Artemisia annua is the source of artemisinin, an antimalarial drug which is effective against multidrug-resistant strains of plasmodium, the malarial parasite. Malaria has serious effects on morbidity and mortality thus negatively impacting on agricultural production and food security. Although artemisinin has been found to be a useful medicine; its production is very low in comparison with what is actually needed to treat the worldwide threat of malaria. On the other hand, the lower content (0.01% - 0.8%, dry weight) of artemisinin found in leaves and flowers of A. annua has seriously limited its commercialization. Currently there are only two varieties of A. annua present in Kenya; hence there is a need to increase its diversity. The objective of the current study was to determine the effect of mutation on agronomical traits and artemisinin production by parents and mutant (M 2 ) A. annua plants in Kenya Agricultural and Livestock Research Organisation (KALRO) Njoro and the University of Eldoret (UoE). Seeds of two varieties of Artemesia annua varartemis and varanamed were sent to Vienna Austria for irradiation at the International Atomic Energy Agency (IAEA) at a dosage of 150 gray. The M 1 seeds were multiplied at the University of Eldoret farm. The harvested seeds were planted in replicate at the University of Eldoret and Njoro (KALRO). The results showed that mutation had significant effect on agronomical traits (P-value < 0.001). Mutant varieties and lines showed wide variation in terms of agronomical traits (crown length, stem length, plant height and stem diameter) and yield of artemisinin. The following lines showed superiority in artemisinin production; Artemis line 1, 2 and 9 with an average mean of 58.843, 58.393 and 69.192 μg/l, respectively while Anamed line 2, 3, 5 and 8 with an average of 56.268, 51.704, 53.557 and 54.448 μg/l, respectively. The effect of mutation on both agronomical traits and production of the artemisinin content appeared to occur randomly and was also dependent on environmental factors in the different ecological zones. Higher means in agronomical traits was observed in UoE while production of artemisinin content was enhanced in Njoro. The artemisinin yield in A. annua crops was negatively correlated with leaf traits, shoot and stem characteristics. Leaf traits had positive correlations with shoot and stem characteristics. It is recommended that superior lines be advanced in generations for further stability and evaluation of its efficacy in treatment of malaria.
Artemisia annua L. is also referred to as annual wormwood, sweet wormwood and sweet annie. It is a highly aromatic annual herb of Asiatic and eastern European origin. It is widely dispersed throughout the temperate region [
The study was conducted at Kenya Agricultural and Livestock Research Organisation (KALRO) Njoro and the University of Eldoret (UoE), (0˚30'0'' North, 35˚15'0'' East), 10 Km from Eldoret town, in Uasin Gishu county, Kenya. It is located at an altitude of 1280 m above sea level; it consists primarily of an agro-ecological zone Lower Highland 3 (LH3). The University of Eldoret receives a unimodial rainfall which begins in March. The average annual range is between 900 mm and 1100 mm and mean annual temperature of 16.6˚C. The soils are shallow, ferralsol, well drained, non humiccambisols with low nutrient availability and moisture storage [
One gram of M0 (non-mutated seeds) of both Artemisia annua varartemis and Artemisia annua varanamed, obtained from ICRAF (World Agroforestry Centre, Nairobi), Kenya, were sent to International Atomic Energy Agency (IAEA) in Vienna Austria and subjected to gamma radiation at an irradiation dose of 150 gy (gray) to obtain M1 (mutated seeds that gives rise to the first generation of mutants). This is the first generation of irradiated seeds which was designed as M1.
Irradiated seeds were sown under controlled conditions. Germination rates of the seeds were determined from the 7th day after sowing until the 14th day. The seeds were exposed to irradiation of between 50 gy and 450 at intervals of 50. The effective dose was determined by measuring the dose that decreases germination rate down to 50% hence 150 gy.
The nurseries were prepared in the greenhouse and the seeds spread and left uncovered according to the world agroforestry Centre recommendations. The seeds germinated after 7 days although some could be seen germinating after 4 days. The seedlings were transplanted in poly-tubes (75 by 140 by 37 mm) after the appearance of four leaves. After 7 weeks the seedlings were hardened off through the exposure to full sunlight for a week before transplanting to the experimental field site. The field site was prepared in advanced. The seedlings were planted at spacing of 1 m by 1 m between and within row. The field were kept weed free until the Artemisia were fully established. At harvest the chimeric and deformed plants were not selected. The harvested seeds from each plant were put into individual envelope and labeled. One group was planted at the university of Eldoret experimental field while the corresponding groups were planted at Njoro. The seeds from each plant were designated as M2.
