TITLE: Plant minerals deficiencies. AIM: To investigate the effect of plant mineral deficiencies.

s. To develop problem solving and experimental skills, for example, information is accurately processed, experimental procedures are planned, designed and evaluated properly, and producing valid results and recording results. To develop careful observing skills on the development changes occurred on Lemna sp. leaves. INTRODUCTION: LEMNA

Figure 1: Lemna minor Image source: Google images

Lemna sp. are small, free-floating aquatic water plants found in ponds. It is from the Duckweed family. The name Lemna is originated from the Greek which means water-plant, star-grass. Lemna sp. plants reproduce asexually by vegetative propagation, where two daughter plants bud off from the adult plant. When they are big enough, they will separate from the mother leaf and can reproduce themselves. Sometimes, Lemna sp. plants can have up to 3 or 4 buds. This form of growth is a very rapid colonisation of new water. Duckweeds are flowering plants, nearly all of them known to reproduce sexually, flowering, and

producing seed under appropriate conditions. Like normal plants, they grow through a process called photosynthesis where they can use sunlight, minerals, carbon dioxide and water synthesise the food they need. When Lemna sp. invades a waterway, it can be removed mechanically, by the addition of herbivorous fish (e.g. grass carp) or treated with a herbicide. The rapid growth of duckweeds finds application in bioremediation of polluted waters and as test organisms for environmental studies. It is also being used as an expression system for economical production of complex biopharmaceuticals. Duckweed meal (dried duckweed) is a good cattle feed. It contains 25-45% proteins (depending on the growth conditions), 4.4% fat, and 8-10% fibre, measured by dry weight. Lemna sp. has been used for testing toxicity of certain chemicals. Lemna sp. can be transformed by molecular biologist to express proteins of pharmaceutical interest. Lemna sp. is genetically engineered to produce in the growth medium at high yield, and thus reduce the manufacturing costs. It is easily grown in garden ponds, where it removes excess nutrients from water and providing sheds to inhibit algae growth. Their high fat and protein content makes them a source of food for animals and poultry.1 CHLOROSIS

Figure 2: Plants with chlorosis Image source: Google images

In botany, chlorosis is a condition in which leaves produce insufficient chlorophyll. As chlorophyll is responsible for the green colour of leaves, chlorotic leaves are pale, yellow, or yellow-white. The affected plant has little or no ability to manufacture carbohydrates through photosynthesis and may die unless the cause of its chlorophyll insufficiency is

http://en.wikipedia.org/wiki/Lemna

treated, although some chlorotic plants, such as the albino Arabidopsis thaliana mutant ppi2 are viable if supplied with exogenous sucrose. Chlorosis can be noted by yellowing and decolourising of the leaves. This is because the breakdown of chlorophyll a and chlorophyll b (both green in colour) may reveal other photosynthetic pigments. They are xanthophylls (yellow) and carotene (orange). Necrosis happens due to chlorosis. The leaves may have brownish or blackish patches. Plant symptoms may appear in leaves, stems, roots, flowers, fruits, and seeds. Chlorosis is typically caused when leaves don't have enough nutrients to synthesise all the chlorophyll they need. It can be brought about by a combination of factors including:

a specific mineral deficiency in the soil, such as iron; a soil pH at which minerals become unavailable for absorption by the roots poor drainage (waterlogged roots) damaged and/or compacted roots pesticides and particularly herbicides may cause chlorosis, both to target weeds and occasionally to the crop being treated. exposure to sulphur dioxide

Of course, the exact conditions would be different for different plants. For example, Azaleas prefer unusually acidic soil and rice isn't troubled by waterlogged soil.2 PHOTOSYNTHESIS

Figure 3: Chlorophyll involves in photosynthesis process Image source: Google images

Photosynthesis is a process that converts carbon dioxide into organic compounds, especially sugars, using the energy from sunlight. Photosynthesis occurs in plants, algae, and many species of Bacteria, but not in Archaea. Photosynthetic organisms are called photoautotrophs, since they can create their own food. In plants, algae and cyanobacteria
2

http://en.wikipedia.org/wiki/Chlorophyll

photosynthesis uses carbon dioxide and water, releasing oxygen as a waste product. Photosynthesis is vital for life on Earth. As well as maintaining the normal level of oxygen in the atmosphere, nearly all life either depends on it directly as a source of energy, or indirectly as the ultimate source of the energy in their food (the exceptions are chemoautotrophs that live in rocks or around deep sea hydrothermal vents). The amount of energy trapped by photosynthesis is immense, approximately 100 terawatts: which is about six times larger than the power consumption of human civilization. As well as energy, photosynthesis is also the source of the carbon in all the organic compounds within organisms' bodies. In all, photosynthetic organisms convert around 100,000,000,000 tonnes of carbon into biomass per year.3 PROBLEM STATEMENT: Do minerals deficiencies in plant will affect its growth? HYPOTHESIS: Plant grows healthy in complete solution and shows symptoms when lacking certain mineral. VARIABLES: INDEPENDENT DEPENDENT FIXED : The deficiencies of different types of minerals. : Growth of Lemna sp. : The initial number of Lemna sp. leaves in culture solution. The type of plants. The initial volume and concentration of minerals present in each culture solution. Light intensity, surrounding temperature and humidity.

