**1. Introduction**

Artemisinin is a natural sesquiterpene lactone containing an unusual peroxide bridge (**Figure 1**) [1]. It is present mainly in the leaves of *Artemisia annua L***.** (family Asteraceae) by storing in the glandular trichomes, which are tiny specialized hairlike epidermal cells found on the epidermis of leaves [2]. This traditional Chinese herb is a wild growing species with relatively low artemisinin content, ranging from 0.01 to over 1% of the plant dry weight, depending on the geographical origin, seasonal, and somatic variations [3, 4] and density of glandular trichomes in the leaves and aerial parts [5]. At present, the only commercial source of artemisinin is by extraction from field-grown leaves and flowering tops of the plant although many attempts to obtain higher artemisinin yield have been made from using simple breeding programs to complicated biotechnological approaches (for review, see [6]). Total synthesis of the compound has been reported [7, 8], but many chemical steps are required and the yields are low. In vitro cultures of *A. annua*, such as cell suspension and callus [9], shoot [10, 11] and hairy root cultures [12–15], have also been established for studying their potentials of producing artemisinin, but in vitro culture for artemisinin production has yet to prove commercially feasible. Therefore, the whole plant of *A. annua* is still the most economic source of artemisinin, and the development of high-producing plants of *A. annua* seems to be the main direction to obtain large quantities of relatively cheap artemisinin.

Ionizing radiation has been recognized as a powerful technique for plant improvement, especially in crop plants [16–18] and medicinal plants (for review, see [19]). This technique creates genetic variability in plants, which can be screened for desirable characteristics. So far, very little is known about the effect of gamma irradiation on the potential of artemisinin biosynthesis in *A. annua*, which involves several steps in its pathway [6]. Previously, we have reported a method for establishing in vitro plantlet variants of *A. annua* using low-dose gamma irradiation (less than 10 Gray) [20]. The survived plantlet variants maintained under the in vitro conditions for more than 6 months appeared to have stable content of artemisinin. However, it remained unknown whether the changes in artemisinin biosynthesis in in vitro plantlets would be maintained when the plants are grown ex vitro in a greenhouse or an open field. This question prompted us to investigate the process of acclimatization of the plantlets, followed by evaluation of the artemisinin content in the resulting whole plants.

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**Figure 2.**

*Gamma Irradiation Causes Variation and Stability of Artemisinin Content in Artemisia annua…*

In this study, mature seeds of *A. annua* were first surface sterilized and germinated on the MS medium, supplemented with 3% sucrose and solidified with 0.8% agar. The cultures were incubated for 2 months at 25°C with an exposure to 16 h light (ca. 3000 lux) and 8 h dark cycle. The obtained plantlets were then subcultured for five times before their shoot tips were excised and treated with gamma radiation. Practically, 1000 shoot tips (ca. 5 mm) excised from the in vitro plantlets were placed onto the same MS medium and irradiated with gamma rays generated

Atomic Energy for Peace (OAEP), Bangkok, Thailand). With this dose rate, the amount of irradiation energy absorbed by the shoot tips from 1 to 10 Gray (Gy) was conducted using the irradiation times from 7 to 70 s. After the irradiation, the exposed shoots were transferred to the fresh hormone-free MS medium. The shoots with subsequent active growth were subcultured every six weeks for four times on the same hormone-free MS medium. All the cultures were grown under the same conditions. After the forth passage, each vigorous shoot was cultured on the hormone-free MS medium in a 230-ml glass bottle. After culturing for six weeks,

**Figure 2** shows the morphology of *A. annua* plantlets derived from shoot tips gamma irradiated with a low dose range from 1 to 10 Gy. The plantlets depicted are representative of populations irradiated with the indicated gamma ray doses that survived four subsequent passages over a period of more than 6 months. The lowest dose of 3 Gy appeared to promote the growth of the plantlets, whereas doses above 5 Gy led to significant growth and morphological abnormalities. As shown in **Figure 2**, the 8-Gy dose gave rise to plantlets with pale green, fully expanded leaves,

In terms of survival rate, the results showed that there was a continual reduction in the survival percentage of the in vitro plantlets with increase in gamma

and the 10-Gy dose resulted in dwarf plantlets with no root differentiation.

*Effects of low dose of gamma irradiation on the morphology of A. annua plantlets.*

(using the facilities at the Office of

**2. Application of the gamma irradiation technique for potential** 

**2.1 Effects of gamma irradiation on the morphology and survival** 

**increase of artemisinin accumulation in** *A. annua*

*DOI: http://dx.doi.org/10.5772/intechopen.82385*

by Cobalt-60 at the dose rate of 8.56 Gy min<sup>−</sup><sup>1</sup>

survival percentage and regrowth ability were recorded.

**of** *A. annua* **plantlets**

**Figure 1.** *The structure of artemisinin [1].*

*Gamma Irradiation Causes Variation and Stability of Artemisinin Content in Artemisia annua… DOI: http://dx.doi.org/10.5772/intechopen.82385*