The nurseries were prepared in the greenhouse and the seeds spread and left uncovered. The seeds germinated after 7 days. The seedlings were transplanted in poly-tubes (75 by 140 by 37 mm) after the appearance of four leaves. After 7 weeks the seedlings were hardened off through the exposure to full sunlight for a week before transplanting to the experimental field site. A field experiment was then established. The land was disc ploughed suitable for A. annua planting. The experiment was laid out in a randomized completely block design (RCBD) replicated three times. The spacing between and within plants was 1 m by 1 m. The experimental units were separated by 1.5 m by 1.5 m wide alleyways within and between blocks, respectively. The fields were kept weed free until the Artemisia were fully established.
The agronomic traits and artemisinin content for each of the M2 plants in the different zones were measured 4 months after establishment. For artemisinin analysis the leaves found in the upper parts of the A. annua plant, were harvested just before full flowering.
Leaves were obtained from the plant during the flowering stage and air dried at 27˚C. merceration was performed in Erlenmeyer with a magnetic stirrer speed of 700 rpm using ethanol as a solvent. This process was done many times until the methanol layer was colorless. The extract was then evaporated using a rotavapor vacuum at a temperature of 40˚C until the extract volume was 100 ml.
Spectrophotometric quantification was performed using slightly modified methods described by Sreeviydaand Narayana [
All solutions were prepared with double distilled water. Chemicals were of analytical grade. Hydrochloric acid (5M), Potassium iodide (2%), Sodium acetate (2 M) and 0.001% solution of Safranin were used.
A 1000 µg・ml−1 standard drug solution was prepared by dissolving 0.1 g of ART in ethanol and diluting to the mark in 100 ml calibrated flask.
Different aliquots (16.0 - 112.0 µg/l) were prepared. 10 ml of standard ART and sample extract was transferred to calibrated flasks by means of micro burette, then 1 ml each of 2% potassium iodide and 5 M HCL was then added followed by 2 ml of 2 M sodium acetate solution then the mixture was shaken for 5 mins. The contents were then diluted to the mark with distilled water and mixed well. The absorbance of each solution was then measured at 521 nm against the corresponding reagent blank. The reagent blank was prepared by replacing the analyte (ART) solution with distilled water. The absorbance of the corresponding to the bleached color, which in turn corresponds to the analyte (ART) concentration, was obtained by subtracting the absorbance of the blank solution by that of the test solution.
The data was analyzed using Genstat 13th edition and means separated using Tukeys Multiple Range Test where applicable. Correlation was done using Genstat Pearson correlation Coefficients.
There was significant difference in crown length, plant height, and stem diameter between parent and mutant plant sat P < 0.001 at UoE study site (
Traits | Site | Mean/Variety (units in cm) | P-value | |
---|---|---|---|---|
Par Artemis | Mutant Artemis | |||
Crown length | UoE | 67.41 ± 6.39 | 105.96 ± 6.39 | <0.001 |
Njoro | 71.77 ± 5.15 | 84.27 ± 5.15 | 0.017 | |
Stem length | UoE | 18.59 ± 14.17 | 75.83 ± 14.17 | <0.001 |
Njoro | 42.53 ± 12.73 | 62.80 ± 12.73 | 0.019 | |
Plant height | UoE | 17.83 ± 1.48 | 15.48 ± 1.48 | 0.115 |
Njoro | 98.39 ± 7.50 | 116.38 ± 7.50 | 0.018 | |
Branches | UoE | 21.00 ± 4.61 | 27.39 ± 4.61 | 0.168 |
Njoro | 18.21 ± 4.45 | 31.72 ± 4.45 | 0.003 | |
Stem diameter | UoE | 1.84 ± 0.21 | 3.00 ± 0.21 | <0.001 |
Njoro | 2.00 ± 0.15 | 2.20 ± 0.15 | 0.179 | |
Par Anamed | Mutant Anamed | |||
Crown length | UoE | 77.94 ± 6.39 | 127.62 ± 6.39 | <0.001 |
Njoro | 83.88 ± 5.73 | 80.864 ± 5.73 | 0.600 | |
Stem length | UoE | 29.82 ± 14.86 | 102.65 ± 14.86 | <0.001 |
Njoro | 44.41 ± 10.23 | 46.03 ± 10.23 | 0.875 | |
Plant height | UoE | 103.25 ± 5.98 | 145.56 ± 5.98 | <0.001 |
Njoro | 112.52 ± 6.75 | 111.15 ± 6.75 | 0.839 | |
Branches | UoE | 24.47 ± 4.82 | 35.52 ± 4.82 | 0.022 |
Njoro | 29.18 ± 4.22 | 35.04 ± 4.22 | 0.170 | |
Stem diameter | UoE | 2.11 ± 0.17 | 3.14 ± 0.17 | <0.001 |
Njoro | 2.24 ± 0.16 | 2.11 ± 0.16 | 0.410 |
length, stem length and Stem diameter in Artemis plants with means of 67.41 ± 6.39, 18.59 ± 14.17 and 1.84 ± 0.21 for parent plants and 105.96 ± 6.39, 75.83 ± 14.17 and 3.00 ± 0.21 for mutant plants respectively, while in Anamed plants there was significant difference in crown length, stem length, Plant height and Stem diameter with means of 77.94 ± 6.39, 29.82 ± 14.86, 103.25 ± 5.98 and 2.11 ± 0.17 for parent plants and means of 127.64 ± 6.39, 102.65 ± 14.86, 3.14 ± 0.17 and 145.56 ± 5.98 for mutant plants respectively. None of the agronomical characteristics analyzed were significantly different at Njoro in the two plant varieties.
