Radiation Physics and Chemistry 80 (2011) 833836

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Radiation Physics and Chemistry

journal homepage: www.elsevier.com/locate/radphyschem

Enhancing the growth, photosynthetic capacity and artemisinin content in

Artemisia annua L. by irradiated sodium alginate
Tariq Aftab a,n, M. Masroor A. Khan a, M. Idrees a, M. Naeem a, Moinuddin a, Nadeem Hashmi a,
Lalit Varshney b
a
b

Plant Physiology Section, Department of Botany, Aligarh Muslim University, Aligarh 202002, India
ISOMED, Radiation Technology Development Section, Radio-Chemistry and Isotope Group, BARC, Mumbai 400085, India

a r t i c l e i n f o

abstract

Article history:
Received 6 December 2010
Accepted 3 March 2011
Available online 10 March 2011

Degrading the natural bioactive agents by ionizing radiation and then using them as growth promoting
substances is a novel emerging technology to exploit the genetic potential of crops in terms of growth,
yield and quality. Polysaccharides, such as sodium alginate, have proven to be wonderful growth
promoting substances in their depolymerized form for various plants. The effect of depolymerized form
of sodium alginate, produced by irradiating the latter by 60Co gamma rays, was studied on Artemisia
annua L. with regard to growth attributes, physiological and biochemical parameters and artemisinin
content. The study revealed that the irradiated sodium alginate (ISA), applied as leaf-sprays at a
concentration of 20120 mg L  1, improved the growth attributes, photosynthetic capability, enzyme
activities and artemisinin content of the plant signicantly. Application of ISA at 80 mg L  1 increased
the values of the attributes studied to the maximum extent. The enhancement of leaf-artemisinin
content was ascribed to the ISA-enhanced H2O2 content in the leaves.
& 2011 Elsevier Ltd. All rights reserved.

Keywords:
Artemisia annua L.
Artemisinin content
Growth attributes
H2O2 content
Irradiated sodium alginate (ISA)

1. Introduction
The oligomers, generated by depolymerization of alginates,
have been reported to stimulate the plant growth, seed germination and shoot elongation in plants. Biologically active oligosaccharides are known to act as signal molecules that regulate plant
growth and development as well as the defense reactions in
plants by regulating gene expression. Hu et al. (2004) reported
that the oligomers of irradiated sodium alginate enhanced the
plant growth as well as productivity of Zea mays, suggesting the
role of degraded sodium alginate as plant growth promoter in
agriculture.
Artemisia annua turned into a valuable crop in medicinal
agriculture after the recommendation of World Health Organization (WHO) declaring artemisinin as a component of ACT (artemisinin-based combination therapies) for malaria. The enhanced
production of the artemisinin content in the whole plant is highly
desirable (Aftab et al., 2010). Therefore, present study was taken
up on A. annua L. to assess the effect of irradiated sodium alginate
(ISA) on various growth, physiological, biochemical, yield and
quality attributes and to nd out the changes in artemisinin

Corresponding author. Tel.: 91 99976 16302.

E-mail address: tarik.alig@gmail.com (T. Aftab).

0969-806X/$ - see front matter & 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.radphyschem.2011.03.004

content at different plant growth stages as affected by the ISAtreatments.

2. Materials and methods

The experiment was conducted using earthen pots according
to randomized complete block design in the natural conditions.
Graded levels of irradiated SA (0 (control), 20, 40, 80 and
120 mg L  1) were employed as treatments. Each treatment was
replicated ve times and each replicate pot carried one plant.
Growth and biochemical attributes were determined at preowering (90 DAS), owering (120 DAS) and post-owering
(150 DAS) stages.
Sodium alginate (SA) samples were irradiated by gamma rays
at 520 kGy using 60Co as irradiation-source. Different concentrations of irradiated sodium alginate (ISA), viz. 0 (control), 20, 40, 80
and 120 mg L  1, were nally prepared to use in the present study.
The clean and blot-dried plants were used to record shoot
length and then they were oven-dried at 80 1C for 48 h to
determine the plant dry weight. Net photosynthetic rate (PN),
stomatal conductance (gs) and internal CO2 concentration (ci)
were measured with the help of an Infra Red Gas Analyzer (IRGA,
LI-COR 6400 Portable Photosynthesis System, Lincoln, Nebraska,
USA) device. Total chlorophyll and carotenoids contents in the
leaves were estimated by the method of Lichtenthaler and

