BIOLOGY. Word count: IB Exam May 2017

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1 The allelopathic effect of extracts of Vaccinium myrtillus, Empetrum nigrum and Sphagnum girgensohnii on the germination and growth of Lepidium satvium IB Exam May 2017 BIOLOGY Word count: 3814

2 ABSTRACT Allelopathy regulates the density and biodiversity of plant communities by the plants release of allelochemicals into the soil. Allelochemicals have harmful effect on the growth and metabolism processes in plants, thereby decreasing the productivity of forestry and lowering crop yield as well as the quality which additionally leads to economic losses. Other allelochemicals have beneficial effects. Interest in allelopathy has increased during recent years because knowledge and awareness of the mechanisms can increase yields productivity. The aim of this study was to investigate if Vaccinium myrtillus, Sphagnum girgensohnii and Empetrum nigrum have allelopathic tendencies. In the present investigation the effects of different extracts of Vaccinium myrtillus, Sphagnum girgensohnii and Empetrum nigrum on growth of Lepidium satvium seeds was examined. Seed germination, length of seedling, root hair development, opening of the leaves of Lepidium satvium seeds harvested in different concentration of extract (0.00 g/10 cm 3, 0.05 g/10 cm 3, 0.10 g/10 cm 3, 0.50 g/10 cm 3 and 1.00 g/10 cm 3 ) was recorded after 10 days. The results obtained show that all three species had adverse effects on the germination, length of seedling, root hair development and opening of the leaves of Lepidium satvium seeds. The allelopathic effects was found to increase with concentration of extracts. The scale of increasing toxicity of extract in this study was the following: Sphagnum girgensohnii < Empetrum nigrum < Vaccinium myrtillus. Allelopathy is an eco-friendly way of increasing agricultural production, and includes both advantages in health and environmental aspects, however allelopathy requires further research for applications in agricultural production worldwide. Future studies would include larger range of seeds and seedlings to examine the relative effectiveness of plants used in this study. Word count: 273 1

3 TABLE OF CONTENTS 1. INTRODUCTION Allelopathy Allelochemicals Transport of allelochemicals Ecological importance and use in agriculture Vaccinium myrtillus Empetrum nigrum Sphagnum girgensohnii Lepidium satvium AIM, HYPOTHESIS AND RESEARCH QUESTION MATERIAL, METHOD AND VARIABLES Material Method Preparation of extract Preparation of Petri dishes Variables Dependent variables Independent variables Uncontrolled variables Controlled variables RESULTS Raw data Effect on germination Length of seedling Root hair development Opening of the leaves CONCLUSION DISCUSSION Limitation and improvements REFERENCES

4 1. INTRODUCTION 1.1 Allelopathy The word allelopathy is derived from the Greek words allelon, ''of each other'' and pathos, ''to suffer''.the phenomenon is defined as the direct or indirect biochemical interaction of one plant on another, by the production of allelochemicals which are released by plants into the environment in respond to stress. Allelopathy is often a result from competition. [6] Competition is the event in which one plant removes or limits sources from the environment such as light, water, nutrients, etc, and thereby limits the survival and growth of a neighbouring plant. [2] Allelopathy can be explained to be a protection mechanism that ensures the survival of a plant. The plant will act by releasing allelochemicals to limit the population of the species that threatens them. Allelopathy contributes to adverse impacts to the plant ecology as it affects the growth, productivity, diversity and structure of communities. [8] However, there are also allelochemicals that have beneficial impacts on other species. [6] Allelochemicals Allelochemicals are substances produced by plants that have inhibitory effect on growth, metabolism and population biology of neighbouring organisms. [10] They can affect a plant indirectly by altering soil property, nutrition and population activity of microorganisms, or directly by altering plant growth, cell division, respiration, mineral uptake, protein synthesis, germination, photosynthesis, membrane permeability, inhibition of enzymes, metabolism etc. [2,6] Agents identified to have allelopathic effects can be divided into two categories; (1) storage compounds such as organic acids, amino acid, carbohydrates, fats and proteins, and (2) secondary compounds such as alkaloids, tannins and pigments. [3] 3

5 1.1.2 Transport of allelochemicals Allelochemicals are released from the donor plant to the soil and is absorbed by the receiver plant. The donor plant releases allelochemicals through different mechanisms such as root exudation, biomass decomposition, volatilization and as leachates (Figure 1). [7] The allelopathic effect depends also on the environment in which the allelochemicals are released into. Some allelochemicals are reactive and can transform into less harmful or more toxic substances. Furthermore, their solubility will affect how they move in the soil or water, their vapour pressure will affect their volatility in the air, and their structure will affect their degradability. Their mobility in soil will further be affected by particle size, ph and change in ion concentration in the soil. [6] Figure 1. shows how allelochemicals move in soil from the donor to the receiver plant. 4

