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Abstract Plants suffering from lacking of mineral grow poorly and manifest various signs of the deficiency in their physical appearance. In this experiment, we will grow Lemna sp. Plantlets under controlled conditions. Following range of nutrient solutions to explore were chosen : nitrogen, phosphorus, magnesium, potassium, calcium, iron and sulphate. The plantlets were then placed in a medium that were lacking in selected nutrient and also two more of plantlet sets were placed in fully absent and present of all-nutrient medium. They were then, observer for 14 constituent days for symptoms and recorded. INTRODUCTION 1. Lemna sp. (1) Lemna sp. or commonly known as duckweed among society is an aquatic plant that float freely on or just beneath the water surface. Most are small, not exceeding 5mm in length. This species grow abundantly forming colonization in ponds and lakes in large number mainly by vegetative reproduction, specifically through budding. Lemna sp. are flowering plant and most of them produce sexually. Duckweed meal also high in protein, fats and fibres and its a good cattle feed.

Figure 1 : Lemna sp. (2)

Figure 2 : Budding of Lemna sp. (3)

Since Lemna sp. grow rapidly in short time, it is used as a model system for studies in community ecology, basic plant biology, in ecotoxicology, in production of biopharmaceuticals, and as a source of animal feeds for agriculture and aquaculture so that no ethical issues will rise.

When Lemna sp. invades a waterway, it can be removed mechanically, by the addition of herbivorous fish 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.

2. Chlorosis (4) Chlorosis is a condition in which leaves become pale, yellow or yellow-white due to insufficient production of chlorophyll. The affected leaves will unable to perform photosynthesis thus, unable to manufacture carbohydrate which will lead death of the plant due to insufficient nutrients. Chlorosis may caused by a specific mineral such as iron, nitrogen and magnesium deficiency in the soil, poor drainage damaged or compacted root exposure to sulphur chloride and many more.

Figure 3 : Chlorosis of leaf (5)

3. Nutrients (6)

Sixteen elements are known to be important to a plant's growth and survival. They are carbon, hydrogen, oxygen, nitrogen, phosphorous, sulphur, potassium, calcium, magnesium, copper, zinc, molyhdenum, boron, iron, chloride and manganese.

Each of these nutrients has a critical function in plants and is required in varying amounts in plant tissue. Plants show symptoms being unhealthy when they experience insufficient nutrients. Too little or too much of any one nutrient can cause problems. Plant nutrients fall into 2 categories: macronutrients and micronutrients.

As the name suggest, macronutrients are those elements that are needed in relatively large amounts while micronutrients are those elements that plants needed in small amount. Both macronutrients and micronutrients are naturally obtained by the roots from the soil. Macronutrients are further divided into two groups : primary and secondary nutrients. The primary nutrients are nitrogen (N), phosphorus (P), and potassium (K). These major nutrients usually are lacking from the soil because plants use large amounts for their growth and survival. The secondary nutrients are calcium (Ca), magnesium (Mg), and sulfur (S).

Figure 4 : Essential Plant Nutrients(7)

Figure 5 : Image of plant that experience deficiency of nutrient(8)

Table 1 : Some of the nutrients physiological role and deficient symptom (9)

Objective To investigate the effect of plant mineral deficiencies. Problem Statement How do different mineral deficiencies affect the growth of the Lemna sp. plantlets? Hypothesis Plants need both macronutrients and micronutrients in right proportion to achieve optimal growth and development. Absence of one or more of these nutrients can lead to mineral deficiencies in plants and these mineral deficiencies can be found out using several symptoms that are showed up in the plant.

Variable : Types of Variables Manipulated Variable: Type of culture solution used Use different type of culture solution of same amount measured using small beakers. Ways to control the variables

Responding Variables: Growth condition of the Lemna sp. Plantlets All the symptoms and conditions (number of plantlets, number of dead and green leaves, and root length) were observed, measured and noted. Fixed Variables: Type of Lemna sp. used All the Lemna sp. Plantlets were taken from same pond area and were kept in same solution in laboratory. Lemna sp. With 2 leaves (that were almost identical in colour and size) were used throughout the experiment. Volume of culture solution used (ml) Use small beakers to measure 15ml of each nutrient solutions before the Lemna sp. Is cultured.

