TESTED STUDIES

FOR LABORATORY
TEACHING

Proceedings of the Tenth Workshop/Conference of the

Association for Biology Laboratory Education (ABLE)

Edited by

R. W. Peifer
Associate Education Specialist and Laboratory Coordinator
General Biology Program
College of Biological Sciences
University of Minnesota
P-180 Kolthoff Hall
225 Pleasant St. S. E.
Minneapolis, MN 55455
(612) 625-9048
Copyright © 1989 by the Association for Biology Laboratory Education (ABLE)
All rights reserved. No part of this publication may be reproduced,
stored in a retrieval system, or transmitted, in any form or by any
means, electronic, mechanical, photocopying, recording, or otherwise,
without the prior written permission of the copyright owner.

Printed in the United States of America

This volume is dedicated to Joseph R. Larsen, former Director, School of Life
Sciences, University of Illinois, and current Director of the Division of
Rehabilitation Education at the University of Illinois, for his many years of
contributions to science laboratory education, and his continuing work to provide
those with disabilities an opportunity to experience the wonders of science.
 Contents
Forward ix
Preface xiii
Address xv
Chapter
1 Rapid-Cycling Brassicas (RCB's) in Hands-on Teaching of Plant Biology
Paul H. Williams 1
Key Words: botany, Brassica rapa, life cycle, Fast Plants.
Wisconsin Fast Plants is a novel technology involving the use of rapid cycling genetic
stocks (35 days seed to seed) of Brassica rapa and related species and includes simple,
inexpensive, self-contained growing systems suitable for the classroom or laboratory at
all levels from kindergarten to college. Participants are introduced to the uses of these
plants in teaching: development, reproduction, genetics, physiology, and ecology.
2 Professional Telecommunications: How to Get the Most from the NABT
Electronic Bulletin Board and Other Useful Databases
Steven P. Lanphear 31
Key Words: electronic bulletin board, NABT, computer services, electronic mail,
database, communication tools.
This workshop demonstrates on-line use of the national electronic bulletin board,
complete with electronic mail started in 1987 by the National Association of Biology
Teachers. Once on-line, 14 special interest areas are available, such as AP-Biology,
magazine and book reviews, ABT Journal, NABT membership services, question and
answer forum, software reviews, and swap/sale of used equipment. Also available for
downloading onto your computer are extensive files of labs, graphics, and handouts.
Discussions of this and other databases will emphasize the power of these new
professional communication tools. [Note: This workshop was not submitted for
inclusion in this Proceedings Volume.]
3 Nerve Conduction in Frogs and Humans
Elizabeth Vizsolyi 33
Key Words: vertebrate physiology, nerve conduction, frog, human, nerve trunk, sciatic
nerve, biphasic action potential, refractory period, electromyogram.
These exercises are taken from a vertebrate physiology course, and use either a human
subject or a dissected frog, thus providing relatively simply alternatives that may suit
your needs. Nerve conduction velocity can be measured in the frog sciatic nerve with
recordings of the biphasic action potential on the outside of the nerve trunk. Absolute and
relative refractory periods can also be determined. Conduction velocity in the human can
be obtained from electromyograms taken from the fourth and fifth fingers following
stimulation of the ulnar nerve.
4 Electron Flow in Photosynthesis
R. Patrick Harrison 43
Key Words: chloroplasts, photosynthetic pigments, absorption spectrum, electron flow,
photosynthesis.
The fascinating concept of electron flow is explored with simple equipment in an exercise
for first-year students. Students use a spectrophotometer to generate an absorption
spectrum for spinach chloroplasts, and then make a prediction about the effect of
wavelength of light on the rate of photosynthesis. Students design their own carefully
controlled experiments to test their predictions

v
5 Introduction to Electron Microscopy
Ellen Rosenberg and Michael Weis 59
Key Words: electron microscope, scanning EM, Transmission, EM.
This tutorial/demonstration focuses on image formation in the scanning and transmission
electron microscopes. In this exercise, students are introduced to the principles involved,
and then tour the Electron Microscope Facility for observations of the microscope in
operation.
6 Meiosis in Rye and Sordaria
Ramesh Bhambhani and Ann M. Schramm 67
Key Words: meiosis, cereal rye, Secale cereale, Sordaria brevicollis, cytology, genetics,
gene mapping, centromere mapping.
In the morning session, participants will be shown the use of anthers of cereal rye (Secale
cereale)for the cytological demonstration of the salient features of meiosis. Irrespective
of the academic level (high school, college, or university) at which introductory genetics
is taught, rye anthers have many advantages. Participants in the afternoon session will
discover that another organism, the fungus Sordaria brevicollis, is ideal for the study of
gene and centromere mapping in introductory college or university genetics labs. [Note:
This workshop was not submitted for inclusion in this Proceedings
Volume.]
7 Modelling Population Structure
Robert E. DeWreede 69
Key Words: population, population modelling, Leslie Matrix, fecundity, mortality,
computer simulation, intrinisic rate of increase, age distribution, ecology.
The Leslie Matrix is a model used commonly to predict future age or stage distributions of
a plant or animal population. In this exercise designed for students with some
background in ecology, the model is run on APPLE II series computers. Using data for
real or imagined populations, students study the effects of altered fecundity and mortality
on population structure, calculate the intrinisic rate of increase, analyze the model's
sensitivity, and study the requirements for a stable and constant age distribution in the
population.

8 Thermoregulation in Vertebrates Studied by Telemetry

David W. Osgood 79
Key Words: radio telemetry, thermoregulation, physiology, body temperature, wildlife.
This exercise, which is adaptable for introductory biology students or advanced
physiology classes, uses a simple radio telemetry system for measuring body temperature
from unrestrained and undisturbed animals. The advantages of this system over
traditional probe-type thermometers are enormous. The laboratory will include practice in
calibration of the transmitters, instruction in inserting the transmitter into the animals, and
experience in interpreting the data obtained.

9 -
The Botanical Garden A Tool to Teach systematics, Physiology and a Lot
More
Gerald B. Straley and Iain E. P. Taylor 93
Key Words: botanical gardens, teaching methods, plant systematics, morphology,
physiology, adaptation.
The UBC Botanical Garden will be used to demonstrate the wide range of possibilities
for teaching using materials that are available in sins or freshly collected. An exercise in
general systematics will use materials from the British Columbia Native Garden; the uses
of plants as chemical sources will be examined with materials from the Physick Garden;
the diversity of morphology will be examined using plants from the Food Garden;
environmental and physiological adaptations will be seen in the Alpine Garden plants.

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10 Microbial Ecology of the Oral Cavity
Barbara Dill and Heather Merilees 107
Key Words: bacteria, oral cavity, isolation, identification, classification,aerobic,
anaerobic, techniques.
Using simple microbiological techniques, this exercise will introduce students to the
variety of bacterial type present in their mouths. Enriched, selective, and differential
media will be used to isolate the major aerobic and anaerobic species. Visual
observations of the bacteria enhance the students' appreciation for the complex microbial
world of the mouth. [Note: This workshop was not submitted for inclusion
in this Proceedings Volume.]
11 Mapping Genes in C. elegans
Denise Clark and Bob Johnsen 109
Key Words: nematode, gene mapping, linkage group, markers, Caenorhabditis elegans,
mutation, culturing, genome.
The advantages of using Caenorhabditiselegans in introductory genetics courses will be
demonstrated in this exercise in which an "unknown" visible mutation will be assigned to
a linkage group and mapped relative to known markers. The nematode can be cultured
easily, has a short generation time, reproduces either as self-fertilizing hermaphrodites or
in outcrosses yields large numbers of progeny, and has a simple genome.
Trail-Following in Snails: A Behavior and Statistical Laboratory Exercise
Sandra Millen 119
Key Words: behavior, snail, intertidal littorine snails, land snails, experiments, trail
following, statistical analysis.
It is well documented that many snails follow the trails of other snails. A variety of
simple experiments can be designed around trail following that teach experimental design,
observation and data collection, and statistical analysis of results. The easily obtained,
intertidal littorine snails can be used in areas with access to the ocean, while land snails or
slugs can be substituted in inland areas. The statistical emphasis can be modified to make
this exercise suitable to a variety of levels.
13 Recording Action Potentials From Cockroach Mechanoreceptors
Tom Linder 125
Key Words: active transport, potassium ion, midgut, tobacco hornworm larva.
Readily available from suppliers, this larva has a large midgut that specializes in
transporting excess potassium (from its plant food) into its lumen. The exercise is
offered as an alternative to the traditional study of frog skin. In this exercise, the midgut
is mounted on a perfusion tube, and the electrical potential difference across the wall of
the midgut is measured with an oscilloscope which provides data on the rate of potassium
transport. The basic set-up is amenable to the study of a variety of interesting questions.
14 Plant Hormones: Bioassay for Gibberellin
Sandra L. Biroc 131
Key Words: gibberellic acid, bioassay, plant hormone, barley seed, endosperm, starch
breakdown, development.
This simple assay makes use of the ability of the plant hormone GA3 to induce starch
breakdown in the endosperm of a barley seed from which the embryo has been removed.
The effect of the hormone is clear and repeatable. This exercise can be used in
introductory biology courses to demonstrate a basic plant process, or can be modified and
used to investigate more sophisticated questions in a developmental biology course.

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15 The Genetics of Eye Color in Drosophila melanogaster
Carol Pollock 141
Key Words: mutation,pterin, eye pigments, chromatography, Mendelian inheritance,
metabolic pathway,fruitfly, Drosophila melanogaster.
This exercise has been designed to help first-year biology students understand Mendelian
inheritance. The pterin (red) eye pigments of wild type and mutant strains are separated
using a simple paper chromatography system, and the patterns are analyzed to determine
where the metabolic pathway is blocked in each mutant. Crosses of these strains are
followed for two generations to provide data that students analyze to determine the mode
of inheritance of each mutation, as well as the relationship between each mutant
phenotype and the enzyme in the pathway which is affected by the mutation.

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 Forward
Jon C. Glase

This volume contains the complete materials for the major workshops given at the tenth annual
workshop/conference of the Association for Biology Laboratory Education (ABLE) held at the
University of British Columbia, June 13-17, 1988. Paul G. Harrison and R. Pat Harrison, of the
Biology Department at U. B. C., were co-organizers of the event. Thanks to the carefully
planning of messieurs Harrison and their staff and the competence and enthusiasm of the 15 major
workshop and 16 mini-workshop presenters, the meetings were a great success. The participants
left U. B. C. with many new ideas for enriching their own laboratory programs and with a deep
appreciation for the beauty of the campus and the Vancouver area.
As an organization dedicated to the improvement of biology laboratory education, ABLE has
grown substantially over the years. Starting with fewer than 50 members during its first year of
existence, ABLE now includes over 350 dues-paying members. The first workshop/conference
held at the University of Calgary in 1979 had 49 participants. Recently, workshop/conferences
include from 100 to 150 participants, depending on the facilities of the host institution.
Unfortunately, it has become common for more people to apply than can be accommodated. In
1985, the board of directors decided that current ABLE members should be given preference for
admission to the annual meeting and a reduced registration fee relative to non-members.
ABLE was originally conceived as an organization to promote sharing of information among
college and university faculty concerned with teaching biology in a laboratory setting. Despite its
growth, this primary mission of ABLE has not changed. Few would disagree that the unique
character of the workshop/conference, where participants get hands-on experience with up to six
new laboratories and numerous mini-workshops, is the single most important feature contributing
to the success of ABLE as an organization. The location of each workshop/conference has
depended on a willing member from a supportive institution with adequate staff and facilities to
serve as host. The organization has also attempted to site the meetings in a geographically
representative manner, as the following list of ABLE's first decade of meetings suggests:
University of Calgary (1979), University of Illinois (1980), State University of New York at
Stony Brook (1981), University of Washington (1982), Clemson University (1983), Memorial
University, Saint John's, Newfoundland (1984), University of Nevada-Las Vegas (1985), Cornell
University (1986), University of Minnesota (1987), and University of British Columbia (1988).
This year's meeting is scheduled for 12-16 June 1989, at the University of New Brunswick in
Fredericton, N. B.
Some members have voiced concern that ABLE may become too big and, as a result, lose its
ability to develop a sense of community among academicians concerned with laboratory teaching.
My own observations are that despite the increased numbers of participants at ABLE meetings,
people still quickly form common interest groups and communication does not seem diminished.
However, the increased size and complexity of workshop/conferences may limit the number of
individuals who are able or willing to host a workshop/conference in the future. Yet growth is
obviously important to the continued improvement of the laboratory cumculum. ABLE needs to
attract individuals who have developed new approaches and laboratory materials if it is promote the
dissemination of this information.
Many an innovative lab exercise had its origin as a spin-off from someone's research program.
In an attempt to improve the awareness of ABLE among the research community, Bette Nicotri
(University of Washington), past president and liaison officer, sent a letter describing ABLE and
its workshop/conferences to 50 biological sciences societies, requesting communication with
scientists who have developed their research interests in ways applicable to use in student
laboratories. This first attempt has already lead to contacts with members of organizations who
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potentially have much to offer to the improvement of laboratory teaching. We need to continue
fostering communication with this important group of scientists if our laboratory programs are to
remain current and representative of contemporary biology.
This volume is dedicated to Professor Joseph R. Larsen. As past director of the School of Life
Sciences at the University of Illinois and an established member of the research community, Joe
Larsen was critically important to the success of ABLE during its formative years. He volunteered
to host the second ABLE conference and found the needed resources to carry it off. Joe
shepherded several important early projects of the organization, such as the ABLE Library. Joe
Larsen's keynote address at the ABLE banquet focused on the important work he is now doing as
Director of the Division of Rehabilitation Education Services at the University of Illinois.
Although, the academic program at an ABLE workshop/conferencesis very important, I think
there are other considerations that keep people coming back year after year. Camaraderie and the
chance to share your own enthusiasm for laboratory teaching with others of similar inclination is an
important factor. The meeting at U.B.C. was rich with opportunity for formal and informal
gatherings, tours of scientific and scenic places, and memorable events related to the ABLE's tenth
anniversary. The native American dance performance, preceding the banquet at the historic Brock
House overlooking English Bay, was for many of us the highlight of a very full week.
Thanks to the hard work and determination of editor R. W. Peifer (University of Minnesota), I
think those of you who missed the meetings will find this book to be "the next best thing to having
been there". I predict that this volume will become a valuable reference for all of us.

x
xi
Row 1: Ron Chmielewski. Chuck Curry, Angela Gloss, Karen Romanyk, Hazel Skinner, Neysa Wiens, George Knox, Nancy Goodyear, Linda Berg, Anna
Wilson, Lynn Hodgson. Lucy Dyer, Judy Shepherd, Marsha Fanning, Seth Lubega
Row 2: Paul Harrison, Mary-Jane Turnell, Ken Perkins, Bill Sumner, Ruth Smith, Charlotte A. Candelaria, Corey Goldman, Anne Karpala, Carol Pollock,
Linda Cholewiak, Patricia Waller, Bill Glider, Mary Ball
Row 3: Pat Harrison, Barb Newman, Cairine Miner, Yousuf Ebrahim, Ray Brown, Ruthanne B. Pitkin, Jane Ouellette, Sandra Biroc, Jean Dickey, Judy
Morgan, Elizabeth King
Row 4: Forrest Bent, Jerald Oldham, Grant Doering, Allan Hawryzki, Donald Serva, Leona Truchan, Roberta Ellington, Roberta Williams, Elizabeth Godrick,
Ruby Littlepage, Gene Braun
Row 5: Karen Moms, Donna Daugherty, Leland Johnson, Ann Wilke, JoAnn Lane, Gay Ostarello, Jon C. Glase Bill Leonard, Don Igelsrud, Virginia
Hodgkinson, Ruth St. John, Paul Monson
Row 6: Keith Grisham, Janice Coffey Swab, Dennis Cartwright, Rick Peifer, Bob Kull, Trevor Chandler, Ellen Rosenberg, Michael Weis, Steve Lanphear,
Ted Swensen, Bette Nicotri, Peter Wood, Paul Williams
xii
 Preface

This Proceedings volume contains the major workshops presented at the annual Workshop/
Conference of the Association of Biology Laboratory Education (ABLE) held at the University of
British Columbia, June 13-17, 1989. The Workshop/Conference marked the tenth anniversary of
the organization, and with the publication of this volume reaffirms the dedication and commitment
of hundreds of ABLE members to the betterment of biological education through the laboratory.
As editor of this, and the ninth Proceedings volume, I've attempted to bring the volumes to
press more quickly than in the past by establishing certain criteria for the submission of
manuscripts. This volume marks the first time that all manuscripts were submitted to the editor on
computer floppy disks in ASCII format. This has allowed me, and should allow future editors, to
more easily manipulate text into a consistent format throughout the volume, and to produce a
professional looking product, while significantly reducing the cost compared to having the
volumes professionally typeset. Much work remains to be done by future editors before a final
format for the Proceedings volume is selected, but hopefully this volume can act as the nucleus for
change. Presenters at future Workshop/Conferences will be given a list of guidelines for
submitting their manuscripts to the editor, and hopefully in the near future an appendix section
containing these guidelines can be included in each Proceedings volume.
As in previous volumes, the workshops presented at UBC are organized by chapters. The
volume contains twelve of the fifteen workshops that were presented. Unfortunately, three
workshops are missing because they were not submitted. The tenth anniversary conference was
represented by a great diversity of workshops. Four chapters cover topics from plant biology,
while three chapters are devoted to topics in physiology. Genetics is represented by two chapters,
while behavior, ecology, population biology and cell biology are represented by single chapters.
Finally, there are single chapters dealing with professional telecommunication in biology and
electron microscopy. Each chapter in the Table of Contents contains a list of key words and an
abstract.
I would like to thank Bruce Fall for his editorial suggestions and comments while I assembled
the manuscript. Most of all, I would like to thank the many authors for their patience and
cooperation.
Minneapolis, Minnesota R. W. Peifer
February, 1989

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xv
 Banquet Address

The Importance of Laboratory Education in Life Sciences

Joseph R. Larsen

It is great to be with you at this most significant tenth anniversary of ABLE. We mark some
milestones in life and certainly this is one worthy of demarcation. ABLE has survived ten years,
and that is most significant. We had dinner last night at the "Kettle of Fish" and I am delighted to
say with Rick Peifer as our host that the spirit of ABLE is alive and well. Some of the things I say
tonight are to the wrong people. You are already dedicated or you probably wouldn't be at these
meetings. But they need to be said and you need to be the crusaders.
Carl Sagan once said, "Science is a way of thinking much more than it is a body of
knowledge." To me, science is exploring, searching for and answering questions, solving
problems, understanding principles and processes together with their causes and consequences. If
science is more than facts--information--the student experience with science should result in more
than that also.
Science is a dynamic, exciting search for the understanding of patterns, regularities and
principles. In no other place does this happen better than in the biology laboratory. In no other
place can it be more indelibly impressed on the minds of fledgling scientists than in an introductory
course in Life Sciences. I was extremely concerned in my former role as Director of Life Sciences
with the number of students who come to Illinois as transfer students who had no laboratory
experience in a biology course.
Recently a National Commission on Excellence in Education in the U.S. generated a renewed
interest in education, particularly in relation to scientific literacy. This is an opportune moment for
us as teachers of biology to examine our biology courses and to look at our curricula and ask if our
courses in introductory biology, or any level of biology, reflect science as a process as it can be
studied in the laboratory. It is so essential to introduce the student to living systems, to teach them
to ask how and why, which can only be done when they are looking at living material.
I would like to share with you an experience I had in my early years as a professor at the
University of Illinois. Having been assigned to help develop a new course in introductory
biology, I went to the laboratories to take an inventory of existing equipment which I will share
with you in its entirety. The inventory consisted of one rusty ring stand, two dozen finger bowls,
two Bunsen burners, and 200 gallons of formaldehyde. As we developed this new course which
was a wedding of an old general zoology and botany course, the laboratory was the first thing
developed. To me it was the most important element in that course. This is a course that runs for
an entire year, 5 semester hours each semester. One-half of the entire credit or grade is attributed
to the laboratory. It is not 4 hours on lecture and one hour on lab, but half of the entire grade
depends on the student's performance in the laboratory.
I made a pledge then that the laboratories would, and did indeed, become a living experience.
Our students would know that biology did not come out of a pickle barrel filled with
formaldehyde. We worked with living plants, we worked with living animals, we brought in
marine specimens; we invited the students to take them out, dissect them, look at them, watch
them, and have a hands-on experience with living material. Bob Tuveson, who is with you at the
meetings this year for the first time, now runs that course and I hope he will perpetuate the ideal
that we started some 24 years ago.
We tried to make our laboratories dynamic, constantly adding new laboratory material and new
equipment. This is what made my commitment to ABLE so easy and logical. I realize that this

