18 shoots per explant were obtained when medium was again used with 10 mM KNO₃ and 75 mg/l of casein hydrolysate. The plantlets were successfully transferred to the field and produced viable seeds.

Joshi and Dhawan (2007) described the micropropagation of Swertia chirayita through axillary shoot multiplication from 4 weeks old seedling derived nodal explants. 4.5 fold multiplication was obtained after every 4 weeks on MS medium supplemented with 4µM BAP and 1.5µM 2ip. Rooting was optimized on modified MS medium supplemented with 1µM NAA and 500 mg of activated charcoal which showed 94% of rooting.

A protocol for plant regeneration through indirect organogenesis was established by Bisht et al. (2008) for Swertia angustifolia Buch.-Hams. Callus was induced on MS basal medium supplemented with cytokinin (Kinetin or BA) and auxin (2,4-D/IBA/NAA) from leaf, petiole and stem explants. Higher concentration of Kinetin and 2,4–D (2.5-3.0 mg/l ) exhibited best callusing in leaf and better in petiole explants. BA and NAA in the range of 1.5-2mg/l exhibited fast proliferation in callus mass in both explants.Shoots were regenerated on MS medium containing BA (1.5– 2.5 mg/l) and IBA or NAA (0.5-1.5 mg/l). Rooting was obtained with full or half MS media with IBA or NAA (0.5 -1.5 mg/l).

Balaraju et al. (2009) established rapid system for micropropagation of Swertia chirayita Buch. Hams. ex Wall. using shoot tip explants derived from in vitro grown seedlings. MS medium containing BAP (1.0 mg/l) and Kinetin (0.1 mg/l) along with 2% sucrose induced highest number of multiple shoots per explants. Micro proliferated shoots were transferred to elongation medium amended with 0.1 mg/l GA₃. The highest frequency of rooting was obtained in half MS medium supplemented with 0.1 mg/l NAA.

Wang et al. (2009) investigated the effects of phyto hormones on shoot regeneration from the leaves of field grown Swertia chirayita .The best result obtained in MS medium supplemented with 13.2µM 6- BAP and 0.54 µM á- NAA. The highest rate of shoot regeneration was 96.5% on the medium with 0.54 µM NAA. Adventitious shoots were transferred on the rooting medium. Rooting was optimized on MS medium supplemented with NAA 5.40µM. Pant et al. (2010) developed an efficient protocol for in vitro propagation of Swertia chirayita. Axillary shoot bud multiplication was achieved using nodal segments as explants. A combination of BAP 4.4 µM + IAA 2.85 µM + Adenosine sulphate 271.45 µM proved to be the best giving 11.8 fold multiplication with average shoot length of 1.9 cm after 4 weeks and 18.5 fold multiplication with mean shoot length 2.6 cm was observed. Best rooting was observed on MS medium with IBA 4.90µM. Maximum mean number of root per shoot 35.3 was observed after 8 weeks.

Pant et al. (2011) described procedure for regeneration of complete plantlets of Swertia chirayita via indirect organogenesis. Callus was obtained from in vitro regenerated roots on MS medium supplemented with varying concentrations of BAP and 2,4-D. BAP (13.32 μM) in combination with 2,4-D (0.90 μM) proved to be the most effective concentration for callus induction, multiplication and adventitious shoot regeneration from callus surface. The optimal hormone combination for shoot multiplication was shown to be BAP (8.88 μM), IAA (2.85 μM) and 271.45 μM adenine sulphate (Ads). Individual elongated shoots were rooted on half-strength MS medium supplemented with varying concentrations of auxins. Best rooting was obtained with MS Medium supplemented with 4.90 μM IBA. In vitro raised plantlets with well developed shoots and roots were acclimatized successfully.

Jha et al. (2011) carried out in vitro propagation and conservation of Swertia bimaculata Hook. f. & Thomas. Seeds were germinated aseptically with low concentration of BA (2.22 µM) or Kinetin (2.32 µM). The best response of in vitro grown shoots was obtained on MS medium with BA (2.22 µM), Kinetin (2.32 µM ) and NAA (0.54µM). The number of shoots were increased to 20.6 on addition of 10 mM potassium nitrate (KNO₃) in the medium. Isolated shoots induce 100% rooting on basal medium with in 5 weeks. Rooted plants were hardened and transplanted in soil with 80-90% survival rate.

