This breakthrough allows researchers to visualize where individual genes are active at the cellular level—information that has long been missing due to technical limitations in tea biology. By enabling accurate characterization of gene expression heterogeneity, the method offers a powerful new tool for functional genomics and may accelerate advances in tea breeding, metabolic pathway engineering, and stress-response research.
Tea plant research has progressed rapidly in recent years, driven by major questions surrounding the biology, metabolism, and genetic diversity of this globally important crop. Although numerous functional genes have been identified, most expression data remain at the tissue level because stable genetic transformation in tea plants is technically challenging. Existing approaches—such as promoter–reporter constructs, single-cell isolation paired with qPCR, and RNA in situ hybridization—are difficult to apply broadly or require specialized probes. These limitations make it difficult to determine which specific cell types express key genes, hindering studies on signaling pathways, metabolite transport, and root development. To overcome these challenges, developing an accessible, reliable method to map in vivo gene expression at cellular resolution is urgently needed.
A study (DOI: 10.48130/bpr-0024-0033) published in Beverage Plant Research on 09 January 2025 by Shupei Zhang’s team, Anhui Agricultural University, presents an efficient in situ RT-PCR method for mapping gene expression at the cellular level in tea plant roots, providing a valuable tool for understanding gene function in tea plant biology.
The research team designed and optimized an in situ RT-PCR method to detect gene expression in specific tea plant root cells. This technique involves a series of steps including tissue preparation, fixation, embedding, sectioning, proteinase K treatment for digestion, DNA removal, reverse transcription, PCR amplification, immunoassay, and microscopy imaging. For tissue preparation, young, tender tea plant roots were chosen to ensure ease of reagent penetration. Fixation was achieved using a formaldehyde-acetic acid-ethanol solution, with agarose embedding used to preserve tissue integrity, as it provides better RNA stability compared to paraffin embedding. Proteinase K digestion increased cell permeability, enabling effective PCR amplification. The primers designed for PCR were specific for gene fragments of interest, ensuring high specificity and reliable detection. Reverse transcription was carried out to convert mRNA into cDNA, using either random or specific primers based on gene expression levels. PCR amplification included a DIG-labeled probe for in situ detection, with optimized conditions for magnesium concentration and cycle numbers to prevent nonspecific amplification. To confirm the gene expression patterns, negative and positive controls were included, and the final results were visualized under a microscope. The method was validated using the expression of CsGL3, CsCAT2, and CsAAP4 genes in tea plant roots. CsGL3 was expressed in epidermal cells, while CsCAT2 was found in pericycle, cortex, and endodermis cells, consistent with previous studies. CsAAP4 was not expressed, confirming the method's sensitivity. These results demonstrate the effectiveness of the in situ RT-PCR technique in accurately mapping gene expression at the cellular level in tea plant roots.
Overall, this study provides the first comprehensive and tea-specific protocol for in situ RT-PCR, filling a major methodological gap in tea plant biology. By enabling rapid, cost-effective visualization of gene expression at cellular resolution, the method will strengthen functional analyses in root development, nutrient transport, and secondary metabolism.
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References
DOI
10.48130/bpr-0024-0033
Original Source URL
https://doi.org/10.48130/bpr-0024-0033
Funding information
This work was supported by the National Natural Science Foundation of China (32072624), Anhui Provincial Major Science and Technology Project (202103b06020024) and Anhui Educational Committee Excellent Youth Talent Support project (gxyqZD2022018).
About Beverage Plant Research
Beverage Plant Research (e-ISSN 2769-2108) is the official journal of Tea Research Institute, Chinese Academy of Agricultural Sciences and China Tea Science Society. Beverage Plant Research is an open-access, online-only journal published by Maximum Academic Press. Beverage Plant Research publishes original research, methods, reviews, editorials, and perspectives that advance the biology, chemistry, processing, and health functions of tea and other important beverage plants.