6 6.1 Ecological importance and use in agriculture Farmers and gardeners have observed allelopathy for over 2000 years, but the phenomenon was not conducted until the 1900 hundreds. [2] Allelopathy has a very important impact on the ecosystem. It regulates the density and biodiversity of plant communities, as the dominant species tend to limit the population size of minor species. [8] Furthermore it has been discovered that most plants develop resistance to the allelochemicals of species which they coexist with, but not to toxins of species which they do not coexist with and thereby create ecological stability in a plant community. On the other hand, it can also lead to, if a plant in a community lack resistance, this may result in disruption of the existing plant community as the dominant species will limit the population of minor species. [15] Moreover, it has been found that allelopathy affects the nitrogen cycle due to it inhibitive effect on plants and microorganisms. The nitrogen cycle demonstrates how nitrogen moves between plants, animals, bacteria, the air, and soil. The life of every living organism is dependent on nitrogen supplement. Nitrogen is important component of amino acids, proteins, DNA and chlorophyll in plants. Allelopathy limits nitrification in many ecosystem by the loss of nitrogen in the soil due to plant removal, leaching and vitalization. [2] Scientists have made research about allelopathy to use it for their advantage. Allelopathy can be used to increase crop yield through the avoidance of negative impacts, suppress pest (weed, insects, nematodes, pathogens) and the use of allelochemicals as growth regulators. Allelochemicals act on pest in many ways. They prevent them from infesting the plant, killing them, or decreasing the effect of damage on the plant. [7] Allelopathy is also used to develop rotation systems, such as maintenance of soil fertility, soil structure, plant nutrients, etc. [6] Allelopathy is an eco-friendly way of increasing agricultural production without increasing farm inputs. It includes both advantages in health and environmental aspects. [6] 5

7 1.2 Vaccinium myrtillus Figure 2. Vaccinium myrtillus. Vaccinium myrtillus belongs to the Ericaceae family. It is a 20-40cm high bush that carries blue-like berries. The plant bloom in May-June, and the berries do not mature until July- August. [9] Vaccinium myrtillus is one of Sweden's most common plants (Figure 3). It has a wide geographical spread and is dominant in the habitants where it occurs. It is most commonly found in forests and on mountains. Furthermore, it covers on average 17% of Swedish forests and the rhizome of a single seedling can cover an area of 5,5cm 3 [1, 9]. Earlier observations have shown that it is hard to plant new trees close to areas with Vaccinium myrtillus seedlings. This was later justified by Andreas Jäderlund, professor at SLU in Umeå reported that Vaccinium myrtillus has suppressive impact on the germination and growth of tree seedlings by the production and release of phenols into the environment. [1] The Figure 3. The geographical expansion of Vaccinium myrtillus in northern Europe. 6

8 1.3 Empetrum nigrum Figure 4. Empetrum nigrum. Empetrum nigrum belongs to the Ericaceae family. It is approximately cm high, and carries black-like berries. The plants bloom in April-June. [12,13] Empetrum nigrum is the dominant plant in Götaland and Svealand (Figure 5). Its subspecies Empetrum hermaphroditum is the dominant specie in Norrland. Empetrum hermaphroditum has larger berries, and 52 chromosomes, compared to Empetrum nigrum who has only 26 chromosomes. [13] Furthermore, Empetrum nigrum and Empetrum Hermaphroditum have expanded in Swedish forests over time. This has become serious problems to the ecosystem because of its aggressive impact on surrounding species sharing the habitant. The leaves of Empetrum nigrum release a toxic which prevents germination and growth of species. According to Marie-Charlotte Nilsson, professor at SLU in Umeå, this may be the reason for why new forest in Norrland are difficult to grow. [14] Furthermore, Empetrum nigrum has the ability to compete for limiting resources like nutrients, water and light and this additionally favours the plants survival. [3] Figure 5. The geographical expansion of Empetrum nigrum in northern Europe. 7

9 1.4 Sphagnum girgensohnii Figure 6. Sphagnum girgensohnii. Sphagnum girgensohnii belongs to the Sphagnaceae family. 10% of Swedish surface is covered with sphagnum. It is mainly found in mires, mosses and marsh. It grows close to, or on water. This has a great impact on ecosystem as it makes oxygen inaccessible for other plants, and thereby suffocates them. [10,21] Additionally, Sphagnum girgensohnii releases organic acids which make the surrounding environment acidic. Many plants and other organisms cannot survive in acidic environment. Sphagnum girgensohnii is thereby inhibiting the growth of other species. Additionally, it is discover that Sphagnum girgensohnii expand vigorously in acidic environment. [10] Sphagnum girgensohnii violent seed dispersal mechanism has also a great importance for its enormous expansion. Each spore capsule can hold a capacity of spores which can reach up to twenty centimetres. [16] 8

10 1.5 Lepidium satvium Figure 7. Lepidium satvium. In this experiment, Lepidium satvium seeds were cultured to investigate if Vaccinium myrtillus, Empetrum nigrum and Sphagnum girgensohnii shows allelopathic tendencies on the germination and growth of the seedlings. Lepidium satvium (garden cress) is a quick growing seed that can be harvested after 10 days. It is a cool season plant and grows in early spring. [11] Lepidium satvium is suitable to be grown outdoors and indoors. It grows best in sunny locations with moist and well-drained soil. The plant requires a lot of water and must therefore be kept moist. Its tolerate ph is between 6 to 7. [11] 9

