Biotechnol. Appl. Biochem.
(2003) 38, 283–288 (Printed in Great Britain)
Biological effect of radiation-degraded alginate on flower plants
in tissue culture
Le Q. Luan*, Nguyen Q. Hien†, Naotsugu Nagasawa‡, Tamikazu Kume‡, Fumio Yoshii‡1
and Tomoko M. Nakanishi*
*Graduate School of Agricultural Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, †Nuclear
Research Institute, Vietnam Atomic Energy Commission, 01 Nguyen Tu Luc, Dalat, Vietnam, and ‡Takasaki Radiation
Chemistry Research Establishment, Japan Atomic Energy Research Institute, 1233 Watanuki, Takasaki, Gunma 370-1292,
Japan
Alginate with a weight-average molecular mass (Mw )
of approx. 9.04 × 105 Da was irradiated at 10–200 kGy
in 4 % (w/v) aqueous solution. The degraded alginate
product was used to study its effectiveness as a growth
promoter for plants in tissue culture. Alginate
irradiated at 75 kGy with an Mw of approx. 1.43 × 104 Da
had the highest positive effect in the growth of flower
plants, namely limonium, lisianthus and chrysanthe-
mum. Treatment of plants with irradiated alginate
at concentrations of 30–200 mg/l increased the shoot
multiplication rate from 17.5 to 40.5 % compared with
control. In plantlet culture, 100 mg/l irradiated alginate
supplementation enhanced shoot height (9.7–23.2 %),
root length (9.7–39.4 %) and fresh biomass (8.1–
19.4 %) of chrysanthemum, lisianthus and limonium
compared with that of the untreated control. The sur-
vival ratios of the transferred flower plantlets treated
with irradiated alginate were almost the same as the
control value under greenhouse conditions. How-
ever, better growth was attained for the treated
plantlets.
Introduction
Alginate, a block polymer of manuronic and guluronic acid
attached through random 1,4-glycoside linkages, has been
widely used in the preparation of pharmaceutical products,
cosmetics, biotechnology, agriculture and other industries
[1]. Oligoalginate, a degraded product of alginate, was found
to have several novel feature that can be useful in agriculture.
It was reported that this oligosaccharide had successfully
acted as a plant growth promoter, a phytoalexin-induction
trigger and also an enhancer of the activity of enzymes
towards plants [2–6]. Adachi et al. [2] reported that the
addition of oligoalginate into the growth medium had a
strong effect in increasing the leaf weight (114 %) and tuber
weight (324 %) of Japanese radish. Yonemoto et al. [3]
showed that oligoalginate with weight-average molecular
283
mass (Mw ) of approx. 1.8 × 103 Da had a strong effect on
shoot elongation of rice, grains and tobacco. Germination
and root growth rates of barley were positively affected by
a supplement of 800 mg/l oligoalginate [4]. Oligoalginate is
recognized not only as a factor enhancing the activity of
some enzymes such as alcohol dehydrogenase and lactate
dehydrogenase, but also as a promoter of the induction of
antibiotic enzymes, e.g. 5
-phosphodiesterase and chitinase
[4–6]. Oligoalginate is usually produced by using an alginate-
degrading enzyme such as alginate lyase [3]. Recently, a
lot of attention has been paid to the use of radiation
processing technology to obtain degraded alginate, owing
to the superiority of this method over the conventional
enzymic degradation process. The main advantages are the
following: (i) the degradation reaction can be performed
at room temperature (25 ◦ C); (ii) after processing, the
degraded alginate can be used without purification; (iii) the
simplicity of controlling the whole process; and, above all,
(iv) large-scale application. Although degraded alginate was
reported to affect plant growth, it is yet to be shown in plant
tissue culture [7,8]. In the present study, we determined the
biological effect of irradiated alginate for in vitro propagation
of the commercial flower plants limonium, lisianthus and
chrysanthemum.
Materials and methods
Preparation of irradiated alginate
Alginate with an Mw of about 9.0 × 105 Da (supplied by
Kishida Chemical Co. Ltd., Osaka, Japan) was kept overnight
in water at room temperature to allow swelling and then
Key words: alginate, growth promotion, irradiation, oligoalginate, plantlet.
Abbreviations used: DP, degree of polymerization; GPC, gel-permeation
chromatography; IBA, indole-3-butyric acid; LSD0.05 , least significant
difference at 5 % probability level; MS medium, Murashige and Skoog’s
medium; NAA, 1-napthylacetic acid.
1
To whom correspondence should be addressed, at Department of
Materials Development, Takasaki Radiation Chemistry Research
Establishment (e-mail yoshii@taka.jaeri.go.jp).

