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Radiation Physics and Chemistry 78 (2009) 796–799

Contents lists available at ScienceDirect

Radiation Physics and Chemistry

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

Enhancement of plant growth stimulation activity of irradiated alginate

by fractionation
Le Quang Luan a,, Naotsugu Nagasawa b, Vo Thi Thu Ha a, Nguyen Quoc Hien c, Tomoko M. Nakanishi d
a
Center for Nuclear Techniques, Vietnam Atomic Energy Commission, 217 Nguyen Trai, 1st District, Hochiminh City, Vietnam
b
Quantum Beam Directorate, Japan Atomic Energy Agency, 1233 Watanuki, Takasaki, Gunma 370-1292, Japan
c
Research and Development Center for Radiation Technology, Vietnam Atomic Energy Commission, 202A, Str. 11, Linh Xuan Ward, Thu Duc District, Hochiminh City, Vietnam
d
Graduate School of Agricultural and Life Sciences, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

a r t i c l e in f o a b s t r a c t

Article history: Alginate with the weight-average molecular weight (Mw) approximately 900 kDa and ratio of M
Received 23 January 2009 (mannuronate)/G (guluronate) about 1.3 was irradiated by gamma Co-60 in aqueous solution at doses
Accepted 8 May 2009 up to 200 kGy. The irradiation dose was shown to be a function for reducing Mw and molecular weight
distribution of irradiated alginates. The distribution of oligomer fractions in irradiated products was
Keywords: also investigated by separation using ultrafiltration membranes. The irradiated alginate with Mw
Alginate approximately 14.2 kDa was found to have a positive influence for growing of barley and soybean.
Degradation The irradiated oligoalginate fraction with Mw ranging from 1 to 3 kDa displayed the strongest effect on
Fraction the growth and development of the mentioned plants at low concentration (20 ppm). It is suggested
Growth stimulation
that oligoalginate with Mw in the range 1–3 kDa is a trigger for the growth and development of plants.
Irradiation
& 2009 Elsevier Ltd. All rights reserved.
Oligoalginate

1. Introduction addition, the alginate oligomer fractions with the degree of

Alginate, a linear polymer composed of (1-4)-linked
b-D-mannuronate and a-L-guluronate units arranged in homo-
or hetero-polymeric sequences, is extracted from cell wall
of marine molluscs, bacteria and brown algae (Boy and Turvey,
1977; Gacesa, 1988; Haug et al., 1966; Linker and Evans, 1984;
Tombs and Harding, 1998). Recent studies have reported oligoal-
ginate as an increasingly important bioactive polysaccharide.
Other reports have described their physiological activities on
plant growth (Ariyo et al., 1997; Gacesa, 1992; Hien et al., 2000;
Iwasaki and Mastsubara, 2000; Kume et al., 2002; Natsume et al.,
1994; Tomoda et al., 1994; Xu et al., 2003; Yonemoto et al., 1993).
Similar to enzymatically degraded alginate, irradiated alginate is
also reported to have a novel feature as plant growth stimulator
(Kume et al., 2002; Hien et al., 2000). In our previous study,
irradiated alginate with an average molecular weight (Mw)
approximately 14  103 showed a strong growth-promotion effect
on plant in vitro (Luan et al., 2003). Radiation is a convenient
and efficient tool for degradation of natural polysaccharides.
Therefore, much attention has been paid recently for the use of
radiation for the production of oligosaccharides (Hien et al., 2000;
Kume et al., 2002; Luan, et al., 2003; Nagasawa et al., 2000). In
Abbreviations: ANOVA, Analysis of variance; M, Mannuronate; G, Guluronate
 Corresponding author. Tel.: +84 8 38356568; fax: +84 8 8367361.
E-mail address: lequangluan@gmail.com (L.Q. Luan).
polymerization from tetramer (3) to hexamer (6) have been
reported to show a special growth-promotion effect for plant
(Iwasaki and Mastsubara, 2000). In the present study, the
separated fractions of irradiated alginate were used for testing
the plant growth-promotion effect.
2. Materials and methods
Alginate was supplied by Kisida Chemical Co. Ltd., Japan.
Alginate lyase from Flavobacterium sp. with Catalogue no. A-1603
and Lot 083K0695 (38,730 unit/g solid) was supplied from
Sigma—Aldrich Co., USA and ultrafiltration membranes used for
the study were regenerated cellulose membranes in the series of
YM supplied from Millipore Corporation, USA.
Alginate with Mw approximately 900 kDa and M/G ratio (the
ratio between Mannuronate content and Guluronate content in
alginate sample) approximately 1.3 was kept overnight in water at
room temperature for swelling and then stirred for 5 h to obtain
the solutions of 4% (w/v). The alginate solutions were then
irradiated at room temperature by gamma-rays from a Co-60
source at the doses of 10, 30, 50, 75, 100, 150 and 200 kGy with
the dose rate of 10 kGy/h for degradation and a volume of 150 ml
was devised into three bottles and applied for each treated
dose to obtain a dose distribution of 5% for each irradiated sample
(Dmax/Dmin1.05).

