Results very less amount and this could

Results and discussionExplant selection and cultures establishmentPhenolic exudation:The survival frequency of fresh/juvenile explants was significantly greater (P< 0.05) than old/woody explants (Fig. 2). Survival of the old/woodyexplants was lesser because they produced a higher amount of phenolic exudatesthat could have suppressed further tissue proliferation. According to Minochaand Jain (2000), phenolic exudation/leaching is a common problem during cultureof mature explants that can notably influence the success of cultureestablishment/subsequent proliferation. In contrast, fresh/juvenile explantsproduced phenolic exudates in very less amount and this could be overcome bytreating the explants with sterile antioxidant solution of ascorbic acid andcitric acid. Similarly, beneficial results of use of antioxidant solutionduring culture establishment have also been reported by other researchers (Shekhawatet al.

1993; Cördük and Aki 2011; Lodha et al. 2014).Explanting season: The effect ofexplanting season on culture establishment is a key factor, especially in caseof explants derived from a mature plant. The frequency of axillary shootdifferentiation was significantly higher (?<0.5) in spring (87.

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3%)and autumn (81.6%) in comparison to that of summer (39.0%) and winter (60.3%)seasons (Fig.

3). Spring was found the best explanting season for Prosopiscineraria (Shekhawat et al. 1993) and Populus tremula (Peternel etal. 2009), whereas autumn proved the most suitable season for Crataevaadansonii (Sharma et al. 2003) and Lawsonia inermis (Ram andShekhawat 2011) culture establishment. In contrast, explants collected during springand autumn both seasons were found suitable for culture establishment in T.cordifolia.

Our results are in consonance withTimofeeva et al (2014) in Laburnum anagyroides. It should be pointed outthat T. cordifolia does not exhibit pronounced dormancy/growthretardation during off seasons.Bud breaking: Shoot bud inductionwas observed from the fresh/juvenile nodal explants within 10-14 days on themedium containing cytokinins (BAP or Kin).

The optimum axillary shoot bud inductionwas observed on MS medium containing BAP – 2.0 mg l-1. On thismedium, 3.8 ± 0.

63 shoots of mean length 2.55 ± 0.60 cmwere differentiated per explant (Table 1; Fig. 1b).

Earlier, Gururaj et al (2007)observed 3.4 ± 0.62 shoots on Kin–13.94 µM, Choudhury et al (2013) 2.23 shootson BAP–2.0 mg l-1, Sivakumar et al (2014) 1.8 ± 0.

1 shoots on Kin–4.36µM and Sharma et al (2015) 3.5 ± 0.

15 shoots on BAP–2.0 mg l-1. Inthe present study, BAP supplemented medium proved more effective for shoot budinduction than Kin as reported in Caralluma edulis (Patel et al. 2014a),Cadaba fruticosa (Lodha et al.

2015) and Couroupitaguianensis (Shekhawat and Manokari 2016). Explant inoculated cultures wereinitially kept in diffused light conditions (20–25 µmol m?2s?1PFD) for bud breaking and then transferred to high light intensity (45–50 µmolm?2s?1 PFD) for further growth/differentiation of shoots.According to Lodha et al. (2014), initial incubation of explants in diffused lightfor certain period can enhance morphogenesis through promoting early axillarymeristem activation. Similar trends were also reported in Leptadeniareticulata (Rathore et al. 2013a) and Caralluma edulis (Patel et al.2014a).Multiplication and maintenance of shoot culturesRepetitive transferof original explants: Inthe first approach of shoot multiplication, original explants were repetitivelycultured on the fresh medium containing comparatively lower concentrations ofBAP (0.

25, 0.5, 1.0 or 2.0 mg L-1). Of these, MS medium supplemented with BAP (1.0mg L?1) formed the maximum number ofshoots (9.2 ± 1.03 per explant) after the second repetitive passage (Table 2).

Duringthe next two passages (third and fourth), lesser number of shoots were producedwith continuous decrement at each passage. Shoot multiplication through repetitivetransfer of original explants is an economic way to produce new flush of shootsin shorter period by reinvigorating and rejuvenating the basal dormantmeristematic cells (Sánchez et al. 1997). This approach of shoot amplificationhas also been applied in Leptadenia reticulata (Rathore et al. 2013a), Cadaba fruticosa(Lodha et al. 2015) and Blyttia spiralis (Patel et al. 2016).

Subculturing of in vitro raised shoots:In the second approach ofshoot amplification,in vitro raised shoots were subcultured to MS medium supplemented with three concentrations(0.25, 0.5 or 1.0 mg l-1) of cytokinins (BAP and Kin) alone/incombination with an auxin (IAA–0.1 mg l-1). Initial stages ofmultiplication were carried out in 150 ml Erlenmeyer flasks (Fig.

1c), then in250 ml flasks and finally in 420 ml screw cap culture bottles. Of the various concentrationsof PGRs tested, MS medium having a combination of BAP and Kin (0.5 mg l-1each) and IAA (0.1 mg l-1) gave the optimum regeneration response(?10-12 shoots/culture vessel). In comparison to only cytokinins (BAP or Kin)containing medium, greater number of shoots were obtained on medium containinga combination of cytokinins (BAP and Kin) and an auxin (IAA).

Incorporation ofIAA (0.1 mg l-1) in subculturing medium showed synergistic effect onshoot multiplication and increased the shoot number by promoting axillary branching.The probable reason for this synergism may be the cross-talk mechanism ofcytokinin(s) and auxin, where cytokinin(s) promotes the stem cell proliferationin central meristematic region and auxin initiates organ primordium formationfrom the surrounding region and this in turn results in shoot meristemdevelopment and shoot branching (Shimizu-Sato et al. 2009; Su et al.

2011).This type of auxin-cytokinin(s) synergism has also been reported in many other plantspecies (Rathore et al. 2013a; Lodha et al. 2015; Shekhawat and Manokari 2016),including few climbers such as Leptadenia reticulata (Patel et al. 2014b)and Blyttia spiralis (Patel et al. 2016).


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