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Soluble sugar content. The foliar content of soluble sugars in the tested tea leaves was determined using a test kit for plant soluble sugar content (No. A145-1-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The basis for this determination is that when concentrated sulfuric acid solution and sugar are combined, the resulting furfural or hydroxymethyl furfural can react with anthrone, and a change in the absorbance value is formed at 630 nm.
Soluble protein content. The foliar content of soluble sugars in the tea leaves that were tested was determined using a test kit for plant soluble sugar content (No. A145-1-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The anions in Coomassie brilliant blue can react with the amino termini in proteins to turn the solution blue when the dye is added to a sample that contains proteins. The amount of protein in the sample or standard solution can then be determined through measuring the absorbance.
Free fatty acid content. The foliar content of free fatty acids in the tea leaves tested was determined using a free fatty acids assay kit (A042-1-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Free fatty acids can form a copper salt of fatty acids by combining with copper ions and dissolving in chloroform, and a color change occurs.

The fully expanded mature leaves of diploids and tetraploids were selected for physiological parameter measurements. For the determination of the catalase (CAT) activity, peroxidase (POD) activity, malondialdehyde (MDA) content, total protein content, and soluble sugar content, their corresponding assay kits (Catalog No. A007-1-1, A084-3-1, A003-1-2, A045-2-2, and A145-1-1, Nanjing Jiancheng Bioengineering Ins., Nanjing, China) were used according to the manufacturer’s protocols.

Approximately 100 mg of fresh leaves and roots was homogenized for determination of proline content following the manufacturer’s instructions (A107-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and the absorbance was measured at 520 nm. For the analysis of soluble sugars, approximately 0.1 g of fresh samples was homogenized in 1 ml ddH₂O with a glass homogenizer. The tubes were boiled at 95°C for 10 min and cooled with tap water. After cooling, the homogenate was centrifuged at 4,000 × g for 10 min. Thereafter, the supernatant was diluted with ddH₂O at 1:9. The diluted extracts were used for determination of soluble sugar content using a commercially available test kit (A145-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Finally, the absorbance was measured at 620 nm and the soluble sugar concentration expressed and the results expressed in the fresh weight (FW) basis (Dai et al., 2018 (link); Kumar et al., 2021 (link)).