DEPARTMENT OF BIOLOGICAL AND ENVIRONMENTAL SCIENCES

IDENTIFICATION OF SALT TOLERANT WHEAT LINES An analysis of EMS-mutagenized spring wheat through root/shoot assays and hydroponic cultivation

Josefin Winberg

Degree project for Master of Science with a major in Environmental ScienceES2500, 30 HECAdvanced levelSpring term 2019Supervisor: Henrik AronssonExaminer: Johan UddlingReport number:

AbstractSoil salinization is a growing environmental issue, limiting agricultural yields in countries all over the World. One of the most severely affected regions are coastal areas of Bangladesh, a densely populated part of the country where soil salinity imposes a great threat towards food security. The situation is projected to be further exacerbated with climate change, raising an urgent need for suitable salt-tolerant crops and vegetables available for exposed farmers. Wheat is a commonly known crop in Bangladesh, the second most important source of calories but the leading crop as a source of protein. To increase the salt-tolerance of the Bangladeshi spring wheat BARI Gom-25 variety, a non-transgenic method called induced mutational breeding have been used in a current research project at University of Gothenburg. Wheat seeds were treated with ethyl methanesulphonate (EMS) to induce random mutations and then screened to identify lines with characteristics like salt tolerance. 70 lines have been identified as more salt tolerant in germination tests with 200 mM NaCl, and these lines have in this thesis been subject for further tests to investigate their root and shoot development when germinated in 50 mM, 100 mM, and 150 mM NaCl. The results show that the mutagenized wheat lines on average produce significantly longer roots than the control for all tested salinities. Three lines, OA5, OA44, and OA52, stood out as they were found among the groups with best root and shoot development in at least two out of three salt concentrations. These lines are recommended to be prioritized in future work of the research group. Furthermore, the results suggest that hydroponic cultivation can be an efficient method for testing long-term salt tolerance. The comparison of solutions indicated that the so-called Rahman et al. solution was the best adapted to be used in a non-circulated, aerated hydroponic system, and the germination time of wheat seedlings is suggested to range from 4-7 days. When evaluating the salt-tolerance of the mutagenized wheat seedlings, measurements of chlorophyll levels in shoots and photosynthetic capacity have proven to be good measurements. The findings all together indicates that EMS-mutagenized wheat is one step closer towards an accessible salt tolerant food crop that is not genetically modified, a product that is important for food security in currently salinized areas and in regions that are likely to become saline with a changing climate.

SammanfattningFörsaltning av jordar är ett växande miljöproblem, något som begränsar jordbruksskördar i länder runt om i världen. En av de värst drabbade regionerna är Bangladeshs kustområden, en tätbefolkad del av landet där matsäkerheten hotas av saltskadad jordbruksmark. Situationen är beräknad att förvärras genom klimatförändringar, något som påskyndar det redan brådskande behovet av salttåliga grödor som är lämpliga och tillgängliga för de utsatta bönderna. Vete är en välkänd gröda i Bangladesh och rent näringsmässigt är det den näst viktigaste källan till kalorier samt den gröda som ger mest protein. För att öka salttoleransen hos den bangladeshiska vårvetesorten BARI Gom-25 har så kallad mutationsförädling inducerats av en aktiv forskningsgrupp på Göteborgs universitet. Vetefrön har behandlats med etylmetansulfonat (EMS) vilket har gett upphov till slumpmässiga mutationer, och dessa mutanter har sedan testats för att identifiera egenskaper som salttålighet. 70 linjer har pekats ut som mer salttoleranta i grobarhetsförsök med 200 mM NaCl och dessa linjer har använts i denna uppsats för att för att vidare undersöka rot- och skottutvecklingen när fröerna gror i 50 mM, 100 mM och 150 mM NaCl. Resultaten visar att de muterade vetelinjerna generellt sett producerar signifikant längre rötter än kontrollen för alla testade salthalter. Särskilt tre linjer, OA5, OA44 och OA52, utmärkte sig då de återfanns bland grupperna med bäst rot- och skottutveckling för minst två utav de tre salthalterna. Dessa linjer rekommenderas att bli prioriterade i forskningsgruppens framtida arbete. Vidare föreslår studiens resultat

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hydroponisk odling som en effektiv metod att testa långsiktig salttolerans. Jämförelsen av näringslösningar antydde att den lösning som baserats på studien av Rahman et al. (2016) var bäst anpassad att användas i icke-cirkulerande, syresatta hydroponiska system och att veteskotten bör gro mellan fyra till sju dagar. När salttoleransen hos de muterade vetelinjerna ska testas har mätningar av klorofyllhalter i skotten samt fotosyntesens kapacitet visat sig vara behjälpliga. Slutsatsen från denna studie indikerar att EMS-muterat vete är ett steg närmare en salttålig och ätbar gröda som inte är genmodifierad. Detta är en viktig produkt ur matsäkerhetsperspektiv i områden som i dag är försaltade, men också i områden som riskerar att få saltskadade jordar i ett förändrat klimat.

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Table of contentAbstract......................................................................................................................................1

Sammanfattning........................................................................................................................1

1. Introduction.......................................................................................................................41.1 Soil salinization and salt stress......................................................................................................41.2 Salinization and agriculture in Bangladesh...................................................................................51.3 Reclaiming saline soils...................................................................................................................71.4 Induced mutational breeding using ethyl methanesulphonate (EMS)...........................................71.5 Research objective.........................................................................................................................8

2. Methodology......................................................................................................................82.2 Root and shoot assay......................................................................................................................82.3 Hydroponic experiments................................................................................................................92.4 Measurement of plant performance.............................................................................................11

2.4.1 Chlorophyll concentration in leaves.....................................................................................112.4.2 Measurements of photosynthetic performance.....................................................................11

2.5 Data analysis................................................................................................................................12

3. Results..............................................................................................................................123.1 Root and shoot assay....................................................................................................................123.2 Hydroponic experiments..............................................................................................................16

3.2.1 Comparison of solutions.......................................................................................................163.2.2 Germination time of seedlings..............................................................................................183.2.3 Hydroponic salt stress...........................................................................................................18

4. Discussion........................................................................................................................204.1 Root and shoot assay....................................................................................................................204.2 Hydroponic solutions...................................................................................................................21

5. Conclusion.......................................................................................................................23

6. Acknowledgements.........................................................................................................23

7. References........................................................................................................................24

8. Appendix..........................................................................................................................268.1 Hydroponic solutions...................................................................................................................26

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1. IntroductionThis thesis has been conducted in an on-going research project at the University of Gothenburg and Lund University that aims to develop salt tolerant bread wheat (Triticum aestivum) varieties using chemical mutagenesis. From analyses of the market, research and development infrastructure, salinized agricultural area, and political stability, Bangladesh has been selected as a country of special interest and potential for the research project. By creating new, salt tolerant and locally adapted wheat varieties, saline soils can be reclaimed, and even restored, for agricultural production and contribute to increased food security.

1.1 Soil salinization and salt stressSoil salinization is a rapidly increasing environmental problem that disables or decreases food production in all parts of the world. Salinization is the accumulation of soluble salts in the root zone of a soil, mainly neutral salts like sodium chloride (NaCl) and sodium sulphate (Na2SO4) (FAO & ITPS, 2015). A soil is classified saline when the electric conductivity (EC) exceeds 4 dS/m, equivalent to 40 mM NaCl (FAO & ITPS, 2015; Rahman et al., 2016). As seen in Fig. 1, salinization occurs at all continents and in all climates but is more common in coastal areas and on irrigated lands in arid and semiarid areas where rainfall is insufficient to leach salts out of the soil (SRDI, 2010). Globally, more than 800 million ha are affected by salinization, which account for nearly 7% of the total land area in the world (FAO, 2018). When only considering irrigated land, 45 million ha out of the 230 million ha worldwide have so far been salinized (Hanin et al., 2016).

