BANGLADESH RESEARCH PUBLICATIONS JOURNAL

ISSN: 1998-2003, Volume: 10, Issue: 2, Page: 214-222, July - October, 2014

SELECTION OF EMS INDUCED TOMATO VARIANTS THROUGH TILLING

FOR POINT MUTATION

A.S.M. Nahiyan1, L. Rahman1, S. Raiyan2, H. Mehraj3 and AFM Jamal Uddin3*

A.S.M. Nahiyan, L. Rahman, S. Raiyan, H. Mehraj and AFM Jamal Uddin (2014). Selection of EMS Induced Tomato
Variants through Tilling for Point Mutation. Bangladesh Res. Pub. J. 10(2): 214-222. Retrieve from
http://www.bdresearchpublications.com/admin/journal/upload/1410027/1410027.pdf

Abstract
An experiment was conducted at Horticulture farm, Sher-e-Bangla Agricultural
University, Bangladesh to create EMS induced new variants of tomato through
TILLING for point mutations. Mutation induction is considered as an effective way to
enrich plant genetic variation. Seeds of two tomato lines (LINE-1 and LINE-2) were
treated with different concentrations (0.0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%
and 1.0%) of chemical mutagen EMS. Tomato plants showed wide range of
variation concerning their studied characters. Among 216 plants 27 plants were
selected having variations in traits concerning crop duration, yield contributing
characters and shelf life the requirement of the farmer’s yield and customer choice.
Number of fruit, individual fruit weight, yield/plant and shelf life of the 27 plants
ranged from 51.0-104, 41.0-80.4 g, 2.1-4.5 kg and 8.0-31.0 days respectively.

Key words: Tomato, EMS, Tilling, Point mutations, Variants.

Introduction
Mutagenesis has been widely used as a potent method of enhancing variability for crop
improvement (Subuthi et al., 1991) and has brought a lot of benefits in the modern
agricultural production as a method for crops improvement and addition of new valuable
traits into the existing varieties (Ahloowalia et al., 2001). The use of induced mutations for
the improvement of crop plants has yields several mutants which have been used directly
as new cultivars (Gottschalk and Wolf, 1983). Mutation is accomplished by chemical or
physical treatments followed by selection for heritable changes of specific genotypes
(Micke et al., 1985). Alkylating agents were the first class of chemical mutagens
(Auerbach and Robson, 1946). Alkylating agents such as mustard gas, Methyl Methane
Sulphonate (MMS), Ethyl Methane Sulphonate (EMS) and nitrosoguanidine have several
effects on DNA. The alkyl group of an alkylating agent reacts with DNA which may lead to
a change in the nucleotide sequence and hence leads to point mutation (like C-to-T
changes) as well as loss of chromosome segments or deletions (Alcantara et al., 1996).
EMS is more effective than physical mutagens (Dhanayanth and Reddy, 2000; Bhat et al.,
2005). It can proficiently induce chemical modification of nucleotides, which results in
various point mutations such as nonsense, missense and silent mutations (McCallum et al.,
2000a and 2000b). EMS is a potent chemical mutagen that has been found to be
mutagenic in wide a variety of genetic test systems. EMS reacts with DNA in variety of
ways, a broad spectrum of mutagenic effects are manifested in the population. Because
of its potency and ease with which it can be used, EMS is the most commonly used
chemical mutagen in plants. Tomato (Solanum lycopersicum) belongs to Solanaceae
family is one of the most important vegetable plants in the world. Its fruits are end
products both for the fresh market and food processing industry. A large number of
mutations in plants and cultivars have been achieved by the use of EMS, e.g. resistance to
herbicides (Jander et al., 2003) and male sterility (Van der Veen and Wirtz, 1968). In
tomato, EMS showed very high efficiency by inducing different morphological and

