Plant Module 1 Final

Laboratory 1 (There are 4 parts to this lab) Introduction & Plant Growth Biology 106 Plant Module: LAB 1 of 3 ( This document has been modified from the original protocol written by Dr. Lisa Carloye and her group of Biol 106 instructors at WSU ) 1. Plant Module Case Study – Introduction 2. Scientific investigation – designing experiments to answer questions about the role of nutrients in increasing plant biomass for a canola farm. 3. Data management: ± Using spreadsheets to organize data. ± Analyzing data using statistics ± Visually representing data using bar graphs. 4. Getting started – experiment set up. PART 1: PLANT MODULE CASE STUDY Trade wars with China have dominated the political and economic news lately as President Trump slaps tariffs on Chinese goods and China retaliates with tariffs on targeted US goods. Amidst this economic bad news for farmers, one crop produced here on the Palouse may benefit from the restrictions created by this trade war: Rapeseed ( Brassica napus ). One of the commodities hard hit by China’s tariffs is US-grown soybeans, so China has been increasing their demand for rapeseed to help offset the lost oil they extract from soybeans. Rapeseed is grown for the oil produced in the seeds. In the spring, the intense yellow of rapeseed ( Brassica napus ) covering select fields around Pullman is a stunning display, attracting photographers from around the world. Depending on the variety grown, rapeseed is harvested for use in cooking (canola oil), and as an ingredient in margarine and soap. Its oils have long been used as an industrial lubricant for jet engines and marine engines due to its ability to work at high temperatures. It is also used in plastics such as bread wrappers and garbage bags. It can be made into biodiesel as well. Furthermore, after the oil is extracted, the plant residue can be used in feed for livestock and the plant can be grown as forage for cattle to graze. Rapeseed is a highly versatile commodity. Q. Based on this list of uses, categorize the ways in which we use rapeseed. Give between 3 and 4 types (categories) of uses (e.g., “Food production”): In order to make the most of this increased demand for rapeseed in China (and around the world), local producers want to maximize their yield and produce a high-quality product. Plants, like animals, require resources from the environment to grow and produce healthy offspring. Over the next three labs you will investigate factors important for plant growth and maximizing yield. You will then draw conclusions and write a report to communicate your findings to the Canola Council of Canada (and earn a grade for Bio 106). _ cooking (canola oil) _ , ___ Jet engine lubrican __ , __ Plastics (wrappers ____ , Forage for cattle ___________

Biology 106: 2022 Summer 2 ± Please list below 4 resources you think plants require for growth: PART 2: SCIENTIFIC EXPERIMENTATION Because one common way farmers produce quality crops is to increase soil nutrients by applying fertilizers, we will focus our efforts in BIOL 106 on determining just how much fertilizer is necessary to impact plant biomass and, potentially, increase yield. Furthermore, we will investigate whether increasing access to nutrients also increases herbivory on the plant tissues since this might just negate any benefits seen with add fertilizer to the growing crop. In this lab, you will gather data to answer two key questions using a fast-growing relative of Brassica napus , Brassica rapa (the data you will use in this experiment was obtained by Dr. Lisa Carloye and her group of Bio 106 instructors who previously conducted the study to produce data for your use) : ± Question 1: Will adding nutrients (fertilizer) to the growth medium increase Brassica growth? If so, how much is optimal? ± Question 2: Will increased nutrients affect the amount of feeding on the leaves by primary consumers (herbivores)? In order to answer these questions, we need to know how to design and conduct a valid, scientific experiment. Exercise 1: The scientific approach to answering questions Step 1: Formulate a testable hypothesis, often referred to as the alternative hypothesis, (also formulate its companion null hypothesis). In science, questions are investigated through the process of hypothesis testing. A hypothesis is a testable explanation for a set of observations based on the available data and guided by inductive reasoning (Campbell Biology, 2014). Note that hypotheses are testable explanations constructed as statements, not if/then sentences which are more like predictions. For example, “Water is necessary to rehydrate carbohydrates in seeds in order to trigger germination,” is a hypothesis (we know water is necessary for seeds to germinate and think it is logical they facilitate seed germination by freeing up carbohydrates to use for growth). Experiments are conducted and the information collected (data) is used to test a pair of complementary hypotheses: the null hypothesis ( H 0 ) and the alternative hypothesis ( H 1 ). For question #1 above, the null hypothesis (H 0 ) would be “adding nutrients will have no effect on plant yield”. The alternative hypothesis (H 1 ) could be, “adding nutrients will increase plant yield.” Note how the alternative hypothesis is the plausible hypothesis you have developed and want to test. 1. Sunlight 2. Water 3. Nutrients 4. CO2 e 3. t How can w e t whether or not the items listed above are important? As scientists, we do this by developing testable 4. hypotheses and designing experiments to test them.

