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Salt tolerance in wheat

A way to avoid starvation

Emma Hamfeldt Teaching Education Program oriented towards upper secondary school, Biology and mathematics

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Degree project: 15 hp Course: LGBI1G Självständigt arbete (examensarbete) 1 för gymnasielärare

i biologi Institution: Institutionen för biologi och miljövetenskap Göteborgs Universitet Level: Basic Term/year: HT/2015 Instructor: Henrik Aronsson

Instutionen för Biologi och miljövetenskap, OlsAro Crop Biotec AB/ Göteborgs Universitet

Examinator: Bernard Pfeil Institutionen för biologi och miljövetenskap, Göteborgs universitet Code: HT15-3130-002-LGBI1G

Key words: Salt, wheat, Bangladesh 1 Abstract Bangladesh is one of the world’s 40 poorest countries. It is estimated that as much as 1.02 million ha, or 70% of all agricultural land in Bangladesh, is contaminated with salt. Wheat is the second most important cereal of Bangladesh. Using EMS to mimic natural occurring mutations in wheat helps to avoid the strict laws regarding GMOs and acceptation of the product. The aims of this project are to isolate fourth generation (M4) wheat lines of the EMS-treated Bangladeshi wheat species Gom-25, which has high salinity tolerance for NaCl, KCl and MgCl2, and to harvest new M5 lines that will be tested further. Methodical tests were performed to establish what method should be used for identifying salt tolerant lines. The use of 200 mM NaCl on petri dishes (9 cm Ø) with filter paper and no sealing was found to be the best method. Screening tests for NaCl revealed that 40 lines (out of 142) showed a germination level of 50% or higher and 4 lines showed germination levels of 80-90%. Screening tests also showed that KCl negatively affected germination more than NaCl, and that MgCl2 was shown to influence germination (and root production) the most. Further studies needs to be performed and lines showing very high germination frequency should be carefully selected and tested in the forthcoming M5 generation. However, the goal of hindering Bangladesh from starvation by producing a salt tolerant wheat species is definitely viable.

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2 Sammanfattning Bangladesh är ett av världens 40 fattigaste länder. Landytan är 147570 km2 och det upppskattas att 1,02 miljoner ha, ca 70% av all jordbruksmark, är saltkontaminerad. Vete är det näst viktigaste sädesslaget i Bangladesh. Genom att använda EMS för att efterlikna naturliga punktmutationer hos vete slipper man strikta GMO lagar och underlättar acceptansen av produkten. Målet för detta projektet är att isolera linjer från fjärde generationens (M4) EMS behandlat vete från Bangladesh hos sorten Gom-25 som redan har hög tolerans för salterna NaCl, KCl och MgCl2, och även att skörda nya M5 linjer för framtida tester. Metodtester genomfördes för att ta reda på vilken metod som skulle användas för identifiering av salttoleranta linjer och 200 mM NaCl i petriplattor (9 cm Ø) med filterpapper var det bästa. Screeningtester för NaCl visade att 40 linjer (av totalt 142) hade groddfrekvenser på 50% eller mer, och 4 linjer hade groddfrekvenser på 80-90%. Screeningtester visade också att KCl hade större negativ påverkan på groddfrekvensen än NaCl, och att MgCl2 hade störst påverkan (även på rotbildning). Det finns mycket kvar att göra och linjer med väldigt höga groddfrekvenser bör selekteras och testas i kommande M5-generation, men det övergripande målet att minska svälten i Bangladesh genom att odla fram ett salttolerant vete är absolut genomförbart!

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3 Table of contents 1 Abstract ................................................................................................................................... 1

2 Sammanfattning ..................................................................................................................... 2

3 Table of contents .................................................................................................................... 3

4 Introduction ............................................................................................................................ 4

4.1 Bangladesh ...................................................................................................................... 4

4.2 Wheat .............................................................................................................................. 4

4.3 Different salts .................................................................................................................. 4

4.4 Non-GMO ....................................................................................................................... 5

4.5 Aims ................................................................................................................................ 5

5 Materials and methods .......................................................................................................... 6

5.1 Method Test .................................................................................................................... 6

5.2 Tape Test ........................................................................................................................ 6

5.3 Standard Curves .............................................................................................................. 6

5.4 NaCl-Screening ............................................................................................................... 7

5.5 KCl- and MgCl2-Screening ............................................................................................. 7