*Corresponding Author Email: jamal4@yahoo.com

1Advanced Seed Research and Biotech Center, ACI Limited, Dhaka
2International University of Business Agriculture technology, Dhaka-1230, Bangladesh
3Department of horticulture, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh
Selection of EMS Induced Tomato Variants 215

functional mutations (Watanabe et al., 2007). In recent years, TILLING (Targeting Induced
Local Lesions IN Genomes) (Colbert et al., 2001 and McCallum et al., 2000a and 2000b) a
new emerging technology that doesn't rely on genetic transformation techniques, allows
systematic functional genomic studies. Therefore TILLING strategy was applied to tomato
for the development of tomato lines carrying desirable traits of demand.
Materials and Method
Experiment was conducted at Horticultural Farm under 2abiotech, Sher-e-Bangla
Agricultural University, Dhaka, Bangladesh during October 2013 to April 2014. Tomato
seeds of (LINE-2 and LINE-1 cultivars which were selected in previous year) were treated
by EMS @ 0.0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7% and 1.0% for point mutations. EMS
treatments were done at Advance Seed Research & Biotech Centre (ASRBC), ACI limited.
15 seeds were treated in each percentage and after that treated seeds were sown in the
seed bed at 14th November, 2013. After taking the data of germination percentage,
seedlings were transplanted on the main field at 10th December, 2013. Manures and
fertilizers were applied as recommended by Bangladesh Agricultural Research Institute
(Mondal et al., 2011). The plant spacing was 40 cm and 60 cm in rows. Each
concentration of EMS treatments of both varieties contained 12 plants and each plant
was considered as an individual treatment in main field. Firstly these 12 plants were
marked by numerically from 1 to 12. Data were taken from each plant on plant height,
crop duration, number of fruit, individual fruit weight, fruit length, fruit diameter, yield/plant
and shelf life. Plants which were not able to provide all of the desirable characters
including shape, size, uniformity and disease infected were discarded. Plants which
fulfilled the demand of traits (Table 1) and varied from 0.0% EMS concentration were
selected as mutagenic tomato cultivars. After that selected plats were marked by using
(a), (b), (c), (d) etc. and plant which did not fulfill the demand of traits (Table 1) were
discarded. Data of the discarded plats were not shown.
Table 1. Demand of traits for tomato to achieve the selected goal
Plant Yield Shelf
Varieties Duration Fruit Single fruit
height (kg) life Fruit shape
(Days) number weight (g)
(cm) /plant (Days)
Square/round/o
blong/ellipsoid/r
Ranges 70-120 80-120 ≥ 50 ≥ 40 ≥2 ≥7
ectangular/ox-
heart
Goal Fulfill the requirement of the farmer’s yield and customer choice

Results
Plant height: Plant height of EMS treated selected plants LINE-1 and LINE-2 was
ranged in 75.0-118.0 cm and 75.0-91.0 cm respectively (Table 2) which follow the demand
of traits (Table 1). In both LINE-1 and LINE-2, all plants were found to increase in plant
height as compared to LINE-1 0.0% (68.9 cm) and LINE-2 0.0% (66.2 cm) due to application
of different concentrated EMS (Table 2).
Crop duration: Crop duration of EMS treated selected plants LINE-1 and LINE-2 was
ranged in 92.0-111.0 days and 89.0-117.0 days respectively (Table 2) which follow the
demand of traits (Table 1). In both LINE-1 and LINE-2, plants were tends to earlier in crop
duration as compared to LINE-1 0.0% (133.7 days) and LINE-2 0.0% (121.6 days) (Table 2).
Fruit length: Fruit length of both EMS treated selected plants LINE-1 and LINE-2 was
varied in each treatments and it was increased or decreased as compared LINE-1 0.0%
(50.9 mm) and LINE-2 0.0% (49.6 mm) (Table 3). Fruit length was ranged from 42.5-54.3 mm
and 47.4-56.9 mm in LINE-1 and LINE-2 treated with different concentrated EMS.
Fruit diameter: Fruit diameter of both EMS treated selected plants LINE-1 and LINE-2
was varied in each treatments and it was increased or decreased as compared to the
LINE-1 0.0% (46.5 mm) and LINE-2 0.0% (50.4 mm) (Table 3). Fruit diameter was ranged from
41.5-48.4 mm and 41.8-54.1 mm in LINE-1 and LINE-2 treated with different concentrated
EMS.