http://en.wikipedia.org/wiki/Photosynthesis

WAYS TO CONTROL VARIABLES: INDEPENDENT : Use 8 different types of culture solution (all nutrients

present(normal), lacking iron, lacking calcium, lacking sulphur, lacking potassium, lacking phosphorus, lacking magnesium, and lacking nitrogen). DEPENDENT : Observe, count and record the changes in Lemna sp. plants within 10 days of observation. FIXED : Put only 4 pairs of Lemna sp. plants in each culture solution. Use the same species of plant which is Lemna sp. Use 15ml for each culture solution. Put all the Petri dishes containing Lemna sp. under the same condition.

APPARATUS: 8 Petri dishes with covers, dropper, a pair of forceps, 10 ml measuring cylinder, paper towel sticker, tray. MATERIALS: Lemna sp. plantlets, distilled water, complete nutrient solution, range of nutrient solutions lacking of nitrogen, phosphate, potassium, magnesium, iron, sulphate and calcium.

PROCEDURES: 1. 8 Petri dishes were rinsed and washed using distilled water and dried using paper towel. 2. These Petri dishes were then labelled using sticker.

Figure 4: Labelling the Petri dish

3. Using a 10ml measuring cylinder and dropper, 15ml of solution containing all nutrients or normal solution was measured and poured into the Petri dish.

Figure 5: Measuring volume of culture solution

4. Next, 4 pairs of Lemna sp. plantlets were taken from a container full of Lemna sp. plants by using a pair of forceps.

Figure 6: Choosing the best Lemna sp.

5. The Lemna sp. plantlets were placed onto the surface of the nutrient solutions.

Figure 7: Placing Lemna sp. in Petri dish

6. The Petri dish was covered and placed in the tray which exposed to sunlight.

Figure 8: Arranging the Petri dish containing Lemna sp.

7. Steps 3 to 6 were repeated by replacing the normal solution with nutrient solutions lacking nitrogen, potassium, calcium, phosphate, magnesium, iron and sulphur. 8. Observations regarding number of leaves present, number of plantlets, colour of leaves, size of plantlets, and presence of root were recorded. The observations were repeated for everyday within 10 days. The results were recorded in a table for a comparison to be made between different conditions of plants. RESULTS:
Solution Observation Day 1 Full Solution No. of Leaves Colour Presence of Roots Present Without Calcium Colour Green Green Green Green Pale Green Presence of Roots Pale Green Pale Green Pale Green Pale Green Pale Green No. of Leaves 8 Present 10 Present 15 Present 19 Present 25 Present 29 Present 34 Present 38 Present 46 Present 51 8 Green Day 2 13 Green Day 3 26 Green Day 4 48 Green Day 5 59 Green Day 6 68 Green Day 7 77 Green Day 8 86 Green Day 9 95 Green Day 10 101 Green

Present

Present

Present

Present

Present

Present

Present

Present

Present

Present

Without Nitrogen

No. of Leaves Colour

8 Green

9 Pale Green

9 Pale Green

9 Yellow

9 Yellow

9 Colourle ss

9 Colourle ss

9 Colourles s

9 Colourless

9 Colourles s

Presence of Roots Present Without Potassium Colour Presence of Roots Present Without Magnesium Colour Green Green Green Pale Green Presence of Roots Present Without Phosphorous Colour Presence of Roots Present Present Present Present Present Present Present Present Present Present Green Green Green Green Green Green Green Green Green Green No. of Leaves 8 Present 14 Present 19 Present 26 Present 33 Present 39 Present 47 Present 56 Present 67 Present 78 Pale Green Pale Green Pale Green Pale Green Pale Green Pale Green No. of Leaves 8 Present 12 Present 17 Present 24 Present 28 Present 33 Present 39 Present 45 Present 52 Present 59 Green Green Green Green Green Green Green Green Green Green No. of Leaves 8 Present 9 Present 14 Present 17 Absent 22 Absent 27 Absent 34 Absent 39 Absent 46 Absent 53

Without Sulfur

No. of Leaves Colour Presence of Roots

8 Green

10 Green

16 Green

20 Green

25 Green

33 Green

41 Green

48 Green

54 Green

62 Green

Present Without Iron Colour Green No. of Leaves 8

Present 15 Green

Present 21 Green

Present 27 Green

Present 34 Pale Green

Present 41 Pale Green

Present 50 Pale Green

Present 62 Pale Green

Present 73 Pale Green

Present 81 Pale Green

Presence of Roots Present Present Present Present Present Present Present Present Present Present

Table 1: Observations on Lemna sp. plants based on number of leaves, colour of leaves and presence of root within 10 days.

