Tree Genetics and Molecular Breeding 2025, Vol.15, No.5, 192-201 http://genbreedpublisher.com/index.php/tgmb 197 components (Cornejo et al., 2018). At present, there are a large number of SNP resources and genomic annotation data, which provide a basis for the development of molecular markers related to high theobromine content, superior flavor and disease resistance (Cornejo et al., 2018). Through these markers, breeders can more quickly screen out new materials that have both good flavor and health value to meet the demands of high-end chocolate and functional foods (Wickramasuriya and Dunwell, 2017). 7.2 Biotechnological strategies for metabolic engineering of theobromine levels With the development of genomics and metabolomics, metabolic engineering has become another means to regulate theobromine levels. By using gene editing and transgenic techniques, key enzyme genes (such as 7-methylxanthine synthase) can be targeted and regulated, thereby increasing or decreasing theobromine content (Koyama et al., 2003). In addition, techniques such as in vitro embryogenesis and genetic transformation have been applied to the genetic improvement of Theobroma cacao, providing a platform for the precise regulation of alkaloid metabolism. These methods can not only help cultivate specialized varieties with high or low theobromine, but also carry out multi-trait improvement in combination with goals such as disease resistance and stress tolerance (Wickramasuriya and Dunwell, 2017). 7.3 Industrial implications for chocolate quality, pharmacology, and sustainability The level of theobromine directly affects the flavor, bitterness and functionality of chocolate, thereby determining the market positioning and consumer experience of the product. Cocoa beans with a high theobromine content can produce chocolate with a stronger flavor and higher health value, meeting the demands of high-end and functional markets (Cortez et al., 2023). Meanwhile, theobromine and its derivatives have antioxidant and cardiovascular protective effects in the fields of health care and pharmacology, and have also expanded the application prospects of Theobroma cacao industry (Wickramasuriya and Dunwell, 2017). Increasing theobromine levels through genomics and metabolomics helps improve raw material utilization and product added value, and promotes the sustainable development of the industry (Wickramasuriya and Dunwell, 2017; Cornejo et al., 2018). Meanwhile, precision breeding and biotechnology improvement can also reduce dependence on the environment, enhance production efficiency, increase the income of small-scale farmers, and promote the green transformation of the global Theobroma cacao industry (Wickramasuriya and Dunwell, 2017). 8 Challenges and Research Gaps 8.1 Complexity of alkaloid regulation and redundancy in gene families The synthesis of theobromine involves multiple purine metabolic pathways and also requires the joint action of various enzymes. This regulatory network is very complex. Research has found that there are often multiple homologous genes and repetitive enzyme activities in related pathways, so the metabolic patterns of different tissues and developmental stages vary greatly. For instance, the theobromine content in young fruits and tender leaves is relatively high, but it will significantly decrease as tissues mature. Moreover, the activities of synthetic and degradation pathways in different tissues also vary (Koyama et al., 2003; Zheng et al., 2004). Transcriptome analysis also revealed that multiple transcription factors and signaling pathways interact, which makes it more difficult to identify key regulatory nodes. The redundancy of gene families also indicates that it is difficult to precisely regulate theobromine content through single gene editing (Gallego et al., 2021). 8.2 Integration difficulties between metabolomic and genomic datasets Although metabolomics and genomic technologies have provided a wealth of data, the integration of the two remains highly challenging. The dynamic changes of metabolite abundance are often inconsistent with the expression of genes at different times and in different tissues, so it is difficult to directly establish the correspondence between genes and metabolites (Gallego et al., 2021). Environmental factors, such as light exposure and fermentation treatment, can also simultaneously affect metabolites and gene expression, increasing the complexity of integration (Cortez et al., 2023). At present, there is a lack of a unified analytical framework and standardized methods, which limits the in-depth understanding of the regulatory mechanism of theobromine synthesis (Gallego et al., 2021).
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