1.5 Scientific and practical significance 1.5.1 Scientific significance Through hydroponics and tissue culture technique in combination with somaclonal variation selection and mutagenes
Trang 1MINISTRY 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
Trang 2THE DISSERTATION WAS COMPLETED AT
CAN THO UNIVERSITY
Scientific advisor 1: Assoc Prof Dr Nguyen Bao Toan
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
Trang 3LIST 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 2333), vol Special issue of Agriculture (4), pp 179-188
1859-2 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
Trang 42
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
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
- Evaluating the genetic difference in salt-selected soybean lines by molecular technique
- Acclimatizing and experimentally cultivating soybean lines after selected were in the greenhouse
Trang 51.5 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
Trang 64
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
Trang 7Experiment 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
Trang 86
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 Co 60 and 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 Co 60 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
Trang 9Two factors comprising five Co 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 760-
4 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
Trang 108
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 rate (%)
Survival rate (%) at NaCl 4 g/L
Plant height (cm)
Internode number on main stem
Root length (cm) Varieties
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) NaCl 4 g/L (d)
Trang 11Growth 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
rate (%)
Plant height (cm)
Internode number on main stem
Root length (cm)
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
Trang 1210
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
760-4 had callus formation rate with no difference between concentrations of 2,760-4-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
2,4-D concentration (mg/L) Callus formation rate
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
Trang 13is 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
concentration
(mg/L)
Root formation rate (%)
Root number
Root length (cm)
Shoot height (cm)
Leaf number
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