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T. Aftab et al. / Radiation Physics and Chemistry 80 (2011) 833836

Buschmann (2001). Nitrate reductase (E.C. 1.6.6.1) and carbonic

anhydrase (E.C. 4.2.1.1) activities in the leaves were determined
by the method given by Jaworski (1971) and Dwivedi and
Randhawa (1974), respectively. The content of H2O2 in the leaves
was determined according to the method of Mukherjee and
Choudhuri (1983).
1 g of the dry leaf material was used for the estimation of
artemisinin content, using the method of Zhao and Zeng (1986).
Standard curve was prepared using 1 mg of the standard artemisinin (SigmaAldrich, USA) dissolved in 1 mL of HPLC-grade
methanol. Artemisinin content was quantied against the artemisinin standard curve, using an HPLC. The data were analyzed
statistically using SPSS-17 statistical software (SPSS Inc., Chicago,
IL, USA) according randomized complete block design. Mean
values were compared using Duncans Multiple Range Test
(DMRT) at Po0.05 level.

3. Results and discussion

3.1. Growth attributes
Various ISA concentrations applied improved the plant growth
attributes of Artemisia annua signicantly (Po0.05) at pre-owering, owering and post-owering stage of crop growth. There
was a progressive increase in values with the increase in the
applied ISA level up to 80 mg L  1. Thereafter, the values declined
signicantly (Table 1). As compared to the control, application of
ISA at 80 mg L  1 increased the shoot length by 38.02% and plant
dry weight by 25.97% at pre-owering stage (Table 1). In conformity with these results, Kume et al. (2002) reported signicant
improvement in plant growth attributes due to the application of
radiation-derived oligosaccharides of alginate. Khan et al. (2010)
also conrmed the growth promoting effect of irradiated sodium
alginate in Papaver somniferum.
3.2. Physiological and biochemical attributes
Compared to the control, ISA application at 80 mg L  1
enhanced the net photosynthetic rate by 36.23%, 27.27% and
20.73% at pre-owering, owering and post-owering stage,
respectively. At owering stage, ISA at 80 mg L  1 also increased
the stomatal conductance and internal CO2 concentration by

16.71% and 15.38%, respectively. Additionally, ISA at 80 mg L  1

enhanced the stomatal conductance and internal CO2 concentration by 14.72% and 14.50% at pre-owering stage, and by 13.35%
and 14.11% at post-owering stage, respectively (Table 1). In this
context, Hien et al. (2000) observed signicant enhancement in
net-photosynthesis and CO2 assimilation as a result of application
of depolymerized SA in rice.
As compared to the control, application of ISA at 80 mg L  1
enhanced the total chlorophyll and carotenoids content at owering stage by 17.18% and 11.53%, respectively (Table 2). Both the
photosynthetic pigments were also signicantly improved by the
ISA application at pre-owering stage, with 80 mg L  1 enhancing
the chlorophyll and carotenoids content by 14.77% and 11.18%,
respectively (Table 2). At post-owering stage, the total chlorophyll
content was signicantly increased by the ISA applied at
80 mg L  1, while the total carotenoids content was unaffected.
Since the important role of SA-oligosaccharides (degraded alginate)
has been recognized in inducing cell signaling in various plants,
leading to stimulation of various physiological processes (John
et al., 1997), the application of ISA might improve photosynthetic
capacity and photosynthetic pigment contents in this study.
The highest activities of NR and CA enzymes were recorded at
an ISA application of 80 mg L  1 at all the three growth stages.
Thereafter, the values were declined signicantly. Compared to
the control (water spray treatment), 80 mg L  1 of ISA resulted in
20.24% and 17.75% increase in NR and CA activity, respectively
(Table 2). The ISA application at 80 mg L  1 also proved optimum
for both the enzymes at pre-owering and post-owering stages,
surpassing the control by 15.87% and 13.69% regarding NR
activity and by 14.79% and 12.62% regarding CA activity, respectively. In this regard, our ndings are similar to those of Akimoto
et al. (1999), who reported the synthesis of certain enzymes in a
tissue culture study after addition of alginate-derived oligomers.
Application of ISA at 80 mg L  1 increased the content of H2O2
by 46.88% as compared to the non-treated plants (control) at the
owering stage (Fig. 1). At an ISA application of 80 mg L  1, the
increase in H2O2 content was 41.73% and 35.90% compared to the
control at pre-owering and post-owering stage, respectively
(Fig. 1). Thus, the application of ISA could induce the formation of
H2O2 in the plant cells. Likewise, Pu et al. (2009) and Wallaart
et al. (2000) have reported an increase in H2O2 content when
A. annua plants were treated with salicylic acid or they underwent
the night frost.

