Artemisinin is an anti-malarial sesquiterpene lactone isolated from Artemisia annua L., a traditional Chinese herb of the family Asteraceae. The plant contains relatively low artemisinin content, ranging from 0.01 to 0.8% of the plant dry weight, depending on the geographical origin, seasonal, and somatic variations. Ionizing radiation has been recognized as a powerful technique for plant improvement, especially in crop plants. This technique creates genetic variability in plants, which can be screened for desirable characteristics. Very little is known about the effect of gamma irradiation on the potential increase of artemisinin production in A. annua. In this study, 130 shoot tips excised from the population of in vitro A. annua plantlets (with an average leaf artemisinin content of 0.18 ± 0.09%) were exposed to 5 Gy 60Co gamma irradiation and subsequently transferred to a suitable medium for in vitro development of plantlets. The resulting 90 stable survived after four passages appeared to have a wide variation of artemisinin content, ranging from 0.02 to 0.68% of dry weight. All the viable plantlets were then transferred from the in vitro cultures to ex vitro conditions both in a greenhouse and an open field. A significant correlation was observed between artemisinin content among individual pairs of the vitro plantlets and ex vitro mature plants, with the correlation coefficient (R2) values of 0.915 for the greenhouse plants and 0.797 for the open field plants. Among these, the highest artemisinin-containing plant appeared to accumulate 0.84% artemisinin of dry weight in the open field, which is almost five times higher than the original plants. These results suggest that gamma irradiation with 5-Gy dose can produce viable variants of A. annua that can maintain the biosynthetic capability of artemisinin throughout the in vitro-ex vitro transfer and development of the first generation of mature plants.
- Artemisia annua L.
- gamma irradiation
- in vitro plantlets
- ex vitro plants
Artemisinin is a natural sesquiterpene lactone containing an unusual peroxide bridge ( Figure 1 ) . It is present mainly in the leaves of
Ionizing radiation has been recognized as a powerful technique for plant improvement, especially in crop plants [16, 17, 18] and medicinal plants (for review, see ). 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
2. Application of the gamma irradiation technique for potential increase of artemisinin accumulation in
2.1 Effects of gamma irradiation on the morphology and survival of
In this study, mature seeds of
Figure 2 shows the morphology of
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 irradiation dosage from 1 to 10 Gy ( Figure 3 ). The lethal dose of gamma rays that causes 50% survival reduction (LD50) was 8 Gy. Again, the doses lower than this LD50 value showed essentially normal morphology of the survived plantlets, whereas the higher doses seemed to cause significant abnormalities as shown by dwarf plantlets with pale leaves ( Figure 2 ). This LD50 value was obtained from the in vitro plantlets survived for at least 6 months (four subsequent subcultures) after the irradiation. It is, therefore, likely that they are genetically stable variants.
2.2 Effects of gamma irradiation on artemisinin accumulation in
A. annuain vitro plantlets
To perform a rapid analysis of artemisinin in a large population of the irradiated plantlets, we used our own developed simple and sensitive TLC-densitometric method for artemisinin analysis which was reported previously . Practically, fresh leaves obtained from various in vitro plantlets were collected, dried at 60°C, and ground to fine powder in a grinder. Each powder sample (100 mg) was extracted under reflux in 10 ml hexane (70°C) for 1 h. The extract was then filtered, and a 10 μl aliquot was spotted onto a pre-coated silica gel TLC plate. Up to 15 samples could be applied onto each plate which was developed using the solvent system of hexane:ethyl acetate:acetone, 16:1:1. The plate was dried and exposed for 2 h with saturated ammonia vapor (in a closed TLC tank) for complete derivatization of artemisinin. The TLC plate was then taken from the tank, air dried, and observed in a light box under the wavelength of 366 nm which could be seen variation of artemisinin band intensity among various extract samples ( Figure 4A
Using this TLC-based technique, the surviving plantlets (obtained after 8 Gy treatment and four subsequent subcultures) were analyzed for their ability to accumulate artemisinin. The results showed a wide variation of artemisinin content, ranging from 0.03 to 0.70% dry weight ( Figure 5 ). The control plantlets derived from the shoot tips not exposed to gamma rays showed their artemisinin levels of as low as 0.18 ± 0.09% of dry weight. In terms of content distribution, almost 80% of the irradiated plantlets showed artemisinin content less than 0.3%, and approximately 5% of the population showed higher than 0.5% of dry weight ( Figure 5 inset). Thus, it was clear that there were variations in the potential of artemisinin biosynthesis among the irradiated plantlet population.