5.6 Harvest of M5 line .......................................................................................................... 7

6 Results ..................................................................................................................................... 7

6.1 Method Test .................................................................................................................... 7

6.2 Tape Test ........................................................................................................................ 8

6.3 Standard Curves .............................................................................................................. 8

6.4 NaCl-Screening ............................................................................................................. 10

6.5 KCl- and MgCl2-Screening ........................................................................................... 11

7 Discussion .............................................................................................................................. 11

8 Conclusion ............................................................................................................................. 12

9 Future perspectives .............................................................................................................. 13

10 Pedagogical angle of approach ............................................................................................ 14

11 Acknowledgements ............................................................................................................... 15

12 References ............................................................................................................................. 15

13 Appendix A ........................................................................................................................... 17

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4 Introduction 4.1 Bangladesh The land area of Bangladesh is 147,570 km2, and 70% out of the total land area is agricultural land (The World Bank, 2015). The most saline affected areas of the country are along the sea coast, where floods and tide water have a large effect on the salinity levels in the soil. Salinity surveys have shown that around 1,02 million ha (~70%) of the agricultural land is affected with different degrees of soil salinity (Haque, 2006). With a Gross Domestic Product (GNP) per capita of $1,092 USD between the years 2010-2014, Bangladesh ranks among the 40 poorest countries in the world (The World Bank, 2015). The high percentage of salinity-affected soils, the low GNP per capita, increasing dependence on wheat, along with the previous and existing contacts with Bangladesh, made this country an ideal target for this project.

Figure 1. Map of the coastal regions of Bangladesh. Illustrating the different degrees of salinity in the soil in 2009. Source: http://www.srdi.gov.bd/

4.2 Wheat Rice is the most important cereal crop in most parts of Asia, and wheat comes second after rice in Bangladesh (Rahman & Hasan, 2007). All plants differ greatly in their tolerance of salt, where lettuce and Arabidopsis are among the more sensitive plants and cereals are typically more tolerant. Within the cereals, barley can stand the highest levels of salt and rice the lowest levels. Wheat has a fairly high tolerance for salts and can survive on NaCl concentrations of 150-200 mM in lab tests (Munns & Tester, 2008). In addition, the salt tolerance of wheat has been shown to increase as the plant ages (Yamaguchi & Blumwald, 2005). Plants within one specific line have the same genotype. 4.3 Different salts Salt stress affects plants in two different ways. High levels of salts inside the plant are toxic to most plants. The exact toxic effect that high salt concentrations have on plants is not completely known. High salinity levels in the ground will make it harder for the roots to soak up water. Salts within the plant take a long time to reach toxic levels, whereas salts in the soil affect plants almost immediately, limiting cell growth (Munns & Tester, 2008). Both effects will lead to limited growth of the plant. Zhu (2001) specifies that “The integrity of cellular membranes, the activities of various enzymes, nutrient acquisition and function of

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photosynthetic apparatus are all known to be prone to the toxic effects of high salt stress”. He also mentions that a likely cause for the damage on the inside of the plant are reactive oxygen species (ROS), which are generated from salt stress. Immediately after the concentration of salts has reached a certain threshold, the growth rate of the shoots decreases considerably. Germination frequency of the seeds is affected both by the osmotic pressure, i.e., the total amount of dissolved salts, and also by the specific type of salt (Ryan, Miyamoto & Stroehlein, 1975). Tavakkoli, Rengasamy and McDonald (1997) mentions a range of different salts that can be found dissolved in saline soils, among which are the salts NaCl, KCl and MgCl2 that were used in different tests. 4.4 Non-GMO Genetically Modified Organisms (GMOs) can be defined as organisms that have received genetic material from other organisms, through recombinant DNA technology (Brooker, 2012). GMOs are also called transgenic organisms, where transgenic is derived from the term transgene, meaning a gene that has been introduced from one species to another. To induce mutations in Gom-25, the Bangladesh wheat variety used in this project as starting material, the chemical mutagen ethyl methanesulfonate (EMS) was used. EMS is an alkylating agent used to produce mutations and changes in nucleotides, resulting in base changes and mispairing (Kim, Schumaker & Zhu, 2006). In almost every case, using EMS will lead to C-to-T changes in the DNA strain, which in turn will result in C/G to T/A substitutions. This type of mutations occur naturally in the different cells of any organism and are called spontaneous mutations (Brooker, 2012) Since Gom-25 hasn’t received any new genetic material from another organism, but instead had its own DNA altered, it is not classified as a GMO. This fact is very beneficial to the project, since one does not have to follow the strict laws and guidelines that apply to use and handling of GMO plants. Another advantage is that the final product will be accepted in Bangladesh, because it will not be labeled as GMO plants. With the example of Golden Rice (a GMO plant with increased level of vitamin A), developing countries have a history of not accepting GMO plants, most likely due to scientific ignorance (Potrykus, 2001). 4.5 Aims My aims for this project are to:

