Nghiên cứu biến dị tế bào soma và xử lý tia gamma trong chọn tạo các dòng đậu nành (glycine max (l ) merrill) chống chịu mặn tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY

SUMMARY OF DOCTOR THESIS
Major: Crop Science
Code: 62 62 01 10

LE HONG GIANG

STUDY ON SOMACLONAL CELL
VARIATION AND GAMMA TREATMENT
IN SELECTION FOR SALT TOLERANT
SOYBEAN LINES (Glycine max (L.) Merrill)

Can Tho, 2019


THE DISSERTATION WAS COMPLETED AT
CAN THO UNIVERSITY

Scientific advisor 1: Assoc. Prof. Dr. Nguyen Bao Toan
Scientific advisor 2:

The dissertation will be defended in front of the doctoral thesis
examination board of Can Tho University
At: Doctoral Dissertation Defense Auditorium, Can Tho
University
Time: 8:00 am
Date: 02nd August, 2019

Reviewer 1: Prof. Dr. Tran Nhan Dung

Reviewer 2: Dr. Dang Minh Tam

Further information of this dissertation can be found at:
Learning Resource Center, Can Tho University.
The National Library Vietnam.


LIST OF PUBLISHING
1. Le Hong Giang and Nguyen Bao Toan, 2014. Evaluation on salinity tolerance
ability of some soybean cultivars. Can Tho University Journal of Science (ISSN 18592333), vol. Special issue of Agriculture (4), pp. 179-188.
2. Le Hong Giang, Tran Thi Tuyet Lan and Nguyen Bao Toan, 2016. Selection for salt
tolerant callus lines of soybean cultivar MTD 760-4 by gamma ray treatment. Can Tho
University Journal of Science (ISSN 1859-2333), vol. 45b, pp. 39-48.
3. Le Hong Giang, Huynh Thi Minh Thi and Nguyen Bao Toan, 2016. Selection for
salt tolerant callus of MTD 760-4 soybean by in vitro culture technique. Can Tho
University Journal of Science (ISSN 1859-2333), vol. Special issue of Agriculture (3),
pp. 47-54.

1


Chapter 1: INTRODUCTION
1.1 Rationale
Soyean (Glycine max (L.) Merrill) is a food crop which has high econonic
value and is an alternate one which considerably improves soil (Pham Van Bien et
al., 1996). Today, salinity problem in the Mekong Delta has become more serious
effecting the productivity of many crops. Soybean is considered as a sensitive
species to salinity (Lauchli, 1984). The production of soybean varieties which are
susceptible to salt strongly reduces under saline conditions (Chang et al., 1994;
Katerji et al., 2003). Therefore, to greatly cultivate and expand the area of soybean

in the Mekong Delta, using salt tolerant varieties is one of solutions. Applying the
method of creating somaclonal variation and gamma irradiation through tissue
culture can select salt tolerant crop lines. These methods are successfully used in
many crops such as rice (Dang Minh Tam and Nguyen Thi Lang, 2003; Saleem et
al., 2005; Zinnah et al., 2013), sugarcane (Patade et al., 2008), wheat (El-Sayed et
al., 2007), potato (Yaycili and Alikamanoglu, 2012) and so on. For this reason, the
study to select salt tolerant soybean lines to adapt to the adverse environment as
salinity in the Mekong Delta is necessary.
1.2 Objective
The thesis was conducted to determine the method to select the soybean line
that has salinity tolerance.
1.3 Study contents
Content 1: Determing the salt tolerance ability of some soybean varieties
which were popular in the Mekong Delta.
Content 2: Determing the tissue culture medium of soybean suitale for
obtaining initial sources for selection methods.
Content 3: Evaluating the ability of selection for salt tolerant soybean lines
by somaclonal variation creating and gamma irradiation method.
1.4 Research object and scope
1.4.1 Research object
Some soybean varieties which were popularly cultivated in the Mekong
Delta.
1.4.2 Research range
- Determing the salt tolerance ability of soybean varieties by hydroponic
method was conducted in the greenhouse.
- Determing the tissue culture medium of soybean suitale for obtaining initial
sources for selection methods and selecting for salt tolerant soybean lines were
done in the lab with one variety evaluated the degree of salt tolerance in the
greenhouse before.
- Evaluating the genetic difference in salt-selected soybean lines by

molecular technique.
- Acclimatizing and experimentally cultivating soybean lines after selected
were in the greenhouse.

2


1.5 Scientific and practical significance
1.5.1 Scientific significance
Through hydroponics and tissue culture technique in combination with
somaclonal variation selection and mutagenesis by gamma irradiation, the thesis
determined the salt tolerance ability of some soybean varieties which were popular
in the Mekong Delta, the suitale tissue culture medium for MTD 760-4 soybean, a
salt sensitive variety, to obtain initial sources for selection methods and produced
salt tolerant soybean line which was identified by molecular makersr of ISSR. The
study results determined the method to produce salt tolerant soybean line of MTD
760-4 variety which supplies the literature for other science studies and the
premise for following studies and serves in education.
1.5.2 Practical significance
The thesis result obtained one MTD 760-4 soybean line that was salt tolerant
to NaCl concentration of 5 g/L by somaclonal variation creating method on
embryo axes. From that, it can be experimentally cultivated and released new lines
to natural condition, especially in saline lands of Mekong Delta.
1.6 New points of thesis
- The study result determined the salt tolerance ability of ten soybean
varieties which were popular in the Mekong Delta. Among that, MTD 748-1, DH
4 and MTD 720 had the high salt tolerant ability at 4 g/L NaCl when evaluated by
hydroponic method. MTD 878-3 variety was sensitive to salt and MTD 760-4
completely died at this salt concentration.
- The study obtained callus and plantlet lines which were salt tolerant from

the salt sensitive variety of MTD 760-4.
- In selection methods to achieve salt tolerant soybean lines, creating
somaclonal variation on embryo axes of MTD 760-4 soybean obtained one
soybean plantlet line that was salt tolerant at NaCl of 5 g/L. There was genetic
difference in DNA structure of the salt tolerant shoot compared to the control with
non-salt treatment when analyzed by molecular marker of ISSR22. MTD 760-4
soybean plantlets after selected with 5 g/L NaCl normally grew after 5 weeks
acclimatized under saline water irrigating condition in the greenhouse.
- Both methods of creating somaclonal variation and Co60 gamma irradiation
combined with NaCl salt selection on callus achieved two salt tolerant callus lines
to NaCl dose of 5 g/L at none irradiated explants and irradiated explants with
gamma dose of 10 Gy. Genetic analysis with ISSR22 marker in these two callus
explants showed that there was no appearance of DNA band 450 bp compared to
control explants.
Chapter 2: LITERATURE REVIEW
Soybean is considered as a sensitive species to salinity (Lauchli, 1984). The
production of soybean varieties which are susceptible to salt strongly reduces
under saline conditions (Katerji et al., 2003). Damage by salinity in soybean is due
to the accumulation of Cl- ions in stems and leaves and is expressed by leaf