Number of leaves in on Lemna sp. plantlet

Apparatus 9 petri dishes with lids, small beakers, forceps, labeling stickers, cling film, paper, black pen. Materials Lemna sp. plantlets (from pond water), distilled water, a range of culture solutions (15cm 3) containing : All nutrients Lack of nitrogen Lack of phosphorus Lack of potassium Lack of magnesium Lack of calcium Lack of iron Lack of sulphur Lack of all nutrients

Procedure 1. Nine petri dishes were rinsed with distilled water and labeled with stickers indicating nutrient absent in each of their culture medium, as following: All nutrients Lack of nitrogen Lack of phosphorus Lack of potassium Lack of magnesium Lack of calcium Lack of iron Lack of sulphur Lack of all nutrients

2. Using a small beaker, 15cm3 of the culture solution containing all nutrients (nitrogen, phosphorus, potassium, magnesium, calcium, iron, sulphur) is measured using a small beaker and was poured into its respective petri dish. 3. Five pair of Lemna sp. each containing two buds and roots was picked up using a pair of forceps and gently transferred into the respective petri dish. 4. Steps two and three were repeated for other petri dishes. 5. The petri dishes were then covered with cling film was used to prevent the solution from dripping out of the petri dish. 6. After all the petri dishes were placed in the same brightly lit area, the cling film was removed gently.

7. The experiment was allowed to proceed for fourteen days and the growth of Lemna sp. plantlets was observed and examined every day with any changes observed noted. 8. The growth of Lemna sp. plantlets were measured using a scale that was drawn on a paper using black ink pen. Since the petri dish is transparent thus, it was placed above the scale paper and adjusted to obtain the reading. 9. The data was collected were based on the number of platelet, the number of green leaves, the number of dead leaves (that are white or yellow), and the average root length.

Safety precaution In order to avoid any accident or injury during the experiment in laboratory, the precautionary steps should be taken and applied. Wearing lab coat and a pair of suitable shoes are

compulsory when conducting an experiment in the lab at all times to protect the skin and clothing from spillage of any substance. Washing hands thoroughly with soap and water before and after conducting experiment is vital to avoid contamination. Furthermore, the glassware such as small beakers should be handled with full care because they are fragile. The apparatus such as forceps is also sterilized to prevent infection of microorganism and should be used with care to avoid any injury. After using all samples and apparatus at the end of experiment, they should be discarded properly and returned back to their places to avoid injuries and unnecessary accidents that may result fatal results.

Risk Assessment The Lemna sp. plant should be choose carefully as they are very fragile little creature. Other than that, once apparatus (such as small beakers) was used, they were washed and placed in their place. The petri dishes were rinsed with distilled water to remove any impurities and microorganism that may cause disruption to the plants health and growth.


Solution All Nutrients Present

Observations 0 5 10 1.70 5 10 0.76 5 10 1.20 5 10 1.04 2 5 10 1.80 5 10 0.76 5 11 1.25 5 11 1.04 4 6 14 1.58 5 10 0.72 5 11 1.48 6 11 1Y 1.33 6 6 15 -

Days 8 7 15 1.65 4 7 2Y, 1W 0.61 4 9 1W 1.01 6 10 2W 1.36 10 8 18 1.52 4 5 3Y, 2W 0.62 4 9 1.25 5 8 1Y 1.24 12 10 19 1Y 1.50 3 2 2Y, 2W 0.59 4 8 2W 1.18 3 7 2Y 1.25 14 13 19 1W 1.49 3 2 2W 0.55 4 8 2W 0.99 3 7 2Y 0.78