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requires a commitment from your administration which it is not always easy to come by. But it is
important for you who are gathered here as biology teachers to perfect your craft, to go back and
convince your administration that you need money for laboratory equipment. The cost of the
laboratory course at Illinois is roughly $45-$50 per student. This includes personnel, TA's,
hourly help, supplies and equipment. There is a total annual budget in excess of $27,000 for
Introductory Biology. It is a majors course required of all undergraduates in Life Sciences. I felt
gratified in knowing that all of our students would go away understanding that biology is a living
science.
College faculty involved in teaching biology in a laboratory setting share many common
challenges--the need to develop interesting, reliable laboratory activities; to identify reliable
suppliers of biological material; to maintain and manage living laboratory organisms--and this often
does stretch tight operating budgets. We have a role to train assistants for laboratory teaching and
to develop relevant laboratory materials. Recognizing these commonly shared concerns a group of
biologists met at the University of Calgary in June, 1979 and founded an organization of
laboratory biology teachers called the Association for Biology Laboratory Education or ABLE.
ABLE'sprimary purpose is to facilitate communication between teachers actively involved with
laboratory instruction in the various areas of biology. With approximately 40 charter members,
ABLE has now grown to over 500 members from the United States and Canada. During its 10
years of existence, ABLE has tried to improve biology laboratory education mainly by identifying
successful laboratory exercises and materials. Each year they publish a dozen or so laboratory
exercises that have grown out of the workshop conferences where participants have an opportunity
to get hands-on experience with new kinds of laboratories.
It is important to know that there is a large group of biologists out there who are making an
effort to develop good laboratory material. We have developed through ABLE an extensive
bibliography of 250 annotated volumes in a library of laboratory manuals. We have made an effort
to identify papers from the research literature that can be modified into teaching laboratories. We
have found that there are many current experimental procedures that adapt themselves well to an
introductory laboratory education. If we can encourage more of our colleagues to specifically
identify some of their research as having potential for a laboratory exercise, we would all benefit a
great deal.
An editorial recently published in The Chicago Tribune stated, "The distressing fact is that the
overwhelming majority of our population lives in a state of debilitating scientific illiteracy." I think
that no other place is that more evident than in those students who go through a biology c o m e
without a laboratory experience. With a sharp decline in laboratory teaching during the last 10 to
15 years, what have we harvested? I think we will have a generation of young biologists who
have memorized textual knowledge and who can master our machine-designed examinations to
prove it. However, they do not know how to do an experiment and even more damaging, they
have little or no interest in learning. They feel little or no compulsion to apply principles of a
controlled experiment in their search for understanding. They do not appreciate their heritage from
research, they have become biologists who fail to recognize their dependence on research for their
own ability to effectively search into the unknown.
One example that might help students realize the importance of laboratory education is that
virtually all that is known in the modern handling of cardiovascular disease has evolved within our
lifetime. Our generation has seen the emergence of almost 90% of what is known in modem
medicine, thus accounting for almost all of a modem physician's ability to diagnose and treat
disease. This has all come about as a result of laboratory research. I wonder if we can convey this
message to our students. I wonder if we help them realize the importance of learning to function in
the laboratory. Do we infect our students with a sense of excitement for a stimulation of new
knowledge? Do we stimulate their natural curiosity about the biological complexities they
encounter everyday? For some I am afraid the answer to these questions is a resounding NO.
Have we as a profession abrogated our reliance on the laboratory as the place to learn biology,
responsibility for teaching the principles of the scientific method? Haven't we succumbed to the
teaching the principles of the scientific method? Haven't we succumbed to the temptation to
emphasize handouts, reward the student for memorizing lecture notes? What is happening in the

xvi
biology laboratory? How many students get through all the basic sciences today without seeing a
vigorously beating heart surrounded by inflating and deflating lungs.
I recall my first course in physiology at Hopkins when we dissected a dog and studied
circulation. What an exciting experience it was to feel the strength and coordinated rhythm of
ventricular pumping! How many of you have personally experienced the dynamic changes in the
ventricular pumping action at the onset of fibrillation? Compare and contrast in your own mind the
impact of just reading about fibrillation in a textbook with that elicited by holding the heart in your
own hand at the instant fibrillation is induced. This was a living system, we should be excited and
our excitement should be transmitted to the student. They will then gain that natural,
overwhelming curiosity to find out why it works. We have, in many cases, opted for the easier
way of teaching. Some may argue that new ways are better; some will defend the teaching
machine, the slide/tapes, the problem programmed computer. But if these are substitutes for the
teaching laboratory, I cannot accept the argument. I accept these things as teaching aids, and they
can be superb. Some of the materials that Don Igelsrud has developed and shared with me at the
meetings today were exciting, but they can never totally replace the living laboratory. I do
acknowledge with conviction that laboratory teaching is hard work; it is expensive in terms of time,
effort, and money; it requires total commitment from the best teachers; and it demands time away
from your own research.
When I served as Director of Life Sciences, I deliberately assigned myself to teach the
laboratories in the introductory course in biology. Our very best talent needs to be teaching in
those laboratories. Our students may get through basic sciences with virtually no research
experience, often no laboratory at all. They are forced to depend entirely on sheer memory for all
they know. It would be impossible for us to design a more intellectually, stupefying, stultifying
framework in which to learn biology.
What are some of the tangible rewards for good laboratory teaching in biology? There are many
but let's look at one or two examples. Eighty-five percent of all biology majors at Illinois are pre-
professional, it may be higher--it may be as high as 90% in some years. It may be different at your
schools, but I doubt it. Of that 85% less than 50% will get into the professional school whether it
be medicine, dentistry, veterinary medicine, or allied medical sciences. As Director of Life
Sciences for many years, I worried a great deal about these students and what they would do with
their lives. In counseling with biology students, many times I have said to them in a group, "Not
all of you are going to get into medical school." And one can see etched on their countenances the
internal response to themselves, "Well, I certainly feel sorry for the person to my right or may left,
but he's certainly not talking about me." But the honest fact of the matter is that less than 50% of
them will be accepted into the professional school. I was told by many people that there was no
job market for someone with a biology degree. I refused to accept that and after careful nurturing,
searching, and casting about for potential job markets we have found that there is indeed a viable
job market for people with a biology major. We have gone to academic institutions, we have gone
to industry, and we have gone to the government at the local, state and federal levels. All of them
have job descriptions and positions which utilize people with a good sound degree in biological
sciences. This brings me to the point I want to make about the payoff for good laboratory teaching.
Every major corporation or government agency that has come to Illinois to recruit our students has
said before they ever come, "We want laboratory trained individuals. Please do not put anyone on
your interview list who has not been trained in the laboratory." There is a career, there are jobs,
but there is no substitute for laboratory experience. They have to know how to use the
spectrophotometer, an analytical balance, an ultra-centrifuge. Give them hands-on experience in
the laboratory and they will find jobs! In a real sense, we are training them not only in a storage of
knowledge and information but in practical techniques that they will take with them into the
professional school or into the job market.
Another payoff for good laboratory teaching is found in the potential of bringing undergraduate
students into your own individual research laboratory as assistants. If you can generate in them the
excitement and interest in biological systems through their experiences in basic biology
laboratories, they will be filled with the excitement of laboratory experimentation. This can be
extended into your own personal research. Collin Pittendrigh, one of the world authorities on

xvii
circadian rhythms, during his years as a professor at Princeton University had many post-doctorals
working in his laboratory. But all of the research on circadian rhythms and eclosion was done
with undergraduate students who had been excited about biology and who came to work on
individual projects in Pittendrigh's laboratory. I recall the first time I visited his laboratory at
Princeton, he took me into a room with 4 or 5 cots and all kinds of experiments on the laboratory
benches. These students who were working on eclosion were sleeping in the laboratory, living,
eating and breathing the excitement that was going on in a research laboratory. Many of them
published joint papers with Pittendrigh,. Can you imagine the potential of developing such a
program in your own laboratory? Each student is a potential pair of hands with not only a desire
but an excitement to work in your area of research. A few years ago I was at Brigham Young
University in the laboratories of an old post-doctoral student of mine, Dr. Gary Booth. He was
with you at the Las Vegas meetings. He has at least 15 or 20 undergraduate students standing in
line by his office door every semester begging to go to work for him because they have been
excited about their introductory course. They are excited about the biology laboratory. He has
tremendous manpower and can assign each one of those students a portion of a research project
and generate a tremendous amount of work. I know you carry heavy teaching loads and there are
administrative responsibilities, committees and duties at a university that take you away from
research in biology. These students can become a powerful asset in your own individual programs.
You really cheat yourself if you don't utilize that potential. They are eager, they are anxious to be
involved, and I suspect I shouldn't dwell on this very much, you don't need to pay them anything.
You can have them sign up for research credit; they can work in your laboratory and for their
efforts go away with 3, 4, 5, hours credit and they are ecstatic because they have had an
opportunity to work in a research experience.
These are just two of the many by-products, if you will, or rewards for good laboratory
teaching. The more general rewards are obvious. The students who do well in the laboratory will
do well in all of their courses and they are the ones who will be accepted in the professional
schools. They are the ones who will be successful in their careers.
I am no longer Director of the School of Life Sciences. I am currently Director of the Division
of Rehabilitation Education at the University of Illinois, and so for the last part of my remarks I
would like to impress on you the responsibility I feel that each one of you has to get students who
have disabilities involved in science. For the past 14 years, I have been involved in the National
Science Foundation and AAAS in developing programs around the concept that more individuals
with disabilities should enter the field of science. For a long time, there has been a stigma that one
should not go into the laboratory or expect to do bench science if indeed a person has a disability.
It is important for you as teachers and I make a ernest plea that you encourage students with
disabilities to become involved in biological science. Teach them science, let them know that they
can function. I will never forget Joe Lilienthal at Johns Hopkins, my physiology professor when I
returned to graduate school after breaking my neck some 34 years ago, calling me into his office
and sitting squarely in front of me and putting his nose in my face, saying, "Larsen, I am sorry
you broke your neck, but if you're going to take my class you're going to do everything everyone
else does in the lab and I don't give a damn about your wheelchair." Well, I don't know whether
he was trying to make me mad or whether that's how he really felt, but he was successful. He
angered me to the point where my gut reaction was, "I'll show you, S .O.B." I went into the
physiology laboratory that afternoon and saw the typical laboratory bench normally found in a dog
lab. Tables high enough for "Kilroy" to peek over, I could have worked under the table more
conveniently in my wheelchair. That night I returned to the laboratory with one of my friends and
a saw. I took a laboratory table and cut 18" off every leg of one of the benches. The next day
Lilienthal came into the lab. We had our dog at lap level and were carrying out an experiment in
our group. He walked over and saw that his table had been cut down to my size and said, "What
in the hell is going on?" Now I had a turn to look him straight in the eye and say, "You take care
of the course and I'll take care of my responsibilities." There was an embarrassing moment of
silence, he walked out of the lab and never said another word. I believe in expediency, there is
always a saw and hammer in my laboratory and whatever needs modified will be modified. It is
important as you work with people that have disabilities that you encourage them to adapt

xviii
themselves to the maximum. When they have reached maximum adaptation of self, then you
modify the environment as necessary to meet their needs. You will find that if you will be willing
to make reasonable modifications that will not detract from that laboratory setting for anyone else,
you will generate an excitement in many young minds who have been previously denied.
Foundation for Science and the Handicapped is an organization that has been formed as a result
of the activity in developing science for the handicapped. Out of this organization has come a
number of scholarships for disabled people who are pursuing graduate research in any of the
sciences including biological sciences. There is a large group of role models and peers out there
who are encouraging and smving to develop programs for those individuals with disabilities. I
encourage you to make whatever modifications are necessary to allow people who have physical
disabilities to come into your laboratory. You will have a great, rewarding experience.
Well, I am sure I've exceeded my time. It's been good to be with you. I have enjoyed sharing
with you my enthusiasm for laboratory teaching. It's great to be here and share the 10th
anniversary of ABLE. I want to say just a few words about Don Igelsrud. His dream, his
dedication and enthusiasm, devotion almost, to the concept has been a monumental force in
formation of ABLE. There are many people who should be recognized over the years and while I
am sure that if I tried to mention them all I would make an error, but certainly Jon Glase, Roberta
Williams, Jim Waddell, Anna Wilson, Karen Morris, Betty Nicom, Don Fritch, and the list goes
on. All have done so much to make ABLE a success. Remember that biology is an experimental
science and could not be so if it were not for the laboratory. Therefore do not cheat your students
at that most crucial point in their educational pathway of the laboratory experience in biology.
There they will truly realize that biology is an experimental experience where they can gain hands-
on excitement of life's processes.

xix
 Chapter 1

Rapid-Cycling Brassicas (RCB's) in Hands-on

Teaching of Plant Biology

Paul H . Williams

Department of Plant Pathology

Russell Laboratories
University of Wisconsin-Madison
Madison, WI 53706

Paul H. Williams received his B.S.A. degree in Plant Science from

the University of British Columbia in 1959, and his Ph.D. in Plant
Pathology/Botany from the University of Wisconsin-Madison in
1962. He is currently Professor of Plant Pathology at the
University of Wisconsin-Madison. Williams is the Principal
Investigator of the NSF funded Wisconsin Fast Plants Instructional
materials Development Program. His research interests include the
development of multiple resistance (MDR) screening technology,
genetics of and breeding for MDR in Brassica and Raphanus, and
the development and distribution of rapid-cycling stocks of
crucifers, including Brassica, Raphanus, and Arabidopsis through
the Crucifer Genetics Cooperative.

1
 Educational Uses of the Rapid-cycling Brassicas

INTRODUCTION
The development of rapid-cycling brassicas (RCB's) as model organisms for research and
education is profoundly influencing the quality of science education at all levels by bringing
dynamic living materials into the classroom. Most biology courses lack convenient living materials;
many use animals predominantly. General and advanced courses in biology, botany, science
education and applied plant sciences usually lack suitable living plant material that would permit
students to explore plant growth and development, physiology, reproduction, genetics, evolution
and ecology. These speedy relatives of mustard are particularly amenable to classroom settings
because they show remarkably rapid development (Figure 1), they flower in 13 to 18 days, they
are small, and they can reproduce at high densities (up to 2500 plants per square meter) under
fluorescent lighting in a classroom. The ease with which RCB's can be grown and pollinated,
together with the wide array of interesting variants available in the rapid-cycling type, make these
plants particularly attractive to teachers and students. RCB's have far-reaching educational
potential, from kindergarten through college. Teachers at all levels can help students learn more
about plant biology through hands-on exploration with these rapidly responding plants.

0 1 2 3 4 7 Days after sowing Scale is 1/2

Figure 1. Growth of Rapid-cycling Brassica rapa cultivar RCBr showing growth stages at various
times from seeding until 28 days.
3
 With support from the Educational Materials Development Program of the National Science
Foundation, the Wisconsin Fast Plants Program was initiated to develop a Wisconsin Fast Plant
(WFP) kit consisting of 1) specialized genetic seed stocks of rapid-cycling Brassica rapa (RCBr)
tailored especially for classroom use and 2) self-supporting systems for growing and
experimenting with RCB's.
WFP growing systems (Figure 2) are designed to be used in various experiments. The basic
growing unit is a 'quad' pot containing four cells, each cell supporting one plant. Seed is sown in a
specially tested soil mix to which a slow-release balanced fertilizer is added. A small wick
protrudes from the bottom of each cell of the quad, providing a moisture conduit to a water mat
lying on a platform over a water containing reservoir. One edge of the water mat extends into the
water in the reservoir. The depth of the reservoir is sufficient to provide water to the plants for 3-4
days. Once the seed is sown and watered, only water is added to the reservoir.

Figure 2. Wisconsin Fast Plants growing system.

Accompanying the seed and growing system is a WFP manual, comprising many exercises
which have been developed and tested by teachers participating in the WFP program. These
materials address the educational goals of: 1) teaching basic concepts of biology; 2) stimulating
inquiry and problem solving; 3) increasing the impact of genetics teaching; and 4) bringing new
excitement into the classroom.
The WFP educational materials have been developed to provide teachers and students with the
opportunity to investigate a wide range of higher plant biology. Table 1 presents some central
topics that can be addressed through the WFP materials.

4
Table 1. Educational topics that can be addressed using RCB's.
1. Growth and development
a. Growth; seed germination (plants up in 2 days), leaf formation, stem elongation, flowering
(13-16 days), fruit (pod) and seed (embryogenesis) maturation
b. Growth responses; (plant bends up in 2 hours)
c. Development/morphology; root, stem, leaf, flower
2. Reproductive biology
a. Flower development; male and female parts of flower
b. Pollen and pollination; control of pollination, bee sticks
c. Fertilization
d . Embryogenesis
3. Genetics; Mendelian and lion-Mendelian
a. Mendelian; gene expression, dominance, interaction
b. Mendelian; gene assortment, independence, linkage, F1, F2 test cross
c. Non-Mendelian; maternal inheritance
d . Selection
e. Evolution
4. Physiology; underlying mechanisms of growthand developmerit
a. Using numerous physiological mutants: gsowt h hormone responders
b. Photosynthesis; radiant energy utilization
c. Nutrition; effects of major and minor elementso n growth and reproduction
d. Water relations; excesses and deficiencies
e. Photoresponses; light intensity, photoperiodand flowering, tropism, etc.
5. Ecology; the plant responding to its environment
a. Influencesof acid rain on plant growth and development
b. effects of air pollution;pollution-sensitive mutant stocks
c. Chemicals in the plant environment;salt injury, herbicide effects
d. Effects of pests and diseases; disease resistance, microbe-plant interactions
RCB's are suitablefor introducing students to all aspects of growth and developmentfrom
germination through to the harvesting of seed. Germinating in less than 12 hours, RCB's emerge
in 48 hours, flower buds appear in 7-8 days and flowers begin to open in 12-13 days.
With the initiation of flowering, many aspects of reproductive biology can he learned. Floral
morphology and its intimate relationship with the honey bee (Apis mellifera) provide an excellent
example of coevolutionary interdependence between two organisms. An understanding of the
relationships between the bee and the flower can be gained through the dissection and close
observation of the parts of the flower and the honey bee. Followingthe dissection, students can
explore the remarkably efficient pollen collecting ability of the bee by making a beestick from a
dead bee and using it as a pollination device for their plants (Figure 3). By investigating pollination
and the control of pollen germination, the mechanismsfor ensuring outbreeding of the species can
be understood. Following double fertilization, the exploration of endosperm and embryo
development through dissections under a microscope can be a challenging and exciting experience
in learning.

5
Figure 3. Bees, beesticks, and cross pollination of brassica flowers using a beestick.
An understanding of reproductive biology provides a useful setting in which to present
genetics. With many interesting phenotypes and mutants available, Mendelian, cytoplasmic and
population genetics can be explored. Ongoing research in the scientific community will soon make
available cytogenetic stocks, molecular markers, physiological mutants, cytoplasmic hybrids and,
eventually, transformed RCB's. Rapid-cycling Brassica rapa can be crossed readily with turnip
and Chinese cabbage (see accompanying exercise). The progeny and subsequent F2 generation of
such crosses can provide exciting materials for students interested in evolution, domestication and
plant breeding.
Underlying the expression of the phenotype in growth and development is the domain of
physiology. The RCB's are well suited for exploring how plants respond to physical and chemical
stimuli in their environment. Various physiological mutants are available with which to investigate
the influence of light, nutrients and hormones on plant growth and photosynthesis.
Exploring how the RCB's respond to changes in their environment can provide the basis for
interesting experiments in ecology. Variation in the acidity of precipitation, the salinity of water and
the chemical composition of the soil and atmosphere in which the plants are growing all are
excellent avenues for exploratory learning. By growing the RCB's in cages or jars, the effects of
various pests on plant growth can be examined. Modifying the chemical, physical and biological
environments in which the RCB's grow provides virtually unlimited opportunities for independent
investigations and learning by students.
The Need for Good Lighting
The major requirement for successful use of the RCB's is adequate lighting. These plants have
been selected to perform best under continuous bright cool-white fluorescent lighting. Adequate
lighting can be obtained at 5-10 centimeters distance from banks of six 4-foot cool-white bulbs
spaced at approximately 10 centimeters apart. Eight or ten closely spaced cool-white fluorescent
bulbs will result in even better plant growth. Providing they have adequate light, the plants grow
very well in classrooms and hallways; frequently they do better in these open areas than in the
confines of small plant growth cabinets where other environmental parameters such as relative
humidity and air velocity are difficult to control.

6
 Taxonomy of Rapid-cycling Brassica rapa (RCBr)
Species in the genus Brassica belong to the mustard family or Cruciferae (also known as
Brassicaceae) so named for the cross-shaped form of its four petals (crux= Latin for cross). Higher
up the taxonomic ladder (taxis= Greek for arrangement or order), the crucifers are part of the
Order Papaverales in the subclass Dicotyledonae (flowering plants having two cotyledons and
netted leaf venation). All flowering plants are in the class Angiospermae. Angiosperms are in the
subdivision Spermatophyta (seed bearing plants) in the division Tracheophyta indicating the
presence of vascular tissue (trachia= Latin for artery) (Table 2).
The naming of Brassica species has been in a state of confusion for more than a hundred years.
Because of the great diversity of forms of brassicas, even within a single species, early
taxonomists described many of the major forms as separate species. Within B. rapa, many forms
exist (Figure 4). For instance, wild forms were called B. campestris denoting that they were found
in fields as weeds (campestris =Latin for field). B. rapa was the name given to turnip by the
Romans and has persisted until now (rapa=Latin for root forming). B. pekinensis was the heading
Chinese cabbage, B. chinensis, or pak choi was the large petioled type of oriental brassica.
Table 2. Phylogeny of Brassica rapa
KINGDOM-Plantae
-plants have cell walls and chlorophyll
--other kingdoms are: Monera (bacteria), Protista (protozoans), Fungi and Animalia
DIVISION-Tracheophyta
-vascular plants
SUBDIVISION-Spermatophyta
-seed plants
CLASS-Angiosperms
-flowering plants
SUBCLASS-Dicotyledonae (dicots)
-two cotyledons, branching veins in leaves
ORDER-Papaverales
-special anatomy of fruit and embryo
-contains several families
FAMILY -- Cruciferae or Brassicaceae (e.g., mustards and cabbages)
-4 petals, 4 sepals, 6 stamens, ovary consists of two carpels
-contains 375 genera and 3200 species
GENUS-Brassica
--fruit a silique, embryos conduplicate
SPECIES--rapa (formerly campestris)
--chromosome number 2n=20
--subspecific groups
SUBSPECIFIC OR CULTIVAR GROUPS--e.g., chinensis (pak choi), pekinensis (Chinese
cabbage), rapifera (turnip), oleifera (turnip rape)
--cultivar=cultivated variety name --domesticated through selection and breeding

7
B. nipposinica has many small shoots and is found in Japan whereas B. parachinensis was an
early flowering form with succulent edible leaves, petioles, stems and flower buds. During the first
part of this century cytogenetics determined that all of these "species" contained 20 chromosomes
and that all could be intercrossed to produce fertile progeny, primary requisites for denoting a
single species. The distinctive form-species were therefore designated as subspecies of the
common species B. campestris. Thus, Chinese cabbage became B. campestris ssp. pekinensis etc.
More recently however, Dutch taxonomists investigating 18th century specimens and records of
Brassica campestris found that the first authentic description of the 2n=20 chromosome species
was actually that for B. rapa. B. rapa therefore has been adopted as the official name for this
species with B. campestris considered to be a synonym. Occasionally you may still see the old
terms such as B. pekinensis, or B. parachinensis used for what should now be B. rapa.

a b c d e

1/6 1/10 1/1 0 1/8 1/20

Scale at this magnification

Figure 4. Forms of Brassica rapa representing various cultivar groups; a. B. rapa, turnip group; b.
B. rapa, Chinese cabbage group; c. B. rapa, pak choi group; d. B. rapa, saichin group; e. B. rapa,
turnip rape group; f. B. rapa, rapid-cycling.
Scientists are still unsure as to whether to give subspecies names to each of the forms of B.
rapa. Because the cultivated varieties (cultivars) of various forms can be crossed so easily, many
intermediate forms are being produced by plant breeders. Rather than designating each form as a
subspecies, many scientists are in favor of categorizing cultivars into broad cultivar groups. Thus,
cultivars appearing more like Chinese cabbage than any other form are grouped in the B. rapa
Chinese cabbage group; turnip-like types are in the B. rapa turnip group; oil seed types are in B.
rapa turnip rape group and so forth (see Table 3). Within each cultivar group are many cultivars. A
cultivar is given a name by plant breeders and seed companies to differentiate one from other
distinctly different cultivars. Cultivar designations can be names, or code numbers. The proper
designation for a turnip commonly grown in the United States would be as follows: genus-
Brassica; species - rapa; cultivar group - turnip; cultivar - Purple Top White Globe.

