Materials and methodsExplant preparation and surface sterilizationPlants of T. cordifolia (more than 10 yearsold) were marked from different places near and around Jodhpur (a district of westernRajasthan, India). A morphologically/phenotypically healthy plant was selectedas mother plant for culture establishment (Fig. 1a). Two types of explants i.

e.old/woody nodal segments and fresh/juvenile nodal segments were used forculture initiation. For obtaining fresh/juvenile nodal explants, the motherplant of T. cordifolia was lopped/pruned (during winters) and watered regularlyto maintain continuous sources of explants. Both types of explants were collectedthroughout the year to evaluate the effect of different seasons on cultureestablishment. The explants were pre-treated with 0.1% (w/v) each of Bavisitin(a systemic fungicide, BASF India Limited, Mumbai, India) and antibiotics(Tetracycline and Streptomycin) for 15 minutes.

This was followed by surfacesterilization with 0.1% (w/v) aqueous solution of HgCl2 for 3-4minutes and finally rinsed 5-7 times with sterile water under a Laminar AirFlow Hood. Finally the explants were kept in sterile, chilled aqueous solutionof antioxidants (100 mg L-1 of ascorbic acid and 50 mg L-1 ofcitric acid) for 15 min, prior to inoculation.Culture establishment and culture conditionsThe surface sterilized explants were placedvertically in culture tubes on MS (Murashige and Skoog 1962) medium supplementedwith various concentrations (1.0, 2.0, 3.

0 or 4.0 mg l-1) ofcytokinins (BAP or Kin) and additives (50 mg l-1 ascorbic acid, 25 mg l-1 eachof citric acid, L-arginine and adenine sulphate). The pH ofmedium was adjusted to 5.8 ± 0.02 before autoclaving at pressure 1.

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06 kg cm?2,temperature 121?C for 15 min. After inoculation, cultures were initially(for 1-2 days) incubated in diffused light intensity (20–25 µmol m?2s?1PFD) for bud breaking and thereafter shifted to culture room maintained at regularlight intensity (45–50 µmol m?2s?1 PFD), photoperiod 16/8h d-1, temperature 26 ± 2?C and RH 55-60% for furthergrowth.Multiplication and maintenance of shoot culturesAfter bud breaking, invitro raised shoots were multiplied by two ways: (a) the original/motherexplants (after harvesting the first crop of sprouts) were repetitivelytransferred to fresh MS medium supplemented with comparatively lowerconcentrations (0.

5 or 1.0 mg l-1) of BAP for up to four passages, after aninterval of 4 week each; and (b) in vitro raised shoots (after making a clumpof 4-5 shoots) were subcultured to MS medium with different concentrations(0.5, 1.0, 1.5 or 2.0 mgl-1) of cytokinins (BAP orKin) alone or in combination with IAA (0.1 mg l-1).

After optimizing the best combination of PGRs,cultures were transferred to different nutrient media to study the effect ofmedia compositions on growth and development of shoots. For this, cultures weretransferred to Modified MS (MMS; Shekhawat et al. 1998), WP Medium (WPM; Lloydand McCown 1981) or MS½ medium. The shoot cultures of T. cordifolia weremaintained for two years with subculturing every 5th to 6thweek.

Concurrentex vitro rooting and acclimatization (CEVRA)For ex vitro root induction under green houseconditions, individually harvested shoot’s base was dipped in/pulse-treatedwith various concentrations (50, 100, 200, 300 or 400 mg l-1)of freshly prepared auxins (IBA or NAA) for different (1, 3, 5 or 7 min) timedurations. The pulse-treated shoots were transferred to the bottles containingautoclaved Soilrite® amix of expanded perlite (horticulture grade), Irish peat moss and exfoliatedvermiculite (1:1:1); Keltech Energies Limited, Bengaluru, India and irrigated with quarterstrength of MS basal salts. The bottles containing ex vitro inoculated shootswere incubated in the greenhouse initially near the pad section (continuouslydrenched with water for maintaining high humidity and low temperature) andsubsequently transferred towards the fan section (having heavy duty axial flowfan for maintaining low humidity and high temperature) in order to CEVRA of theplantlets. In addition, caps of the bottles were also gradually unscrewed overa period of 2-3 weeks and eventually removed.

Soil transfer of acclimatized plantletsThe acclimatized plantlets were transplanted to the polybags (LodhaPlastic Industries, Jodhpur, India) containing a mixture of garden soil, organicmanure and sand (1:1:1). These were kept in the greenhouse for next 2-3 weeksand finally shifted to the nursery.Experimental design and statistical analysisAll the experiments were conducted in a completely randomisedblock design (RBD) for single factor experiments (Compton and Mize 1999). Aminimum of 20 replicates were taken for each treatment and all the experiments wereperformed thrice.

The data for shoot bud induction, multiplication and CEVRA werescored over a period of 2 to 5 weeks depending upon the experiment.Thepercentage response was calculated as the number of explants exhibited responseout of the total number of explants cultured for a particular treatment. Thedata were statistically analysed by one-way Analysis of Variance (ANOVA) andprocessed using SPSS ver. 17 (SPSS Inc., Chicago, IL). Thesignificance of differences among the mean values was calculated by Duncan’s MultipleRange Test (DMRT) at significance level (P < 0.05; Duncan1955) and results were presented as mean ± SD of three independent experiments.