• find M4 wheat lines that are able to tolerate high concentrations of sodium chloride (NaCl)

• from the M4 wheat lines that were highly tolerant of NaCl find lines that are also tolerant of potassium chloride (KCl) and magnesium chloride (MgCl2)

• harvest and collect seeds from the M5 wheat lines

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5 Materials and methods 5.1 Method Test A test was performed to find out what method would be best to use in the screening tests later on. The methods tested were:

1. 10 ml water with 200 mM NaCl and filter papers in a petri dish (9 cm Ø) 2. Nutrient agar with 200 mM NaCl in a petri dish (9 cm Ø) 3. 10 ml distilled water and filter papers in a petri dish (9 cm Ø) 4. Water agar in a petri dish (9 cm Ø)

To each of the methods, 5 petri dishes were placed with 5 seeds of Dacke (a Swedish spring wheat variety commonly used by Swedish farmers) on each. The same procedure was performed with Diskett (another Swedish spring wheat variety commonly used by Swedish farmers), Gom-25A, Gom-25B and Gom-25C (letters indicate different mutated lines of Gom-25 seeds). One petri dish for each method was placed with 5 seeds of the wild-type Gom-25 variety on each plate. The plates were placed in growth chambers at 21 degrees Celsius and screened for results after 6 days. Only seeds showing sprouts were counted as germinated. 5.2 Tape Test A tape was performed to find out if it was necessary to seal the petri dishes before incubation, i.e., to avoid contamination by bacteria. Ten petri dishes were placed with 10 ml water with 200 mM NaCl and 9 cm filter papers. Five seeds of Gom-25A were placed on each of the petri dishes. Five of the petri dishes were sealed with surgical tape and 5 were left unsealed. A total of 10 petri dishes were placed in growth chambers and screened for results after 7 days. 5.3 Standard Curves Standard curves were performed to evaluate which NaCl concentration should be used later on in the screening tests. With 5 seeds of Dacke on each dish, 10 petri dishes were placed with 9 cm filter papers and 10 ml water with 50 mM NaCl. The same procedure was performed using concentrations of 100 mM, 150 mM, 200 mM, 250 mM and 300 mM NaCl. This was repeated with seeds from Diskett. It was also repeated with wild type (WT) Gom-25, but with only 2 plates per concentration instead of 10 due to less seeds stored of Gom-25. The same test was repeated using the same range of concentrations, 50-300 mM, of KCl and MgCl2. Tests were later performed using concentrations of 400 mM and 500 mM for NaCl.

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5.4 NaCl-Screening Two petri dishes were placed with 10 ml water and 200 mM NaCl on 9 cm filter papers. Five seeds from each EMS treated Gom-25 M4 lines were put on each of the 2 dishes. The dishes containing the seeds were placed in growth chambers and screened for germination after 5-7 days.

Figure 2. Growth chamber with petri dishes for screening. Petri dishes with wheat seeds were incubated for 5-7 days, depending on the different tests. The seeds were grown at 16 hours day and 8 hours night cycles, temperature was 22°C, and lights were 250 μmol m-2s-1.

5.5 KCl- and MgCl2-Screening Fifteen wheat lines showing 50% or higher rates of germination on 200 mM NaCl were selected for a similar test using KCl and MgCl2 instead. For each of those lines, 2 petri dishes were placed with 10 ml water and 200 mM KCl on 9 cm filter papers and 5 seeds were put on each dish. The dishes were then placed in growth chambers and screened for germination after 7 days. The same test was performed on 200 mM MgCl2, with the same wheat lines. 5.6 Harvest of M5 line Around 700 lines of M5 mutated Gom-25 were harvested from a field where they were planted in the spring of 2015, in Borgeby just outside the city of Lund. Scissors were used to cut off the stems and axes of the plants, which were then put into individual bags along with a stick labeled with the line number. The bags were left for threshing to be returned later to us for screening. 6 Results 6.1 Method Test Results from the Method Test showed best germination on distillated water and 9 cm filter papers and slightly less germination on water agar. With added salt, germination frequency was significantly higher on 10 ml water, 200 mM NaCl and 9 cm filter papers than on nutrient agar with 200 mM NaCl (see Appendix A for tables of the results).

