The evaluation of agronomic characters and artemisinin content of EMS-mutated artemisia lines

The cases of malaria in Indonesia are high, especially in Eastern Indonesia, while the basic raw materials for malaria drugs are still imported. Artemisia (Artemisia annua L.) produces artemisinin, a sesquiterpene lactone compound, to treat malaria. Artemisinin content of Artemisia growing in Indonesia is low, therefore technological support is needed to produce new varieties of artemisia that have high artemisinin content. In vitro mutagenesis could increase plant genetic diversity which is the main requirement in plant breeding. The application of Ethyl Methane Sulphonate (EMS) to the artemisia cell population has been carried out and nine 3rd generations of artemisia mutant lines were obtained. This study aims to evaluate the growth of artemisia mutant lines based on their agronomic characters and artemisinin content. The research was conducted in Lembang, Bandung in July - December 2022. A completely randomized block design was used. The results showed that EMS affected growth characteristics that correlated with plant productivity, i.e. plant height, number of branches, stem diameter, and artemisinin content. Of the nine tested lines, at least 7 had higher plant height, number of branches, wet weight, and dry weight than the control. The artemisinin content of the mutants ranged from 0.67 to 1.02%, while the artemisinin content of the control was 0.4%. The increasing artemisinin content of these mutants reached 1.5 times compared to the control, and the mutant lines’ yields reached 2 times that of the control. The diversity of plants can also be seen from the differences in the color of the stems.


Introduction
Malaria is an infectious disease caused by Plasmodium and is transmitted through the bite of the female Anopheles mosquito.Malaria cases in Indonesia are high, especially in eastern Indonesia.Artemisinin, a sesquiterpene lactone compound obtained from the Artemisia annua plant, is a new substance for malaria drugs to overcome chloroquine resistance.These compounds and their derivatives are effective in inhibiting the growth of Plasmodium falciparum both in vitro and in vivo.World Health Organization (WHO) has recommended the use of artemisinin as the best treatment for malaria.
Artemisia (Artemisia annua L.) is a source of artemisinin and essential oils such as artesunate and artemether.Artemisinin is a secondary metabolite product that can act as an antimalarial drug without causing serious side effects such as quinine or chloroquine [1].This compound is a group of sesquiterpene lactones with endoperoxide bridges which are rarely found in nature.Secondary metabolites have a more complex structure than primary metabolites, making them more difficult to synthesize chemically.The problem encountered in developing artemisia as a raw material for medicine is the low content of active ingredients, which ranges from 0.01 -1% depending on cultivar, cultivation, and environmental conditions [2].The low content of artemisinin will not be profitable if developed on an industrial scale.
Mutagenesis is an alternative method in creating genetic variability in plants and modulating metabolic pathways towards enhanced production of a targeted metabolite [3], using physical or chemical mutagens, leading to genetic alterations in biological genomes.Ethyl methyl sulphonate (EMS) is an alkylating agent that can cause point mutations at the DNA level and has been reported to be an effective mutagen for the improvement of crop plants [4].The advantages of using mutagens in improving plant characters depend on the effectiveness of the mutagens used [5].
This study aimed to evaluate the growth of artemisia mutant lines based on their agronomic characters and artemisinin content.

Methods
The plant materials used were 9 third-generation mutant lines which were obtained from callus-derived artemisia leaves treated with the chemical mutagen (Ethyl Methane Sulphonate/ EMS) at a concentration of 0.1%; 0.3%; and 0.5% for 1 and 3 hours.The surviving calli were transferred to the growing medium, regenerated into plantlets, and acclimatized in the greenhouse.Mutant seedlings have been grown in the field for 2 generations (2020-2021), the selection was made based on growth characteristics (plant height, stem diameter, fresh weight, dry weight), and artemisinin content.Nine mutant genotypes were obtained which were tested in this activity.
The research was conducted in 2022.Field testing was carried out at the Manoko experimental, Lembang which is at an altitude of 1100 m above sea level.Seeds from 9 Artemisia lines and the wild type as control were sown in a mixture of soil and compost with a ratio of 1:1.After the seedlings have 4 leaves (2 weeks after planting) they were transferred to plastic polybags filled with a mixture of soil and manure with a ratio of 1:1.Seedlings were planted in the field after they reach 15-20 cm high.The spacing used was 1 m x 1 m, while the distance between beds was 50 cm.NPK fertilizer was given at a dose of 40:40:40 kg/ha.P 40kg/ha, N 20kg/ha, and K 20kg/ha given one week after planting.The second fertilization, N 20 kg/ha and K 20 kg/ha, was given one month after planting.Observations were made on plant height, the number of branches, stem diameter, and stem color.Data collection was carried out on a randomly selected sample of 5 plants per plot.Harvesting was done when the plants enter the generative phase or when 10% of the plants were flowering, by cutting all parts of the plant, separating the stems and leaves, and weighing the wet weight.The stems and leaves were dried and weighed for dry weight.Furthermore, an analysis of the artemisinin levels of each genotype was performed.
Quantitative data were analyzed using analysis of variance test (ANOVA) at 5% level.If the test results show a significant effect, then a further test is carried out using Duncan's Multiple Range Test (DMRT) with a level of 5%.