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284 L. Q. Luan and others

stirred for 5 h to obtain a 4 % (w/v) solution [7]. The alginate

solution was then irradiated by γ -rays from a 60 Co source
at doses up to 200 kGy with a dose rate of 10 kGy/h.

Mw determination
Mw of the irradiated alginate was measured at 40 ◦ C by
GPC (gel-permeation chromatography) (model CO-8020;
Tosho Co. Ltd., Tokyo, Japan), equipped with three TSKgel
PWXL columns (300 mm × 7.8 mm) in series G6000PWXL ,
G3000PWXL , G2500PWXL (Tosho Co. Ltd.) combined with
a TSK guard column PWXL (40 mm × 6.0 mm). They were
eluted at 40 ◦ C with 0.1 mol/l sodium nitrate solution at a
flow rate of 0.5 ml/min. The eluent was monitored by an
RI-8020 differential refractometer at 40 ◦ C and the alginate
concentration was 0.1 % (w/v). Mw was determined from a Figure 1 Change in the Mw of alginate on its γ -irradiation in aqueous
solution
calibration curve using seven poly(ethylene glycol) standard
samples with Mw values in the range 2 × 102 –6 × 103 Da and Mw was measured by GPC using poly(ethylene glycol) and pullulan standards.
eight pullulan standard samples with Mw values in the range
5.9 × 103 –7.9 × 105 Da (Wako Co. Ltd., Osaka, Japan) as were then transferred to pots containing a mixture of soil
described elsewhere [9]. and fertilizer and cultivated for 30 days in a greenhouse.

Plant growth test in vitro Data collection and statistical analysis

Flower plants used in the present experiments were
limonium (Limonium latifolium), lisianthus (Eustoma grandi-
florum) and chrysanthemum (Chrysanthemum morifolium). To
investigate the effect of irradiation dose for alginate
on the growth of the shoot cluster, 20 shoots of
flower plants were cultured in a vessel containing
MS medium (Murashige and Skoog’s medium) [10,11],
supplemented with 3 % sucrose, 0.8 % agar, 0.3 mg/l 6-
benzylaminopurine, 0.1 mg/l NAA (1-napthylacetic acid)
and 50 mg/l alginate that had been irradiated at various
doses. To investigate the effect of concentration on the
plant growth promotion, alginate irradiated at 75 kGy,
i.e. with Mw ≈ 1.43 × 104 Da [DP (degree of polymeri-
zation) ≈ 76], was supplemented into the culture medium
at a concentration ranging from 5 to 200 mg/l.
For plantlet culture, 20 plant shoots were cultured in
a vessel containing half-strength MS medium, 3 % sucrose,
0.8 % agar and 100 mg/l alginate irradiated at various doses
for chrysanthemum and supplemented with 1 mg/l IBA
(indole-3-butyric acid) for limonium and either 1 mg/l IBA or
0.01 mg/l NAA for lisianthus. To determine a suitable con-
centration, irradiated alginate with Mw ≈ 1.43 × 104 Da was
supplemented at 5–200 mg/l. Before autoclaving at 121 ◦ C
for 15 min, the pH of the medium was adjusted to 5.8. All
cultures were incubated in a light culture room at 25 + ◦
−1 C
with a photoperiod of 12 h/day.
Transfer to soil
The plantlets (100 in number) with well-developed roots
were removed and washed gently under tap water. They