3


burning (Abel and MacKenzie, 1964; Essa, 2002). However, different genotypes
in term of vulnerability levels indicate genetic diversity in salinity tolerance.
The tissue culture induces variation in regenerated plants, called somaclonal
variation (Larkin and Scowcroft, 1981). In the world, in vitro selection technique
or creating somaclonal variation has been applied on many crop cultivars to
produce tolerance to stresses. Salt tolerant plants can be produced by using
selection agent of NaCl in culture medium. To the present, a lot of cultivars has

been studied for salt tolerance by using this technique such as rice, wheat,
sugarcane, potato, tomato, strawbery, citrus, cabbage family, sweet potato,
sunflower and so on (Rai et al., 2011).
Beside that, there are many salt tolerant crops have been made by applying
the combination of mutagenesis with gamma ray and in vitro selection such as rice
(Saleem et al., 2005), wheat (El-Sayed et al., 2007), sugarcane (Patade et al.,
2008; Nikam et al., 2014), potato (Yaycili and Alikamanoglu, 2012), sweet potato
(He et al., 2009) and so on. However, researchs on applying mutagenesis method
by gamma irradiation and selection on saline medium to produce salt tolerant
cultivars are not reported in soybean. ISSR (Inter-Simple Sequence Repeat) has
been used for detection of genetic polymorphism and discrimination between
soybean cultivars (Yang et al., 1996). This technique has also been applied to
identify markers associated with salt tolerance in soybean (Mahgoub et al., 2016),
wheat (Lang et al. 2001), rice (Kaushik et al. 2003), barley (Khatab and Samah,
2013), sorghum (Khalil, 2013) and sugarcane (Markad et al., 2014).
Chapter 3: STUDY METHODS
3.1 Experiment materials
- Ten popular soybean varieties of Mekong Delta are MTD 176, MTD 748-1,
MTD 760-4, Nhat 17A, OMDN 29, DH 4, MTD 720, MTD 860-1, MTD 878-3
and MTD 885-2 which were colected in the Deparment of Genetic and Crop
Breeding, College of Agriculture, Can Tho University and Van Duc company
(Dong Hoa Village, Song ThuanCommune, Chau Thanh District, Tien Giang
Province).
- One of these ten varieties was used to select for salt tolerant line.
- Chemicals were used for experiments of determing the salt tolerance ability
of soybean varieties, for plant tissue culture, for proline analysis, DNA extraction,
PCR and electrophoresis.
3.2 Research periods and locations
Research contents were carried out from March 2014 to May 2019 at
Agriculture Experiment and Research Farm, Tissue Culture Lab and greenhouse of

Plant Physiology and Biochemistry Deparment, College of Agriculture, at
Biotechnology Research and Development Institute of Can Tho University and
Dalat Nuclear Research Institute.

4


3.3 Methodology
3.3.1 Content 1: Determing the salt tolerance ability of some soybean
varieties which were popular in the Mekong Delta
Experiment 1: Effect of NaCl on growth of soybean vaieties MTD 176,
MTD 748-1, MTD 760-4, Nhat 17A and OMDN 29
The experiment was arranged in a randomized complete design (RCD) with
two factors including five soybean cultivars of MTD 176, MTD 748-1, MTD 7604, Nhat 17A and OMDN 29 (factor 1) and four NaCl levels of 0, 1, 2 and 4 g/L
(factor 2). There was 10 replications for one treatment which was a spongy bucket
planted two plants/cultivar. Observed parameters were the survival rate, plant
height, internode number on main stem and root length.
Experiment 2: Effect of NaCl on growth of soybean varieties DH 4, MTD
720, MTD 860-1, MTD 878-3 and MTD 885-2
The experiment was carried out similar to Experiment 1 with the material
were five soybean cultivars of DH 4, MTD 720, MTD 860-1, MTD 878-3 and
MTD 885-2.
3.3.2 Content 2: Determing the tissue culture medium of soybean suitale
for obtaining initial sources for selection methods
Material for researching selection methods was soybean MTD 760-4, a salt
sensitive cultivar but having good growth chracteristics selected from the results of
Experiment 1 to aim to enhance salt tolerance of this non-salt tolerant cultivar.
Experiment 3: Effect of 2,4-D and BA on callus formation from
cotyledon of soybean MTD 760-4
The experiment was designed completely randomized with two factors

including 2,4-D (1.25, 2.5, 5 and 10 mg/L) and BA concentrations (0, 0.5 and 1
mg/L) with 10 replications. Each replication was a jar (cultured four cotyledon
explants). Callus and root formation were observed.
Experiment 4: Effect of NAA and macronutrient on root formation from
internode of soybean MTD 760-4
The experiment was set up as RCD in a factorial arrangement with two
factors consisting NAA concentrations (0, 0.1, 0.2 and 0.4 mg/L) and
macronutrient contents (MS and 1/2 MS). Each treatment was replicated 10 times
with one jar cultured three explants. Root parameters as formation rate, number,
length and shoot height, number of leaf were estimated.
Experiment 5: Effect of substrates on acclimatization of in vitro soybean
plantlets in greenhouse condition
Five treatments of substrates including coco (fiber) dust, straw manure, coco
(fiber) dust + straw manure (1:1), coco (fiber) dust + straw manure + rice husk ash
(1:1:1) and coco (fiber) dust + rice husk ash + soil (1:1:1) was set in RCD with
five replications. Each replication consisted two plantlets. Growth parameters were
collected as survival rate, increased shoot height and leaf number.