Fig. 1 Extent of saline and sodic soils in the World (Zheng, 2014).

Salinization of soils can occur naturally from rock weathering, marine sediments, tidal inundation, and atmospheric deposition from sea or dry lake beds (FAO & ITPS, 2015). It can also be induced by human activities like poor drainage that cause the ground water table to rise, irrigation with brackish water, seawater intrusion resulting from overuse of ground water

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in coastal areas and from sea level rise, poor irrigation and water management techniques, and land use changes that lead to the rise of saline ground water (FAO & ITPS, 2015).

High salt concentrations in soil is a major abiotic stressor for plant growth and development, resulting in decreased agricultural crop productivity and quality (Liang et al., 2018). Plants are typically most sensitive to salt during germination and early seedling growth, a limitation for the establishment of plants on saline soils (Hussain et al., 2013). Salt stress mainly exposes the plant to increased osmotic imbalance caused by reduced ability to take up water (the water potential is balanced by losing water) and to ion toxicity from high influx of Na+

and Cl- to the plant cell, which in turn can cause oxidative stress and a series of other secondary stresses (Liang et al., 2018; Hanin et al., 2016). The complex stress responses all together inhibit photosynthetic processes and plant growth, leading to a reduction in root length, plant height, seed germination and fruitification of plants (Liang et al., 2018). To manage the consequences of salt stress, plants have to activate several physiological and biochemical responses like changes in the morphology, anatomy, water balance, photosynthesis, hormones, biochemical adaptation, and toxic ion distribution (Acosta-Motos et al., 2017).

1.2 Salinization and agriculture in Bangladesh Soil salinity is one of the major environmental constraints for farmers in the coastal districts of Bangladesh (Clarke et al., 2015). Low-lying coastal areas cover more than 30% of the cultivable lands in the country (SRDI, 2010), and about half of these are affected by salinity of varying degrees caused by tidal flooding during wet season, direct inundation by saline or brackish water, and intrusion of saline ground and river water during dry season (Haque, 2006). According to the Soil Resources Development Institute (SRDI, 2010) the extent of soil salinity in coastal areas has had an average increase of 0,74% per year, resulting in 223 000 ha of new land becoming salinized between 1973 and 2009 (Fig. 2). Seawater inundation is already a great problem for traditional farming in coastal areas of Bangladesh, contributing to an important reduction of the yields (IPCC, 2014). Future prospects suggest that soil salinization will be further exacerbated in coastal Bangladesh with climate change as temperatures, rainfall, sea level, and riverine flows from the Himalayas are altered (Dasgupta et al., 2015).

Agriculture is a major sector of the economy in Bangladesh (21% of national GDP) and serves as the main source of employment for almost half of the population (UNSD, 2017). The saline coastal areas already experience much lower crop yields, production levels, cropping intensity, and quality of livelihood than other parts of the country, at the same time as food demand of these areas is increasing with population growth (SRDI, 2010). As a consequence, many salinized agricultural regions have been turned into shrimp farms to profit from the salinity (Haque, 2006). However, this measure is not only decreasing the agricultural lands, but also increase soil salinity ever further (Khanom, 2016). As one of the most densely populated countries in the world, Bangladesh already struggles to feed its 164 million inhabitants (UNSD, 2017). Worsened soil salinization will have significant socio-economic implications and food security issues for these areas and their inhabitants (Clarke et al., 2015). In addition, it will also negatively affect the national economy.

During the winter (November-February), which is the dry season in Bangladesh, soil salinity becomes more severe as the soil desiccates (Haque, 2006). If the land is not in fallow, the winter crops grown are commonly wheat, boro rice, potato, and winter vegetables (Hossain & Teixeira da Silva, 2012). Wheat is, after rice, the second most important grain crop and source

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of calories in Bangladesh, but the leading crop as a source of protein (Hossain & Teixeira da Silva, 2012).

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Fig. 2 Increase in areas affected by different levels of soil salinity in coastal Bangladesh from 1973-2009 (source SRDI, 2010).

1.3 Reclaiming saline soils To halt the spreading of salinization and to reclaim soils that already has been salinized, sustainable management is required. Salinization can be prevented through methods like embankments, levelling of land, improved irrigation/drainage, and more sustainable farming methods (SRDI, 2010; Haque, 2006). For the already salinized soils measures like direct leaching of salts (for example excess irrigation), chemical treatments (e.g. gypsum and acids), soil organic improvements (addition of organic matter), phytoremediation (cultivation of plants that take up salt from the soil), domestication of native wild halophytes for use in agro-pastoral systems, or planting salt tolerant varieties are suggested (FAO & ITPS, 2015).

Increased salt tolerance of crops has drawn more attention in the last years as it can lessen the costs and resources needed from remediation as well as increase the production on saline soils, and it is seen as one of the most important challenges for modern agriculture (Hanin et al., 2016). The difficulty lies in that salt tolerance is a multi-gene genetic feature that commonly is regulated by multiple genes and dozens of physiological traits (Liang et al., 2017). According to Munns & Tester (2008) there are three primary responses that contribute to plant salt tolerance: Na+ exclusion, tissue tolerance, and osmotic stress tolerance. In other words, the plants either survive high salt concentrations by getting rid of the Na+ from the leaves, by compartmentalize Na+ and Cl- to protect the cytoplasm from toxic ion levels, or by reducing the responses to osmotic stress. For wheat, the responsible genes that control these traits and the salt tolerance are not fully mapped yet as wheat is hexaploid (the wheat cell has six copies of its seven chromosomes) with a large and complex genome (the largest among common agricultural crops) (Gill et al., 2004). The approaches to improve the salt tolerance of wheat is many, with examples like exploitation of natural genetic variation, crossings with more salt-tolerant progenitors, hybridization with halophytic wild relatives, and usage of transgenic techniques to introduce new genes or to alter expressions of existing genes (Yamaguchi & Blumwald, 2005; Colmer et al., 2005). Conventional plant breeding has contributed to the development of stress tolerance for wheat varieties but have had limited success to develop a crop with the necessary salt tolerance traits that is commercially available (Hanin et al., 2016). Biotechnological approaches have therefore caught attention as it can accelerate the evolutionary process to express new traits and in the case of wheat, where the genes controlling the salt tolerance is not fully known, a method called Targeting Induced Local Lesions IN Genomes (TILLING) offers several advantages (Slade & Knauf, 2005). TILLING is a non-transgenic and reverse genetics method that uses chemical mutagenesis followed by screening of mutants to detect variations in genes of interest (Dong et al., 2009).

1.4 Induced mutational breeding using ethyl methanesulphonate (EMS)

7Fig. 3 EMS-induced mispairing of guanine and thymine (from Griffiths et al., 2000). Treatment with EMS causes mispairing when guanine (G) is alkylated to O6-ethylguanine, which can pair with thymine (T) but not with cytosine (C). When the DNA is replicated the G-C pair is switched to an A-T pair.