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6.2 Tape Test For the Tape Test, none of the tested petri dishes showed any significant difference in water levels between the different methods. The level of contamination by bacteria did not differ significantly between the two methods. The non-sealed only showed a total of 10 small and 2 large colonies of bacteria, where the sealed dishes showed 6 small and 2 large colonies of bacteria. The different levels of contamination were defined as: small when it was thin but visible, medium when the mycelium was more compact and more visible in color, and large when there was more volume or the seed was overgrown with mould. The frequency of seeds that had sprouted roots was also measured, though it will not be taken in to consideration during the screening tests. With tape the dishes showed that 36% of all seeds had produced roots, as opposed to 76% of the dishes without tape.

Figure 3. Germination of wheat seeds on petri plates with or without being sealed with tape. Bar chart displaying the difference in germination frequency (%) between petri dishes that had been sealed with surgical tape and dishes that were not sealed. The narrow bars represent the standard deviation. Those with tape showed a germination frequency of 28% (SD ≈ 0,55) and those without showed a frequency of 62% (SD ≈ 1,64)

6.3 Standard Curves As shown in Fig.4, the length of the sprouts differed significantly between the different MgCl2 concentrations applied. However, this was not taken in to consideration during the screening, just added as a comment. All seeds showing some form of green sprouts were recorded as germinated, no matter how big or small the actual sprouts were.

Figure 4. Germination of Gom-25 grown on MgCl2. WT Gom-25 seeds were grown on different concentrations of MgCl2. The concentrations were: A=50 mM, B=100 mM, C=150 mM, D=200 mM, E=250 mM and F=300 mM.

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On NaCl, Gom-25 showed a 100% germination frequency on 0-150 mM, a strong decline to 50% germination on 200 mM and then down to 10% on 250 mM. On KCl, Gom-25 displayed high levels of germination on 0-150 mM, ranging between 90-100%. Gom-25 showed a germination frequency of 80% on 200 mM and weakened to 40 % on 250 mM. On MgCl2, Gom-25 showed germination frequencies of 100% on 0-50 mM, 80 % on 100 mM, peaking to 100% germination on 150 mM, down to 60 % on 200 mM and further declined to a germination frequency of 25 % at 250 mM. Diskett declined to 0 % germination at 250 mM on all salts and had a germination frequency of around 50-60 % on 150 mM. Dacke followed similar patterns as those of Diskett, with slightly higher frequencies most of the time.

Figure 5. Standard curves using different concentrations of salts and wheat lines. Line charts displaying standard curves of germination frequency (%) for Dacke, Diskett and WT Gom-25 on NaCl, KCl and MgCl2. None of the different wheat species showed any germination on 300 mM of any of the salts.

Though not taken in to consideration during the screenings, frequency of root production was also measured during the standard curve tests. For NaCl and KCl, root production exceeded germination levels on all concentrations and for all three species of wheat. However, for MgCl2 germination levels surpassed root production on both Dacke and Diskett, but not on WT Gom-25.

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Figure 6. Frequency of sprouts and roots at different MgCl2 concentrations. Line charts showing the frequency (%) of sprouts and roots of the different wheat species Dacke, Diskett and WT Gom-25 on 0-300 mM concentrations of MgCl2. The bars show standard error.

6.4 NaCl-Screening Out of 142 tested mutated lines, 38 different lines had no germination, and no lines showed 100% germination (Fig. 7). As mentioned in section 5.5, 15 lines that showed a 50% germination frequency, or higher, on NaCl were also tested on KCl and MgCl2. The lines were selected in chronological order from the data of NaCl-tested lines. Fifty percent is thought to be a good germination frequency because of the high salinity level that the seeds were grown on. The standard curve for NaCl show that both WT Gom-25 and Dacke have a germination frequency of 50% on 200 mM NaCl (Fig.5). The NaCl screening revealed that 40 lines showed a germination level of 50% or higher, which is about 28% of all tested lines. Four lines, or ~2,8% of all lines, displayed very high germination frequencies of 80-90% (Fig.7).

