全 文 ：不同浓度 EMS 对苦荞种子发
芽的影响
温日宇 1*，郭耀东 1，刘建霞 2，郑联寿 1，樊丽生 1，
武小平 1 （1.山西省农业科学院玉米研究所，
山西忻州 034000；2.大同大学农学与生命科学
院，山西大同 037009）
摘 要 [目的] 确定荞麦种子发芽的适宜甲
基磺酸乙酯（EMS）诱变剂量，为进一步获得荞
麦优良性状并培育符合 EMS 诱变育种需求的
新品种打下坚实的基础。 [方法] 以山西省当
地的苦荞 [Fagopyrum tataricum（L.）Gaertn] 品
种晋荞麦 4 号种子为材料， 用不同浓度梯度
（0.3%、0.5%、0.7%、1.0%、1.5%、1.7%）EMS 诱
变处理不同时间（4、8、12 h），分析苦荞种子发
芽率和发芽势。[结果]随着 EMS 诱变剂浓度的
增加， 晋荞麦 4 号种子发芽势、相对发芽势和
发芽率、相对发芽率均呈下降趋势，当 EMS 浓
度为 0.3%～1.0%时与对照没有显著差异 ，当
EMS 浓度为 1.5%和 1.7%时与对照差异极显
著 EMS 处理 48 h 对晋荞麦 4 号种子发芽率、
相对发芽率都没有大的影响， 当处理时间为
12 h 时，与对照有显著差异；对于发芽势和相
对发芽势，这 3 个处理时间没有显著差异。 [结
论 ] EMS 诱变苦荞的半致死的适宜浓度为
1.7%，适宜的处理时间为 12 h。
关键词 苦荞；EMS；发芽率；发芽势
基金项目 山西省农业科学院玉米研究所攻
关项目（SGG2014-3）。
作者简介 温日宇（1976-），男，山西朔州人，
助理研究员， 主要从事荞麦育种与栽培工作，
E-mail：riyuwen@163.com。 * 通讯作者。
收稿日期 2015-01-10
修回日期 2015-03-13
Effects of Different-concentration EMS on
Germination of Tarary Buckwheat [Fagopyrum
tataricum (L.) Gaertn] Seeds
Riyu WEN1*, Yaodong GUO1, Jianxia LIU2, Lianshou ZHENG1, Lisheng FAN1, Xiaoping WU1
1. Maize Research Institute, Shanxi Academy of Agricultural Sciences, Xinzhou 034000, China;
2. College of Agriculture and Life Sciences, Datong University, Datong 037009, China
Supported by Research Project of Maize Research Institute of Shanxi Academy of
Agricultural Sciences (SGG2014-3).
*Corresponding author. E-mail: riyuwen@163.com
Received: January 10, 2015 Accepted: March 13, 2015A
Agricultural Science & Technology, 2015, 16(5): 877-879
Copyright訫 2015, Information Institute of HAAS. All rights reserved Agronomy and Horticultrue
A t current, the studies on tararybuckwheat mainly focus ongenetic diversity, taxonomy
and other basic aspects of biology [1].
The studies on breeding of tarary
buckwheat are extremely important.
Among them, both the traditional and
modern breeding techniques all play
very important roles. Reproductive
problem is one of the important limiting
factors for improvement of tarary
buckwheat yield. Therefore, how to
overcome these adverse features,
such as breeding excellent traits using
EMS mutagenesis, will be the key to
make outstanding progresses in
breeding of tarary buckwheat.
The selection of mutagenic parent
material and position is an important
part of EMS mutation breeding. Gen-
erally, the organs that are easy to be
treated and sensitive to mutagenesis
are selected as mutagenic positions.
Seed is the earliest and most com-
monly used material for EMS mutage-
nesis. Although the seed mutagenesis
by EMS is restricted by many factors,
it still has huge advantages that other
materials do not have[2]. Seed is an in-
dependent organ of plant, and it can
maintain its vitality and preserve the
mutagenized gene for a long time
across the mutagenesis treatment [3].