11 2. AIM, HYPOTHESIS AND RESEARCH QUESTION From earlier studies, and investigations it has been found that Vaccinium myrtillus and Empetrum nigrum have harmful effects on the germination and growth of near growing seedlings. This has become to a problems in many Swedish ecosystems, especially in forestry as large economical values get lost as allelopathic species like Vaccinium myrtillus inhibit the growth of newly planted forest seedlings. It has also been reported that Sphagnum girgenohnii shows similar inhibitory characteristics on aqueous plants. Allelochemicals have adverse effect on the growth and metabolism processes in plants, hence decreasing productivity of forestry and lowering the yield produced as well as their quality which additionally lead to great economic losses. Aim: The aim was to investigate if Vaccinium myrtillus, Sphagnum girgensohnii and Empetrum nigrum have allelopathic tendencies. This was explored by observing the development of growth (germination, root hair development, opening of the leaf and length of seedling), of Lepidium satvium seeds, exposed to different concentrations of leaf extracts. Hypothesis: Vaccinium myrtillus, Empetrum nigrum and Sphagnum girgensohnii will show inhibitory effects on the germination and growth of Lepidium satvium seeds. Research question: Does extracts of Vaccinium myrtillus, Empetrum nigrum and Sphagnum girgensohnii have allelopathic impacts on the seed germination, growth, root hair and leaf development on Lepidium satvium seeds at different concentrations of extract ( 1.00 g/10 cm 3, 0.50 g/10 cm 3, 0.10 g/10 cm 3, 0.05 g/10 cm 3 and 0.00 g/10 cm 3 ). 10

12 3. MATERIAL, METHOD AND VARIABLES 3.1 Material Plants Empetrum nigrum Vaccinium myrtillus Sphagnum girgensohnii Lepidium satvium seeds Equipments Scissor Mortar and pestle 100 cm 3 measuring cylinder (±1cm 3 ) Conical flask 500 cm 3 Reagent bottles Glass rod Markers Funnel Filter paper Petri dishes Tweezers 3 cm 3 Pipette (± 0.5cm 3 ) 3.2 Method This experiment was separated into two different stages. The first stage was the preparation of extract. Extracts of Empetrum nigrum, Vaccinium myrtillus and Sphagnum girgensohnii were prepared using the same method. The second stage was the preparation for germinating and cultivating of Lepidium satvium. 11

13 3.2.1 Preparation of extract 1. The extracts of Empetrum nigrum, Vaccinium myrtillus and Sphagnum girgensohnii, were made by crushing 50 g plant parts, leaves and stem, with a mortar. 2. Tap water was added successively in small volumes to the crushed plant parts of Empetrum nigrum. In total 500 cm 3 of water was added to 50 g crushed plant parts. This was repeated for Vaccinium myrtillus and Sphagnum girgensohnii. 3. The extracts were placed in the dark for 24 hours, and then each extract was filtrated. 4. Four different concentrations of each extract was prepared by diluting the stock solution with tap water into final concentrations of 1.00g/10cm 3, 0.50g/10cm 3, 0.10g/10cm 3 and 0.05g/10cm 3 according to Table 1. Table 1. The table shows how the four concentrations of extract were prepared by diluting the stock solution with water. The final volume of each dilution of the extracts was 250 cm 3. Concentration (g/10cm 3 ) Volume of H 2O (cm 3 ) Volume of extract (cm 3 ) The solutions were poured in reagent bottles, and reached a level of 250 cm 3 each. Figure 8. The figure above shows the conical flasks with extracts of Empetrum nigrum, Vaccinium myrtillus and Sphagnum girgensohnii. The extract of Empetrum nigrum and Vaccinium myrtillus had an orange-like colour and had a strong smell, while the extract on Sphagnum girgensohnii was colourless and had a mild smell. 12

14 3.2.2 Preparation of Petri dishes cress seeds were placed on filter papers in Petri dishes cm 3 of extract with different concentration was added to each Petri dish. The Petri dish was then covered with a lid to avoid vaporisation, and placed on the windowsill. Three parallels were run for each extract. Additionally, three control trials were watered with only water cm 3 of extract was added every 24 hours, and the Petri dishes were rearranged randomly. 4. Germination, growth, root hair and opening of the leave was recorded after 10 days. Figure 9. The figure shows the distribution of seeds on the Petri dishes. 13

15 3.3 Variables Dependent variables In this investigation several aspects of germination and growth were taken in account: The number of seeds that germinated were counted. The number seeds that developed root hair were counted. The number of seeds that opened their leaf were counted. Lengths of seedlings were measured Independent variables The seeds were watered with extract form three different extracts: Empetrum nigrum Vaccinium myrtillus Sphagnum girgensohnii The extracts were at different concentrations to see if the allelopathic affect was concentration dependent Uncontrolled variables The variables that cannot be controlled are that we cannot know if dormant seeds of Lepidium satvium were present. Dormant seeds will not germinate even under environmental conditions which normally are favourable for germination. One more variable that is uncontrolled, is that the plants used to make extract may not have been under stress, as the stress level of plants varies through different seasons and time in life cycle. 14

