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 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 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 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 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 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 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 and MTD 720 had the high salt tolerant ability at 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 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 g/L NaCl normally grew after 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 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 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 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, and 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 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 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, and 10 mg/L) and BA concentrations (0, 0.5 and 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 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, and 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 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 Treatments were four NaCl concentrations of 0, 2.5, 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 and The experiments were put in RCD with one factor consisting four treatments which were NaCl concentrations 0, 2.5, 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 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, 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, and 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 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 and The factor was NaCl concentration including three levels considered as treatments (0, 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 and g/L NaCl (result of Experiment 6) and callus irradiated at dose of 10 Gy + g/L NaCl (result of Experiment 8) and eleven shoot explants at g/L NaCl (one explant), 2.5 g/L NaCl (six explants) and g/L NaCl (four explants) from the result of Experiment 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 g/L) in combination with saline water irrigating of 0, 2.5 and 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 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 g/L NaCl concentration and MTD 7604 variety is most sensitive to salinity with plants completely died at weeks after planted NaCl concentrations of and g/L gave significantly different survival rates at 1% compared to those of and g/L NaCl Table 4.1: Growth of soybean varieties MTD 176, MTD 748-1, MTD 760-4, Nhat 17A and OMDN 29 affected by NaCl at weeks after planted Survival Internode Root Survival Plant height rate (%) at number on length rate (%) (cm) NaCl 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 91.0a NaCl 73.0b NaCl 25.0c NaCl * 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 weeks planted NaCl g/L (a) NaCl g/L (b) NaCl g/L (c) NaCl g/L (d) 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 and MTD 720 had high salinity tolerance at 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 and to improve salinity tolerance of non-salt tolerant variety The MTD 760-4 did not survive at 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 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 97.5 33.3b 2,4-D 10 93.3 33.3b BA concentration (mg/L) BA 97.5 100a BA 0.5 96.9 12.8b BA 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 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 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 weeks after cultured NAA Root Root Root Shoot Leaf concentration formation number length height (cm) number (mg/L) rate (%) (cm) NAA 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 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 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, and 3, the concentration of 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 selections, the study achieved one callus line of soybean MTD 760-4 which was 12 tolerant to salinity at a concentration of g/L Callus stabilized vitality at this concentration during the selection time The highest proline content of callus was at 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 weeks after cultured in the selection time 1, 2, and NaCl Proline content Selection Selection Selection Selection concentration (µmol/g fresh time time time time (g/L) weight) 100a 100a 100a 100 1.48b a a a 2.5 100 100 100 100 1.51b b b b 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 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 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 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 weeks after cultured in the selection time 1, and NaCl concentration (g/L) 2.5 7.5 Mean F CV (%) Selection time 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 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 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 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 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 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 10.0 1.90c 60 6.7 2.45b 7.5 60 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 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 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 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 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 weeks after cultured in the selection time 1, 2, and Selection Selection Selection Selection Irradiation dose time time time time Gy 79.1ab 92.9a 84.1a 60.7a a bc ab 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 a a a NaCl 2.5 94.4 98.8 99.2 95.2a 66.4b 75.5b 65.2b 51.0b NaCl 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 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 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 weeks cultured in the selection time Gy + NaCl g/L (a) 10 Gy + NaCl 2,5 g/L (b) 10 Gy + NaCl 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 selection times Irradiation dose NaCl concentration(g/L) Mean (Gy) 2.5 7.5 0.85e 1.10e 3.62a 1.85 e e 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 selection times, it could get callus lines of soybean MTD 760-4 which were salt tolerant to NaCl concentration of 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 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 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 weeks after culturedin the selection time Survival rate Shoot formation Shoot height Irradiation dose (%) rate (%) (cm) a a 60.9 25.0 1.58 Gy 20 Gy 76.6 18.8a 0.34c a 40 Gy 57.8 12.5 0.45b 60.9 0.0b - NaCl NaCl 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 and In the selection time and 3, shoot growth was still very low and almost no growth (Figure 4.4) After 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 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 weeks after cultured in the selection time and Irradiation dose and Selection time Selection time NaCl concentration (g/L) Gy + NaCl 5.88a 5.91a b Gy + NaCl 0.10 0.20b b 20 Gy + NaCl 0.45 0.25b b 20 Gy + NaCl 7.5 0.23 0.13b b 40 Gy + NaCl 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 Gy + NaCl g/L (a) 20 Gy + NaCl 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 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 g/L and callus irradiated with 10 Gy combined with salt treatment of 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: Gy + NaCl g/L (control); well 2, 5: NaCl g/L; well 3, 6: 10 Gy + NaCl 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 selection times, the results achieved one callus line which was tolerant to NaCl of 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 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 g/L (Control); well 2-7: NaCl 2.5 g/L; well 8-11: NaCl g/L To sum up, after selection times, the experiment result obtained one plantlet line of MTD 760-4 soybean that was tolerant to salinity of 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 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 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 + Tap water 19.67a 3.00a c Line NaCl + Saline water NaCl 2.5 9.53 2.33bc c Line NaCl + Saline water NaCl 9.14 2.20c b Line NaCl 2.5 + Saline water NaCl 2.5 16.58 2.75abc ab Line NaCl + Saline water NaCl 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 weeks after planted Line NaCl + Tap water (a) Line NaCl + Saline water NaCl (b) Line NaCl 2.5 + Saline water NaCl 2.5 (c) Line NaCl + Saline water NaCl (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 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 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 and MTD 720 had the high salt tolerant ability at 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 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 + Creating somaclonal variation method on embryo axes obtained one soybean plantlet line that was salt tolerant at NaCl of g/L There was genetic difference in DNA structure of the salt tolerant shoot in comparison with the control with none-salt treatment when analyzed by molecular marker of ISSR22 MTD 760-4 soybean plantlets after selected with g/L NaCl normally grew after weeks acclimatized under saline water irrigating condition in the greenhouse + Co60 gamma irradiation method combined with salt selection with NaCl on callus achieved two salt tolerant callus lines to NaCl dose of 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 + To embyro axes which were gamma irratiated and salt selected, there was not obtained salt tolerant lines 5.2 Suggestion It can be applied the somaclonal variation creating method on embryo axis explants of soybean MTD 760-4 to achieve salt tolerant lines MTD 760-4 plantlets which were salt tolerant to NaCl g/L should be constinuously multiplied for many generations and experimentally cultivated in the saline lands of Mekong Delta to evaluate the genetic stability of salt tolerance as well as observe further other agronomical characteristics such as disease tolerant ability, nodule formation and so on 24 ... University Journal of Science (ISSN 1859-233 3), vol Special issue of Agriculture ( 3), pp 47-54 Chapter 1: INTRODUCTION 1.1 Rationale Soyean (Glycine max (L. ) Merrill) is a food crop which has high econonic... 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... selection time Gy + NaCl g/L (a) 10 Gy + NaCl 2,5 g/L (b) 10 Gy + NaCl 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