**4. Conclusion**

*Use of Gamma Radiation Techniques in Peaceful Applications*

primarily at the biochemical and physiological levels.

the artemisinin content in *A. annua*.

low-content leaves found in greenhouse conditions.

content remains unclear.

greenhouse, with R2

In terms of formation of secondary products, little information is available regarding the use of gamma irradiation for yield improvement. To our knowledge, the only related report characterized the effect of low-dose gamma irradiation (2–16 Gy) on the increased production of shikonin derivatives in callus cultures of *Lithospermum erythrorhizon* [28]. In agreement with these results, we also found a significant effect on artemisinin content caused by a similar low-dose range of gamma irradiation. However, in the present study, we observed these effects in mature plants, rather than in disorganized tissues. Our results show that treatment with either 8 Gy [19] or 5 Gy can create a population of plantlets with a high range of artemisinin contents. Due to the minimal associated morphological effects, the 5-Gy dose was chosen to produce *A. annua* variants that were presumably affected

The specific genes affected by the low-dose gamma radiation were observed to

For the ex vitro acclimatization of the plants, we have previously characterized the conditions and supporting material important for photoautotrophic growth of *Eucalyptus camaldulensis* plantlets, both in vitro and ex vitro [23]. Adoption of this protocol resulted in a survival rate of 67% for *A. annua* plants after the in vitro-ex vitro transfer and a survival rate of 38% after 6 months of ex vitro growth to obtain mature plants. Clearly, the stresses generated in weakened irradiated plants during the process of acclimation lead to significant mortality. However, whether the mortality is more prevalent among in vitro individuals with low or high artemisinin

For the 23 surviving mature plants, we observed an individual correlation in artemisinin content between the in vitro plantlets and the ex vitro mature plants. This one-to-one correlation was strongly positive for plants grown in the

 = 0.797. These results suggest that the capability for artemisinin biosynthesis in each in vitro plantlet is maintained throughout the in vitro-ex vitro transfer and the subsequent development into a mature plant. With respect to the greenhouse plants, although the correlation coefficient value was quite high, the high-yield plants did exhibit a reduction in artemisinin content. This observation is likely due to the high biomass weight per leaf for high-content leaves, which clearly appear thicker than

The differences in biomass associated with artemisinin content are not so obvious among the established in vitro plantlets, resulting in a decrease in the

= 0.915, and relatively positive for field-grown plants, with

be at least on the gene of ADS of the biosynthetic pathway of artemisinin in *A. annua* plantlets. More than half of the variant population appeared to have a high correlation coefficient value (R = 0.922) between artemisinin content and ADS enzyme activity. The reason why ADS gene is particularly sensitive to the irradiation is still not clear. However, it might be that the low doses of 5–8 Gy of the gamma irradiation are just mild enough to affect this ADS gene. In principle, some lesions of ADS gene caused by the irradiation are likely to be repaired through the action of intracellular DNA repair process, while ADS gene of some other samples might remain unpaired or misrepaired, giving rise to permanent changes in the affected ADS gene. This would lead to a cellular response including a wide range of the enzymatic systems, as observed in this case with the variable ADS enzyme activities in *A. annua*. In the literature, there has been a report supporting our results. That is the case of low-dose irradiated callus cultures of *Lithospermum erythrorhizon* in which the enzyme activity of p-hydroxybenzoic acid geranyltransferase involving in the shikonin biosynthesis is boosted after the gamma irradiation [28]. Thus, it was suggested that the creation of plant variation through gamma irradiation has significant effect on ADS gene which is likely to be related to the enhancement of

**244**

R2

Based on these results, we conclude that the technique of gamma irradiation can produce viable variants of *A. annua* that are capable of maintaining changes in gene expression associated with the artemisinin biosynthetic pathway (such as ADS) throughout the in vitro-ex vitro transfer process and, at minimum, through the first generation of mature plant development. Relatively low doses of gamma irradiation (ca. 3–8 Gy) can be effective for yield enhancement of artemisinin in *A. annua*. A mechanistic understanding of the increased biosynthesis of artemisinin in response to gamma irradiation is important for the development of a production-scale operation.