16 3.3.4 Controlled variables The variables that are not changed for this experiment are the number of seeds (30), culturing time (10 days), sunlight, temperature, watering, oxygen and carbon dioxide supplement. Variable Why How Number of It is important to use the same numbers of Each trial was run with the seeds seeds in each trial to be able to compare same number of seeds (30 Culturing time Sunlight Temperature Watering Oxygen Carbon dioxide the results. Culturing time affect germination and growth. If a plant is given more time to culture, more seeds may germinate and the plant may grow more. Sunlight is necessary for photosynthesis to occur. Photosynthesis is the plants own production on nutrients. The nutrients who are produced are essential for plant growth. [22] Temperature affects germination, growth and the rate of photosynthesis. The rate of photosynthesis increases with increased temperature. Hence, too hot environments can also decrease the rate of photosynthesis. [18] The optimal temperature for seed germination changes between species. Water is also essential for germination. Germination occurs only when seeds have absorbed enough water to create a force strong enough to break the seed coat. [5] Plants do also require water for photosynthesis. [22] Additionally, many metabolic reaction takes place in water. If there is insufficient supply of water this can cause dehydration and the plant dries out. Oxygen is one of the factors required for cell respiration. Plants need to respire to release the energy in the carbohydrates produced by photosynthesis. [18] Carbon dioxide is one of the factors required for photosynthesis. A plant cannot carry out photosynthesis if there is insufficient carbon dioxide. [18] seeds). Every trial was performed under a period of 10 days. All trials received the same amount of sunlight. Petri dishes were placed on the window shell during the period of the experiment. The Petri dishes were moved randomly every 24 hours. All the trials were carried out at the same temperature. The experiments for all trials were performed in the same room at during the same period of time. All trials were kept moist, by adding 2 ml of extract every day. All trials were supplied with the same air containing oxygen. All trials were supplied with the same air containing carbon dioxide. 15

17 4. RESULTS Seedlings of Lepidium satvium were grown in different extract and concentrations. Three trials were run for each concentration. Germination and growth of seedlings was recorded after 10 days. Figure 10. Seedlings watered with only water. Germination started in between 24h hours after culturing. Figure11.Seedling watered with extract of Sphagnum girgensohnii (1.0g/10cm 3 ). Germination started after 24 hours after culturing. Figure 12. Seedling watered with extract of Empetrum nigrum (1.0g/10cm 3 ). Germination started after 3 days. Figure 13. Seedling watered with extract of Vaccinium myrtillus (1.0g/10cm 3 ). Germination was fully inhibited. 16

18 4.1 Raw data Table 2. The table shows the number of Lepidium satvium seeds that germinated after 10 days. The seeds were cultured in extracts of Vaccinium myrtillus, Empetrum nigrum and Sphagnum Girgensohnii, at different concentrations ranging from g/cm -3. The control was watered with regular tap water and had therefore the concentrations 0.00g/cm 3. Concentration of Vaccinium myrtillus extract (g/cm 3 ) Concentration of Empetrum nigrum extract (g/cm 3 ) Concentration of Sphagnum girgensohnii extract (g/cm 3) Number of germinated Lepidium satvium seeds Table 3. The table illustrated the number of Lepidium satvium seedling that developed root hair after 10 days, after cultured in extracts of Vaccinium myrtillus, Empetrum nigrum and Sphagnum Girgensohnii, at different concentrations ranging from g/cm -3. The control was watered with regular tap water and had therefore the concentrations 0.00g/cm 3. Concentration of Vaccinium myrtillus extract (g/cm 3 ) Concentration of Empetrum nigrum extract (g/cm 3 ) Concentration of Sphagnum girgensohnii extract (g/cm 3 ) Number of Lepidium satvium seeds that developed root hair Table 4. The table shows the number of Lepidium satvium seedling that opened their leaves after 10 days of culturing in extracts of Vaccinium myrtillus, Empetrum nigrum and Sphagnum Girgensohnii, at different concentrations The control was watered with regular tap water and had therefore the concentrations 0.00g/cm 3. Concentration of Vaccinium myrtillus extract (g/cm 3 ) Concentration of Empetrum nigrum extract (g/cm 3 ) Concentration of Sphagnum girgensohnii extract (g/cm 3 ) Number of Lepidium satvium seedlings that opened their leaves

19 Table 5. The table illustrates the length of Lepidium satvium seedlings that were cultured in different concentration of Vaccinium myrtillus extract. Length of seedling (mm) ± 1mm Concentration of Vaccinium Myrtillus extract (g/cm 3 ) Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial

20 Table 6. The table illustrates the length of Lepidium satvium seedlings that were cultured indifferent concentration of Empetrum nigrum extract. Length of seedling (mm) ± 1mm Concentration of Empetrum nigrum extract (g/cm 3 ) Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial

21 Table 7. The table illustrates the length of Lepidium satvium seedlings that were cultured indifferent concentration of Sphagnum girgensohnii extract. Length of seedling (mm) ± 1mm Concentration of Sphagnum Girgensohnii extract (g/cm 3 ) Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial 3 Trial 1 Trial 2 Trial