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Nahiyan et al. 216
Number of fruits: Number of fruits of EMS treated selected plants LINE-1 and LINE-2
was ranged in 53.0-104.0 and 51.0-149.0 respectively (Table 4) which followed the
demand of traits (Table 1). In both LINE-1 and LINE-2, all plants were found to increase in
number of fruits as compared to LINE-1 0.0% (33.0) and LINE-2 0.0% (39.4) due to
application of different concentrated EMS (Table 4).
Table 2. Performance of selected EMS treated tomato plants for plant height and crop
duration
Plant height (cm) at 70 DAT Crop duration (Days)
EMS treatments
(a) (b) (c) (d) (a) (b) (c) (d)
LINE-1 0.1% 105.0 92.0 - - 92.0 95.0 - -
LINE-1 0.2% 101.0 104.0 102.0 - 97.0 90.0 99.0
LINE-1 0.3% 102.0 118.0 114.0 76.0 108.0 93.0 102.0 106.0
LINE-1 0.4% 75.0 - - - 104.0 - - -
LINE-1 0.5% 97.0 - - - 96.0 - - -
LINE-1 0.6% 108.0 98.0 - - 111.0 97.0 - -
LINE-1 0.7% 107.0 - - - 98.0 - -
LINE-1 1.0% - - - - - - - -
LINE-2 0.1% 75.0 89.0 - - 91.0 89.0 - -
LINE-2 0.2% 87.0 75.0 - - 101.0 108.0 - -
LINE-2 0.3% 87.0 73.0 - - 106.0 109.0 - -
LINE-2 0.4% 76.0 - - - 113.0 - - -
LINE-2 0.5% 79.0 - - - 115.0 - - -
LINE-2 0.6% 75.0 82.0 - - 117.0 107.0 - -
LINE-2 0.7% 91.0 74.0 78.0 - 107.0 105.0 101.0 -
LINE-2 1.0% - - - - - - - -
Here, average plant height and crop duration: LINE-1 0.0% = 68.9 cm and 133.7 days; LINE-2
0.0% = 66.2 cm and 121.6 days;
Range of plant height and crop duration was 73.0-118.0 cm and 89.0-117.0 days
respectively
Single fruit weight: Single fruit weight of EMS treated selected plants LINE-1 and
LINE-2 was ranged in 38.0-67.0 g and 38.9-80.4 g respectively (Table 4) which follow the
demand of traits (Table 1). In LINE-1 all plants were found to increase in single fruit weight
as compared to LINE-1 0.0% (37.2 g) while in LINE-2 all plants except LINE-10.3%(a) (38.0 g)
was found to increase the single fruit weight in comparison to LINE-2 0.0% (39.4 g) due to
application of different concentrated EMS (Table 2).
Yield: Yield of EMS treated selected plants LINE-1 and LINE-2 was ranged in 2.1-4.1
kg and 2.2-4.5 kg respectively (Table 5) which followed the demand of traits (Table 1). In
both LINE-1 and LINE-2, all plants were found to increase in yield as compared to LINE-1
0.0% (2.0 kg) and LINE-2 0.0% (1.8 kg) due to application of different concentrated EMS
(Table 5). However, among these plants, maximum yield was found from LINE-2 0.1%(b)
(4.5 kg) followed by LINE-1 0.1%(a) (4.1 kg) (Table 5).
Shelf life: Shelf life of EMS treated selected plants LINE-1 and LINE-2 was above 7.0
days to about 26.0 days (Table 5) which followed the demand of traits (Table 1). In both
LINE-1 and LINE-2, all plants were found to increase in shelf life as compared to LINE-1 0.0%
(6.3 days) and LINE-2 0.0% (5.7 days) due to application of different concentrated EMS
(Table 5). However, some of the plants showed more than 20 days shelf life like LINE-1
0.2%(a) (26.0 days), LINE-1 0.3%(c) (21.0 days), LINE-1 0.4%(a) (22.0 days), LINE-1 0.5%(a)
(31.0 days) and LINE-2 0.3%(a) (26.0 days) (Table 5).
Fruit shape: Fruit shapes of the plants were also varied. The different fruit shapes of
selected EMS treated tomatoes were shown in Plate 1a and 1b. Plate 1a represented the
fruits for LINE-1 while Plate 1b for LINE-2. Different plants treated with same concentrated
EMS of same line showed different fruit shape like as LINE-1 0.3%(a) and LINE-1 0.3%(b) had
round shaped fruits whereas LINE-1 0.3%(c) and LINE-1 0.3%(d) had rectangular shaped
fruits (Table 6). Similarly two plants those were fulfilled the demands of traits of LINE-2
treated with 0.1% EMS showed two different shaped of the fruits, one was ellipsoid and
another was rectangular while two plants from 0.3% EMS treated of LINE-2 showed
rectangular and ox-heart shaped (Table 6).