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The number of shoots was determined by counting the
shoots longer than 0.5 cm after incubating for 25 days.
The shoot height and root length were measured by a
millimetre ruler and fresh biomass was weighed after 15 days
incubation for chrysanthemum and 20 days for limonium and
lisianthus. For each experiment, 100 samples were used
and experiments were repeated three times. Data were
statistically analysed by variance analysis (ANOVA) and
LSD0.05 (least significant difference at 5 % probability level)
and means + − S.D. were determined. The degree of growth
promotion was calculated by the following formula:
Growth promotion degree (%)
= 100 × (evaluation of the treated bed)/
(evaluation of the untreated bed)
Results and discussion
Change in molecular mass of alginate on irradiation
The use of radiation technology to degrade alginate has been
studied. It has been shown that the degree of degradation de-
pends on the irradiation dose and the alginate concentration
in aqueous solution [7,8,12]. In the present study we used
a 4 % solution of alginate and irradiated it at various doses.
The effect of irradiation on Mw was determined by GPC,
and the results are shown in Figure 1. The results indicate
that there is a decrease in Mw of alginate with an increasing
dose of irradiation. A rapid decrease in Mw of alginate was
observed with an irradiation dose of up to 50 kGy (from
9.04 × 105 to 1.43 × 104 Da). Then the Mw of alginate
 Effect of irradiated alginate on flower plants 285

Table 1 Effect of irradiated alginate degradation on shoot multiplication

rate of flower plants a
Samples were incubated for 25 days in MS medium supplemented with 3 %
sucrose, 0.8 % agar, 0.3 mg/l 6-benzylaminopurine, 0.1 mg/l NAA and 50 mg/l
alginate irradiated at various doses.

Number of shoots

Irradiation dose (kGy) Chrysanthemum Lisianthus Limonium

Controla 2.87 +
− 0.03 3.37 +
− 0.13 2.62 +
− 0.10
0 2.77 +
− 0.16 3.60 +
− 0.09 2.82 +
− 0.35
10 2.95 +
− 0.18 3.85 +
− 0.13 2.68 +
− 0.12
30 2.90 +
− 0.11 3.58 +
− 0.13 3.01 +
− 0.11
50 3.34 +
− 0.23 4.30 +
− 0.10 3.20 +
− 0.09
75 3.41 +
− 0.05 4.67 +
− 0.18 3.65 +
− 0.13
100 3.21 +
− 0.16 3.87 +
− 0.15 3.37 +
− 0.26
150 3.14 +
− 0.12 3.88 +
− 0.10 3.31 +
− 0.17
200 3.42 + 3.87 + 3.42 +
b
− 0.35 − 0.15 − 0.25
a
Without supplementation of irradiated alginate.

decreased gradually on increasing the irradiation dose. The

results are in agreement with the previous reports of Hien
et al. [7] and Nagasawa et al. [12].

Effect of the irradiation dose on alginate for plant

growth in tissue culture
Nowadays, ornamental flowers such as chrysanthemum, c
lisianthus, limonium etc. attract considerable interest among
horticulturists because of their popularity and commercial
importance. Tissue-culture techniques have been used as
a useful method for propagation in vitro, to obtain high-
quality young plants for planting. The technique requires
a suitable medium, supplementation with phytohormones,
suitable conditions for culture etc., but, with careful planning,
it ensures high profitability. In addition, oligosaccharides
have been reported to have biological effects on plant
growth, particularly on morphogenetic processes [13],
root and shoot elongation, promotion of induction of
antibiotic enzymes and enhancement of enzyme activity Figure 2 Effect of alginate irradiation dose on the growth of flower plants
[4,8,14–17]. The Mw or DP of polysaccharides plays
(a) Chrysanthemum was incubated for 15 days in half-strength MS medium
a key role in enhancing biological activity [7,12,15,18].
supplemented with 3 % sucrose and 0.8 % agar; (b) lisianthus was incubated
This experiment was performed to investigate the most for 20 days in half-strength MS medium supplemented with 3 % sucrose, 0.8 %
appropriate irradiation dose required for the degradation of agar, 1 mg/l IBA and 0.01 mg/l NAA; (c) limonium was incubated for 20 days
in half-strength of MS medium supplemented with 3 % sucrose, 0.8 % agar and
alginate to obtain a suitable Mw , which can be effective in
1 mg/l IBA. Black bar, shoot height; hatched bar, root length; white bar, fresh
plant propagation in vitro. biomass.
Alginates irradiated with various doses were used to
grow the shoot cluster, and the effect was scored by
observing the shoot multiplication rate. The results in
Table 1 show that the supplementation of the growing promotion of shoot cluster of the flower plants in tissue
media with alginate irradiated at 50–200 kGy increased culture was approx. 75 kGy; the Mw of alginate irradiated at
the number of shoots of chrysanthemum (12.0–19.2 %), this dose was approx. 1.43 × 104 Da (DP ≈ 76).
lisianthus (14.9–38.6 %) and limonium (14.9–39.5 %). The Previous studies revealed that oligoalginate had high
dose for degradation of alginate for the most efficient growth positive effects on the increase in the root length of barley