22 4.2 Effect on germination Table 8.The table shows the mean percentage of seeds germinated, when cultured in extract of Vaccinium myrtillus, Empetrum nigrum and Sphagnum girgensohnii of different concentrations (1.00g/10cm 3, 0.50g/10cm 3, 0.10g/10cm 3 and 0.05g/10cm 3 ). The control was watered with regular tap water and had therefore the concentrations 0.00g/cm 3. First the percentage of seeds that germinated was found for each trial. If 30 seeds corresponds to 100%, then 15 seeds equal to = 50%. The mean germination was found by the 30 formula: mmmmmmmm = TTTTTTTTTT nnnnnnnnnnnn oooo gggggggggggggggggggg ssssssdddd TTTTTTTTTT nnnnnnnnnnnn oooo ssssssssss 100 Mean percentage of seeds germinated (%) Concentration (g/10 cm 3 ) Vaccinium myrtillus Empetrum nigrum Sphagnum girgensohnii Table 9. The table illustrates the Standard deviation for the mean percentage of seeds that germinated at different extracts and concentration(1.00g/10cm 3, 0.50g/10cm 3, 0.10g/10cm 3 and 0.05g/10cm 3 ). To find the standard deviation, the percentage germinated for each trial was first found, and then the standard deviation was calculated by excel. SS. DD = (XX xx )2 nn 1, where xx is the mean of the sample, and n represents the number of values in the sample. S.D Concentration (g/10 cm 3 ) Vaccinium myrtillus Empetrum nigrum Sphagnum girgensohnii

23 Mean % of seeds germinated Concentration of extraxt (g/cm 3 ) Vaccinium myrtillus Control (water) Mean % of sseeds germinated Concentration of extract (g/cm 3 ) Empetrum nigrum Control (water) Graph 1. The graph shows the effect of different concentration of Vaccinium myrtillus extract on the germination of Lepidium satvium seeds. The error bars indicate standard deviation. Graph 2. The graph shows the effect of different concentration of Empetrum nigrum extract on the germination of Lepidium satvium seeds. The error bars indicate standard deviation. Mean % of seeds germinated Sphagnum girgensohnii Control (water) Mean % of seeds germinated Vaccinium myrtillus Empetrum nigrum Sphagnum girgensohnii Concentration of extract (g/cm 3 ) Concentration of extract (g/cm 3 ) Graph 3. The graph shows the effect of different concentration of Sphagnum girgensohnii extract on the germination of Lepidium satvium seeds. The error bars indicate standard deviation. Graph 4. The graph shows the effect of different concentration of Vaccinium myrtillus, Empetrum nigrum and Sphagnum girgensohnii extract on the germination of Lepidium satvium seeds. 22

24 4.3 Length of seedling Table 10. The table demonstrates the effect of different extracts on the mean length of Lepidium satvium seedling measured in millimetres, at different concentration of extract (1.00g/10cm 3, 0.50g/10cm 3, 0.10g/10cm 3 and 0.05g/10cm 3 ). The control was watered with regular tap water and had therefore the concentrations 0.00g/cm 3.The length of the seedling was measured from the tip of the root to the tip of the shoot. The mean length of seedlings was found by the formula; mmmmmmmm = SSSSSS oooo tthee llllllllllh oooo aaaaaa ssssssssssssssssss TTTTTTTTTT nnnnnnnnnnnn oooo ssssssssssssssssss 100 The seedlings that did not germinate, and had therefore a length of 0 mm were not calculated into the mean. Mean length of seedling (mm ±1) Concentration (g/10 cm 3 ) Vaccinium myrtillus Empetrum nigrum Sphagnum girgensohnii Table 11. The table illustrates the Standard deviation for the mean length of seedlings, at different extracts and concentrations (1.00g/10cm 3, 0.50g/10cm 3, 0.10g/10cm 3 and 0.05g/10cm 3 ). The control was watered with regular tap water and had therefore the concentrations 0.00g/cm 3.Standard deviation was calculated with excel. The general formula for standard deviation which excel uses is, SS. DD = (XX xx )2, where xx is the mean of the nn 1 sample, and n represents the number of values in the sample. S.D Concentration (g/10 cm 3 ) Vaccinium myrtillus Empetrum nigrum Sphagnum girgensohnii

25 Mean length of seedling (mm) Concentration of extract (g/cm 3 ) Vaccinium myrtilus Control (water) Length of seedling (mm) Concentration of extract (g/10 cm 3 ) Empetrum nigrum Control (water) Graph 5. The graph shows the effect of different concentration of Vaccinium myrtillus extract on the length of Lepidium satvium seedlings. The error bars indicate standard deviation. Graph 6. The graph shows the effect of different concentration of Empetrum nigrum extract on the length of Lepidium satvium seedlings. The error bars indicate standard deviation. Length of seedling (mm) Concentration of extract (g/cm 3 ) Sphagnum girgensohnii Control (water) Length of seedling (mm) Concentration of extract (g/cm 3 ) Vaccinium myrtillus Empetrum nigrum Sphagnum girgensohnii Graph 7. The graph shows the effect of different concentration of Vaccinium myrtillus extract on the length of Lepidium satvium seedlings. The error bars indicate standard deviation. Graph 8. The graph shows the effect of the length of Lepidium satvium seedlings, cultured in different concentration of Vaccinium myrtillus, Empetrum nigrum and Sphagnum girgensohnii extract. 24