Figure 7. Germination frequency of mutated M4-lines. Bar chart showing the number of mutated M4-lines in relation to the germination frequency (%) of seeds grown on 200 mM NaCl. N=142.

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6.5 KCl- and MgCl2-Screening All mutated Gom-25 lines apart from G, germinated better on NaCl than the other salts tested. Therefor 93% of all tested lines showed highest germination levels on NaCl. With the exception of lines A, C and F, 80% of all lines also germinated better on KCl than MgCl2 (Fig.8). Figure 8. Frequency of germination on different salts. Bar chart displaying the germination frequencies (%) of 15 different lines (A-O) on 200 mM NaCl, 200 mM KCl and 200 mM MgCl2. 7 Discussion Results showed a significant difference in germination frequency of seeds that had been grown in petri dishes on 9 cm filter papers and 10 ml water (with and without salt) compared to seeds grown on agar (with and without nutrient and salt). It was discovered that the recipe for the nutrient agar had not taken the salt of the nutrient in to consideration when mixing nutrient agar with 200 mM NaCl, but simply calculated the concentration of NaCl with the molecular weight of the salt compared to the amount of water used. It was difficult to calculate how much effect the nutrient salts actually had on the germination, along with the effect of NaCl , so that the amount of NaCl could have been decreased to fit the 200 mM concentration. Total concentration of salts was calculated to be around 249 mM. However, in the control tests with only water compared to water agar, the seeds germinated better without the agar. This should indicate that the presence of agar limits the ability of the seeds to germinate. The limiting of germination on agar is why we chose to do our tests on filter papers and water instead, along with being less time consuming. The Tape Test showed results in favor of the less time consuming alternative of not sealing the petri dishes as method. The germination frequency of those that had been sealed was far greater than those that had not, 68% compared to only 28% (Fig. 3) and the small difference in contamination was not significant enough to want to seal the dishes anyway. Even though there was a little more contamination on the dishes that had not been sealed (4 small mole colonies more), it evidently did not affect the germination since those seeds had a more than twice as high germination frequency than those seeds that were grown on petri dishes that had been sealed. Results from the Standard Curve tests were expected, with less germination on higher concentration levels of salt. This was true for all three types of salt. WT Gom-25 showed some unexpected patterns with 100% germination on concentrations 0-150 mM NaCl, and with a peak of 100% on 150 mM MgCl2, when it was only 80% on 100 mM MgCl2. However, there cannot be too much read in to this since only 10 seeds of Gom-25 were tested on each concentration, as appose to 50 seeds of each for Dacke and Diskett. The reason the tests were

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performed with such low number of seeds is simply, as earlier mentioned, that fewer wild type seeds from Bangladesh were available at the start of this project. All the WT Gom-25 seeds that germinated were planted after screening for the results, so that new seeds will be available in a few months. When those seeds are mature it would be a good idea to re-do the standard curves for Gom-25 to get an even better accuracy of the data. The ability to produce roots was also measured during the standard curve tests. As expected, more roots that sprouts were produced on NaCl and KCl (Fig. 6). On MgCl2 in contrast, the seeds of both Dacke and Diskett had higher frequencies of germination than of root production. WT Gom-25 stood out in these results, with the same expected results as with NaCl and KCl (Fig. 6). A striking trend is visible in the results – Dacke and Diskett germinated better on NaCl than KCl and better on KCl than MgCl2, whereas Gom-25 showed better germination frequency on MgCl2 with higher concentration than the other salts. In Alkali Sacaton (Sporobolu airoides) it has been shown that the effect of specific ions, in particular Mg2+, is more important than the effect of the total amount of salts (Ryan, Miyamoto & Stroehlein, 1975). Thus, it appears that Gom-25 might have a more adapted protective system against salt stress of MgCl2 than against any of the other tested salts. The results of lower root production could also indicate that Mg2+ targets the growth of roots more than the rest of the plant and that Gom-25 has developed a defence against this. In the NaCl screening test, 40 lines out of 142 tested showed high germination frequencies of 50% or more (Fig.7). If you can assume that the rest of the lines that were not tested by us follow the same pattern, as much as 28% of all EMS treated M4 Gom-25 would show high germination frequencies. Four lines even showed very high germination frequencies of 80-90%, which would mean that 2,8% of all lines could show very high frequencies. This low number makes it important to select seeds from lines with very high germination frequencies of 80% or more, so that even stronger lines can be collected from the next generation. Different from the standard curve tests, the EMS treated Gom-25 lines showed lowest germination frequency on MgCl2, which according to Ryan et al. (1975) would be expected since Mg2+ has such a strong effect on plants. These EMS treated Gom-25 was originally selected for tolerance against NaCl, which is why it is not that unexpected to see that the different lines germinate better on NaCl than on the other salts (Fig. 8). Maybe the EMS treatment resulted in a higher tolerance for NaCl, but at the same time diminishing the original potential defense against Mg2+. 8 Conclusion The first part of this project consisted of different method tests to develop the best suitable method for the Standard Curve- and screening-tests. From the Method Test results, it was concluded that the different wheat species germinated better without agar, i.e., future tests should be performed with 10 ml water, 9 cm (in diameter) filter papers in petri dishes with or without added salt depending on the assay. The process of making petri dishes with agar is more time consuming than growing the seeds on filter papers and water, the agar alone would take at least 1-1,5 hour to make (compared to 5 minutes for the salt water) and the dishes would also need time to set. This is another reason for choosing not to use nutrient agar, even though that was the method previously used for this project (Mahmud Zada, 2015). Previously in this project, seeds were cleaned from any contamination using ethanol, but without convincing results compared to those not cleaned with ethanol. It appeared that the ethanol also harmed the seeds in addition to reducing the contaminating factor (Erlandsson,

