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Production of Soybean Mutants with Pale-Green-Leaf
Phenotype by Ion Beam Irradiation
S. Arase a) , J. Abe a) , S. Nozawa b) , Y. Hase b) , I. Narumi b) and A. Kanazawa a)
a) Research Faculty of Agriculture, Hokkaido University,
b) Radiation-Applied Biology Division, QuBS, JAEA
Soybean (Glycine max) is an important crop in terms of
production of food, oil, and forage. However, existing
mutant lines of soybean is very limited, which is a constraint
on performing a genetic study and breeding of this plant.
Soybean is considered to have derived from ancestral
plant(s) that have a tetraploid genome, and as a consequence,
more than 90% of nucleotide sequence in the soybean
1)
genome is duplicated . It is conceivable that such a
duplicated nature of the genome brought about a low
frequency of mutant production by conventional methods
for mutagenesis such as γ-ray or X-ray irradiation as well as
chemical treatments. In these circumstances, we have
started to examine whether ion beam irradiation is effective
in producing a mutant in soybean because ion beam
irradiation is expected to cause genomic changes that are
more drastic than those induced by conventional
mutagenesis.
We have previously analyzed the effects of ion beam
irradiation on plant growth and morphology in soybean by
exposing dried seeds to the 320 MeV carbon ions with the
range of 0.2 – 25 Gy 2) . The irradiated seeds were sown on
soil and plants were grown for three weeks in a greenhouse.
We found that irradiation doses higher than approximately
5 Gy affected plant growth rate. In order to establish a
plant population that can be available for screening mutants,
we further examined the effects of irradiation by growing
plants in a field. We found that both plant height and the
ratio of the number of plants that survived until seed-setting
per the number of seeds sown in the field depended on the
doses of irradiation. We tentatively concluded that
irradiation doses around 2.5 Gy are suitable for producing
mutants 3) .
Based on these results we grew plants from irradiated
seeds in a large scale to obtain a population of M2 seeds:
3,200 seeds and 3,320 seeds irradiated at 5 Gy and 2.5 Gy,
respectively, were sown in the field and seeds were
harvested. The harvested M2 seeds were sown in the field
next year and generation of individuals with visibly altered
4)
phenotypes was examined .
We detected plants with a visibly altered phenotype.
The observed change was a chlorophyll deficiency, as
evidenced by pale-green leaves (Fig. 1). Frequency of this
type of change was 0.10% (n = 1,911) and 0.19% (n = 1,038)
for M2 plants irradiated with 2.5 Gy and 5.0 Gy, respectively.
We confirmed that this phenotypic change was heritable by
growing the progeny of M2 plants with the altered
phenotype. The M3 progeny of four out of six M2 plants
maintained the altered phenotype (Fig. 1; the progeny of
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‘D-2,’ ‘B-1,’ ‘B-2,’ and ‘C-2’ plants). Thus, at least these
four M 2 plants had stable heritable change(s) that caused the
altered phenotype, demonstrating that ion-beam irradiation
at 2.5–5.0 Gy can induce mutation.
The frequency of the chlorophyll-deficient phenotype
was comparable to that observed in other plants. For
example, the frequency of the mutation in petunia was 0.4%
when irradiated at half the dose that caused a decrease in
survival rate but which generated various mutants 5) . Thus,
we consider that the irradiation conditions, including doses,
to obtain mutants for use as breeding materials were
effectively optimized in our study.
References
1) R. C. Shoemaker et al., Genetics 144 (1996) 329.
2) A. Kanazawa et al., JAEA Takasaki Ann. Rep. 2006
(2008) 88.
3) A. Kanazawa et al., JAEA Takasaki Ann. Rep. 2007
(2008) 71.
4) A. Kanazawa et al., JAEA Takasaki Ann. Rep. 2008
(2009) 68.
5) Y. Hase et al., Plant Biotechnol. 27 (2010) 99.
M3 generation
Fig. 1 Inheritance of the pale-green-leaf phenotype.
Four progeny plants from each M 2 plant are shown
(except D-2, for which one progeny plant is
shown): from left to right, non-irradiated control,
progeny of M 2 plant with no visible change,
progenies of D-2, B-1, B-2, and C-2 plants.