5


3.3.3 Content 3: Evaluating the ability of selection for salt tolerant
soybean lines by somaclonal variation creating and gamma irradiation
method
Experiment 6: Effect of NaCl on callus growth of soybean MTD 760-4
+ Experiment 6a, 6b, 6c and 6d: Effect of NaCl on callus growth of soybean
MTD 760-4 in the selection time 1, 2, 3 and 4
The experiments were performed in RCD with one factor including five NaCl
treatments of 0, 2.5, 5, 7.5 and 10 g/L. Each treatment had 10 replications with
five callus explants cultured in a jar. Surviving callus after 5 weeks on saline

medium of the prior experiment were cultured on the medium with same NaCl
concentrations to select. Observed parameters included the survival rate and
proline content of callus which was estimated by the method of Bates et al. (1973).
Experiment 7: Effect of NaCl on shoot formation and growth from
embryo axes of soybean MTD 760-4
+ Experiment 7a: Effect of NaCl on shoot formation and growth in the
selection time 1
Treatments were four NaCl concentrations of 0, 2.5, 5 and 7.5 g/L which
were set in RCD with 15 replications including a jar cultured four embryo axis
explants. Shoot formation rate and height were recored.
+ Experiment 7b and 7c: Effect of NaCl on shoot growth in the selection time
2 and 3
The experiments were put in RCD with one factor consisting four treatments
which were NaCl concentrations 0, 2.5, 5 and 7.5 g/L. A jar containing two shoot
explants which were the surviving one of the prior experiment was set as a
replication with 10 times. Observed quotas included increased shoot height and
leaf number, salt tolerant plantlet number and proline content.
Experiment 8: Effect of gamma irradiation Co60and NaCl on callus
growth of soybean MTD 760-4
+ Experiment 8a: Effect of gamma irradiation Co60 and NaCl on callus
growth of soybean MTD 760-4 in the selection time 1
The experiment was laid out in RBD in a factorial arrangement with two
factors. The first consisted five Co60 gamma irradiation dose (0, 5, 10, 20 and 40
Gy) and the second was four concentrations of NaCl (0, 2.5, 5 and 7.5 g/L) with 10
replications. Each included five callus cultured in a jar. The survival rate and
proline content of callus were observed.
+ Experiment 8b, 8c and 8d: Effect of gamma irradiation Co60 and NaCl on
callus growth of soybean MTD 760-4 in the selection time 2, 3 and 4.
Experiments were conducted in the same way as Experiment 8a with the
material was the callus surviving from the prior experiment.

Experiment 9: Effect of gamma irradiation Co60 and NaCl on shoot
formation and growth from embryo axes of soybean MTD 760-4
+ Experiment 9a: Effect of gamma irradiation Co60 and NaCl on shoot
formation and growth in the selection time 1

6


Two factors comprising five Co60 gamma irradiation dose (0, 20, 40 and 60
Gy) and four concentrations of NaCl (0, 5, 7.5 and 10 g/L) were arranged in RBD.
Each treatment had four replications by a jar containing four embryo axes. The
survival rate of explants, shoot formation rate and height were recorded.
- Experiment 9b and 9c: Effect of gamma irradiation Co60 and NaCl on shoot
growth in the selection time 2 and 3
The factor was NaCl concentration including three levels considered as
treatments (0, 5 and 7.5 g/L). Treatments were put RCD with four replications as a
jar cultured two explants. The surviving shoots of the prior experiment were used
as material. Increased shoot height and leaf number were observation.
- Evaluating the genetic difference of salt tolerant soybean lines
Materials were three callus explants selected with 0 and 5 g/L NaCl (result of
Experiment 6) and callus irradiated at dose of 10 Gy + 5 g/L NaCl (result of
Experiment 8) and eleven shoot explants at 0 g/L NaCl (one explant), 2.5 g/L
NaCl (six explants) and 5 g/L NaCl (four explants) from the result of Experiment
7. DNA was extracted by CTAB process (Rogers and Bendich, 1988). PCR
amplified with 10 ISSR primers (Karuppanapandian et al., 2010) for callus
explants and one ISSR primer which resulted in salt tolerance for shoot explants.
Experiment 10: Evaluating the growth and development of salt tolerant
soybean lines in the greenhouse
The experiment was arranged in RBD with one factor consisting four
treatments of NaCl concentrations (0, 2.5 and 5 g/L) in combination with saline

water irrigating of 0, 2.5 and 5 g/LNaCl. Each treatment was replicated 12 times
with one plantlet put in a pot. Observation were increased shoot height and number
of internode.
3.3.4 Data collection and analysis
Data was processed by Microsoft Excel software and statistically analyzed by
SPSS version 20.0. It was performed by analysis of variance (ANOVA).
Significant differences between means were determined using the Duncan’s
multiple range test at level of 1% and 5%. The percentage data varying from 0 to
100% was transferred into form of Arcsin√x (Gomez and Gomez, 1984).
Chapter 4: RESULTS AND DISCUSSION
4.1 Content 1: Determing the salt tolerance ability of some soybean
varieties which were popular in the Mekong Delta
4.1.1 Experiment 1: Effect of NaCl on growth of soybean vaieties MTD
176, MTD 748-1, MTD 760-4, Nhat 17A and OMDN 29
Results in Table 4.1 showed that MTD 748-1 variety presented the highest
salinity tolerance (70.0% survival rate) at 4 g/L NaCl concentration and MTD 7604 variety is most sensitive to salinity with plants completely died at 5 weeks after
planted. NaCl concentrations of 2 and 4 g/L gave significantly different survival
rates at 1% compared to those of 1 and 0 g/L NaCl.

7


Table 4.1: Growth of soybean varieties MTD 176, MTD 748-1, MTD 760-4, Nhat
17A and OMDN 29 affected by NaCl at 5 weeks after planted
Survival
Internode
Root
Survival
Plant height
rate (%) at

number on
length
rate (%)
(cm)
NaCl 4 g/L
main stem
(cm)
Varieties
68.8b
25.0e
43.1c
7.4d
18.9b
MTD 176
a
cd
c
c
83.8
70.0
52.4
9.8
42.8a
MTD 748-1
65.0b
0.0e
77.3a
12.5a
41.4a
MTD 760-4

b
e
b
b
70.0
10.0
55.4
10.1
38.8a
Nhat 17A
73.8ab
20.0e
58.2b
10.5b
42.1a
OMDN 29
NaCl concentration (g/L)
100a
NaCl 0
91.0a
NaCl 1
73.0b
NaCl 2
25.0c
NaCl 4
*
Fvarieties
**
FNaCl
**

Fvarieties x FNaCl
CV (%)

84.0a
67.2b
46.7c
19.7d
**
**
ns

35.3

21.8

11.8a
10.6b
9.7c
7.0d

43.6a
37.2b
36.9b
26.4c

**
**
**
13.6

**
**
ns
17.6

Data of percentage was tranferred into form of Arcsin√x before statistically analyzed. Means sharing
the same letters within column are not significantly different from each other; (ns): not significantly
different; (*) and (**): statistically significant difference at 5% and 1%.