Biology 106: 2022 Summer 4 ± Label each component to make it clear what you would use, how you would use it, and what it will tell you. Q. Can you get meaningful results from just using 1 plant per treatment? Why or why not? Discuss the importance of replication in an experiment. It would not be as meaningful because the treatment working for one plant could be an outlier. The more plants you have the more valid the results. Being able to replicate the study helps determine its validity. Q. For this experiment, each pot will house 4 seeds. Why do you think we chose 4 instead of fewer, or more? 4 Seeds were chosen in order to determine replicability of the experiment however still give the seeds room to grow and receive nutrients. If there was less than 4 seeds the study may not be as valid and reliable. Step 3: Make a prediction! Predicting results provides a critical analysis of the experimental design. A prediction is always based on a particular experiment designed to test a specific hypothesis. Predictions are written in the form of if/then statements: “ If the hypothesis is supported, then the results of the experiment will be…” Please write an if/then statement for the alternative hypothesis (H 1 ) above and your predicted outcome: Exercise 2: Preparing the Plants Brasica rapa seeds were planted in pots containing nutrient-free soil and watered with deionized (DI) water by BIOLOGY 106 Lab Manager Kara . Over the course of 3 weeks, Kara photographed the plants to record their growth. METHODS: ± Each pot was planted with 6-8 Brassica rapa seeds. ± After seed germination, Lab Manager Kara identified the 6 healthiest looking plants, and removed all other seedlings, leaving these 6 plants undisturbed. You will measure 4 of these and they will be your experimental subjects. ± At the start of the study each pot was randomly selected for a given treatment and fitted with 1 label tag: 1. Four pots were labeled “ +fertilizer-HIGH ” 2. Four pots were labeled “ +fertilizer-MEDIUM ” 3. Four pots were labeled “ +fertilizer-LOW ” 4. Four pots were labeled “ ±± fertilizer-CONTROL ” ± Each pot was then photographed at eye-level. These photographs capture the seedlings before they receive their experimental or control treatment and serve as the baseline. ± One plant from each pot was measured (in mm) and that number was recorded. This measurement allowed Drs. Hellmann and Carloye to scale the

