*4.5. Determination of Lignin Content*

The plant materials were collected from the greenhouse of Zhejiang Agriculture and Forestry University, which is situated at 30◦15 2 N/119◦43 37 E, by upright *L. indica* 'Shaoguifei' (Figure 6A) and weeping *L. indica* 'Xiariwuniang' (Figure 6B). They were relocated into a habitat with stable temperature and favorable growing circumstances. By keeping track of processes since the time of budding, the growth condition of branches was studied.

The whole branches of 'Shaoguifei' and 'Xiariwuniang' were weighed 0.5 g at the early growth stage, growth stage, and the end of the growth stage, respectively. Further, three samples of each were collected and ground in liquid nitrogen and the ground powder was packed into 5 mL of an eluent. We then, centrifuged at 12,000 rpm for 20 min after 30 min of shaking the sample at 28 ◦C, then discarded the supernatant. After adding 100% methanol, the mixture was shaken for 30 min before being centrifuged at 12,000 rpm for 20 min, with the supernatant being discarded. There were four iterations of this stage. After that, they were dried in an oven set to 80 ◦C for an overnight period.

Accurately weigh 10 mg (can be recorded repeatedly) of the powder (washed and dried) into a 10 mL tube (the total weight of the powder after drying should also be recorded): first add 1 mL of 2 M HCL, then add 0.1 mL of thioglycolic acid, mix it upside down and evenly, then place it in a boiling water bath and heat it for 8 h. Then it was cooled on ice and centrifuged at 12,800 rpm/4 ◦C for 20 min and the supernatant was discarded. The precipitate was washed twice with distilled water and the precipitate was dried overnight after centrifugation. Then the precipitate was resuspended in 2 mL of 1 M NaOH, mixed evenly, and slightly shaken at 28 ◦C to react for 18 h. The precipitate was centrifuged at 12,800 rpm for 20 min. Next, 0.5 mL of the supernatant was put into a new glass test tube, 100 μL of concentrated hydrochloric acid was added to each tube, and the solution was placed in a refrigerator at 4 ◦C for 4 h (this operation was to precipitate the thioglycolate-bound lignin). The solution was centrifuged at 12,800 rpm/4 ◦C for 20 min to precipitate 1 mL of 1 M NaOH.

After dilution, a UV spectrophotometer was used to determine the absorbance at A280 nm. Each sample underwent three biological replicates, with NaOH solution serving as the blank control. This lignin determination method is referenced by Xu et al. [21].