Table 1
Effect of different concentrations of irradiated sodium alginate (ISA) on growth and photosynthetic parameters of Artemisia annua L. Means within a column followed by
the same letter are not signicantly different (p r 0.05). The data shown are means of ve replicates 7 SE.
Parameters

Growth stages

ISA concentrations
Control

20 mg L  1

40 mg L  1

80 mg L  1

120 mg L  1

Shoot length per plant (cm)

Pre-owering
Flowering
Post-owering

65.06 7 2.29c
85.47 7 2.92d
94.73 7 3.73d

70.23 7 2.40bc
92.17 7 3.21cd
101.20 7 4.11cd

77.107 2.58b
102.807 3.26b
111.467 4.21b

89.80 7 2.84a
116.467 3.54a
125.20 7 4.58a

71.707 2.51b
96.67 7 3.17bc
107.707 4.36b

Dry weight per plant (g)

Pre-owering
Flowering
Post-owering

74.70 7 3.54d
87.80 7 3.86d
94.73 7 4.65d

79.50 7 3.71c
92.10 7 3.97cd
101.20 7 4.86b

86.107 3.94b
98.507 4.22b
111.467 4.88b

94.10 7 3.98a
107.40 7 4.53a
125.20 7 5.21a

84.207 3.51bc
95.507 4.19bc
107.707 4.36b

Net photosynthetic rate (mmol CO2 m  2 s  1)

Pre-owering
Flowering
Post-owering

12.16 7 1.11d
13.84 7 1.27d
11.13 7 1.07d

12.73 7 1.24c
14.46 7 1.48c
11.87 7 1.13c

13.86 7 1.41b
16.607 1.56b
12.53 7 1.32b

15.40 7 1.55a
18.82 7 1.72a
13.46 7 1.39a

13.58 7 1.52b
15.64 7 1.51bc
12.12 7 1.25bc

Stomatal conductance (mol m  2 s  1)

Pre-owering
Flowering
Post-owering

Internal CO2 (mmol m  2 s  1)

Pre-owering
Flowering
Post-owering

0.29 7 0.002d
0.28 7 0.002d
0.28 7 0.002d
262.0 7 1.15d
273.6 7 1.26d
255.0 7 1.17d

0.31 7 0.002c
0.34 7 0.003c
0.29 7 0.002c
275.6 7 1.21c
284.3 7 1.36c
261.0 7 1.14c

0.327 0.003b
0.367 0.004b
0.307 0.003b
283.6 7 1.31b
298.07 1.37b
279.07 1.18b

0.34 7 0.004a
0.39 7 0.005a
0.32 7 0.003a
299.3 7 1.42a
315.6 7 1.54a
291.0 7 1.34a

0.317 0.003c
0.357 0.004c
0.287 0.002d
279.6 7 1.39bc
296.07 1.48b
264.07 1.27bc

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835

Table 2
Effect of different concentrations of irradiated sodium alginate (ISA) on total chlorophyll content, total carotenoid content, nitrate reductase and carbonic anhydrase activities
of Artemisia annua L. Means within a column followed by the same letter are not signicantly different (pr0.05). The data shown are means of ve replicates 7SE.
Parameters

Growth Stages

ISA concentrations
20 mg L  1

Control

40 mg L  1

80 mg L  1

120 mg L  1

Total chlorophyll content (mg g  1 FW)

Pre-owering
Flowering
Post-owering

1.047 0.004d
1.127 0.004d
1.017 0.004c

1.09 70.004c
1.17 70.005c
1.11 70.003b

1.13 70.003b
1.26 70.005b
1.12 70.004b

1.21 70.004a
1.30 70.006a
1.15 70.002a

1.157 0.004b
1.247 0.003b
1.127 0.002b

Total carotenoid content (mg g  1 FW)

