Introduction
Tomato (Solanum lycopersicum
L.) is an important crop in terms of production and consumption (Maharatha et al.
2019). It is widely cultivated in tropical and subtropical regions of the world
(Sualeh et al. 2016; Yao et al.
2019). Conventional breeding has been applied to improve the yield and quality
of tomatoes for many years, severely limiting its genetic diversity (Das et al. 2019). One solution for breaking
the limitation is inducing effective mutations in the tomato genome (Cheng et al. 2019). Mutation breeding in crop plants is an effective tool for
introducing novel traits, especially in crops having narrow genetic base (Liman
et al. 2018). The effectiveness
of ethyl methane sulfonate (EMS) as a mutagen has been demonstrated in many
horticulture crops, including pepper (Cheng et
al. 2019), cucumber (Xue et al. 2016) and cabbage (Huang et
al. 2016). It can induce random point mutations with significant
effects on the coding or regulatory domain of key genes in plant tissues/organs by converting
complementary base pairing (G:C to A:T) ( Shirasawa et
al. 2016), which is important to overcome barriers of conventional breeding
and broaden the genetic background of crop plants.
Generally, EMS dose resulting in
the death of 50% seeds (50% lethality, LD50)
is an important indicator for evaluating the balance
between plant variation and seed germinability (Shah et al.
2015; Ke et al. 2019). Higher EMS doses raise the genetic
variations in plant tissues while inhibiting plant
development. In contrast, lower EMS doses result in a lower mutation frequency,
but ensures seed germinability (Shah et al. 2015). The effective dose of EMS
varies depending on the active level, presoaking time and method, plant species, and
temperature (Sayed et al. 2012;
Ruicheng et al. 2017). Therefore, it
is challenging to identify the effective LD50 dose of EMS.
Several genetic studies have
verified that all mutations cannot be identified with standard molecular
analysis in the first generation
due to no segregation of allelic genes (M1 generation) (Pasternak 2005; Shah et al. 2015). However, the mutation site is isolated during meiosis
in the M2 generation, creating recessive homozygotes (Dicenta et al.
2007). At same time, some mutants with stress resistance or phenotypic mutation
can be identified by stress treatments or phenotype screening. Mutants with
desired characters like stress resistance, plant height, leaf-color changes, or growth period can be selected in
the M2 generation (Espina et al. 2018; Cheng et al. 2019).
A few studies have identified many causal mutations in
tomato EMS populations. However, the response of the M1 generation Solanum lycopersicum L. to EMS during
seed germination remains unknown. This study aimed to explore physiological and
biological changes in seeds when exposed to EMS and
optimize EMS dose for inducing new mutants
in the tomato cultivar ‘Moneymaker’. Further, the induced effective mutations were screened in the M2
generation. This study will provide a basis
for germplasm innovation in tomatoes in the future.
Materials and Methods
Plant material
The experiment was performed using
the seeds of tomato cultivar ‘Moneymaker’ provided by the School of Agriculture, Ningxia University (W 106.1’ N
38.5’), Yinchuan, Ningxia 750021, P. R. China.
Experimental procedures
The experiment was conducted as described by Arisha
et al. (2014) with a few
modifications. The untreated tomato seeds (M0 seeds) were
soaked in water for 4 h at 28℃ and then treated with different doses of EMS (Table 1).
The treated seeds (M1 seeds) were
incubated at 28℃ on a shaker (110 rpm) for 12 h to
infiltrate EMS into seeds. The M1 seeds were transferred to
fume cupboard and washed with running water for 4 h to remove residual EMS on
seed surface. M1 seeds without residual EMS were incubated at 28℃ for germination and then sown into pots placed in
a chamber with a cycle of 16 h light/8 h dark. The control seeds were subjected
to the same conditions as the treated seeds except treated with water instead of EMS. Each treatment consisted
of three replicates, containing 200 seeds. Standard cultural practices were
performed uniformly during the growth of plants.
Generation analysis
M1 generation: The number of germinated M1
seeds was counted daily until the 7th day. Seeds with
visible radicles (1 mm in length) were considered to have germinated. The
number of surviving seedlings was recorded 7 days after the M1 seeds
sown in the pots. Germination percentage and survival ratio were calculated
according to formula:
%20332-340_files/image002.png){kind=small}
%20332-340_files/image004.png){kind=small}
The abnormal plants were counted 3 days after survival seedlings were
planted in greenhouse and the frequency of abnormal plants was calculated by
the formula:
%20332-340_files/image006.png){kind=small}
Furthermore, owing to the
importance of LD50 for mutations observed in M2. The
experiment was performed following a completely randomized design with three replicates.