The means of agronomical characteristics per plant line at UoE and Njoro sites (P < 0.001) are shown in
Artemi | Njoro (units in cm) | UoE (units in cm) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Lines | C. Lgt | S. Lgt | P. hgt | branch | s dter | C. Lgt | S. Lgt | P. hgt | branch | s dter | |
Parents | 71.7 | 42.5 | 98.4 | 18.2 | 2.0 | 67.4 | 18.6 | 17.8 | 21.0 | 1.8c | |
1 | 83.4 | 68.8 | 109.6 | 33.1 | 2.6 | 102.4 | 119.4 | 137.4 | 41.3 | 3.5a | |
2 | 85.7 | 38.0 | 103.6 | 29.3 | 2.0 | 109.9 | 54.1 | 132.6 | 24.8 | 3.0b | |
3 | 83.0 | 52.1 | 103.0 | 30.0 | 2.5 | 99.2 | 49.8 | 132.2 | 20.9 | 3.1b | |
4 | 70.5 | 48.6 | 106.5 | 24.0 | 1.8 | 102.9 | 56.3 | 136.6 | 27.1 | 2.6b | |
5 | 85.6 | 51.3 | 116.1 | 32.6 | 1.8 | 113.3 | 95.4 | 136.8 | 31.4 | 2.6b | |
6 | 87.7 | 60.5 | 122.7 | 34.7 | 2.1 | 97.4 | 98.0 | 137.8 | 30.7 | 2.4c | |
7 | 79.4 | 79.2 | 122.4 | 31.8 | 2.5 | 105.1 | 98.7 | 122.0 | 34.6 | 2.7b | |
8 | 90.4 | 81.8 | 130.6 | 38.3 | 2.2 | 91.1 | 91.6 | 146.9 | 27.4 | 2.2c | |
9 | 93.4 | 75.9 | 124.0 | 37.3 | 2.5 | 125.9 | 51.0 | 145.1 | 18.7 | 3.9a | |
10 | 88.9 | 48.0 | 110.1 | 31.7 | 2.4 | 100.2 | 74.9 | 105.4 | 32.8 | 3.1b | |
11 | 88.0 | 65.1 | 113.9 | 26.4 | 1.8 | 122.8 | 93.6 | 147.7 | 31.0 | 3.7a | |
12 | 66.7 | 69.1 | 123.4 | 25.1 | 2.2 | 102.0 | 36.8 | 151.7 | 11.4 | 3.0b | |
P-value | 0.247 | 0.772 | 0.266 | 0.757 | 0.02 | 0.123 | 0.048 | 0.003 | 0.052 | <0.001 | |
Anam | Njoro | UoE | |||||||||
Parents | 83.9 | 44.0 | 112.2 | 29.2 | 2.2b | 77.9d | 29.8 | 103.3d | 24.5 | 2.1 | |
1 | - | - | - | - | - | - | - | - | - | - | |
2 | 74.4 | 33.5 | 116.2 | 38.7 | 1.7c | 146.1a | 122.7 | 165.4a | 43.1 | 3.2 | |
3 | 86.5 | 76.9 | 102.1 | 39.6 | 2.1b | 120.7b | 106.0 | 136.0b | 36.3 | 3.3 | |
4 | 77.9 | 52.1 | 120.8 | 33.6 | 2.1b | 120.7b | 111.3 | 147.6b | 39.3 | 3.3 | |
5 | 79.2 | 27.8 | 117.8 | 43.6 | 2.1b | 142.1a | 122.4 | 159.6a | 36.8 | 3.9 | |
6 | 89.4 | 57.2 | 114.9 | 32.9 | 2.1b | 148.6a | 102.8 | 146.9b | 37.1 | 2.9 | |
7 | 68.8 | 35.1 | 97.9 | 21.8 | 1.6c | 107.8c | 75.8 | 140.6b | 27.3 | 2.9 | |
8 | 85.4 | 40.7 | 104.0 | 29.2 | 2.7a | 109.1c | 73.1 | 128.4c | 29.4 | 2.8 | |
9 | - | - | - | - | - | 126.1b | 107.2 | 139.9b | 36.3 | 2.8 | |
P-value | 0.519 | 0.04 | 0.162 | 0.06 | <0.001 | <0.001 | 0.49 | <0.001 | 0.648 | 0.003 | |
Means followed by the same letter in a column are not significantly different.