8
 Diversity, Biology and Production of Brassicas
Although brassicas are known in the United States mainly as highly nutritious
vegetables--e.g., cabbage, cauliflower, broccoli, collards, kale, mustard greens and Chinese
cabbage--their potential value as oilseed crops and animal fodder is beginning to be recognized.
Crucifer oil, known as rapeseed oil, is the third most commonly traded vegetable oil in the world.
Rapeseeds contain 40% oil, which is pressed from the seeds, leaving a high-protein seed meal of
value for animal feed and nitrogenous fertilizer. Most Northern European countries produce
rapeseed as their main edible oil crop. Salt-tolerant rapeseed is one of the first crops grown on
reclaimed polder land in Holland. China and India each grow rapeseed on over 3 million hectares.
An important component of some rapeseed varieties is a 22-carbon unsaturated fatty acid, erucic
acid (22:1). Erucic acid is a component of resins and lubrication oils for jet engines and is used in
steel manufacturing. Since it interferes with mammalian metabolism, only plants containing little or
no erucic acid are grown for human and animal consumption.
Brassicas are also grown for animal fodder in regions too cool to grow maize, or during winter
months when grasses grow slowly. Large acreages of turnips, rutabagas, leafy forms of cabbage
and kales with thickened succulent stems provide winter grazing for sheep and cattle in Northern
Europe and New Zealand.
Brassica oil and vegetables are an essential part of the diets of many developing nations. The
Chinese consume 0.25 kilogram of crucifer vegetables per capita daily; in Korea consumption is
even higher. Radish (genus Raphanus), a close relative of Brassica, is grown as a vegetable in
China, Korea, Japan and India, where many large root types are dried, brined, pickled, cooked or
fed to animals.
The six major Brassica species of economic importance exist in a natural relationship that was
described by the genetic and cytogenetic work of U and Morinaga (Figure 5). Three diploid
species, B. nigra (bb), B. rapa (aa), (syn. B. campestris) and B. oleracea (cc) are the progenitors
of the naturally occurring allotetraploid species B. juncea (aabb), B. napus (aacc) and B. carinata
(bbcc). Diploid B. rapa (aa) has 20 chromosomes and allotetraploid B. juncea (aabb) has 36
chromosomes.

B. nigra

B. juncea B. carinata Raphanobrassica

(ABaabb)

B. rapa B. napus B. oleracea R. sativus

(Aaa) ( ACaacc) (Ccc) (Rrr)
a=10 ac=19 c=9 r=9

Figure 5. The cytogenetic interrelationships among six Brassica species and Raphanus sativus.
Intergeneric crosses between R. sativus and other Brassica species are also possible. Cytoplasmic
genome is designated by capitals. Nuclear genome is designated by lower case letters, where a =
10 chromosomes; b = 8 chromosomes; c and r = 9 chromosomes.

9
 Within each of the species there is a range of forms that are a result of divergent selection during
domestication (Table 3). Within B. oleracea are "cole crops" such as cabbage, cauliflower, curly
kale, kohlrabi, Brussels sprouts and the bizarre tree cabbage, or Jersey kale. Tree cabbage, which
may be up to 3 meters tall, is grown in Portugal and on the Channel Islands, where the leaves are
used in a nutritious vegetable soup and as winter cattle feed. The remaining stalks are cut and dried
for manufacture of walking sticks. As well as Chinese cabbage, turnip, pak choi and a host of
other forms representing vegetables consumed in various Oriental cultures, several oilseed types
are found within B. rapa. Brassica juncea, generally known as mustard, also displays a wide
divergence of form and is used as a source of oilseed in India and Pakistan, and as a vegetable in
western central China. The sharp mustard flavor is imparted by high levels of the mustard oil, allyl
isothiocyanate, in seed and leaf tissue. The genes controlling mustard oil synthesis are contributed
to B. juncea (aabb) largely through the genome of B. nigra (bb), black mustard. Brassica napus
varieties are used for oilseed, fodder, and as a vegetable, rutabaga. Both wild and cultivated forms
of B. carinata are major sources of leafy greens and cooking oil in Ethiopia.

Table 3. Names of subspecific taxa of agriculturally important brassicas and radish. considerable
taxonomic confusion exists in the literature for Brassica. (n) is the haploid complement of
chromosomes; a=10, b=8; c and r=9.
Species Subspecies Cultivar group
(genome) or variety or Common Name
Brassica
nigra (bb=16) Black mustard
oleracea (cc=18)
acephala Kales
alboglabra Chinese kale
botrytis Cauliflower, Heading broccoli
capitata Cabbage
costata Portuguese cabbage
gemmifera Brussels sprouts
gongylodes Kohlrabi
italica Broccoli, Calabrese
medullosa Marrow stem kale
palrnifolia Tree cabbage, Jersey kale
ramosa Thousand-head kale
sabauda Savoy cabbage
sabellica Collards
selensia Borecole
rapa (aa=20)
(syn. campestris) chinensis Pak c hoi
narinosa Taatsai
nipposinica Mizuna
oleifera Turnip rape, Toria
parachinensis Saichin, Choy sum
pekinensis Chinese cabbage, Petsai
perviridis Tendergreen, Komatsuna
rapifera Turnip
trilocularis Yellow sarson
utilis Brocoletto, Broccoli rab

10
Table 3 - contd.
carinata (bbcc=34) -- Ethiopian mustard

juncea (aabb=36)
capitata Head mustard
crispifolia Cut leaf mustard
faciliflora Broccoli mustard
lapitata Large petiole mustard
multiceps Multishoot mustard
oleifera Indian mustard, Raya
rapifera Root mustard
rugosa Leaf mustard
spicea Mustard
tsa-tsai Big stem mustard
Subspecies, variety
Species (n) or group Common name
napus (aacc=38) Fodder rape
oleifera Oil rape
rapifera Swede, Rutabaga
Raphanus
sativus (rr = 18) radicola Radish, dikon
oleifera Oil radish
caudatus Rat tail radish

The potential for exchange of useful genetic information between brassicas and the closely
related radish was demonstrated in the 1920's by the Russian geneticist, Karpechenko. To
combine the large root of Raphanus sarivus (radish) with the heading form of cabbage, he created
the synthetic genus Raphanobrassica (Figure 5). As with many such wide crosses in domesticated
plants, neither the attributes of radish nor of cabbage were attained. Rather, raphanobrassicas are
vigorous plants used for sheep and cattle fodder and green manure. Raphanobrassica can serve as a
bridge for the transfer from radish into brassicas of useful traits such as cytoplasmic male sterility
and disease and nematode resistance (Figure 5).
The relative ease with which diploid and tetraploid species may be intercrossed has permitted
the resynthesis of the amphidiploid species, as well as the production of new types with varying
numbers of chromosomes. An artificial B. napus (aacc), known as "hakuran," has been derived
from Chinese cabbage and cabbage, and is a new vegetable and fodder crop. The transfer of
resistance to clubroot and blackleg diseases into susceptible species has also been achieved by
interspecies crosses.
Seed production cycles of different brassicas and radishes can be annual, winter annual or
biennial cycles that require a few days to several months of cool temperatures) (<5º C) to induce
flowering. Flowering may also be under the control of photoperiod. Seed of vegetable and fodder
crucifers is produced in regions with mild winters where flower induction takes place. After late
summer and fall sowing, flowering occurs in the spring and seed harvest in late summer. Seed of
vegetable and fodder crucifers is produced in Australia, China, Europe, India, Japan, Korea, New
Zealand and the United States. The mild Pacific-coast states of Washington, Oregon and California
are ideal for crucifer seed production because dry summers minimize seed-borne diseases caused
by the fungi Alternaria and Leptosphaeria, and by Xanthomonas bacteria.

11
 The Development of RCB's
In order to understand the genetic basis for the diversity of forms found in brassicas and to
incorporate more efficiently traits of economic importance such as disease and pest resistance, an
ideal model plant type or ideotype (ideo=Greek for idea) was needed to speed research in genetics
and plant breeding. Of major importance was rapid flower and seed production, faster than the
normal reproductive time of six months to one year for the various crop groups. From a world
collection of over 2,000 brassicas obtained from the United States Department of Agriculture's
National Plant Germplasm System, a few plants were observed to flower in a significantly shorter
time than others. By combining the genes of early flowering types from various sources, plants
were bred for reduced reproductive time. These faster flowering individuals could then be used to
develop a population that would be tailored to suit the experimental ideotype needed for growing
large numbers of plants under standardized laboratory or classroom conditions. To do this, fast
flowering plants of various Brassica species were grown at 24º C in multipots at plant densities of
(880 plants/m2) in a standardized soil mix, irrigated with a balanced liquid nutrient solution (0.5
x Hoagland's solution) and illuminated continuously with bright light from cool-white fluorescent
bulbs [250 micro Einsteins per second per square meter (250 mEs-1m-2) of PAR]. Criteria used in
selecting individuals for successive generations were: 1) minimum time from sowing to flowering;
2) rapid seed maturation; 3) absence of seed dormancy; 4) small plant size and 5) high female
fertility. Populations of 288 or more were grown at each cycle of reproduction and the 10% of the
population that flowered earliest was selected and mass pollinated to produce the next generation.
In each successive generation the plants flowered in less time than the previous one. When the
reduction in the average days to flowering became stabilized and when greater than 50% of the
population flowered within a 2-3 day period, selection on the populations was discontinued. The
resulting model plant of B. rapa flowered in an average of 16 days, was 12 cm to the first flower
and averaged 78 seeds per plant (Table 4).
Table 4. Phenotypic characterization of rapid-cycling brassica and radish base populations grown
at 24º C under continuous high light. Nuclear genome is designated by lower case: a=10
chromosomes; b=8 chromosomes; c and r=9 chromosomes. When grown under lower
temperatures and light, development may be delayed. Data are expressed as mean (SD=standard
deviation).
Genome & Days Length
chromosome to (cm) to Seeds Days for Cycles
Species number flower first flower per plant cycle per
year
B. rapa aa=20 16(1) 11.9(3.1) 78(54) 36 10
B. nigra bb=16 20(2) 27.1(4.9) 69(49) 40 9
B. oleracea cc=18 30(3) 22.6(5.3) 18(21) 60 6
B. juncea aabb=36 19(1) 29.6(4.0) 107(46) 39 9
B. napus aacc=38 25(2) 35.3(7.1) 76(53) 55 6
B. carinata bbcc=34 26(2) 41.7(6.6) 67(46) 56 6
R. sativus rr=18 19 48 7

This stock was capable of cycling (seed-to-seed) ten times per year. The seed stocks of the
rapid-cycling base population of each species were given code numbers and made available to
researchers throughout the world via the Crucifer Genetics Cooperative (CrGC). Many scientists
are using the RCB's as model plants for research in genetics, molecular biology, plant breeding,
cell biology and physiology. Through the CrGC, new information and new genetic stocks are
shared among more than 1000 scientists from 45 countries.

12
 In addition to the development of the rapid-cycling population of B. rapa (RCBr), rapid-cycling
populations of five other related Brassica species, B. nigra, B. oleracea, B. juncea, B. napus and
B. carinata and of radish, Raphanus sativus were developed (described in Table 4). More than 100
distinctive genetic traits are being studied in the RCB's. Genetic mapping of the chromosomes is
under way using distinctive morphological markers, physiological and disease resistance markers,
isozymes and restriction fragment length polymorphisms (RFLP's). Various quantitative traits and
cytoplasmically-inherited phenotypes are being incorporated into the rapid-cycling stocks.
Early in their development, stocks of RCBr from the CrGC were sent to Cornell University, the
University of California-Davis, the University of Guelph and various other institutions where they
were used in plant genetics and plant breeding courses. Today the potential for wider use of the
RCB's as model organisms for hands-on learning in the classroom is being realized.

REFERENCES
Williams, P. H. and Hill, C. B. 1986. Rapid-cycling populations of Brassica. Science 232: 1385-
1389.

13
 Growing Instructions for Fast Plants
Since the growing conditions for rapid-cycling Brassica rapa differ from most traditional
classroom plant growing, it is strongly recommended that you grow a cycle of the plants before
your students begin experiments. Continuous bright fluorescent lighting and a constant water
supply are critical for the rapid-cycling of these plants. By explicitly following these guidelines,
you and your students will enjoy a successful growing experience.
Read these guidelines completely before you begin planting.
Before Planting
1. Become familiar with the materials in the Fast Plant Kit:
a. Brassica rapa seed-It's small and needs to be handled with care.
b. quads -- 4-celled planting units in which you will grow one plant to maturity in each cell.
c. fertilizer pellets-slow-release source of nitrogen (N), phosphorous (P) and potassium
(K).
d . potting mix
e. wicks-conduct water from water mat to soil in cell of quad.
f. water mat--conducts water from reservoir to wicks.
g. pipets-to water cells from above when necessary.
h. plant labels-see "Records and Terminology" section for labeling suggestions.
i. dried honeybees-used to make beesticks.
j. small water mat squares (blue)-contain copper sulfate to prevent algae growth in reservoir.
k. wooden stakes and plastic support rings-to support the plants if necessary.
2. Lighting:
Assemble a light bank and the rack to support it. To complete the growth cycle in 40 days, a
bank of six or eight, 4-foot cool-white fluorescent bulbs (40 watts/bulb) is necessary (Figure
1). This arrangement can be constructed by fastening lights to a frame. Use a plug adapter
and an extension cord. Choose a light-weight light so that your light bank can be moved and
transported easily. Suspend the light bank from a wooden rack. This arrangement will permit
you to adjust the height of the light bank as the plants increase in height. Plants and quad will
need 30-40 cm of space below the bulbs at maturity.

Figure 1. Light bank and watering system.

14
 Keep growing tips of plants about 5-10 cm from the bulbs throughout the life cycle.
The plants will complete their life cycle in 40 days only if they are given 24 hour of light per
day and the recommended light intensity. If your light source has less than six 40 watt bulbs
or you allow more than 5-10 cm from growing tip to bulb, plants will grow tall and spindly.
In addition, the time to complete the life cycle will be extended several days.
As an alternative to raising the light bank, set the light bank 40 cm above the table surface
and raise the reservoirs initially so that the plants are 5-10 cm from the bulbs. Gradually
lower the reservoirs as plant height increases.
If a growth chamber is used, it must be in optimal operating condition.
Planting

1. Plan to begin a Fast Plant cycle by planting on a Monday or Tuesday. This schedule will allow
you consecutive school days for watering from above for the first three days and align
flowering with weekdays.
2. Water
Prepare the water mat for use. Soak the mat in water, then squeeze water out. Repeat this
process two more times. After the final soaking, do not squeeze the water out, but simply lay
the mat in the correct position on the platform (Figure 2). Smooth out the mat on the platform,
leaving no air pockets under the mat. Fill the reservoir with water.
This watering system is based on capillary action. Once the water mat is wet, it continues to
draw water from the reservoir. Wicks in the bottom of each cell draw water into the potting
mix. The reservoir holds enough water for 2-3 days.

Figure 2. Watering system.

3 . Place copper sulfate squares in the reservoir water.

4 . Moisten the potting mix until it is slightly damp.

15
5 . Drop one wick into each cell so that the tip extends 1-1.5 cm out the hole in the bottom
(Figure 3).

Wicks
Bottom of quad

Figure 3. Quad and wicks.

6 . Fill each cell halfway with potting mix (Figure 4).

Wick

Figure 4. Planting.

7. Add 3 fertilizer pellets to each cell.

8. Add more potting mix to fill each cell at the top. Do not pack soil. Use your finger to make a
0.5 cm depression on top of each cell.
9 . Drop 3 seeds into the depression in each cell.
10. Cover seeds with just enough potting mix so that the seeds are no longer visible.

16
11. Water gently with pipet until water drips from each wick tip (Figure 5). Place the quad on the
water mat. The top of the quad should be 5-10 cm from the bulbs of the light bank.

Water Dripping
Figure 5. Initial watering.

12. Label each quad by inserting a pot label which has been correctly marked with a waterproof
pen (Figure 6). See Records and Terminology section for a method of labeling.

Figure 6. Labeling.

After Planting
1. Be sure to water gently from above with pipets for the first three days to insure adequate
moisture for germination.
2. Check the water reservoir daily and keep it filled. Completely fill the reservoir Friday before
leaving for the weekend.
3. Check the water mat and potting mix in each cell daily. Both should be moist at all times. If the
mat has dry spots, remove all quads and soak the mat again before returning quads to the mat.
If potting mix in a cell appears dry, check the wick. Occasionally a wick dries out because of air
pockets in the cell. Add water from above with a pipet until water drips from the wick.

17
4. Drought
If the worst happens (e.g., you forgot to fill the reservoir on Friday) and the plants are wilting
(but not yet crispy), you may be able to save your plants. Fill reservoirs with water and float the
quads in the water while adding water from above with pipets. Allow the quads to float on the
water until plants are turgid again. Re-soak the water mat and return the quads to the mat.

5. Thin Plants
Thin to one plant per cell. Use scissors or tweezers. Transplant extra seedlings to cells without
plants (Figure 7).

Figure 7. Thinning.

6. As the plants grow, remember to maintain the 5-10 cm spacing between the growing tip and the
bulbs.
Although insects are not usually a problem, check plants for insect damage. The most common
sources of insects are other plants in the room that are infested or insects such as aphids that
attach to clothing outdoors and are then inadvertently brought into the room. Three methods are
recommended for insect control:
-First, simply remove the insects from your plants by hand and crush them.
-Second, spray plants with a solution of an insecticidal soap which can be purchased from
a local garden store. Be sure to follow instructions on the label.
-The third method will vividly demonstrate the effects of nicotine on insects. Place a large
metal waste basket inside a large plastic trash can liner. Place the quads with the infested
plants at the bottom of the metal waste basket. Light a cigarette and place it about three
inches from the quads and close the plastic liner (Figure 8). After 1-2 hours, remove the
quads and check for insects.

18
Figure 8. Insect control.

8. As the Fast Plants grow, you may use small wooden stakes and plastic support rings to support
the plants (Figure 9). Gently hold plant next to the stake, open the plastic ring and slip ring
around both.

Plastic

Figure 9. Staking plants.

Pollination
1. Prepare beesticks 1-2 days in advance of pollination (Figures 10 and 11). The volatile chemicals
in the glue are toxic to the pollen grains and will prevent pollen germination if the bee sticks are
used immediately after being constructed. Build and store freshly made beesticks in a different
area than where the plants are growing.

Figure 10. Dissect bees. Figure 11. Glue thorax to toothpick

19
2. Pollinate with beesticks by rotating the bee thorax over flowers to pick up and distribute pollen.
Transfer pollen back and forth among different plants (Figures 12 and 13). Brassica rapa plants
must be cross-pollinated, except if you use a bud pollination method (see section "Self-
pollinating rapid-cycling Brassica rapa").

Figure 12. Remove wings and Figure 13. Cross pollinate.

pollinate.
3. Pollinate daily for 2-3 days.
4. On the last day of pollination, pinch off all other unopened buds (Figure 14) and mark the date
on the pot label.

Figure 14. Remove unopened buds.

20
After Pollination
1. Seed pods and seeds develop. Seed pods will begin to elongate within 3-5 days, and will
mature in 20 days (Figure 15).
2. During days 18-36 of the life-cycle, continue to pinch off new flower buds. The plant will then
direct its resources to the developing seed pods.
3. Beesticks loaded with pollen may be stored in a glass screw-cap vial with an indicator silica gel
desiccant capsule (Figure 16). At 4º C the pollen will remain viable for several months.
Periodically check the color of the capsule and replace it if it turns from blue to pink.

Figure 15. Mature seed pods. Figure 16. Beestick storage vial.
Seed Harvest
1. Remove plants from water 20 days after the last pollination. Dry for 5 days. To cut the drying
time to 3 days, cut off seed pods, place in brown paper bags and set the bags on top of the light
bank with the lights on.
2 . Harvest seed by gently rolling dry seed pods between hands over a collecting pan. Place the
seed in an envelope like the one you received in the Fast Plant Kit.
3. Seed envelopes should be kept in a cool dry place. Seed stored in your desk retains its viability
for about 4-6 months. For longer storage, place seed envelopes in a screw-cap bottle with an
indicator silica gel desiccant in a small paper envelope or gelatin capsule. Store the bottle at 4º
C. Periodically check the color of the silica gel and replace it if it turns from blue to pink.
Indicator silica gel may be reactivated (pink color will return to blue) by drying overnight in an
oven at minimum heat (65º C).
Before The Next Cycle
1. Reservoirs, platforms, water mat, quads and wicks should be soaked in a 10% chlorine bleach
solution for at least 15 minutes. Then, scrub quads with a brush and rinse all materials
thoroughly with water. Let all materials dry completely before reusing.

