Discussion questions

How is corn used as a food? What part(s) of the plant is consumed?

Why is aseptic technique so important in this lab?

Corn seeds obviously do not germinate and grow in an aseptic environment in nature. How do they manage?

Could this experiment be done with a dicot such as the bean? How might the approach and the results differ? What about using a pine seed? A fern spore?

Objectives of this week’s lab

To observe the major anatomical features of seeds.

To understand the differences between dicot and monocot seeds.

To study the development of an isolated plant embryo into a seedling..

To introduce you to in vitro plant culture techniques, especially the aseptic culture of living embryos.

To investigate some of the nutritional and hormonal factors that control development from embryo to seedling in corn by manipulating the contents of the culture medium on which the isolated embryos are grown.

Reading

Campbell et al. 6th edition: pp. 790-794 and 806-817.

Lab 6-1

Lab 6 ** LAB PRACTICAL EXAM**SEED GERMINATION EXPERIMENTS

OCTOBER 18-22, 2004

LAB OVERVIEWSEED GERMINATION EXPERIMENTATION

The Big Picture:In angiosperms, the new sporophyte generation begins with the zygote, which develops into an embryo within a mature seed. Because it would be disadvantageous for seedlings to germinate while attached to the maternal

plan(although this can and does happen), maturation almost always involves arresting development of the embryo inside the seed. Today we begin studying hormonal and nutritional factors that regulate this arrested

state and the early phases of seedling growth.

BEFORE CLASS

Prepare for your midterm lab practical exam!The exa, will occupy less than one hour at the beginning of your lab. Following the exam, there will be a short break, followed by the pre-lab quiz and the lab itself.

Attend recitation and read today’s lab handout and pages from Campbell in preparation for the prelab quiz.

DURING LAB THIS WEEK

1—lab practical exam conisting of 18 one-minute stations (including 2 rest stops) and a five-minute period to complete answers at the end.

2—short break

3—pre-lab quiz

4—s tudy the anatomy of the dicot and monocot seed and seedlings

5--dissect embryos from corn seeds and set up an in vitro germination experiment

AS HOMEWORK Complete a worksheet predicting the outcome of your experiments.

DURING LAB NEXT WEEKObserve the embryos that you dissect today, collect data, and contribute your data to a pool from the entire class. As homework for your lab report, analyze class data and write report.

LAB REPORTDue by NOON, Friday 11/5

You will prepare bar graphs showing how the four variables responded to different experimental manipulations. Using a list of questions, you will prepare a lab report about this experiment. This is not a group project. You and your group members will write your reports individually.

Lab 6-2

Introduction to the angiosperm seed

The mature seed of an angiosperm is composed of three main parts: 1) seed coat; 2) embryo; and 3) stored food. Seeds are embedded in tissue derived from the ovary; this tissue is the fruit. The seed coat is derived from a layer of maternal tissue that surrounded the egg sac. The embryo is the young sporophyte generation formed by the fusion of the first sperm nucleus and egg nucleus during double fertilization. (We will cover this in much greater detail next week). As the embryo develops, one or two seed leaves, called cotyledons, begin to form.

The number of cotyledons is a characteristic that is used in naming the two majors groups of flowering plants:

monocots: plants whose embyos bear one cotyledon dicots: plants whose embryos bear two cotyledons.

The stored food in a seed either consists of or is derived from endosperm. The endosperm is a triploid tissue formed from the union of the second sperm nucleus and the two polar nuclei at the time of fertilization. Remember that there is a double fertilization: one 1n sperm nucleus fertilizes the 1n egg to form the 2n zygote, and the other 1n sperm nucleus fuses with the two 1n polar nuclei to form the 3n endosperm (keep in mind that many plants are polyploid, in which case “n”, the number of copies of each chromosome, are adjusted accordingly).

In many dicots, as the embryo develops, the cotyledons absorb nutrients from the endosperm and expand into the space once occupied by the endosperm. No endosperm remains in the mature seed, and the cotyledons are thick and fleshy and occupy most of the volume of the seed. In monocots, endosperm tissue serves the food-storing function in most mature monocot seeds and embryos possess a single cotyledon that serves as a food-absorbing, rather than a food-storing, structure.

Follow the directions below for dissecting the seeds of bean and corn and identify the three main components in each type of seed. Read the descriptions, and label all of the structures indicated on the accompanying drawings.

SAFETY : It should go without saying, but PLEASE be very careful with razor blades. They are EXTREMELY sharp and it is surprisingly easy to slice off the tip of your finger if you are not paying attention (your instructor has done it before) !!!!!

Bean Seed

Remove the seed coat (the outermost covering) of a bean seed that has been soaked in water overnight. Most lima beans will subsequently separate into the two cotyledons. If they do not separate, use a scalpel or razor blade to cut the bean seed in half longitudinally (as depicted in the following figure) and note the two large cotyledons, or seed leaves. You will not see any endosperm because it has been digested by the growing embryo and absorbed into the cotyledons. Identify: the epicotyl (epi- “above”, and coytl- “cotyledon), the region above the attachment of the cotyledons and the plumule, the tip of the epicotyl consisting of a shoot tip and two young leaves. The shoot apical meristem is at the apex of the epicotyl. The hypocotyl (hypo-“below”) lies below the attachment of the cotyledons and is terminated by the embryonic root or radicle. The root apical meristem is at the apex (or tip) of the radicle.

Lab 6-3

Corn Seed

Using a scalpel or razor blade, cut the broad side of a soaked corn kernel in half longitudinally. Notice that approximately half of the seed is filled with endosperm, which occupies a triangular area with the widest portion at the top of the seed. The corn embryo consists of both shoot and root structures. The plumule includes the shoot tip and embryonic leaves, enclosed in a hood-like sheath called the coleoptile. The shoot apical meristem is at the apex of the shoot tip. On the shoot there is also a scutellum, the single modified cotyledon, located between the endosperm and the embryo, which absorbs nutrients from the surrounding endosperm. The embryonic root, or radicle is enclosed within a sheath of tissue called the coleorhiza; the root apical meristem is at the apex of the radicle. The external layer of this structure is the inner layer of the ovary wall (labeled as ‘pericarp’ in the diagram below). In other words, this corn “seed” is technically a corn fruit.

Lab 6-4

Starch in seeds

The endosperm and cotyledons characteristically contain large quantities of stored carbon in the form of starch that supports the growth and development of the seedling until it can establish itself as a photosynthetically competent plant. Note that some seeds contain large quantities of fats/oils instead of starch—these can be pressed to create vegetable oils (like olive oil, soybean oil, or canola oil).

Determine where starch is stored in the bean and corn seeds by placing your dissected seeds in a petri dish and add a few drops of I2KI solution, a starch-specific stain, to them [note that this solution will stain your skin and clothing, so please be careful—wash with soap and water if you get it on your skin]. Let the seed sit for some time (approximately 5 minutes) before making your observations. Record your observations below (you will not turn these in, but you will be responsible for the information on your final exam).

Do both seeds store starch in the same places?

Where on each seed did the I2KI solution react with starch to form a purple/black color?

Is starch present in the cotyledons of both types of seeds?

How is the function of the cotyledon in corn different from that of the cotyledons in beans?

Lab 6-5

Seed maturation and seedling germination

Once the egg sac inside of an angiosperm flower is fertilized, the triploid endosperm and the diploid embryo develop in a coordinated manner until the seed and fruit are mature.

Although mature seeds are alive, they may not seem so at maturity and prior to germination. This is because the embryo within a mature seed enters an arrested state of development that involves removal of most of the water from the tissues. Plant hormones regulate the initiation and maintenance of this arrested state.

Mature seeds contain as little as 5-10% of their total weight as water. The embryo can resume growth only after the seed imbibes (absorbs) water, which is required for metabolic activity. When a seed is rehydrated and other requirements of oxygen and appropriate temperature are met, enzymes are synthesized and activated to metabolize stored nutrients. Stored food reserves provide the nutrition and energy for germination and development of the embryo until the seedling becomes self-sufficient when the immature leaves expand and become photosynthetic.

In corn seeds, the plant hormone gibberellic acid (GA), produced by the embryo, promotes germination by stimulating the aleurone layer to synthesize and secrete enzymes that are necessary to break down the starch, the stored food reserve in the endosperm, to glucose. Glucose is then absorbed by the scutellum and supplied to the developing seedling.

Some seeds germinate as soon as they are in a favorable environment. Others are dormant and will not germinate. They require prolonged cold exposure or leaching or abrasion of the seed coat before they can germinate. Some even require a trip through the digestive tract of a specific species of animal.

The plant hormone abscisic acid (ABA) has been implicated in the process of developmental arrest and dormancy. In certain concentrations, ABA has also been found to antagonize the action of GA, preventing mobilization of the stored endosperm reserves, therefore preventing germination.

In the Laboratory this week

One technique used to study plant embryos is to remove them aseptically from the surrounding tissues and grow them on a defined nutrient medium — in vitro embryo culture technique allows one to perform experiments to investigate, for example, the nutritional or hormonal factors regulating the growth and development of the embryo, development arrest, and the resumption of growth and germination of the embryo under controlled conditions.

In vitro (from the Latin “in glass,” meaning in a dish, plate, or test tube) contrasts with in vivo (from the Latin “in life,” meaning in a normal living context). Such experiments are difficult to perform with embryos surrounded by other tissues in the ovary or in the seed. Although in vitro experiments disrupt the normal relationship of the embryo to the rest of the plant or seed, they nevertheless provide information that is useful in learning about embryo development in the intact plant and seed.

We will culture embryos from corn kernels that are relatively mature but that have not yet had their water content reduced. Such embryos normally would not yet germinate. Because the seeds are slightly immature, the embryos occupy a somewhat smaller volume of the seed.

In this laboratory exercise, you will be investigating the effects of two plant hormones, GA and ABA, on the emergence of a seedling corn plant from its embryonic state. You will also observe the dependence of the corn seedling on a supply of glucose, a source of energy. The endosperm of the seed is the natural food supply during germination, but in vitro culture will separate the embryo from that resource.

Lab 6-6

You should recognize that the process of germination is very complex, especially in a grain such as corn. Basically, the tiny embryo is “all meristem.” It lacks true leaves and roots, does not carry out photosynthesis, and has very little capacity for absorbing mineral nutrients. Until functioning shoots and roots form, a complex regulation of development takes place. It involves growth of both the shoot and the root ‘fed’ by nutrition transferred from the endosperm. (In most dicots, the fleshy cotyledons ‘feed’ the earliest phases of seedling development. How would this process work in a conifer, which is a seed plant but not an angiosperm?) One thing you will want to think about is whether and how an isolated seed embryo can grow into a seedling.

This laboratory will require two sessions for completion. During the first session this week, you will aseptically excise corn embryos from their kernels and transfer four embryos to each of eight different media plates. These plates will then be incubated for one week. During the second session next week, you will assess the influence of each medium on growth and development of the embryos by weighing the embryos and by measuring the length of the shoot and root systems.

After you do the set-up this week, and you will have a worksheet to complete as a homework assignment. The worksheet will guide you in making predictions about how glucose, ABA, and GA will affect germination and subsequent seedling development and growth. The data you collect during lab next week will be contributed to a pool of data from all laboratory sections. Each student will write up an individual lab report based on the comparing her predictions with her group’s own data and the pooled class data.

In the experimental part of this lab:

This week: You will work in groups of four to dissect corn fruits and remove embryos, separating them from the nutritious endosperm tissue and the protective seed coat. You will investigate how the embryo inside of a seed emerges from its arrested state of development and begins to grow. Three factors will be investigated:

(1) glucose;(2) abscisic acid (ABA); and (3) gibberellic acid (GA).

We will work with embryos on sterile Petri plates filled with plant growth media that contains the nutrients necessary for embryo growth. The media in the Petri plates also contain glucose and the hormones GA and ABA.

During the week next week: Embryos on Petri plates will be incubated at controlled temperatures (25 º C) under constant fluorescent lighting.

Next week: If your sterile technique was successful, it should be possible to observe seedlings with roots growing into the media in the plates and green shoots growing above the media under the plate covers. You will collect data about the total weight of the embryos, the length of shoots and roots, and the number of lateral roots formed. If your sterile techniques were sloppy, you will find fungi and/or bacteria growing on the growth media—these contaminants are likely to kill your embryos.

Lab 6-7

FIRE SAFETY

Please make note of the location of the fire extinguisher(s) before beginning this lab:______________________________

You may be tired after finishing your lab practical exam. However, you must remain alert and be very careful throughout the rest of the lab period. You will be using Bunsen burners to maintain sterile conditions for your corn embryos. Bunsen burners can be dangerous. Please tie back your hair before this lab; students have set their hair on fire before. Use extreme caution when working near the Bunsen burner—it is on fire and can severely burn you. Sometimes the flame can be very difficult to see, so if you are not careful, you or a classmate could be seriously hurt. NEVER leave a Bunsen burner unattended.

You will be dipping your instruments into small beakers containing ethanol. When you briefly pass the instrument through the flame of your Bunsen burner, the alcohol will catch on fire. Be very careful about how you hold the instrument. If done improperly, the alcohol can drip down the instrument and onto your hand. When the alcohol is ignited, your skin could be too. ALWAYS hold the instrument so it is pointing down, away from your hand and clothing. DO NOT put an instrument that is dripping with alcohol through the flame; it can then drip BURNING alcohol onto you, your clothing, the lab bench, your lab notebook, etc. BE VERY CAREFUL.

If a drop of burning alcohol should happen to drip from your instrument, do not panic. There are glass lids available that you can place over the flame and it will extinguish quite quickly if it is on the lab bench (which is covered with a non-flammable surface). If something else has been caught on fire by the dripping alcohol, calmly ask for assistance from your lab instructor. It is quite easy to accidentally set the alcohol in your beaker on fire, and it may be very difficult to see the flame. Note that it is possible to be seriously burned by this flame. PLEASE BE CAREFUL.

Aseptic Technique

Sterile media and sterile technique must be used to prevent contamination of your experimental embryo cultures. The embryo inside an intact, undamaged seed is sterile. Thus, if you sterilize the outside of the seed and use sterile tools to remove the embryo in an aseptic environment you should be able to culture the embryos successfully without any contamination. Note that bacteria and fungi like to grow on the plant growth media even though they are not plants. If you are not VERY careful when setting up your experiment, your embryos are likely to be killed by contaminating bacteria and/or fungi.

In the laboratory, when unwanted microbes enter sterile material, they will grow and multiply. Every motion, every manipulation performed with aseptic materials is likely to introduce bacteria and fungi from your hands, from the air, from the microscopic droplets of saliva you expel while talking, from your clothing, from the bench top, from the lid of the aseptic media plate that you removed and laid on the bench top, etc., etc., etc.

To prevent the entry of unwanted microorganisms into your aseptic materials, attentively follow these guidelines and proceed to handle your materials as demonstrated by the laboratory instructor.

1. Wash your hands and wrists with soap and water.

2. Pick up only one aseptic tool or media plate at a time!

3. To alcohol-sterilize small, heat-resistant instruments, first pour a small amount of alcohol into a beaker. Next dip the instrument (e.g. spatula, glass spreader, forceps) into the alcohol and ignite the alcohol in a Bunsen burner. Do not hold the instrument in the flame, quickly pass the instrument through the flame; the burning alcohol is sufficient to sterilize the instrument. Be careful not to allow burning alcohol to drip and ignite the alcohol in the beaker. If this happens, cover the beaker with a glass plate available in the laboratory.

Lab 6-8

4. After sterilizing the instruments, do not touch the instruments to another surface other than what you are investigating. Once the instrument has touched anything else, it is no longer sterile. If you doubt whether any instrument or media is aseptic, assume it is not.

5. Remove and replace lids of the media plates in a timely fashion. Don’t leave the lid off the media plates while aseptically removing another embryo. Only keep the lid off when transferring an embryo and immediately replace the lid to prevent “others” from being transferred along with it.

6. Believe in the ubiquity of bacteria and fungal spores (review question: are these spores haploid, diploid, or dikaryotic?).

Isolation and Culture of Embryos

1. Wash your hands and wrists with soap and water. Dry them with a paper towel. Dip your fingers into the ethanol beaker and allow them to air dry (your fingers won’t be sterile, but they will be cleaner).

2. Working in groups of four: Break an ear of corn into halves or thirds so that you can remove individual kernels from the center of the ear of corn. Kernels must be removed with the base intact in order to maintain the sterility of the embryo. Discard any kernels that have broken tips or walls. To obtain embryos similar to each other in size, remove all your kernels from one area of the ear. Holding each kernel lightly in forceps, dip the kernel briefly into ethanol and lay it on a “clean” paper towel (note that this won’t be sterile) to drain and dry.

3. Place a kernel of corn on a piece of paper towel with one of its flat sides up. Look for a small, opaque protuberance from the surface at the base of the kernel; this is the embryo.

4. Dip a scalpel into alcohol and ignite the alcohol by passing it quickly through the flame of a Bunsen flame to sterilize the cutting edge (do not hold the instrument in the flame; it will get very hot!).

Similarly prepare a pair of forceps. With the embryo side of the kernel facing upward, remove the top third of the kernel with the sterile scalpel.

Holding the kernel between your thumb and forefinger, gently squeeze and push up the embryo out far enough to remove it with your sterilized forceps. Because the embryo is positioned against one face of the kernel, it is easier to squeeze it out if that face is against your thumb. Your instructor will show you how to locate and position the kernel in your hands. You may want to practice first to get the hang of it.

Knowing that although your hands are “clean,” they are not sterile—do not allow the embryos themselves to touch your fingers. If they do, they are likely to become contaminated with bacterial and/or fungal spores.

5. Using a freshly flamed spatula, carefully remove the embryo from the forceps and place it rounded side down on the surface of agar medium; the flat side of the embryo should face up.

Remember to keep the plate closed at all times except when introducing an embryo (don’t forget sterile technique “rule” number 4).

Lab 6-9

6. Excise and place four embryos on each of the media plates A-H (a total of 32 embryos—8 embryos for each of the 4 students in your group).

7. Seal each plate with a strip of Parafilm so that the agar will not dry out during incubation. All cultures will be incubated at 25oC under constant fluorescent light for one week.

8. Excise several more embryos and determine the initial weight per embryo by weighing several embryos (around 10) and calculating an average weight. Record you data in the table provided.

Observations and Collection of Data

During next week’s laboratory period, you will observe your growing embryos and quantify four variables pertaining to seedling growth and development for each embryo: 1) weight; 2) shoot length; 3) length of the primary root; and 4) number of lateral roots. Record your own data in the tables provided and follow the instructions from your instructor on how to pool your group’s data with other student investigators in your section and in other sections.

Formalize your hypotheses: HOMEWORK ASSIGNMENTSince we are pressed for time during today’s lab, and since this is a rather complex experiment, you will be completing a homework assignment before coming to lab next week. This worksheet assignment will help you to formalize your thinking about data from this experiment.

You and your lab partners may confer about your hypotheses, but each of you will complete and hand in a separate hypothesis worksheet.

In this experiment, there are three different factors being investigated:

1. Glucose at two levels – absent or present

2. The hormone gibberellic acid (GA) at two levels – absent or present

3. The hormone abscisic acid (ABA) at two levels – absent or present

This is a complex experimental design because all three factors are present as single factors and as factors in combination with one or two other factors. If we knew absolutely nothing about the three factors, conducting such an experiment would be unwise. It would make much more sense to conduct simpler experiments manipulating just one factor at a time.

But the effects of the three factors are somewhat predictable.

- it should be possible to predict the general effects of glucose, a source of nutrition

- gibberellic acid (GA) generally promotes cell elongation.

- for many plant species, the arrested state of development of a seed within an embryo is somehow regulated by abscisic acid (ABA)

Lab 6-10

You should read more about the effects of GA and ABA in your text (pp. 806-817 in the 6th edition of Campbell, especially Table 39.1). You will need to understand GA and ABA in order to form your predictions

about their effects; you will need to get this information on your own from your textbook.

Before proceeding with predictions, let’s formalize the experimental design as in the two tables below:

Glucose absent Glucose present

Plate AABA absentGA absent

Plate CABA presentGA absent

Plate EABA absentGA absent

Plate GABA presentGA absent

Plate BABA absentGA present

Plate DABA presentGA present

Plate FABA absentGA present

Plate HABA presentGA present

Medium Contents Medium Contents

A No additions E 2.5% Glucose

B 10-5 M GA F 2.5% Glucose + 10-5 M GA

C 10-4 M ABA G 2.5% Glucose + 10-4 M ABA

D 10-5 M GA +10-4 M ABA H 2.5% Glucose + 10-5 M GA + 10-4 M ABA

Gibberellic Acid = GA; Abscisic Acid = ABA

Within this complex experimental design, there are specific comparisons that can be made. Such comparisons will allow you to look at the effects of single factors and at specific combinations of factors.

Lab 6-11

TABLE 1

Consider Table 1 on the Hypothesis Worksheet. This provides you with a place to write down how you predict Plate E will compare to Plate A. This is a very specific comparison within the complex experimental design. It tells you about just the effect of adding glucose to the embryo, without adding any hormones. What do you predict will be the difference?

You may want to predict no difference between the two Plates. A prediction of “no difference” is often called a null hypothesis. Make this prediction unless you have some prior biological knowledge about the factor being examined (in this case, glucose). (In a short-hand notation, you might write A = E, to indicate that you don’t expect much of a difference between Plate A and Plate E.)

Logically, there are always one or more alternative hypotheses to the null hypothesis. For example, embryo weight could be greater in Plate A than in Plate E. (As a short-hand, you could write A > E.) Yet another alternative hypothesis is that embryo weight could be much less in Plate A than in Plate E. (Again, using shorthand, you might write A < E.) Biological knowledge about the factor being examined should allow you to propose a specific alternative hypothesis to the null hypothesis.

Enter your hypotheses for how glucose will affect all four traits.

Is there a biological reason why are you making those predictions? You may want to make a few notes below, especially if you are proposing something other than a null hypothesis. These notes will help you later with your lab report.

Notes:

Lab 6-12

TABLE 2

Next, consider Table 2 on the Hypothesis Worksheet. One of the main advantages of our experimental design is that it lets us examine interactions. What exactly do we mean by “interact” or “an interaction”? It means that the effect of a given experimental factor (say, glucose) differs, depending on a second experimental factor (say, ABA). In our experimental design, we have a set of Plates where glucose is absent, and another four Plates where glucose is present. So, it is possible to examine whether or not glucose interacts with either (or both) hormones. (With our experimental design, we could look at more complex three-way interactions, but we won’t.)

In this table, we’ll look at the effect of the presence or absence of glucose, and whether and how ABA interacts with it.

First, predict how Plate E will compare to Plate A. This is the same comparison as in Table 1. Enter your predictions in the “Presence or Absence of glucose, absence of ABA” column on the left. Next, predict how Plate G will compare to Plate C. This is the effect of the glucose, but now when ABA is present. Fill in your predictions in the “Presence or Absence of glucose, presence of ABA” column in Table 2, on the right.

Are the predictions in the two columns similar or different? In other words, are you predicting that the effect of glucose will interact with the effect of ABA? When conducting an experiment to investigate interactions, the typical null hypothesis is “No interaction.” Alternative hypotheses would include various form of interaction. Record your predictions about whether or not there will be an interactions in the place provided below Table 2. Do this for each of the four variables. Ideally, if you are predicting an interaction, the details of the interaction should be specified.

Notes:

Lab 6-13

TABLE 3

Finally, consider Table 3 on the Hypothesis Worksheet. This table provides you with a place to make predictions about how GA will affect seed germination and seedling development, and whether and how GA interacts with ABA. For simplicity, we will ignore the four Plates where glucose is present and focus only on Plates in which glucose is absent (Plates A-D).

First, think about Plate B in comparison to Plate A. This is the effect of GA when ABA is absent. Make predictions and record them in the left column of Table 3. Remember, if you don’t have a biological rationale, a null hypothesis of “no difference” is appropriate.

Next, think about Plate D in comparison to Plate C. In both these Plates, ABA is present. So, this comparison tells you about the effect of the presence or absence of GA when ABA is also present. Make a prediction and describe it in the right column of Table 3.

Are your predictions in the two columns of Table 3 different? In other words, does the effect of GA depend on whether or not ABA is also present? In the place provided, indicate whether or not you predict the null hypothesis of no interaction, or some specific type of interaction. In anticipation of your lab report, record a few notes here justifying your predictions. Notes:

Lab 6-14

Individual Data TablesFirst day (average weight per embryo):

**Important: Be sure your units are in mg, not grams.***

weight of embryos / no. of embryos weighed = mean weight per embryo

______________mg__ / _________________ = ________________mg____

One week later: Table 1. Embryo weight

Embryo cultivated on medium:Embryo # A B C D E F G H

1234

Mean:

Table 2. Shoot Length Embryo cultivated on medium:

Embryo # A B C D E F G H1234

Mean:

Table 3. Root Length Embryo cultivated on medium:

Embryo # A B C D E F G H1234

Mean:

Table 4. Number of Lateral Roots Embryo cultivated on medium:

Embryo # A B C D E F G H1234

Mean:

Lab 6-15

DATA ENTRY INSTRUCTIONS

1. Gather your data and enter it in the data sheet (attached). Use two digits after the decimal point for all values.

2. After your data have been entered on the data sheet, go to the following web address: http://160.39.101.211/embryo/index.html

3. Log in with your group ID. Your group ID will be assigned by your lab instructor.

4. On the next screen, enter your username. Make CERTAIN it is the same as your Group ID.

5. Enter your embryo weight data, making CERTAIN that your weight units are mg. Double check your data and submit.

6. Enter your data into the online version of the worksheet. Make CERTAIN your length measurements are in mm. When you are finished, click on submit once only.

7. Double-check your data on the verification page. If there are any mistakes, use the “Back” button to return to the previous page and make any necessary corrections.

8. After your data are verified, click “submit” again ONLY once. This will input your data into the group pool. Once your data have been submitted, you cannot get them back. An output page will be shown. You should print this form in landscape format. Include this in your lab report.

9. Your instructor will collect your completed written data sheets. You will not receive credit for your lab if you fail to turn in your data sheets.

ASSIGNMENT

Lab 6-16

Begin your experiment during lab week 6 (Oct.18-22)

Collect data during lab week 7 (October 25-29)

Pooled data will be available beginning of week 8 (Monday, Nov. 1)

Reports to be turned in to Altschul, Room 911 NO LATER than NOON Friday, November 5—NOTE THAT THIS IS THE FRIDAY OF FALL BREAK WEEK.

Because of fall break, there are no recitations on Friday, Oct. 29 and Monday, Nov. 1.

if results do not support the hypothesis,

revise hypothesis or develop a new one

if results do support the hypothesis, make more

predictions and test them

The Scientific Inquiry Method

Scientists use the scientific inquiry method to further scientific knowledge. The steps in this method are the following:

make observations

ask question

develop hypothesis

make prediction

design experiment

collect data

analyze data

draw conclusions

The scientific method begins with the process of making observations. From these observations, you ask a question about what you see. Next, you develop a hypothesis, often defined as an educated guess to answer that question. You can make a prediction based on whether or not your hypothesis is true, and then design and carry out an experiment to test your hypothesis and prediction. Data (remember that “data” is plural, “datum” is singular) must be collected and analyzed. From the results of your experiment, you can draw conclusions about whether your hypothesis is or is not supported. You will use all the steps in the scientific method in lab this week.

Please note that you can NEVER “prove” a hypothesis to be true, you can only have your hypothesis supported by data. NEVER, EVER use the word “prove” in a scientific setting. Really, NEVER.

Because of time constraints, this embryo growth experiment has been pre-designed for you. You will be working a bit backwards to develop your own hypotheses about the effects of glucose, ABA, and GA on embryo growth. You will need to look up, read, and understand the information about these hormones and about what is present in the corn kernel from which you separated the embryo. You will collect and analyze data to test your hypotheses and draw conclusions. Note that if your data do NOT support your hypothesis, you should NOT go back and change what your hypothesis was to make them match. The scientific method involves developing and revising hypotheses multiple times as a greater approximation of the “truth” is achieved.

Results are never “good” or “bad.” Instead, they either support or don’t support your original hypothesis.

You will use this experiment as the basis for writing a lab report. Your lab report needn’t be extremely long, but you MUST follow the given instructions VERY carefully. If you do not follow the instructions, your grade will be marked down. Your first source for instructions on how to write a lab report should be A Short Guide to Writing About Biology (a recommended text available from the Columbia Bookstore and on reserve in the Barnard Library). The instructions on the following page should be considered supplemental. Your instructors will use the instructions in A Short Guide to Writing About Biology when grading your reports.

Lab 6-17

WRITING A LABORATORY REPORT

By filling out your Lab 6 worksheets, you have made it relatively easy for yourself to write a laboratory report based on your experiment. Your lab report does not need to be very long in length, but it does need to follow the instructions in A Short Guide to Writing About Biology very carefully (secondarily, you may use the guidelines below). Laboratory reports follow the same general format as articles in scientific journals. In learning to write a lab report, you are learning a skill that will be valuable when you become a professional scientist. However, even if your life’s goal is not to become a scientist, the process of writing a lab report is useful AND FUN! (Well, maybe that’s stretching it a tiny bit, but give it a try….)

The following instructions on writing a lab report are slightly modified from those at the Academic Writing Center at Hamilton College. The original version can be found at http://www.hamilton.edu/academics/resource/wc/bio_lab.html.

For a sample annotated lab report written using these instructions, can be found at

http://www.hamilton.edu/academics/resource/wc/sample_bio.html.

Lab Reports for Biology

FormatPlease follow the instructions given below when writing lab reports for this course. Don't hesitate to ask if you have questions about form or content. Above all, remember to write with precision, clarity, and economy.

WritingYour writing should be in full sentences and easily understood. It should conform to the conventions of standard written English (sentence form, grammar, spelling, etc.). Good writing is as important in science as it is in other disciplines because one's ideas have little impact, no matter how important they may be, if they are not well communicated. While style is mostly an individual characteristic, everyone should strive for presentations that are easily understandable as well as grammatically correct. One reason for emphasizing clarity is that writing and thinking are closely related; as many people have said, "fuzzy writing reflects fuzzy thinking." When people have difficulty translating their ideas into words, they generally do not know the material as well as they think they do.

StyleScientific writing is usually in the past tense because one reports on experiments that have been completed. The writing should not be too self-referential (e.g., "I ground up the..."), although you may use the word "I" if doing so makes the writing easier to read. Writing that is predominantly in the passive voice is deadly to read (e.g., "acorns were eaten by the squirrels"), so use the active voice as much as possible (e.g., "squirrels ate the acorns"). Remember: PAST TENSE, ACTIVE VOICE.

Lab 6-18

PresentationThe first page of a lab report should be a title page with the title of the report, your name, the date, the course (e.g., BC1001 Revolutionary Concepts Laboratory), your lab section (e.g., Monday 1-4pm), your lab instructor, and your lab partners. There should then follow text that is a minimum of three pages and a maximum of six double-spaced, typewritten pages in length (tables, figures, and references do not count in this total). All writing should be on only one side of the page, and the reports should be stapled in the upper left-hand corner. The best length is shorter than the maximum, so don't expand a shorter report to reach a five page limit. It is important to write concisely. The report must be typed or word-processed. Neatness and clarity of presentation are almost as important as clarity of thought. Please do not use an interesting font or (especially!) font size to try to cheat on the length; nor should you use interesting margins. 12-point Times is a standard font most often used in academic settings.

AudienceWrite the report as if you were writing to other students who are taking a similar course but have not done this experiment. Assume that they have some familiarity with the subject matter but no expertise. Do not write specifically for the instructor.

CollaborationYou may talk about the lab exercise as much as you like while in the laboratory. However, it is essential that you write your own report in your own words. You may and are encouraged to discuss the experiment itself with anyone at anytime to ensure that you have understood it.

ReferencesIf you use outside sources (including your textbook and your lab handout), and you should, then cite those sources in the body of the report and list the references in a literature cited section. Citations should be made with a standard scientific format (not by footnotes); cite the author and date of publication only, so that a quick look at the Literature Cited can provide the reader with all necessary information. When there are more than two authors, simply list the first author and et al., along with the date. You should not use direct quotations from the references; paraphrase information and give credit to the source of the idea. The following are sample citations:

"Garrett (1989) showed that a gene in yeast ...""... is found in the urinary bladder of the turtle (Gapp et al., 1990).""... as reported recently (Miller, 1986; Pfitsch & Pearcy, 1989)."

You should list a reference for every idea not your own. Plagiarism is more than copying material word for word; it is also using someone else's ideas or phraseology without giving reference to the other work or other person. Fortunately, the reference format is so simple that it is very easy to include references to all the work that one has used (Williams, 1983). If the idea is not published but is provided by a lab partner or someone else, give the reference as a personal communication (N. Cutler, pers. comm.). Be aware of the difficulties that arise when one uses material from another source and changes only a word here or there without acknowledging the source. Such actions are plagiarism, even though the statement may not be word-for-word the same as in the original. Just remember the basic rule: list a reference for every idea or statement that is not your own. FormatThere are four fundamental sections to a scientific report, with

Lab 6-19

acknowledgments, literature cited, and appendices being additional sections. An underlined heading should be given at the beginning of each section (optional for the introduction). Keep in mind that the lab report is parallel to the experimental process (D. Flynn, 1988):  Experimental Process  Lab Report What is the problem?  Introduction How did I solve the problem?  Materials and Methods What did I find out?  Results What does it mean?  Discussion

TitleYour title should be DESCRIPTIVE, explaining what you did, how you did it, and what you found as much as possible (while it sounds contradictory, you should still be concise.) Bad title example: ABA and Corn. Good title example: Milk Decreases the Release of Carbon Dioxide from Goldfish, as Measured Indirectly Though the pH Indicator Phenolphthalein.

