Lab Manual

Investigative Lab. 1. Morphology of Plants

Examine the plants in the lab and identify the parts found in the terms below. Make you own identification dictionary either with drawings or descriptions.

apical meristem

terminal bud

leaf scar

leaf primordium

node

internode

axillary bud

leaf

stipule

petiole

lamina

roots

lateral roots

fibrous roots

tap roots

peduncle

pedicel

sepal

calyx

petal

corolla

stamen

filament

anther

androecium

pistil

carpel

gynoecium

placenta

stigma

style

ovary

fruit

ovule

seed

pollen

Investigative Lab. 2. Reviewing Anatomy

Look at the slides and identify the parts given in the terms below. Make you own identification dictionary either with drawings or descriptions. Then section portions of the shoots and roots, stain them with a variety of stain and again identify many of the parts.

structure stem:

epidermis

cortex

primary xylem

primary phloem

pith

structure root:

endodermis

pericycle

root hairs

root cap

primary meristems:

shoot apical meristem

root apical meristem

protoderm

procambium

ground meristem

root apical meristem

secondary meristems and tissues:

cambium

secondary phloem

secondary xylem

periderm

wood

rays

growth ring

Methods of hand-sections of living material

Hand-sectioning is a skill that you should become accomplished at. The object is to make as thin a section as possible. The technique will be demonstrated and you will need:

1. razor blade

2. dropper bottle with water

3. material to be sectioned

When sectioning, remember that it is a slicing motion. DO NOT attempt to just push the razor blade through.

If the section is thick

1. wet both the material to be sectioned and the razor blade with a drop of water.

2. hold between the thumb and forefinger of one hand and the razor blade in the other.

3. rest the razor blade flat on the forefinger and, in a slicing motion, slide it towards you.

4. make several sections and allow them to accumulate on the razor blade.

OR USE HANDHELD MICROTOME

1. open jaws of microtome and place specimen inside.

2. rotate to lowest setting.

3. place plant part inside with portion to be section just above the cutting stage, and clamp specimen in place.

4. section and then rotate to raise specimen and section again.

If the section is thin.

1. wet the razor blade. Place the thin plant part on a hard surface with a hard edged ruler on top, trim edge to be cut.

2. cut along the hard edge angling the cutting edge of the razor blade away from the hard edge.

3. cut again, angling the edge of the razor blade in the opposite directing.

4. repeat several times to get a number of sections.

To wet mount.

5. gently transfer 1 or 2 of the best sections to a glass slide.

6. Start by adding 1 to 3 drops of water to the specimen. Then select a coverslip and hold it by the edges. Place one edge to one side of the water surrounding the specimen and gently lower it down so that no air bubbles are formed. If the area under the coverslip is partly dry, touch the water dropper to the edge of the coverslip and squeeze gently until the slide is filled. If the coverslip is floating, there is too much water and should be blotted with a towel.

How stains can enhance details of structure.

Stains are specialized chemicals which will preferentially color certain organs, organelles or structures in plants. A list of these are given below.

To stain, add one drop of stain first, then let sit. Then follow we mount procedure.

Hand section several plant parts trying all three techniques. Try Neutral Red and one other type of stain.

Stains for sectioned materials.

There are a series of stains that are especially valuable when you are using fresh materials neither killed nor fixed. Utilize these stains and their special characteristics to help you analyze your sections.

A. Toluidine Blue. This is a metachromatic dye that is blue in aqueous solution, but that reacts with cations in the plant materials to give several different staining reactions. For example, compounds containing pectic acids are stained a pinkish-purple. The dye reacts with polyphenolic compounds such as lignin and tannins) to give a green, greenish-blue or bright blue color. Thus lignified walls, either primary or secondary, will show one of these color reactions. Note that the lignified secondary wall of phloem fibers stain bright blue, while those of xylem are a greenish color. Sieve cells, sieve tube elements and companion cells generally stain pinkish-purple. This stain does not stain cellulose or starch and leaves meristematic cells unstained.

B. Phloroglucinol and HCL. This reagent is prepared by adding 1 part stock concentrated HCL to one part water (slowly while cooling in a water bottle) and saturating the resulting solution with phloroglucinol. This reagent is almost a specific test for lignin, resulting in a bright red color in lignified walls. Sections can be placed directly in the reagent and a cover slip added. Or the section may be removed from the stain and mounted in a glycerine jelly. The stain is not permanent, but usually lasts for a matter of months. Care should be taken not to get any on the person or on the microscope. The fumes, too, are quite potent and material should not be examined on the stage of the microscope without a coverslip and without the removal of any excess stain on the slide.