Starting from the commonly grown Bangladeshi spring wheat BARI Gom-25, which already is fairly well adapted to heat and salt stress, 2700 lines have been raised with a mutated genome from ethyl methanesulphonate (EMS) treatment. EMS cause randomly distributed changes in the wheat genome, mainly by inducing mispairing through alkylation (G pairs with T) followed by base changes (C-to-T), as illustrated in Fig. 3 (Kim et al., 2006). The DNA code is built up by C (cytosine)-G (guanine) base pairs and A (adenine)-T (thymine) base pairs, and with EMS mutagenizes several C-G pairs are randomly switched into A-T pairs, giving a new order of the DNA code with new traits like for example salt tolerance (Kim et al., 2006).

The initial EMS mutagenized population (M1) was allowed to self-pollinate for five generations (M5) to scale up the number of seeds and to allow for continued mutations and to wait out gene silencing. From the fourth (M4) and fifth (M5) generation did 70 lines indicate salt tolerance when germinated at 200 mM NaCl on petri dishes. These lines are currently subject for screening and further tests in lab and in field trials, not only for salt tolerance but also for other desired characteristics like high protein content and slowly degradable carbohydrates. As an indicator of salt tolerance root and shoot development has been used. Simultaneously, work is on-going in the research group to identify the genes responsible for the salt tolerance in the mutagenized wheat.

In the screening of the salt-tolerant lines hydroponic cultures has been identified as a research method of interest as the salinity of the growing media is easy to adjust in comparison to soil. Hydroponic cultivation is a fundamental and recognized research tool to study plant response and tolerance to abiotic stresses like salt stress (Shavrukov et al., 2012). However, the research group have yet no established method for hydroponic cultivation of wheat to investigate the salt stress tolerance.

1.5 Research objectiveThe aim for this thesis is to:

Identify the lines with best root and shoot development under salt stress from EMS-mutated GOM-25 wheat seeds from the 5th, 6th and 7th generation through seed germination experiments.

Develop a methodology for testing salt tolerance of the mutagenized lines of spring wheat through hydroponic cultivation.

2. Methodology

2.1 Plant materialThe wheat seeds used in the experiments of this thesis are the Bangladeshi spring wheat BARI Gom-25, the Swedish spring wheat Dacke, and 70 different lines of EMS-mutagenized Gom-25. The mutagenized lines are from the 5th, 6th and 7th generation of plants grown in greenhouses in Lund and Gothenburg. The wheat has reproduced through self-pollination.

2.2 Root and shoot assayBy germinating wheat seeds in seed germination pouches (PhytoTC, Medium size) under different levels of salt stress, the influence of salt on root and shoot development during germination was tested. The pouches were prepared with 20 ml of NaCl-solution (NaCl dissolved in deionized water) with salt concentrations of 50 mM, 100 mM, or 150 mM. Each pouch contained five seeds placed with the embryo facing down. The prepared pouches were

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placed vertically in racks in a growth chamber (Master Plant, CLF Plant Climatics with settings: temperature day: 21°C/night: 19°C, humidity: 70%, photoperiod: 16 h light/8 h dark, light intensity: 230 μmol/m2/s) to germinate for seven days (Fig. 4). The pouches were then removed from their plastic cover and photographed together with a meter-stick as a reference of distance.

The number of roots per seed plus root and shoot length of each seed were measured with segmented lines in the software ImageJ 2.0 (Fig. 5). The root number and root length were combined to a measurement called root development, and this indicated the total root length of each seed (mean root length*number of roots). This way the lines with both long roots and many roots per seed would be seen as the strongest lines. The scale of measurement was set by using the meter-stick in the image as a reference to indicate how many pixels that were equivalent to a known distance. The number of roots and the root length per seed was combined to a joint root measurement named root development (number of roots*mean root length).

Initially, Gom-25 seeds were used in five replicates of each NaCl concentration to test the experiment design. The experiment was later conducted with the same design but with the 70 lines of EMS-mutagenized wheat and Gom-25 as control. For some lines there were very few seeds available, and in that case the higher salt concentrations were prioritized for testing. That is why 66 lines were tested for 50 mM, 69 lines for 100 mM, and 70 lines for 150 mM NaCl. The lines were not tested with 0 mM NaCl as this data already existed from earlier experiments in the research group and all lines germinated well. Due to space limitations in the growth chamber, all lines could not be tested at once. Instead five rounds of experiments were carried out, each time with a new control of Gom-25. When comparing the performance of mutagenized lines to the control, the average of the five Gom-25 results was calculated and used as control.

2.3 Hydroponic experimentsIn hydroponic systems plants are grown in a nutrient solution instead of soil, a method that makes it easier to control the salt concentration of the growth medium. A variety of systems and nutrient solutions can be used, and here four different types of solutions have been tested in non-circulated systems. One of the solutions has earlier been used in the research group but was originally composed for Arabidopsis plants, and is therefore called Arabidopsis solution. However, when this solution was used in initial hydroponic tests the seedlings wilted and turned yellow. This initiated the search after a more suitable hydroponic solution and the

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Fig. 5 Manual measurement of roots in ImageJ 2.0Fig. 4 Prepared seed pouches germinating in growth chamber.

Arabidopsis solution was thereafter used diluted to half the strength. Another solution tested was a ready-made NPK mixture with micronutrients from IKEA (Växer) mixed according to instructions for hydroponic growing. The two other solutions were based on two different research papers, Rahman et al. from 2016 that have tested different types of Bangladeshi wheat under salt stress in hydroponic culture, and Shavrukov et al. from 2012 that have developed a protocol specifically adapted to hydroponic growth of wheat and barley. The Arabidopsis, Rahman, and Shavrukov solutions were prepared according to instructions in the lab at University of Gothenburg. The content of each solution can be found in Attachment 1, whereas in Table 1 the distribution of the main essential macro- and micronutrients are presented. The concentration in Table 1 is expressed in molarity (moles/L), which for the Arabidopsis, Rahman, and Shavrukov solutions could be read from their protocols. For the IKEA solution, the main nutrients were only expressed in percentage, and these were converted into molarity based on the indicated density of the solution (1,26 g/ml).

Table 1 Concentration of macro- and micronutrients in the hydroponic nutrient solutions tested.Elements Arabidopsis

solutionDiluted Arabidopsis solution

IKEA Växer solution

Rahman et al. solution

Shavrukov et al. solution

N 7,20 mM 3,60 mM 4,50 mM 2,00 mM 9,60 mMP 0,80 mM 0,40 mM 0,27 mM 0,10 mM 0,10 mMK 4,80 mM 2,40 mM 2,14 mM 0,10 mM 5,13 mMCa 1,20 mM 0,60 mM - 0,50 mM 2,00 mMMg 0,80 mM 0,40 mM - 0,20 mM 2,00 mMS 0,81 mM 0,41 mM - 0,20 mM 2,00 mMSi 0,50 mM 0,25 mM - - -Cl 800,00 μM 400,00 μM - 11,10 μM 29,00 μMFe 0,08 μM 0,04 μM 11,28 μM 2,00 μM 100,00 μMB 37,00 μM 18,5 μM 11,65 μM 11,00 μM 12,50 μMMn 8,05 μM 4,00 μM 2,29 μM 2,00 μM 2,00 μMZn 0,61 μM 0,31 μM 0,58 μM 0,35 μM 3,00 μMCu 0,26 μM 0,13 μM 0,40 μM 0,20 μM 0,50 μMMo 0,55 μM 0,28 μM 0,13 μM 0,70 μM 0,10 μMNi - - - - 0,10 μM

Spring wheat seeds from the Swedish type Dacke and the Bangladeshi type GOM-25 were placed in Petri dishes with 9 cm Ø filter paper to germinate (Fig. 6). Each petri dish was prepared under sterile conditions in a fume hood with five seeds and 10 ml of distilled water and was sealed with surgical tape. The seeds were germinated for four or seven days in a plant growth chamber (Master Plant, CLF Plant Climatics) with the following settings: temperature day: 21°C/night: 19°C, humidity: 70%, photoperiod: 16 h light/8 h dark, minimum light intensity: 200 μmol/m2/s.