0969-806X/$ - see front matter & 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.radphyschem.2009.05.001
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To obtain enzymatic-degraded product, alginate was hydro-
lyzed by using enzyme alginate lyase according to the method
of Iwamoto et al. (2001). A portion of 0.5 g of alginate was
dissolved in 100 ml of phosphate buffer at pH 7.0, followed by an
addition of 2 ml of alginate lyase (1 mg/ml) (about 77.5 units). The
reaction was carried out at 37 1C in 10 h.
For fractionation of radiation-degraded alginate, a stirred
ultrafiltration cell model 8400 was employed for fractionation of
radiation-degraded alginate. The regenerated cellulose ultrafiltra-
tion membranes with a diameter of 63.5 mm, namely, YM1 (Mw
cut-off 1000), YM3 (3000), YM10 (10,000) and YM30 (30,000)
were used in the cross-flow mode. Deionized water was used as a
solvent and a pressure of 0.1 MPa was applied for fractionation.
The oligoalginate content in fractionated solution was determined
by method of Dubois et al. (1956).
The growth-promotion effect of degraded and fractionated
alginate was tested on barley and soybean. For barley test, 10
germinated seeds were cultivated in 500 ml solution containing
0.1% hyponex and degraded or fractionated alginate. The test on
soybean was carried out by cultivation of 3 germinated seeds
in 500 ml of 15 Stainberg nutrient solution supplemented with
degraded or fractionated alginate. The controls were performed
at same conditions but without supplementation of alginate. All
cultures were incubated in a Biotron with a photo period of 12 h
per day. The experiment was carried out with 3 blocks with three
replicates for each treatment. Data were statistically processed by
analysis of variance (ANOVA).
3. Results and discussion
The Mw and molecular weight distribution of alginate in
4% solution irradiated at various doses are shown in Fig. 1. It
can be seen that the higher the dose the lower the Mw and
the narrower the molecular weight distribution (the molecular
weight distribution is defined as the ratio between weight-
average molecular weight (Mw) and number-average molecular
weight (Mn)). The radiation degradation yield (G-value) of alginate
in 4% solution was calculated according to the equation described
by Charlesby (1960) to be of 16.35 scissions/100 eV (1.69 mmol/J).
Fig. 1. Molecular weight (Mw) and molecular weight distribution (Mw/Mn) of
alginate irradiated at different doses; (K), Mw; (m), Mw/Mn.
797
This result of G-value was fairly in agreement with the G-value
reported by Nagasawa et al. (2000). In addition, the results in
Fig. 1 also show that a dramatic decrease in alginate Mw is
observed with an increase of the irradiation dose up to 50 kGy and
then alginate Mw decreases gradually with an increase of the
irradiation dose.
All of degraded alginate products irradiated in various doses in
4% solution were fractionated by using four kinds of ultrafiltration
membranes for separating the degraded products into 5 fractions
with the size of molecular weight as follows: fraction 1 (F1) with
Mw below 1 kDa, fraction 2 (F2) with Mw from 1 to 3 kDa, fraction 3
(F3) with Mw from 3 to 10 kDa, fraction 4 (F4) with Mw from 10 to
30 kDa and fraction 5 (F5) with Mw more than 30 kDa. The results
shown in Fig. 2 indicated that the distribution of five mentioned
fractions in alginate products irradiated at various doses in 4%
solution was quite different. The content of high Mw fractions
(F5 and F4) was directly proportional to the increase of irradiation
dose and the content of low Mw fraction (F1) was reciprocal to
the increase of irradiation dose, while the content of F2 and F3 was
highest generated in the product irradiated at the irradiation dose
of 75 kGy.
In our previous study, the alginate product irradiated at
75 kGy in 4% solution showed a strong growth-promotion effect
for plants, namely, chrysanthemum, lisianthus and limonium in
tissue culture (Luan et al., 2003). The other studies also reported
that degraded alginate significantly stimulated the growth and
development of rice, lettuce and komatsuna (Ariyo et al., 1997;
Hien et al., 2000; Iwasaki and Mastsubara, 2000; Kume et al.,
2002; Yonemoto et al., 1993). In the present study, the degraded
alginate products were used to study its effectiveness as a growth
promoter for barley and soybean. The results in Fig. 3 showed that
the degraded alginate irradiated at 75 kGy in 4% solution also
displayed the highest effect on the growth of barley and soybean.
According to Iwasaki and Mastsubara (2000), the oligoalginate
fraction with degree of polymerization of 6 (hexasaccharide) had
an especially strong stimulation activity on the growth of lettuce.
In the present study, the results from the test on the growth-
promotion effect of separated fractions (see Figs. 4 and 5) show
that the fraction F2 (Mw from 1 to 3 kDa) strongly promoted the
growth of the tested plants. The results of optimum concentration
of fraction F2 and irradiated alginate with and without separation
for the growth promotion of barley were showed on Fig. 6. It
can be seen that for non-separated sample, the appropriate
70
60
50
Fraction content, %
40
30
20
10
0
25 50 75 100 200
Irradiation dose, kGy
Fig. 2. Fraction content of alginate irradiated in 4% solution at various doses; (’),
Mwo1 kDa; ( ), Mw: 1–3 kDa; ( ), Mw: 3–10 kDa; (&), Mw: 10–30 kDa; ( ),
Mw430 kDa.
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2.8
Fresh biomass, g/10 seedlings