Pre-owering
Flowering
Post-owering

0.447 0.001d
0.487 0.002d
0.417 0.001d

0.46 70.002c
0.49 70.002cd
0.42 70.002d

0.48 70.002b
0.51 70.003b
0.44 70.002c

0.50 70.003a
0.53 70.003a
0.48 70.002a

0.477 0.001bc
0.507 0.002bc
0.467 0.002b

Nitrate reductase activity (nM NO2 g  1 FW h  1)

Pre-owering
Flowering
Post-owering

298.7 7 6.14d
312.6 7 6.27d
292.3 7 5.87d

319.5 76.32c
332.3 76.49c
296.1 75.94c

324.8 76.46bc
344.3 77.12b
315.2 76.27b

346.4 76.61a
375.2 77.54a
332.6 76.35a

329.5 7 5.52b
341.4 7 6.52b
317.3 7 6.24b

Carbonic anhydrase activity (mmol CO2 kg  1 FW s  1)

Pre-owering
Flowering
Post-owering

209.67 3.24d
239.2 7 3.43d
206.57 3.02d

216.1 73.29c
248.9 74.03c
212.3 73.21c

225.4 73.38bcc
262.6 74.21b
221.3 73.39b

240.6 74.14a
281.7 74.53a
232.2 73.73a

231.3 7 3.64b
267.1 7 4.42b
223.9 7 3.27b

80

40

60
d

800

a
b

bc

20

Artemisinin content (g g-1 DW)

H2O2 content (nmol g-1 FW)

Pre-flowering
Flowering
Post-flowering

Pre-flowering
Flowering
Post-flowering

c
600

d e
d

Control

20 mg

L-1

L-1

40 mg
80 mg
ISA concentrations

L-1

120 mg

3.3. Artemisinin content

The artemisinin content reached the highest extent in the
plants treated with ISA at 80 mg L  1 at owering stage, exceeding
the control by 25.36% (Fig. 2). Similar increases in artemisinin
content were observed due to ISA application at 80 mg L  1 both
at pre-owering and post-owering stages, outshining the control
by 20.30% and 21.51%, respectively. Wallaart et al. (2000) suggested that dihydroartemisinic acid might act as a scavenger of
reactive oxygen species (ROS) (H2O2 in this case) that are
generated in plant cells when they are exposed to oxidative
stress. During the reaction, dihydroartemisinic acid hydroperoxide (DHAA-OOH) is produced that is ultimately converted to
artemisinin. We have also noticed the relationship between
H2O2 and artemisinin content in some of our previous studies
(Aftab et al., 2010).

4. Conclusion
It could be concluded that ISA application might enhance the
growth, photosynthetic efciency, enzyme activities and artemisinin

200

Control

L-1

Fig. 1. Effect of different concentrations of irradiated sodium alginate (ISA) on

H2O2 content of Artemisia annua L. Bars showing the same letter are not
signicantly different at p r0.05 as determined by Duncans multiple range test.
Error bars (>) show SE.

400

a
b

20 mg L-1 40 mg L-1 80 mg L-1 120 mg L-1

ISA concentrations

Fig. 2. Effect of different concentrations of irradiated sodium alginate (ISA) on

artemisinin content of Artemisia annua L. Bars showing the same letter are not
signicantly different at p r 0.05 as determined by Duncans multiple range test.
Error bars (>) show SE.

content of A. annua, with 80 mg L  1 concentration of ISA resulting in

the maximum response. However, further investigations are
required to comprehend the mechanism and mode of action of
alginate-derived oligomers in plants. The experiments in the eld
are underway and the results are very encouraging that will be
reported soon.

Acknowledgments
Authors are indebted to Mr. M. Ram (SRF-CSIR) of Jamia
Hamdard (Hamdard University, New Delhi, India) for his kind
help in HPLC analyses of artemisinin. The nancial support
provided to the rst author in the form of Research Assistantship
(CST/D-3539) by the Council of Science and Technology, Lucknow,
UP, India, is also gratefully acknowledged.
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