a

b

c

d

Figure 4.1: Effect of NaCl on the survival and growth of five soybean
varieties Nhat 17A, MTD 748-1, MTD 176, MTD 760-4, OMDN 29
(from right to left) after 5 weeks planted
NaCl 0 g/L (a)

NaCl 1 g/L (b) NaCl 2 g/L (c)

8

NaCl 4 g/L (d)


Growth parameters such as plant height, number of internodes on main stems

and root length differed between varieties and decreased with the increasing of
NaCl salt concentration (Figure 4.1). The study of Hamayun et al., (2010) also
proved that plant height, biomass and yield parameters significantly reduced at the
NaCl concentrations of 4.1 and 8.2 g/L. MTD 760-4 had more prominent
agronomic properties than others such as plant height at 77.3 cm, internode
number on main stem at 12.5 internodes, and rather long root length with 41.4 cm.
4.1.2 Experiment 2: Effect of NaCl on growth of soybean varieties DH 4,
MTD 720, MTD 860-1, MTD 878-3 and MTD 885-2
Table 4.2 indicated that varieties of DH 4 and MTD 720 were less affected by
salinity (survival rates were 76.3 and 75.0%, respectively). MTD 878-3 was the
least salt tolerant variety with the survival rate reducing to 58.8%. Plant height and
number of internodes on main stems were significantly influenced by salt and
these had differences between varieties.
Table 4.2: Growth of soybean varieties DH 4, MTD 720, MTD 860-1, MTD 878-3
and MTD 885-2 affected by NaCl at 5 weeks after planted
Plant
Internode
Survival
Root length
Varieties
height
number on
rate (%)
(cm)
(cm)
main stem
a
a
a
76.3

60.6
6.9
39.2
DH 4
ab
bc
a
75.0
55.1
7.4
38.3
MTD 720
bc
d
b
61.3
37.3
5.3
37.2
MTD 860-1
d
b
a
58.8
45.5
6.5
36.4
MTD 878-3
61.3bc
58.6ab

5.6b
38.4
MTD 885-2
NaCl concentration (g/L)
NaCl 0
NaCl 1
NaCl 2
NaCl 4
Fvarieties
FNaCl
Fvarieties x FNaCl
CV (%)

100a
90.0a
43.0b
33.0b
*
**
ns
47.5

83.2a
53.1b
38.6c
30.9d
**
**
**
23.0

8.3a
6.7b
5.6c
4.8d
**
**
ns
19.7

40.5a
39.0ab
35.1c
37.0b
ns
**
ns
9.8

Data of percentage was tranferred into form of Arcsin√x before statistically analyzed. Means sharing
the same letters within column are not significantly different from each other; (ns): not significantly
different; (*) and (**): statistically significant difference at 5% and 1%.

According to Abel and MacKenzie (1964) and Chang et al. (1994), the
agronomic characteristics of soybeans could be severely affected by high salinity
such as reducing of plant height, internode number and biomass. Results of
Kondetti et al. (2012) also indicated that salinity had detrimental effects on
germination and all physiological parameters of soybean.

9

The objective of this content was to determine salinity tolerance of some
common soybean varieties in the Mekong Delta. The results identified high salt
tolerant varieties and salt sensitive varieties. Among that, MTD 748-1, DH 4 and
MTD 720 had high salinity tolerance at 4 g/L NaCl concentration, MTD 878-3
was sensitive to salinity and MTD 760-4 completely died at this salt concentration.
In addition, the results of the two experiments also determined the most sensitive
variety to salinity which was MTD 760-4 to use as material in selective researchs
on salinity tolerance in content 2 and 3 to improve salinity tolerance of non-salt
tolerant variety. The MTD 760-4 did not survive at 4 g/L NaCl, while the MTD
878-3 was able to save 30.0% at this concentration. Moreover, the experiment
results also showed that MTD 760-4 was the cultivar which had more prominent
agronomic and growth characteristics as plant height, number of internodes on
main stems and root length in comparison with others.
4.2 Content 2: Determing the tissue culture medium of soybean suitale
for obtaining initial sources for selection methods
4.2.1 Experiment 3: Effect of 2,4-D and BA on callus formation from
cotyledon of soybean MTD 760-4
Experimental results presented that cotyledon explants of soybean MTD 7604 had callus formation rate with no difference between concentrations of 2,4-D and
BA, obtaining from 93.3-100%. The explants rooted best on medium
supplemented with 2,4-D at lowest concentration of 1.25 g/L and without BA
(Table 4.3).
Table 4.3: Callus and root formation rate (%)from cotyledon of soybean MTD
760-4 affected by 2,4-D and BA at 4 weeks after cultured
Callus formation rate Root formation rate
2,4-D concentration (mg/L)
(%)
(%)
2,4-D 1.25

100
53.9a
2,4-D 2.5
96.7
36.4b
2,4-D 5
97.5
33.3b
2,4-D 10
93.3
33.3b
BA concentration (mg/L)
BA 0
97.5
100a
BA 0.5
96.9
12.8b
BA 1
96.3
5.0c
F2,4-D
ns
**
FBA
ns
**
F2,4-D x FBA
ns
**

CV (%)
13.3
28.3
Data of percentage was tranferred into form of Arcsin√x before statistically analyzed. Means sharing
the same letters within column are not significantly different from each other; (ns): not significantly
different; (**): statistically significant difference at 1%.

The callus formation reaction is common in tissue culture, especially in MS
medium with the strong growth regulator as 2,4-D. The principle of callus reaction

10


is the cell differentiation process (Sun et al., 2000). Structure and colour of callus
in treatments supplemented with 2,4-D single concentrations from 1.25-10 mg/L
were spongy, relatively friable and yellowish green. The combination of 2,4-D and
BA formed callus which were compact and yellow-green in colour. The results
proved that the medium supplemented with 5 mg/L 2.4-D obtaining spongy friable
callus and biomass was also superior to those of other treatments. Therefore, callus
in this treatment was chosen to multiply and later to use as material for Content 3.
4.2.2 Experiment 4: Effect of NAA and macronutrient on root formation
from internode of soybean MTD 760-4
Results in Table 4.4 demonstrated that the highest rooting rate achieved on
medium supplemented with NAA 0.2 mg/L and with non-reducing MS content
(84.1% and 71.8%, respectively). The number of roots, root length, shoot height
and leaf number were also higher than that of remaining treatments.
On Indian soybean Glycine max (L) Merr. cv. CO3, Radhakrishnan and
Ranjithakumari (2007) rooted shoots regenerated from callus on B5 medium
supplemented with 14.7 µM IBA. Akitha Devi et al. (2012) used triacontanol
(TRIA) which had the highest number of roots and root length (6.3 ± 0.5 and 21.5

± 0.5). The results of this experiment showed that MS medium supplemented with
0.2 mg/L NAA was suitable for rooting of soybean shoots of MTD 760-4.
Table 4.4: Root formation and shoot growth of soybean MTD 760-4 affected by
NAA and macronutrient at 4 weeks after cultured
NAA
Root
Root
Root
Shoot
Leaf
concentration
formation number
length height (cm)
number
(mg/L)
rate (%)
(cm)
NAA 0
37.7c
2.6d
3.1d
5.7c
1.8b
c
c
c
b
NAA 0.1
37.0
4.5

4.4
6.5
1.9b
a
a
a
a
NAA 0.2
84.1
7.5
7.8
8.4
2.7a
b
b
b
b
NAA 0.4
67.0
5.5
6.8
6.8
2.0b
Macronutrient content (M)
MS
71.8a
5.3a
5.8a
7.0a
2.1

b
b
b
1/2 MS
41.2
4.8
5.2
6.7b
2.0
FNAA
*
*
*
*
*
Fmacronutrient
*
**
*
**
ns
FNAA xFM
**
*
*
*
ns
CV (%)
34.9
17.8

8.4
7.0
20.1
Data of percentage was tranferred into form of Arcsin√x before statistically analyzed. Means sharing
the same letters within column are not significantly different from each other; (ns): not significantly
different; (*) and (**): statistically significant difference at 5% and 1%.

4.2.3 Experiment 5: Effect of substrates on acclimatization of in vitro
soybean plantlets in greenhouse condition
Table 4.5 proved that the highest survival rate (80%) obtained in coco (fiber)
dust substrate and mixture of coco (fiber) dust + rice husk ash + soil which were

11


not different from that of coco (fiber) dust + straw manure + rice husk ash (70%)
at 4 weeks of acclimatization. Plantlets did not live on a single straw manure
substrate. The highest height achieved on the coco (fiber) dust + rice husk ash +
soil mixture (6.2 cm) and the lowest was on the coco dust and coco (fiber) dust +
straw manure (2.9 and 1.7 cm respectively). The increased number of leaves did
not differ between treatments.
Some acclimatization technique on soybean were also reported. The authors
used many different kinds of substrate as a mixture of vermiculite and soil
(Ranjitha Kumari et al., 2006), soil (Radhakrishnan and Ranjithakumari, 2007;
Janani and Ranjitha Kumari, 2013), steriled clay and sand (1:1) (Zia et al., 2010).
Plantlets could be covered with nylon bags to retain moisture or not and put in
room condition for a time before transferred to the greenhouse. However, the
details of these results were not showed.
Table 4.5: Growth of soybean plantlets MTD 760-4 affected by substrates after 4
weeks acclimatized

Survival
Increased shoot
Increased
Substrates
rate (%)
height (cm)
leaf
number
Coco (fiber) dust
80.0a
2.9c
1.8
b
Straw manure
0.0
Coco (fiber) dust + straw
20.0b
1.7c
2.0
manure
Coco (fiber) dust + straw
70.0a
4.4b
2.0
manure + rice husk ash
Coco (fiber) dust + rice husk
80.0a
6.2a
2.0
ash + soil

Mean
50.0
4.3
1.9
F
*
*
ns
CV(%)
53.0
22.8
31.0
Data of percentage was tranferred into form of Arcsin√x before statistically analyzed. Means sharing
the same letters within column are not significantly different from each other; (ns): not significantly
different; (*): statistically significant difference at 5%.

4.3 Content 3: Evaluating the ability of selection for salt tolerant soybean
lines by somaclonal cell variation creating and gamma irradiation method
4.3.1 Experiment6: Effect of NaCl on callus growth of soybean MTD
760-4
The Table 4.6 results showed that in the selection time 1, 2 and 3, the
concentration of 5 g/L NaCl salt had a significant effect on the survival rate of
soybean callus. The rate decreased sharply when the concentration increased to 10
g/L. At high salt concentrations, callus did not grow, decreased in size, changed
colour into dark brown and then died. According to Kowles (2010), when the
environment outside the cell has a higher solute concentration than that in the cell,
the water inside the cell moves out, resulting in the cell shrinking. Through 4
selections, the study achieved one callus line of soybean MTD 760-4 which was

12

tolerant to salinity at a concentration of 5 g/L. Callus stabilized vitality at this
concentration during the selection time 4.
The highest proline content of callus was at 5 g/L salt concentration with 2.78
µmol/g fresh weight and significantly differed from the control (1.48 µmol/g fresh
weight). The increase of proline content in saline tolerant callus with NaCl had
also been reported on soybean in the study of Liu and Staden (2000) and on many
other cultivars such as peanut (Jain et al., 2001), barley (Chaudhuri et al., 1997),
rice (Basu et al., 2002), sugarcane (Gandonou et al., 2006) and so on.
Table 4.6: Survival rate (%) and proline content of soybean callus of MTD 760-4
affected by NaCl at 5 weeks after cultured in the selection time 1, 2, 3 and 4
NaCl
Proline content
Selection Selection Selection Selection
concentration
(µmol/g fresh
time 1
time 2
time 3
time 4
(g/L)
weight)
0
100a
100a
100a
100
1.48b
a

a
a
2.5
100
100
100
100
1.51b
b
b
b
5
62
76
94
94
2.78a
c
c
7.5
44
17.5
10
26d
F
**
**
*
ns
*

CV (%)
46.1
20.8
10.6
10.9
26.3
Data of percentage was tranferred into form of Arcsin√x before statistically analyzed. Means sharing
the same letters within column are not significantly different from each other; (ns): not significantly
different; (*) and (**): statistically significant difference at 5% and 1%.

Summarized results proved that through somaclonal variation method by
constantly culturing on selective medium with NaCl, callus of soybean MTD 7604 were able to tolerant to salinity at the NaCl concentration of 5 g/L. Similarly, in
the selection of soybean cultivar cv. Acme., Liu and Staden (2000) also achieved
one callus line tolerant to NaCl 5.8 g/L.
4.3.2 Experiment 7: Effect of NaCl on shoot formation and growth from
embryo axes of soybean MTD 760-4
Table 4.7 demonstrated that in the first selection, shoot formation rate and
height of shoots decreased starting from NaCl concentration of 2.5 g/L and
achieved lowest at 7.5 g/L (25.0% and 0.66 cm, respectively) compared to the
control (65.0% and 3.51 cm). In the selection time 2 and 3, NaCl concentration of
2.5 g/L also had a significant effect on shoot growth. The increased shoot height
and leaf number reduced sharply when the concentration increased to 7.5 g/L. The
results showed that after 3 selections, soybean shoots of MTD 760-4 still
maintained viability and growth to NaCl concentration of 7.5 g/L, although shoot
growth was low when cultured at high salt concentrations. Soybean shoots grew
slowly but were still alive, a trait showed that soybean shoots were able to
maintain growth under adverse conditions. According to Queiros et al. (2007),
reduced growth was a common phenomenon of saline stress plants observed in
organs, tissues, or cells cultured on medium with NaCl supplementation.

13


Table 4.7: Shoot formation rate (%) of embryo axes and shoot growth of soybean
MTD 760-4 affected by NaCl at 3 weeks after cultured in the selection time 1, 2
and 3
NaCl
concentration
(g/L)
0
2.5
5
7.5
Mean
F
CV (%)

Selection time 1
Shoot
Shoot
formation height
rate (%)
(cm)
65.0a
36.7b
38.3b
25.0c
41.3
**
29.4

3.51a
2.47b
1.52c
0.66d
2.10
**
33.6

Selection time 2
Increased
Increased
shoot
leaf
height
number
(cm)
6.51a
2.15a
2.04b
1.30b
0.92c
0.55c
d
0.28
0.30c
2.43
1.08
**
**

24.2
26.6

Selection time3
Increased
Increased
shoot
leaf
height
number
(cm)
7.53a
2.25a
2.47b
1.30b
0.53c
0.85c
c
0.16
0.40d
3.03
1.31
**
**
16.9
21.4

Data of percentage was tranferred into form of Arcsin√x before statistically analyzed. Means sharing
the same letters within column are not significantly different from each other; (**): statistically
significant difference at 1%.

Soybean shoots selected on saline medium were subcultured into MS
medium supplemented with 50 ml/L coconut water and 0.2 mg/L NAA but without
NaCl salt to multiply. Results showed that some saline treated shoots grew
normally (average height >= 2.0 cm) after 2 weeks cultured. Table 4.30 proved
that shoots on NaCl salt treatment medium of 7.5 g/L were still alive but its growth
was very slow. Plantlets were unable to grow on non-saline MS medium (0%). At
NaCl concentration of 5 g/L, some plantlets had a rapid recovery and growth but
the rate was also very low (6.7%), reaching only four plantlets/60 explants. At a
salt concentration of 2.5 g/L, the number of plantlets obtained higher with six
plantlets (10.0%). Shoots at 5 g/L NaCl had the highest proline content with 3.10
µmol/g fresh weight, different from that at 2.5 g/L (2.45 µmol) and the 0 g/L
concentration gave the lowest proline content of shoots (1.90 µmol). Shoots at 2.5
g/L NaCl also had significantly different proline content compared to the control
treatment.
Table 4.30: Number of salt tolerant soybean at NaCl concentrations
Treated Salt tolerant Rate (%)
Proline content
NaCl concentration
embryo axis
plantlet
(µmol/g fresh
(g/L)
number
number
weight)
2.5
60
6
10.0

1.90c
5
60
4
6.7
2.45b
7.5
60
0
0.0
3.10a
F
**
CV (%)
9.0
Means sharing the same letters within column are not significantly different from each other; (**):
statistically significant difference at 1%.

Experimental results presented that soybean shoots after selected had the
tolerant ability to salinity at NaCl concentrations of 2.5 and 5 g/L (Figure 4.2).

14


a

b

Figure 4.2: Soybean lines after selected for salt tolerance were
mutiplicated on MS + 50 ml/L coconut water + 0.2 mg/L NAA

Line NaCl 2.5 g/L (a)

Line NaCl 5 g/L (b)

4.3.3 Experiment 8: Effect of gamma irradiation Co60 and NaCl on callus
growth of soybean MTD 760-4
Results of Table 4.8 revealed that after 4 selection times, both irradiation
dose and NaCl concentration affected the survival rate of callus. In the selection
time 4, irradiation dose of 40 Gy resulted in significant reduction in callus vitality
(50.5%). Salt concentration increasing from 5 g/L considerably reduced this
parameter. The result was lowest at NaCl concentration of 7.5 g/L (4.6%) (Figure
4.3).
Table 4.8: Survival rate of callus (%) affected by gamma irradiation Co60 and
NaClat 5 weeks after cultured in the selection time 1, 2, 3 and 4
Selection
Selection
Selection
Selection
Irradiation dose
time 1
time 2
time 3
time 4
0 Gy
79.1ab
92.9a
84.1a
60.7a
a
bc

ab
5 Gy
86.5
80.8
80.6
70.4a
ab
ab
a
10 Gy
79.8
86.8
82.7
65.2a
bc
c
ab
20 Gy
70.0
74.0
72.8
60.2a
c
c
b
40 Gy
64.5
74.0
67.8
50.5b

NaCl concentration (g/L)
99.6a
100a
100a
98.8a
NaCl 0
a
a
a
NaCl 2.5
94.4
98.8
99.2
95.2a
66.4b
75.5b
65.2b
51.0b
NaCl 5
c
c
c
NaCl 7.5
43.5
53.2
45.9
4.6c
FIrradiation dose
**
**

*
**
FNaCl
**
**
**
**
FIrradiation dose x NaCl
**
**
ns
*
28.9
24.0
30.3
27.2
CV (%)
Data of percentage was tranferred into form of Arcsin√x before statistically analyzed. Means sharing
the same letters within column are not significantly different from each other; (ns): not significantly
different; (*) and (**): statistically significant difference at 5% and 1%.

