Tea, derived from the evergreen Camellia sinensis tree, is a widely consumed non-alcoholic beverage processed from young leaves and is recognized for its stimulant, antioxidant, and anti-cancer properties, particularly, the green tea (Mondal et al. 2003; Vasisht 2003). Tea leaves contain various metabolites, including polysaccharides, essential oils, vitamins, minerals, purines, flavonoids, alkaloids like caffeine, and polyphenols like catechins. Phytochemicals, carbohydrates, and polyphenols constitute the major portion of tea composition and, contribute to its flavor, aroma, and potential health benefits (Soni et al. 2015). The effectiveness of tea, containing polyphenols like catechins, has been demonstrated in inhibiting tumor-cell proliferation, inducing apoptosis, and preventing angiogenesis and tumor cell invasiveness (Lambert and Yang 2003; Zaveri 2006). Tea also contains theobromine and theophylline, compounds known for their ability to relax coronary arteries, improving circulation. Moreover, tea is abundant with flavonoids and antioxidant polyphenols, which were acknowledged for their potential in reducing the risk of coronary heart disease and stomach cancer (Reyes and Cornelis 2018).
Despite these health benefits, tea contains caffeine, a key quality determinant but with potential adverse effects on cardiovascular health, palpitations, and insomnia (Schaffer et al. 2014). In pregnant women, caffeine tends to linger longer in the body due to slower metabolization, which potentially lead to issues such as infertility and birth defects (Miyagishima et al. 2011).
Consuming eminent amounts of caffeine, especially beyond 200 mg, can result in negative effects such as nervousness and anxiety, particularly in non-regular caffeine consumers. Excessive caffeine intake is also known to potentially elevate blood pressure, cause irregular heart rhythms, posing risks for those with pre-existing heart conditions (Schaffer et al. 2014). Those experiencing adverse reactions to caffeine are generally advised to discontinue caffeinated beverage consumption. To address these concerns, decaffeinated tea products are being promoted as a healthy alternative. This has led to a demand for tea production without caffeine or with reduced caffeine content, a process known as decaffeination (Miyagishima et al. 2011). However, this is a costly process as it requires skilled labour and sophisticated infrastructure.
India is the producer as well as the largest consumer of tea in the world. India produces nearly 23% of total world production and consumes about 21% of total global consumption of tea. Nilgiris tea products, in particular, have distinct demands in the world market for tea. Hence opting for low caffeine Nilgiris tea would be desirable to enjoy the complete health benefits of caffeine. Considerable progress in conventional breeding has led to the release of successful tea cultivars suitable for the Indian production system (Jain and Newton 1990; Mondal et al. 2004; Wachira 1990). However, challenges including perennial nature of tea plants, coping with extended gestation periods, managing issues of high inbreeding depression, handling self-incompatibility, encountering difficulties in finding distinct mutants for various biotic and abiotic stresses, lacking clear selection criteria, facing a low success rate in hand pollination, working with the short flowering window (2–3 months) of tea plants, enduring the lengthy period required for seed maturation (12–18 months), and managing variations in flowering time and fruit-bearing capacity among different tea clones (Mondal 2014) in improving the multigenic traits (such as those that govern tea yield, and quality) greatly limit the progress within a shorter period. Particularly, breeding for low-caffeine tea cultivars has not been a primary focus due to limited genetic information on caffeine synthesis.
Consequently, there is a need for an alternative and effective method to sustain tea plantations with high remuneration. In light of this, genome engineering or genome editing has emerged as a new advance technique with the ability to edit the genomes of plants (Feng et al. 2014; Jiang et al. 2014) and such advanced biotechnological tools are being employed to manipulate various traits in tea (Ashihara et al. 1995; Mondal et al. 2004; Takeda 1994). Conversely, it is essential to identify the key genes that regulate caffeine biosynthesis to evolve novel varieties with low caffeine content, preferably in the range of 1–2%. Furthermore, to realize their full potential, all these strategies require a common effective tissue culture protocol. Ensuring the genetic fidelity of tissue-cultured plantlets is also crucial in this process, and recent advancements in genetic fidelity testing rely on molecular markers. DNA-based marker techniques such as restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), simple sequence repeats (SSR), and amplified fragment length polymorphism (AFLP) are commonly employed in plant science for ecological, evolutionary, taxonomical, phylogenetic, and genetic investigations (Agarwal et al. 2008).
Among them, SSRs are highly favored for fidelity testing of in vitro grown plants due to their unique characteristics (including minimal repetition at each genetic location, even distribution across the genome, stable inheritance, and substantial genetic diversity (Boopathi and Boopathi 2020)). Thus, developing novel SSRs within the target genes (such as TCS investigated in this study) facilitates authentic genetic testing in large-scale production of tissue-cultured plantlets.
Gas Chromatography-Mass Spectrometry (GC-MS) serves as a highly effective analytical instrument for precisely identifying and quantifying the diverse array of metabolites found in tea. This facilitates a comprehensive understanding of tea's chemical composition, encompassing bioactive compounds such as catechins. Furthermore, GC-MS enables the assessment of concentrations of various health-promoting compounds in tea including polyphenols, flavonoids, and amino acids. Thus, GC-MS plays a crucial role in investigating the potential health effects of tea consumption by analyzing bioactive compounds, while also quantifying the volatile compounds that contribute to the aroma and flavor of tea. For example, a non-targeted metabolomic analysis was conducted on white, green, and black teas (all produced from the same tea leaves), which unveiled substantial distinctions in amino acids, catechins, dimeric catechins, and aroma precursors among their metabolomic profiles (Dai et al. 2017).
Results reported in this paper employed GC-MS analysis to gain a detailed understanding of the metabolites present in both in vitro and in situ grown tissues of the UPASI 9, a widely cultivated Nilgiris tea clone. Additionally, this study provided anatomical evidence for the support of carbendazim, which can be used for tea explant sterilization and offered novel TCS specific SSR markers that can be employed to test the genetic consistency of in vitro-cultivated callus.