## 3 Results and Discussion
### Effect of Cold Plasma Treatment on Endogenous Hormone Regulation
#### 3.2.2 Effect of Cold Plasma Treatment on Endogenous Hormone Regulation and Radicle Growth  
To study how different RNS/ROS ratios in the plasma jet affected the regulation of the endogenous growth hormone GA\({}_{3}\), seeds were treated by plasma jet with different tube lengths, and their radicle growth was monitored up to 96 h. The optimal plasma treatment time of 10 min obtained from the previous section was applied in all cases. We followed a previous extraction protocol to extract GA\({}_{3}\) from untreated and plasma-treated seeds [19] as described in the methods. Figure S2 shows liquid chromatograms of the extracted liquid vs. the calibration curve of the standard GA\({}_{3}\). The retention time of GA\({}_{3}\) was from 7 to 8 min, peaking at 7.9. To confirm whether the substance obtained after LC from the extraction solution was GA\({}_{3}\), we measured the MS spectrum of the extracted liquid from 7 to 8 min retention time. Figure 6a shows the MS spectrum of GA\({}_{3}\) separated from the extraction solution. In addition to a peak at 345.1 (\(m/z\)) corresponding to [M\(-\)H]\({}^{-}\) ion, we obtained a peak at 239.1 (\(m/z\)) corresponding to [M\(-\)H-COO-COO-H2O]\({}^{-}\) ion of  
Figure 5: (**a**) Different downstream tube lengths. (**b**) Optical emission spectra in 270–900 nm range. (**c**) Dependence of optical emission intensities from N\({}_{2}\) SPS (337 nm), OH radical (309 nm), and NO concentration with different downstream tube lengths. GA\({}_{3}\). These peaks agreed with a previously published MS spectrum of GA\({}_{3}\)[25]; further confirming our successful extraction and separation of GA\({}_{3}\). For quantitative analysis of GA\({}_{3}\), a calibration curve was constructed with the standard GA\({}_{3}\) at seven different amounts (0.1; 0.5; 1; 2; 3; 5; 10) \(\upmu\)g based on LC spectrum (Figure S3). The amount of gibberellic acid (GA\({}_{3}\)) in the extracted solution was determined based on the integrated area of the LC spectrum. Based on the quantitative calibration curve of GA\({}_{3}\), through three average measurements, we determined the amount of GA\({}_{3}\) (\(\upmu\)g) in 1 g bean sprout powder in each sample, as shown in Figure 6b (red, above). We observed that the shorter downstream tube length--hence the higher RNS/ROS ratio--led to upregulating GA\({}_{3}\) in the treated seeds. The amount of GA\({}_{3}\) reaches a maximum value of 1.34 \(\pm\) 0.07 \(\upmu\)g per 1 g bean sprout powder (which is \(\sim\)2.8 times more than untreated seeds) with the 3-cm plasma downstream tube length (T3). This result was the first direct measurement to show that cold plasma treatment can induce upregulation of the natural  
Figure 6: (**a**) Mass spectrum (MS) and the chemical structure (above) of GA\({}_{3}\) from the extracted solution. (**b**) The amount of GA\({}_{3}\) (red, above) extracted per 1 g mung bean; the average length of the radicles (blue, below), and (**c**) a photo of the radicles after 96 h under no plasma and plasma treatment with different downstream tube lengths. The ruler on the right is 20-cm long. endogenous hormone GA\({}_{3}\), which leads to enhancing germination and radicle growth of treated seeds. Interestingly, the plant growth of the radicles correspondingly followed the change in the regulated GA\({}_{3}\) (Figure 6b). The average length of the radicles also reached its maximum value of 13.5 \(\pm\) 1.6 cm (which is 2.7 times more than in the untreated seed) with T3 plasma jet. This result suggested that NO-induced upregulation of GA\({}_{3}\) by gas-phase cold plasma could be the dominant phytochemistry pathway responsible for enhancing the radicle growth of mung bean. Therefore, for the highest induced GA\({}_{3}\) concentration in seeds and the longest plant growth of the radicles, we recommend NO concentration between 20-95 ppm, which corresponds to the plasma downstream tube length from 7 cm (T7) to 1 cm (T1) (Figure 6c).