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2014). Our Tape Test indicated that the contamination level did not necessarily affect the germination frequency negatively. Thus, sealing the petri dishes with tape to try and keep the contamination lower is not necessary, especially since the seeds germinated better in the non-sealed dishes. From the percentage of lines with very high germination frequencies of 2,8% (Fig. 8) on high salt concentration one could conclude that there is still a lot of work to be done. The lines with high salt tolerance need to be carefully selected and tested in the forthcoming M5 generation. In addition, it is important to identify those lines that have a high tolerance for NaCl and that also tolerate higher concentrations of KCl and MgCl2, since the salt contaminated soils does not only consist of NaCl. If there will be continuous experiments and development of the lines that show high germination levels on 200 mM salt it is likely that one will be able to produce a species of wheat that can be used on the salt contaminated soils of, e.g., Bangladesh. This would mean that more seeds could be produced, resulting in a higher yield, which in turn would be especially beneficial for the people of Bangladesh, since it is such a poor country. 9 Future perspectives For my part of the project there are some factors I would like to develop further, the number one being that more seeds of WT Gom-25 needs to be produced and tested for standard curves, since only Gom-25 showed unexpected results. I need to know whether that was due to different stress responses or simply due to chance, since only 10 seeds were tested. I would also like to try and develop a method of cleaning the seeds from mould spores that later contaminate the petri dishes. Since all seeds were set on the petri dishes in the sterile room and instruments such as forceps were sterilized before use, we concluded that it must be the seeds themselves that contaminate the tests. That I want to eliminate. I would also like to test for MgCl2- and KCl tolerance on lines with lower germination frequencies on NaCl. Since KCl and MgCl2 seem to affect germination more, I would like to see if one could start from that direction instead and later select those that also show higher tolerance for NaCl. As there was a significant difference in length of the sprouts, I would also like to try and create a better screening method that can differentiate between different levels of germination. A seed with a sprout of 5 cm in one week is probably more likely to survive in the wild than a seed with only a 0,5 cm sprout in the same time, which is why it would be important to distinguish between them and not simply count both as germinated. Since I am part of a bigger project, there is more to be done in the future. The M5 lines that were harvested will need to be tested for salt tolerance and screened too, to confirm salt tolerant lines, or to find additional salt tolerant lines being masked in the M4 population. EMS-treated Gom-25 will be crossed with Swedish wheat lines, to ensure that it will be possible to cross Gom-25 with other locally adapted wheat lines, and with new lines from other countries of interest.

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Hydroponic growth of wheat lines will be started, so that any change of gene expression in the wheat could be registered, with or without salt present in the hydroponic solution. Finally, for our own understanding of the biology behind the product, mutation frequency of the EMS treated lines will be established. Another interesting thing is the overlap between salt- and arsenic contamination in Bangladesh (Fig.9). When compared to a map visualizing the salt contamination (Fig.1), it is clear that the most salt affected areas are also the most arsenic affected areas. This suggests that a screening for arsenic tolerance could be useful to conduct in the future.