Number of Plantlets Number of Green Leaves Number of Dead Leaves (Yellow/White) Average Root Length (cm) Lack of Number of Nitrogen Plantlets Number of Green Leaves Number of Dead Leaves (Yellow/White) Average Root Length (cm) Lack of Number of Phosphorus Plantlets Number of Green Leaves Number of Dead Leaves (Yellow/White) Average Root Length (cm) Lack of Number of Potassium Plantlets Number of Green Leaves Number of Dead Leaves (Yellow/White) Average Root Length (cm)

1.60 5 8 2Y 0.71 5 10 1Y 1.04 6 12 1Y 1.35

Lack of Number of Magnesium Plantlets Number of Green Leaves Number of Dead Leaves (Yellow/White) Average Root Length (cm) Lack of Number of Calcium Plantlets Number of Green Leaves Number of Dead Leaves (Yellow/White) Average Root Length (cm) Lack of Number of Iron Plantlets Number of Green Leaves Number of Dead Leaves (Yellow/White) Average Root Length (cm) Lack of Number of Sulphur Plantlets Number of Green Leaves Number of Dead Leaves (Yellow/White) Average Root Length (cm) Lack of all Number of nutrients Plantlets Number of Green Leaves Number of Dead Leaves (Yellow/White) Average Root Length (cm)

5 10 1.32 5 10 1.30 5 10 1.16 5 10 1.88 5 10 1.30

5 10 1.20 5 10 1.40 5 10 1.26 5 10 1.38 5 10 1.08

5 9 1Y 1.25 5 8 2Y 1.33 6 12 1.18 6 11 1.24 4 9 2Y 1.20

4 8 1Y 1.54 4 7 2Y 0.87 7 14 1.23 7 13 1.23 3 8 2Y, 1W 1.23

4 8 1W 1.63 3 5 4W 0.73 7 14 1Y 1.05 7 14 1.15 2 7 3W 0.82

4 7 2Y 1.15 2 4 4W 0.69 8 16 1.32 9 17 1W 1.30 2 7 2Y, 1W 0.63

4 5 2Y 1.24 2 4 3W 0.70 8 17 1.05 11 18 1.14 2 5 2W 0.54

3 5 1Y, 2W 0.99 1 2 3W 0.53 8 17 1W 1.34 11 20 1.53 2 3 3Y 0.32

Data Interpretation(10) This experiment was conducted to study the effect of various mineral deficiencies on the Lemna sp. plantlet or also known as duckweed. Lemna sp. plantlets were used in this experiment because they are found abundantly in pond environment, easily obtained and have less ethical issues . but most importantly, the effects of any deficiency of mineral can be seen quite clearly in short amount of time as this plant only possesses short life span. Amount of sunlight (by placing all petri dishes containing medium nearby window), air and temperature for the Lemna sp. plantlets were kept in control for all the cultures. This is to ensure that the only factor that will affect the growth of the Lemna sp. plantlets were the different culture solution used. As discussed in procedure, by manipulating the absence of the minerals in culture solution, the effect of mineral deficiencies that were shown physically (number of plantlets, number of green leaves, number of dead leaves that are yellow or white or being in different state or colour and average root length) by the Lemna sp. plantlets which are responding variables in this experiment were noted. A culture medium containing all necessary nutrients were used as a control in this experiment to show the actual growth and development that should be achieved theoretically by Lemna sp. plantlets. This controlled culture medium sample was then used as comparison with other mineral deficient cultures to point out the effect resulted by specific mineral deficiency.

Control Medium Looking at the result , it can be concluded that presence of all the macronutrients namely nitrogen, phosphorus, potassium, magnesium, calcium and sulphur and micronutrient iron results in increase in both number of plantlets and number of green leaves. Increase in number of plantlets is due to their vegetative reproduction (budding) since there is enough availability of source and nutrients needed for their development. Average of the root length also seems to be constant and this indicates good root development despite the increase in number of the plantlets. Other that this, it also noted that only one of the green leaves turn to yellow and then white and this may due to the competition in obtaining nutrients among the plants.

Figure 1 : Some of the Lemna sp. plantlets in control medium at the end of the observation day.