crown length and plant height. In crown length, line 5 had the highest mean of 148.6 while line 6 had the lowest mean of 107.8. In plant height, line 1 had the highest mean of 165.4 while line 7 had the lowest mean of 128.4. In Njoro Anamed lines, there was a significant difference in stem diameter only with line 8 having highest mean of 2.7 while line 7 had the lowest mean of 1.6.
The results for means of comparison of artemisinin production between UoE and Njoro sites (P-value 0.001) are shown in
There was significant difference in artemisinin production by Artemis and Anamed at Njoro at P < 0.001 (
The results for means of artemisinin production per plant line in UoE and Njoro sites (P < 0.001) are shown in
The estimated correlation among agronomical traits in mutant Artemis is represented in
Variety | UoE mean ± SE (Units in µg/l) | Njoro mean ± SE (Units in µg/l) | P-value |
---|---|---|---|
Par Artemis | 21.80 ± 2.04 | 22.40 ± 2.04 | 0.783 |
Artemis | 24.93 ± 2.39 | 44.92 ± 2.39 | <0.001 |
Par Anamed | 23.70 ± 2.00 | 22.80 ± 2.00 | 0.644 |
Anamed | 25.71 ± 3.31 | 43.67 ± 3.31 | <0.001 |
Site | Mean/variety (units in µg/l) | P-value | |
---|---|---|---|
Par Artemis | Mutant Artemis | ||
UoE | 21.788 ± 2.583 | 24.925 ± 2.583 | 0.227 |
Njoro | 22.355 ± 5.754 | 43.166 ± 5.754 | <0.001 |
Par Anamed | Mutant Anamed | ||
UoE | 22.665 ± 3.636 | 25.908 ± 3.636 | 0.375 |
Njoro | 22.805 ± 6.323 | 45.484 ± 6.323 | <0.001 |
Line | Artemis (units in µg/l) | Line | Anamed (units in µg/l) | ||
---|---|---|---|---|---|
UoE Njoro | UoE Njoro | ||||
Parents | 21.800 | 22.400d | Parents | 25.700b | 43.700a |
1 | 23.565 | 58.843b | 1 | - | - |
2 | 20.037 | 58.393b | 2 | 23.300b | 56.268a |
3 | 28.636 | 48.648c | 3 | 39.200a | 51.704a |
4 | 30.143 | 47.293c | 4 | 25.900b | 27.009b |
5 | 17.274 | 43.253c | 5 | 24.500b | 53.557a |
6 | 25.408 | 29.774d | 6 | 9.200c | 20.192b |
7 | 19.898 | 39.327c | 7 | 27.600b | 45.401a |
8 | 25.97 | 47.578c | 8 | 28.800b | 54.448a |
9 | 24.035 | 69.192a | 9 | 27.200b | - |
10 | 30.076 | 41.843c | 10 | - | - |
11 | 29.526 | 44.222c | 11 | - | - |
12 | 24.748 | 25.949d | 12 | - | - |
P-value | 0.144 | <0.001 | 0.001 | <0.001 |
Means followed by the same letter in a column are not significantly different.