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Table 3. Performance of selected EMS treated tomato plants for fruit length and diameter
Fruit length (mm) Fruit diameter (mm)
EMS treatments
(a) (b) (c) (d) (a) (b) (c) (d)
LINE-1 0.1% 49.8 52.3 - - 45.6 44.4 - -
LINE-1 0.2% 51.0 53.2 52.5 - 48.3 47.9 47.9 -
LINE-1 0.3% 42.5 50.9 51.6 48.3 41.5 46.9 44.4 44.7
LINE-1 0.4% 47.8 - - - 47.2 - - -
LINE-1 0.5% 54.3 - - - 48.1 - - -
LINE-1 0.6% 47.9 50.7 - - 46.9 48.4 - -
LINE-1 0.7% 48.4 - - - 43.2 - - -
LINE-1 1.0% - - - - - - - -
LINE-2 0.1% 54.0 50.6 - - 47.4 41.8 - -
LINE-2 0.2% 48.2 45.3 - - 47.8 47.7 - -
LINE-2 0.3% 52.5 52.8 - - 52.6 48.2 - -
LINE-2 0.4% 56.9 - - - 46.1 - - -
LINE-2 0.5% 58.2 - - - 45.6 - - -
LINE-2 0.6% 56.2 56.0 - - 54.1 45.0 - -
LINE-2 0.7% 49.0 47.4 52.5 - 49.6 52.7 51.7 -
LINE-2 1.0% - - - - - - -
Here, average fruit length and diameter: LINE-1 0.0% = 50.9 mm and 46.5 mm; LINE-2 0.0% =
49.6 cm and 50.4 mm;
Range of fruit length and diameter was 42.5-58.2 mm and 41.5-54.1 mm respectively
Table 4. Performance of selected EMS treated tomato plants for number of fruits and single
fruit weight
Number of fruit Single fruit weight (g)
EMS treatments
(a) (b) (c) (d) (a) (b) (c) (d)
LINE-1 0.1% 79.0 76.0 - - 56.0 41.0 - -
LINE-1 0.2% 68.0 74.0 60.0 - 60.3 56.0 54.0
LINE-1 0.3% 104.0 90.0 53.0 72.0 38.0 54.4 48.6 47.4
LINE-1 0.4% 74.0 - - - 54.0 - - -
LINE-1 0.5% 58.0 - - - 67.0 - - -
LINE-1 0.6% 77.0 64.0 - - 45.8 49.7 - -
LINE-1 0.7% 59.0 - - - 48.8 - - -
LINE-1 1.0% - - - - - - - -
LINE-2 0.1% 70.0 149.0 - - 61.1 38.9 - -
LINE-2 0.2% 51.0 75.0 - - 61.1 48.6 - -
LINE-2 0.3% 62.0 112.0 - - 56.5 49.4 - -
LINE-2 0.4% 70.0 - - - 53.6 - - -
LINE-2 0.5% 66.0 - - - 50.5 - - -
LINE-2 0.6% 58.0 98.0 - - 80.4 47.2 -
LINE-2 0.7% 88.0 59.0 75.0 - 57.0 67.1 49.1 -
LINE-2 1.0% - - - - - - - -
Here, number of fruits and average single fruit weight: LINE-1 0.0% = 33.2 and 37.2 g; LINE-2
0.0% = 39.4 and 55.6 g;
Range of number of fruits and single fruit weight was 51.0-149.0 and 38.0-80.4 g respectively