Figure 9. Map of Bangladesh. Illustrating the contamination levels of arsenic in the ground. Source doi: 10.1016/S0883-2927(02)00018-5

10 Pedagogical angle of approach In the curriculum for the Swedish upper secondary school you can find different abilities that the students are supposed to, with guidance from the teacher, develop and learn for the different subjects. One specific ability for biology is to plan, to perform, to interpret and to present field studies, experiments and observations, with the addition of being able to handle materials and equipment (Skolverket, 2011). This project, or a similar one, can easily be “translated” in to the school world. To test germination frequency on different salts, or maybe even different essential plant nutrients or toxins would be a great way for the students to learn how to conduct scientific experiments and to learn to handle different materials and equipment. From their results they could later discuss conclusions of how much of the different substances that can be allowed in the environment and how much is actually necessary for the plants to reach as high germination frequencies, sprout- or root lengths as possible to generate the largest yield possible. Another ability for biology is that the students should develop knowledge about the significance of biology for the individual person as well as for society. As White (2006) points out, I believe that emotions play a big role in the students ability and desire to learn. Since this is a big project where the main goal boils down to being able to help starving people in less developed countries (starting with Bangladesh), I think that a lot of emotions could be evoked. That in turn could lead to a higher aspiration to learn among the students and an actual understanding for the impact that this, and other biological projects, can have on society as well as on individuals. In the core content for the subject biology one can read that the students should learn “Ecologically sustainable development, locally and globally, and different ways of contributing to this” and “Natural and man-made disturbances in the ecosystem linked to questions about bearing capacity and biological diversity” (Skolverket, 2011). As mentioned

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above, if my project were to be translated into a school environment, I think that it would give the students great opportunities to learn those two aspects of the core content for biology. As with the specific abilities, the core content also mentions different strategies and methods that the students should learn, that they should be able to interpret results and so on. This again I think that the students could gain from my project. 11 Acknowledgements I would like to thank my instructor Henrik Aronsson for excellent guidance and for all the help that he has given me during my project. I would also like to thank Research Assistant Johanna Lethin for her assistance and help throughout our shared part of the project. Another thank you to my lab-partners Petter Larsson and Sandra Bains for a good co-operation. 12 References Brooker, R. J. (2012). Genetics – Analysis & Principles Fourth Edition. New York: McGraw-Hill Companies, Inc. Elandsson, E. (2014). Praktiska tillämpningar på EMS-muterat vete. Bachelor dissertation, University of Gothenburg. Haque, S. A. (2006). Salinity problems and crop production in coastal regions of Bangladesh. Pakistan Journal of Botany, 38(5), 1359-1365. Kim, YS., Schumaker, S. & Zhu, J-K. (2006). EMA Mutagenesis of Arabidopsis. Methods in Molecular Biology, 323, 101-103. doi: 10.1385/1-59745-003-0:101 Mahmud Zada, T. (2015). Salttolerant vete – ett medicinskt hopp för ett fattigt land. Bachelor dissertation, University of Gothenburg. Munns, R. & Tester, M. (2008). Mechanisms of Salinity Tolerance. Annual Review of Plant Biology, 59, 651-681. doi: 10.1146/annurev.arplant.59.032607.092911 Potrykus, I. (2001). Golden Rice and Beyond. Plant Physiology, 125(3), 1157-1161. doi: http://dx.doi.org/10.1104/pp.125.3.1157 Rahman, S. & Hasan, M. K. (2007). Impact of environmental production conditions on productivity and efficiency: A case study of wheat farmers in Bangladesh. Journal of Environmental Management, 88(4), 1495-1504. doi: 10.1016/j.jenvman.2007.07.019 Ryan, J., Miyamoto, S. & Stroehlein J.L. (1975). Salt and specific ion effects on germination of four grass. Journal of Range Management, 28(1), 61-64. doi: 10.2307/3897581 Skolverket. (2011). Ämne – Biologi. Collected 2015-10-15 from www.skolverket.se Smedley, P.L. & Kinniburgh, D.G. (2002). A review of the source, behavior and distribution of arsenic in natural waters. Applied Geochemistry, 17(5), 517-568. doi: 10.1016/S0883-2927(02)00018-5