2.6

2.4

2.2

2.0
Control 0 10 30 50 75 100 150
Irradiation dose, kGy

Fig. 3. Effect of irradiation dose for alginate on the growth of barley.

Fig. 6. Effect of the concentration of irradiated alginate (75 kGy) with and without
separation on the growth of barley; (&), separated fraction; ( ), non-separation.

Fig. 4. Effect of irradiated alginate fractions on the growth of barley.

Fig. 7. Effect of the concentration of enzymatic-degraded alginate with and

without separation on the growth of barley; (&), separated fraction; ( ), non-
separation.

from Fig. 7 that compared to control, enzymatic separated fraction

Fig. 5. Effect of irradiated alginate fractions on the growth of soybean.
concentration was found at 100 ppm, while the optimum
concentration of separated fraction was 20 ppm. The main
reason may be due to the difference of the content of fraction F2
in the degraded samples.
In addition, the alginate degraded by enzyme alginate lyase
was also carried at 10 h to obtain enzymatic-degraded alginate
with Mw approximately 14.4 kDa. This product was then used for
separating and testing the growth-promotion effect. It can be seen
stimulates 11.3% growth of barley seedling at 50 ppm (optimum
concentration), while the non-separated enzymatic-degraded
alginate stimulates 8.8% growth of barley seedling at 300 ppm.
The above results suggest that the oligoalginate fraction F2 with
Mw from 1 to 3 kDa was seemed to be a trigger for the growth and
development of mentioned plants. The results from Figs. 6 and 7
also show another interesting result that the growth stimulation
effect of irradiated degraded alginate fraction on barley seedling
(17.6%) is higher than that of enzymatic-degraded alginate
fraction (11.3%). And the optimum concentration of irradiated
products is quite lower than that of enzymatic-degraded product.
These results indicated that gamma-ray irradiation for degrada-
tion of alginate to induce plant growth promotion is a suitable
tool.
4. Conclusions
Gamma-ray irradiation is a useful tool for degradation of
alginate. The irradiated alginate with Mw approximately 14.2 kDa
showed a positive effect for the growth of barley and soybean and
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the fraction F2 with Mw from 1 to 3 kDa was a trigger for the
growth and development of the mentioned plants.
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