C. Neutral red. This is one of the most useful stains. It stains primary cell walls and is especially useful on fresh material having transparent cell walls. Sections can be mounted directly in a 0.1% solution.

D. Sudan IV. This reagent stains cutinized and suberized cell walls as well as the outer cuticular layer. Add a drop of the reagent to the material to be stained. It is sometimes necessary to wait a minute or two or even to slightly warm the section before the red stain appears.

E. I₂KI. This is one which is sensitive to starch. Add a drop and the presence of starch is indicated by the blue-black color.

Investigative Lab 3. Life Cycles, Ovules, and Seeds.

.

For the life cycle terms, draw them on a X life cycle chart below.

Life Cycle

zygote

sporophyte

sporangium

spore mother cell

spores

gametophyte

archegonium

egg

antheridium

sperm

syngamy

meiosis

Now examine the prepared slides of ovules and identify the parts found in the terms below. Make you own identification dictionary either with drawings or descriptions.

Ovule

funiculus

micropyle

integument(s)

megasporangium = nucellus

megaspore

Embryo Sac = Megagametophyte

egg

synergid

antipodals

polar nuclei

zygote

endosperm

sperm

tube nucleus

pollen tube

Embryo

suspensor

filamentous

globular

heart-shaped

dicotyledonous

monocotyledonous

Seed

seed coat - testa

endosperm

embryo

epicotyle

hypocotyle

radicle

See the hand out on Scientific Problem Solving

Background.

The two major classes of angiosperm are the Monocotyledonous plant and Dicotyledonous plants. A number of characteristics distinguish these groups from each other including generally different leaf venation patterns, number of petals, and arrangement of primary vascular bundles. Another set of differences are found in the seeds

Problem: How do the seeds of Monocotyledonous plant and Dicotyledonous plants differ in their structure including the amount of endosperm and number of leaves, and are the initial germination patterns similar?

Hypothesis or Statement:

Hypothesis Test:

Fruits or seeds have been provide which have been that have been germinated in sterile water for a variety of days. Record the types of seeds and the experimental conditions. Use what ever technique will best test your hypothesis.

Results:

Debriefing

Investigative Lab. 4. Shoot Apex

Examine the prepared slides and identify the parts found in the terms below. Make you own identification dictionary either with drawings or descriptions.

Tunica

Corpus

Phyllotaxy

Examine a variety of the apices provide and dissect and section to reveal the structure.

Investigative Lab 5. Chimeral Analysis of Stems and Leaves

Background

By now you have an idea of the structure of apical meristems in angiosperms. They are frequently composed of L1 (epidermis), L2 (subdermal) and L3 (remaining central region) with the first two forming the tunica and the latter from the corpus. You also know about chimeras and the resulting cell lineages. Lets use this knowledge together to understand the 3D structure of the apex of a chimeral plant.

Problem

What type of cell lineage would you expect if a single cell of the tunica or a single cell of the corpus was marked? Would it matter where in these regions the cell is marked and would it effect the size cell clone?

Hypothesis or Statement

Hypothesis Test

We have provide a number of chimeric plants. These have cell lineages with or without chlorophyll production. Examine and dissect with care. Please try to preserve at least part of the plant for future classes.

Results

Debrief

Investigative Lab 6. Leaf initiation and phyllotaxy

Problem Solving

Background

The shoot apical meristem produces two major types of structure leaves and axillary shoots. The leaves are initiated first, followed by by the axillary buds. The production of leaves is very systematic and with the new leaves initiated in specific places based on the placement of the previous leaves.

Look at the experimental plant provided. First look at the phyllotaxy, the placement of the leaves on the stems at the nodes. Is the arrangment opposite, alternate (or spiral) or whorled? If alternate, how many times around do the leaves go before one is above another and how many leaves are produced before the pattern is produced. If it is once arround and three leaves the plant is said to have a 1/3 phyllotaxy.

Problem

How are the leaves initiated on the apical meristem? What is the order and pattern when the next node is produced?