Conservation of Swertia Chirayita

Over exploitation of plant sources is a normal occurrence due to its increasing demand. It is mostly used as traditional drug. The demand of this plant is on rise at both national and international level due to its multiple uses which leads to increase over harvesting of wild populations and ultimately in reduction of population. According to the International Union of Conservation of Nature (IUCN) criteria, S.chirayita conservation status has been categorized as “critically endangered” (Joshi and Dhawan, 2005). There are limitations in the use of seed propagation, due to low viability, and low germination percentages (Badola and Pal, 2002; Chandra et al., 2012).

Biotechnology offers new means of conservation of Swertia chirayita. Synthetic seed production is one of them.In this method somatic embryos are encapsulated in a suitable matrix like sodium alginate along with insectisides, fungicides and herbicides. Kumar et al. (2014) reported on synthetic seed production and plant regeneration of S.chirayita from somatic embryos. However, studies are required to improve this technology so that it can be used on large scale.. Cryopreservation this is also one another method of conservation. In this method, the cells are preserved in the frozen state. The germplasm is stored at a very low temperature using liquid nitrogen (at-196ºC). The cells stay in completely inactive state and thus can be conserved for long periods. Certain compounds like- DMSO (dimethyl sulfoxide), glycerol, ethylene, propylene, sucrose, mannose, glucose, praline, acetamide are added during the cryopreservation. These are called cryoprotectants and prevent the damage caused to cells by reducing the freezing point and super cooling point of water., (Ara et al., 2000; Sharma et al., 2013a; Perveen and Anis, 2014; Gantait et al., 2015)

Table 1: list of important bioactive constituents isolated from Swertia chirayita
Active constituents	Biological activities	References
Amarogentin (chirantin)	Topoisomerase inhibition, chemo-preventive and antileishmanial effects .	[Ray 1996), [Saha and Dass 2005), [Phoboo et al. 2013]
Amaroswerin	Gastro-shielding	[Niiho 2005], [Phoboo et al. 2013]
Gentianine	Anti-inflammatory, anesthetic, antihistaminic, anticonvulsant properties, hypotensive, antipsychotic, lenitive, diuretic, antimalarial, antiamoebic and antibacterial properties.	[Song Zhen Yu 1958; Geng Tao 1959; Kwak 2005]. [Bhattacharya 1974], [Mansoor and Malghani MAK, 2005] , [Natarajan et al., 1974, [Phoboo et al. 2013]
Swerchirin	Antimalarial, hypoglycemic, hepatoprotective, pro-heamatopoitic, and weak chemo preventive pharmacological effects.	[Arino 1997], [Bajjpai 1991], [Saxena 1996], [Ya 1999] [Hirkawa1987], [Phoboo et al. 2013].
Swertiamarin	Analgesic property	[Lei 1982], [Phoboo et al. 2013]
xanthones, flavonoids, iridoids and secoiridoid glycosides	Therapeutic properties	Joshi and Dhawan 2005

Conclusion and Future Perspectives

This review article revealed the morphogenetic potential of leaves of Swertia chirayita as a source for micropropagation. The explants can be easily and regularly obtained from established shoot cultures and do not require disinfection treatment hence being ideal for germplasm exchange and cryopreservation. Normal leaf culture establishment for a number of plant species have the ability to accumulate secondary metabolites and plays important role in pharmaceuticals. In vitro conservation was done by cryo conservation. The government has imposed total ban on collection or removal of planting materials of this important species from their natural populations but this is not possible without the support of local healers. There should be awareness among local peoples to control the overexploitation. Scientists cannot conserve this species without the help of local healers. There are many institutes and universities where the research work is going on but that work should be explored at higher level so that other researchers gain experience from that and help in conserving endangered medicinal plants otherwise this species also become extinct. Biotechnological approaches are required in future also to promote its medicinal use.

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