*2.6. DNA Extraction and Molecular Characterization*

Genomic DNA was isolated from young leaves of the donor plant as well as micropropagated plants of *P. amboinicus* using the CTAB (cetyltrimethylammonium bromide) method [58]. Quantification and DNA purity was determined by a NanoDrop 200c Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The DNA samples were diluted in Ultrapure Milli-Q water to a final concertation of 25 ng/μL and stored in a laboratory refrigerator at 4 ◦C until further use. Five DAMD (Directed amplifications of minisatellite-region DNA; Table 1) primers (Metabion International AG, Planegg, Germany) and nine ISSR (Inter-simple sequence repeat; Table 2) primers (GeneLink, Inc., Orlando, FL, USA) were used for PCR.

**Table 1.** DAMD primers screening for the genetic stability of *Plectranthus amboinicus*.


**Table 2.** ISSR primers screening for the genetic stability of *Plectranthus amboinicus*.


PCR amplification was carried out in 20 μL volumes containing 2.0 μL of 10× PCR buffer, 1.2 μL MgCl2 (25 mM), 0.4 μL dNTPs (10 mM), 1 μL (10 pmole) primers, 0.2 μL Taq polymerase (3 Unit), and 1.2 μL Template DNA (25/μL ng). Both DAMD and ISSR reactions were carried out with a Bio-Rad thermal cycler (T100; Bio-Rad Laboratories, Inc., Hercules, CL, USA) with initial denaturation for 2 min (1 cycle) at 94 ◦C, denaturation for 5 min (35 cycles) at 94 ◦C, annealing for 2 min at 47–57 ◦C, extension for 1 min at 72 ◦C, and a final extension for 7 min at 72 ◦C. The amplified DNA fragments were separated and resolved on 1.5% (*w*/*v*) agarose gel (Sigma Aldrich, Inc., St. Louis, MO, USA) containing 4 μL ethidium bromide (Sigma Aldrich, USA) in a horizontal electrophoresis system (Biometra, Göttingen, Germany) with 1× TBE (Tris-Boric acid-EDTA). The electrophoresis was run for 2 h and the DNA bands were visualized on a UV Gel-Documentation System (G:BOX F3, Syngene, Cambridge, UK). Each sample were run thrice and only intense and reproducible bands were scored.
