## 4 Discussion  
In this study, _S. marianum_ seeds were irradiated by a He-Ne laser with a wavelength of 632.8 nm for various durations (2, 4, 6, 8, and 10 min). The seeds were then grown, and the vegetative growth, fruit yield, photosynthetic pigments, silybin (A + B) content, and expressions of chalcone synthase genes in the mature plants were evaluated. The positive plant growth and crop yield impacts as a result of pre-sowing irradiated seeds have been documented in various plant species [1; 2; 7; 20; 24; 25; 26]. Consistent with reports from other plant species, we also observed similar enhancement effects in the growth, yield, and photosynthetic pigment contents of the _S. marianum_ plants derived from seeds irradiated for different durations. Our observations further support the positive laser seed treatment effects observed by [PERSON] and [PERSON] [15] on _S. marianum_ seeds and seedlings. The increases in photosynthetic pigment (Chl a and carotenoids) contents were highly correlated with improvements in growth and yield. The 10-min pre-sowing irradiation treatment showed the highest enhancement among the five irradiation durations examined in this study. It was reported that the effective range of laser treatments varied among plant species, and when the irradiation limit was reached, negative effects were observed [27; 28]. In this study, we observed that the 8-min irradiation treatment showed the lowest enhancement in plant height, number of leaves, number of capitula, and contents of all photosynthetic pigments. In addition, the 8-min treatment also negatively impacted the dry weight of roots and aerial plant parts. However, the 10-min treatment increased values in all growth and yield parameters as well as photosynthetic pigment content compared with 8-min of irradiation. Therefore, we could not conclude that 8 min was the irradiation limit for _S. marianum_ seeds. Increased bioactive compounds have been reported in plants, such as fennel, coriander, anise, cumin, and curcumin, derived from laser-irradiated seeds or vegetative reproductive organs [20; 29; 30]. Similarly, in our study, in addition to laser-enhanced growth and yield, increased silybin (A + B) contents were also observed in the 6-, 8-, and 10-min laser treatment groups as compared with the control. For short irradiation durations (2 and 4 min), decreases in silybin (A + B) contents were observed despite the high growth rates and fruit yields. Despite having the highest growth and yield, plants from the 10-min-irradiated seeds were found to have lower silybin (A + B) contents as compared with those from the 6-min-irradiated seeds. This is consistent with the fact that secondary metabolites are produced in plants with lower growth and yield due to the redirection of plant resources from primary metabolism to secondary metabolism. Furthermore, in the present study, a strong correlation was also observed between the silybin (A + B) contents and expressions of the three chalcone synthase (_CHS1_, _CHS2_, and _CHS3_) genes. The stimulating effect of He-Ne laser irradiation on _CHS_ gene transcription observed in the present study could likely be the contributing factor that led to the high silybin (A + B) contents, similar to the observed increase in phenolic compounds and flavonoids previously reported in _Celosia argentea_ plants [31]. Using _D. discoideum_ as a cellular model confirmed the fruit extract bioactivity differences between plants derived from the 6-min laser-irradiated _S. marianum_ seeds and those derived from the non-laser-irradiated _S. marianum_ seeds. Increasing the content of silybin (A + B) via a 6-min laser treatment seems to inhibit _D. discoideum_ cell proliferation. A significant decrease in _D. discoideum_ cell growth was observed after 24 h of incubation with laser-treated _S. marianum_ extract at concentrations of 300 ug and 600 ug (Figure 2B,C). The significant inhibition of cell growth was also observed during the 72-h incubation with laser-treated _S. marianum_ extract.