When germinated (t=0 days), the seedlings were transferred to Styrofoam trays with cut Eppendorf tubes (Fig. 7). The seeds with the most developed sprouts and roots were selected. The Styrofoam trays were placed in 10-liter plastic tanks filled with the hydroponic solution. To prevent algae growth, the tanks where covered with aluminium foil. The tanks were kept in a plant growth chamber (Percival, CLF Plant Climatics) with the following settings: temperature day: 21°C/night: 19°C, humidity: 70%, photoperiod: 16 h light/8 h dark, minimum light intensity: 200 μmol/m2/s. The hydroponic solutions were kept oxygenated with air bubbling through the system. To keep the concentration of different salts stable a few deciliters of distilled water was added every other day. The pH of each solution tested was

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monitored during the trial (at start and after five days) and was adjusted if needed to a pH between 6 and 6,5 using a NaOH-solution.

After five days in the hydroponic tank (t=5 days) the chlorophyll concentration in the leaves and photosynthetic performance was measured. The seedlings were hydroponically grown for five more days in the same way as described above. On the last day (t=10 days), the same measurements were made again, before finishing the experiment. The measurements from the fifth and the tenth day were first analyzed together and then separately in the comparative analysis.

The solution that gave the best results in the comparative analysis was tested for a final round with four different levels of salt concentration; 0, 50, 100 and 150 mM of NaCl. The methodology was the same as described above, however, after the measurements were taken on the fifth day (t=5 days), the solution was changed to a newly mixed solution of the same type, and salt was added. The final measurements were taken after five additional days (t=10 days).

2.4 Measurement of plant performance

2.4.1 Chlorophyll concentration in leavesTo investigate the level of stress the chlorophyll concentration of the flag leaf of each wheat seedling was measured using a SPAD-502 meter (Konica-Minolta, Tokyo, Japan). For each measurement a numerical SPAD value was calculated by the SPAD-502 meter, proportional to the amount of chlorophyll in the leaf. Each seedling was measured at three places (top, middle and bottom of leaf) with the adaxial leaf side facing the emitting window of the instrument, and an average for each seedling was calculated with the SPAD-502 meter.

2.4.2 Measurements of photosynthetic performanceTo quantity the well-being of the wheat seedlings, a chlorophyll fluorimeter can be used that measures the efficiency of photosystem I and II. Here a Pocket PEA (Hansatech Instruments, Norfolk, United Kingdom) was used for measuring the parameters Fv/Fm and Performance Index (PI). Fv/Fm is the ratio of variable fluorescence over the maximum fluorescence value

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Fig. 7 Seedlings in the hydroponic system.

Fig. 6 Seeds germinating in Petri dishes in growth chamber.

and indicates the maximum quantum efficiency of Photosystem II. The Performance Index or PI gives an indication of plant vitality by reflecting the efficiency of both Photosystem I and II. The Pocket Pea leaf clip was used to dark-adapt the samples for approximately 15 minutes before measurements were made. One measurement per seedling was taken from the adaxial leaf side of the flag leaf. The values that were obvious outliers or were error measurements according to the Pocket PEA have been removed from the analysis.

2.5 Data analysisThe results from the seed pouch experiments were analyzed in Microsoft Excel and statistically tested using the SPSS statistical package. The data was tested for normal distribution with a Shapiro-Wilk’s test. For analyzing variance among performance in the different salt concentrations an independent-samples Kruskal-Wallis test was used. An independent-samples Mann-Whitney U-test was used to compare variance in root and shoot development between mutagenized lines and the non-mutagenized control. A one-way ANOVA were used for testing the variance of Fv/Fm, PI, and SPAD measurements between the different hydroponic solutions, as well as in the final test of hydroponic salt stress. Independent-sample t-tests were used to compare the variance among Gom-25 and Dacke in the hydroponic salt stress experiment.

3. Results3.1 Root and shoot assayA Shapiro-Wilk’s test (p>0.05) and a visual inspection of the statistics for the root length showed that the data was not normally distributed for any of the salt concentrations [50 mM (M = 27.65, SE = 0.61), 100 mM (M = 16.24, SE = 0.49), 150 mM (M = 8.61, SE = 0.34)]. The non-parametric independent-samples Kruskal-Wallis test was therefore used, and its output showed that there is a statistically significant effect (p<0.05) of the salt concentration on the root development (number of roots*mean root length) of all tested wheat lines.

When the same tests were done for shoot development (length of shoot) a similar result was given. The data for shoot development did not follow normal distribution for any of the salt concentrations [50 mM (M = 4.25, SE = 0.12), 100 mM (M = 2.24, SE = 0.09), 150 mM (M = 1.11, SE = 0.06)]. The independent-samples Kruskal-Wallis test showed that salt concentration has a statistically significant effect (p<0.05) on shoot length. All together the results show, as expected, that salt has a negative effect on the root and shoot development of the control and the EMS-mutagenized wheat.

When independent-samples Mann-Whitney U-tests were conducted to compare root development of non-mutagenized and EMS-mutagenized wheat, there was a significant difference (p<0.05) for all three salt concentrations; 50 mM [U = 2670, p = 0.003], 100 mM [U = 3010, p = 0.012], and 150 mM [U = 3332, p = 0.046). In other words, the mutagenized wheat lines had significantly better root development than the control (Table 2, Figure 8).

Table 2 Comparison of mean root and shoot development between mutagenized and non-mutagenized wheat lines for three different salt concentrations. NaCl conc. Group Mean root

dev. (cm)Std deviation

Sign. (U-test)

Mean shoot dev. (cm)

Std deviation

Sign. (U-test)

50 mM Mut. 28,13 11,39 0,003* 4,294 2,232 0,210Not mut. 21,36 10,14 3,606 1,645

100 mM Mut. 16,57 9,49 0,012* 2,278 1,743 0,102

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Not mut. 11,77 8,78 1,778 1,686150 mM Mut. 8,77 6,616 0,046* 1,138 1,208 0,273

Not mut. 6,39 6,173 0,773 0,790* Significant difference p<0,05

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Fig. 8 Variation in a) root development and b) shoot development for three different salt concentrations. Bar 1 (left) include the mutagenized lines and Bar 2 (right) is the control/not-mutagenized line.

a)

b)

When the same tests were run for shoot development, there was no significant difference (p>0.05) between the mutagenized and non-mutagenized wheat lines for any of the salt concentrations; 50 mM [U = 3506, p = 0.210], 100 mM [U = 3468, p = 0.102], and 150 mM [U = 3801, p = 0.273]. This indicates that the mutagenized lines do not produce significantly longer shoots than the control.

The most salt tolerant mutagenized lines were identified by statistical analysis of the mean root development and mean shoot development for each line. In total, 56 lines out of total 66 lines had a higher mean for root development than the control for 50 mM, for 100 mM NaCl it was 54 lines out of 69, and 48 lines out of 70 for 150 mM NaCl. This data was assumed to be normally distributed. All lines with better root development than the control (non-mutagenized Gom-25) were statistically analyzed with independent-samples t-tests for each salt concentration. The results show that 19 lines had significantly better (p<0.05) root development than the control for 50 mM, 14 lines for 100 mM, and 11 lines for 150 mM (Table 3).

