Abstract developed a concurrent ex vitro rooting and

AbstractTinospora cordifolia (Willd.)Miers, is an important medicinal climber of family Menispermaceae. This plantis principally known for its medicinal properties in Ayurvedic system of medicines.Poor seed setting coupled with low seed germination limit its naturalpropagation through seeds. This paper reports an improvised micropropagationsystem of T. cordifolia using nodal explants.

The maximum numbers ofaxillary shoots (3.8 ± 0.63) were differentiatedon MS medium containing 6-benzylaminopurine (BAP; 2.0 mg l-1).

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Thenumbers of shoots were further amplified using repetitive transfer of originalexplants and through subculturing techniques. The greatest numbers of shoots(15.3 ± 1.05) of an average length (5.5 ± 0.84 cm) were achieved on 0.8%agar-gelled Modified MS (MMS) medium supplemented with 0.

5 mg L?1each of BAP and kinetin (Kin), and IAA (0.1 mg L?1) in screw-capculture bottles, within 5-6 weeks. In rooting experiments, we developed a concurrentex vitro rooting and acclimatization (CEVRA) method for the first time in T.

cordifolia. About 87.6% of the micropropagated shoots rooted-ex vitro onpulse-treatment with indole-3-butyric acid (IBA; 200 mgl-1) for 3 min. More than 80% of the concurrently ex vitro rootedand acclimatized plantlets were successfully hardened in a greenhouse andtransplanted to the soil. The discussed micropropagation system can be used forthe large-scale offsite propagation of this medicinally important perennialclimber, T. cordifolia.

Keywords: Tinospora cordifolia;concurrent ex vitro rooting and acclimatization (CEVRA); micropropagation; Menispermaceae;modified MS medium  Abbreviations:BAP                6-benzylaminopurineCEVRA          Concurrentex vitro rooting and acclimatization IAA                 Indole-3-aceticacidIBA                 Indole-3-butyricacidKin                  KinetinMS                  Murashige andSkoog (1962)NAA               ?-naphthaleneacetic acidPFD                 Photon fluxdensityPGRs               Plant growthregulatorsRH                  RelativehumidityIntroductionTinospora cordifolia (Willd.)Miers, is a large, deciduous climbing shrub, belongs to the familyMenispermaceae and commonly known as Giloya (a Hindu mythological term whichmeans to save celestial beings from old age and kept them eternally young). Thisplant is an important drug of Ayurvedic system of medicines (Sinha et al.2004). T. cordifolia extracts exhibit anti-arthritic, anti-allergic, anti-diabetic,anti-inflammatory, anti-neoplastic, anti-malarial, anti-microbial, anti-osteoporotic,anti-oxidant, anti-periodic, anti-pyretic, anti-stress, anti-spasmodic, diuretic,hypotensive, hepatoprotective and immunostimulant/immunomodulatory properties (Regeet al. 1984; Nayampalli et al. 1982; Pathak et al.

1995; Jagetia et al. 1998; Singhet al. 2003; Abiramasundari et al. 2012; Aranha et al. 2012; Duraipandiyan etal. 2012).

Maliyakkal et al. (2013) reported that ethanolic extracts of T.cordifolia exhibit potential anti-cancer activities against human breastcancer cells, and it also showed less cytotoxic activity against’non-cancerous’ cells; and therefore can be used in the development of novelcancer chemotherapeutic drugs. The stem extracts are used in the treatment ofdebility, dyspepsia, chronic and intermittent fever, jaundice, neurological disorders,respiratory illness, rheumatism, skin ailments and urinary diseases (Nadkarni1954; Gupta et al. 1967; Warrier et al.

1996; Raghu et al. 2006). Roots of theplant are powerful emetic and used in visceral obstructions and leprosy (Nayampalliet al. 1982; Sarma et al.

1995). The decoction of leaves is used in the treatmentof gout (Sinha et al. 2004). Also, leaves are rich source of proteins (11.2%)and have good amount of calcium and phosphorus (Singh et al. 2003).

The pharmaceuticalsignificance of Giloya  isdue to the presence of various bioactive compounds such as Tinosporin (alkaloid);Tinocordiside, Tinocordifolioside, Cordioside, Cordifolioside A and B, CordifolisideA, B, C, D and E (glycosides); Tinosporon, Tinosporides (diterpenoid lactones);Giloinsterol (steroids); Tinocordifolin (sesquiterpenoid); Tinosporidine, Cordifol,Cordifelone, Giloin, Giloinin and Tinosporic acid (miscellaneous compounds),etc (Singh et al. 2003).T. cordifolia is distributed in northwestIndia, Nepal, Bangladesh, Myanmar, Vietnam and Sri Lanka (Catalogue of Life 2017).This plant flowers during the summer and fruits during the winter seasons (Singhet al. 2003).

T. cordifolia naturally propagates through seeds; however poorseed set followed by low seed germination limit its natural propagation. Though,stem cuttings are useful for vegetative propagation, but are dependent upon theweather conditions for proper growth (Raghu et al.

2006). Owing to its numerousmedicinal values, pharmaceutical companies have been over-exploiting this plantfrom their natural/wild stocks, thus threatening its future status. Therefore,there is an urgent need to develop alternative methods for its multiplication/propagationand ex-situ conservation. During the last three to four decades, plant tissueculture techniques have provided a number of viable methods for offsitepropagation and germplasm conservation of important medicinal and threatened plants(Gonçalves et al. 2017).

Among these,micropropagation is the most important (economical and practical) method owingto its mass multiplication capacity (Teixeira da Silva et al. 2015). It canalso be used for various crop improvement programs through GMO and non-GMOtechniques (Rathore et al. 2015).The present paper reports an improvised protocol foroffsite propagation of T. cordifolia.

The shoot multiplication rateachieved in the present study is greater than the all previous reports on the sametaxon. To the best of our knowledge, this is the first report on concurrentex vitro rooting and acclimatization (CEVRA) of micropropagated T. cordifolia.Thus, the present study was undertaken with the objectives: (i) to develop animprovised shoot amplification system over the existing protocols and (ii) to standardizethe most favourable conditions for CEVRA in micropropagated shoots of Giloya.

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