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Tavakkoli, E., Rengasamy, P. & McDonald, G.K. (2010). High concentrations of Na+ and Cl- ions in soil solutions have simultaneous detrimental effects on growth of faba bean under salinity stress. Journal of Experimental Botany, 61(15), 4449-4459. doi: 10.1093/jxb/erq251 The World Bank. (2015). Data. Collected 2015-10-12 from http://data.worldbank.org/ White, R.T. (2006). Dimensions of Content – Need for a Theory of Content. In P.J. Fensham, R.F. Gunstone & R.T. White (Ed.), The Content of Science: A Constructivist Approach to its Teaching and Learning (p.255-262). London: The Falmer Press Yamaguchi, T. & Blumwald, E. (2005). Developing salt-tolerant crop plants: challenges and opportunities. TRENDS in Plant Science, 10(12), 615-620. doi: 10.1016/j.tplants.2005.10.002 Zhu, K-J. (2001). Plant salt tolerance. TRENDS in Plant Science, 6(2), 66-71. doi: 10.1016/S1360-1385(00)01838-0

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13 Appendix A G = Germination R = Roots C = Contamination

Plates 1 2 3 4 5 Average StDG 2 2 3 1 0 1,6 1,140175R 2 2 4 1 0 1,8 1,48324C 0 3S 2S 3S 1SG 4 3 2 2 1 2,4 1,140175R 4 4 2 2 2 2,8 1,095445C 1S 0 1M 1M 2S,1MG 0 0 1 1 0 0,4 0,547723R 0 0 1 1 0 0,4 0,547723C 2S,1M 1S 1S 1S,1M 3S,1MG 3 0 2 2 1 1,6 1,140175R 3 2 2 2 1 2 0,707107C 0 1M 2M 1S 1SG 0 0 0 2 1 0,6 0,894427R 0 0 0 2 1 0,6 0,894427C 3S,1M 2S,1M,1L 3M 3S 2M,2LG 5R 5C 0

MS medium + agar + 200 mM NaCl

5acke

5iscett

DOa-25A

DOa-25B

DOa-25C

WT DOa-25

Plates 1 2 3 4 5 Average StDG 5 5 5 5 3 4,6 0,894427R 5 5 5 5 5 5 0C 0 0 0 0 0G 3 3 3 4 4 3,4 0,547723R 5 3 3 5 5 4,2 1,095445C 0 1M 1S 1S 0G 2 3 4 2 4 3 1R 1 3 4 2 4 2,8 1,30384C 2S,1M 3S 1M 1S,1M 0G 2 5 5 5 2 3,8 1,643168R 3 5 5 5 2 4 1,414214C 1S,2M, 1L 0 2S 1S,1M 2S,2M,2LG 2 1 1 2 2 1,6 0,547723R 3 2 1 2 2 2 0,707107C 1S 3S 2M 1S,1M 2S,2LGRC

Missing

5iscett

DOa-25A

DOa-25B

DOa-25C

WT DOa-25

10 ml water + 200 mM NaCl + 9 cm filter paper

5acke

18

Plates 1 2 Average StDG 5 5 5 0R 5 5 5 0C 1M 0G 5 5 5 0R 5 5 5 0C 0 0 0G 5 4 4,5 0,707107R 5 4 4,5 0,707107C 0 0G 4 5 4,5 0,707107R 4 5 4,5 0,707107C 2M 0G 4 3 3,5 0,707107R 4 4 4 0C 2S,1M 1SG 5R 5C 0

10 ml Water (no NaCl) + 9 com filter paper

Dacke

Discett

GOM-25A

GOM-25B

GOM-25C

WT GOM-25

Plates 1 2 Average StDG 3 5 4 1,41421R 3 5 4 1,41421C 1L 0G 5 5 5 0R 5 5 5 0C 0 0G 3 3 3 0R 3 3 3 0C 1M 1M,1LG 5 4 4,5 0,70711R 5 4 4,5 0,70711C 1S 1LG 4 2 3 1,41421R 4 2 3 1,41421C 1S 1S,1MG 5R 5C 0

WT GOM-25

Water + agar (no NaCl)

Dacke

Discett

GOM-25A

GOM-25B

GOM-25C

See section 6.2 Tape Test for definitions of the different levels of contamination.