Metabolomic Profiling of in vitro and in situ Grown Nilgiris Tea Unraveled Unique Signatures of Caffeine Related Metabolites

Abstract

Abstract In this study, UPASI 9, a Nilgiris tea clone (Camellia sinensis), commonly cultivated using environmentally harmful and economically impractical cutting methods, has been reconsidered for propagation through a more sustainable approach using tissue culture. It was begun by establishing an in vitro culture and optimizing various factors such as explant source materials, disinfection procedures, and the composition of the culture medium. Anatomical studies demonstrated that the use of lower carbendazim concentration for sterilization (0.2%) produced viable and healthy explants for callus initiation, which is a key limiting factor in the success of tea tissue culture. To confirm the genetic consistency of the regenerated plants, gene-specific SSR markers were developed and utilized to ensure that the proliferated callus preserved the same genetic characteristics as that of the mother plants. Gas chromatography-mass spectrometry (GC-MS) was employed to analyze volatile metabolites extracted from callus, stem, micro shoots, and leaves of the UPASI 9 tea genotype. The results revealed distinct compositions of metabolites in each sample: callus showed unique metabolites like keto acids and derivatives, organic sulfonic acids, and their derivatives, and oxanes. Leaves stood out with imidazopyrimidines and prenol lipids, micro shoots with purine nucleotides and sulfinic acids, and stems with indoles and derivatives, pyrans, and tetrazines. However, eight classes of metabolites were consistently present in all four UPASI 9 samples. More interestingly, caffeine was exclusively detected in leaf samples but absent in all other investigated tissues, despite the presence of Tea Caffeine Synthase (TCS) gene-specific SSRs. Thus, this study provided unique information on the absence of caffeine in the in vitro grown Nilgiris tea clone, UPASI 9, which can be utilized further to create a decaffeinated tea product with minimal cost that has a unique demand in the global market.