DISCUSSION: The using of miniature Lemna sp. plant is ideal for investigating the effect of plant mineral deficiencies. Lemna sp. can easily acclimatize them to the new environment therefore; it is easy to grow them in Petri dish containing nutrient solution. Furthermore, Lemna sp. plants can be cultured in large amounts for reliable statistical data to be collected. The amount of exposure to sunlight is controlled by placing the Petri dishes at the same places where the daily exposure to sunlight is nearly a constant. The Petri dishes are covered to maintain the temperature, the carbon dioxide concentration, and humidity surrounding the plants in the Petri dishes. The volume of the culture solutions is maintained in the Petri dishes to ensure that the rate of photosynthesis is not affected in each Lemna sp. plants. The initial concentration of culture solution should be the same for all Petri dishes because the amount of nutrients available can affect the growth of the Lemna sp. plants. There must be the same number of Lemna sp. available in each Petri dish because the same level of competition should be maintained for all Petri dishes so that this factor will not affect the level of Lemna sp. plant growth. There are 13 species of Lemna plants. Therefore, the same species of Lemna plants are selected in the experiment because different species of Lemna plants may result different levels of competition. Furthermore, healthy plants of the same age should be chosen to maintain the fixed variable. The Petri dishes are rinsed with water and wiped dry to prevent the fungal or bacterial contamination. The contamination may damage the plant or making the plants deprived of nutrients. 15ml of solution is added in order to give a constant supply of nutrients to the plants. 4 pairs of Lemna sp. plants are scattered as far as possible in each Petri dish so that the competition for space can be omitted. The Petri dishes are covered to prevent bacterial and fungal contamination. The Petri dishes are put under a shed to ensure a moderate intensity of sunlight can reach for the Lemna sp. plants. The changes in colour on the Lemna sp. leaves can be difficult to observe because the leaves are small and small spots of decolourising are easily neglected. This problem can be overcome by using a stereo microscope where a magnification of 2X and 10X are available for checking against your observations and fungal contamination. The Petri dishes

can be placed directly under the microscope for observations to be made. Furthermore, high resolution photographs can be taken each time the observation is made for future reference. Sodium hydrogen carbonate can be added to the nutrient solution to order to saturate the air the Petri dishes with carbon dioxide. It is ideal that to prevent salt accumulation and nutrient depletion, nutrient solutions were changed every week and distilled water was added periodically to maintain solution volume. EVALUATION: The experiment is valid and reliable because we have considered all the possibilities where the absence of different minerals is investigated. Furthermore, the observations are done consistently every day. The fixed variables are well controlled. Besides, controlled experiments have been set up to investigate what happens to Lemna sp. plants when immersed into complete nutrient solution. This is ensuring that abnormal plants are due to certain mineral deficiencies. Replicates of the experiment are needed to minimize the errors of the results and to ensure the results are true. EXPLANATION AND ANALYSIS OF RESULTS: From the results, it is obvious that the lacking of iron has the least effect on the growth of Lemna sp. plants whereas the lacking of nitrogen has the most effect on the growth of Lemna sp. plants. This is because at the end of our observation, we found out that there are only 9 Lemna sp. plants left after 10 days for nutrient solution lacking nitrogen whereas there are 81 Lemna sp. plants remaining for nutrient solution lacking iron. This means that iron is most probably a micronutrient for the Lemna sp. plant while for nitrogen, it is the macronutrient. Therefore, nitrogen is the most important nutrient required by plants. The lacking of nitrogen can cause chlorosis (yellowing of leaves) because nitrogen is essential for the formation of amino acids, proteins, and chlorophyll. Chlorophyll is the pigment that keeps plants green. The loss of chlorophyll can cause the colour of the leaves to turn yellow and eventually decolourises. Furthermore, the growths of plants are retarded due to the lack of protein which is essential for growth. Furthermore, the roots are also

