JAEA-Review-2010-065.pdf:15.99MB - 日本原子力研究開発機構

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Phenotypic Improvement of Bradyrhizobium japonicum
USDA 110 into a High Temperature Tolerant Strain in
terms of Ion-beam Microbial Mutation-breeding
Technology
K. Takeda a) , K. Tejima b) , K. Satoh b) , I. Narumi b) and T. Yokoyama c)
a) Graduate school of Agriculture, Tokyo University of Agriculture and Technology,
b) Radiation-Applied Biology division, QuBS, JAEA,
c) Institute of Agriculture, Tokyo University of Agriculture and Technology
In order to supply food to increasing population in Asia,
agricultural production should be increased and a large
amount of nitrogen fertilizer is required. Most of the
nitrogen fertilizers are chemical nitrogen fertilizers. They
are produced by oil and natural gas in terms of the
Haber–Bosch process, which exhausts a large amount of
CO 2. Furthermore, the economically minable period of
the fossil resources is estimated to be around 40 years,
therefore a reduction of chemical nitrogen fertilizer input is
required in agricultural practices.
In Asian countries, in order to increase crop yield under
low input of chemical nitrogen fertilizers, many researchers
in agricultural institutes are trying to develop biofertilizers,
which contain beneficial soil microorganisms and increase
availability of plant nutrients from soils. However,
several researchers point out constraints on application of
biofertilizers. Major constraint of biofertilizer utilization
in agricultural practice is a serious deterioration of qualities
in biofertilizers under both storage and transportation
conditions. In other words, exposing high temperature
and drought stress causes a viability loss of beneficial
microorganisms in biofertilizers. This becomes a serious
problem on the dissemination of biofertilizer in
southeastern Asia. Therefore, in order to prevent
deterioration in variability of inoculants in biofertilizers
exposed to high temperature, we are trying to improve a
phenotypic character concerning high temperature tolerant
in Bradyrhizobium japonicum USDA 110, which is a
worldwide superior inoculant to soybean. In this study,
ion-beam microbial mutation-breeding technology is used
to generate high temperature tolerant mutants.
B. japonicum USDA 110 was cultivated in YM liquid
medium until they reached to a concentration of
10 8 cells/mL. Cells were harvested by centrifugation and
resuspended in a solution containing 10% skim milk and
1.5% sodium glutamate. The suspension was then
subjected to vacuum-freeze drying (Fig. 1) to minimize the
effect of water radiolysis during irradiation. Freeze-dried
cells of B. japonicum USDA 110 were irradiated with
carbon ion beams (220 MeV 12 C 5+ ) at TIARA in JAEA.
Irradiation dose was ranged from 0 to 800 Gy. Survival
rates were changed as follows: 10% at 100 Gy, 0.7% at
300 Gy, and 0% at 800 Gy. Aliquots of irradiated cells
were proliferated on YM-agar plate in order to fix
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mutations. Mutant frequency was determined by
measuring the number of rifampicin resistant colonies
(Fig. 2; rifampicin resistance is conferred by a mutation in
rpoB gene encoding RNA polymerase subunit) in total
number of viable colonies. The highest frequency value
of mutants was obtained from cells irradiated at 300 Gy.
In general, B. japonicum USDA110 cannot survive at 40 o C.
In order to obtain high temperature tolerant mutants, the
irradiated cells were put on the YM agar plate and keep for
over 1 month at 45 o C in several times. But, we couldn’t
find any proliferated colony. Therefore, we developed a
new method to screen survival cells at 45 o C. In the new
method, the irradiated cells were incubated in YM liquid
medium at 45 o C for 5 days, and the cultures were put on
YM-agar. Consequently, we obtained high temperature
tolerant mutants of B. japonicum USDA 110, which can
survive at 45 o C for 5 days. Now, we are characterizing
these mutants in terms of differences in several phenotypic
properties.
Fig. 1 Preparation of freeze-dried cells of B.
japonicum USDA 110.
Fig. 2 Refampicin resistant
colonies of B. japonicum
USDA 110 on YM-agar
plate supplemented by
400 µg/mL of rifampicin.