The estimated correlation among agronomical traits in mutant Anamed is represented in
In the present study mutant varieties and lines showed wide variation in terms of agronomical traits (crown Length, Stem length, Plant height and Stem diameter) and yield of artemisinin content. A total of 108 Artemis and 72 Anamed plants were planted at UoE and KALRO Njoro sites respectively. At UoE site, only 3 plants in each variety (Artemis and Anamed) had a production of more than 60 µg/ml Artemisin concentration while at Njoro, 46 Artemis and 19 Anamed plants had a production of more than 60 µg/l artemisin concentration. Parent Artemis had an average artemisinin production of 21.788 at UoE and 22.355 at Njoro while parent Anamed plants had an average artemisinin production of 22.665 at UoE and 22.805 at Njoro. Concentrations levels of Artemisinin that are above 60 µg/l, is the commercially viable level [
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
---|---|---|---|---|---|---|---|---|
1) Crown length | - | |||||||
2) Stem length | 0.1563** | - | ||||||
3) Leaf length | −0.0372 | 0.0696 | - | |||||
4) Leaf width | 0.0713 | 0.043 | 0.1493** | - | ||||
5) Plant height | 0.5501* | 0.3718** | 0.008 | 0.0745 | - | |||
6) No. branches | 0.0797 | 0.7508* | 0.0872 | −0.0208 | 0.2578** | - | ||
7) Stem diam | 0.6389* | 0.2322** | 0.0333 | 0.1534** | 0.5038* | 0.1223** | - | |
8) Artemisinin | −0.2828** | −0.1492** | 0.0709 | 0.0164 | −0.3642** | −0.0701 | −0.3599** | - |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
---|---|---|---|---|---|---|---|---|
1) Crown length | - | |||||||
2) Stem length | 0.5588* | - | ||||||
3) Leaf length | 0.0307 | 0.1031** | - | |||||
4) Leaf width | 0.2003** | 0.0380 | 0.0497 | - | ||||
5) Plant height | 0.7896* | 0.5359* | 0.0630 | 0.1203** | - | |||
6) No. branches | 0.2256** | 0.6852* | −0.0139 | 0.0235 | 0.2212** | - | ||
7) Stem diam | 0.7204* | 0.5273* | 0.0912 | 0.2202** | 0.6384* | 0.1679** | - | |
8) Artemisinin | −0.2838** | −0.2217** | −0.1601** | −0.0167 | −0.3127** | −0.0499 | −0.1881** | - |
**Significant at P < 0.05; *Significant at P < 0.01.
**Significant at P < 0.05; *Significant at P < 0.01.
at the two study sites. At UoE, Line 2 had the highest mean of 39.2 while Line 5 had the lowest mean of 9.2; while at Njoro, Line 2 had the highest mean of 56.26 while Line 6 had the lowest mean of 20.19 (
The coefficients of correlations between all the pairs of variables are presented in
Mutant varieties and lines showed wide variation in terms of agronomical traits (crown length, stem length, plant height and stem diameter) and yield of artemisinin content. Some lines showed superiority over other mutant lines and control (parent plants). There was a significant difference in production of artemisinin by Artemis lines at Njoro site only where Line 1 had the highest mean of 58.843 and Line 12 with the lowest mean of 25.949 while in Anamed lines, significant difference in production of artemisinin was observed at the two study sites. At UoE, Line 2 had the highest mean of 39.2 while Line 5 had the lowest mean of 9.2; while at Njoro, Line 2 had the highest mean of 56.26 while Line 6 had the lowest mean of 20.19. The effect of mutation on both agronomical traits and production of the artemisinin content occurs randomly. Environmental factors appear to have an influence in expression of mutated genes related to agronomical characteristics and artemisinin production. Higher agronomical traits were observed in UoE while production of medicinal (artemisinin) content was enhanced in Njoro. Plants that produce artemisinin concentration ≥ 60 µg/l are economically viable. Artemisinin yield in A. annua crops was negatively correlated with leaf traits and stem characteristics. Leaf traits had positive correlations with stem characteristics. The study recommends that continual screening of Artemis lines 1, 2 and 9 and Anamed lines 2, 3, 5 and 8 be done and stabilization of these mutant lines through double haploid techniques and backcrossing to reduce the effects of mutations.
We are grateful to the International Atomic Energy Agency (IAEA) Vienna Austria for funding the project, World Agroforestry Centre (ICRAF), Nairobi, Kenya for providing the seeds and The University of Eldoret for hosting the project.
Mutai Raymond,Kinyua Miriam,Kiplagat Oliver,Mutai Edwin,Kimno Stephen, (2015) Enhancement of Artemisinin in Artemisia annua L. through Induced Mutation. Open Access Library Journal,02,1-11. doi: 10.4236/oalib.1102189