Initiation of aseptic culture and shoot induction
The young terminal shoot tip explants (2.0-3.5 cm) of S. mollis were collected from the plants conserved in the Experimental Botanical Garden and identified with the help of herbarium specimen (BSD 123495) for the further in vitro and cytological studies. Explants were initially washed under running tap water for 30 min followed by Tween-20 (Himedia Laboratories, Mumbai, India) to remove dirt particles, traces of soil, and followed by fungicide treatment (1% bavistin) for disinfection of the explants for 30 min. Thereafter, explants were disinfected with different surface sterilizing agents, i.e., ethanol (70%), sodium hypochlorite (6%) (Merck & Co., USA) and mercuric chloride (0.1%) (Himedia Laboratories, Mumbai, India) with different time duration of 2, 5, and 8 min, respectively. After each treatment, explants were washed thrice with sterilized double distilled water. Properly disinfected shoot tip explants were further used for the organogenesis experiments. The pH of the medium was adjusted to 5.7 before autoclaving at 121 °C for 15 min and then sterilized shoot tip explants were inoculated onto basal MS medium [18] supplemented with agar (6%), sucrose (30 g l−1), and various PGRs alone or in combinations. Cultures were maintained in the culture room at 24±2 °C, under a 16/8 h light and dark cycle with a light intensity of 47.29 μmol m−2 s−1 provided by white fluorescent PAR lights (40 W; Wipro, India). All the plant growth regulators (PGRs) applied was procured from Himedia Laboratories, Mumbai, India, and glass wares used (conical flask: Borosil 4980, 250 ml, and 85×140 mm; culture tubes: 38×200 ml) were from Borosil, India.
Explants (size, 1.5-2.0 cm) were inoculated onto basal MS medium (control) and MS medium augmented with different concentrations of cytokinins, viz., 6-benzylaminopurine (BAP) (2.2 to 11.1 μM), N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (thidiazuron/TDZ) (2.27 to 6.8 μM), and kinetin (2.32 to 9.3 μM). Subsequently, the optimal concentration of BAP (8.9 μM), TDZ (4.54 μM), and kinetin (6.9 μM) were further tested in combination with different concentrations of naphthalene acetic acid (NAA) (0.53-2.65 μM) to observe the synergistic effect of both the PGRs on shoot induction and proliferation.
After shoot initiation, shoot proliferation was performed in MS medium supplemented with BAP (8.9 μM) having 0.5% agar (Himedia Laboratories, Mumbai, India). Shoot proliferation cultures were sub-cultured at regular interval of 3 weeks. Shoot multiplication rate was calculated on the basis of percentage of explants with positive response, number of total shoots per explant and shoot height after 8 weeks of incubation.
Root induction
In vitro developed single shoots/shoot cluster of 2-3 cm length were inoculated onto MS and modified MS medium (half strength and quarter-strength). Further, half-strength MS medium supplemented with different concentrations of auxins, viz., indole-3-acetic acid (IAA) (5.71, 11.42, 17.13, and 22.84 μM), indole-3-butyric acid (IBA) (4.9, 7.36, 9.8, 12.26, and 14.7 μM), and naphthalene acetic acid (NAA) (5.3, 10.60, 15.90, 21.20, and 23.85 μM) were used for the root development. Cultures were incubated under the same conditions as above and rooting percentage, number of roots and root length were recorded after 6 weeks of incubation.
All the experiments were conducted in triplicates and each set of experiment was carried out with 20 explants. Analysis of variance and mean separation was carried out using Duncan’s multiple range tests (DMRT) utilizing the SPS software.
Hardening and transplantation
Plantlets with properly developed roots were taken out from the culture tubes/flasks after 6-weeks of incubation and washed gently under running tap water to detach the traces of the medium from the roots. Initially to optimize the hardening conditions, the regenerated plantlets were transferred into two set of plastic cups (8×7 cm), one set was filled with a mixture of soil and sand in equal ratio (w/v) while another set was containing only sand. All the plantlets were maintained in the green house at 25±2 °C. Initially to maintain the humidity, plants were covered with transparent polythene sheet and removed after 1 week. After 2 months, plantlets were shifted to nursery black polybags (4.5×8 inch) containing soil and maintained in the poly house. Plantlets were provided half-strength modified Hoagland solution [19] at 3 days interval. In order to acclimatize plants to field conditions, plantlets were transferred to poly bags containing compost enriched soil after 4 weeks and maintained in the open.
Meiotic studies
For chromosome counts and male meiosis, suitable-sized floral buds were fixed in Carnoy’s fluid (absolute alcohol: chloroform: glacial acetic acid in a ratio of 6:3:1 (v/v)). Samples (BSD 123495) for the study were collected from the plant conserved in the Experimental Botanical Garden. Young and emerging anthers from unopened buds were squashed in 1% acetocarmine and meiotic preparations were made. In each case, 50-100 meiocytes were observed under light microscope at different stages of meiosis for chromosome counts and detailed meiotic course. For microsporogenesis, 100-200 sporads were analyzed in each case. Pollen fertility was assessed through stainability tests by crushing the completely developed anthers in glycerol-acetocarmine mixture (1:1). Well-filled pollen grains with totally stained cytoplasm and nuclei were counted as fertile whereas shriveled and those with stainless/incompletely stained cytoplasm were noted as sterile. Photomicrographs of meiocytes, sporads, and pollen grains were taken from temporary preparations using Nikon microscope fitted with a digital camera.