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Table 5. Performance of selected EMS treated tomato plants for yield and shelf life
Yield (kg) Shelf life (days)
EMS treatments
(a) (b) (c) (d) (a) (b) (c) (d)
LINE-1 0.1% 4.1 3.1 - - 16.0 16.0 - -
LINE-1 0.2% 3.2 3.4 3.4 - 26.0 15.0 18.0 -
LINE-1 0.3% 2.8 3.5 2.1 2.5 12.0 14.0 21.0 14.0
LINE-1 0.4% 3.0 - - - 22.0 - - -
LINE-1 0.5% 3.4 - - - 31.0 - - -
LINE-1 0.6% 2.6 2.5 - - 14.0 16.0 - -
LINE-1 0.7% 2.1 - - - 11.0 - - -
LINE-1 1.0% - - - - - - - -
LINE-2 0.1% 3.2 4.5 - - 15.0 12.0 - -
LINE-2 0.2% 2.6 2.2 - - 17.0 11.0 - -
LINE-2 0.3% 2.2 3.9 - - 26.0 14.0 - -
LINE-2 0.4% 3.0 - - - 17.0 - - -
LINE-2 0.5% 2.7 - - - 13.0 - - -
LINE-2 0.6% 3.0 2.7 - - 19.0 15.0 - -
LINE-2 0.7% 3.0 3.4 2.7 - 10.0 17.0 8.0 -
LINE-2 1.0% - - - - - - - -
Here, yield/plant and shelf life: LINE-1 0.0% = 2.0 kg and 6.3 days; LINE-2 0.0% = 1.8 kg and
5.7 days
Range of yield/plant and shelf life was 2.1-4.5 and 11.0-31.0 respectively

Table 6. Fruit shape of the selected EMS treated tomato plants

Fruit shape
EMS treatments
(a) (b) (c) (d)
LINE-1 0.1% Ellipsoid Ellipsoid - -
LINE-1 0.2% Rectangular Rectangular Round -
LINE-1 0.3% Round Round Rectangular Rectangular
LINE-1 0.4% Ellipsoid - - -
LINE-1 0.5% Round - - -
LINE-1 0.6% Round Round - -
LINE-1 0.7% Round - - -
LINE-1 1.0% - - - -
LINE-2 0.1% Ellipsoid Rectangular - -
LINE-2 0.2% Rectangular Ellipsoid - -
LINE-2 0.3% Rectangular Ox-heart - -
LINE-2 0.4% Ox-heart - - -
LINE-2 0.5% Ellipsoid - - -
LINE-2 0.6% Ellipsoid Rectangular - -
LINE-2 0.7% Round Rectangular Round -
LINE-2 1.0% - - - -

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 Selection of EMS Induced Tomato Variants 219

LINE-1 0.2%
LINE-1 0.1%(a) LINE-1 0.1%(b) LINE-1 0.2% (a) (b) LINE-1 0.2% (c) LINE-1 0.3% (a)

LINE-1 0.4%
LINE-1 0.3% (b) LINE-1 0.3% (c) LINE-1 0.3% (d) (a) LINE-1 0.5% (a) LINE-1 0.6% (a)

LINE-1 0.7%
LINE-1 0.6% (b) (a)
Plate 1a. Fruit shape of the selected EMS treated tomato plants of LINE-1