M2 generation: All M2 seeds from the M1
individuals were harvested, mixed and germinated in a chamber at 28℃ for 7 days. After
germination, the survival M2 seedlings were distributed averagely into three groups. To evaluate the stress
resistance of the seedlings (4-leafed-6-leafed stage), each group was subjected
to either cold (4oC), salt (NaCl, 300 mM), or drought stress
(polyethylene glycol, PEG 15%, g/v) for 12 h. These seedlings were put in
triangular flasks with the above-mentioned solution. All mutations observed
were recorded throughout the entire period of life. In addition, the frequency
and segregation ratio of the mutant plants out of total number of seedlings in
the same M2 group was calculated according to the formula.
Measurement of physiological
parameters
Antioxidants activities and the malonaldehyde
(MDA) content: A total of 0.2 g seeds were sampled and ground in a mortar with 5 mL phosphate buffer to determine malondialdehyde (MDA) content and the
activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). These substances were quantified spectrophotometrically at optical
densities (ODs) and calculated as described by Dionisio-Sese and Tobita (1998).
Seed electrical conductivity: A total of 0.2 g seeds were sampled and immersed
in 10 mL ddH2O for 24 h. Afterwards, the seed conductivity was
measured using a Radiometer (Copenhagen) CDM80 conductivity meter with
automatic temperature correction to 25°C. The conductivity value was
calculated according to the method described by Sun et al. (2019).
RNA extraction and gene expression
Total RNA was isolated from the study of Meng and Feldman
(2010) using the Trizol method. Then, the first strand of cDNA was
synthesized using the SYBRGreenPCR Master Mix
(Takara, Bio, Japan). Primers were designed using PRIMER5 software (version 5.0, Premier
Company, Canada). qRT-PCR was performed on a qRT-PCR equipment (qTOWER3, Germany). The ubiquitin-conjugating gene SlUbi3 was used as the reference gene (Hoffman et al. 1991). Relative gene
expression was calculated using the 2−△△CT method (Xiaojie 2012).
Statistical
analysis
Data were analyzed using the SPSS
software (version 19.0, SPSS, Inc., USA). The analyzed data
are presented as means ± SE (standard error) of two replicates in all measured
parameters except for physiological and biological parameters, which was done
using three replicates. Statistical significance was inferred at P < 0.05.
Results
Germination percentage (%) and LD50 of M1 generation
The EMS dose of 0.25 and 0.5% decreased the number of germinated seed each day in comparison to control
(Fig. 1A–C). Interestingly, the
number of germinated seed each day was enhanced significantly along with increase of EMS dose, and reached to peak at 3rd
or 4th days (Fig. 1D–I).
The results of seed lethality under different EMS doses were also observed
in the study (Table 2). The EMS dose of 1.25% was an ideal concentration to screen tomato mutant due to 47.33% seed
lethality (LD50) The moderate or high EMS dose promoted the capacity
for seed germination, no significant difference was observed between the
control and EMS treatments of 0.25, 0.50, 0.75 and 1.00%. Only higher doses of
EMS (> 1.25%) resulted in the reduction in germination significantly, with
the seeds treated with 2.00% EMS dose
presenting the worst germinating capacity (17.33%). These findings show that
EMS toxicity only play a key role in inhibition of tomato seeds at high dose.
Physiological responses and gene
expression
The activities of antioxidants (SOD, POD and CAT) in seeds treated with
lower or moderate EMS doses (0.25, 0.50, 0.75, 1.00 and 1.25%) were higher or
equal to those treated with higher EMS doses (1.50, 1.75 and 2.00%) (Fig. 2A–D).
An opposite trend was observed in the MDA content (Fig. 2E). Interestingly, no
significant difference in the electrical conductivity of seeds was observed
among the treatment, even though it ranged from 61 to 72%. These data revealed
that EMS do not damage cells severely, and oppositely induce synthesis of
antioxidants.