26 4.4 Root hair development Table 12. The table shows the mean percentage of Lepidium satvium seedlings that developed root hair, when cultured in extract of Vaccinium myrtillus, Empetrum nigrum and Sphagnum girgensohnii of different concentrations (1.00g/10cm 3, 0.50g/10cm 3, 0.10g/10cm 3, and 0.05g/10cm 3 ). The control was watered with regular tap water and had therefore the concentrations 0.00g/cm 3. First the percentage of seedlings that developed root hair was found. If 14 of 25 seedlings developed root hair, then 25 = 100%,and, the percentage is % = 56%. Finally, the mean was calculated with the formula; mmmmmmmm = TTTTTTTTTT nnnnnnnnnnnn ooff ssssssssssssssssss tthaaaa dddddddddddddddddd rrrrrrrr haaaaaa TTTTTTTTTT nnnnnnnnnnnn oooo ssssssssssssssssss 100% Mean percentage of seedlings that developed root hair (%) Concentration (g/10 cm 3 ) Vaccinium myrtillus Empetrum nigrum Sphagnum girgensohnii Table 13. The table illustrates the Standard deviation for the mean percentage of seedlings that developed root hair at different extracts and concentration (1.00g/10cm 3, 0.50g/10cm 3, 0.10g/10cm 3 and 0.05g/10cm 3 ). The control was watered with regular tap water and had therefore the concentrations 0.00g/cm 3.The standard deviation was calculated with excel, SS. DD = (XX xx )2, where xx is the mean of the sample, and n represents the number of values nn 1 in the sample. S.D Concentration (g/10 cm 3 ) Vaccinium myrtillus Empetrum nigrum Sphagnum girgensohnii

27 Mean percentage of seedlings that developed root hair (%) Concentration of extract (g/cm 3 ) Vaccinium myrtillus Control (water) Mean percentagge of seedlings that developed root hair (%) Concentration of extract (g/cm 3 ) Empetrum nigrum Control (water) Graph 9. The graph shows the effect of different concentration of Vaccinium myrtillus extract on the development of root hair of Lepidium satvium seedlings. The error bars indicate standard deviation. Graph 10. The graph shows the effect of different concentration of Empetrum nigrum extract on the development of root hair of Lepidium satvium seedlings. The error bars indicate standard deviation. Mean percentage of seedlings that developed root hair (%) Concentration of extract (g/cm 3 ) Sphagnum girgensohnii Control (water) Mean percentage of seedlings that developed root hair (%) Concentration of extract (g/cm3) Vaccinium myrtillus Empetrum nigrum Sphagnum ggirgensohnii Graph 11. The graph shows the effect of different concentration of Sphagnum girgensohnii extract on the development of root hair of Lepidium satvium seedlings. The error bars indicate standard deviation. Graph 12. The graph shows the effect of the development of root hair of Lepidium satvium seedlings, cultured in different concentration of Vaccinium myrtillus, Empetrum nigrum and Sphagnum girgensohnii extract. 26

28 4.5 Opening of the leaves Table 14. The table shows the mean percentage of Lepidium satvium seedlings that opened their leaves, when cultured in extract of Vaccinium myrtillus, Empetrum nigrum and Sphagnum girgensohnii of different concentrations (1.00g/10cm 3, 0.50g/10cm 3, 0.10g/10cm 3, and 0.05g/10cm 3 ). The control was watered with regular tap water and had therefore the concentrations 0.00g/cm 3. First, the percentage of seedlings that opened their leaves were found, then the mean was found by the formula; mmmmmmmm = TTTTTTTTTT nnnnnnnnnnnn oooo ssssssssssssssssss tthaaaa oooooooooooo ttheeeeee llllllllllll TTTTTTTTTT nnnnnnnnnnnn oooo ssssssssssssssssss 100% Mean percentage of seedlings that opened their leaves (%) Concentration (g/10 cm 3 ) Vaccinium myrtillus Empetrum nigrum Sphagnum girgensohnii Table 15. The table illustrates the Standard deviation for the mean percentage of seedlings that opened their leaves, at different extracts and concentration (1.00g/10cm 3, 0.50g/10cm 3, 0.10g/10cm 3 and 0.05g/10cm 3 ). The control was watered with regular tap water and had therefore the concentrations 0.00g/cm 3.The standard deviation was calculated with excel, SS. DD = (XX xx )2, where xx is the mean of the sample, and n represents the number of values nn 1 in the sample. S.D Concentration (g/10 cm 3 ) Vaccinium myrtillus Empetrum nigrum Sphagnum girgensohnii