Table 3 Mean root development and standard deviation for the mutagenized lines with significantly better (p<0.05) root development than the control. The asterisk (*) indicates the lines that are significantly better for all three salt concentrations.

50 mM 100 mM 150 mMLine Mean root

dev. (cm)Std dev. Line Mean root

dev. (cm)Std dev. Line Mean root

dev. (cm)Std dev.

Control 21,35 10,14 Control 11,77 8,78 Control 6,39 6,17OA45 33,14 10,70 OA36 21,10 11,34 OA64 12,29 2,88OA5* 33,27 8,63 OA64 21,71 8,74 OA16 12,90 5,62OA2 33,42 2,97 OA62 21,73 7,54 OA50* 13,17 5,76OA71 33,48 8,60 OA3 22,61 4,58 OA29 13,26 7,09OA24 33,82 10,41 OA40 22,85 9,71 OA6 13,87 3,41OA63 34,52 15,40 OA45 23,26 15,15 OA14 14,49 5,44OA52* 35,04 3,83 OA53 23,47 13,68 OA8 15,66 5,48OA16 35,06 13,30 OA5* 23,77 12,37 OA52* 15,69 5,30OA10 35,73 7,77 OA50* 25,17 7,85 OA10 17,82 8,21OA49 36,15 10,68 OA31 25,83 12,98 OA44* 18,39 7,10OA31 36,73 12,99 OA70 26,61 5,92 OA5* 19,63 8,78OA19 36,96 17,63 OA44* 28,20 5,06OA39 37,97 9,96 OA52* 28,33 6,96OA66 38,09 9,18 OA43 30,26 6,36OA43 38,65 12,06OA28 38,79 12,64OA37 39,35 13,42OA50* 41,50 2,64OA44* 48,53 11,99

Four lines stood out that had significantly better root development than the control for all three salt concentrations (OA5, OA44, OA50 and OA52). OA44 was the only line that for all salt concentrations performed among the third best for root development. In addition, it stood out as it showed markedly better root and shoot development than the control (Fig. 9).

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When comparing the shoot development of the lines with longer average shoot length than the control the results look somewhat different. Out of the 66 lines that were tested for 50 mM NaCl 33 lines had longer mean shoot length than the control, for 100 mM it was 32 out of 69 lines, and 46 out of 70 lines for 150 mM. These lines were assumed to be normally distributed and statistically analyzed as described above with independent-samples t-tests to find the lines that had significantly better shoot development than the control. The results show that 14 lines had significantly longer shoots than the control for 50 mM NaCl, 11 lines out of the 32 lines for 100 mM, and four lines out of the 46 lines for 150 mM (Table 4). However, no lines had significantly longer shoot length for all three salt concentrations. The lines that showed significantly better shoot development for two of the concentrations are OA5, OA8, OA16, OA39, OA43, OA44, OA52, and OA71.

Table 4 Mean shoot development and standard deviation for the mutagenized lines with significantly better (p<0.05) shoot development than the control.

50 mM 100 mM 150 mMLine Mean shoot

dev. (cm)Std dev.

Line Mean shoot dev. (cm)

Std dev.

Line Mean shoot dev. (cm)

Std dev.

Control 3,60 1,65 Control 1,78 1,69 Control 0,77 0,79OA5 5,48 1,13 OA43 3,53 1,89 OA39 2,23 1,09OA66 5,50 1,79 OA71 3,77 1,33 OA8 2,30 1,16OA49 5,62 2,92 OA3 3,78 1,28 OA6 3,24 1,01OA46 5,65 0,95 OA16 3,91 2,59 OA5 3,42 2,02OA43 5,95 1,94 OA36 4,03 2,10OA16 6,07 2,44 OA52 4,06 1,12OA28 6,09 2,01 OA31 4,11 2,29OA52 6,34 1,59 OA8 4,17 1,04OA50 6,37 0,94 OA45 4,43 2,72OA62 6,38 2,71 OA44 4,70 1,55OA39 6,71 2,55 OA70 5,05 1,14OA71 7,42 0,86OA44 8,02 2,13

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Fig. 9 Root and shoot development of the a) control and b) line OA44 for 50, 100 and 150 mM NaCl.

a) b)

OA10 8,30 1,51

3.2 Hydroponic experiments

3.2.1 Comparison of solutionsThe measured SPAD values of chlorophyll concentration in leaves were assumed to be normally distributed and were run in a one-way ANOVA (F(3,148)=0.435, p=0.728). The results showed that there was no significant difference (p>0.05) between the SPAD values of the four solutions when the 5th and 10th day measurements were analyzed together. The post hoc Tukey HSD test confirmed the same result. However, a comparison of the means (see Table 5) show that the Rahman et al. solution gives the highest SPAD value (M=35.458, SD=5.459) followed by the Shavrukov et al. solution (M=35.047, SD=5.835). The lowest values were seen for the weak Arabidopsis solution (M=34.018, SD=7.079). When the last day (day 10) measurements were analyzed separately with a one-way ANOVA test (F(3,72)=0.949, p=0.422) the results look somewhat similar, but the averages for the IKEA solution (M=35.247, SD=5.606) and the Rahman et al. solution (M=36.732, SD=6.055) have had a noteworthy increase, and so has the standard deviation for these values.

Table 5 Comparison of average and standard deviation of SPAD, Fv/Fm, & PI values for all solutionsHydroponic

solutionMeasurement

dayAverage SPAD

Std dev.

Average Fv/Fm

Std dev.

Average PI

Std dev.

IKEA Växer 5+10 10

34,5235,25

4,825,61

0,800,80

0,010,01

2,572,88

0,920,92

Diluted Arabidopsis

5+10 10

34,0233,33

7,087,30

0,790,78

0,020,02

2,032,19

0,660,68

Rahman et al. 5+10 10

35,4636,73

5,466,05

0,800,81

0,020,02

3,103,90

1,791,24

Shavrukov et al. 5+10 10

35,0535,47

5,846,05

0,780,78

0,020,02

2,062,14

0,690,73

For the Fv/Fm measurements, normal distribution was assumed as well, and a one-way ANOVA (F(3,148)=11.474, p=0.000) followed by a post hoc Tukey HSD test showed that the IKEA solution (M=0.801, SD=0.014) gave significantly higher (p<0.05) Fv/Fm values than both the weaker Arabidopsis solution (M=0.786, SD=0.016) and the Shavrukov et al. solution (M=0.780, SD=0.015) when the measurements from day 5 and 10 were analyzed together. The Rahman et al. solution (M=0.795, SD=0.021) gave only significantly better Fv/Fm results than the Shavrukov et al. solution. The measurements from day 10 follow the same pattern in the one-way ANOVA (F(3,72)=13.813, p=0.000) and the values has not changed much as seen in Table 5. However, the post hoc Tukey HSD show that the increase in the Fv/Fm values for the Rahman et al. solution (M=0.807, SD=0.018) and the decrease of the weak Arabidopsis solution (M=0.782, SD=0.020) resulted in that the Rahman et al. solution gave significantly better (p<0.05) Fv/Fm values than both Shavrukov et al. solution (M=0.780, SD=0.015) and weak Arabidopsis solution. The IKEA solution (M=0.801, SD=0.014) continued to give significantly higher (p<0.05) Fv/Fm values than both the Shavrukov et al. solution and the weak Arabidopsis solution.