Seed mutagenesis is not needed to be
performed under aseptic conditions,
which largely reducing the processing
Abstract [Objective] This study aimed to determine the optimum ethyl methane sul-
fonate (EMS) concentration for germination of tarary buckwheat seeds so as to lay
a solid foundation for obtaining excellent traits of tarary buckwheat and breeding
new varieties that meet the requirements by EMS mutation breeding. [Method] The
seeds of Jinqiaomai No.4, a local tarary buckwheat variety [Fagopyrum tataricum
(L.) Gaertn] in Shanxi Province, were used as the material. They were mutagenized
by different concentrations (0.3%, 0.5%, 0.7%, 1.0%, 1.5%, 1.7%) of EMS for differ-
ent times (4, 8, 12 h). Then the germination rate and germination vigor were ana-
lyzed. [Result] With the increase of EMS mutagenic agent concentration, the germi-
nation vigor, relative germination vigor, germination rate and relative germination rate
are all trended to be decreased. When the EMS concentrations ranged from 0.3%
to 1.0%, there were no significant differences between treatment and control groups
(P>0.05). When the EMS concentrations were 1.5% and 1.7%, significant differences
were shown between treatment and control groups (P<0.01). The 4 and 8 h treat-
ment of EMS all had no significant effects on germination rate and relative germina-
tion rate of Jinqiaomai No.4, but the 12 h treatment of EMS showed significant ef-
fects. Among the three treatment times, there were still no significant differences in
germination vigor and relative germination vigor between treatment and control
groups. [Conclusion] The optimum median lethal dose and treatment time of EMS
for mutagenizing Jinqiaomai No.4 were 1.7% and 12 h, respectively.
Key words Tarary buckwheat; EMS; Germination rate; Germination vigor
DOI:10.16175/j.cnki.1009-4229.2015.05.005
Agricultural Science & Technology 2015
steps. Although the effect of seed mu-
tagenesis can not be shown immedi-
ately and some of the traits will even
be expressed in the second and third
generation, the new traits obtained
from mutagenesis can still be passed
on to offspring, omitting the subse-
quent identification works. To this end,
the seeds of tarary buckwheat were
used as the material for EMS mutage-
nesis in this study so as to investigate
the effects of different-concentration
EMS on germination of tarary buck-
wheat seeds.
Material and Methods
Material
Plant material The seeds of Jin-
qiaomai No.4, a local tarary buck-
wheat variety in Shanxi Province,
were provided by the Crop Research
Institute of Shanxi Academy of Agri-
cultural Sciences.
Reagents (1) EMS mother liquor.
Ethyl methane sulfonate (EMS), with
purity >99%, was purchased from the
Beijing Solarbio Science & Technology
Co., Ltd.
(2) Phosphate buffer. The phos-
phate buffer (0.2 mol/L, pH 7.0) was
prepared by mixing disodium hydro-
gen phosphate (Na2HPO4, 0.2 mol/L)
and sodium dihydrogen phosphate
(NaH2PO4, 0.2 mol/L) (v/v, 61/39).
(3) Sodium thiosulfate solution.
Certain amount (24.8 g) of sodium
thiosulfate (NaS2O3·5H2O, 0.1 mol/L)
was weighed and dissolved in 500 ml
of cooled boiling water. Subsequently,
certain amount (0.2 g) of Na2CO3 and
500 ml of cooled boiling water were
added.
(4) Different-concentration EMS
solutions. The different-concentration
EMS solutions were prepared as
shown in Table 1.
Methods
Treatment of tarary buckwheat by
EMS (1) EMS concentrations and
mutagenesis times. The concentra-
tions of EMS were arranged as 0.3%,
0.5% , 0.7% , 1.0% , 1.5% and 1.7% .
The seeds of Jinqiaomai No.4 were
treated for 4, 8 and 12 h, respectively.
Thus a total of 21 treatments were ar-
ranged. The seeds of Jinqiaomai No.4
that were not treated with EMS were
treated as control (CK).
(2) Seed selection and soaking.