Biology 106: 2022 Summer 5 online ruler you will use to measure the plants. The ruler is found in the Powerpoint slides showing the potted plants from which you will obtain your measurements – do not resize the ruler so that you obtain accurate measurements! Exercise 3: Data Collection PROCEDURE : 1. Open the document “ Lab 1 Brassica Plants, bio 106, f2022.pptx , ” posted on Canvas. This has pictures of the plants. 2. Look carefully at the little seedlings that have sprouted in the pots. Q. How many plants are growing in the Control-Pot 2 (the second photo in the row)? __ 5 _____ Q. How many plants are growing in the LOW-pot 1 (the first photo in the row)?__ 6 ____ 3. Use the ruler provided on the page to measure each of the 4 plants indicated in each CONTROL pot in millimeters. ± The ruler should be able to be “grabbed” and moved to each plant in turn. ± Measure from the vermiculite surface to the tallest part of the plant as best you can. ± Record the heights for your potted plants here, and give the units: Plant 1: 1mm Plant 2 2mm Plant 3 2mm Plant 4 1.8mm Q. What factors make it challenging to accurately measure these plants? List them all: Accurately measuring size, leaf size, measuring consistently across plants, using power point software, making sure to measure from the 0 of the ruler, distance distortion 4. Open the Plant Data Excel Spreadsheet posted on Canvas, file, “ Plant Module lab 1 Excel Data Sheet, bio 106, f2022 master.xlsx .” Record the data by entering the plant heights into the appropriate cell, one plant height per cell (only numbers, do not include units ), in the “Raw Data” sheet, based on the treatment, pot and plant: Type in the number you measured for the Control treatment, Pot 1, and one of the plants. Then continue on until you have entered all of the plants in all of the treatments with one plant per cell

Biology 106: 2022 Summer 7 o High variance indicates the data points are spread out fairly far from the mean, both above and below the mean, and from each other. How to calculate Variance : 1. First calculate the Mean by adding the data points for a given treatments and dividing by the total number of data points used. ( 1 cm + 2 cm + 3 cm + 4 cm + 5 cm )/5 plants = 15/5 = The mean height is 3 cm. 2. Then, for each individual data point, subtract the Mean and then square the result and sum them up. Ex: (Plant 1 height – mean height) 2 + (Plant 2 height – mean height) 2 + (Plant 3 height – mean height) 2 … This is the sum of squared differences from the mean. To square a number, just multiply it by itself: 3 2 = 3x3 = 9 3. Then work out the average of those squared differences. Which altogether looks like this: [ (Plant 1 height – mean height) 2 +(Plant 2 height – mean height) 2 + (Plant 3 height – mean height) 2 … ] / [Total number of plants] Write out the calculation for variance in the LOW treatment for the 4 plants in Pot 1 just to get a feel for it. For the denominator, use the total number of plants you used in the calculation (4). Now, dry your tears from this painfully long calculation and hope that Bill Gates has created some wonderful tool to help ease your pain. You’re in luck because he did! It’s called Excel . You will recalculate the variance using all of the plants in the Low Treatment using Excel (and then your denominator will be for all 16 plants instead of just the 4 you used in your hand calculation above). Exercise 4: Using Excel to manage your data and calculate your statistical analysis Procedure: a. Enter a formula to calculate the Mean and Standard Deviation. To enter a formula, highlight a cell, then type an equal sign (=) into the cell or in the formula bar. You then follow the equal sign with the name of the calculation you want it to perform. (If, for some reason, you can’t get this to work, i.e., give you a numeric value, Google, “how to calculate average, or variance,” for your version of Excel). Follow the directions below to perform the following calculations: Calculations : 3.5 Cell Worksheet Formula

Biology 106: 2022 Summer 8 1. Mean: In the cell below the last data cell (should be cell B19 for the controls, C19 for Low fert., …), type =AVERAGE( … then use the mouse to highlight all of the data in the column (rows 3-18). After highlighting the data, press “Return.” If you return to cell in which you calculated the average, it should look like this: “=AVERAGE(B3:B18)” in the bar above the column headings (letters: A, B, …). The average of the highlighted data will be calculated in this cell of the spreadsheet. ± Repeat for each of the treatments. Q. Is the mean calculated by Excel the same as the mean you calculated by hand? YES / NO If not, double check for typos in your original hand calculation and in the Excel spreadsheet to figure out where the discrepancy comes from. 2. Variance: Use the cells below where you calculated the averages to calculate the variances. Be sure to label this row of cells on the left, e.g., in cell A20 enter “Var.” - make similar labels for your means (cell, A19) and standard deviations (below). ± Variance: =VAR.S(B3:B18) Record the calculated variance as calculated by Excel: Q. What is the LOW Fertilizer treatment’s variance? __ 3.39 ___________ Q. What is the MEDIUM Fertilizer treatment’s variance? _____ 10.13 ___ Q. What is the HIGH Fertilizer treatment’s variance? ____ 7.13 ____ Q. What is the Control treatment’s variance? __ .25 ___________ ± Standard deviation = i s a measure used to quantify the amount of variation in a set of data values that is expressed in the same units as the mean . This is similar to variance but because the units match those of the mean standard deviation can be plotted on the same graph as the means while variance cannot; variance is useful in complex statistical calculations. o Low standard deviation indicates that the data points tend to be very close to the mean. o High standard deviation indicates that the data points are spread out over a wider range of values. How to calculate Standard deviation (SD) as calculated by Excel :