21
 Records and Terminology

When carrying out any investigation it is important to keep neat, accurate and complete records
of what you do. In this way you will be able to analyze and interpret what you have done and
communicate it to others. A scientific investigation is only completed when the results of the
investigation have been thoughtfully interpreted and clearly communicated. By understanding the
specialized terminology for each branch of science and using it accurately, you will be able to
communicate effectively your results and ideas. Each part of science has its own specialized set of
terms used to describe things, processes and relationships. Many of these terms have been derived
from Greek or Latin and often have parts of them that sound familiar to some commonly used
English words. In genetics, the core science in biology, symbols are used as a sort of shorthand to
designate certain characteristics, processes and relationships among the units being experimented
with. This is just the same as is done in chemistry or physics where special symbols are used to
designate the chemical elements or various forms of energy. In genetics, the accurate use of
terminology is important so that proper analyses and interpretation can be given to the results of an
experiment. At the molecular level, the arrangement of nucleotides making up the DNA provides a
fundamental understanding of the gene. How the various nucleotide sequences are arranged in the
gene and how the genes are expressed in the vast range of phenotypic characteristics within
organisms largely is unknown and remains one of the fascinating areas for investigation in
biology.
Some of the important terms needed to understand the basic principles of genetics are given
below.

allele - one of two or more alternate forms of a gene occupying the same locus on a particular
chromosome or linkage structure and differing from other alleles at the locus at one or more
mutational sites.
dominance- refers to the expression of genetically controlled characters (phenotypes) and their
corresponding alleles when they are in the heterozygous condition.
1. Dominance and recessiveness are not properties of the genes per se, but the result of the
action of the genetic locus in question within the total reaction system of a particular
genotype.
2. Complete dominance and complete recessiveness are the extreme cases between which all
transitional degrees of expression are possible.
gene -a particular sequence of nucleotides along a molecule of DNA (or in some viruses, RNA)
which represent a functional unit of inheritance.
genotype -
1. The genetic constitution in respect to the alleles at one genetic locus under observation
2. The sum total of the genetic information (genes) contained in chromosomes (linkage groups)
of pro- and eukaryotes. The genotype determines not a unique phenotype but a range of
phenotypic expression referred to as the individual's reaction norm.
heterozygous- in diploid organisms the condition of having different alleles at one or more loci
(genes) in homologous chromosome segments, in contrast to homozygous, having identical alleles
at these loci.

22
locus - the position of a gene on a genetic map. Allelic genes are situated at identical loci in
homologous chromosomes.
phenotype - the observable properties (structural and functional) of an organism, produced by
the interaction between the organism's genetic potential (its genotype) and the environment in
which it finds itself. The term phenotype can be applied either to the totality of expressions of the
genotype or to only a part; i.e., to particular characters or traits. The phenotypic range or
expression is referred to as its reaction norm.
recessiveness - the absence of expression of genetically controlled characters and their
corresponding alleles when they are in the heterozygous condition.
wild type - refers to an organism or gene chosen to be the standard for comparing other
phenotypes or genotypes.

In genetics, different shorthand schemes have been developed for recording genetic
characteristics, processes and relationships of different organisms. This has led to difficulty in
communication between scientists. Recently, geneticists have been working to develop more
standardized terms for their particular organisms so that they could more easily communicate with
other scientists.
Since brassicas are emerging as model organisms, the opportunity exists to adopt genetic
terminology for brassicas that will be in conformity with the terminology used by some of the other
important model organisms. Thus, in designating symbols to define particular genotypes or
phenotypes we suggest using the guidelines of the Crucifer Genetics Cooperative for Brassica.
Guidelines for Using Genetic Svmbols

In the case of the rapid-cycling Brassica rapa, Wisconsin Fast Plants has designated the cultivar
name RCBr to the basic fast cycling stock which has been developed for the WFP kits. For most
traits the phenotype of RCBr represents the wild type. Various other mutant stocks have been
developed in the common genetic background of RCBr. Each of these is accompanied by
genotypic or phenotypic symbols that designate the uniqueness of the particular stock.
1. All gene symbols should consist of three letters.
2. Underline genotypes
3. The genotype designation of wild type is capitalized (e.g. ROS. YGR).
4. The genotype of mutant alleles is in lower case (e.g. ros=rosette; ygr=yellow-green plant).
5. Alleles are designated by a dash followed by a number (e.g. mey-1, mev-2. mey-3). If no
allele is specified it is assumed to be allele number 1 (e.g. mey is equivalent to mey-1).
6. Genes with mutant alleles of similar phenotype can (but need not) be given the same three
letter designation followed by a different number (e.g. ygr, ygr2, ygr3). y
7. Phenotypes are designated by the gene symbol which is not underlined but has the first letter
capitalized (e.g. Ygr), a + or - may follow the phenotype symbol to designate expression (+)
or absence of expression (-). Degree of expression of a quantitative phenotype may be
designated on a 0-9 scale in parentheses following the symbol [e.g. An1 (7)].
8. Uniparentally inherited phenotypes are enclosed in parentheses [e.g. (Var)=cytoplasmically
inherited variegation].

An example of the name and description of an RCBr mutant stock would be as follows: RCBr,
ros/ros; RCBr=rapid-cycling Brassica rapa WFP stock; ros/ros=homozygous for the recessive
mutant gene, ros, conditioning deficiency in gibberellin and resulting in a rosette plant form with
extreme internode compression. The rosette phenotype can be "corrected" by the application of
exogenous gibberellin to the plant.

23
Labeling. Tagging Record Keeping
Accurate labeling and recording of data is vital to an experiment. To insure that you and your
students conduct a successful experiment, the following tips are suggested:
1. Obtain planting labels, waterproof marking pens, seed envelopes, and construct data tables
before beginning an experiment.
2. Number each plant in a quad.
3. Label the planting label with:
seed type
planting date
student's initials
nature of experiment or treatment to be performed
4. If two or more different genotypes/phenotypesare planted in the same quad, use separate
planting labels for each.
5. Seed envelopes should be labeled with:
name of grower
date of harvest
type of seed stock
treatments done to plants
generation (genetics)
6. In recording a cross between two parents, the female genotype is always written first. In
designating the heterozygous condition of a gene, the allele coming from the female is
always placed first, separated by a slash (/) followed by the allele from the male, e.g. ros/ROS
is heterozygous for rosette. The female parent was homozygous for the rosette allele
(ros/ros)and was phenotypically rosette. The male parent was wild type for the rosette allele
(ROS/ROS) and, therefore, was normal height. In the case of the rosette, the wild type allele
ROS is expressed and, therefore, dominant over the mutant ros allele.
7 . Instruct students to return their quads to the same location on the reservoir each day after
making observations and recording data.
8. Use data tables provided or construct your own. More recorded information from daily
observations is always better than less.
9. Keep a record of class data. You will more than likely want to repeat your experiments year
after year and it's handy to look at the data from previous years before you begin again.

24
 Self Pollinating Rapid-cycling Brassica rapa

To obtain self-pollination in self-incompatible Brassica rapa stocks you must overcome the
pollen-stigma incompatibility mechanism. To accomplish this requires manipulation of the flower
buds. Observations from growing Brassica rapa plants will allow you to predict when the flower
buds will open. You must be able to predict when the buds are one to three days from opening.
The timing of this procedure is critical. The whorl of developing flower buds is ideal for examining
which stages of buds are receptive to "self" pollen. In RCBr each successive swollen bud is
approximately 8 hours younger than the next sized one. A new bud will develop approximately
every 8 hours. If buds are too young and the stigma too immature, pollen grains will not remain
viable long enough to ensure fertilization. If the buds are opening, the incompatibility mechanism
will be operational and the pollen tube will be prevented from forming.

Figure 1. Orientation of buds in apical whorl of RCBr.

1. One to three days prior to the buds opening, use forceps to gently pry open the bud (Figure
2). You may want to pull off the sepals and petals. The immature stigma should be exposed
(Figure 3).

Figure 2. Open bud. Figure 3. Exposed stigma.

25
 Transfer pollen from a mature anther on the same plant to the immature stigma. The pollen
may be transferred by beestick (Figure 4) or by using an excised anther (Figure 5).

Figure 4. Pollen transfer with beestick. Figure 5. Pollen transfer with excised anther.

3. Examine the pistil for the next 3-5 days (Fig. 6). Elongation and swelling of the pistil
indicates that fertilization has taken place.

Figure 6. Examine pistil for indications of successful fertilization.

26
 How Can Plants That Look So Different Be the Same?
An Introduction to Plant Breeding

INTRODUCTION
Turnips, Chinese cabbage and Wisconsin Fast Plants (RCBr) look very different. Yet, they
actually belong to the same species. This means that they have the same number of chromosomes
and they can cross breed and produce fertile offspring. But how can you prove that plants that
look so different are really the same species? The following investigations will allow you to
explore this question.

TIME REQUIRED
Stage 1 -- 4-6 weeks, requiring no tending
Stage 2 -- 2-3 weeks
Stage 3 -- approximately 20 days
MATERIALS
turnips, Chinese cabbage, Wisconsin Fast Plant (RCBr) seeds and kit supplies
2 liter soda bottles
rooting powder (e.g. "Rootone")
soil mixture (peat moss and vermiculite)

EXPERIMENT I
1. Purchase a turnip with some small buds or shoots at the crown and Chinese cabbage from your
local grocery store. With a sharp knife, mm most of the large leaves from the Chinese cabbage
leaving about 1 cm of each leaf attached to the core. Use the leaves in your cooking. Trim off
leaves until you have a core about 6-10 cm long. Place the turnip and cabbage core in a plastic bag
in the refrigerator for 4-6 weeks (Figure 1). This cold treatment, called vernalization, simulates
over-wintering and the plants will convert from a vegetative to a flowering stage.

Figure 1. Turnip and cabbage core in plastic bag.

2. When small leaves begin to grow from the top of the turnip, make two growing containers from
2-liter soda bottles. (e.g. Fill bottle with hot tap water, replace cap. Hold the bottle firmlyand
twist off the opaque bottom. Seal holes on bottom with black electrical tape.) Fill containers with
a mixture of equal parts of peat moss and vermiculite (or sand).

27
3. Remove turnips and Chinese cabbage cores from refrigerator. Cut a thin slice off the bottom of
each. Put a little rooting powder on the newly cut surface to help rooting.
4. Place vegetables on growth medium in the containers. Keep the soil moist at all times. Place
them in good light, keep cool and partly covered to prevent excess wilting. (e.g. Cover with the
top portion of a 2 liter soda bottle which has been cut to be about 8" high (Figure 2).

Figure 2. Growing containers.

5. Within 2-3 weeks the plants should begin to produce flowers. At the time the first buds appear,
plant at least six quads of Fast Plant (RCBr) seeds. In two weeks all three types of Brassica rapa
should be flowering.
6. Follow the instructions for pollination found in the Fast Plant "growing instructions." Cross
pollinate each of the vegetables to the RCBr as shown in Figure 3. Use separate beesticks for each
cross.

RCBr Turnip or Chinese cabbage

(seed parent) X
(pollen parent)

F1 Hybrid

Figure 3. Cross pollination of Turnip or Chinese cabbage to RCBr.

7. After 20 days you should have mature seed pods on you RCBr plants.

Questions
1. Were you able to produce seeds on your RCBr plants?
2. Is this final proof that the different plants are the same species?

28
 EXPERIMENT II
Save the seed you have produced from Experiment I and plant them in soda bottle growing
containers in class or in the garden over the summer. These plants constitute the first generation.
1. What is your hypothesis about how these intraspecific (within a species) hybrids will look?
(Hint: if the plants get too large, transplant them into a larger container.)
2. If the plants flower do they produce pollen?
3. Can you produce seed by interpollinating two or more hybrid plants?

EXPERIMENT III
If you get seed from the intraspecific hybrid, try sowing this out in your garden (or in class.) What
do the plants from this generation look like? IF YOU GET THIS FAR YOU ARE WELL ON
YOUR WAY TO BECOMING A PLANT BREEDER and you will understand the riddle of how
things that look so different can be the same.

29
30
 Chapter 2

Professional Telecommunications: How to Get the

Most From the NABT Electronic Bulletin Board
and Other Useful Databases

Steven P. Lanphear

James Madison Memorial High School

201 S. Gammon Rd.
Madison, WI 53717

Note: This workshop was not included in this Proceedings

Volume because it was not submitted.

31
32
 Chapter 3

Nerve Conduction in Frogs and Humans

Elizabeth Vizsolyi

Department of Zoology
University of British Columbia
Vancouver, British Columbia V6T 2A9

Elizabeth Vizsoyi received her BSc degree from Eotvos Lorand

University in Budapest Hungary, and MSc and PhD degrees from
the University of British Columbia in Vancouver, in the field of fetal
endocrinology. She has been a lecturer in Zoology 303/Biology
353, a third year vertebrate physiology course for 14 years.

33
34
 Nerve Action Potential and Conduction Velocity

INTRODUCTION
A transmembrane potential is one of the characteristic features of the living cell. Its source
resides in a differential distribution of ions across the membrane with high K+ inside and high Na+
and Cl - outside. This distribution depends on the permeability properties of the cell membrane and
the presence of ion pumps. In "resting cells", this potential is about 75 to 90 millivolts (mv) with
the outside of the nerve fiber positive with respect to the inside. The potential can be readily
recorded by inserting a fine electrode into the interior of a cell while another electrode touches its
outer surface; the constant potential thus recorded is called the resting potential.
When a cell is excited (muscle cell contracts, nerve cell transmits, gland cell secretes), there is a
characteristic change in this potential (electrogenesis) known as the action potential. One of the
most useful methods of monitoring activity in cells is the measurement of these action potentials.
The action potential in the nerve is the nerve impulse.
In this exercise action potentials will be recorded during the stimulation of the frog sciatic nerve.
It must be remembered that the sciatic nerve as a nerve trunk, contains the axons of many neurons;
consequently the record obtained in your experiment will show a composite action potential. In
addition, placing the electrodes on the outside of the nerve trunk will result in a diphasic recording,
as opposed to the monophasic action potentials recorded by transmembrane electrodes. For further
reference consult Ruch & Patton.
EQUIPMENT
Your equipment consists of a nerve chamber, stimulator, amplifier and an oscilloscope
(Figure 1). Before you dissect the nerve, you should set up your apparatus, ready to record.
1. Connect stimulator output to the stimulating electrodes on nerve chamber (A and B on
diagram.)

2. Connect the first recording electrode (C) to the reference terminal of amplifier, and the second
and third recording electrode (D to H) to the input of the amplifier.
3. Connect amplifier output to "y" input of oscilloscope and "sync out" of stimulator to "x" input
of oscilloscope.

Dissect a long sciatic nerve preparation from a double pithed frog (diagrams provided). Use
only glass probes in handling the nerve and keep the nerve wet with amphibian Ringer's
throughout the dissection. Tie a piece of thread around one of the spinal nerves which leads into
the sciatic nerve and cut the nerve free just beyond the thread. Tie another thread onto the distal
end of the sciatic nerve and sever the nerve beyond the thread. Lay the free nerve on a piece of
filter paper and blot gently free of water. Now mount the nerve in the nerve chamber, passing the
nerve over and under successive electrodes as shown in the diagram. The proximal (spinal) end of
the nerve should be in contact with the two stimulating electrodes. Without overstretching the
nerve, extend it sufficiently to ensure good contact with all of the electrodes. Pass the thread
through the holes in the plexiglass at the two ends of the chamber and secure with plasticine.
Moisten the nerve with Ringers solution, and cover chamber with microscope slide.
If the other sciatic nerve of your frog is not being used by another pair of students, dissect it
and store in cold aerated Ringer's.

35
Figure 1. Nerve chamber, stimulator, amplifier and oscilloscope.

38
 EXPERIMENTAL PROCEDURE
Slowly turn up the stimulus voltage, until you see a "stimulus artifact" (or "shock artifact")
appear as a small vertical deflection at the beginning of the sweep. Do not exceed 0.1 volts of
intensity at this stage. If there is no trace on the screen, you have to adjust the position of the beam
in the following manner:
Turn the intensity dial fully clockwise, depress beam finder button, centre trace on screen using
the vertical and horizontal position knobs. Release beam finder knob and make fine adjustments to
position until the complete trace is visible on the screen.
After the trace is properly centered, start again with a voltage of 0.0, and increase the intensity
of stimulus until you see the stimulus artifact. The stimulus artifact is really an electrical leakage
from the stimulating electrodes, which is conducted around the outside of the nerve and picked up
by the recording electrodes. This artifact marks the exact time that the nerve is shocked. Continue
to gradually increase the stimulus voltage; note that the size of the artifact also increases. Then, a
new, small, delayed deflection will appear - the nerve action potential. Increase the voltage until
the nerve response reaches maximum height. What is the voltage applied when the response is
maximal? Do not exceed this voltage by more than 10-20 mV, to avoid injuring the nerve. Note
that the nerve impulse appears as a diphasic wave. Why? Record the voltages for threshold and
maximal responses of the nerve trunk. Why should the threshold voltage differ from that which
causes maximal response.
To calculate conduction velocity, you should store the trace of the action potential. Connect
recording electrode D & E and obtain a tracing of maximal response. Now, set oscilloscope
triggering mode to single sweep, depress store button, push erase, and press triggering mode to
reset. Allow mode to return to single sweep position, and you should now have a single trace on
the screen. To obtain recording from the second point along the nerve, disconnect recording
electrode D & E and connect electrode G & H. Repeat procedure for storing trace. You should
now have two traces of action potentials on the screen, at two different distances from the stimulus
artifact. Measure the distance along the nerve for each trace between the stimulating and the
recording electrodes. Read the time between stimulus artifact and action potential for both traces
from oscilloscope, using the setting of your time base dial. (dl & d2 on diagram). Subtract time
for shorter distance from that of the greater distance and divide this value into the distance between
the two recording electrodes on the nerve chamber. The value obtained from this calculation is the
velocity of the impulse conduction.

Calculate nerve conduction velocity in meters/sec.

REFERENCES
Florey, E. 1966. An introduction to general and comparative physiology. Saunders,
Philadelphia.
Katz, B. 1952. The nerve impulse. Sci. Am. 187 (5): 55-64.
Katz, B. 1966. Nerve, muscle and synapse. McGraw Hill, N.Y.
Keynes, R. D. 1958. The nerve impulse and the squid. Sci. Am. 199.
Ruch, T. C. and Patton, H. D. 1965. Physiology and Biophysics. Saunders, Philadelphia.

37
 Refractory Periods

Set up a nerve preparation and equipment as described in the previous exercise, but in place of
the regular stimulator, connect the GRASS S 48 twin pulse stimulator. The twin pulse stimulator
allows the delivery of two shocks at pre-set intervals, where the time lapse between the two stimuli
is regulated by a time delay switch. To set the stimulator; place Stimulus switch to ON position,
Pulses-DC to pulses, Function switch to Single position and Duration to 0.15 msec. To assure a
square wave stimulus, a Stimulus Isolation Unit (SIU) is connected to your stimulator. Set the
voltage multiplication dial on the S 48 to X10 (SIU), and leave it in this position throughout the
experiment. On the SIU, set Voltage Input multiply dial to required range, Coupling switch to
capacity and Polarity to normal. Regulate voltage range during the experiment using the dial on the
SIU unit.
To determine Refractory Period, turn stimulus mode to repeat at 30 pulses/sec., voltage range
from SIU to 0.1, and pulses to single. Turn voltage up slowly, using the control on your
stimulator, until you just obtain maximal response. Switch to twin pulses and increase delay time
gently until you record a second Action Potential. The delay in msec.s represents the total
Refractory Period of the nerve trunk. Now turn the voltage to 4-5 times threshold strength and
obtain delay time as before. The time obtained using suprathreshold stimulus is the end of the
Absolute Refractory period. To determine Relative Refractory Period subtract Absolute Refractory
Period from total Refractory Period.

38
 Nerve Conduction Velocity Measurements in Human

INTRODUCTION
Nerve conduction velocity in human can be easily determined by stimulating a motor nerve and
monitoring the response of its associated muscles. The time interval between the stimulation and
the response - latent period - is recorded from two different points along the nerve. The difference
between the two latent periods is the time required for the nervous impulse to travel from point A to
point B.
In your exercise you will stimulate the ulnar nerve and record muscle contractions in the fifth
finger. Before coming to the laboratory, you should consult an anatomy book to familiarize
yourself with the location of the ulnar nerve. For further reference, make use of the diagrams on
your laboratory benches.

EXPERIMENTAL PROCEDURE
Location of the Ulnar Nerve
1. Set Grass stimulator to a frequency of 1/sec.; duration of 20 msec.; voltage 50 V; monophasic;
+ polarity; and the mode switch to repeat.
2. Connect "sync out" of stimulator to X input of oscilloscope.
3. Connect Grass stim. output to pulse generator input.
4. Plug recording electrodes into amplifier outlets. The first one, which will be the active
electrode into RA outlet, the second one (indifferent electrode) into LA and the ground into RL
outlet.

5. Cover surface of recording electrodes with contact media and secure them to your hand. The
active electrode should be midway along the lateral border of the large muscle above the fifth
finger (hypothenar eminence); the indifferent electrode is placed at the base of the proximal
phalanx of the fifth finger, and the ground electrode to the palmar surface of the hand. (see
diagrams in laboratory)

6. Turn the output level of the pulse generator all the way down, and touch your arm with the
stimulating electrode. Now increase the output level of the pulse generator until you feel a
strong muscle contraction.
7. Move the stimulating electrode to the approximate location of the ulnar nerve. ALWAYS
PLACE THE CATHODE OF THE STIMULATING ELECTRODE NEAREST TO THE
MUSCLE BEING STIMULATED. Locate the ulnar nerve at the wrist, elbow and upper arm,
by watching for contraction of the little finger. Mark the location of the nerve at these points
with a ball point pen. Now you are ready to record.
Recording

1. Turn oscilloscope ON, Store "off" (button in an out position). Set triggering to ext., mode to
norm., and turn CHANNEL 1 on. (Left hand side control panel, and button to be pushed in.)

39
 Use an 0.2 volts/div., a time base of 1 msec/div.
The oscilloscope should now produce a trace only when the Grass stimulator produces a pulse.
If this is not happening, it may be necessary to adjust the trigger level on the oscilloscope. If no
trace at all is visible on your screen, the position of the beam will have to be adjusted in the
following manner:
Turn intensity dial fully clockwise, depress beam finder button, centre trace on screen using the
vertical and horizontal position knobs. Release beam finder knob and make fine adjustments to
position until the complete trace is visible on the screen.
2. Change oscilloscope triggering to "line". A continuous trace should now be seen. (It may be
necessary to readjust trigger level.)
3. Flex the little finger and adjust the gain of the amplifier until substantial trace deflection is seen.
Do not proceed until an EMG is observed. If no deflection can be seen on the oscilloscope,
check all the connections, particularly the recording electrodes. Return triggering to "EXT".
4. Stimulate the nerve at the points marked earlier. The resulting waveform should appear as
follows:

"stimulus artifact"

The first deflection is the stimulus artifact. The stimulus artifact is an electrical leakage from the
stimulating electrodes, which is conducted along the surface of the arm and picked up by the
recording electrodes. It may be necessary to adjust the horizontal position for the stimulus artifact
to appear on the display. If the artifact trace disappears off scale, and adjusting the vertical position
does not bring it back, reduce the sensitivity (Volts/div.).
The second deflection represents the contraction of the muscle. The time delay between the
stimulus and the response represents the latent period. To produce a suitable trace it may be
necessary again to make some adjustment to the time base or sensitivity.
Velocitv Measurements

1. Set oscilloscope triggering mode to single swp, STORE-on and press erase.

2. Hold probe on the first mark on your arm and momentarily depress mode switch to reset.
When a trace is obtained, repeat procedure on the next point marked on your arm.