Proline content got the highest at salt concentration of 5 and 7.5 g/L (2.32
and 2.42 mol/g fresh weight, respectively) which was indifferent from each other
but different from the control (1.36 mol). Non-irradiated callus and callus

15


irradiated with 10 Gy and selected with salinity of 5 g/L NaCl had the highest
proline content with 3.62 and 2.86 mol, respectively.

a

b

c

d

Figure4.3: Survival level of soybean callus MTD 760-4 after 5 weeks
cultured in the selection time 4
0 Gy + NaCl 0 g/L (a) 10 Gy + NaCl 2,5 g/L (b) 10 Gy + NaCl 5 g/L (c) 10 Gy + NaCl 7,5 g/L (d)

Table 4.9: Proline content of callus (mol/g fresh weight) affected by gamma
irradiation Co60 and NaCl after 4 selection times
Irradiation dose
NaCl concentration(g/L)
Mean
(Gy)
0
2.5
5
7.5
0
0.85e
1.10e
3.62a
1.85
e
e
5

1.05
0.76
1.52cde
2.42bcd
1.44
10
1.28de
1.61cde
2.86ab
1.91
20
2.49bc
1.30de
1.31de
1.70
40
1.12e
1.69cde
2.33bcd
1.71
Mean
1.36b
1.29b
2.32a
2.42a
FIrradiation dose
ns
FNaCl
**
FIrradiation dosex NaCl

**
CV (%)
40.7
Means sharing the same letters within column are not significantly different from each other; (ns): not
significantly different; (**): statistically significant difference at 1%.

Briefly, by Co60 gamma irradiation method, after 4 selection times, it could
get callus lines of soybean MTD 760-4 which were salt tolerant to NaCl
concentration of 5 g/L from explants irradiated with 10 Gy dose as well as from
explants with non-irradiated.
4.3.4 Experiment 9: Effect of gamma irradiation Co60 and NaCl on shoot
formation and growth from embryo axes of soybean MTD 760-4
Experiment 9a: Effect of gamma irradiation Co60 and NaCl on shoot
formation and growth in the selection time 1
As shown in Table 4.10, the survival of embryo axes did not affect by
irradiation dose while shoot formation rate and shoot height influenced. At
irradiation of 60 Gy, explants did not form shoots. NaCl concentration of 5 g/L

16


began to reduce the survival rate of embryo axes and shoot formation as well as
growth.
Table 4.10: Shoot growth of soybean MTD 760-4 affected by gamma irradiation
Co60 and NaCl at 6 weeks after culturedin the selection time 1
Survival rate
Shoot formation
Shoot height
Irradiation dose
(%)

rate (%)
(cm)
a
a
60.9
25.0
1.58
0 Gy
20 Gy
76.6
18.8a
0.34c
a
40 Gy
57.8
12.5
0.45b
60.9

0.0b

-

NaCl 0
NaCl 5

92.2a
71.9b

31.3a

12.5b

1.19a
0.45b

NaCl 7.5
NaCl 10

40.6c
51.6c

9.4bc
3.1c

0.38b
0.30b

ns
**
**

**
**
**

**
**
**

42.9

79.7

23.3

60 Gy
NaCl concentration (g/L)

FIrradiation dose
FNaCl
FIrradiation dose x NaCl
CV (%)

Data of percentage was tranferred into form of Arcsin√x before statistically analyzed. Means sharing
the same letters within column are not significantly different from each other; (ns): not significantly
different; (**): statistically significant difference at 1%.

Experiment 9b and 9c: Effect of gamma irradiation Co60 and NaCl on
shoot growth in the selection time 2 and 3
In the selection time 2 and 3, shoot growth was still very low and almost no
growth (Figure 4.4). After 3 selection times, the surviving shoots from these saline
treatments were transferred into MS medium without salt to get shoot
multiplication. However, all of these explants still could not recover and grow
normally. Thus, by the Co60 gamma irradiation method with doses of 0, 10, 20, 40
and 60 Gy combined with saline treatment through 3 selective cycles, the results
did not obtain saline tolerance lines. The reason may be because the number of
explants processed was not much while the frequency of mutation was low causing
the mutant lines had not been collected.

17

Table 4.11: Increased shoot height affected by gamma irradiation Co60 and NaCl at
6 weeks after cultured in the selection time 2 and 3
Irradiation dose and
Selection time 2
Selection time 3
NaCl concentration (g/L)
0 Gy + NaCl 0
5.88a
5.91a
b
0 Gy + NaCl 5
0.10
0.20b
b
20 Gy + NaCl 5
0.45
0.25b
b
20 Gy + NaCl 7.5
0.23
0.13b
b
40 Gy + NaCl 5
0.25
0.15b
b
40 Gy + NaCl 7.5
0.25

0.23b
Mean
2.66
2.79
F
**
**
CV (%)
22.9
10.4
Means sharing the same letters within column are not significantly different from each other; (**):
statistically significant difference at 1%.

a

b

c

Figure 4.4: Effect of gamma irradiation Co60 and NaCl on shoot
growth of soybean MTD 760-4
0 Gy + NaCl 5 g/L (a)

20 Gy + NaCl 5 g/L (b)

20 Gy + NaCl 7.5 g/L (c)

4.3.5 Evaluating the genetic difference of salt tolerant soybean lines
4.3.5.1 The genetic difference of salt tolerant callus lines of soybean MTD
760-4 after selected by somaclonal variation creating and Co60 gamma

irradiation method
Analysis results of ISSR-PCR technique with 10 primers indicated that five
primers of ISSR02, ISSR03, ISSR13, ISSR19 and ISSR27 showed no genetic
difference between two callus explants treated with 5 g/L NaCl using somaclonal
variation creating and Co60gamma irradiation method compared to the untreated
control. There were four primers of ISSR31, ISSRK1, ISSRK2 and ISSRK3 that
did not give PCR products. Only one primer of ISSR22 (well 1-3) expressed a
clear difference between the two saline-selected callus lines compared to the
untreated control with a DNA band of about 450 bp did not appear in the selected

18


callus explants with NaCl of 5 g/L and callus irradiated with 10 Gy combined with
salt treatment of 5 g/L NaCl while the control had the appearance of this band
(Figure 4.5). The result proved that there was a change in genetic structure of
MTD 760-4 callus after selected with NaCl salt.

Figure 4.5: Electrophoresis photograph of PCR product with ISSR22
primer (well 1-3) and ISSR27 (well 4-6)
L: standar 100 bp; well 1, 4: 0 Gy + NaCl 0 g/L (control); well 2, 5: NaCl 5 g/L; well 3, 6:
10 Gy + NaCl 5 g/L

ISSR has been used for detection of genetic polymorphism and
discrimination between soybean cultivars (Yang et al., 1996). Mahgoub et al.
(2016) reported that ISSR markers associated with salt tolerance in different
soybean cultivars under salinity stress condition.
Therefore, after 4 selection times, the results achieved one callus line which
was tolerant to NaCl of 5 g/L by somaclonal variation method and one line by Co60
gamma irradiation (dose of 10 Gy).

