Collection of plant material, surface disinfection, and preparation of explants
Fresh apical leaves (1.5–2 cm) were collected from a 60-day-old plant growing in a polyhouse. The collected explants underwent surface disinfection (under Laminar Air-Flow chamber) through consecutive treatments utilizing 0.05% (w/v) Bavistin®, 10% (v/v) Tween 20® (polysorbate 20), 20% (v/v) sodium hypochlorite, 1% (w/v) cetrimide (alkyltrimethylammonium bromide), 70% (v/v) ethyl alcohol, and 0.1% (w/v) mercuric chloride (all from Merck Life Sc. Pvt. Ltd., India). Exposure duration of the explants to the majority of the sterilant was 5 min each, except ethyl alcohol (for 30 s). Following completion of the disinfection process, the explants were washed with sterile water.
Culture media and culture conditions
The Murashige and Skoog (MS) semisolid medium  was prepared with the addition of ready-MS salt (HiMedia Laboratories Pvt. Ltd., India) fortified with 3% (w/v) sucrose and 0.7% agar, to serve as basal medium. For the induction of callus and subsequent regeneration of multiple shoots and roots, the MS medium was further fortified with different plant growth regulator (PGR) sources (Sisco Research Laboratories Pvt. Ltd., India) at variable concentrations maintaining the medium pH at 5.7. The growth room was set with 25 ± 1°C temperature, 60% relative humidity, 50 μmol/m2/s photosynthetic photon flux density (using cool white fluorescent lights), and 16 h photoperiod. However, during callus induction, initially, the inoculated leaf explants were kept under zero irradiance for 72 h, prior to their transfer to the above-mentioned culture condition.
Callus induction and proliferation
For induction of callus, the leaf explants were inoculated in the MS medium fortified with 0.5, 1, and 1.5 mg/l 2,4-D, picloram, and NAA, individually. The MS medium without any PGR served as “control”. Explants were horizontally scraped with a scalpel before their inoculation in order to enhance the probabilities of callus induction. Observations were recorded on daily basis for days to callus induction, whereas weight (mg) of fresh and dried (under hot-air oven for 36 h at 45°C) calli were recorded after 6 weeks of culture.
Shoot initiation from callus
For indirect shoot regeneration, fresh organogenic calli (induced in 1 mg/l picloram-supplemented medium), each weighing 500 mg, were inoculated in the MS medium supplemented with 0.5, 1, and 1.5 mg/l BAP, kinetin, or thidiazuron, wherein the MS medium devoid of PGR served as “control”. Observations were recorded on daily basis for days to fresh shoot initiation, whereas the number and length (cm) of shoots, as well as leaf numbers, were recorded after 6 weeks of culture.
Rooting of shoots
In vitro shoots were individually inoculated in the MS medium fortified with 0.5, 1, and 1.5 mg/l indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) for initiation and elongation of roots. The MS medium without any PGR served as “control”. Days taken to root initiation, number, and length of roots were recorded after 4 weeks from the day of inoculation.
Acclimatization of in vitro regenerants
In vitro regenerated complete plantlets with well-developed shoots and roots were shifted for acclimatization in sand for 2 weeks and then to an equi-volume mixture of soil, sand, tea leaf waste, and cow urine for the next 4 weeks before those were eventually planted in the polyhouse. During initial acclimatization, a transparent polythene sheet was used to cover the whole set-up and frequent spraying of water was done to ensure high humidity. During the initial as well as advanced acclimatization phases, the survival rate of the plantlets was documented. Following complete acclimatization, the plantlets were transferred to larger earthen pots (filled with garden soil) for subsequent growth and flowering.
Clonal fidelity assessment
To assess the genetic fidelity of the in vitro regenerated and acclimatized plantlets, genomic DNA was collected from leaves of randomly selected plantlets with the aid of GSure® Plant Mini (DNA extraction) Kit (GCC Biotech, Kolkata, India). Following the extraction of DNA, a polymerase chain reaction (PCR) was performed using a thermocycler system (Applied Biosystems® by Life Technologies™, Singapore) for amplification of the fragments. Each 25 μl of PCR mixture included 40 ng template DNA, 0.1 μM of primer [5′(AG)8T3′, 5′(CA)8AG3′, 5′(AC)8C3′, 5′(GGA)53′, and 5′A(GGA)53′, individually], 200 μM of dNTP, 2 mM MgCl2, 5 μl 1X Taq polymerase assay buffer, and 0.5 μl Taq polymerase, along with sterile water. PCR was commenced with preliminary denaturation at 94°C for 5 min, and then 35 cycles of denaturation at 94°C for 1 min, 1 min at annealing temperature (40–58°C based on melting point), an extension of 1 min at 72°C, and final extension at 72°C for 10 min. Next, PCR products were resolved using 1.5% (w/v) agarose gel electrophoresis alongside a 100-bp ladder. The gels were then visualized under UV light in the Gel-Doc system (Labmet Asia Pvt. Ltd., Chennai, India), and the amplified bands were scored as “1” for the presence and “0” for the absence for each of the PCR products.
A completely randomized design was adopted for all the experiments, each of which comprised three replications and 20 samples per replication were involved. Collected data were statistically analyzed using SPSS software package (version 17.0, SPSS Inc., Chicago, IL, USA), and a one-way analysis of variance was formulated to assess statistical significance. Successively, mean±standard error data were compared with Tukey’s test at P = 0.05.