IntroductionBegin with broad statements, including enough background information (with reference to outside sources—most likely your textbook in this case) to set the stage for your experiment. You’ll want to include the information about seeds, glucose, ABA, and GA that you used as the basis for developing your hypotheses. Then narrow down to your particular study, explaining why it is of interest. Specify the objectives of the experiment, and make your hypotheses clear. One to three paragraphs is usually sufficient. Do not regurgitate the lab handout; write your own introduction.

Materials and Methods (or just Methods)Briefly summarize the entire process that was followed and the materials that were used, and then refer to the lab directions and to any flow charts you have included for the details. Don’t forget to cite the lab handout! Do note any differences in the procedures you actually followed from what was specified in the lab directions. Anyone who reads your report should be able to duplicate the experiment. This section should be a small part of the report, so don't expand endlessly. Do not include results here.

ResultsThe data and results are given here in summary form. All results should be described in a narrative; don't just list measurements. One of the commonest mistakes beginning students make is to omit the narrative in the results section. The narrative should be more than just saying, "Table 2 shows the percentage of students with different blood types." You should state and explain the actual results, e.g., "Most students had type O blood, while the fewest had type AB (Table 2)." Data must be presented in figures (graphs) and in carefully planned tables, rather than as raw data

All tables and figures should be titled and numbered sequentially, and the axes should be well labeled with clearly marked units. In addition to the title, each table and figure should have a legend (1 to 3 sentences) that explains what is being presented. A sample table and figure can be seen in the example paper online. If the whole thing can be typed, it is a table; if lines have to be drawn, then it is a figure. Each table and figure should be put on a separate page and referred to by number in the results narrative. Tables and figures follow the text of the report (after the literature cited). Sample calculations may be included in an appendix at the end of the report.

Lab 6-20

DiscussionIn this section the results should be interpreted and their significance explained. Begin the discussion by interpreting your specific results and end it more broadly by placing your results in context. Don't declare the experiment a success or failure; evaluate the results in view of the purpose of the experiment. If erroneous results were obtained, discuss the results you expected as well as those you received. You may also compare methods or discuss difficulties, but if you list sources of error, you should estimate how important each source of error may be. If you were to do the experiment again, what if anything, would you do differently? It is inappropriate to include statements such as "I learned a lot from this experiment..." The discussion is a very important section; it is your chance to show how well you understand the ideas and techniques involved and to relate your results to the ideas expressed in outside sources (the literature cited).

AcknowledgmentsThe acknowledgments section is optional. If you wish to thank someone, such as a lab partner or a tutor at the Writing Center, for help in understanding the experiment or in organizing the report, you do so here. Scientists regularly acknowledge others for helping with experiments or commenting on written drafts.

Literature CitedList any publications referred to in your paper alphabetically by first author; do not number them. Every item in your bibliography should be referred to in the body of your paper, or it shouldn't be listed at all. If you use information from an intermediary source, you should list the original reference but should also note the intermediary: "...cited in...". We will use the following standard forms (some journals use variations of these), shown in order for (1) an article with one author, (2) an article with more than one author, (3) a book, and (4) a chapter from an edited volume:

Reynolds, P.D. 1992. Mantle-mediated shell decollation increases posterior aperture size in Dentalium rectius Carpenter 1864 (Scaphopoda: Dentaliida). Veliger 35:26-35.

Gapp, D.A., R.N. Taranto, E F. Walsh, P.J. Favorito, and Y. Zhang. 1990. Insulin cells are found in the main and accessory urinary bladders of the painted turtle, Chrysemys picta. J. Exp. Zool. 254:332-337.

Stokes, D., L. Stokes, and E. Williams. 1991. The Butterfly Book. Little, Brown and Co., Boston. 96 pp.

Pearcy, R.W., and W.A. Pfitsch. 1994. Consequences of sunflecks for photosynthesis and growth of forest understory plants. Pages 343-359 in E.D. Schulze and M.M. Caldwell, editors. Ecophysiology of Photosynthesis. Springer Verlag, New York.

Lab 6 Handout: Seed Germination Experiments. Biology 2003. Biodiversity Laboratory, Fall Semester 2004. Barnard College, New York, New York.

AppendixAppendices are optional. You may use them to include your laboratory handout, sample calculations, sets of raw data, etc. (Not optional in this report is the inclusion of your data sheets.)

Final CheckThe last thing to do before turning a report in is to read it. Correct all typographical errors and other mistakes, and ensure that you have said what you wanted to say!

[These lab report instructions (not the whole lab handout) were written by E.H. Williams, Hamilton College, with modifications by D. Gapp, N. Cutler, and E. Cuebas-Incle. [with further modifications by M. Olney, Barnard College.]

Lab 6-21

Your report should answer the following questions in the appropriate sections:

1. Why are the actions of the hormones GA and ABA on embryos inside of seeds significant for plant propagation? Why is worthwhile to also investigate the action of glucose on embryo growth? (~1 paragraph)?

2. In words, state the hypotheses outlined in the three tables on the Hypothesis worksheet. (Attach your hypothesis worksheet to your lab report as an appendix.) (~1 paragraph).