reducing in size and dying. At the end of our observation, we found out that the leaflets decolourised and started to decompose and there arent any roots visible. As for plantlets in a normal solution which contain all the minerals needed, they grow successfully. The number of leaves increases dramatically, the colour of the leaves remain green and the roots is present within that 10 days. The plantlets were grown in an ideal condition. As for plantlets grow in other nutrient solutions such lacking of potassium, calcium, phosphorus, sulphur, magnesium, the number of leaves is still increasing but not that much. The colour of the leaves also changes only from green to pale green for certain lacking of minerals and the roots are still present within that 10 days. This also shows that the presence of iron may be needed by the plants in very small quantity but it can survive for a long time without it. However, some yellow spots and white are noted on young leaves due to chlorosis. This is most probably because those minerals especially iron still has some minor roles in the formation of chlorophyll besides responsible for the respiration and metabolism of the plant. Iron is a component of ferredoxin 4 (iron sulphur proteins) that mediates electron transfer in light dependent reactions in plants. Sulphur is an essential element for plant and animal health, and is needed in approximately the same quantities as phosphorus. In plants, sulphur is involved in the synthesis of proteins, especially the amino acids cysteine and methionine.5 However, sulphur seems to give effects more on the plants compared to phosphorus. SAFETY PRECAUTIONS AND RISK ASSESSMENT: In this experiment, a few steps should be taken into consideration when conducting the experiment. Firstly, the Lemna sp. plants should be arranged to maximum distance within the Petri dish to ensure minimum competition. Place all the Petri dish at same location so that all the Lemna sp. plants in all the culture shall receive the same sunlight, temperature and humidity. Avoid placing the Petri dish on top of another. Next, choose the Lemna sp. plants of the similar size and similar intensity of chlorophyll by checking them first
4

http://en.wikipedia.org/wiki/Ferredoxin http://en.wikipedia.org/wiki/minerals

under stereo microscope. This can ensure the age of the Lemna sp. plants used. Observe all the plants at regular interval carefully to make sure the right changes of colour and size of Lemna sp. plants during the experiment. Ensure that the Lemna sp. plants do not adhere to the wall of the cover. If it is stick to the wall of the cover, use a sterilised forceps to transfer it back to the nutrient solution. Use sterilised forceps to transfer the plants and avoid your finger from touching the nutrient solution and the plant to prevent contamination. After a few days of observations, some water droplets may be found condensed on the cover. Wipe the cover dry to give a clearer observation. ERRORS AND MODIFICATIONS: There are some anomalies in the results because we have chosen plants of different ages. We may even choose plants with poor health conditions. Hence, choose the Lemna sp. plants of the similar size and chlorophyll intensity. Furthermore, light intensity may not be constant because weather patterns are fluctuating. On a rainy day, there may be not sunlight. Furthermore, the nutrient solution in the Petri dishes can dry out at different rates. Carbon dioxide concentration and temperature can fluctuate due to changing weather patterns. To overcome this errors, all the Lemna sp. plants must be place at the same location so that if any change in the temperature, carbon dioxide concentration, humidity and sunlight, all the plants would experience the same change, hence, would not affect the overall results. Besides, each plant has its own rate of photosynthesis. They have period where the activity of photosynthesis is very high (usually in the afternoon) and a period of low photosynthesis rate (usually at dawn or dusk). In addition, the anomalies can be caused by our wrong judgments on the colour of the leaves. During our observations, we can see leaves with pale green and dark green colour. This could be minimised as we can assume the size and the colour of Lemna sp. plants under the microscope. However, we cannot judge which leaf is healthy. Some slides which are contaminated by fungal or bacterial contamination can influence our results too. Admittedly, the nutrient in the Petri dish may be use up by plants where we can see the plants in the complete culture solution has some white spots on them. So, make sure all the slides and Petri dish used has been cleansed or even sterile first before conducting the experiments.

CONCLUSION: Plants need nutrients such as nitrogen, calcium, potassium, and magnesium for their optimal growth. These macronutrients are imperative for the formation of vital components of plants such as nitrogen for the formation of amino acids, protein and chlorophyll. Besides, iron is the least important nutrient in plants and it is only needed in small amounts. In a conclusion, plant grows healthy in complete solution and shows symptoms when lacking certain mineral. The hypothesis is accepted by this experiment. REFERENCES: 1. http://www.123helpme.com/view.asp?id=120325 2. Lee, C. 2007. Longman Pre-U Text STPM Biology.448. Shah Alam: Pearson Malaysia Sdn. Bhd. 3. http://www.hort.wisc.edu/cran/pubs_archive/proceedings/1994/minpet.pdf 4. http://www.rook.org/earl/bwca/nature/aquatics/lemna.html 5. http://4e.plantphys.net/article.php?ch=t&id=289 6. http://en.wikipedia.org/wiki/Chlorophyll 7. http://en.wikipedia.org/wiki/Ferredoxin 8. http://en.wikipedia.org/wiki/Lemna 9. http://en.wikipedia.org/wiki/Photosynthesis 10. http://en.wikipedia.org/wiki/minerals