From the results that were obtained, it was shown that the Lemna sp. plantlets were affected in several ways due to the following mineral deficiencies : Without Nitrogen From Table 1, it can be seen that the number plantlets stays the same for six days before it reduces into four and three by the end of observation period. Number of green leaves also noticeably reduce although it remained constant for four days. Number of dead leaves (9 yellows and 7 white leaves) in this medium is also at high rate and the average length of root linearly decreases with observation days. These may be due to inability of the plantlet to synthesis proteins, nucleic acids, chlorophyll and enzymes for photosynthesis and respiration continuously thus, halting the leaf growth and root development. The plantlets also seemed to be having stunted growth as no distinct differences can be seen in its shoot development. The green leaves also become paler until the end of the observation day none of the leaves were in actual green colour. Thus, it is concluded that disruption in chlorophyll production lead to chlorosis.

Figure 2 : Some of the Lemna sp. plantlets in nitrogen lacking medium at the end of the observation day.

Without Phosphorus In this culture medium, number of Lemna sp. plantlets stays constant for 6 days as five and reduced to four until the end day. The number of green leaves, although seem to increase at first, but it also reduces throughout the experiment but in a more stable manner; and there is no apparent changes in colour of the leaves. Number of dead leaves also in lower rate and average root length varied along the time. Since phosphorus plays an important role in synthesizing nucleic acids, adenosine triphosphates (ATP)and phospholipids of plasma membrane and also acts as a coenzyme in both photosynthesis and respiration, thus it can be concluded that absence of this phosphorus resulted in poor root formation and lower metabolism rates (leads to reduced photosynthesis and reproduction processes).

Figure 3 : Some of the Lemna sp. plantlets in phosphorus lacking medium at the end of the observation day.

Without Potassium In this medium, it was noted from Table 1 that the number of Lemna sp. plantlets and number of green leaves to increase but then dramatically fall on day 12. The number of dead leaves ( 7 yellow leaves and 2 white leaves) also can be said to be high and the average root length seem to be vary. Pottasium plays role in osmosis regulation but the impact caused on growth rate is the function of potasium in opening stomata. Without enough potassium ions to regulate the opening of stomata, the rate of photosynthesis is affected thus directly affecting the growth of the Lemna sp. plantlets, hence explaining the reduction of both the number of plantlets and green leaves on the end of the observation day. Some green leaves turn into pale colour throughout the experiment indicate symptoms of chlorosis .

Figure 4 : Some of the Lemna sp. plantlets in potassium lacking medium at the end of the observation day.

Without magnesium From both Table 1, it can be seen that the number of plantlets in magnesium lacking medium decreases into three at the end of observation day. Number of green leaves seem to decrease quiet steeply and number of dead leaves also can be said to be high, which is about 10 leaves. The average root length seems to be varied too and the green leaves themselves fade in colour throughout the experiment period. Magnesium is an important macronutrient in chlorophyll synthesis and absence of this nutrient result in lower production of chlorophyll thus reduces the rate of photosynthesis. This, without any doubt will affect both root and shoot growth and full development of the plantlet. Some symptoms of chlorosis can be seen on the leaves where the regions between the veins turn yellow. And magnesium being the key to turn on other plant enzymes and being involved in carbohydrate metabolism, absence of this nutrient slows down the growth of plant as translocation of photosynthase is slowed down and thus affect the uptake of other elements.

Figure 5 : Some of the Lemna sp. plantlets in magnesium lacking medium at the end of the observation day.

Without calcium

From Table 1 it can be seen that number of plantlets stay constant for six days but fall down to one on the final day, while number of green plant decreases across the period dramatically. The number of dead leaves in calcium lacking medium is the highest among all other mediums where it has 4 yellow leaves and 14 white leaves, which some of them decompose before the end day. The average root length also deteriorates across the time line showing that the condition of the plantlet is the worst among the bad. Calcium is the major constituent of the middle lamella of cell walls and key for the formation of spindle fibre during cell division. Without calcium ion, cell division cannot occur at all resulting in the death of the plantlets in long term run. Since the permeability of the cell wall also not regulated by calcium in this medium, so some of vital living processes fail to take place leading towards cell dying and decaying , explaining the reduction of root length and green leaves.

Figure 6 : Some of the Lemna sp. plantlets in calcium lacking medium at the end of the observation day.

Without Iron Number of plantlets in this iron lacking medium slowly increase and the same goes with number of green leaves, but the leaves become lighter in colour across time. There is no much variance in average root length. Absence iron affects the production of chlorophyll leads to lower photosynthesis activity, which in turns slows down the growth of the plant. Although iron is a micronutrient, the absence of this nutrient still give impact to plants growth and development (slow and stunted growth).

Figure 7 : Some of the Lemna sp. plantlets in iron lacking medium at the end of the observation day.

Without sulfur Based on Table 1 , number of plantlets and green leaves increases without much disruptions while only one leave was noted to turn white. Other than that, the average length of root doesnt vary much but have its own small dramatic changes. The colour of green leaves noted to be slightly yellowish but not fully, thus it was counted under number of green leaves. Deficiency in sulphur halts the synthesis of certain amino acids, nucleic acids, vitamin B and other coenzymes. This results in yellowing of leaves and delayed maturity..

Figure 8 : Some of the Lemna sp. plantlets in sulphur lacking medium at the end of the observation day.

Lack of all nutrients Without any essential nutrients, the number of plantlets, number of green leaves and average length of root goes down the slope. The vice versa happens for the number of dead leaves and eventually none of the plantlet survive till the final day. This is because all the metabolic activities are restricted thus the plants cant produce any food to survive thus resulting in zero survival chance.

Figure 9 : Some of the Lemna sp. plantlets in all nutrient lacking medium at the end of the observation day

Limitations There are several limitations that have been identified throughout this experiment. The Lemna sp. plantlets may be infected earlier before it is culture into the medium. This may lead to inaccurate results as it may change the symptoms of the mineral deficiencies . During picking up the Lemna sp. plantlet, the number of leaves was controlled to be two. But, there are possibilities that at that very moment, the plantlet is in the middle of having another new leaves. Thus, this will affect both the number of green leaves and number of plantlets , leading to misinterpretation about certain mineral deficiencies. Since water vapour formation raising difficulties in counting the number of plants, the petri dishs lid was opened up and wiped. At this moment, the plantlets in the medium are very vulnerable for the microorganism in the air to attack. Furthermore, this unintentionally provides carbon dioxide for the plantlets in various amounts that may contribute to different development. For medium of lacking all nutrients, distilled water was used. This may alter the result as the distilled water was placed in container and exposed to the surrounding for long time may contain microorganisms or minerals from air.

Sources of errors Several sources of error in this experiment were identified and steps were taken to minimize these errors to make the result more accurate. Floating Lemna sp. plantlets tend to clump together whenever they were given even the slightest force. So, they were arranged to maximum distance within the petri dish to ensure minimum competition. To avoid contamination, a pair of sterilized forceps was used gently to place the plantlets. The solution medium may split out when the petri dishes were brought back to the window side. Thus, by placing cling film around the petri dish, any alteration in volume of solution is prevented.

Conclusion Plants need both macro (Nitrogen, Phosphorus, Potassium, Magnesium, Calcium and Sulphur) and micro (Iron) nutrients to achieve full growth and development. Deficiency in any one of the mineral can be spotted through the symptoms that will display on its physical appearance. Different type of mineral deficiency results in different effects on the growth of Lemna sp. plantlets. Thus, the hypothesis is accepted.

Further Investigation Another experiment can be carried out by replacing Lemna sp. plantlets with barley seedlings using water culture technique. The seedlings are grown in culture solution (same with the above procedure) but in a test tube. The test tube should be covered by foil to exclude light, preventing algae growth. The seeds are moisten to germinate a week before use. Plants should inspect regularly for general growth, shape of leaves, length of leaf growth, colour of upper leaves, length of root growth, colour of lower leaves and mass of the seedling (before and after the experiment).

Figure 10: Set up of apparatus for further experiment (11)

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