Materials and methods

Explant preparation and surface sterilization

Plants of T. cordifolia (more than 10 years
old) were marked from different places near and around Jodhpur (a district of western
Rajasthan, India). A morphologically/phenotypically healthy plant was selected
as mother plant for culture establishment (Fig. 1a). Two types of explants i.e.
old/woody nodal segments and fresh/juvenile nodal segments were used for
culture initiation. For obtaining fresh/juvenile nodal explants, the mother
plant of T. cordifolia was lopped/pruned (during winters) and watered regularly
to maintain continuous sources of explants. Both types of explants were collected
throughout the year to evaluate the effect of different seasons on culture
establishment. 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 surface
sterilization with 0.1% (w/v) aqueous solution of HgCl2 for 3-4
minutes and finally rinsed 5-7 times with sterile water under a Laminar Air
Flow Hood. Finally the explants were kept in sterile, chilled aqueous solution
of antioxidants (100 mg L-1 of ascorbic acid and 50 mg L-1 of
citric acid) for 15 min, prior to inoculation.

Culture establishment and culture conditions

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The surface sterilized explants were placed
vertically in culture tubes on MS (Murashige and Skoog 1962) medium supplemented
with various concentrations (1.0, 2.0, 3.0 or 4.0 mg l-1) of
cytokinins (BAP or Kin) and additives (50 mg l-1 ascorbic acid, 25 mg l-1 each
of citric acid, L-arginine and adenine sulphate). The pH of
medium was adjusted to 5.8 ± 0.02 before autoclaving at pressure 1.06 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?1
PFD) for bud breaking and thereafter shifted to culture room maintained at regular
light intensity (45–50 µmol m?2s?1 PFD), photoperiod 16/8
h d-1, temperature 26 ± 2?C and RH 55-60% for further

Multiplication and maintenance of shoot cultures

After bud breaking, in
vitro raised shoots were multiplied by two ways: (a) the original/mother
explants (after harvesting the first crop of sprouts) were repetitively
transferred to fresh MS medium supplemented with comparatively lower
concentrations (0.5 or 1.0 mg l-1) of BAP for up to four passages, after an
interval of 4 week each; and (b) in vitro raised shoots (after making a clump
of 4-5 shoots) were subcultured to MS medium with different concentrations
(0.5, 1.0, 1.5 or 2.0 mg
l-1) of cytokinins (BAP or
Kin) 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 of
media compositions on growth and development of shoots. For this, cultures were
transferred to Modified MS (MMS; Shekhawat et al. 1998), WP Medium (WPM; Lloyd
and McCown 1981) or MS½ medium. The shoot cultures of T. cordifolia were
maintained for two years with subculturing every 5th to 6th

ex vitro rooting and acclimatization (CEVRA)

For ex vitro root induction under green house
conditions, individually harvested shoot’s base was dipped in/pulse-treated
with 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) time
durations. The pulse-treated shoots were transferred to the bottles containing
autoclaved Soilrite® a
mix of expanded perlite (horticulture grade), Irish peat moss and exfoliated
vermiculite (1:1:1); Keltech Energies Limited, Bengaluru, India and irrigated with quarter
strength of MS basal salts. The bottles containing ex vitro inoculated shoots
were incubated in the greenhouse initially near the pad section (continuously
drenched with water for maintaining high humidity and low temperature) and
subsequently transferred towards the fan section (having heavy duty axial flow
fan for maintaining low humidity and high temperature) in order to CEVRA of the
plantlets. In addition, caps of the bottles were also gradually unscrewed over
a period of 2-3 weeks and eventually removed.

Soil transfer of acclimatized plantlets

The acclimatized plantlets were transplanted to the polybags (Lodha
Plastic Industries, Jodhpur, India) containing a mixture of garden soil, organic
manure and sand (1:1:1). These were kept in the greenhouse for next 2-3 weeks
and finally shifted to the nursery.

Experimental design and statistical analysis

All the experiments were conducted in a completely randomised
block design (RBD) for single factor experiments (Compton and Mize 1999). A
minimum of 20 replicates were taken for each treatment and all the experiments were
performed thrice. The data for shoot bud induction, multiplication and CEVRA were
scored over a period of 2 to 5 weeks depending upon the experiment.
percentage response was calculated as the number of explants exhibited response
out of the total number of explants cultured for a particular treatment. The
data were statistically analysed by one-way Analysis of Variance (ANOVA) and
processed using SPSS ver. 17 (SPSS Inc., Chicago, IL). The
significance of differences among the mean values was calculated by Duncan’s Multiple
Range Test (DMRT) at significance level (P < 0.05; Duncan 1955) and results were presented as mean ± SD of three independent experiments.


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