40
3. You should have a composite display of several delay times remaining on the screen. To
calculate conduction velocity, record delay times from oscilloscope, and measure the distance
between the point of stimulus and the first recording electrode on the arm. Plot the distance
along the nerve vs. time delay. The slope of the line will give the conduction velocity of the
nerve impulse in the ulnar nerve. Express this velocity in meters/sec., and compare your results
to those in the literature.
OR:
Read time delay between peaks from oscilloscope, and record distance between points of
stimulus on the ulnar nerve. To calculate conduction velocity, use the following formula:
Distance between points of stimulus/time delay = conduction velocity in cm/msec.
Convert the obtained value to meters/sec.

REFERENCES
Rhodes, R.M.G. Larrabee and W. Gennan. 1948. The human electromyogram in response to
nerve stimulation and the conduction velocity of motor axons. Arch. Neur. Psych.
60:340-365.
Lundervold, A., H. Bruland and P. Stensrud. 1965. Conduction velocity in peripheral nerves.
Acta Neur. Scand. 41: (Suppl.13): 259-262.
O'Connell, A.L. and E.B. Gardner. 1963. The use of electromyography in kinesiological
research. Res. Quart. 34:166-184.

41
42
 Chapter 4

Electron Flow in Photosynthesis

R. Patrick Harrison

Biology Program
University of British Columbia
Vancouver, British Columbia V6T 2B 1

R. Patrick Harrison has been a lecturer for the past eleven years in
the first year biology program at the University of British Columbia.
He received his B.A. in Zoology from the University of Montana-
Missoula in 1967, and his M.A. in Botany from the University of
Montana-Missoula in 1976.

43
44
 Leaf Structure and Pigmentation

1. To make leaf cross-sections and become familiar with leaf structure.

2. To separate and identify the pigments present in leaves.
3. To determine the absorption spectra of a plant and an alga.

READ the lab outline before coming to class. Background information can be found in the
following references in Keeton and Gould.
Leaf structure: pp. 213-214,274-278
Chromatography: p. 62
Absorption spectra: pp. 199-202

INTRODUCTION

In our study of plants, we have yet to consider their most obvious characteristic, that being their
ability to harness the sun's energy to build complex molecules from simple ones - something that
animals are unable to do. Before examining this process, called photosynthesis, in detail, we want
to look at some features of plants which enable this process to occur.
The leaf is the plant's organ of photosynthesis. It is the structure which captures sunlight and
makes it available for the chemical reactions of photosynthesis. Leaves come in all shapes and
sizes depending on the particular species of plant. Because the leaf is also the site of water loss for
the plant, each plant has evolved a compromise between maximizing the capture of sunlight and yet
minimizing its loss of moisture. In some environments, such as the desert, the need for water is
so great that the leaves have been reduced to non-photosynthetic spines and the stem has taken
over the role of capturing sunlight. In the conifers, the leaves take the form of needles which have
a lower rate of water loss compared to the broad-leaved trees which occur more frequently in areas
of moderate to heavy rainfall.

EXERCISES

A. LEAF STRUCTURE (work in pairs)

We will study the leaves of Photinia, an evergreen shrub found locally on campus. Its general
leaf structure is typical of broad-leaved trees and shrubs.

45
1. Make a cross-section of the Photinia leaf (do both a green and a red leaf) by placing it on a
glass slide and, while using another slide as a 'ruler', making very thin slices using a sharp
razor blade. Float the sections on water in a watch glass. To be able to see the structure of the
leaf, VERY THIN SECTIONS will have to be made. (If you are not able to make them thin
enough, obtain some sections from someone else.) Once you have succeeded in making good
sections, place two or three of them on a glass slide and make a wet mount. Observe the
cross-section with your compound microscope.
In Photinia, when new leaves are produced in the spring they are reddish-purple in color,
rather than green.
Make a sketch of the cross-section of each leaf below.

Label the diagrams with the following structures/tissues and state their function below (Keeton
& Gould: p. 213):

Cuticle
Upper epidermis
Palisade mesophyll
Spongy mesophyll
Vascular bundle
Lower epidermis
How is the leaf well-adapted to its photosynthetic role?

Is there any difference between the older green leaf and the younger red leaf with respect to:
cuticle thickness?
size and number of chloroplasts?

Where is the red pigment mainly concentrated in the younger leaves? (This water-soluble
pigment is called anthocyanin and is contained within the vacuoles of the cells.)

46
3. As the leaves of this plant age, the walls of certain cells near the vascular bundles become
impregnated with lignin, a material which makes the cell walls thick and rigid. This results in a
stiffened leaf, giving it more structural support. This effect is especially noticeable along the
central vein of the leaf. These lignified cells are called sclerenchyma (sclare-rank-ke-ma).
Make some more cross-sections of both leaf types and stain these sections with
phloroglucinol. This stain is specific to lignin which is only found in xylem cells within the
vascular bundles, and in sclerenchyma. Xylem cells can be identified by their large circular
spaces - these are the passageways through which water moves.
CAUTION: Phloroglucinol is made up in strong acid. Do not touch the stain or your slide.
Also, make sure phloroglucinol does not get on the microscope lenses as it is
very corrosive.
Leave the phloroglucinol on the sections for about two minutes, then use an eye dropper to
wash the stain off the slide. Make a wet mount of the stained sections and observe under the
compound microscope.
Where in your preparation do you find sclerenchyma tissue? Can you give a reason why it
might be situated as it is?
Compare your preparation of the green leaf with the younger leaf that has been stained with
phloroglucinol. Does the younger leaf contain any sclerenchyma?
If you were a herbivore, on which leaf would you prefer to browse? What might the purple
anthocyanin pigment have to do with this?

B. LEAF PIGMENTS

Based on your background reading you should be aware that chloroplasts contain a variety of
different pigments. Chlorophyll a, chlorophyll b, and the carotenoids are the major pigments
associated with photosynthesis. Each pigment is responsible for capturing light of specific
wavelengths and making it available for further photosynthetic reactions.
When we look at a leaf, our eyes can distinguish only a general green color; however, by using
the process of chromatography, we can separate the pigments using filter paper and a solvent. We
will use Coleus leaves since they contain ample quantities of the photosynthetic pigments, as well
as the purple anthocyanin pigment.

1. Cut the Coleus leaf into thin strips which are equal in length to half the width of the filter paper
and approximately 2 mm in width. Place the strip 2.5 cm from one end of the filter paper and
roll the handle of your scissors over the strip, crushing the leaf material and allowing the
pigments to be absorbed by the filter paper (see figure below). Remove and discard the leaf
strip. Repeat this procedure five times on each side of the folded filter paper, using 10 fresh
strips of Coleus leaf, making sure to place each strip directly on top of where the last one was
crushed. Put your name on the top right hand corner of the filter paper and place it in one of the
beakers containing solvent on the side bench. Do not touch the solvent!

47
 At this point you may begin the last exercise of the lab.

2. After 30 minutes remove your chromatogram from the solvent beaker and immediately proceed
to distinguish the different pigments, as the color of the pigments will fade rapidly when
exposed to light. Holding the filter paper up to the light will aid you in discriminating among
the pigments; you may wish to outline the bands of pigment using a pencil. Identify the
pigments by their colors:
Chlorophyll a - blue-green
Chlorophyll b - grass-green
Carotenoids - yellow
Anthocyanins - purple (this pigment does not dissolve in the solvent
and so will not move from its original spot)

What is the order of pigments on the filter paper? What does this indicate about their relative
solubility in this solvent? What are the roles of the pigments in photosynthesis?

48
C. THE ABSORPTION SPECTRA OF A SPINACH CHLOROPLAST SUSPENSION AND A
RED ALGA SUSPENSION

In the photosynthesis lab you will be carrying out an experiment to determine how effective
light of different wavelengths is at enabling a spinach chloroplast suspension to carry out
photosynthesis. Before doing that experiment, we want to formulate an hypothesis concerning its
results. One clue regarding the effectiveness of different wavelengths would be the absorption
spectrum of the suspension. What does an absorption spectrum tell you? In this lab we
will generate the absorption spectrum of a spinach chloroplast suspension and compare it to the
absorption spectrum of a red alga suspension. Before generating the spectra make a rough sketch
below outlining your prediction of the results. The color of the spinach and the alga should help.

1. Zeroing of the Spectrophotometer (work in groups of 6). See guide by the spectrophotometer.
Why is zeroing a type of control?
2. Once you have zeroed the spectrophotometer,determine the absorption spectrum as follows:
a. Fill one spectrophotometer tube with 5 ml of chloroplast suspension and a second one with
5 ml of red alga suspension.
b. Measure the absorbance of the suspensions at each of the wavelengths in Table 1 on the
next page (you MUST zero the spectrophotometer with the blank at each wavelength
BEFORE measuring the suspension - work as a team on this).
Graph your results in class. Do the absorption spectra correspond with your predictions?
What is the major difference between the two spectra? What does this suggest to you about the
pigments contained in the red alga? Might there be a reason for the different absorption spectrum
of the red alga? Where do red alga commonly grow? (Caution - not a hard & fast rule.) What is
the difference (if any) between an absorption spectrum and an action spectrum? Think about this
before next week's lab on Photosynthesis.

SUMMARY

By the end of this exercise you should be able to:

1. Make a cross section of a leaf and identify its basic structural components, relating each to its
function.

2. Make and analyze a chromatogram of the pigments found in a leaf.

3 . Use the spectrophotometer to determine the absorption spectrum of plant or algal species.

49
Table 1. Absorbance of the chloroplast and red alga suspension recorded at wavelengths in the
visible spectrum.

50
 PRE-LAB ASSIGNMENT FOR PHOTOSYNTHESIS EXERCISE

Before corning to class, you must design your experiment, including which controls and
replicates you will use. The following questions will help you do this:
1. What is the specific question this experiment is trying to answer?

2. List the different factors involved in this experiment.

3. The most obvious control in an experiment is the one where the experimental variable has been
removed. What would be the control in this experiment?

4. When you measure absorbance change in your experimental tubes, you assume it is due to the
DCPIP absorbing electrons from the photosynthetic process. How can you be sure that the dye or
the chloroplast suspension do not change in absorbance all by themselves? What tests or further
controls could you carry out to show that the results of your experiment are not due to changes in
the dye or chloroplast suspension?

5. If the latter tests (controls) do show changes in absorbance, how could you take this into
account when reporting your experimental results?

6. What does it mean to oxidize a substance? To reduce a substance?

51
 Photosynthesis

To determine the effect of light quality on the energy-capturing reactions of photosynthesis.

Prelab Preparation
READ the lab outline. You must understand not only the procedures but also the theory behind
the exercise. You can find useful information in the following texts:
Keeton & Gould: pp. 195-210
Curtis 4: pp. 210-224
Complete the pre-lab assignment found at the end of the previous exercise prior to coming to
lab.
New terms to learn from this lab exercise:
Light energy
Pigments
Energy-capturing reactions
Oxidized
Reduced
Light quality
Buffer
Nanometer (nm)

INTRODUCTION

Photosynthesis is the major process by which external energy (derived from the sun) is made
available to the living world. Light energy striking pigments in the chloroplast is transformed
first to electrical energy (excited electrons) and then to chemical energy bonds in the molecules
ATP and NADPH2. Some of these bonds are subsequently broken down and in the process energy
is released which is used to drive the enzymatic reactions which change atmospheric carbon
dioxide, a low energy molecule, into sugars. Although photosynthesis is restricted to chlorophyll-
containing organisms (plants and some protists) and some bacteria, the sugars they produce can be
used by all living organisms, via glycolysis and respiration, to provide chemical energy for living
processes.
The overall photosynthetic reaction is:
6C02 + 12H 20 + light + photosynthetic -------- > C6H1206+ 6O 2 + 6H2O
energy pigments (sugar)

In this exercise you will be dealing only with the energy-capturing reactions. These
reactions occur only in the presence of light. Energy is released as electrons move along electron
transport chains (series of molecules held in membranes) after being 'energized' by photons of

52
 light. You will measure photosynthetic activity by determining the extent of color loss of
the dye DCPIP. This dye intercepts the flow of electrons in the process of
photosynthesis. When it accepts electrons, it becomes reduced and changes color, from blue
(oxidized form) to colorless (reduced form) (see Figure 1). This color change can be
measured with a spectrophotometer. The amount of color lost is proportional to the number of
electrons activated during photosynthesis, which is proportional to photosynthetic activity.
The white light emitted from the sun consists of a range of wavelengths (colors). In this
exercise you will design an experiment to determine the effect of light quality (wavelength)
on the photosynthetic (energy-capturing) activity of a chloroplast suspension isolated from spinach
leaves.

LIGHT

---- CELL MEMBRANE

CYTOPLASM

----- CHLOROPLAST MEMBRANE

STROMA

THYLAKOID electron
MEMBRANE acceptor
decrease in energy-
C

Figure 1. DCPIP interrupts the flow of electrons in Photosystem II and turns from blue to
colorless as it is reduced.

EXERCISES

A. PREPARATION OF SPINACH CHLOROPLASTS

Preparation of the spinach chloroplasts will be done for you before the lab begins. The
chloroplasts are obtained as follows:

53
1. Blend spinach leaves and sorbitol (a pH-buffered sugar solution). Why is a buffer used?
2. Filter mixture through cheese cloth, discard residue. What does the residue contain?
3. Dilute filtrate to appropriate concentration and keep on ice. Why keep it cool?

B. REDUCTION OF DCPIP - a measure of photosynthetic rate.

In the previous lab you determined the absorption spectrum for spinach chloroplasts, so that
you now know which wavelengths of light are absorbed most effectively by the chloroplast
suspension. In this lab we want to determine which wavelengths cause the highest rate of
photosynthesis: an action spectrum. (Would you expect a relationship between the two?) As
explained in the introduction, we will measure rate of photosynthesis by change in color of DCPIP
The spectrophotometer will be used to monitor color change (change in absorbance) of DCPIP.
The peak absorbance of DCPIP occurs at 600 nm (see Figure 2); however, since this
wavelength lies on the border between the red and blue light spectrophotometers, we will record
absorbance by DCPIP at 620 nm using the "red light" spectrophotometer (what would be the
outcome if we set the spectrophotometer at 420nm & use the blue bulb?). This will allow each of
the four groups to do the following experiment.

WAVELENGTH, nanometers

Figure 2. Relative absorbance spectrum of DCPIP in sorbitol solution.

54
 Before doing this section turn off the lights in the lab room and close the blinds. Why are these
procedures necessary?
Procedure
1. To a spectrophotometer tube add 5 ml of chloroplast suspension
2. Then add 2 drops of DCPIP
3. Cover with a piece of parafilm and invert to mix
4. Record the absorbance of the mixture at 620 nm (this is time = 0 reading)
Place this tube in a 250 ml beaker 68 cm from a 150 watt desk lamp. The lamp should be
resting on the bench top. After 5 minutes (exactly) in the light, record the absorbance of the
chloroplast suspension in Table 1 below (remember to zero the machine before measuring the
absorbance).

TABLE 1. Use of the dye DCPIP to measure rate of photosynthesis of a chloroplast

suspension. (Is this a control?)
Time (min) Absorbance

t=0
t=5
Change in absorbance

How do you account for the change in absorbance? Discuss this within your group.
You should now have established that the chloroplast suspension can reduce the dye and that
the amount of reduction can be measured quantitatively. The oxygen produced during
photosynthesis can reoxidize the dye very rapidly after the dye has been reduced (see previous
equation). What precautions should you take when measuring the reduction of the dye?

C. AN EXPERIMENT TO DETERMINE THE EFFECT OF LIGHT QUALITY

(WAVELENGTH) ON THE RATE OF PHOTOSYNTHESIS
Work in the same four groups as before.
Materials

Each group of students is provided with the following lab materials:

150 watt light bulb
meter stick
spectrophotometer
six 250 ml beakers 24 Spec-20 tubes
150 ml diluted chloroplast suspension (kept cold)
DCPIP in dropper bottle
50 ml of .4M sorbitol solution
2 , 5 m graduated cylinders

55
 Constant Intensity (determined
Acetate filters Wavelengths transmitted Distance by a
photometer)
Blue 440 nm 16 cm
Green 520 nm 12 cm
Red 620 nm & longer 38 cm

* At these distances the intensity of light passing through the different filters is constant (is this
a control?)
Note: A blue filter transmits blue light unlike a blue sweater which reflects blue light.

Based upon your absorption spectrum of isolated chloroplasts, formulate an hypothesis

regarding the possible effects of light quality on the energy-capturing reactions of
photosynthesis. Decide which steps your group will take to test this hypothesis.

Once you have decided which experimental tubes you will use you will have to deal with the
following questions:
1. What controls do you need?
2. How will you make sure that there is no change in absorption occurring in the chloroplast
solution or in the dye itself, which are not caused by photosynthesis?
3. If your controls show change in absorbance, how will you take this into account when
reporting your experimental values?
4. Which tubes should be replicated and how many replicates should there be?
Once your group has answered the questions, proceed with the experiment. You should have
time to repeat the entire procedure. If you come up with new ideas, these can be incorporated in
a second run.

Analvsis of the Results of this Experiment

Discuss in your groups what this experiment demonstrated about the following: which portion
of photosynthetic process was observed in this experiment; experimental procedure; action spectra
of different wavelengths of light; the controls used; the purpose of replicates. Then discuss your
results with another group comparing your outcome with theirs. Then each student should
independently write up the results, discussion, and conclusion (see assignment sheet) and hand
this in before leaving the lab.

56
 SUMMARY

By the end of this exercise you should be able to:

1. Describe the relationship between photosynthetic (energy-capturing) activity and light

quality.
2. Use the spectrophotometer to measure the absorbance of a sample.
3 . Design, carry out, and write up an experiment to test the effects of isolated factors on
photosynthetic activity.

57
 IN CLASS ASSIGNMENT
(Photosynthesis Exercise)
(Turn in at end of period)
NAME:
LAB SECTION:

RESULTS: (append tables/figures)

DISCUSSION:

CONCLUSION:

58
 Chapter 5

Introduction to Electron Microscopy

Ellen Rosenberg
and
Michael Weis

Biology Program
University of British Columbia
Vancouver, British Columbia V6T2B1

Ellen Rosenberg has been a permanent lecturer at the University of

British Columbia since 1983. She is in charge of the Cell Biology
labs at the second, third and fourth year levels. Michael Weis, who
is in charge of the Biological Sciences E.M. facility at U.B.C., has
cooperated in this program and generated the figures in this paper.

59
60
 BACKGROUND

This is a lab-tutorial designed to introduce second-year cell biology students to the principles of
image formation in electron microscopy. Students have already completed a unit on light
microscopy that included a theoretical consideration of resolution and the factors governing the
limit of resolution.
In both the light microscopy and the electron microscopy tutorials the emphasis is on the
interpretation of micrographs. The fact that the image seen is a product of the image-forming
system is stressed. Many different light microscope images of the same specimen are considered
... phase contrast, Nomarski interference, fluorescent as well as bright field. In this tutorial, we
will consider scanning electron microscopy as well as transmission electron microscopy. We will
also consider the effects of different specimen preparation techniques ... negative staining, metal
shadowing, etc.
The goal is for the student to look at a micrograph and recognize what technique was used and
what the advantages and limitations of that technique are. This critical ability will be valuable even
in reading their textbook; definitely if they go further into the literature. For the future cell
biologist, this is the beginning of a basic vocabulary of tools that will be available for their
research.
The tutorial begins with the electron microscope section of the Nature of Things program
"Microscope: Making it Big", which gives a historical perspective as well as images of specimen
preparation. An introductory lecture follows which includes a consideration of why electrons are
used, the basic principles of microscope construction, specimen preparation and a comparison of
SEM, TEM and STEM.
The following pages are excerpted from the Biology 200 lab manual and form the core of
material for this introductory talk.

INTRODUCTION
Why the Electron Microscope?

From last Unit it was shown that the resolution of a microscope depends on 2 factors:
wavelength of the illumination source ( λ ) and the numerical aperture of the lens (N.A.):

limit of resolution = 0.61 λ

N.A.
The maximum value of N.A. for light microscope is approx. 1.4; it is obvious, therefore, that
even the short blue light ( λ = 436 nm) of the visible spectrum will yield a resolution of only 190
nm. The electron microscope, however, utilizes electrons for illumination. Electrons have the
characteristics of both particles and waves. The wavelength of an electron beam is about 100,000
times less than that of visible light and hence the resolution of an electron microscope is far
superior to that of the light microscope.

61
 THE TRANSMISSION ELECTRON MICROSCOPE

Construction of the Microscope:

We store an old, non-functioning Hitachi TEM in our lab. It is used as a comparison with a
light microscope. Similarities in the construction of the two microscopes are pointed out starting
with the filament as a source of electrons or light, that is focussed onto the specimen by condenser
lenses. The illumination penetrates the specimen and objective lenses magnify the image.
Projector or ocular lenses produce the image on the fluorescent screen or in the eye. Each of the
components is discussed in turn.
The Illumination Source
The Illumination Source or "Electron Gun": The Electron beam is generated by the electron gun
located at the top portion of the microscope column. The gun consists of a V-shaped tungsten
filament surrounded by a cathode shield with a circular hole in the center, During operation, a high
voltage is applied between the filament (-) and the anode (+), while an electric current is regulated
through the filament causing it to emit electrons. These electrons are attracted by the + anode but
are forced through the hole of the cathode shield. The negative charge around the hole forces
electrons from the filament into a very narrow beam.
The electron beam, as seen below, is accelerated through a potential difference of voltage
between the filament and the anode. The greater the voltage (V), the higher is the speed of the
electrons and the shorter the wavelength ( λ ), as shown in this formula:

SELF-BIASED ELECTRON GUN

RESISTOR

VOLTAGE
SUPPLY

to condenser lenses

62
Wavelength of the Electron Beam
In a microscope using a routine voltage of 50 KV, the wavelength will be approximately
0.0054 nm. (1/100,000 wavelength of visible light). This value when applied to the resolution
formula will yield a resolution limit about .84 nm or 8.4 Angstroms.
The Electron Lenses
The electron beam from the electron gun can be focussed and defocussed by a series of electro-
magnetic lenses. Similar to the light microscope, the "Condenser Lenses" concentrate the beam
onto the specimen. Electrons passing through the specimen will be focussed by the "Objective" &
"Intermediate" lenses to form an intermediate image. The "Projector lens" enlarges this image into
a final image on the fluorescent viewing screen at the bottom of the microscope column.
Each lens is basically a circular electro-magnet. A variable electric current through the lens will
produce a magnetic field of variable strengths which will deflect or bend the electron beam passing
through.
The Vacuum System

It is important to remember that the electron beam must be generated in and traverse through the
microscope column under a high vacuum condition. The presence of air molecules will result in
the collision and scattering of the electrons from their path. In the electron microscope the vacuum
is maintained by a series of highly efficient vacuum pumps.
* THE VACUUM FACTOR: Biological material must be properly fixed and preserved.
Image Formation in the TEM
The basis of image formation in the TEM is the scattering of electrons caused by collisions
between the beam electrons and the atoms of the specimen. The scattering results in a shadow on
the viewing screen or photographic film.
Material with high atomic numbers (large number of electrons around the proton) will cause
more scattering and produce a deep shadow. Such material is termed "electron dense" and has
high image contrast. Biological material has low electron density and is known generally as
"electron transparent". Hence, an inherent low contrast image is formed.
* BIOLOGICAL MATERIAL must, therefore, be STAINED with heavy metal salts. It must
also be SLICED into very thin sections because electrons have very low penetrating
power.