The seeds were soaked in a breaker
with water and stirred moderately. The
empty seeds, broken seeds, grass
seeds and impurities floating on the
water surface were removed. Then a
total of 5 000 large, plump and size-
uniform tarary buckwheat seeds were
selected so that the germination rate
and germination vigor would be im-
proved. The selected seeds were
soaked in clear water at 25℃ for 14 h.
(3) Treatment of seeds by EMS.
The selected seeds were distributed
evenly in 24 labeled dishes, including
21 treatments and 3 controls. A total
of 100 tarary buckwheat seeds were
placed in each of the 24 dishes. Sub-
sequently, 13 ml of EMS solution at a
certain concentration was added to
each of the 21 dishes. Correspond-
ingly, 13 ml of clear water was added
to each of the three controls. Similar-
ly, another 24 dishes were arranged
as above. All the dishes were placed
in a constant temperature incubator
(25 ℃).
(4) Termination of mutagenesis
treatment. After the treatment, the re-
maining EMS in the dishes was
drained. Subsequently, 10 ml of
Na2S2O3 (0.1 mol/L) was added to
each of the dishes to rinse the seeds.
The rinsing was repeated three times.
After the three times of washing with
Na2S2O3, the seeds will be rinsed with
clean water. The seeds were then
transferred in a new dish laid with two-
layer filer paper. The seeds were
evenly distributed in the new dishes.
After adding certain amount of water,
all the dishes were placed in a con-
stant temperature incubator (25℃).
(5) Transplanting of germinated
seeds. After the seeds germinated,
they were all transplanted in soil.
Indicator measurement and meth-
ods Germination rate = Number of
germinated seeds within 7 days/Test-
ed seeds×100%;
Relative germination rate = Num-
ber of germinated seeds in treatment
groups within 7 days/Number of ger-
minated seeds in control groups within
7 days×100%;
Germination vigor = Number of
germinated seeds within 3 days/Test-
ed seeds×100%;
Relative germination vigor =
Number of germinated seeds in treat-
ment groups within 3 days/Number of
germinated seeds in control groups
within 3 days×100%.
Data statistics and analysis
Multiple comparisons of various
factors were performed with Duncan’s
new multiple range method using
SPSS software.
Table 1 Preparation of different-concentration EMS solutions ml
EMS concentration∥% EMS mother liquor Phosphate buffer
0.3 0.3 99.7
0.5 0.5 99.5
0.7 0.7 99.3
1.0 1.0 99.0
1.5 1.5 98.5
1.7 1.7 98.3
2.0 2.0 98.0
Table 2 Effects of different-concentration EMS on germination of Jinqiaomai No.4
EMS concentra-
tion∥%
Germination
vigor∥%
Relative
germination vigor
Germination
rate∥%
Relative
germination rate
0 (CK) 85.0 aA 1.00 aA 96.0 aA 1.00 aA
0.3 77.7 aA 0.91 aA 89.7 abA 0.94 abA
0.7 76.3 aA 0.90 aA 89.3 abA 0.93 abA
0.5 74.7 aA 0.88 aAB 88.7 abA 0.93 abA
1.0 68.7 aAB 0.81 aAB 74.3 bcB 0.78 bcB
1.5 46.7 bBC 0.55 bB 68.0 cB 0.71 cBC
1.7 27.3 cC 0 cC 45.3 dC 0.47 dC
Different lowercase letters in the same column indicated significant differences at the 0.05
level; different capital letters in the same column indicated significant differences at the
0.01 level.
878
Agricultural Science & Technology2015
Responsible editor: Tingting XU Responsible proofreader: Xiaoyan WU
Results and Analysis
Effects of EMS on germination of
Jinqiaomai No.4 seeds
Effects of different-concentration
EMS on germination of Jinqiaomai
No.4 seeds Table 1 showed that
with the increase of EMS mutagenic
agent concentration, the germination
vigor and germination rate of Jinqiao-
mai No.4 all trended to be decreased.
