Molecular differentiation of the green and purple Tulsi (Ocimum tenuiflorum L.) and its application in authentication of market samples

Tulsi (Holi Basil, Ocimum tenuiflorum) is extensively used in herbal medicine, and it includes two distinct subtypes; namely green Tulsi and purple Tulsi. Both types have similar medicinal properties. However, purple Tulsi contains a significantly higher amount of methyleugenol, which is genotoxic, and its daily intake is restricted. We developed a polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) method to identify purple Tulsi. For this purpose, we selected a C > T single nucleotide polymorphism (SNP) in the ycf1 gene of the chloroplast genome that overlapped with a DdeI restriction site. The primers and PCR conditions were optimized to amplify a 797 bp DNA encompassing the C > T SNP, specifically from O. tenuiflorum. After restriction digestion of the PCR product with DdeI, green Tulsi was identified by two fragments (539 bp and 258 bp), and purple Tulsi was recognized by a single fragment (797 bp). Analysis of 40 Tulsi market samples revealed that only 36 (90%) were derived from O. tenuiflorum. The majority of the market samples were purple Tulsi (60%) or a mixture of green and purple Tulsi (27.5%), with some mixed samples containing up to 50% purple Tulsi.