It should be noted that more than 50% of the plantlet population accumulates artemisinin in the content higher than the original untreated plants (0.18 + 0.09% of dry weight). This is probably due to the use of low-artemisinin containing plants as a starting material, which allow higher artemisinin-containing variants be obtained more easily upon the irradiation. The observed quantitative and some extent of qualitative variations indicate that the secondary metabolism in the irradiated plantlets of
2.3 Correlation between artemisinin accumulation and enzyme activity of amorpha-4,11-diene synthase in irradiated plantlets
The observed variation of artemisinin accumulation in the irradiated plantlets raised a question on the possible site genes of mutation, especially of the genes involved in the biosynthetic pathway of artemisinin. Since amorpha-4,11-diene synthase (ADS) has been known as one of the key enzymes of the pathway in
The enzyme activity of ADS was then determined by modifying the radio-isotopic method described previously . The reaction mixture contained [1-3H(N)] farnesyl diphosphate (100,000 dpm), 5 mM Mops buffer, pH 7.0, 10%(v/v) glycerol, 10 mM ascorbic acid, 10 mM MgCl2, 2 mM DTT, and 10 mM Na2MoO4 in a total volume of 70 μl. After 30 min of incubation at 30°C, the reaction mixture was extracted with 1 ml hexane, taking the hexane layer to evaporate followed by spotting onto a TLC plate (aluminum sheet, silica gel 60 F254, 0.25 mm thickness). The resulting TLC plate was then developed in a solvent system of hexane:ethylacetate:acetic acid (25:7:1) and was scanned to obtain radio-chromatograms by a TLC-radioscanner. The area under amorpha-4,11-diene peak of each radio-chromatogram was then used for calculating the synthase activity. Figure 6 shows typical TLC-radiochromatograms of the reaction mixtures catalyzed by enzyme preparations obtained from some irradiated plantlets. It can be seen that the radioactive peaks of amorpha-4,11-diene (Rf value of 0.55) could be clearly detected with different peak sizes from different samples, suggesting that the TLC-radio assay worked well for determining the enzyme activity of ADS.
Subsequently, 18 plantlet samples with different artemisinin contents were assayed for their ADS activity. Again, it was found that the selected samples showed high variation in the enzyme activity, ranging from 0.02 to 0.18 pkat mg−1 protein. When the results of the enzyme activity and artemisinin content were plotted together ( Figure 7 ), it was found that the 18 plantlets showed their poor value of the correlation coefficient at R = 0.300 ( Figure 7A ). However, by excluding only two outliners with extremely high ADS activities, the correlation coefficient appeared to be much better, with R = 0.717 ( Figure 7B ). Among these, 11 of the 16 samples showed quite high value of the correlation coefficient, R = 0.922 ( Figure 7C ). This suggested that the gamma irradiation did affect the gene of ADS, and thus the biosynthetic capability of artemisinin in the mutant plants.