29 Mean percentage of seedlings that opened their leaves (%) Concentration of extract (g/cm 3 ) Vaccinium myrtillus Control (water) Mean percentage of seedlings that opneded their leaves (%) Concentration (g/cm 3 ) Empetrum nigrum Control (water) Graph 13. The graph shows the effect of different concentration of Vaccinium myrtillus extract on the development of leaves Lepidium satvium seedlings. The error bars indicate standard deviation. Graph 14. The graph shows the effect of different concentration of Empetrum nigrum extract on the development of leaves Lepidium satvium seedlings. The error bars indicate standard deviation. Mean percentage of seedlings that opened their leaves (%) Concentration of extract (g/10 cm 3 ) Sphagnum girgensohnii Control (water) Mean percentage of seedlings that opened their leaves (%) Concentration of extract (g/cm 3 ) Vaccinium myrtillus Empetrum nigrum Sphagnum girgensohnii Graph 15. The graph shows the effect of different concentration of Sphagnum girgensohnii extract on the development of leaves Lepidium satvium seedlings. The error bars indicate standard deviation. Graph 16. The graph shows the effect of the development of leaves on Lepidium satvium seedlings, cultured in different concentration of Vaccinium myrtillus, Empetrum nigrum and Sphagnum girgensohnii extract. 28

30 5. CONCLUSION In the present study the allelopathic effect of extracts of Vaccinium myrtillus, Empetrum nigrum and Sphagnum girgensohnii on the germination, root hair development, opening of the leaves, and length of Lepidium satvium seeds was studied. The obtained result showed that extracts of Vaccinium myrtillus showed the highest inhibitory effect on germination of Lepidium satvium seeds (Graph 4). 1.0 g/cm 3 concentration of Vaccinium myrtillus extract completely inhibited germination of Lepidium satvium seeds. This is in line with previous studies [1]. Empetrum nigrum also showed as strong inhibitory effect on germination in the same range as Vaccinium myrtillus (Graph 4). Sphagnum girgensohnii only showed a very small inhibitory effect not at all in the range of extracts of Vaccinium myrtillus and Empetrum nigrum (Graph 4).For all extracts germination was dose-dependent, i.e. the inhibitory effect on germination was much more pronounced the higher the concentration of the extract. This is because allelochemicals generally affect cellular processes like mineral uptake, protein synthesis mineral uptake, protein synthesis, respiration, membrane permeability, cell division and metabolism of species at higher concentrations than at lower. [2] The obtained results also showed that Vaccinium myrtillus and Empetrum nigrum had the most vigorous effect on the length of Lepidium satvium seedling, where Vaccinium myrtillus showed the highest inhibitory effect on length (Graph 8). Sphagnum girgensohnii only inhibited the lengths to a lower extent (Graph 8). At highest concentration of extract of Sphagnum girgensohnii. The length was only inhibited 12% compared to the control. Throughout the experiment it was found that Vaccinium myrtillus and Empetrum nigrum had the greatest impact on root hair development of Lepidium satvium seedlings (Graph 12). At the highest concentration of Vaccinium myrtillus no seeds germinated so no roots could develop. The highest concentration of Empetrum nigrum extract showed vigorous effects on root hair development. Only 23% of Lepidium satvium seedlings developed root hair (Table 29

31 12).This is in line with previous studies which showed that Empetrum nigrum has the effect to completely inhibits root hair development [3]. Through the investigation it was also found that extracts of Vaccinium myrtillus had the most harmful effect on opening of the leaves (Graph 16). Empetrum nigrum and Sphagnum girgensohnii did also show inhibitory effects on opening of the leaves to a slightly smaller extent. Vaccinium myrtillus, Empetrum nigrum and Sphagnum girgensohnii all showed harmful effects on the germination and growth of Lepidium satvium seedlings. Taken together, if placing the allelopathic effect of the different plant extracts on Lepidium satvium seeds on a scale in decreasing order: Vaccinium myrtillus, Empetrum nigrum and Sphagnum girgensohnii. 6. DISCUSSION The experiments showed that Vaccinium myrtillus and Empetrum nigrum exerted the greatest inhibitory effect on Lepidium satvium. (Graph, 4,8,12 and 16). The results with Empetrum nigrum showed greater variation in the degree of germination at 0.10 and 0.5 g/cm 3 of extract, this is shown by a larger value on the standard deviation (Table 9, Graph 2). Extracts of Sphagnum girgensohnii did only inhibit the germination to a very limited degree (Graph 3). The data obtained from measuring the length of seedlings showed a greater variation (see error bars in Graph 5-7). The trend was however that Vaccinium myrtillus inhibited the length the most, as there was no germinated seeds in the extracts watered with the highest concentration of Vaccinium myrtillus, and therefore no seedlings were to be measured. Empetrum nigrum inhibited the length of the seedlings the same pattern out to a lower degree. Sphagnum girgensohnii did not inhibit the length of the seedlings very much, and the 30

32 variation in the data varied like Sphagnum girgensohnii has no effect on the length of the seedling. A reason for why sphagnum girgensohnii was observed to have small allopathic effects of growth on Lepidium satvium seeds in this study could be because Sphagnum girgensohnii was not exposed to stress during the spring season when it was picked. The frequency of allelochemicals produced varies through the life cycle of a plant and it is also dependent on environmental conditions. A decrease in the stress level of a plant can reduce the allelopathic potential of the donor plant [7]. The Sphagnum girgensohnii used in this study might not have been under stress. It might also have been the case that the life of the plant was not threatened by other species in the particular habitant, and therefore did not produce large amounts of inhibitive chemicals. Another explanation for why Sphagnum girgensohnii showed small allelopathic effects is that it might not be allelopathic. It is hard to separate allelopathy from competition as they work together. Sphagnum girgensohnii grows on and covers small lakes. This makes oxygen inaccessible for other plants in the surrounding, and suffocates them. [21] Sphagnum girgensohnii has characteristics which favours its reproduction and survival in the nature. Firstly, Sphagnum girgensohnii releases acids which makes the soil and the water acidic. [11] Plants and other organisms cannot live in the acidic environment and die. The extract of Sphagnum girgensohnii prepared might have been acidic, and thereby inhibited the germination and growth of Lepidium satvium seeds. Secondly, Sphagnum girgensohnii can procreate itself very fast as it has the ability to disperse thousand of seeds. [16] This explains that Sphagnum girgensohnii can cover large areas without allelochemicals being involved. These aspects could not be studied in the present investigation. 31