Biology 106: 2022 Summer 10 Which type of graph? ± Bar graphs are often used when comparing values from two or more groups or categories. For example, the number of plants in a population with Red and White flowers. ± Line graphs are often used to show data that is part of a continuous process. For example, we have plotted the mean plant height over a period of days after planting. The X- and Y-axis are both continuous. ± The “error bars“ in the Line graph indicate the level of variation in plant height between individuals in a given treatment. We will discuss this way of representing variation more later. ± The independent variable goes on the x -axis (bottom) and is the one you manipulate . ± The dependent variable goes on the y -axis (left side) and is what you measure . ± Each axis must have an appropriate label . ± An axis may include the results of a measurement, including the units (Plant height in cm). Or an axis may contain categorical data, which is essentially a classification label (Flower color). ± A descriptive title, i.e. one that specifically describes what information is in the graph, is helpful. The title is placed at the top of the graph. For example, for the bar graph: “The number of red and white flowers” and for the line graph: “Change in plant height over time“. Bar graph: Q. If you wanted to compare the battery life of different brands of batteries, would the brands be categorical data or numeric data? Highlight one: Numeric / Categorical Q. Would the amount of time the battery lasts be Numeric / Categorical ? Q. Could a category be represented by a number? YES / NO Q. For the Bar Graph shown above, indicate the following: Q. Which variable is on the x-axis and which is on the y -axis? Line graph: Q. For which type of data would you use a line graph? Highlight one: Numeric / Categorical Explain: Dependent variable: _____ Independent variable________ x -axis = ______________ y -axis = _______________ Explain: Dependent variable: _____ Independent variable________ It would be number because it is the actual number included in the data x y Flower Color number of flowers Line graphs display changes in numbers and data on the x and y axis x y

Biology 106: 2022 Summer 11 Q. For the Line Graph above indicate the following parameters: Q. Which variable is on the x-axis and which is on the y -axis? Exercise 6: Now consider the data set from the Brassica growth experiment. Now apply your understanding of graphing to the data you collected on your plants today. Q. Would a bar or line graph be more appropriate for graphing these data? Highlight one: Bar graph / Line graph . ± Before doing it in Excel, do a quick sketch of what your graph should look like and show it to your TA (optional). Don't worry about depicting the actual values, just the general shape and components. Exercise 7: Making Graphs in Excel In science, your job as the research scientist is to analyze your data and then report your findings. A common mistake made by introductory students is to report every single data point rather than reporting the overall picture as indicated by the data analysis (mean, standard deviation). Your next job is to graph your data to create a visual representation of your overall results. Q. Should you graph each individual replicate, or should you graph your mean? Highlight one: Replicates / Mean The following are basic instructions for using Microsoft Excel 2013+ to make Column graphs. If you use a different version of Excel you may need to Google “how to” instructions for your version. Column Graph: Use this type of graph when your data represent a single measurement for each replicate (rather than measuring the same plant repeatedly over time). Step 1: Highlight the data you want to chart. Then click on the “Insert” tab at the top of the workbook. Step 2: Click on the “Column” option and then select the “2-D Clustered Column” – it is the first one in the row. A graph will appear with your mean data. Step 3: Format your graph by selecting the “ Plus ” icon to the side of your chart when the chart is selected. Use this to update the chart title and the axis titles. x -axis = _______________ y -axis = _______________ independent dependent

Biology 106: 2022 Summer 13 ± To mix the fertilizer, use the ratio of fertilizer per 1 liter of deionized water indicated for each treatment. Q. How many grams of fertilizer per liter will you use for each treatment? Before “mixing up the solutions”, pause and brainstorm what variables you need to control (i.e, the “controlled variables”) to ensure the only thing that is different between treatments is the amount of fertilizer: Making up the fertilizer solution (this is what Kara did for you, and, thus, you don’t do) 1. Measure 90 mL of deionized water in the graduated cylinder labeled “ + fertilizer ”. 2. Place this water into your beaker. 3. Weigh out the fertilizer amount you calculated above. 4. Do NOT place the fertilizer directly on the scale-weighing pan. Use “weigh boats” to hold your materials. 5. Turn on the scale. Find the white, plastic weigh boat and place it on the scale. 7. Tare (zero) the scale. 8. Add the fertilizer to the required amount. 9. Dissolve your fertilizer in the 90 mL of deionized water in the beaker. 10. Once crystals are dissolved return the liquid to the graduated cylinder and top up to 100 mL. Q. ( DO ANSWER THIS QUESTION ) Why do you think you don’t want to begin with the full 100ml? Step 2: Apply the treatments to the plants Step 3: Place plants under natural lighting allowing them to grow for 1 week. Treatments: Control: 0 g/ L – deionized water only Low: 0.75g/L Medium: 1.25g/L High: 2g/L Grams of Fertilizer to measure per Liter: Control: _____ g Low: _____ g Medium: _____ g High: _____ g Controlled variables: YOU ARE NOW READY FOR THE EXIT QUESTIONS The Exit Questions are what will be graded, although there are points associated with the completeness of the entire lab 0 .75 1.25 High: 2g/L Deionized water sprayed the same way as the fertilizer Same bottles, soil, pots used across all plants You may not want to start with the full 100 mL so you can properly add the crystals and dissolve allowing for extra space that the fertilizer takes up.

Biology 106: 2022 Summer 14 Lab 1 EXIT QUESTIONS Participation in and Completeness of lab: [5 pts] _________ Experimentation 1 What is the difference between a dependent and independent variable? [2 pt] 2. What does standard deviation show for a set of data? [1 pt] The Plant Nutrients Experiment 3a. In your own words, explain the real-world context motivating the experiment we are conducting for BIO 106:[1 pt] 3b. What is the question our experiment today seeks to answer? [1 pt] 4a. For the nutrient experiment you set up today, what are your dependent and independent variables? [1 pt] 4b. What is the experimental control treatment? [1 pt] 5.Why is it important to have replicates in this and other experiments? [1 pt] 6. What are the 3 key ingredients of the fertilizer you used to supply nutrients? [1 pt] 7. Calculate the mean for the follow dataset: [1 pt] Replicate Value 1 12.8 2 8.6 3 10.4 4 7.8 5 11.1 Mean: The dependent variable is what you measure, this variable stays the same The independent variable is what is changing (water vs fertilizer amounts) The standard deviation shows how the data variability is. A lower standard deviation is closer to the mean, and higher is farther away. A higher standard deviation means the data plots are not close together. In the real world people often do have gardens but it is up for debnate what to give plants. This experiement shows if we should be not using any fertilizer, some, or even a lot of fertilizer to help our plants grow and yield more. Our experiment seeks to answer if adding nutrients affects plant yield Dependent- Size if plants Independent- Pot and Plant number Deionized water To ensure validity of the experiment, if it can be replicated then we can find out the accuracy of claims and hypothesis Nitrogen, Phosphorus , Potassium 41.82