SPECIMEN

HEAVY METAL ATOMS

TISSUE OR EMBEDDING
MEDIA ATOMS

IMAGE

63
 PREPARATION OF BIOLOGICAL MATERIAL FOR THE TEM

As mentioned before, biological tissues must be fixed and well preserved. It is necessary also
to slice them into ultrathin sections which are then stained The following is a conventional
method for the processing of tissues & cells for the TEM. It is known as "The Thin Sectioning
Method".
Thin Sectioning Method
1. Fixation - Material is killed and preserved as life-like as possible with a chemical fixative such
as glutaraldehyde and osmium tetroxide.
2. Dehydration - Water in the tissues is removed by graded alcohol or acetone solutions to allow
the penetration of a supporting medium which is not miscible with tissue water.
3. Embedding - The material is embedded in a supporting medium, usually an epoxy plastic resin
which when polymerized, facilitates thin sectioning.
4. Ultramicrotomy - The block of plastic containing the material is sectioned into very thin slices
of 50-100 Angstroms thick.
5. Staining of the section - Sections thus obtained are first mounted onto copper grids and then
stained in high electron density metal salts such as lead or uranium salts to increase the image
contrast.
6. Viewing and recording of the images - The copper grids containing stained sections are viewed
in the TEM. The images are recorded on photographic films and then reproduced.

THE SCANNING ELECTRON MICROSCOPE

In this section we will discuss the SEM and how it differs from the TEM.

Constructionof the SEM

The upper portion of the SEM (electron gun and condenser lenses) is similar to that of the TEM.
As in the TEM, the SEM condenser lenses focus the electron beam onto a small spot on the
specimen surface.
Image formation in the SEM is different from the TEM in that the SEM image is a result of
secondary electrons emitted from the spot on the specimen where the primary beam from the
condenser strikes the specimen's surface. The event is illustrated.
After the impingement of the primary electrons on the specimens, secondary electrons as well
as other forms of radiation are emitted. But only the secondary electrons will be collected by the
signal detector. In the detector these electrons strike a scintillator and the light produced is
converted to electric signals by a photomultiplier at the far end of the detector. The electric signal
is then amplified and displayed on the cathode ray tube (CRT).
In the SEM the electron beam is rapidly scanned back and forth in an orderly pattern across the
specimen surface. What you see then is a composite of many individual image spots similar to the
image formed on the TV screen. The SEM has a specimen stage that allows the specimen to move
freely so that the surface of the specimen can be viewed from all angles.

64
Some features of the SEM
Magnification is determined by the ratio of the viewing screen/scan area of the specimen.
Magnification range is from 20x to 100,000x. Magnification can be changed by changing the
scanning area.
Resolution of the SEM is equal approximately to the spot size of the primary electron beam; at
present it lies between 4 to 20 nm.
Depth of focus is great when compared with the light microscope. The reason is that the
secondary electron beam emitted is in relation to the angle of the specimen surface or the
topography of the specimen.

Effects of
electron
beam bom-
bardment of
a specimen.

Sample scanning
pattern of
electron beam.

65
 SCANNING TRANSMISSION ELECTRON MICROSCOPY (STEM)

This is a recent technological advance in the field of Electron Microscopy. The beam of
electrons scans the specimen, as it does in scanning electron microscopy. However, it is the
transmitted electrons that are collected and amplified and form an image on a cathode ray tube. The
small spot size of the beam allows different areas of the specimen to be discriminated and analyzed.
A major use of STEM is in X-ray analysis which allows the elemental composition of the specimen
to be mapped.

ACTIVITIES

Following this introductory talk, tours of the EM facility begin. Students visit this facility in
groups of 7-8. Other students are left with activities in the lab.
The most difficult process for second-year students is visualizing a three-dimensional object
from a two-dimensional micrograph. Understanding the plane of section of a TEM micrograph
requires practice. We provide posters as well as fruit and knives and stamp pads. Students are
challenged to produce as many different images as possible by slicing the fruit (apple, pear, etc.) in
different ways, inking the section and printing it.
In order for students to understand the importance of specimen preparation to TEM, a
demonstration is set up. Each step in the process has an actual object accompanying it. Fixation
and dehydration have specimens in vials. Students can handle the epon blocks with embedded
specimens. Trimmed and untrimmed blocks are shown. Microtomes and glass knives are
available for the sectioning demo. Grids are floated on stain. Finally, EM negatives are shown.
The heart of this demonstration tutorial is in booklets of micrographs with accompanying light
microscope slides. The booklets are organized as a review of prokaryotic vs. eukaryotic
organisms, as well as plant vs. animal cells. There are both SEM and TEM micrographs of each
organism, as well as light microscope slides. Organisms include gram negative and gram positive
bacteria, blue-green algae, euglenoids, rat liver, higher plant cells and yeast.
In summary, this is a demonstration tutorial designed for second year cell biology students. It
has four components:
1. Introductory talk
Principles of electron microscopy
TEM, SEM, STEM
Specimen preparation - including TEM specimen preparation
demonstration
2. Light microscope exercise
Students are given folders that contain SEM and TEM views of prokaryotic and eukaryotic
cells. These accompany light microscope slides of the same organisms.
3. Tours of the EM facility
Small groups of students are given demonstrations of SEM, TEM, STEM and image
enhancement.
4. Planes of section-fruit printing
Additional reference material and posters are available.

66
 Chapter 6

Meiosis in Rye and Sordaria

Ramesh Bhambhani
and
Ann M . Schramm

Department of Genetics
G216 Biological Sciences Centre
University of Alberta
Edmonton, Alberta, Canada T6G 2E9

Note: This workshop was not included in this proceedings

volume because it was not submitted.

67
68
 Chapter 7

Modelling Population Structure

R. E. De Wreede

Department of Botany
The University of British Columbia
Vancouver, B.C. V6T 2B1

R. De Wreede is Associate Professor of Botany, specializing in algal

ecology and population studies of marine algae, especially of the
large kelps. He was born in Holland, and moved to the United
States where he did his undergraduate and graduate studies at
Western Michigan University and the University of Hawaii,
respectively. Between undergraduate and graduate studies he spent
two years in the Peace Corps, teaching in Sierra Leone. His
experience includes mariculture research done in the Philippines and
Micronesia, as well as teaching and research at the University of
British Columbia, Vancouver, Canada, where he is currently
employed.

69
70
 "The sciences do not try to explain, they hardly even try to interpret, they mainly make models.
By a model is meant a mathematical construct which, with the addition of certain verbal
interpretations, describes observed phenomena. The justification of such a mathematical construct
is solely and precisely that it is expected to work."
J. Von Neumann. (in Gleick, 1987)

INTRODUCTION

While some biologists may not agree with the first part of the above quotation, many would
agree that modelling such phenomena is a useful way to gain understanding about biological
systems. In this exercise you will use a computer model to learn how fecundity and survival help
to determine the age and stage structure of populations.

WHAT ARE MODELS?

In general terms, a model is any abstraction or simplification of a system, and a system is any
phenomenon having at least two separable components and some interaction between these
components. For example, an ecosystem has various components, such as biotic and abiotic at the
most fundamental level (Hall and Day, eds., 1977). Other definitions of models are:
A. Models are devices for predicting the behavior of a complicated, poorly understood system
from the behavior of parts that are well understood.
B. Models are a formalization of our knowledge about a system.
Each of these definitions include points often alluded to by the critics of models, e.g.,
simplification and undue faith in the model and its predictions. A useful characterization of this
discussion is given by James Gleick, in his book "Chaos":
"The choice is always the same. You can make your model more complex and more faithful
to reality, or you can make it simpler and easier to handle. Only the most naive scientist
believes that the perfect model is the one that perfectly represents reality. Such a model
would have the same drawbacks as a map as large and detailed as the city it represents, a map
depicting every park, every street, every building, every tree, every pothole, every inhabitant,
and every map. Were such a map possible, its specificity would defeat its purpose: to
generalize and abstract...........Whatever their purpose, maps and models must simplify as
much as they mimic the world."

Given the simplification that does occur when models are formulated, the following analogy by
Skellum (1971) is a useful caution to keep in mind:
"The application of mathematics to science bears some analogy to pictorial expression. The pen
sketches out fine lines or the brush on damp paper yields all the gradations of light and shade, but
in all cases much color and detail are lost. Only those who know how to relate what is seen to
what gets drawn, know how to relate what has been drawn to what could have been seen. The
uncertainties are always two-fold."

71
 Perhaps in the context of this exercise models are best seen as an aid to understanding, and as
an aid to testing our understanding of the factors that determine ecological processes. In this
exercise we will construct a model that will help you to understand the components of population
growth and structure and, through analysis of the results, to make an estimate of how well the
system is understood.
There are many types of models, and different individuals classify models in different ways.
Some types of models are Analytic, Dynamic, Matrix, Optimization, Multivariate, Stochastic and
Simulation. In this exercise we will be concerned with a matrix model, which is a deterministic
model in that it always gives the same answer (output), given the same input. This contrasts with a
stochastic model, in which the output will differ with the same input. However, a matrix model
may be made stochastic by generating random terms in place of a specific input. The random terms
may be limited in scope so that they only cover a biologically reasonable range. It should be noted
that many researchers believe that the stochastic model more closely mimics ecological reality, and
some consider the chaotic model (see Gleick, 1987) to be most realistic.

THE MATRIX MODEL IS ONE KIND OF MODEL

What follows is an explanation of what the matrix model is, and how it operates
mathematically. This is followed by an explanation of model construction, using the matrix model
as an example.
A computer program to actually 'run' the model with different inputs and for varying lengths of
time [generations] is available from the author and can be used on the Apple II and IIE, and some
Apple IIGS, computers. However, commercially available programs which allow matrix
manipulation may be equally suitable.
In most general terms the matrix model consists of a transition module and a
current-state-of-affairs module. The transition module will be referred to as a transition matrix,
and the current-state-of-affairs module will be referred to as a column vector, for reasons which
will become apparent shortly. In operation, the model multiplies the column vector by the
transition matrix, resulting in a new column vector which represents the updated state of affairs or,
to put it another way, the state of affairs in the next time interval.
In the example to be used in this exercise, the transition matrix consists of numbers denoting
birth and death rates of a population, usually on an age or stage specific basis, and the column
vector gives the numbers in each age or stage class of the same population. Note: an ape class is a
portion of the population defined by age, and a stage-class is a portion defined by some other
criterion, such as size. When the matrix is "run" the transition matrix is multiplied by the column
vector in a specific manner (outlined below), resulting in a new column vector which contains the
numbers in the next generation.
In this example, a 5x5 matrix (5 columns and five rows) is used which is multiplied by a 1x5
(1 column and 5 rows) matrix. As indicated above, these matrices are called, respectively, the
transition mamx and the column vector. The transition matrix contains the survival and fecundity
values of the population being studied, and the column vector the current age or stage distribution
of the same population at time t. To "run" the model the two matrices are multiplied together; the
product is the age or stage distribution in the next time interval, t+1. The survival and fecundity
values in the transition matrix can be held constant over time, or changed at each new time interval.
For example, one may wish to change survival values over time if there is evidence that survival is
density dependent, and that the population density is changing.
Some definitions of terms used below may be necessary:
Stable age distribution - the same proportions exist in each age class (or stage class) from one
time period to the next time period; the population may be increasing or decreasing when this
condition exists.
Constant age distribution - The same numbers exist in each age class from one time period to
the next time period; the population neither increases nor decreases when this condition exists.

72
L a m b d a (λ) - Denotes the factor by which the population must be multiplied in order to obtain the
next time periods' population.
Finite rate of increase - The rate at which the population increases from one time period to the
next time period; equivalent to (λ).
Intrinsic rate of increase (r) - The rate of population increase over an infinitesimally small
period of time. (r) is also defined as the number of individuals added to the population per
individual per unit of time. When a stable age distribution has been attained, the following
relationship holds between (λ) and (r):
N ( t + l ) = λNt and since N(t+1) also equals erN(t), one can see that (λ) = er

HOW DOES THE MATRIX MODEL WORK?

What follows is an example of how the computer multiplies the transition matrix and the column
vector together:
Transition Matrix Column Vector
A B C D E 1
V O O O O 2
O W O O O 3
O O X O O 4
O O O Y Z 5
A-E = Fecundity values 1-5 designate age or stage classes
V-Z = Survival values
To multiply these two matrices, proceed as follows:
1. Multiply 1xA, 2xB, 3xC, 4xD, and 5xE and sum the products; this sum is the new value for
age (or stage) class 1, at time t+1.
2. Multiply 1xV, 2x0, 3x0, 4x0, and 5x0 and sum the products; this sum is the new value for
the age (or stage) class 2, at time t+1.
3. Continue in this manner until new values have been obtained for each age or stage class. The
sum of the age or stage class values at any time is the size of the population at that time. The
proportion in an age class can be found by dividing the number in each age class by the size
of the population at that time. The finite rate of increase (λ) can be found, once a stable age
distribution has been reached, by dividing population size at time t by the size at time t+1.
The requirement of a stable age distribution is necessary in order for the finite rate of increase
to be applicable for more than the two generations for which it was calculated.
Since Lambda (λ) is the finite rate of increase, the potential harvest that a population can sustain
without decreasing in numbers can be calculated simply as:

H = Harvest = 100 λ - 1
λ

73
 H is the proportion that can be taken from each age class of the entire population. In practice,
more could be taken from an age class with high mortality relative to other age classes, if the
harvest could be done prior to the occurrence of most of the mortality. This is so because by
definition most of the members of this age class will die anyway.

HOW IS A MODEL CONSTRUCTED?

Despite the plethora of models, the process of constructing a model is similar for all, and is as
follows:
1. A conceptual stage
2. A diagrammatic stage
3. A mathematical stage
4. A computer stage
The conceptual stage is one that mentally limits and describes the system. What is preying on
what, which physical factors may be important and which are not, what can be included and
excluded from consideration.
In the diagrammatic stage pencil is put to paper, and the relationships considered in the
conceptual stage are drawn, for example, in block diagrams.
The illustration below is an example of a block diagram for the change in numbers of a
hypothetical seaweed population, over time.

Adu = Adult Juv = Juvenile

In the mathematical stage the numerical relationships between the variables are defined, and this
is followed by the actual construction of the model. These steps are shown below, in the context
of the matrix model.
First, the transition matrix is constructed for a hypothetical species of seaweed:
To determine mortality of the seaweeds, 100 individuals of five age classes were tagged and
monitored every year for three years. Earlier observations had determined that individuals rarely
survived beyond five years. The following data were obtained:

74
 SURVIVORSHIP DATA FECUNDITY DATA1
AGE CLASS TIME PERIOD (Years) (Zygotes per plant)

1 All figures to be multiplied by 1x10 4

Which of these figures are to be used? Year 1, year 2, or should they be averaged? One way
of utilizing the data is to use the figures for year 1, and then see if the model will predict the correct
data for years 2 and 3; the predictions can be checked since the second and third year data are
available.
Why are the survivorship values in the last age class (4-5 years) essentially zero?
Additional work in the seaweed community over subsequent years supplied the following
information on the numbers in the population at times t, t+1, and t+2:
AGE CLASS

Using the previously discussed generalized matrix model as a guide, the figures for the first
year are inserted into the transition mamx and the column vector as follows:
TRANSITION MATRIX COLUMN VECTOR
0 20 75 80 100 500

75
 The results of running the model for three generations are as follows:

HOW IS THE COMPUTER PROGRAM USED?

This is a good time to become familiar with the computer program, by using the above data to
run the model. (The comments below refer to the program available from the author.) Insert the
diskette, start the computer, and follow the instructions on the screen.
1. Insert the data into the model;
2. Practice saving the transition matrix to disk;
3. Try viewing the model when presented with that option;
4. Run the model for three consecutive years and compare your third year data in the column vector
with that of your neighbor or check with the instructor,
5. Run the model for 30 years (using the continuous run option) and again check your data;
6. Explore the option to calculate lambda (λ), and determine whether the population has reached a
stable age distribution.
Questions you might consider are;

1. How can you get rid of the 'pile-up' of organisms in the first age class?
2. What property of the model causes this 'pile-up to occur?
3. Is the 'pile-up' biologically reasonable?
4. What is the biological assumption which, if true, would account for the accumulation of
organisms in that first age class?
5. How accurate is the model in predicting the age structure of the following years population?
6. How close do the predicted numbers have to be to the actual numbers before you consider the
model a success?
7. How would a stage-specific model differ from the above age-specific model?
In attempting to answer the above questions, keep the following assumptions of the matrix
model in mind:
1. Reproduction occurs in an age class before any mortality occurs.
2. Fecundity and mortality are constant over the time period of the model.
3. A relatively simple life history is assumed (gametic or zygotic, as opposed to an alternation of
generations).
All of these assumptions can be ignored with more complex matrix models, but these are not
considered here.

76
 CONSTRUCTING YOUR OWN MODEL

The last step of this exercise is for you to construct a model, based on what you think are
reasonable numbers for fecundity and survivorship, for the organism of your choice. Run it for at
least 40 generations and determine lambda, r, and the potential harvest rate. If there is time, report
results to the class, and include a rational for the numbers you choose for fecundity and
survivorship.
One variant on the above exercise is to start as though your population were colonizing a new
habitat, as in a newly congealed lava flow. Think carefully about the structure of the column
vector! Also, you might wish to test whether your population is more sensitive to changes in
fecundity than survivorship.
LITERATURE CITED
Gleick, J. 1987. CHAOS - Making a new Science. Viking Penguin Inc., New York. Pp. 1-352.
Hall, C.A. and J. W. Day, Jr. 1977. Ecosystem modeling in theory and practice: An introduction
with case histories. John Wiley & Sons, New York. Chapter 1, Pp. 6-36.

ADDITIONAL REFERENCES ON MODELING

Atkinson, J.W. 1985. Models and myths of science: a view of the elephant. Am. Zool. 25:
771-777.
Barnes, D.M. 1986. Lessons from snails and other models. (Research News in Science) Science
231: 1246-1249.
Chapman, A.R.O. 1986. Population and community ecology of seaweeds. In: Advances in
Marine Biology, Baxter, J.H.S. and A.J. Southward (Eds.) 23: 1-161.
Costanza, R. 1987. Simulation modeling on the Macintosh using STELLA. Bioscience 37:
129-132.
Crutchfield, J., J. Farmer, N. Packard and R.S. Shaw. 1986. Chaos. Scientific American
255: 46-57.
De Wreede, R.E. 1986. Demographic characteristics of PTERYGOPHORA CALIFORNICA
(Laminariales, Phaeophyta). Phycologia 25: 11-17.
Ebersole, J.P. 1980. Food density and territory size: an alternative model and a test on the reef
fish EUPOMACENTRUS LEUCOSTICTUS. Am. Nat. 115: 492-509.
Goda, T., M. Naito, S. Ikeda and M. Watanabe (Eds.). 1986. Scope and limit in the application
of ecological models to environmental management. V-VI. (Special Issue). Ecological
Modelling 32:1-241.
Goudey, J.S. 1987. Modeling of the inhibitory effects of metals on phytoplankton growth.
Aquatic Toxicology 10: 265-278.
Hall, C.A.S. and J.W. Day, Jr. 198 . Systems and models: terms and basic principles. In: Hall,
C. and J. Day (Eds.). Ecosystem Modeling in Theory and Practice: An Introduction with Case
Histories. Pp. 6-36.
Herman, P.M.J. and C. Heip. 1986. The predictability of biological populations and
communities: an example from the macrobenthos. Hydrobiologia 142: 281-290.
Jorgensen, S.E. 1986. Fundamentals of ecological modelling. Developments in environmental
modelling 9. Elsevier Publ. Pp. 1-389.
Kolata, G. 1987. Mathematical model predicts AIDS spread. Science 235: 1464-1465.
La Roche, J. and W.G. Harrison. 1987. Compartmental models of nitrogen cycling in tropical
and temperate marine environments. Mar. Ecol., Progr. Ser. 38: 137-149.
Loehle, C. 1987. Applying artificial intelligence techniques to ecological modelling. Ecological
Modelling 38: 191-212.
77
Lough, T.J., J.B. Wilson, A.F. Mark and A.C Evans. 1987. Succession in a New Zealand
alpine succession community: a Markovian model. Vegetatio 71: 129-138.
Matagne, R.F. 1987. Chloroplast gene transmission in CHLAMYDOMONAS REINHARDTII.
A model for its control by the mating-type locus. Current Genetics 12: 251-256.
Meinhardt, H. and M. Klingler. 1987. A model for pattern formation on the shells of molluscs.
J . Theor. Biol. 126: 63-89.
Menge, B.A., J. Lubchenco, S.D. Gaines and L.R. Askenas. 1986. A test of the
Menge-Sutherland model of community organization in a tropical rocky intertidal food web.
Oecologia 71: 75-89.
Mohn, R.K. and R.J. Miller. 1987. A ration-based model of a seaweed-sea urchin community.
Ecol. Model. 37: 249-267.
Moorby, J. 1987. Can models hope to guide change? Annals of Botany 60: Supplement
4:175-188.
Pickett, S.T.A., S.L. Collins and J.J. Armesto. 1987. Models, mechanisms and pathways of
succession. The Botanical Review 53: 335-371.
Pielou, E.C., J.S. Campbell and V.J. Lieffers. 1985. Comparison of the structures of even-aged
aspen stands in three geographic regions. Can. J. Bot. 64: 122-129.
Pirnm, S.L. and J.C. Rice. 1987. The dynamics of multi-species, multi-life-stage models of
aquatic food webs. Theoretical Population Biology 32: 303-325.
Plant, R.E. 1986. A method for computing the elements of the Leslie Matrix. Biometrics 42:
933-940.
Roff, D.A. 1986. Predicting body size with life history models. Bioscience 36: 316-323.
Schaffer, W.M. and M. Kot. 1986. Chaos in ecological systems: the coals that Newcastle forgot.
TREE 1: 58-63.
Schneider, S.M. and H. Fems. 1986. Estimation of stage-specific developmental times and
survivorship from stage frequency data. Res. Pop. Ecol. 28: 267-280.
Shoemaker, C.A. 198 . Mathematical construction of ecological models. In: Hall, C. and J. Day
(Eds.), Ecosystem Modeling in Theory and Practice. John Wiley and Sons. New York. Pp.
76-113.
Spain, J.D. 1982. BASIC microcomputer models in biology. Addison-Wesley Pub. Co. Pp.
1-11.
Straskraba, M. and A. Gnauck. 1985. Freshwater ecosystems - Modelling and simulation.
Developments in Environmental Modelling, 8. Elsevier - Amsterdam. Pp. 1-309.
Usher, M.B. 1972. Developments in the Leslie Matrix Model. In: Jeffers, J.N.R. (Ed.)
Mathematical Models in Ecology. 12th Symp. Brit. Ecol. Soc. (1971). Blackwell Scientific
Publishers, Oxford. Pp. 29-60.
Van Hulst, R. 1987. Invasion models of vegetation dynamics. Vegetatio 69: 123-131.
Westman, W.E. and J.F. O'Leary. 1986. Measures of resilience: the response of coastal sage
scrub to fire. Vegetatio 65: 179-189.
Wetzel, R.L. and H.A. Neckles. 1986. A model of ZOSTERA MARINA L. photosynthesis and
growth: simulated effects of selected physical-chemical and biological interactions. Aquatic
Botany 26:307-324.
Woolhouse, M.E.J. and R. Harmsen. 1987. A transition matrix model of seasonal changes in
mite populations. Ecol. Model. 37: 167-190.

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 Chapter 8

Thermoregulation in Vertebrates
Studied by Telemetry

David W. Osgood

President
Mini-Mitter Co., Inc.
P.O. Box 3386
Sunriver, Oregon 97707

David W. Osgood was born and raised in Oregon, earning a BS in biology from
Portland State University in 1963. He was awarded MA and PhD degrees in
Zoology by Duke University, the latter in 1968. He spent one year at Duke as a
junior faculty member and 14 years as a member of the Zoology Department of
Butler University, leaving as a full professor in 1982. His interest in biotelemetry
began in graduate school when body temperature data from free-ranging snakes
were needed to compare with laboratory results. When these data were published
in 1970, the pressure to build similar equipment for others led to the formation of
Mini-Mitter. By 1982, Mini-Mitter had grown so much that he gave up teaching
and moved the business home to Oregon. He currently oversees the operation of
Mini-Mitter, a task which includes acting as a consultant to literally hundreds of
teachers and researchers worldwide who use the company's products.

79
80
 Reptilian Behavioral Thermoregulation
Studied by Telemetry

INTRODUCTION

Most vertebrates are incapable of maintaining body temperatures much above that of
their surroundings. We speak of these animals as poikilothermic, (variable-temperature) or
ectothermic. The mammals and birds do maintain body temperatures above that of the ambient
air and these are referred to as homeothermic (constant-temperature) or endothermic.
Endothermic animals metabolically generate the heat required to maintain elevated body
temperatures while ectothermic ones must depend on external heat sources to warm themselves to
higher-than-ambient temperatures. Basking in the morning sun and lying on warm asphalt
roadways at night are two of the means employed by different species of reptiles in
achieving behavioral thermoregulation. This exercise will provide you with the opportunity to
investigate the degree to which ectothermic vertebrates (lizards and/or snakes) are able to
maintain a constant body temperature by moving toward or away from a heat source.
Reptiles are found throughout the tropical and temperate portions of the world with the
greatest concentrations in the warmer latitudes. Most species are restricted to habitats with
characteristic patterns of temperature variation. Desert forms are exposed to surface
temperatures as high as 70° C in the daytime while the night temperatures may be near 0º C (even
in summer). Those in wetter environments seldom experience temperatures above 40° C.
The preferred body temperatures of reptiles reflect the thermal conditions of their natural habitat,
and hence can provide clues to the kind of environment in which they are likely to be found. In
this laboratory exercise you will determine the preferred body temperature of several different
species of lizards, and then you should be able to make some predictions about the natural habitat
of each species.

METHODS AND MATERIALS

The lizards will be housed in sand-floored boxes equipped with a light bulb at one end (Fig.
1) providing a photothermal gradient in which the animals will be free to move about, thereby
allowing adjustment of their body temperatures. The body temperature of the lizards is
monitored with a miniature temperature-sensitive radio transmitter (the Mini-Mitter®) that is
fed to the animals at the start of the experiment. Before feeding the Mini-Mitter to the lizard, it
must be calibrated so that it can be used as a radio thermometer.
Transmitter Calibration

The Mini-Mitter produces a low-strength radio signal that can be picked up on any standard
(AM) radio. The effective range varies from receiver to receiver and particularly with the
orientation of the receiving antenna. To tune in the signal, turn on the radio and hold it within a
foot or two of the Mini-Mitter. Adjust the tuning dial to a position between stations and listen for a
clicking sound (the antenna of the radio is quite directional so a stronger signal will be
heard for some orientations of the receiver than for others.) The Mini-Mitter signal is a train of
clicks with the rate of clicking determined by a temperature-sensitive element in its circuit. Since
each Mini-Mitter has its own click rate vs. temperature curve, it must be calibrated before
proceeding with the experiment.

81
 Maximum The

Figure 1. The photothermal gradient box. 1" deep sand on floor.

Place the Mini-Mitter and a thermometer in a 1000 mi beaker of water (about 3/4 full) at about
room temperature. Allow the transmitter to equilibrate with the water for at least two minutes
before taking a reading. Make sure that the water is well stirred during this period so that the
temperature of the transmitter and that of the thermometer bulb are the same. Otherwise, you
will get a faulty calibration and the entire experiment will not work. After the Mini-Mitter has
had time to equilibrate, count the number of clicks in a 60 second interval and record this count on
the chart below. Repeat this procedure at about 5-6º C intervals over the temperature range of
interest (this will vary depending on the particular animals being investigated). After you
have made at least five counts, plot the points on the graph and connect them with a smooth
curve. Connecting adjacent points with straight lines will not reduce accuracy noticeably if they
are within 5 degrees of each other. Remember, however, that your data can be no better than
your calibration curve so make sure that it is as accurate as possible. For very precise
calibration of the transmitter, points should be plotted at 3º C intervals over the desired range of
temperatures. Accuracy may also be improved by timing 100 clicks with a stopwatch rather than
using a wristwatch or wall clock. In that case, data can be converted to clicks/min. or plotted
directly as sec./100 clicks. Either method gives good results. By measuring the click rate and
using your calibration curve you can now determine the temperature of the transmitter (and
hence the deep body temperature of the animal in which you will place it). At this point you are
ready to proceed with the experiment.
Animals
The lizards you will be using have been selected to represent a wide range of preferred body
temperatures. Your instructor will provide you with a specimen and information on its care and
feeding, but not its identify or normal habitat. You will then gently slip the calibrated transmitter
down its throat and into its stomach. After inflicting this indignity on your lizard, allow it to

82
escape into its runway where it will spend the next few days. Do not record any data from the
specimen until several hours after ingestion of the transmitter. The remainder of the exercise will
be done at your convenience during the next several days.

C a l i b r a t i o n Data

Clicks/60 sec. or Sec./100 clicks

DATA TABLE
Computation of Standard D e v i a t i o n

Body Environmental
Time Clicks/ Temperature Temperature
60 Sec. (from curve) Max. Min.

Where: mean)

N = Number o f observations
Σ = sum o f a s e r i e s o f observations
* Same as Body Temperature column
in Data Table

83
 Between now and the next lab period set aside one day to monitor the body temperature of
your lizard. Check its temperature once or twice per hour for the entire day (8 a.m. to 6 p.m.).
At each reading also record the maximum and minimum gradient temperatures as an indication
of the range of temperatures available to the lizard. These data are to be recorded in the table
provided. After completing your readings, plot the results on the graph below. The area
between the maximum and minimum environmental temperatures can be shaded to aid in
visibility.

am 10 am 12 noon
TIME

CALCULATIONS

After completing your observations of body temperature of a lizard, you should prepare the
results for presentation to the rest of the class. This is best done by computing the mean body
temperature and the standard deviation of your observations. The standard deviation table above
is designed to assist in making these computations. A desk calculator that accumulates sums of
squares makes the job even easier, but it is not a necessity.
The lizard's mean body temperature is simply the average of your observations. It is a
measure of the preferred body temperature of your particular lizard. When computed for all
available lizards of that species, the mean body temperature estimates the true preferred body
temperature of the species. A single individual may have a preferred body temperature somewhat
different from that of the majority of animals of the same species, but if observations from
several individuals are averaged ,the resultant mean will be a good estimate of the true population
mean.
Standard Deviation (S.D.) is a statistic that reflects the degree of variability in a set of
observations of a particular characteristic or physiological parameter. Standard deviation
provides a measure of the degree of confidence that one can have in his estimate of the true
population mean. In all collections of data, it is always true that two thirds of the distribution
lies less than 1 standard deviation from the mean (x) and 95% of the distribution lies within 2
standard deviations of the mean. The size of the standard deviation of a group of observations is
influenced by the number of observations, with larger sample sizes yielding correspondingly
smaller standard deviations for a given character. In the present experiment, standard deviations
can be used to determine if two species of lizards have significantly different preferred body

84
temperatures. For example, if species A is found to have a preferred body temperature of 35.4º C
with a standard deviation of 1.2º C and species B has a preferred body temperature of 40.6º C
with a standard deviation of 0.7º C, the two species differ significantly (in the statistical sense) in
body temperature. The intervals enclosed by ± 2 S.D. around their respective mean preferred
body temperatures do not overlap [35.4 + (2 x 1.2) = 37.8; and 40.6 - (2 x 0.7) = 38.81.
Calculations of this type are frequently represented graphically as in the figure below. The
mean of the observations is represented as a short horizontal line with the range of the
observations indicated by a vertical line intersecting it. A hollow box is superimposed over
these lines to indicate the interval ± 2 standard deviations. A separate such diagram is
constructed for each species or population, and the reader can see at a glance which
populations overlap and which are statistically different from each other.

At the next laboratory period we will combine our data with those of other students working
with the same lizard species to compute a mean preferred body temperature for the species
studied. Compare this mean with those observed for the other species of lizards and make
predictions about which species might be desert inhabitants and which would be more likely to be
found in wetter (and hence less thermally variable) habitats.
REFERENCES

Brattstrom, B. H. 1965. Body temperatures of reptiles. Amer. Midland Nat. 73:376-422.

McGinnis, S. M. 1966. Sceloporus occidentalis: preferred body temperature of western fence
lizard. Science 152:1090-1091.
Mackay, R. S . Bio-Medical Telemetry. 2nd ed. John Wiley & Sons, New York. 1970. pp 533.
Osgood, D. W. 1970. Thermoregulation in water snakes studied by telemetry. Copeia
1970:568-571.

85
 TECHNICAL INFORMATION SHEET

Photothermal gradient boxes: Dimensions not critical. Our design utilized plywood and masonite
already available.

Materials
1 - 48" x 8" x 1/2" plywood or board (bottom)
2 - 15-3/8" x 8" x 1/2" plywood or board (ends)
2 - 15-7/8" x 48" x 1/8" masonite (sides) assemble with smooth side in.
1/2" x 1" wood strips (2 - 8-1/2" long, 2 - 49-1/4" long) to form frame for top.
1 - 9" x 50" aluminum window screen (stapled to top frame)
Miscellaneous nails and staples to assemble
Lights: We use gooseneck laboratory lamps with 60 watt bulbs, but any arrangement that
provides sand surface temperatures near 50° C immediately under the bulbs is
satisfactory.

Photoperiod

For best results a regular photoperiod of between 12 and 15 hours of daylight (=heat) should
be maintained for several days prior to and throughout the experiment.

Room temperature
If possible the runways should be set up in a room that will be used for no other purpose.
The temperature should be below 22º C to allow the establishment of an adequate thermal
gradient in the boxes.

Food and water

Water should always be available in the center of the runways. The animals should be fed on a
regular schedule that of necessity will vary from species to species. Consult your animal
supplier for specific feeding instructions.

Animals
Reptiles are sold by a number of suppliers in Florida and the Southwestern states. When
planning a laboratory that requires reptiles, a dealer should be consulted well in advance of
anticipated needs. Most dealers stock relatively few desert forms in the winter months so
orders should be placed in the early fall or the experiment postponed until spring. Lizards are
more readily accepted by students, but snakes will work equally well as subjects for this
experiment. Greater care must be taken to prevent snakes from escaping, but they are able to
swallow the transmitters more easily, making them more desirable in this respect.

86
 Recommended lizard genera are Gerrhonotus, Iguana, Tupinambis, Crotaphytus,
Basiliscus, and Sauromalus; Pituophis and Natrix are good snake genera. In general, the
larger the individual, the easier it will take the transmitter.

Recovery of the transmitter

This can occur spontaneously as some animals will regurgitate it or pass it on through the
digestive tract. Others can be forced to regurgitate it by pressing gently on the abdomen
and forcing the transmitter up into the esophagus. Surgical recovery must be used in many
cases. Most reptiles take ether anesthesia very well and are quite tolerant of surgical
procedures. We recommend that the transmitters not be allowed to remain in the animals more
than two weeks without supplementary coatings to seal out moisture. Polyethylene is not a
perfect moisture barrier and transmitters intended to be left in animals for extended periods
should be dipped in successive layers of Paraffin/elvax. This will provide a good enough seal
to allow the transmitter to function for the 2-3 month life of the battery.

Copyright © 1971 Mini-Mitter Company

87
 The Study of Animal Temperature
Regulation by Telemetry

INTRODUCTION
Animals are generally described as being cold-blooded or warm-blooded. The cold-blooded
animals include all the invertebrates, the fishes, the amphibians, and the reptiles. Only the birds
and mammals are warm-blooded. More correctly, cold-blooded animals should be called
poikilothermic (Greek poikilos, various) and warm-blooded ones homeothermic (Latin homeo,
same). Poikilothermic means that the animal is primarily dependent upon environmental
temperature to determine its own body temperature. Although these animals do produce metabolic
heat, they are largely unable to vary heat production or to control heat loss by physiological
adjustments. Homeotherms on the other hand are able to regulate their body temperature and can
maintain a constant or nearly constant body temperature despite variation in the environmental
temperature. In this exercise you will observe that some poikilothermic animals gain or lose heat
faster than other poikilotherms. You may also see that homeotherms do not maintain a constant
body temperature under all conditions. Try to determine the physical factors that affect the rate of
heat loss or gain.
EQUIPMENT

Each group will be provided with a temperature-sensitive radio transmitter (the

Mini-Mitter®) and an AM radio. (Using the sweep second-hand on a clock or wristwatch to
determine accurate time intervals, you will be able to ascertain the temperature of the transmitter
and hence that of the animal in which it is placed.) The transmitter emits a radio signal that consists
of a series of clicks. The rate of clicking is proportional to the temperature of a thermistor in the
circuit. Since each Mini-Mitter has its own click rate vs. temperature curve, the first step in the
experiment is the calibration of the transmitter.

Copyright 1971, Mini-Mitter Co., Inc.

88
 The temperature signal from the transmitter can be picked up anywhere on the AM radio
broadcast band. However, you will find that certain frequencies will give a clearer signal than will
others. After you have located a frequency with no interference, you are ready to begin the
calibration. This is done by placing the transmitter in a water bath and counting the number of
clicks per minute at several temperatures at least 10º C apart. The calibration curves are nearly
linear and can be plotted as straight lines between any two adjacent points; but to insure accuracy
you should plot at least four points, preferably near 10º C, 20º C, 30º C, and 40° C. The
Mini-Mitters have a time constant (time to reach 99% of a new value) of approximately 2 minutes
so they should be left in the water baths for at least this long before making a reading. Be sure to
stir the water bath adequately so that the temperature is uniform throughout! Plot the points for
your transmitter on the graph. Make sure that your calibration is correct; otherwise the data from
the following experiments will not be valid.

INSTRUMENTING THE ANIMAL

Introduction of the Mini-Mitter into the animal depends upon the kind of animal involved. Rats
must be anesthetized with ether after which the Mini-Mitter is implanted surgically in the body
cavity. Rats recover quickly from this type of operation, and only minimal precautions against
infection are necessary. After the transmitter has been implanted, the animal should be allowed a
couple of days to recover from the anesthetic and surgery. Reptiles and amphibians can be forced
to swallow the transmitters without anesthesia. The alligator's jaws can be held open by coaxing it
to bite on a soft stick and then the transmitter can be inserted back in the throat with a forceps.
Finger pressure from the outside will cause the Mini-Mitter to slide down the esophagus into the
stomach.

GENERAL DIRECTIONS FOR TEMPERATURE EXPERIMENTS

After allowing your animal to remain undisturbed for 15 minutes at room temperature, make a
test count to determine its body temperature. Record ambient (immediate surroundings)
temperature with a laboratory thermometer every time you read body temperature. Be sure the
thermometer bulb is dry when making air temperature measurements.
After measuring body and ambient temperatures at normal room conditions, change to different
temperature conditions and determine how fast body temperature of the animal changes. As an
example, if the animal is at 25º C, place it in a 15º C environment, and follow the body temperature
changes at 2 minute intervals. That is, you should make records at 0,2,4,6 minute (etc.) time
periods. If the rate is slow, change to measurements at greater intervals (i.e., 0,5, 10 minutes).
Each student should do the experiment with one poikilotherm and one homeotherm. Be sure
that you understand the results of experiments involving the other animals used in the experiments.
Temperature Regulation in the Frog

Determine the change in body temperature when a frog is removed from a room-temperature
water bath and placed in a cold water bath. The experiment should continue until equilibrium is
reached. Again, place the frog in the room-temperature bath. Is the rate of warming the same as
the rate of cooling? Next, place the frog in a 40° C (no hotter) water bath. Determine the rate of
change. Graph the changes as a function of time.

89
Temperature Regulation in the Alligator* (or lizard)
This experiment can be done in either air or water. If done in air, make sure the animal is dry
before making any measurements. (Why?)
1. Water: Follow the same procedure as given for the frog. Remember that the alligator, unlike
the frog, is exclusively an air-breather. Don't drown the beast!
2. Air: Dry the alligator and allow the animal to equilibrate with the room temperature. Then
place him in a cold, dry environment to determine the rate of temperature change. A
refrigerator, normally ranging from 3-5º C, is satisfactory. (Don't keep the refrigerator door
open any longer than necessary since the cooling system is not overly efficient and may take
some time to return to its normal temperature.) The air temperature in the refrigerator should
be recorded. After reaching equilibrium at the cold temperature, return the alligator to room
temperature and determine the rate of increase in body temperature. Is the increase linear?
Assuming that the alligator and frog were the same size, would their rates of change be the
same under similar conditions? Why? What factors are involved here?
* South American Caiman
Temperature Regulation in the Snake
Determine the ambient and body temperature.
1. Place the snake in a cold, dry environment to determine the rate of temperature change.
Follow the same procedure as outlined in B-2 above.
2. Place the snake in an incubator or under an infra-red light (in a cloth bag, if necessary) to
determine the rate of increase in body temperature above room temperature. With infra-red
light, only the body surface is warmed - the air temperature does not increase. (Infra-red
sources can generate very high temperatures; don't broil the animal.) What factors are
important in determining how rapidly the body temperature of a snake will change? Why
would an inanimate object of the same shape not change at the same rate as does the snake?
Explain the physical principles involved.
RECORD DATA HERE: POIKILOTHERM USED
Temperature Regulation in Mammals

Here we are measuring the core (deep body) temperature. Will it be the same at room
temperature as at low temperature? At high temperature? The transmitter has been previously
implanted so you need only to make the measurements.
Since the radio signal will not pass through sheet metal, provisions must be made to pick up the
signal from inside the refrigerator or incubator for the corresponding cold or warm environment
experiments. One satisfactory method for doing this is to place a portable radio inside the
refrigerator near the transmitter and use the earphone or a small accessory speaker to carry the
signal to the outside. In an alternate method you can loop an antenna (made from any type of wire)
around the animal cage and run this outside the refrigerator to the antenna rod of the radio (see
Mini-Mitter Technical Data Sheet for further information).
Place the animal in the freezer compartment of the refrigerator. Do NOT put it on a cold, hard
surface (frost-bite). Line the container with a cloth or paper. Follow the temperature change.
How does the rate of change compare with that of the poikilothermic (non-regulating) animals?
Which is faster? Which is greater? What behavioral changes do you notice in the mammal at low
temperature? Do not leave the animal in the freezer for more than 15 minutes. Graph the body
temperatures as a function of time.
Place the mammal in an incubator at 45º C. Determine the rate at which the body temperature
changes. Observe the animal closely to make sure it is not in pain. If it begins to behave
peculiarly, remove it from the high temperature immediately. Do not leave the animal in the
incubator for more than 15 minutes. Does the body temperature increase? Why? What
mechanisms do homeothermic (regulating) animals have to control their body temperatures at both
high and low ambient temperatures?

HOMEOTHERN USED

TIME

91
 APPENDIX 1

EQUIPMENT LIST FOR TELEMETRY LABORATORIES

1. Copies of lab exercises

2. Mini-Mitters with batteries
3. Thermometers
4. 1 or 2 liter beakers
5. 8-12 in. pieces of solid copper wire 18-26 ga. (for holding transmitters in beakers)
6. Magnetic stirrers or stirring motors
7. Stopwatches
8. Portable AM radios (may be supplied by students)
9. Animals, as needed. Frogs are generally available from standard sources. Among reptiles,
snakes are best choices and they are readily available locally in most areas. You may have to
plan ahead to get them for a winter lab. Local kids know where and will usually catch all you
can use for a dollar or two apiece. Rats, hamsters, gerbils, mice are all good subjects, with a
large and a small species allowing comparison of surface/volume considerations.