The qRT-PCR assay showed that expression of SlGID1b was upregulated in seeds treated
with low or moderate EMS dose as comparison to the control (Fig. 3A). Similar
result was also observed in the expression of SlGID1ac gene (Fig. 3B). However, contrary results were observed in
the expression levels of SlABA1 gene:
The treatments with > 1.25 EMS dose possess the higher transcriptional
levels of SlABA1 gene than the
control Table 1: EMS treatments and its classification of tomato seeds
|
EMS concentration (%, v/v) |
Classification |
|
0 |
Low |
|
0.25 |
|
|
0.50 |
|
|
0.75 |
Moderate |
|
1.00 |
|
|
1.25 |
|
|
1.50 |
High |
|
1.75 |
|
|
2.00 |
Table 2: Percentage of germinated seeds with EMS treatments
|
EMS concentration (%, v/v) |
Percentage of germinated seeds (%) |
|
0 |
90.00 ± 17.32a |
|
0.25 |
89.33 ± 5.50a |
|
0.50 |
94.67 ± 9.23a |
|
0.75 |
89.67 ± 8.96a |
|
1.00 |
85.67 ± 4.16a |
|
1.25 |
47.33 ± 12.70b |
|
1.50 |
41.00 ± 3.46b |
|
1.75 |
21.00 ± 6.24c |
|
2.00 |
17.33 ± 9.29c |
The error represents SD for three biological replicates,
and the lowercases showed the significant level at P < 0.05.
Table 3: Survival ratio and the frequency of abnormal plants observed in M1
as comparison to the control
|
EMS dose (%) |
Survival ratio (%) |
Frequency (%) |
|
0.00 |
91.21 |
0.00 |
|
0.25 |
100.00 |
0.00 |
|
0.50 |
92.20 |
0.00 |
|
0.75 |
87.53 |
10.08 |
|
1.00 |
100.00 |
11.30 |
|
1.25 |
83.11 |
23.21 |
|
1.50 |
100.00 |
10.69 |
|
1.75 |
80.30 |
12.11 |
|
2.00 |
70.05 |
13.09 |
M1: the first generation
(Fig. 3C).
Survival and phenotype of the M1
generation
It was found that EMS injury to seed
passed on to seedlings. The survival ratio of seedlings only
had a slight decrease, but frequency in abnormal seedlings was increased
significantly among these treatments with EMS compared with the control (Table 3). The tendency was more and more obvious along with
the increase in EMS dose, and reached to optimal effect when EMS dose was up to
1.25% which ensured not only > 80% survival ratio but also > 20% the
frequency of abnormal plants observed. Some abnormal seedlings or plants
observed in the M1 population, including some types such as cotyledon deformity (Fig. 4A), purple
stem, yellow leaf (Fig. 4B), abnormal lateral branching and fascicular terminal bud (Fig. 4C).
M2
generation observation
EMS induced point mutation did not
cease in the M1 generation but inherited to M2 generation
because under normal conditions the growth in the M2 generation was significantly worse than control. To obtain
stress-resistant mutants from the M2 generation, all individuals
distributed in the three groups with either cold, salt, or drought stress,
%20332-340_files/image008.jpg)
Fig. 1: The speed of seed
germination in tomato as comparison to the control in the 7 days. Percentage of
germinated seeds treated by EMS (A)
0, (B) 0.25 %, (C) 0.50 %, (D) 0.75 %, (E) 1.00 %, (F) 1.25 %, (G) 1.50 %, (H) 1.75 %, and (I) 2.00 %Tomato seeds were treated with different EMS doses in
phosphate buffer solution (pH7.0) after soaked in water for 4 h at 20℃
%20332-340_files/image010.jpg)
Fig. 2: Measurement of
physiological parameters in the treatments treated by EMS dose. (A) SOD: superoxide diamutase;
(B) POD: peroxidase; (C) CAT: catalase and (D) seed conductivity. The experiment
was conducted with three biological replicates and each replicate contained 0.2
g tomato seeds. The error bars represent SD for three biological replicates,
and the lowercases showed the significant level at P < 0.05
Table 4: Mutation frequency and segregation in mutants observed
|
|
Types of mutants |
No. of mutants |
Mutation frequency
(%) |
Segregation ratio |
|
The first family |
Cold resistance |
4 |
11.1 |
8:1 |
|
The second family |
Salt resistance |
3 |
4.1 |
23:1 |
|
The third family |
Drought resistance |
1 |
1.3 |
72:1 |
All M2 seedlings are
distributed in three families, and these individuals were treated by cold, salt
and drought respectively at the 4-leafed-6-leafed stage
%20332-340_files/image012.jpg)
Fig. 3: Analysis of qRT-PCR in expression of key genes involved in hormone
levels. Transcriptional levels of (A)
SlGID1b, (B) SlGID1ac and (C) SlABA1
genes in seeds of different treatments. The expression of these genes were
normalized by that of ubiquitin-con-jugating protein
gene SlUBI3. The experiment was conducted with three biological replicates and each
replicate contained 0.2 g seeds. The error bars represent ± SD for three
biological replicates, and the lowercases showed the significant level at P < 0.05
%20332-340_files/image014.png)
Fig. 4: Abnormal growth
in the first generation (M1) as comparison to the control (Normal
seedling). Observation of phenotype in the M1 generation at the
stage of (A) seedling, (B) vegetative growth and (C) productive growth. The phenotype
observed was recorded at the cotyledon stage, 10 days and 30 days after
tomatoes were planted in greenhouse, respectively
respectively. Among the 72 plants in the cold treatment group,
eight individuals resisting to cold stress was observed with segregation ratio
8:1 (Table 4). Among salt treatment group, only three salt-resistant
seedlings were observed, which exhibited a mutation frequency of 4.1% and a
segregation ratio of 23:1. In addition, one plant with a distinct mutant phenotype exhibited drought
resistance in the drought-stress treatment group which showed a 1.3% mutation frequency and segregation ratio of 1:72 (Named ‘dr-1’).