33 6.1 Limitation and improvements Allelochemicals can be polar and non-polar. Non-polar allelochemicals have difficulties moving through a very moist environment as they are not soluble in water. The receiver plant might not receive a high enough dose of allelochemicals for allelopathic properties to be shown. In this experiment, the solubility of allelochemicals was not taken into consideration. If non-polar allelochemicals are involved, a non polar solvent should have been used to extract the allelochemicals from the leaves. On the other hand, many non-polar solvents (ex, ethanol) act on seed germination and growth of seedlings. If alcohols are in question to be used it should be noted that a longer expose to alcohols may kill the seed, but in some cases, alcohol can break the seed dormancy and speed up germination. [20] Water might still be the best method to extract the allelochemicals as it is used as the solvent in nature. Another imitation in this experiment is the presence of dormant seeds. Dormant seeds, are seeds which are not germinating even under environmental conditions which are normally favourable for germination. Although a dormant seed has received full supply of water, and carries out normal respiration, protein synthesis and metabolism, it might fail to germinate due to environmental factors. These environmental factors include too cold or too warm temperatures and insufficient supply of light. The seeds will not germinate until these requirements have been satisfied. [5] It was very cloudy during the days of harvest. This might have restricted germination. In order to improve this experiment a plant lamp should have been used. A condition which can inhibit germination of seeds is osmotic potential. Water molecules move from a hypotonic solution (more water, less solutes) to a hypertonic solution (less water, more solutes) across the seed coat. Seed absorb water to create a force strong enough to break the seed coat. Extract of Vaccinium myrtillus, Empetrum nigrum and Sphagnum girgensohnii is a hypotonic solution as it contains more solute than water. Therefore, the seeds watered with extract will not absorb enough water the break its coat 32

34 and germination will not occur. In order to improve this experiment the control should be altered to have the same osmotic potential as the extract. Field studies are difficult to replicate in laboratories as the environmental conditions are not the same. Coexisting species that have been exposed to allelochemicals of one plant may adapt to the chemicals and become resistant in the long run. The allelopathic effect on invasive species may therefore reduce over time. [15] Plants have the ability to produce and excrete enzymes that break the harmful allelochemicals into non-harmful substances, indicating that a plant may not express any allelopathic properties. This aspect could not be investigated in the present study. To improve this experiment, different kind of seeds should have been used. The allelopathic tendency should be tested on both coexisting and noncoexisting plants to investigate this phenomenon further. Furthermore, allelopathic tendencies were only tested on seeds and young seedlings. To improve this experiment mature plant should have been used to investigate this phenomenon further. One more improvement is that the seeds should be harvested in a pot instead of Petri dishes to prevent the seeds from moving when watered. A pot experiment is more sustainable for harvesting as the soil conditions are closer to field studies. Previous studies have shown that Empetrum nigrum has the effect to completely inhibit the growth of root hair. In the present study, the highest concentration did not completely inhibit root hair development. In order to improve this experiment a greater variation of different concentrations of extract should be used. Another limitation was the method used to measure the mass of plant used for the extract. 50g of fresh plants part were measured. However, the fresh plant parts contained different amounts of water which could have an effect on the mass. Empetrum nigrum has very small leaves which means that they contain not so much water. On the other hand, Sphagnum girgensohnii grows in water and it soaks in a lot of water making the plant very heavy. In order to improve this experiment the plants should have been dried. 33

35 The systematic errors in this experiment include the inability to spread water evenly in Petri dishes, germination was tricky to measure as the seed were small, limitations on measuring the exact length of the seedlings and the extract vaporizing quickly if the cover was taken off, but with the cover on the seedling might not have been able to reach its potential height. Reasonable results were obtained from this experiment, which means that the systematic errors did not have a large impact on the result. In conclusion, this study showed that Vaccinium myrtillus and Empetrum nigrum has allelopathic impacts on the seed germination and growth on Lepidium satvium seeds. Sphagnum girgensohnii also showed small inhibitory effects on seed germination and growth of Lepidium satvium seeds. However, it was not certain if this is because of allelopathy, or because Sphagnum girgensohnii generally is acidic and thereby inhibits the germination and growth of Lepidium satvium seeds. Allelopathy has a very important impact on the ecosystem. It regulates the density and diversity of plant communities. The phenomenon requires further research for applications in agricultural production worldwide. Future studies would include larger range of seeds and seedlings. Word count: