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Evaluation of influence of plantingdate on growth, artemisinin yield andseed yield of Artemisia annua underBangalore agro-climatic conditionsRakshapal Singh a , K. Puttanna a , E.V.S. Prakasa Rao a , A.K.Gupta b , M.M. Gupta b & S.P.S. Khanuja ba Department of Soil Science , Central Institute of Medicinal andAromatic Plants (CIMAP) , Allalasandra, Bangaloreb Gene Bank, CIMAP , Lucknow, IndiaPublished online: 23 Sep 2009.

To cite this article: Rakshapal Singh , K. Puttanna , E.V.S. Prakasa Rao , A.K. Gupta , M.M. Gupta &S.P.S. Khanuja (2009) Evaluation of influence of planting date on growth, artemisinin yield and seedyield of Artemisia annua under Bangalore agro-climatic conditions, Archives of Agronomy and SoilScience, 55:5, 569-577, DOI: 10.1080/03650340802676293

To link to this article: http://dx.doi.org/10.1080/03650340802676293

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Evaluation of influence of planting date on growth, artemisinin yield and seed

yield of Artemisia annua under Bangalore agro-climatic conditions

Rakshapal Singha, K. Puttannaa*, E.V.S. Prakasa Raoa, A.K. Guptab, M.M. Guptab andS.P.S. Khanujab

aDepartment of Soil Science, Central Institute of Medicinal and Aromatic Plants (CIMAP),Allalasandra, Bangalore; bGene Bank, CIMAP, Lucknow, India

(Received 22 August 2008; final version received 9 December 2008)

Artemisia annua is an important crop grown for producing the anti-malarial drugartemisinin. It has been recently introduced to Bangalore’s agro-climatic conditions ofSouth India. A field experiment was carried out at the Central Institute of Medicinaland Aromatic Plants (CIMAP), Bangalore, from November 2004 to July 2007 tooptimize the planting date for maximum herb, artemisinin and seed yield under thesemi-arid tropical conditions of Bangalore. The study revealed that it was best totransplant seedlings of A. annua in the later part of October to get optimum herb,artemisinin and seed yield under Bangalore’s agro-climatic conditions.

Keywords: Artemisia annua; semi-arid tropics; planting date; growth parameter andartemisinin yield

Introduction

Artemisia annua L. (Asteraceae), commonly known as quinghao, is an annual aromaticplant that is a fast-growing, giant annual with upright, often red stems and bright green,pinnately divided and saw-toothed leaves. Tiny yellow flowers appear in loose panicles insummer. It is a traditional medicinal herb, native to China and widely grown in Asia andEurope (Klayman 1985). Administration of this antibacterial herb destroys malarialparasites, lowers fevers and checks bleeding. The biologically active compound artemisininisolated from the herb of the A. annua plant has been shown to possess anti-malarialactivity against resistant strains of malarial parasite Plasmodium falciparum (Klayman1985; World Health Organization 1986; Elsohly et al. 1987; Anonymous 1992; Wyk andWink 2004). Forty percent of the world’s population is at risk of malaria. Each year, 100million people are infected with malaria and 1 million people die from it (WHO 1993).Artemisinin derivatives are recommended by WHO for resistant and cerebral malaria(Wyk and Wink 2004).

In the present study the plant has been introduced to Bangalore’s climatic conditionsto determine the best date of planting for optimum growth, biomass, artemisinin yield andseed yield under semi-arid tropical conditions.

*Corresponding author. Email: k_puttanna@yahoo.co.in

Archives of Agronomy and Soil Science

Vol. 55, No. 5, October 2009, 569–577

ISSN 0365-0340 print/ISSN 1476-3567 online

� 2009 Taylor & Francis

DOI: 10.1080/03650340802676293

http://www.informaworld.com

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Material and methods

A field experiment was conducted at the Central Institute of Medicinal and AromaticPlants (CIMAP), Resource Center, Bangalore. Bangalore is located at 128580 N, 778350 Eand 930 m above mean sea level. The soil of the experimental field was a sandy loam withpH 6.8, 295 kg ha71 available N, 5.5 kg ha71 available P, 103.7 kg ha71 available K and0.42% organic carbon.

The experiment was started in the first week of November 2004. The treatmentsconsisted date of sowing seeds (DOS) of A. annua, variety ‘Jeevanraksha’ (Kumar et al.1999) and date of transplanting (DOTP) at fortnightly intervals over a period of two years(Table 1). The seeds were obtained from CIMAP, Lucknow. Seeds were sown in1 6 0.5 m flat nursery beds by mixing the seeds with vermicompost, broadcasting over thesurface soil. Watering was carried out with fine rose cans. Transplanting was done whenseedlings attained an age of 60 days. At this stage the seedlings had 5–7 leaves.Transplanting was done in flat beds of 3.5 6 3.5 m with two replicates in a randomizedblock design experiment. The spacing was 50 6 30 cm and the number of plants per bedwas 60. The first transplanting was carried out in the second week of January 2005 and thelast was in the fourth week of December 2006. The beds were irrigated after transplanting

Table 1. Date of seed sowing and transplanting and percent survival of Artemisia annua.

Treatment no.

Date of seed sowing(DOS)

Date of transplanting(DOTP) Percent survival

Meanpercentsurvival

I Year(2004–05)

II Year(2005–06)

I Year(2005–06)

II Year(2006–07)

I Year(2005–06)

II Year(2006–07)

T 1 15.11.04 15.11.05 15.01.05 15.01.06 88.0 90.5 89.3T 2 30.11.04 30.11.05 30.01.05 30.01.06 90.0 91.5 90.8T 3 15.12.04 15.12.05 15.02.05 15.02.06 86.5 83.0 84.8T 4 30.12.04 30.12.05 28.02.05 01.03.06 82.0 80.0 81.0T 5 15.01.05 15.01.06 15.03.05 15.03.06 87.5 82.5 85.0T 6 30.01.05 30.01.06 30.03.05 30.03.06 88.0 83.0 85.5T 7 15.02.05 15.02.06 15.04.05 15.04.06 79.5 78.0 78.8T 8 1.03.05 1.03.06 30.04.05 01.05.06 84.5 82.0 83.3T 9 15.03.05 15.03.06 15.05.05 15.05.06 86.0 83.0 84.5T 10 31.03.05 31.03.06 30.05.05 31.05.06 70.5 70.0 70.3T 11 15.04.05 15.04.06 15.06.05 15.06.06 69.5 70.0 69.8T 12 30.04.05 30.04.06 30.06.05 30.06.06 70.5 70.0 70.3T 13 15.05.05 15.05.06 15.07.05 15.07.06 85.0 88.5 86.8T 14 30.05.05 30.05.06 30.07.05 30.07.06 74.0 70.0 72.0T 15 15.06.05 15.06.06 15.08.05 15.08.06 70.0 70.0 70.0T 16 30.06.05 30.06.06 30.08.05 30.08.06 63.0 60.0 61.5T 17 15.07.05 15.07.06 15.09.05 15.09.06 52.5 50.0 51.3T 18 30.07.05 30.07.06 1.10.05 1.10.06 95.0 93.0 94.0T 19 15.08.05 15.08.06 15.10.05 15.10.06 97.0 95.5 96.3T 20 30.08.05 30.08.06 30.10.05 30.10.06 91.0 91.0 91.0T 21 15.09.05 15.09.06 15.11.05 15.11.06 90.5 90.0 90.3T 22 30.09.05 30.09.06 30.11.05 30.11.06 91.5 91.0 91.3T 23 15.10.05 15.10.06 15.12.05 15.12.06 89.5 88.0 88.8T 24 30.10.05 30.10.06 30.12.05 30.12.06 91.0 90.0 90.5

C.D. (p ¼ 0.01) – – – – 7.01 6.2 4.8

C.D., critical difference; T1–T24, date of transplanting.

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and further irrigations were given as required. No pests were observed in this crop duringthe year. The experiment was completed in July 2007 on maturation of seeds.

The recommended dose of fertilizers, N:P:K at 50:50:50 kg ha71 was applied. P and Kwere applied as a basal dose along with vermicompost at the rate of 3 t ha71. 1/3 N wasapplied 10 days after transplanting and the remaining N was top dressed in two equalsplits at monthly intervals.

In each bed five plants were selected randomly for observations on plant height, plantspread, primary branches, secondary branches and flowering behavior at monthlyintervals until harvest.

The data on days to flowering behavior, seed maturation and date of harvesting (herbbiomass) are presented in Table 2. Half the number of plants in each plot were harvestedwhen 10–15% of the plants flowered by cutting at a height of 60 cm above the ground andthe other half was left until seed maturation for seed yield determination. Attempts toobtain multiple harvests did not succeed in Bangalore. The biomass yield was recorded.The leaves were separated manually; leaf yield was calculated on the basis of leaf/stemratio and the leaves were kept aside for shade drying for 2–3 days. The dried leaf sampleswere stored in plastic bags at room temperature for the analysis of artemisinin content.The artemisinin content was analyzed at CIMAP, Lucknow, as per standard procedure(Gupta et al. 1996). From the value of artemisinin content and from leaf yield, thetheoretical yield of artemisinin in kg per hectare was calculated. The data were evaluated

Table 2. Flowering behavior and seed maturation of Artemisia annua for the years 2005–07.

Treatment no.

Days toflower

initiationDays to

50% flowering

Date ofharvest(herb)

Days toseed

maturation

T 1 110–120 130–150 12.05.06 235–240T 2 100–110 120–130 15.05.06 220–230T 3 90–100 110–120 22.05.06 240–250T 4 80–90 100–120 22.05.06 240–250T 5 100–110 120–130 28.06.06 230–240T 6 130–140 150–160 7.08.06 210–220T 7 120–130 140–160 18.08.06 210–220T 8 110–120 130–150 23.08.06 200–210T 9 120–130 140–150 15.09.06 200–210T 10 100–110 120–140 15.09.06 180–190T 11 105–115 120–130 28.09.06 180–190T 12 105–115 120–130 17.10.06 170–180T 13 90–100 110–120 17.10.06 170–180T 14 105–115 120–130 17.11.06 150–160T 15 90–100 105–115 17.11.06 150–160T 16 100–110 115–125 20.12.06 150–160T 17 105–110 115–120 4.01.07 130–140T 18 180–190 200–210 4.04.07 210–220T 19 190–200 210–220 8.05.07 200–210T 20 180–190 200–210 8.05.07 240–250T 21 170–180 190–200 8.05.07 240–250T 22 150–160 170–180 8.05.07 230–240T 23 140–150 160–170 8.05.07 220–230T 24 120–125 130–140 8.05.07 200–210

T1–T24, date of transplanting.

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statistically by analysis of variance method (Snedecor and Cochran 1989). The resultdiscussed in this paper is based on mean value of two years data.

Artemisinin analysis

Artemisinin

Artemisinin was isolated from A. annua and its identity was confirmed by its spectralanalysis.

Apparatus

HPTLC (CAMAG, Switzerland and winCATS software) with CAMAG TLC Scanner 3and recoated silica gel plates 60F254 (Merck, Germany) with a layer thickness of 0.25 mmwere used.

Standard solution and calibration curve

A stock solution of pure artemisinin (1.0 mg ml71) was prepared in n-hexane and differentamounts of it were applied on TLC plates (20 6 20 cm). Chromatography was carriedout in a glass TLC tank saturated with the mobile phase n-hexane: diethyl ether (1:1) andthe plates were developed to a height of about 15 cm. Plates were taken off, dried andspots were visualized by immersing the plates (CAMAG immersion device) in a freshlyprepared mixture of glacial acetic acid: concentrated H2SO4: anisaldehyde (50:1:0.5),followed by heating of the plates at 1108C for 15 min on a CAMAG TLC plate heater tovisualize a pink color of artemisinin. For quantification, TLC spot, corresponding toartemisinin, was measured at 540 nm. Calibration curve of artemisinin was constructed byplotting concentration versus spot area of the compound.

Estimation of artemisinin in plant samples

Plant material (0.1 g) was sonicated with 5 ml n-hexane for 15 min, filtered, evaporatedand redissolved in 1.0 ml n-hexane. Spots were visualized as above and the quantificationof artemisinin was performed using the calibration graph.

Results and discussion

Survival of plants

The data on survival of plants are presented in Table 1. The survival percentage oftransplanted seedlings was generally more than 80%. Minimum survival of plants wasrecorded when plants were transplanted on 15 September and the maximum survival ofplants was recorded when plants were transplanted on 15 October. Plants that did notsurvive long exhibited early flowering.

Flowering behavior

The data on time of flower initiation, 50% flowering and time for seed maturation asinfluenced by the date of transplanting are presented in Table 2. Flower initiation occurredas early as 80–90 days in February transplanted plants but it took as many as 180–200

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Table

3.

Growth

parametersofArtem

isia

annua.

Treatm

ent

no.

Plantheight(cm)

No.primary

branches

No.secondary

branches

IYear

(2005–06)

IIYear

(2006–07)

Mean

IYear

(2005–06)

IIYear

(2006–07)

Mean

IYear

(2005–06)

IIYear

(2006–07)

Mean

T1

187.50

204.50

196.00

72.50

73.00

72.8

2218.00

2223.00

2220.5

T2

195.00

195.00

195.00

67.50

66.00

66.8

2105.00

2107.50

2106.3

T3

175.00

178.00

176.50

64.00

62.00

63.0

2030.00

2013.00

2021.5

T4

187.50

187.50

187.50

67.50

66.50

67.0

2145.00

2137.50

2141.3

T5

188.50

192.50

190.50

63.50

63.00

63.3

2125.00

2074.00

2099.5

T6

175.00

175.50

175.25

60.00

61.50

60.8

1105.00

1023.00

1064.0

T7

145.00

147.00

146.00

65.00

65.00

65.0

1225.00

1268.00

1246.5

T8

140.00

138.00

139.00

68.00

68.00

68.0

1355.00

1371.50

1363.3

T9

135.00

130.50

132.75

37.50

33.50

35.5

1455.00

1314.00

1384.5

T10

122.50

119.00

120.75

37.50

31.50

34.5

1260.00

1237.50

1248.8

T11

150.00

145.50

147.75

44.00

42.00

43.0

1910.50

1878.50

1894.5

T12

140.50

140.50

140.50

37.50

38.00

37.8

1480.00

1415.00

1447.5

T13

131.00

130.00

130.50

43.00

42.50

42.8

1252.50

1259.50

1256.0

T14

145.00

132.50

138.75

45.00

45.50

45.3

1425.00

1409.50

1417.3

T15

174.50

161.50

168.00

63.00

59.50

61.3

1687.50

1696.50

1692.0

T16

120.00

114.50

117.25

52.00

48.50

50.3

1400.00

1415.50

1407.8

T17

122.50

123.00

122.75

54.50

53.00

53.8

1733.50

1704.00

1718.8

T18

226.50

216.50

221.50

94.00

80.00

94.3

3799.50

2837.00

3843.3

T19

247.50

235.50

241.50

101.50

100.50

101.0

4763.00

4779.50

4771.3

T20

230.00

227.50

228.75

93.50

91.50

92.5

3716.00

3782.50

3749.3

T21

207.50

207.50

207.50

89.50

88.50

89.0

3349.00

3281.00

3315.0

T22

215.00

215.50

215.25

95.00

99.00

97.0

3256.00

3213.00

3234.5

T23

197.50

197.00

197.25

79.50

79.50

79.5

2789.50

2792.00

2790.8

T24

187.50

182.50

185.00

76.50

73.50

75.0

2672.50

2635.00

2653.8

C.D

.(p¼

0.01)

37.16

43.95

11.92

18.93

16.49

3.91

786.22

896.89

92.3

C.D

.,criticaldifference;T1–T24,date

oftransplanting.

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Table

4.

Totalbiomass

andleafyield

ofArtem

isia

annua.

Treatm

ents

Fresh

herbyield

(tha7

1)

Dry

herbyield

(tha7

1)

Fresh

leafyield

(tha7

1)

Shadedry

leafyield

(tha71)

IYear

(2005–06)

IIYear

(2006–07)

Mean

IYear

(2005–06)

IIYear

(2006–07)

Mean

IYear

(2005–06)

IIYear

(2006–07)

Mean

IYear

(2005–06)

IIYear

(2006–07)

Mean

T1

4.7

7.50

6.1

2.2

3.60

2.9

1.5

2.36

1.9

0.7

1.06

0.9

T2

11.5

7.25

9.4

5.5

3.48

4.5

4.7

2.93

3.8

2.1

1.32

1.7

T3

6.6

2.85

4.7

3.2

1.37

2.3

3.0

1.23

2.1

1.4

0.55

1.0

T4

6.8

3.63

5.2

3.3

1.74

2.5

2.8

1.47

2.1

1.2

0.66

1.0

T5

4.2

2.75

3.5

2.0

1.32

1.7

1.9

1.20

1.5

0.8

0.54

0.7

T6

8.5

6.50

7.5

4.1

3.12

3.6

3.8

2.99

3.4

1.7

1.35

1.5

T7

8.2

7.53

7.9

3.9

3.61

3.8

3.7

3.36

3.5

1.7

1.51

1.6

T8

14.8

10.9

12.9

7.1

5.27

6.2

6.3

4.83

5.5

2.8

2.17

2.5

T9

5.2

4.83

5.0

2.5

2.32

2.4

2.9

2.63

2.7

1.3

1.18

1.2

T10

4.8

2.80

3.8

2.3

1.34

1.8

2.4

1.38

1.9

1.1

0.62

0.8

T11

9.4

4.55

7.0

4.5

2.18

3.3

4.5

2.18

3.3

2.0

0.98

1.5

T12

8.8

2.03

5.4

4.2

0.97

2.6

4.5

1.08

2.8

2.0

0.49

1.3

T13

8.4

2.98

5.7

4.0

1.43

2.7

3.3

1.23

2.3

1.5

0.55

1.0

T14

16.4

16.7

16.5

7.9

7.99

7.9

6.7

6.38

6.6

3.0

2.87

3.0

T15

5.5

7.68

6.6

2.6

3.68

3.2

2.3

3.37

2.8

1.0

1.52

1.3

T16

5.5

4.80

5.2

2.6

2.30

2.5

2.8

2.53

2.7

1.3

1.14

1.2

T17

4.6

5.48

5.1

2.2

2.63

2.4

2.1

2.50

2.3

1.0

1.12

1.0

T18

20.6

22.5

21.5

9.9

10.76

10.3

13.3

10.22

11.8

6.0

4.59

5.3

T19

27.3

27.1

27.2

13.1

13.02

13.1

15.9

12.34

14.1

7.2

5.55

6.4

T20

15.8

14.2

15.0

7.6

6.84

7.2

6.5

5.84

6.2

2.9

2.63

2.8

T21

13.3

8.38

10.8

6.4

4.02

5.2

5.2

3.31

4.2

2.3

1.49

1.9

T22

10.6

9.58

10.1

5.1

4.60

4.8

4.6

4.20

4.4

2.1

1.89

2.0

T23

8.75

7.15

8.0

4.2

3.43

3.8

3.7

3.05

3.4

1.7

1.37

1.5

T24

9.25

8.35

8.8

4.4

4.01

4.2

3.5

3.21

3.4

1.6

1.44

1.5

C.D

.(p¼

0.01)

5.31

3.79

4.73

2.5

1.82

2.27

3.3

1.50

2.14

1.5

0.71

1.4

C.D

.,criticaldifference;T1–T24,date

oftransplanting.

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days in October transplanted plants. The same trends were observed for 50% flowering.The time taken for mature seed production by plants was highest with 240–250 days, whentransplanting was done in November. On the other hand, when transplanting was done inSeptember, the time taken for seed production was lowest, being 130–140 days.

Growth parameters

The data on influence of transplanting time on growth parameters of A. annua just beforeharvest are presented in Table 3. Plant height, numbers of primary and secondarybranches were high in October transplanted plants. In the present study the plant height,number of primary branches and secondary branches ranged from 117–242 cm, 35–101and 1064–4771, respectively. In Lucknow, India, the same variety of A. annua, planted inthe middle of January, grew to a height of about 300 cm (Gupta et al. 2002). It wasreported that plant height of the wormwood ranged from 50–200 cm (Baytop 1984;Woerdenbag et al. 1994; Ferreira and Janick 1996). There are a number of factors affectingplant height such as different agricultural practices, climatic factors, soil properties andplant ecotypes. Mert (1999) reported that plant heights of the different wormwoodecotypes grown in Turkey ranged between 177.3 and 217.2 cm.

Table 5. Seed yield and artemisinin content of Artemisia annua.

Treatments

Seed yield (kg ha71) Artemisinin percent

I Year(2005–06)

II Year(2006–07) Mean

I Year(2005–06)

II Year(2006–07) Mean

T 1 312.5 330.00 321.25 0.71 0.73 0.72T 2 330.0 320.00 325.00 0.82 0.84 0.83T 3 297.5 275.00 286.25 0.77 0.76 0.77T 4 322.5 320.00 321.25 0.78 0.78 0.78T 5 252.5 230.00 241.25 0.84 0.82 0.83T 6 345.0 340.00 342.50 0.86 0.85 0.86T 7 302.5 295.00 298.75 0.92 0.94 0.93T 8 342.5 330.00 336.25 0.93 0.90 0.91T 9 220.0 205.00 212.50 0.79 0.77 0.78T 10 267.5 255.00 261.25 0.75 0.74 0.74T 11 257.5 250.00 253.75 0.79 0.78 0.78T 12 312.5 300.00 306.25 0.71 0.66 0.68T 13 212.5 185.00 198.75 0.70 0.75 0.72T 14 395.0 395.00 395.00 0.68 0.52 0.60T 15 290.0 255.00 272.50 0.67 0.61 0.64T 16 175.0 165.00 170.00 0.64 0.57 0.60T 17 250.0 235.00 242.50 0.66 0.61 0.63T 18 427.5 435.00 431.25 0.63 0.63 0.63T 19 525.0 495.00 510.00 0.90 0.67 0.79T 20 372.5 375.00 373.75 0.91 0.92 0.91T 21 282.5 255.00 268.75 0.76 0.51 0.63T 22 287.5 280.00 283.75 0.77 0.59 0.68T 23 310.0 270.00 290.00 0.53 0.43 0.48T 24 257.5 275.00 266.25 0.70 0.53 0.61

C.D. (p ¼ 0.01) 93.99 86.88 28.09 0.26 0.23 0.09

C.D., critical difference; T1–T24, date of transplanting.

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Herb and seed yield

The data on influence of transplanting time on yield of A. annua are presented in Tables 4and 5. It was reported that fresh herbage yields of different ecotypes harvested in varyingperiods varied from 19.7–47.8 t ha71, dry herbage yields were between 15.5 and 26.1 tha71 and dry leaf yields were in the range of 4.2–5.8 t ha71 in Turkey (Mert 1999). But inthe present experiment, fresh herbage ranged from 3.5–27.2 t ha71 and fresh leaf yield was1.5–14.1 t ha71, dry herb and shade dry leaf yields were between 1.7 and 13.1 and 0.7 to6.4 t ha71, respectively. The fresh and dry herb, fresh and shade dry leaf (Table 4) andseed yield (Table 5) exhibited a maximum in October-transplanted plants. In Lucknow,India, yields of A. annua leaf ranging from 1.3–11.4 t ha71, depending on the number ofharvests, have been reported (Kumar et al. 2004). The data presented in this paper, basedon a single harvest, compare well with the above.

Artemisinin content

The artemisinin content in A. annua dried leaves is presented in Table 5. The artemisinincontent showed moderate variation with transplanting time being high in March, Apriland the last week of October-transplanted plants, low in December-transplanted plantsand falling in between in plants transplanted in other seasons. In Lucknow, India,artemisinin content in leaf varied from 0.42–1.12% (Kumar et al. 2004) whereas inBangalore the range was 0.43–0.94%.

Artemisinin yield

The mean of two years’ artemisinin yield data is presented in Figure 1. The data show thatthe artemisinn yield was strongly influenced by the date of transplanting. The artemisinin

Figure 1. Theoretical artemisinin yield (mean of two years, kg ha71).

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yield was mainly determined by leaf yield. The artemisinin yield for the first year was50.6 kg ha71 and for the second year, 37.2 kg ha71. The mean of two years forartemisinin yield was 43.9 kg ha71. Transplanting of A. annua plants in mid-Octoberresulted in the maximum artemisinin yield. Kumar et al. (2004) reported yields ofartemisinin ranging from 11.2–91.6 kg ha71 depending on the number of harvests whereasGupta et al. (2002) reported 18.5 kg ha71 yield of artemisinin in Lucknow, India.

Conclusion

The results clearly show that the seeds of A. annua sown in August and transplanted inOctober produced the maximum herb, artemisinin and seed yield in the semi-arid tropicalconditions of South India.

Acknowledgements

The authors would like to thank the Director, CIMAP, Lucknow, and the Council of Scientific andIndustrial Research, New Delhi (India) who supported this study by providing funds and facilities.

References

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