Hypothesis or Statement

Hypothesis Test

Results

Debriefing

Investigative Lab. 7. Floral apices

Background

The apical meristem can be either indeterminant and for vegetative organs, indeterminant and produce leaves or bracts which then subtend floral or inflorescence meristems or determinant forming bracts, floral or inflorescence meristems. We will look at a number of apices to show the morphology of the developmental change.

Observation

Dissect the apical tips of vegetative and reproductive shoots. Describe the differences including what they produce.

Investigative Lab. 8. Root Anatomy

Observation

Remove some living roots and examine this carefully under the dissecting microscope. Then examine a slide of a root tip whole mount. Get a general feeling of position and relative placement of the root cap, apical meristem and root hairs.

Now examine the longitudinal sections of roots. Look at the development of the xylem based on the size of the cells and the thickness of their walls in the center of the plant. When the vessels have solid think secondary walls the roots can no longer elongate.

Now look at a cross-section of a root. In addition to the normal primary tissues, you should be able to observe the endodermis and the layer of cell inside of these called the pericycle.

Problem Solving

Problem

What is the relationship of the production of root hairs to the development of the primary tissues, specifically the xylem?

Hypothesis or Statement

Hypothesis Test

Results

Debriefing

Investigative Lab. 9. Clonal Analysis of Roots

Background

Examine the assigned manuscript by Taylor, Kinslow and Kirchner.

Observation

The roots have been assayed with GUS which shows the cell lineages which are expressing the gene product. Note that a transposon event begins expression of the gene with all descent cells expressing too.

Describe some of the patterns that you observe. These should include both the cell layers and sectors. Do your results support the findings in the manuscript?

Investigative Lab. 10. Lateral Roots

Problem solving

Background

We have previously look at root morphology and anatomy. Yet roots also make additional root called lateral roots

Problem

Where do lateral roots form, in relation to the root apex? Anatomically, where are they formed and from what tissues or cell layers?

Hypothesis

Hypothesis Test

Results

Debriefing

Investigative Lab. 11. Secondary Structure and Initiation

Background

The secondary meristem develop from existing tissue or primary meristems that have been left behind. So the vascular cambium forms from the procambium left between the primary phloem and xylem and from differentiated parenchyma cell from the pith and cortex. The cork cambia form from parenchyma cells of the cortex as well as the secondary phloem.

Observation

Examine the series of slides from shoots with only primary growth to the initiate of secondary vascular growth to formation of a vascular cambium form a continuous ring. Look at the relative time of secondary growth in different parts and cell types they originate.

Now examine the formation of secondary vascular tissues in the root. Again look at relative timing and where they originate.

Lastly examine the last group of slide which show the formation of the cork cambia. Note the discontinuous formation of the cambia, it rare connects into a ring, the tissues and cells the cambia form from.

Investigative Lab. 12. Living wood

The purpose of this lab is to introduce you so sectioning wood plants. You will pick so examples of living plants and we will sectioning the and look at there anatomy.

Investigative Lab. 13. Floral Diversity

The purpose is to examine the diversity of flowers. In considering development one must consider the diversity of floral organs in angiosperms. Examine the representative types. The list the similarities and homologies (evolutionarily similar parts) in table form. Is there commonality? What could be some share developmental programs?

Investigative Lab. 14. Ephedra and Gnetum

We have review the structure of member of this group. Now you can observe many of the characteristic of the vegetative and more importantly fertile organs.

Observation

Examine the herbarium sheets and shetch the major organs. Then to some detail drawings of the reproductive structures.

Examine any other material available.

Scientific Problem Solving

Purpose: To introduce the student to methods of scientific exploration.

I. Background

Science is based on solving complex problems, a skill that most are not directly trained in. Yet, recent research has shown that thinking and problem solving skills can be enhanced by learning various techniques and by practice. This enhancement is important both for increasing productivity and for increasing the number of creative solutions available for a given problem. Basically these steps are to get you to practice divergent thinking and to think of new associations for solving problems. The Creative Problem Solving (CPS) method created by Osborn and Parnes can be applied to any problem. I have modified this method for specific use for scientific problems and call it Scientific Problem Solving. This will be discussed further later on.

CPS has five (sometimes six) steps (Table 1):

Objective finding- Broad outline of the "mess".

Fact finding- The recording and collection of specifics and details.

Problem finding- Careful statement of problem to entirely encompass problem at hand.

->Restatement of the problem can frequently make the problem and solution more apparent. One such method is to ask why.

Idea finding- The various methods for addressing the problem.