3. Using pooled class data, construct bar graph(s) that will be useful in showing the comparisons and predictions shown on the hypothesis worksheet. You may make graph(s) by hand using graph paper, or by using the ‘Chart’ feature in Excel. There are many options for exactly how to organize and format your bar graph(s). What matters is accurately presenting the data, properly labeling axes on the graph, and use of a legend if appropriate. Each graph should be called a figure, and any figure(s) should be on separate pages attached to the end of your report. There can be multiple bar graphs in a single figure. Any figure(s) should have a number and an appropriate caption. (E.g., “Figure 1. Effects of glucose on four seedling traits.”) Be sure that you properly reference figures as you answer questions 4-7.

4. What effect did the glucose have? Which of the 4 traits did it affect? (~1 paragraph)

5. What effect did ABA have? Which traits did it affect? (~1 paragraph)

6. Did the effect of ABA interact with the effect of glucose? If there was an interaction, explain the specific details of the interaction.

7. What traits did GA affect, if any? Was there an interaction between GA and ABA? What was the specific nature of the interaction?

8. Explain any surprising or notable results of your study, or any limitations of this particular experiment. You may also want to include whether and how the data collected by you and your partner differed from pooled class data, but other topics may also be relevant and appropriate. Use your own judgment about what to discuss.

9. Hormones have various effects, some promote and some inhibit growth. Why is it beneficial for a plant to possess hormones with both promotive and inhibitive effects?

Lab 6-22

Name _________________________________ Lab Day/Time/Intructor: __________________________Biology 2003 Fall 2004

LAB 6: HYPOTHESIS WORKSHEET FOR SEED GERMINATION EXPERIMENT

Detailed instructions for completing this worksheet are found in your lab handout. You may collaborate with your group members to discuss your predictions, but you must each turn in your own hypothesis worksheet.

Recall that there are eight (2 2 2) plates in the experiment:

Glucose absent Glucose present

Plate AABA absentGA absent

Plate CABA presentGA absent

Plate EABA absentGA absent

Plate GABA presentGA absent

Plate BABA absentGA present

Plate DABA presentGA present

Plate FABA absentGA present

Plate HABA presentGA present

Table 1: Predicting the effect of glucose without any added hormones (use =, >, or <).

Trait

Presence or absence of glucose

(Plate E vs. Plate A) [A=E, A>E, or A<E]

embryo weight

shoot length

root length

number of lateral roots

Lab 6-23

Biology 2003 Fall 2004

LAB 6: HYPOTHESIS WORKSHEET FOR SEED GERMINATION EXPERIMENT

Table 2: Predicting the effect of glucose’s presence or absence, and whether ABA interacts. (use =, >, or <)

Trait

Presence or absence of glucose, ABA

absent

(Plate E vs. Plate A)

Presence or absence of glucose, ABA

present

(Plate G vs. Place C)

embryo weight

shoot length

root length

number of lateral roots

Table 3: Predicting the effect of GA’s presence or absence, and whether it interacts with ABA (use =, >, or <).

Trait effect of GA, ABA absent(Plate B vs. Plate A)

effect of GA, ABA present(Plate D vs. Plate C)

embryo weight

shoot length

root length

number of lateral roots

Lab 6-24

Name _________________________________ Lab Day/Time/Intructor: __________________________BC Bio 2003; Fall 2004

OPTIONAL: Results worksheet for Seed Germination Experiment

This worksheet is optional, but using it is likely to improve your lab report (and your grade). The format of this worksheet is almost identical to the hypothesis worksheet that you completed for your homework. There are columns for the data you and your partner collect, as well as columns for the class data. Using this worksheet should make it much simpler to discern whether your predictions were supported by the data.

When filled out, this worksheet may make it easier for you to figure out the best way to construct and format the bar graph(s) that are required for your lab report. Note that when making the graph(s) for your lab reports you will be using class data, not just the data that you collect with your partner.

Recall that there are eight (2 2 2) plates in the experiment:

Glucose absent Glucose presentPlate A

ABA absentGA absent

Plate C ABA presentGA absent

Plate E ABA absentGA absent

Plate G ABA presentGA absent

Plate B ABA absentGA present

Plate D ABA presentGA present

Plate F ABA absentGA present

Plate H ABA presentGA present

Table 1: Effect of glucose, without any added hormone

Trait

Presence or absence of glucose

(Plate E vs. Plate A)

Your data Class data

embryo weight

shoot length

root length

number of lateral roots

Lab 6-25

Table 2: Predicting the effect of glucose’s presence or absence, and whether ABA interacts.

Trait

Presence or absence of glucose, ABA

absent

(Plate E vs. Plate A)

Presence or absence of glucose, ABA

present

(Plate G vs. Place C)

Your data Class data Your data Class data

embryo weight (mg)

shoot length (mm)

root length (mm)

number of lateral roots

Was there an interaction between glucose and ABA? (Are results in the columns 1/3 and 2/4

different?)

Your data (column 1 vs. 3) Class data (2 vs. 4) . embryo weight yes no yes noshoot length yes no yes noroot length yes no yes nolateral roots yes no yes no

Table 3: Predicting the effect of GA’s presence or absence, and whether it interacts with ABA

Trait effect of GA, ABA absent(Plate B vs. Plate A)

effect of GA, ABA present(Plate D vs. Plate C)

Your data Class data Your data Class data

embryo weight (mg)

shoot length (mm)

root length (mm)

number of lateral roots

Was there an interaction between GA and ABA? (Are results in the columns different?)

Your data Class data . embryo weight yes no yes noshoot length yes no yes noroot length yes no yes nolateral roots yes no yes no

Lab 6-26