4.3.5.2 The genetic difference of salt tolerant plantlet lines of soybean
MTD 760-4 after selected by somaclonal variation creating method
The analytical result with ISSR22 primer in Figure 4.6 showed that most
soybean explants obtained after salinity selection at NaCl concentration of 2.5 g/L
(six plantlets) did not express any genetic difference compared to the control
(without NaCl treatment). However, when treated saline at NaCl concentration of
5 g/L, one soybean plantlet (1/4 plantlets) gave the difference with a DNA band of
about 1,250 bp did not appear and a DNA band at 480 bp was found. This proved
that there was a possibility that a DNA fragment of about 770 bp had been mutated
missing. The result indicated that somaclonal variation method performed on
shoots derived from embryo axes had an ability to produce soybean lines tolerant
to NaCl salt.

19


Figure 4.6: Electrophoresis photograph of PCR product with ISSR22
M: standar 1000 bp, well 1: NaCl 0 g/L (Control); well 2-7: NaCl 2.5 g/L; well 8-11: NaCl 5 g/L

To sum up, after 3 selection times, the experiment result obtained one plantlet
line of MTD 760-4 soybean that was tolerant to salinity of 5 g/L NaCl. This
soybean line had a genetic difference compared to the control.
4.3.6 Experiment 10: Evaluation the growth and development of salt
tolerant soybean lines in the greenhouse
Table 4.12 indicated that soybean line of MTD 760-4 that was salt tolerant to
NaCl concentration of 5 g/L normaly grew with no difference in comparison with
salt untreated control plantlets after acclimatized and irrigated with saline water in
the greenhouse. Meanwhile, control two lines were affected on plant height,
internode number and leaf with turning into yellow colour when irrigated with
saline water of NaCl 2.5 and 5 g/L (Figure 4.7).

Table 4.12: Increased shoot height (cm) and internode number affected by saline
water irrigation
Increased shoot
Increased
Plantlet lines + Irrigated water
height (cm)
internode
(NaCl concentration-g/L)
number
Line NaCl 0 + Tap water
19.67a
3.00a
c
Line NaCl 0 + Saline water NaCl 2.5
9.53
2.33bc
c
Line NaCl 0 + Saline water NaCl 5
9.14
2.20c
b
Line NaCl 2.5 + Saline water NaCl 2.5
16.58
2.75abc
ab
Line NaCl 5 + Saline water NaCl 5
17.88
2.83ab
Mean
14.75

2.64
F
**
*
CV (%)
23.8
24.5
Means sharing the same letters within column are not significantly different from each other; (*) and
(**): statistically significant difference at 5% and 1%.

On rice, the study result of Nguyen Quoc Khuong et al. (2018) also presented
that saline water irrigating leaded to the decrease of plant height and shoot number
on pot. Arcoding to De Pascale et al. (2013) and Plaut et al. (2013), saline
irrigation water has an adverse effect on soil–water–plant relations, occasionally
severely restricting the normal physiological activity and productive capacity of

20


the crops. Under high salinity level, the growth of many crops are negatively
affected due to osmotic effect, water deficit, nutritional imbalance, and oxidative
stress (Kim et al., 2008).
a

b

c

d

Figure 4.7: Growth of soybean lines in saline water irrigating
condition at 4 weeks after planted
Line NaCl 0 + Tap water (a)
Line NaCl 0 + Saline water NaCl 5 (b)
Line NaCl 2.5 + Saline water NaCl 2.5 (c)
Line NaCl 5 + Saline water NaCl 5 (d)

In general, through two methods of somaclonal variation creating and Co60
gamma irradiation combined with selection on NaCl supplemented medium on
two explants of callus and embryo axes of soybean MTD 760-4, the results
showed that creating somaclonal variation by culturing on saline medium could
achieve callus lines as well as soybean shoots derived from embryo axes that were
tolerant to salinity at NaCl concentrations of 5 g/L. Both callus as well as shoot
explants had genetic differences compared to the untreated controls when analyzed
by molecular marker of ISSR22. The Co60 gamma irradiation method on callus
explants was also effective for creating callus lines tolerant to salinity of 5 g/L
NaCl at dose of 10 Gy with different genetic analysis results compared to the
control explant, however, the non-irradiated explants also achieved salinity
tolerance at this concentration. Therefore, in terms of effectiveness, it is possible to
choose the method of creating somatic variation because it is easy to implement
and cheaper than the Co60 gamma irradiation method whereas both shares the same
effect.
For selective material, callus is often used because it is more sensitive to
mutant agents due to the ability to create mutation is higher in the early stages of
individual formation and development (young embryos or callus) (Duong Tan

21


Nhut, 2009). However, the use of callus is also limited because it takes time to

prepare the material, the callus is required to regenerate into a complete plant and
the long-lasting maintenance of callus on culture medium may have problems such
as a lack of regeneration (Tal, 1994) and appearing of other somatic variation.
Especially for plants that are hard to regenerate into plantlets from callus explants,
the method of callus selection will face many difficulties. Meanwhile, the
treatment method on the embryo axis explants can regenerate shoots that are
tolerant to salinity with faster time and easier to perform than on callus. Another
material is the shoot tip which is also used extensively in some species such as
potato (Das et al., 2000; Yaycili and Alikamanoglu, 2012), apple (Bahmani et al.,
2012) or seeds on citrus (Ling et al., 2008) and soybean (Kumari et al., 2007).
Therefore, the thesis results suggest that it is possible to use somaclonal
variation creating method on embryo axis explants to produce salt tolerance lines
in soybean MTD 760-4. The summary diagram of this method was described in
Figure 4.22.

22


MS medium + 50 ml/L coconut
water + 0.2 mg/L NAA

Substrates of coco (fiber) dust
+ rice husk ash + soil (1:1:1)

Figure 4.22: Diagram of somaclonal variation creating method to achieve salt
tolerant plantlet lines ofsoybean MTD 760-4 from embryo axis explants
Chapter 5: CONCLUSION AND SUGGESTION
5.1 Conclusion
- Among soybean varieties popularly cultivated in the Mekong Delta, MTD
748-1, DH 4 and MTD 720 had the high salt tolerant ability at 4 g/L NaCl when

evaluated by hydroponic method. MTD 878-3 variety was sensitive to salt and
MTD 760-4 completely died at this salt concentration.
- In selection for salt tolerance, the intolerant variety which was MTD 760-4
formed salt tolerant callus and plantlet lines.
+ The method of creating somaclonal variation on callus achieved one callus
line of soybean MTD 760-4 which had salt tolerance to NaCl dose of 5 g/L, with
results of DNA analysis by ISSR22 marker showed that there was no appearance
of DNA band about 450 bp in this in comparison to control explants with none salt
treatment.

23