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286 L. Q. Luan and others
Figure 3 Effect of the concentration of irradiated alginate (Mw ≈ 1.43 ×
104 Da) on the growth of flower plants
(a) Chrysanthemum plants were incubated for 15 days in half-strength MS
medium supplemented with 3 % sucrose and 0.8 % agar; (b) lisianthus plants
were incubated for 20 days in half-strength MS medium supplemented with
3 % sucrose, 0.8 %agar, 1 mg/l IBA and 0.01 mg/l NAA; (c) limonium plants
were incubated for 20 days in half-strength MS medium supplemented with
3 % sucrose, 0.8 % agar and 1 mg/l IBA. 䊏, shoot height; 䉱, root length; 䊉,
fresh biomass.

C 2003 Portland Press Ltd
seedling (2.4 mm/h) [4], tuber mass of Japanese radish
(324 %) [2] and dried mass of rice (20 %) [7]. In plantlet
culture, the induction and development of root are
important processes to obtain plantlets of high quality.
The irradiated alginate has the ability to affect these two
steps of plant development. A comparison of the growth
promotion effect on chrysanthemumis is presented in
Figure 2(a). Treatment by alginate irradiated at 75–150 kGy
increases the shoot height (9.0–20.5 %), root length
(11.6–12.1 %) and fresh biomass (19.0–32.6 %) of plants.
On the other hand, treatment of plants with alginate
irradiated at doses below 75 kGy or above 200 kGy gave
unstable results and no significant difference was observed
when compared with the untreated control. The growth
promotion effects of irradiated alginate on lisianthus and
limonium are shown in Figures 2(b) and 2(c). Significant
increases in shoot height, root length and fresh biomass
were observed in beds supplemented with alginate irrad-
iated at 50–200 kGy for lisianthus and 50–150 kGy for
limonium. Alginate irradiated at doses below 50 kGy
showed no significant effect on any of the development pro-
cesses in both plants. On the basis of these results,
we conclude that alginate irradiated at 75 kGy, i.e. with
Mw ≈ 1.43 × 104 Da (DP ≈ 76), displays the maximum
growth promotion effect under tissue culture conditions
for all varieties that were tested. Therefore a 75 kGy
irradiated oligosaccharide was selected to investigate the
optimum concentration.
Effect of irradiated alginate concentration on plant
tissue culture
The effect of oligoalginate concentration was studied on
various plants as a function of the mode of treatment.
For foliar spraying, commonly used concentrations were
40–2000 mg/l [2] and for hydroponics 20–3000 mg/l [4,7].
In the present study, the suitable concentration of
irradiated alginate (Mw ≈ 1.43 × 104 Da) for proliferation
of shoot culture was investigated. The results in Table 2
indicate that the supplementation with irradiated alginate
at concentrations of 30–200 mg/l exhibited a considerable
effect on the enhancement of shoot multiplication rate for
all the flower plants.
The induction and development of root from shoot
are quite important for the growth of plantlets. It can
be seen in Figure 3(a) that supplementing the growth
medium with 50–200 mg/l irradiated alginate has a significant
effect on the induction and development of root. Hence
it enhances the shoot height and fresh biomass of
chrysanthemum. Irradiated alginate (50–200 mg/l) treatment
increased the root length, shoot height and fresh biomass
of this plant by 16.9–39.4, 13.1–23.2 and 9.7–19.4 %
respectively when compared with the untreated control.
For lisianthus, the increases in root length (8.9–14.6 %),
 Effect of irradiated alginate on flower plants 287