The PI values were assumed to follow normal distribution. The statistical analysis of the PI values from the 5th and the 10th day combined show, according to the one-way ANOVA (F(3,148)=7.746, p=0.000), that the highest PI values resulted from the Rahman et al. solution. The post hoc Tukey HSD test states that the Rahman et al. solution (M=3.095, SD=1.787) gave significantly (p<0.05) higher values than both the weak Arabidopsis solution

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(M=2.033, SD=0.657) and the Shavrukov solution (M=2.061, SD=0.689). No other differences in means were significant when analyzing the measurements for day 5 and 10 together. The results from the 10th day were also analyzed with a one-way ANOVA

(F(3,71)=14.793, p=0.000) and the results show that the PI values for the Rahman et al. solution (M=3.902, SD=1.242) have increased more than for the other three solutions. The post hoc Tukey HSD test tells that the PI values of the Rahman et al. solution are significantly higher (p<0.05) than all the other solutions. No other differences in means were significant (p>0.05).

When considering the results above, the Rahman et al. solution, followed by the IKEA solution, could be pointed out as the best solutions to use for this methodology in hydroponic growth of wheat, out of the four solutions tested. The statistical tests does not clearly suggest which of these two solutions that are better than the other. However, the results indicate that the Rahman et al. solution gave slightly better values. A visual comparison of the

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performance of the wheats in the two solutions show, as seen in Fig. 10, that the wheat growing in the Rahman solution grew taller than the wheat in the IKEA solution. The IKEA solution on the other hand gave slightly greener wheat and longer roots, when compared to the Rahman et al. solution. However, the length of shoots and roots were not measured so no statistical analysis could support these results.

3.2.2 Germination time of seedlingsTo test if the number of days that the seeds have germinated before being placed in the hydroponic system had any influence on the results above, the variance in results between the seeds that had germinated for four days and the seeds that had germinated for seven days were tested. Independent-samples t-tests were made to compare the SPAD, Fv/Fm, and PI results for the two germination periods but there were no significant difference (p>0.05) between any of the results for four day germination period and seven day germination period; SPAD results [t(150)=-0.830, p=0.408], Fv/Fm results [t(150)=-1.533, p=0.127], and PI results [t(150)=-1.017, p=0.311]. However, when means were compared one could see that the seven day germination period gave slightly higher values; SPAD four days (M=34.39, SD=6.00), SPAD seven days (M=35.17, SD=5.63), Fv/Fm four days (M=0.79, SD=0.02), Fv/Fm seven days (M=0.79, SD=0.02), PI four days (M=2.35, SD=0.95), PI seven days (M=2.54, SD=1.40).

3.2.3 Hydroponic salt stressAs the Rahman et al. solution gave some of the better results in the comparative analysis, this solution was used in an additional test of salt stress for non-mutagenized Gom-25 (a salt tolerant variety) and Dacke seedlings with 0, 50, 100, and 150 mM NaCl. The data was assumed to be normally distributed. As expected, a one-way ANOVA test showed that the salt concentration had a statistically significant effect (p<0.05) on all measurements (Fv/fm [F(3,13) = 25.389, p = 0.000], PI [F(3,13) = 9.955, p = 0.001], SPAD [F(3,16) = 8.424, p = 0.001]) of the Dacke seedlings.

A following Tukey’s HSD post hoc test clarified that the Fv/fm values were significantly lower (p<0.05) for the 150 mM (M= 0.716, SD= 0.018) when compared to all other values (0 mM (M= 0.813, SD= 0.008), 50 mM (M= 0.801, SD= 0.013), and 100 mM (M= 0.775, SD= 0.024)). The measurements for 100 mM were significantly lower than the 0 mM measurements (p= 0.019), whereas no significant difference (p>0.05) occurred between the other concentrations. For the Gom-25 seedlings the salt level had no significant effect on the Fv/Fm values (p>0.05). In Figure 11 (a) the averages of the Fv/fm values for the two wheat varieties are presented separately. As seen, the values for Dacke follow a negative correlation of Fv/Fm with increased salinity, but the values for Gom-25 do not show the same pattern, indicating that the maximum quantum efficiency of Photosystem II is less affected by salinity in Gom-25 than in Dacke. However, the difference in their overall performance is not statistically significant according to an independent samples t-test (t(34)=0.845, p=0.407).

When analyzing the PI values of Dacke seedlings it showed that 100 mM (M= 2.474, SD= 0.918) and 150 mM (M= 0.871, SD= 0.183) gave significantly lower (p<0.05) values than the control 0 mM (M= 4.908, SD= 1.133). Likewise, the 150 mM gave significantly lower (p = 0.007) PI value than the 50 mM (M= 4.152, SD= 1.440). There was no significant difference (p>0.05) between any other concentrations. For Gom-25 the one-way ANOVA test showed that there is an effect of salinity on the PI values (F(3,15)= 3.917, p=0.030). The Tukey’s HSD post hoc test revealed that the significant difference (p= 0.021) occurred between the 0 mM (M=4.375, SD=1.305) and the 50 mM (M=2.190, SD=0.791). No other differences in means were significant (p>0.05).

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When studying Figure 11 (b) and the variance in means of the two types of wheat, the results look similar to the ones for Fv/fm. The efficiency of both Photosystem I and II (PI) of the Dacke seedlings follow a clear trend of being negatively impacted by the increased salinity, but the Gom-25 values fluctuate much more, even if a similar trend can be seen. An independent samples t-test show that the values of the two varieties does not have a significant difference statistically (t(34)=-0.485, p=0.631).

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Fig. 11 Variation in a) Fv/fm values, b) PI values and c) SPAD values for Gom-25 (Wheat 1) and

a)

b)

c)

The SPAD values for Dacke at the NaCl concentrations 150 mM (M= 33.480, SD= 4.492) and 100 mM (M= 39.440, SD= 5.631) showed to be significantly lower (p<0.05) than the 0 mM control (M= 49.980, SD= 4.002). The other concentrations did not show a significant difference (p>0.05) from each other. As seen in Figure 11 (c) the SPAD values of Dacke are slightly higher than the values of Gom-25, and an independent samples t-test show that the difference between the two varieties is significant (t(38)=-2.829, p=0.007). However, when the Gom-25 values are compared to the Dacke values, their chlorophyll content seem to be less negatively influenced by the salinity. A one-way ANOVA test on the Gom-25 values confirm this as it showed that salinity did not have a significant effect on the SPAD values of Gom-25 (F(3,16)=2.560, p=0.091).

4. Discussion4.1 Root and shoot assaySalt stress is a major environmental limitation of plant growth, inhibiting factors like root length and plant height (Liang et al. 2018). The results of this thesis are consistent, showing that salt concentrations of 50, 100, and 150 mM limits the root and shoot development of both the control and the mutagenized wheat when germinated in root pouches. More interestingly, the mutagenized wheat showed significantly better root development than the control for all three salt concentrations. In other words, on average the EMS-treated wheat produces longer and/or more roots. This does, however, not mean that the mutagenized wheat is not affected by increased salt concentration as the development of shoots and roots are decreased with higher salinity, but it does indicate that they produce more root biomass in salt conditions than the control and therefore have a greater likelihood to survive. Acosta-Motos et al. (2017) suggest that an increased root biomass under saline conditions can help the plant to hold the toxic ions below ground and avoid translocation to the aerial parts as well as an improvement in the water and nutrient uptake. An increase in shoot development is more complex as a larger photosynthetic capacity and transpiration increases the water use by the plant, and thereby reduces soil moisture which can contribute to a further rise in soil salinity and exacerbated salt stress (Hanin et al., 2016). However, these functions are the foundation of plant growth and survival, and thereby a necessary trait for a salt-tolerant crop. Further research and close monitoring of how cultivation of salt-tolerant crops affect the salt levels in the soil in a long-term perspective is essential to investigate if these crops is a short-term solution that only contribute to more severe salinity or if they can be a part of the remediation of the soil.