LINE-2 0.3%
LINE-2 0.1% (a) LINE-2 0.1% (b) LINE-2 0.2% (a) LINE-2 0.2% (b) LINE-2 0.3% (a) (b)

LINE-2 0.4% (a) LINE-2 0.5%(a) LINE-2 0.6%(a) LINE-2 0.6%(b) LINE-2 0.7%(a) LINE-2 0.7%(b)

LINE-2 0.7%(c)
Plate 1b. Fruit shape of the selected EMS treated tomato plants of LINE-2

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Nahiyan et al. 220

Discussion
Mutagenesis is a process in establishing mutant for genetic studies. Mutation frequency of
EMS is high in all plant species and appears to be independent of genome size (Greene
et al., 2003; Henikoff and Comai, 2003). It was decided to utilize different doses of EMS,
0.0-0.7% and 1.0% in which plant survival rate is acceptable. Correlation was present
between EMS doses and toxicity, mutation density obtained and frequency of phenotype
alterations like as plant fertility rate was 41% less than plant treated with 0.7% at 1% EMS,
(Minola et al., 2010). Flowering plants are well adapted to random mutagenesis because
large and saturated mutant populations can be generated through chemical
mutagenesis. Such populations can then be screened for the particular phenotypes using
‘reverse screened’ tools, which are conducted based on gene sequence for mutations in
the target gene (Stephenson et al., 2010). From the current study variability was observed
in plant height using EMS in tomato. Similar result was found in Capsicum annum (Jabeen
and Mirza, 2002). Chen and Gottschalk (1970) and Okuno and Kawai (1978) have
reported that mutations were affecting plant height. The result indicated that mutagens
could cause both positive and negative genetic variability in plant height. But plants
posing the plant height in accordance of the demand of traits were sorted out. Mutagens
can change flowering and fruiting period. EMS treatments on tomato seed resulted a
wide range of the crop duration as compared to their control which coincides with a
previous study in linseed (George and Nayar, 1973). Early flowering was also reported in
Lathyrus sativus L (Kumar and Dubey, 1998; Girhe and Choudhary, 2002). Wani and Khan
(2006) in mungbean found early ripening mutants were competitive with or even superior
to their mother varieties with regard to seed production. Fruit length and diameter were
also varied. In the TILLING screens Minola et al. (2010) analyzed seven genes and
discovered a total of 66 induced point mutations. The spectrum of expected mutations in
an EMS-treated population is essentially GC/AT transition because of the frequent
alkylation of guanine residues by EMS (Sega, 1984). Red Setter populations the
percentage of observed GC/AT transition was 60% in the 0.7% EMS population and only
28.6% in the 1% EMS population also identified GC/TA, AT/TA, AT/CG, GC/CG and AT/GC
transversions (Minola et al., 2010). Mutants having higher yield over mother variety were
also reported earlier in rapeseed-mustard (Rahman et al., 1992; Javed et al., 2003; Das et
al., 2004; Barve et al., 2009). Despite these drawbacks, several groups have reported
successes in linking genotypic change to novel phenotypes in a variety of crops on a
specific traits tremendously and TILLING has proven useful in increasing the oleic acid
content through identification of mutations in FAD1, 2, and 3 genes (Dierking and Bilyeu,
2009) and in Sorghum where lignin content has been decreased though mutation of
COMT (Barkley et al., 2008). Current study showed that fruits from some selected plants
posed more than 15 days shelf life and in some case it was up to 31 days in room
condition.
Conclusion
The generation of such new variants depends on a set of reliable data. Current study was
the first step to implement such an open access data to tomato. The next step will be to
clarify mutants and gene functions based on differential of the variants profiles. From the
current study it was found a set of data through the application of EMS for chemical
mutagenesis on tomato for the selection of lines that fulfill the researcher’s demands of
traits. The obtained variants can be used as a source in tomato breeding programs for
better performance.
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