Most of the seedlings treated by
cold stress died after being planted in the greenhouse, while only one seedling
(named ‘cr-1’) with purple leaf and stem phenotype survived to
maturity stage as comparison to the control (Fig. 5A). Although, there was no difference in phenotype
between ‘cr-1’ and the control at
maturity stage, but ‘cr-1’ has less fruits than the control 120 days
after sown (Fig. 6A). Furthermore, in the salt
treatment, three types of phenotypic mutants with salt resistance were
observed, including ‘sr-1’, ‘sr-2’, and ‘sr-3’. Only ‘sr-3’ exhibited better growth at the seedling stage when compared with the control
(complete wilting leaves) (Fig. 5B). The three mutants can complete their life
cycle well, but ‘sr-3’ possessed more fruits than the control 120 days after sown
(Fig. 6B). In the drought treatment group, only one mutant (dr-1’) survived. At the seedling stage, its leaves not wilted, and its growth was
significantly better than the control under drought stress (Fig. 5C). At
maturity, the surviving plant also exhibited differences in growth behavior,
distinguishing it from the control. Especially, ‘dr-1’showed a phenotype
of dwarfism with the characteristic of < 100 cm height and less fruit number
(Table 5 and Fig. 6C).
Discussion
‘Moneymaker’ is a high-yielding tomato cultivar with little resistance
with origin of the Netherlands and bred for field production (Koornneef and Hanhart 1990).
However, the cultivar lacks essential stress resistance genes in its genome,
making it easily threatened by various adverse factors. Given its high fruit
number and susceptibility to adverse environmental conditions, ‘Moneymaker’ is
a suitable candidate for large-scale mutant screening for stress resistance.
Thus, it was selected as a model plant for studying EMS-induced mutagenesis in
this study.
Many studies have focused on the mutagenesis effect of EMS but not the
promotive influence on seed germination. Previous studies reported that the
mutagen must permeate the germinating embryo and reach the meristemic
region if it works during seed germination (Arisha et al. 2014). Therefore, it is no doubt
that the toxic impacts of EMS on the entire seed biology result in low seed
germination by damage of cell constituents, alteration of enzyme activity or
delay, and inhibition of other physiological and biological processes (Talebi 2012; Kumar et al. 2013). However, in present study,
seeds treated by lower or moderate EMS dose exhibited a good germinability as
comparison to the control by promoting accumulation of antioxidants or
expression of key genes. The immune and defense system of plants are triggered
under environmental stress, promoting the metabolism of substances that react
to the injury caused by the adverse factors, but this is not main factors. The
phenomenon may primarily be attributed to EMS inducing point-mutation of negative
regulatory genes such as DAG1 (Gabriele
et al. 2010), RGL2 (Lee et
al. 2002), ACC (Naing et al.
2021) related during seed germination. Mutation of these negative regulatory
genes loss their inhibited function during seed germination, thereby increasing
levels of antioxidants activity or activating expression of key genes like SlGID1b and SlGID1ac. However, this speculation based on the results of
present experiment. The mechanism remains to be unclear and should be studied
further.
Previous studies postulate that LD50 is a key factor in
building mutant library of plant (Nascimento et al. 2015). Selecting an
effective and efficient LD50 in mutation breeding programs is
essential in producing a high frequency of desirable mutations. Yong et al. (2021) evaluated the effects of
0.5% EMS on tomato ‘Improved Apollo’ cultivar and found that it could enhance
the mutation frequency. Similar results were also reported by Just et al. (2013), who found that 0.5% EMS
could enhance the mean mutation frequency (one mutation per 1,710 kb) in tomato
cultivar ‘Micro-Tom’. However, in this study, the ideal EMS dose for mutation
breeding in tomato cultivar ‘Moneymaker’ was 1.25% and not 0.5%, which
contradicts the previous reports in tomato (Gavazzi et al. 1987; Shalaby and El-Banna 2013). The inconsistency in the
results could be due to differences in genetic background and the methods
employed by various studies, including variations in presoaking, treatment
time, temperature, and the pH of the tomato seeds (Arisha
et al. 2014). The findings of present
study will inform future efforts geared towards the building of mutant library
of tomato ‘Moneymaker’ cultivar.