To explore the values within the average of the mutagenized lines each line was statistically analyzed and compared to the control. This way the mutagenized lines with the best root and shoot development were identified. Four lines (OA5, OA44, OA50, and OA52) showed significantly better root development for all three salt concentrations, and eight lines (OA5, OA8, OA16, OA39, OA43, OA44, OA52, and OA71) showed significantly better shoot development for two of the three salt concentrations. The three lines OA5, OA44, and OA52 are found in both the group of best root development and the group of best shoot development. This could be seen as an indication that they grow better than the control in saline conditions. To be certain of these results, replicated test would be needed to exclude sources of error with methodology and materials, together with more long-term experiments so see how these lines are influenced by salinity while they mature and set seeds.

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Fig. 11 Variation in a) Fv/fm values, b) PI values and c) SPAD values for Gom-25 (Wheat 1) and

As the mutagenized seeds are able to grow in salinity of 150 mM NaCl, equivalent to around 16 dS/m (categorized as highly saline), it means that they have the potential to grow in 90% (more than 950 000 ha) of the salt affected coastal areas in Bangladesh (where salinity is less than 16,0 dS/m) according to data from 2009 by SRDI (2010). Hence, land that today is in fallow or abandoned due to salinization could be re-cultivated and produce food for the growing population of Bangladesh. In addition, Bangladesh is one of the most negatively affected countries from climate change, where crop production is stressed from floods, salt intrusion, climate variability and droughts (Arfanuzzaman et al., 2016). The need for salt-tolerant varieties will most likely increase in the future, not only for countries like Bangladesh where salinization is a widespread environmental issue, but also for countries like Sweden where climate change can, and might already, contribute to warmer and dryer conditions in which a need for irrigation with brackish water becomes reality (Qadir et al., 2007; Wimmerberg, 2018).

EMS-mutagenized wheat has the advantage to be non-transgenic (no new genes are inserted) and is based on reverse genetics, meaning that no knowledge of the gene product is needed, and it results in an end product not classified as a genetically modified plant (Dong et al., 2009). In other words, the EMS-mutagenized wheat is currently not included in the strict GM legislation and can therefore be grown as any other conventional crop in most countries. Arfanuzzaman et al. (2016) argue that salt-tolerant varieties that are accessible and affordable for farmers is an important part of successful climate adaptation, and this should be one of the main focuses for the EMS-mutagenized wheat when reaching the market. According to Hossain and Teixeira da Silva (2012), a majority of the households in Bangladesh are landless or own less than 0,2 ha of land, making most farmers a vulnerable group to expensive agricultural changes. Salinization of crop land is generally forcing farmers to abandon native and local species to instead grow high yielding varieties with high input of fertilizers and pesticides, a transition that poor farmer communities have a hard time to cope with (Khanom, 2016). These economic aspects should be included when proceeding with the project, together with ecological aspects on how the EMS-wheat can be grown without having further negative impact on the environment and the soils. Also, the social impacts are of great importance and questions like who will access the seeds, can it be cultivated without being a threat towards the livelihoods of the local farmers, is the wheat culturally appropriate and suitable from a health perspective, to name a few, need to be answered.

The idea of using salt-tolerant varieties as a climate adaptation to salt intrusion and/or inundation, opens for an additional and wider discussion on alternatives for climate adaptation. The Bangladeshi government has mainly focused on protective infrastructure as means of adaptation, known as coastal embankment system, to prevent storm surges and salt water intrusion in the coastal agricultural zone (World bank, 2000). This methodology is considered as a part of community-based adaptation (CBA), which are locally driven adaptation strategies to the impacts from climate change that contributes to development, and that should occur in association with methods like saline-crop cultivation, combined aquaculture and crop production, diversified livelihoods e.g. (IPCC, 2014). Whether these strategies are sustainable and resilient in the long run is debatable, but they do provide a solution to use coastal land for food production. Climate adaptation can also be provided by the use of physical characteristics, ecosystem services, and biodiversity that, for example, comes from salt marshes, mangroves, and oyster reefs (IPCC, 2014). This is known as ecosystem-based adaptation, adaptation based on methods that protect and restore coastal natural habitats, and exploration and development of these methods could efficiently protect coastal areas in Bangladesh from inundation, intrusion and erosion in a long-term perspective.

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4.2 Hydroponic solutionsIn the experiments of hydroponic solutions, the Rahman et al. solution stood out as one of the better solutions when comparing the measured Fv/fm, SPAD and PI values. When comparing the content of the Rahman et al. solution and the IKEA solution, which gave the better results, it could be noted that the IKEA solution had more than twice as high nitrogen (N) content as the Rahman et al. solution. This could explain the fact that the wheat that had grown in the IKEA solution were slightly greener. In addition, the Rahman et al. solution also had a higher content of different micro-nutrients, whereas the IKEA solution lacked several of these. The Rahman et al. solution has been specially adapted to grow Bangladeshi wheat like BARI Gom-25 hydroponically, which could explain the result of the study. However, there is always a risk that the results have been influenced by sources of error, for example in the preparation of solutions, in the cultivation of the seedlings (e.g. with aeration of tanks and quality of seedlings e.g.), and in the measurements. To increase the certainty replicated tests would be needed.

The comparison of germination time for seeds showed that longer germination time (7 days instead of 4 days) gave slightly better values of Fv/fm, SPAD, and PI, but the difference was not significant for any of the measurements. However, the older seedlings are easier to place in Eppendorf tubes, but they suffer an increased risk of drying out in the taped petri dish and being hampered in the growth due to limited space. In addition, the longer time the seeds are left to germinate without any nutrients added, the more likely it is that they face a shortage of nutrients when the nutrient reserves inside the seeds are used up. It was noted during the experiment that the Dacke seedlings germinated faster than the Gom-25 and were thereby larger when placed in the hydroponic system. For testing the mutagenized Gom-25 wheat in the future, the settings of the growth chamber might need to be adapted to see if this changes the time of germination.

In the final hydroponic test with salt stress where the Rahman et al. solution was tested, the solution was exchanged before adding the salt. This was based on the evaluation from the initial solution tests, where it could be noted that many of the seedlings had turned yellow. To rule out the factor of nutrient shortage (as this hydroponic system was non-circulating), the solutions were exchanged to fresh ones after half the time. This change in methodology might have influenced the results and raises the question whether the results of the solution test would have looked the same if the solution had been exchanged after half the time. However, the Rahman et al. solution gave some of the best results for all measurements after both 5 and 10 days in the same solution.

The fact that Dacke seeds germinated faster than the Gom-25 seeds can also explain the larger values in many of the measurements of Dacke seedlings (when compared to the Gom-25) grown in the salt stress experiment with the Rahman et al. solution. A more long-term experiment in saline conditions might have ruled out these differences, but the results still showed that the Gom-25 seedlings were less stressed by the salt than the Dacke seedlings. These results could be expected as Gom-25 is a more salt tolerant wheat variety, likewise it confirms that this method of hydroponic cultivation could be used to show difference in salt tolerance of different wheats. In future hydroponic experiments with mutagenized wheat the same measurements of photosynthetic capacity (PI and Fv/Fm) and chlorophyll content (SPAD) are recommended. In particular, SPAD measurements are of interest as chlorophyll levels in salt-tolerant species is increased or unchanged with salinity, while salt-sensitive species show decreased chlorophyll content, suggesting that chlorophyll fluorescence is an interesting measurement to indicate plant salt tolerance (Acosta-Motos et al., 2017).