->One method to increase the number of ideas is to modify a previous solution, perhaps by adding two together, magnify by suggesting a solution that is broader in scope than intended or minify by suggesting a solution that is narrower in scope.

Solution finding- The listing of criteria to evaluate the ideas, the prioritizing of the criteria and the selection of the "best" solution, based on the criteria.

->It is best to first give all the criteria possible. Then, order the criteria by using each one as the most important and ordering the rest relative to the first one. After you have repeated this for each criteria, select the criteria that were found high on each list.

Acceptance finding- The successful implementation and tallying results of the solution.

->It is important when implementing to consider as many aspects as possible including answering the questions who? why? how? when? and where?

A number of methods have been developed to enhance CPS. In general, try to slow down your thoughts, break your habits, make new associations and go from broad to narrow.

Deferred Judgement- This is the most important principle.

Slow down and let the ideas flow and do not judge them immediately. Some of the ideas may later end up being useful even though their value is not immediately apparent.

List All Ideas- It is important to keep a list at each

stage. Frequently you will forget a previous idea.

Make a Careful Statements at the End of Each Step- It helps

the process to summarize the conclusions of each step so that you know where you have been.

Brainstorming- An important method if you are working with more than one person

a. no criticism of ideas

b. free wheeling

c. quantity important

d. attempt new combinations and improvements

For each step of CPS, you should consider the following aspects:

a. list possibilities (use deferred judgment)

b. list criteria for evaluation

c. select the most important criteria

d. apply the selected criteria to determine the best

possibility

References:

R. B. Noller, S. J. Parnes & A. M. Biondi, 1976. Creative Actionbook. Charles Scribner's Sons, New York.

S. J. Parnes, R. B Noller & A. M. Biondi, 1977. Guide to Creative Action. Charles Scribner's Sons, New York.

G. A. Danis, 1986. Creativity is Forever. 2nd ed. Kendall/Hart Publishing Co., Dubuque, Iowa.

Table 1. Comparison of Creative Problem Solving, Scientific Problem Solving and the procedure in lab

CPS* SPS Lab

(objective finding)

fact finding fact finding background

problem finding problem finding problem

idea finding hypothesis-statement student

formation

solution finding hypothesis testing student

acceptance finding summation of result student

* Based on S. J. Parnes, R. B Noller & A. M. Biondi, 1977. loc. cit.

II. Procedures

We are interest in applying this method in a generalized way towards botanical problems. It is generally not realized that scientist are attempting to solve problems (also call the pursuit of knowledge!) or that creativity is a necessary ingredient. Although the training for science seems to concentrate, sometimes excessively, on memorization, this knowledge is only the first part of investigative studies.

To be an outstanding scientist, one should be creative in your problem solving. A simple, enlightened method for testing a hypothesis is preferable over a laborious one, even if they can both give the same result. Simplicity leads to fewer errors and to a greater chance that the results will be accepted. The method we will use in lab is Scientific Problem Solving (SPS).

SPS has five steps that are similar to those above (Table 1).

Fact finding- Data and ideas from classes, the literature, conferences and previous observations.

Problem finding- Finding a field or topic of interest with the selection of a particular areas which needs to be examined.

Hypothesis-Statement formation- The formation of a hypothesis or a statement of a range of hypotheses. It should:

a. best explain the existing facts or be a statement of the range of possible explanations

b. make predictions

c. be falsifiable (can be tested)

Hypothesis testing- Creating and conducting an observational or theoretical research plan which can corroborate (support) or falsify the proposed hypothesis or choose between the possibilities given in the statement

Summation of results - Gives the particular results for the given protocol, gives whether a hypothesis is corroborated or falsified and may propose a new hypothesis or statement of problem

In this manual, sections of labs or entire labs will be based on SPS (Table 1). Obviously, it is unrealistic at this time to expect you to initiate fact or problem finding since this is an introductory course. The labs topics are written to give structure to these steps, and the information summarized in Background and Problems respectively. Background will include the minimum that you need to know to do the lab including conceptual information, materials available and methodology. The Problems will be given to you for the most part. Yet, the creation of hypotheses-statements and the tests will be up to you, individually or in a group. One important criterion will be whether you have the materials and time to implement your test in lab. When you formulate and implement your test, you will succeed or fail as an active researcher would. The last stage in your lab manual will the Debriefing Summary. In this section you should summarize the problem, hypothesis or statement, and the results.