Table 2 Effect of irradiated alginate concentration on shoot multiplication

rate of flower plants

Samples were incubated for 25 days in MS medium supplemented with 3 %

sucrose, 0.8 % agar, 0.3 mg/l 6-benzylaminopurin, 0.1 mg/l NAA and 50 mg/l
alginate irradiated at 75 kGy (Mw ∼ 1.43 × 104 Da).

Number of shoots
Oligoalginate concentration
(mg/l) Chrysanthemum Lisianthus Limonium

0 2.63 +
− 0.24 3.83 +
− 0.21 4.58 +
− 0.29
5 2.77 +
− 0.16 3.92 +
− 0.08 4.58 +
− 0.25
10 2.95 +
− 0.18 4.48 +
− 0.18 5.07 +
− 0.07
30 3.32 +
− 0.20 4.66 +
− 0.53 5.20 +
− 0.31
50 3.10 +
− 0.18 4.95 +
− 0.09 5.40 +
− 0.39
70 3.70 +
− 0.10 4.33 +
− 0.24 5.47 +
− 0.24
100 3.25 +
− 0.10 4.32 +
− 0.15 5.28 +
− 0.53
150 3.58 +
− 0.16 4.33 +
− 0.13 5.20 +
− 0.29
200 3.42 +
− 0.35 4.29 +
− 0.10 5.11 +
− 0.04

Table 3 Survival ratio of flower plantlets treated with irradiated alginate

after 30 days acclimatizing in greenhouse

Average number of surviving plants per 100

transferred plantlets
Irradiation dose (kGy) Chrysanthemuma Lisianthusb Limoniumc

Controld 84.3 81.4 87.1

0 79.9 75.1 84.0
10 84.6 76.8 92.1
30 75.7 77.1 92.6
50 87.0 83.8 92.5 Figure 4 Growth of transferred flower plantlets treated with irradiated
75 89.7 86.0 91.9 alginate (Mw ≈ 1.43 × 104 Da) after acclimatizing for 30 days in a greenhouse
100 89.9 86.3 91.9
150 81.8 85.2 92.5 (a) Chrysanthemum; (b) lisianthus; (c) limonium.
200 78.6 80.2 91.6
a
LSD0.05 = 7.2.
b
LSD0.05 = 9.6. The survival test of plantlets treated with
c
LSD0.05 = 6.5.
d
Without supplementation of irradiated alginate. irradiated alginate
In plant cells, oligosaccharides were considered to be the
signalling chemicals to induce phytoalexins, which protect
plants from fungal infection [14,15]. Furthermore, irradiated
shoot height (6.2–14.4 %) and fresh biomass (8.1–9.9 %) alginate exhibited not only a remarkable growth promotion
were observed for growing medium supplemented with effect but also a protection effect on plants [7]. After
50–200 mg/l irradiated alginate (Figure 3b). For limonium acclimatizing for 30 days in a greenhouse, plantlets with
(Figure 3c), same concentrations of irradiated alginate irradiated alginate exhibited a similar survival rate as those
enhanced root length (9.7–12.9 %), shoot height (9.3– of untreated control (Table 3). However, a better growth
16.6 %) and fresh biomass (11.7–16.6 %). The most advan- was observed in supplemented plantlets (Figure 4). These
tageous concentration of irradiated alginate for plantlet results suggest that plantlets treated with irradiated alginate
culture of chrysanthemum, limonium and lisianthus was showed a better response to environmental influence due
found to be 100 mg/l. to their acclimation in a greenhouse.
Although the mechanism of influence of irradiated
alginate on growth promotion in plants is still unclear, it was
proved apparently that oligoalginate had some physiological References
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497


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