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5. ConclusionTo combat food insecurity in countries that suffer from soil salinization, wheat lines from the Bangladeshi spring wheat BARI Gom-25 have been mutagenized with ethyl methanesulphonate (EMS) treatment and these are now tested to confirm salt tolerance. The results of this thesis suggest that the tested mutagenized wheat lines are more salt tolerant as they do on average produce more and/or longer roots than the control when germinated in up to 150 mM NaCl. In particular, the three lines OA5, OA44, and OA52 stood out as they were found among the groups with best root and shoot development in at least two out of three salt concentrations (50, 100, 150 mM NaCl). This is a step closer towards finding an accessible salt tolerant food crop that is not genetically modified, a desired product in currently salinized areas and in regions that are likely to become saline with a changing climate.

For further testing of the salt-tolerant lines hydroponic cultures can be recommended. The results of this thesis suggest that the so-called Rahman et al. solution is used in a non-circulated, aerated hydroponic system. The germination time of the wheat seedlings be 4-7 days in a petri dish placed inside a growth chamber, and the settings of this chamber should be adapted to the Bangladeshi spring wheat. When evaluating the salt-tolerance of the mutagenized wheat seedlings, measurements of chlorophyll levels in shoots and photosynthetic capacity have proven to be good measurements.

6. AcknowledgementsI would like to show my gratitude to Lennart Bornmalm, my supervisor Henrik Aronsson and the research team consisting of Johanna Lethin, Ola Nordqvist, and Sameer Hassan, that all supported and helped me during this thesis. Thank you Cornelia Spetea Wiklund, Otilia Cheregi, and Gustav Knutsson for the patient and kind help with hydroponic cultivation and growth chambers, and thanks to Sven Toresson for his assistance with material and tools. I am grateful for the proof reading done by Amber, thank you! Lastly, I would like to thank my partner Joel for endless support, and my friend Florina for providing me with an inspiring workspace, you two made it possible for me to complete the thesis.

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8. Appendix

8.1 Hydroponic solutions

1. Arabidopsis solutionHydroponic solution adapted for Arabidopsis plant experiments. Based on recipe from master thesis of Petter Larsson, 2016, who participated in the Aronsson research group.

Macro solutionSubstance Concentration Molecular weight 1 liter 0,5 literKNO3 1M 101,10 g/mol 101,10 g 50,55 gCa(NO3)2*4H2O 1M 236,14 g/mol 236,14 g 118,07 gKH2PO4 1M 136,09 g/mol 136,09 g 68,05 gMgSO4*7H2O 1M 246,47 g/mol 246,47 g 123, 24 gNH4Cl 1M 53,49 g/mol 53,49 g 26,75 g

Dissolve each substance above in separately with deionized water. Store in separate bottles.

Micro solutionSubstance Molecular weight 1 liter 0,5 literH3BO3 61,83 g/mol 2,860 g 1,430 gMnSO4 151,00 g/mol 1,520 g 0,760 gZnSO4*7H2O 287,56 g/mol 0,220 g 0,110 gCuSO4*5H2O 249,68 g/mol 0,079 g 0,040 gNa2MoO4*2H2O 241,95 g/mol 0,165 g 0,083 gNH4VO3 116,98 g/mol 0,029 g 0,015 g

Add one substance at a time from above and let it dissolve before adding the next one, in deionized water. Store as a solution in a separate bottle. Substance Concentration Molecular weight 1 liter 0,5 literFeNa-EDTA 0,1M 367,05 g/mol 0,0367 g 0,0184 g

Add FeNa-EDTA to deionized water and let it dissolve. Store in a separate bottle.Do not use magnets to mix/dissolve any of the solutions!

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1 l 0,8 0,6 0,5 0,4 0,3 0,2 0,18 0,135

0,10 0,09 0,045

KNO3 400 320 240 200 160 120 80 72 54 40 36 18Ca(NO3)2 120 96 72 60 48 36 24 21,6 16,2 12 10,8 5,4KH2PO4 80 64 48 40 32 24 16 14,4 10,8 8 7,2 3,6MgSO4 80 64 48 40 32 24 16 14,4 10,8 8 7,2 3,6NH4Cl 80 64 48 40 32 24 16 14,4 10,8 8 7,2 3,6FeNa-EDTA

80 64 48 40 32 24 16 14,4 10,8 8 7,2 3,6

μ solution 80 64 48 40 32 24 16 14,4 10,8 8 7,2 3,6H2O 80 64 48 40 32 24 16 14,4 10,8 8 7,2 3,6Total ml 1000 800 600 500 400 300 200 180 135 100 90 45

Dilute this solution to 1:100, i.e. use 1 part of this solution to 99 parts deionized water, to make the finished hydroponic solution. Adjust pH if needed with NaOH or KOH.

2. IKEA Växer nutrient solutionInformation from the product manual accessed from the IKEA webpage (https://www.ikea.com/se/sv/manuals/vaxer-vaxtnaring__AA-1841438-3_pub.pdf)

Solution of NPK fertilizers 5-3-8 with micronutrients Instructions for hydroponic growing, add 4ml of nutrients to 1 litre of water. pH (1 g/l): 5.5 - 6.5Electrical Conductivity (1 g/L): 0,4 mS/mNutrient contentTotal Nitrogen (N): 5 %Nitrate nitrogen: 3.5 %Ammonium nitrogen: 1.5 %Water-soluble phosphorus pentoxide (P2O5): 3%Water-soluble potassium oxide (K2O): 8%Water-soluble MicronutrientsBoron (B): 0.01 %Copper (Cu)*: 0.002 %Iron (Fe)**: 0.05%Manganese (Mn)*: 0.01 %Molybdenum (Mo): 0.001 %Zink (Zn)*: 0.003 %* chelating agent EDTA** chelating agent DTPA

3. Nutrient solution from Rahman et al. 2016Solution used in a study of Bangladeshi wheat varieties grown under salt stress in hydroponic systems.

Sources DosesNH4NO3 500 μM

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Ca(NO3)2 500 μMMgSO4 200 μMKH2PO4 100 μM

FeCl3 2 μMH2BO3 11 μMMnCl2 2 μMZnCl2 0,35 μMCuCl2 0,2 μM

(NH4)6Mo7O4 0,1 μM

All substances were added and dissolved one by one to a glass bottle with distilled water.

4. Nutrient solution from Shavrukov et al. 2012A hydroponic growth solution adapted for cultivation of wheat and barley. Improved composition based on tissue nutrient analysis.

Elements Salts used Final concentration(mM)

NK, NCa, NMg, SP, KSi*

NH4NO3

KNO3

Ca(NO3)2

MgSO4

KH2PO4

Na2SiO3

0,25,02,02,00,10,5

FeB

MnZnCuMoNi

Cl**

NaFe(III)EDTAH3BO3

MnCl2

ZnSO4

CuSO4

Na2MoO3

NiSO4

KCl

(μM)100,012,52,03,00,50,10,125,0

* Silicon is not an essential element and may be omitted from the growth solution, depending on the experiment and plant species grown.** MnCl2 was reduced to 2 M in the improved protocol to optimize Mn nutrition. However, as MnCl2 is the only source of Cl, additional chloride was supplied as KCl to avoid Cl deficiency.

All substances were added and dissolved one by one to a glass bottle with distilled water.

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