When the concentrations of EMS
ranged from 0.3% to 1.0%, there were
no significant differences between
treatment and control groups; but
when the concentration of EMS
reached 1.5% and 1.7%, the germina-
tion vigors of Jinqiaomai No.4 were de-
creased from 85.0% (CK) to 46.7%
(P<0.01) and 27.3% (P<0.01), and the
germination rates were reduced from
96.0% (CK) to 68.0% (P<0.01) and
45.3% (P<0.01). It was indicated that
1.5% and 1.7% of EMS had significant
inhibiting effects on germination vigor
and germination rate of Jinqiaomai
No.4.
At the same time, with the in-
crease of EMS concentration, the rel-
ative germination rate and relative
germination vigor of Jinqiaomai No.4
were all trended to be decreased.
When the EMS concentrations ranged
from 0.3% to 1.0%, no significant dif-
ference was found in relative germina-
tion vigor between treatment and con-
trol groups; but when the EMS con-
centration was increased to 1.5%, the
relative germination vigor of Jinqiao-
mai No.4 seeds was decreased from
1.00 (CK) to 0.55 (P<0.01). When the
EMS concentrations ranged from 0.3%
to 1.0%, there were no significant dif-
ferences in relative germination rate of
Jinqiaomai No.4 seeds between treat-
ment and control groups, but when the
EMS concentrations reached 1.5%
and 1.7% , the relative germination
rates of Jinqiaomai No.4 seeds were
decreased from 1,00 (CK) to 0.71 and
0.47. Therefore, 1.7% was considered
to be the optimum concentration of
EMS for mutagenesis of tarary buck-
wheat.
Effects of different treatment times
on germination of Jinqiaomai No.4
seeds As shown in Table 2, when
the treatment times of EMS were 4 h
and 8 h, there were no significant dif-
ferences in germination rate and rela-
tive germination rate of Jinqiaomai No.
4 seeds between treatment and con-
trol groups; but when the treatment
time was increased to 12 h, the differ-
ences in germination rate and relative
germination rate reached the signifi-
cant levels. However, there were no
significant differences in germination
vigor and relative germination vigor be-
tween treatment and control groups a-
mong the three treatment times.
Conclusions and Discus-
sion
The determination of optimum
concentration of mutagenic agent is
the key for success of mutation
breeding. Different plants require dif-
ferent concentration ranges of certain
mutagenic agent. Only within the opti-
mum concentration range of muta-
genic agent can species maintain their
good traits and generate more new
mutation[4-5].
In this study, the seeds of Jin-
qiaomai No.4, a local tarary buckwheat
variety in Shanxi Province, were used
as the material. They were mutage-
nized with different -concentration
EMS for different time. The results
showed that EMS had significant ef-
fects on germination rate of Jinqiaomai
No.4. When the concentration of EMS
was increased to 1.7% , the relative
germination rate was decreased to
0.46, close to 0.50. Therefore, 1.7%
was considered to be the median
lethal concentration, and was also con-
sidered to the optimum concentration
for mutagenesis[4]. In addition, the mu-
tagenic time should be best more than
8 h.
The control of bacterial infection
was also another important aspect of
this study. During the test, the germi-
nation of tarary buckwheat is easily
infected with microorganisms. The in-
cubating environment is suitable for
growth and propagation of microor-
ganisms, affecting the normal germi-
nation of tarary buckwheat seeds. So
before the starting of the test, the
dishes must be disinfected thoroughly.
In addition, the process of data obser-
vation and statistics also needs to pre-
vent bacterial infection.
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Table 3 Effects of different treatment times on germination of Jinqiaomai No.4
Treatment
time∥h
Germination
vigor∥%
Relative
germination vigor
Germination
rate∥%
Relative germination
rate
4 71.1 aA 0.78 aA 84.7 aA 0.88 aA
8 64.0 aA 0.73 aA 78.1 abA 0.82 abA
12 60.4 aA 0.65 aA 73.4 bA 0.76 bA
Different lowercase letters in the same column indicated significant differences at the 0.05
level; different capital letters in the same column indicated significant differences at the
0.01 level.
879