Mutations in the M2
generation are usually considered more genetically stable. In this study, three
mutation types for specific stress resistance (cold, salt and drought) were
observed during the M2 generation. The visible mutation observed in one tomato plant that exhibited a uniform
purple color in the leaf and stem, characterizing cold resistance. The plant
(named ‘cr-1’) with this mutation
survived to maturity. Previous investigations have shown that a significant
change in anthocyanin accumulation typically results in plant color variation (Cheng
et al. 2019), consistent with our
study. Although its growth is not better than the control, it is significant
and crucial for identifying genes that regulate cold stress or anthocyanins
synthesis. Furthermore, three tomato
mutants with superior salt resistance than the control were also observed in
the M2 generation, and only one mutant ‘sr-3’possessed more fruits than the control. ‘sr-3’is a positive mutation caused by EMS, different from negative mutation
which has to be
%20332-340_files/image016.png)
Fig. 5: Mutants with resistance stress
screened in the M2 generation. (A)
‘cr-1’ mutant with cold resistance, (B) mutants with salt resistance, and (C) ‘dr-1’
mutant with drought resistance. All M2 individuals were distributed into 3
families, and individuals from each family were treated by 4℃, 300 mM NaCl and 0.5 % polyethylene glycol (PEG) at the seedling stage,
and planted in greenhouse until maturity
%20332-340_files/image018.png)
Fig. 6: Phenotype in
mutants screened in the M2
generation at different growth stages (A)
The control, (B) ‘cr-1’, (C) ‘sr-1’, ‘sr-2’, ‘sr-3’and (D) ‘dr-1’. These mutants were investigated
at 100 and 120 days after sown
%20332-340_files/image019.png)
crossed or modified genetically to
be utilized in breeding program. This finding will provide an important genetic
resource for improvement of tomato yield in the future. Also, one mutant exhibited drought resistance and
survived until maturity under drought stress. Mutants with drought resistance
in tomatoes are crucial to understanding the regulatory mechanisms for plant
growth and development (Lamin-Samu et al. 2021). All mutations observed in
this study are not consistent with the classic Mendelian model. The unusual
segregation ratio might be because the altered traits are controlled by
multiple genes, interacting to give the observed mutation. Besides, the unusual
segregation ratios may be because of the fewer number of individuals in the
mutant population, which was not enough to
result in the usual segregation ratio.
Conclusion
This study showed the regulatory effect of EMS on seed germination in tomato cultivar
‘Moneymaker’ and found that low or moderate EMS
dose enhanced the capacity for seed germination by accumulation of antioxidants
and expression of key genes during seed germination. The EMS dose of
1.25% yielded 50% lethality (LD50) of seeds, serving as a crucial
indicator for building mutant library of tomatoes. The effect of EMS on ‘Moneymaker’ did not stop in the M1 generation but continued to the M2
generation. Various stress resistance-related mutants with heritable changes, including ‘cr-1’, ‘sr-3’, ‘dr-1’, were observed in the M2 generation. A detailed understanding of the molecular basis underlying the desired
agronomic traits of tomatoes is necessary for broadening the genetic background
of tomatoes.
Acknowledgments
This work was supported through funding from the National
Key Research and Development Program of China (No.
2021YFD1600302), the Natural Science Foundation of Ningxia (No.
2020AAC03092 and No. 2022AAC05023), the Major science and technology projects
of Ningxia (No. NXNYYZ202001 and No. 2021BBF02024) and the National Natural
Science Foundation of China (No. 31860561).
Author
Contributions
Guo-Xin Cheng
contributed to the conception of the study; Yu-Jing Liu performed the
experiment; Guo-Hua Li, Sheng-Yi Bai, and Fu-Shun Zheng contributed
significantly to analysis and manuscript preparation; Peng-Ze Zhou, and
Hong-Lei Li performed the data analyses and wrote the manuscript; Xiao-Min Wang
helped perform the analysis with constructive discussions.
Conflicts of
Interest
No competing interest
Data Availability
All data was
generated or used during the study appear in the submitted article.
Ethics
Approvals
None
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