Results dan Discussion
The mutant lines had different growth rates (table 1).In general, the tested mutant lines had better growth than the control.Eight of the 9 mutant lines had plant height and number of branches better than the control, and 3 mutant lines had a larger stem diameter than the control (table 1).
The rate of plant growth can be measured from upward growth and the formation of new branches.The M1C1, M2C9, M2C7, and M1C4 lines were the highest among the other lines and the control.The highest number of branches resulted from KI lines which were not significantly different from M2C7, M2C8, and M2C9.The largest stem diameter was produced by the M2C9 line which was not significantly different from M2C7 and KI.
In general, it appears that almost all lines have better growth than the control.Of all the mutant lines tested, it seems that M2C7, M2C9, and KI have the best growth response compared to the other lines.The diversity of the mutant lines can also be seen from the color of the stems.The control plants had green stems, while the color of the stems of the mutant lines varied between green, dark red, greenish red, and reddish green.The varied growth response between mutant lines and the difference in stem color indicated that each mutant genotype had a different genetic background even though they were descended from the same parent.
The application of EMS to chili affects plant growth, including plant height, flowering time, and number of seeds planted [6].EMS caused leaf discoloration in artemisia in vitro culture [7] and causes a diversity of leaf shapes and flower colors in Catharantus roseus [3].This shows that EMS causes changes in plant morphology, including stem color, as well as growth responses, including plant height, number of branches, and stem diameter.
EMS has one or more reactive alkyl groups that can be transferred to other molecules, thus changing guanine to 7-ethylguanine which pairs with thymine [8].The use of EMS can cause point mutations in DNA, causing changes in the arrangement of amino acids which result in changes in plant morphology and physiology [9].Mutations can also be caused due to mRNA aberrations and changes in protein translation [10].The Genetic changes caused by mutagens cause phenotypic changes.Morphological evaluation is one way to detect phenotypic changes [11].
One of the parameters to measure productivity is plant weight.The highest wet weight resulted from M1C1 which was significantly different from the other mutant lines and the control.The highest dry weight was also obtained from M1C1 and the smallest was obtained from KI (figure 1 and 2).Although the KI mutant had a large wet weight (1940 g), it had a small dry weight (460 g), even lower than the control.This indicates that the water content of KI is quite high.

Figure 1. Wet and dry weights of Artemisia mutant lines
Plant selection was carried out by selecting genotypes that had higher wet weights and dry weights than the controls and their artemisinin content.Artemisinin content of the mutant lines ranged from 0.67-1.02%,while the control was 0.4% (table 2).The highest artemisinin content of the mutant lines (1.02%) was obtained from M2C7 which was not significantly different from M1C3, M1C5, M2C9, and KI, the lowest was obtained from M2C8 (0.68%), while artemisinin content of the controls was 0.4%.The increasing artemisinin content of M2C7 reached 1.5 times compared to the control (table 2).lowest was obtained from M1C6 (366.59 g), and the yield from the control was 328.53 g.The yields of the mutant lines reach 2 times that of the control.The results obtained indicated that the application of EMS produced new genotypes that were different from their parents.According to [12], the use of EMS at low concentrations changes genes that affect the biosynthesis of several growth hormones which results in cell division and elongation so that physiology in the body increases which will also have an impact on increasing yields.[13] reported that soaking black soybean seeds in EMS increased seed weight.[14] reported that EMS treatment of A. vulgaris callus produced artemisia mutants that were more tolerant to NaCl than controls.EMS treatment also caused flower color changes in T. Patula [15].Furthermore, [7] stated that EMS caused an increase in the size of the glandular trichomes and increased artemisinin content of A. annua.
The application of EMS in creating a new superior variety has the advantage of not changing the existing superior agronomic characters, the changes only occur in certain characters.Mutagens can increase the frequency of mutations in a short time so that a wider range of genetic variations will be obtained [16].

Conclusion
EMS had an effect on growth characters that correlated with plant productivity, i.e. plant height, number of branches, stem diameter, and artemisinin content.Of the nine tested lines, at least 7 had higher plant height, number of branches, wet weight, and dry weight than the control.The artemisinin content of the mutants ranged from 0.67 to 1.02%, while the artemisinin content of the control was 0.4%.The increasing artemisinin content of these mutants reached 1.5 times compared to the control, and the mutant lines' yields reached 2 times that of the control.The diversity of plants can also be seen from the differences in the color of the stems.

Figure 2 .
Figure 2. The growth performance of 5-month-old artemisia mutant lines

Table 1 .
Phenotypic characters of artemisia mutant lines as compared to control

Table 2 .
Artemisinin contents and yields of artemisia mutant lines