2.4 Variations in artemisinin content in plantlets irradiated with the 5-Gy dose
As mentioned earlier (Section 2.1), the doses above 5 Gy led to significant growth and morphological abnormalities, with the 8-Gy dose giving rise to pale-green and fully expanded leave plantlets, and the 10-Gy dose showing dwarf and no differentiate root plantlets with no root differentiation. Therefore, the 5-Gy dose was chosen for mass irradiation of shoot tips to obtain a population of
The results showed that among 130 shoot tips irradiated with a 5-Gy dose, 90 plantlets (69% survival) were obtained using established in vitro culture conditions. These plantlets were then evaluated for their ability of artemisinin accumulation. The results revealed again wide variation in artemisinin content, ranging from 0.02 to 0.68% dry weight ( Figure 8 ). Among these plantlets, 6 individuals had an artemisinin content greater than 0.5% dry weight, 39 plantlets had an artemisinin content in the range of 0.21–0.50%, and 45 plantlets had an artemisinin content below 0.02%. As control plantlets derived from shoot tips not exposed to gamma rays exhibited an average artemisinin content of 0.18 + 0.09%, treatment with the 5-Gy dose resulted in an artemisinin content above that of the control for approximately half of the irradiated plantlets ( Figure 8 ).
2.5 Ex vitro acclimatization of
A. annuairradiated plantlets
In this study, actively growing shoot tips (length, ca. 5 mm) were excised from in vitro plantlets and stripped of their leaves. The resulting shoot tips (with 130 tips) were then inserted vertically 2 mm in depth into the MS medium containing 3% sucrose and 0.8% agar. Induction of variation in
The development from the step of in vitro irradiated plantlets to ex vitro
2.6 One-to-one correlation of artemisinin content between in vitro plantlets and ex vitro plants
Quantitative analysis revealed that the 13 surviving plants from the in vitro-ex vitro transfer to the greenhouse had their artemisinin content ranging from 0.12 to 0.42% dry weight. This range was slightly narrower than that of the in vitro plantlets, which exhibited artemisinin contents ranging from 0.06 to 0.66%. Interestingly, comparison of in vitro plantlets and ex vitro plants grown in the greenhouse on a one-to-one basis revealed that there was a significant individual correlation between the artemisinin content of paired in vitro plantlets and ex vitro plants grown in the greenhouse, with a very good correlation coefficient (R2) value of 0.915 ( Figure 10a ). For plants transferred to the open field, the 10 in vitro-ex vitro pairs exhibited artemisinin contents for in vitro plantlets ranging from 0.25 to 0.69% and for ex vitro plants ranging from 0.31 to 0.84% ( Figure 10b ). A one-to-one comparison of the artemisinin content of plants grown in the open field with their corresponding plantlets also revealed a good R2 value of 0.797 ( Figure 10b ).
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
The specific genes affected by the low-dose gamma radiation were observed to be at least on the gene of ADS of the biosynthetic pathway of artemisinin in
For the ex vitro acclimatization of the plants, we have previously characterized the conditions and supporting material important for photoautotrophic growth of
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 greenhouse, with R2 = 0.915, and relatively positive for field-grown plants, with R2 = 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 low-content leaves found in greenhouse conditions.
The differences in biomass associated with artemisinin content are not so obvious among the established in vitro plantlets, resulting in a decrease in the degree of the one-to-one correlation observed strictly for high-yield plants. For the field-grown plants, the relatively positive value of the correlation coefficient (R2 = 0.797) may be due to two outliers present among the 10 samples (nos. 308 and 311) that deviate from the rest of the population. A pairwise comparison of the remainder of the samples would result in a higher R2 value, which is reflective of a good correlation in artemisinin content between the in vitro plantlets and ex vitro field-grown plants. In addition, the lower correlation could also be attributed to the less controlled conditions of the open field compared with those of the greenhouse.
To be certain that changes in artemisinin biosynthesis in the ex vitro plants are genetically stable, it is necessary to test the next generation. However, many of the established mature plants could not produce seeds. Therefore, stability tests assessing the next generation through seed germination are not possible. Alternatively, this analysis can be performed through a second round of in vitro-ex vitro transfer. No attempt was made to use this method in the present study due to the high mortality associated with this long process that would have resulted in an insufficient number of pairs of plants for a one-to-one analysis.
Based on these results, we conclude that the technique of gamma irradiation can produce viable variants of
This work was supported by Chulalongkorn University’s Ratchadaphiseksomphot Endowment Fund (to Natural Biotechnology Research Unit) and Thailand National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency (NASDA).
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.