ANIMAL SUPPLIERS

Mammals Reptiles &Amphibians

Ancare Corp. Boreal Labs, Ltd.
47 Manhasset Ave.. 1820 Mattawa Ave.
P.O. Box 354 Mississisauga, ON L4X 1K6
Manhasset, NY 11030 Canada
(516) 627-9292 (800) 387-9379
in US (800) 828-7777
Buckshire Corp.
2025 Ridge Rd. Kons Sci. Co., Inc.
Perkasie, PA 18944 P.O. Box 3
(215) 257-011 Germantown, WI 53022-0003
(414) 242-3636
Charles River Labs., Inc.
251 Ballardvale ST. Wm A. Lemberger Co., Inc.
Wilmington, MA 01887 P.O. Box 2482
(617) 658-6000 2500 Wausau Ave.
Oshkosh, WI 54903
Taconic Farms, Inc. (414) 231-8410
33 Hover Ave.
Germantown, NY 12526
(518) 537-620

92
 Chapter 9

The Botanical Garden - A Tool to Teach

Systematics, Physiology and a Lot More

Iain E. P. Taylor
and
Gerald B. Straley

Department of Botany and

The Botanical Garden
University of British Columbia
Vancouver, British Columbia
Canada, V6T 1W5.

Iain Taylor graduated from the University of Liverpool (B.Sc Hons.

Botany - 1961, and Ph.D. -1964). He taught high school for 2 1/2
years, held a visiting position at the University of Texas at Austin,
and joined the Botany Department at UBC in 1968. His research
interests are in plant cell wall structure and extension, and in the
physiology of micropropagation in conifers, ornamentals and
endangered species. He is Editor of the Canadian Journal of
Botany, a past President of the Canadian Botanical Association, and
on the Executive of the International Union of Biological Sciences.
He was a founding member of ABLE.
Gerald B. Straley, a native of Virginia, has advanced degrees in
both plant taxonomy and ornamental horticulture. He is currently
Research Scientist and Curator of Collections with the Botanical
Garden of the University of British Columbia, and holds adjunct
appointments in the Departments of Botany and Plant Science. His
primary interests are in taxonomy of horticultural plants, especially
herbaceous perennials, floristics of British Columbia and rare and
endangered species. He is the Pacific Northwest editor for the Flora
of North America project.

93
94
 INTRODUCTION
A botanical garden is a living collection of plants that is a valuable resource for a wide range of
in situ teaching activities. However, the accumulation of plants without some organized biological
themes puts severe restrictions upon the value of the collections to teacher, researcher and the
general public. Botanical gardens throughout the world seem to find it easiest to serve plant
systematists and horticulturalists. Recently, we have explored opportunities for teaching a wide
range of biological sub-disciplines from the resources of the University of British Columbia
(UBC) Botanical Garden.

ASSESSMENT OF THE RESOURCES IN A BOTANICAL GARDEN

The taxonomic content of each botanical garden can be generally divided into local flora, special
collections, ecological groupings, and aesthetic display. The nature of these different components
determine the primary opportunities for teaching. Labelling and pamphlets may be adequate for
students to gain some understanding of the garden, but a teacher, even one from the Garden staff,
must become very familiar with the plants and their significance within the garden theme if the visit
or class activity is to be more than a pleasant afternoon's walk to look at plants. The UBC Garden
has an alpine garden, a native garden, a winter garden, a physic garden, a food garden, an Asian
garden, a separate nursery, and a Japanese garden. The systematic and evolutionary garden
remains to be developed. The total area is 45 hectares.
Each component has a particular function. The Physick, Food, and Japanese Gardens are
special display gardens. The Nursery is the technical resource for the garden. The Alpine, Native,
Winter, and Asian Gardens, with their more ecological emphases, provide opportunities to study
form and function relationships. The Systematic and Evolutionary Garden will provide the living
collections for the teaching of systematics.

TEACHING OPPORTUNITIES
Systematics

A botanical garden can be a living laboratory to study diversity within the plant kingdom. The
native garden, or any natural area, can be used for a 'scavenger hunt', which is a useful tool for
students of any age to learn to recognize plants at various levels of taxonomic hierarchy. This will
help them to gain an understanding of systematics as well as ecology, and to look closely at a
microcosm of the biota that is part of their everyday environment. The UBC Garden, on the wet
Pacific coast, contains a small area for growth of plants from the dry interior of British Columbia.
Students and public alike express surprise at the success of these species in what seem like very
wet conditions. The explanation (fast drainage is the key to gardening success for these plants)
directs the student to consider a physiological explanation for the success of this display and for the
natural ecological preferences of these species.
Presentation of native grass species in root constraining concrete tubs allows convenient
comparison of species that are otherwise easily passed over by non-taxonomists. The maintenance
of native habitat also provides good growing conditions for cultivated or spontaneous algae, fungi,
lichens, bryophytes and pteridophytes. The outdoor presentation of these plants that seem
superficially similar to the lay person seems to have greater impact on beginning students than first
exposure in laboratory study. The first experience acts as a catalyst for more penetrating study in
later lab work.

95
Domesticated plants
The Botanical Garden is a good place to learn of the effects that humans have had on selection
of naturally occurring subspecies, varieties and forms. A vegetable, fruit, food or economic
garden is especially useful for teaching morphology, because the various plant parts that are edible
or useful are things to which students can relate, or with which they may already be familiar. One
variable species, Brassica oleracea, shows how selection and breeding, over a long time, can cause
certain parts of the plant to become exaggerated - enlarged terminal buds in cabbage, lateral buds in
Brussels sprouts, flower bud variations in cauliflower and broccoli etc. The challenge to students
is to find food or useful examples representing as many different plant parts as possible.
The food garden also provides a useful tool to teach both the public and the horticultural trade
about potentially new food plants and to display a variety of growing strategies (raised beds,
espaliered trees etc.). The systematics of plants both for ornamental and other uses usually requires
a substantial commitment to a special collection if enough variety is to be available to make the
study critical enough for advanced students, although a small collection can be focussed if the
material is suited for the study of a particular classification problem. The Physick Garden at UBC
is designed and planted in the form of a 16th century European garden. The labels in such a
collection provide much more information than name, family, and origin. The teaching value of
the label is enhanced if the simple rules of presentation are followed; the label must be clearly
visible, legible from a standing position, brief, informative, and interesting. The information must
be accurate.
The recent emphasis on pesticides and drugs of natural origin has led to renewed interest in the
'herbals'. Plant secondary compounds, particularly the alkaloids, are known to have a wide range
of pharmacological and toxicological activity. Well known plants, such as Digitalis sp., continue
to provide medically useful compounds, but there is increasing interest in ethobotanical uses as
clues in the continuing search for new drugs and pesticides.
A relatively infrequent collection in botanical gardens is a grouping of plants that were used by
indigenous peoples. A major opportunity exists, especially in regions where the indigenous
culture is becoming submerged. In areas of North America, for example, much ethnobotanical
information, which was passed on by word of mouth in pre-colonial times, will be lost when the
present generation of elders and medicine men die.
The identity and chemistry of the active principles is relatively easy for teaching purposes.
Solvent extraction, followed by thin-layer chromatography and detection with spray reagents or
UV light, is well within the grasp of the teenage high school student. So much work remains to be
done in the area of useful plant chemicals, that small research projects abound for high school and
undergraduate students, as well as their teachers, with some aptitude for chemistry.
Environmental and Physiological Adaptations

The ecological groupings within a botanical garden provide excellent opportunities to study
adaptations. The most obvious examples emerge in a desert garden or in an alpine collection. The
particular adaptive structures or morphological forms usually survive transplantation, although the
favorable growing conditions provided by cultivation may enhance growth to sizes that exceed
those found in Nature. Demonstration of physiological adaptations may require collection and
transfer to the laboratory. It is sometimes disappointing to find that resources that are readily
available from the Botanical Garden are overlooked in favour of greenhouse or lab-grown material
that is more convenient but provides no better illustration than plants that students can also study
outdoors. The growth of ecophysiology, and the development of several portable versions of
physiological measuring devices, for example to measure leaf gas exchange, nitrogen fixation, and
photosynthetic carbon fixation, provide new opportunities to study plant physiology using plants
that are growing in the Garden. The introduction of hydroponic technology in nursery practice is
yet another opportunity for the Botanical Garden to contribute to the teaching of plant physiology.

96
Micropropagation and Biotechnology
The increasing activity of botanical gardens in collection and study of endangered species
requires the availability of the tissue culture technology that is referred to as micropropagation.
The plant production industry has embraced the technology, and the need for botanical gardens to
produce plants, even if only for their own use, can be directed to teaching not only for plant
physiologists, but also for rare plant workers and for amateur and professional orchid growers.
Other Uses

The largely unrecognized resource of most botanical gardens is the animal life. The absence of
major disturbance and the regulated pattern of human activities are opportunities for birds and
butterflies. Teaching of taxonomic ornithology and entomology can be extended into behaviour and
reproductive biology. The study of horticultural and pesticide management can also be
superimposed in comparative studies between the garden and comparable spaces outside.

CONCLUSIONS
The content and arrangement of plant collections in botanical gardens are easily applied to the
teaching of plant systematics. This paper points to opportunities that exist to teach other
sub-disciplines in Biology. Access to organized collections may not be a complete substitute for
field work, but it allows detailed study in controlled situations. It may also remove some of the
uncertainties that are inherent in the organization of a field excursion that requires advanced
planning with outside agencies. In urban centres, the Botanical Garden may be the only living
resource that is accessible within the logistics of educational timetables. For the non-botanist
teaching in a botanical garden, there is the added benefit of support from professionals who are
actively working with plants. The rate limiting factor is the creativity of the teacher, no matter what
sub-discipline is under study.

97
 APPENDIX 1

PLANTS AS CHEMICAL SOURCES

The use of plants for chemicals and medicines seems to have begun well before recorded
history. The Physick Garden was also developed as a place of particular value, so much so that
early rulers tried to forbid cultivation of such plants having magical powers. Many of the plant
names reflect their medicinal properties, e.g. Papaver somniferum, Lobelia siphilitica, while others
reflect their country of origin, or their use, e.g. Conioselinum chinense, Matricaria chamomilla.
The Physick Garden at the University of British Columbia is modelled on a typical 16th century
herb garden.
Methods are available for the chemical study of the various active, or allegedly active
constituents, and in recent years these have come into the financial range of class teaching budgets.
In addition, students can be introduced to the simpler methods of phytochemistry because
procedures have become relatively quick, easy and sensitive.
The alkaloids are nitrogen-containing secondary compounds that occur in a wide range of plants.
Many are known to have pharmacological and toxicological activity. Approximately 6000
alkaloids are known. Two key journals on alkaloids are Planta Medica and Lloydia, A key
reference book is Medical Botany by W. H. Lewis and M. P. F. Elvin-Lewis (John Wiley, 1977).
A widely-used, standard reference on alkaloid chemistry and analysis is by F. Santavy in
Thin-layer Chrornatography: a laboratory handbook (edited by E. Stahl; pages 421-471,
Springer-Verlag, 1969).
Collection and chemical preparation
Plant material (2-3 leaves) is extracted with 2 x 25 ml 80% methanol in water and filtered. The
pooled filtrate is basified with 0.1M NH4OH and partitioned into chloroform (the lower layer).
Note: There may be some emulsification. This crude material can be concentrated immediately by
rotary evaporation until just dry, or it can be 'cleaned up', with some losses, by back extraction
into 0.1N HCl, followed by rebasification and return partitioning to fresh chloroform. This
chloroform is then removed by rotary evaporation. The solid material taken up in a very small
amount of chloroform is now ready for thin layer chromatography on silica gel.
Chromatography

The diverse chemistry of alkaloids requires different TLC solvent systems for high resolution
separation. Rf values of alkaloids are very sensitive to solvent composition and to atmospheric
saturation in the chamber. Thus, solvents should be kept to 1-3 components, and should be made
freshly before use. In addition, opening and closing of the chromatography chamber should be
minimized. It is also useful to have known standards as reference compounds Cyclohexane-
chloroform-diethylamine (50:40: 10), chloroform-methanol-ammonium hydroxide (60:10:1), and
chloroform-diethylamine (90:10) are useful first solvents. Detection of alkaloids can be very easy
because most fluoresce under UV light (365 nm). Dragendoff s Reagent is still widely used. This
is a bismuth-iodide-acetatereagent and there are several variations.
A word of warning
Nicotine is a commonly used pesticide in gardens. Remember that it is an alkaloid, or you will
have some very odd results!

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 APPENDIX 2

NOTES ON PLANTS IN THE UNIVERSITY OF BRITISH COLUMBIA

PHYSICK GARDEN
The grouping of plants and the preparation of informative labels or a written guide are teaching
tools in themselves. The following are the texts of labels in the UBC Physick Garden. Any small
scale planting on a campus can be used for display and education. The Fern and Primitive
Angiosperm Garden in the Biology building courtyard at UBC was laid out as a teaching resource
and labels provided only scientific identity. A specific-use garden may benefit from labelling with
more information. The cardinal rule, however, is that label information must be attractive,
informative, but not too extensive. A visitor (even a dedicated student!) has a relatively short
attention span. Remember: If you must put a lot on a label, the reader should be comfortable.
Make the letters legible from a STANDING POSITION.
Achillea millefolium Common Yarrow Asteraceae
Achilles used it to staunch his soldiers' wounds. According to Gerard, it grew in churchyards as
a reproach to the dead "who need never have to come there if they had taken their yarrow broth
faithfully every day while living".
Aconitum napellus Monk's-hood Ranunculaceae
Its roots and leaves are so poisonous that Emperor Trajan forbad its growth, and Anglo-Saxon
archers tipped their deadly arrows with it. However, the "hastie poyson" is used today as a cardiac
and respiratory sedative, and in treatment of rheumatism.
Alchemilla vulgaris Lady's Mantle Rosaceae
"The little magical one". Alchemists in the Middle Ages believed the dew drops caught on its
nine-lobed leaves had strong magical qualities.
Althaea officinalis Marsh Mallow Malvaceae
From the Greek 'althino', meaning 'I cure'. Pliny, Virgil and Diascorides all laud its medicinal
virtues. Roots contain a mucilaginous lubricant once valued as a cough medicine and also applied
to burns and animal bites. Sometimes still used for intestinal disorders and bronchial conditions.
Base of confectionery marshmallows.
Amni visnaga Bisnaga Apiaceae
Long used in folk medicine for asthma and angina pectoris. The infusion of its seeds was used to
treat urinary complaints. The flower stalks were sold as tooth picks in ancient Egypt.

Aquilegia vulgaris Columbine Ranunculaceae

This was Shakespeare's "Herb of Venus". It was used in medieval times to cure sore throat and
swollen glands. It has been a popular medicinal plant in Germany ever since it was mentioned by
St Hildegarde, Abbess of Rupertsberg in her "Physica Sacia", dated 1097 A.D.
Arctostaphylos uva-ursi Kinnikinnick Ericaceae
Its leaves were once used in blood infusions. They also have diuretic and antiseptic effects on the
urinary tract.
Argemone mexicana Prickly Poppy Papaveraceae
This plant is poisonous and narcotic. Its milky juice was used for emetic and purgative reasons. It
was also used as a sedative, a cough medicine and for cutaneous diseases.

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Artemisia absinthium Common Wormwood Asteraceae
Oil obtained from this plant is used as a vermifuge tonic. It is also a cerebral stimulant, dangerous
in large doses, for which reason its use in drinks is now prohibited. Originally used as a base for
the French drink 'Pernod', wormwood was held in high repute in medieval times, against
all forms of infection.
Artemisia dracunculus Tarragon Asteraceae
It is a popular seasoning herb in salads and sauces. Tarragon has a stimulating action on the
digestive and urinary systems, and is used in the treatment of gastric and intestinal parasitic
infestations.
Asperula odorata Sweet Woodruff Ru biaceae
A medieval strewing herb. Gives off a scent of new-mown hay when dried and trodden. Used in
"May Wine" and tea and for scenting linen.

Atropa belladona Deadly Nightshade Solanaceae

Renaissance Italian ladies used this juice to dilate their pupils; hence the name 'belladonna'.
Today, atropine, obtained from the roots, is used in eye operations and examinations.
Balsamita Major Costmary Asteraceae
Early uses included the elimination of worms in children, the killing of head lice and the curing of
digestive disorders. "It is good for them to have eaten hemlock" (Gerard).
Betula pendula Weeping Birch Betulaceae
Fluids in this plant have a stimulating effect on several glands. It has been used in urological teas
for kidney and urinary infections, and rheumatism. "...and in our time also the school masters and
parents do terrify their children with rods made of birch" (Gerard).
Centranthus ruber Red Valerian Valerianaceae
This was once thought to be the same as the biblical Spikenard. It was used as a sedative in
hysteria and nervous disorders.
Chamaemelum nobile Roman Chamomile Asteraceae
Its seeds came to Britain with invading Roman legions. "oile of cammomill is exceeding good
against all manner of ache and paine, bruisings, shrinking of sinewes, hardnesse, and cold
swellings" (Gerard). Its foliage and flowers contain aromatic oils; thus it was used as a "strewing
herb" on medieval floors. As a tea, it aids upset stomachs.
Chenopodium ambrosoides Wormseed Chenopodiaceae
Oil is made from these flowers and fruit,and consists of ascadole. This has vermifugal qualities;
for example, it cures round worms and hook worms.
Chrysanthemum cinerariifolium Asteraceae
Tanacetum cinerariifolium Pyrethrum
Comes from the Caucasus, with bright flowers on straight stems. These may be single or double
and are a source of Pyrethrum powder used as an insecticide. Vast quantities are grown for this
purpose in the Kenya highlands. It is non-toxic to man and animals.
Chrysanthemum leucanthemum Ox-eye daisy Asteraceae
Native to North America. Often called the Field Chamomile. Employed to relieve chronic cough,
asthma, and nervous excitability.

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Chrysanthemum parthenium Feverfew Asteraceae
Tanacetum parthenium
In medieval times this plant was used as a cure for "them that are giddie in the head" and "such as
be melancholike".
Colutea arborescens Bladder-senna Fabaceae
This plant has similar properties to Senna. It cures ring-worm, destroys insects, acts as a febrifuge
and as a laxative.
Convallaria majalis Lily-of-the-Valley Liliaceae
The "May Lily", according to Gerard, restored "speech into those that have the dum palfie". Dried
rhizomes of the plant produce the glucoside convallarin. Present day usage includes the treatment
of speech slowness in patients recovering from strokes.
Coriandrum sativum Coriander Apiaceae
When crushed, the seeds have a pungent odor and taste. They are stimulative and relieve colic. If
used too freely, the seeds become narcotic.
Cynoglossum officinalis Hound's-tongue Boraginaceae
The plant smells of mice. The leaves are narcotic and astringent. It was used by the ancients as an
anti-spasmodic. It is also known as the "Herb of Mercury".
Datura stramonium Thornapple Solanaceae
This was used against epileptic fits and madness. All parts are narcotic, and it was once known as
"Devil's Apple". In the 16th century, a woman struck by lightening was saved by a Dature
preparation "when all hope was passed". It is presently used in the treatment of chronic bronchitis
and insomnia.
Digitalis laevigata Foxglove Scrophulariaceae
Glycosides, forming in the second year leaves, yield digitoxin and digitalin which regulate activity
of the heart.
Digitalis lutea Straw Foxglove Scrophulariaceae
This plant was used for many hundreds of years "to cleanse and purge the body both upwards and
downwards" (Culpepper). Since 1775, this plant has been a source of digitalin used in
cardiac treatments.
Digitalis purpurea Common Foxflove Scrophulariaceae
The common name is derived from the Anglo-Saxon word "Foxesglew", an ancient musical
instrument with hanging bells. This is a source of digitalin used as a cardiac stimulant.
Dipsacus fullonium Fuller's Teasel Dipsacaceae
The leaves of this plant trap water, which was once thought to be a remedy for poor eyesight. The
flower heads are hooked, and used to raise the nap on woolen cloth.
Eryngium maritimum Sea Holly Apiaceae
"Roots preserved in sugar have the property of...nourishing the aged and amending the powers of
nature in the younger" (Gerard).
Galega officinalis Goat's Rue Fabaceae
Early herbalists used it as a footbath for people tired with overwalking, and to cure smallpox. It
can also be used in the place of rennet in making cheeses.

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Gaultheria procumbens Wintergreen Ericaceae
The leaves of the plant yield Oil of Wintergreen, and make a drink called "Mountain Tea".
Hamamelis virginiana Witch-hazel Hamamelidaceae
The bark, twigs and leaves of this plant contain tannic acid, which is used to stop bleeding and
prevent inflamation. Its common name comes from use in witching. Branches are used as
divining rods to locate water in order to dig a well.
Hyoscymus niger Black Henbane Solanaceae
This plant is lethal to poultry; hence its common name. The leaves contain lyiscine, a drug used as
a sedative in child birth. This was used by Dr Crippen to murder his wife.
Hypericum perforatum St John's-wort Hypericaceae
This beneficial herb is also called "Grace of God". It has a long Anglo-Saxon history of protecting
houses and churches. Large bunches were hung over doorways to ward off evil powers.
Hyssopus officinalis Hyssop Lamiaceae
A holy herb of ancient times. "Purge me with hyssop and I shall be clean" (Psalms 51:7).
Persians used it in lotions to help the skin. Flavors liquers.
Ipomoea purpurea Morning Glory Convolvulaceae
This plant is known in Mexico as "badoh negro". It is hallucinogenic when used in large
quantities, and a purgative when used in small doses.
Inula helenium Elecampane Asteraceae
Helen of Troy was said to have been digging this plant when she was abducted by Paris. It has
cough easing properties when taken internally. It can also be used to treat wounds.
Iris spuria Iris Iridaceae
Dried rhizomes of certain Iris species are the source of the violet-scented "orris root". Orris was
included among the rare spices of the Egyptians. Romans and Greeks esteemed it for its medicinal
uses, as well as for its perfume. Orris was used to cure ulcers, induce sleep, and as a sovereign
remedy for a "pimpled or saucie face".
Juniperus communis Common Juniper Cupressaceae
The leaves and fruits are carminative, used as an antiseptic and to stimulate diuretic action. It was
also used in "healing leprosy and strengthening the brain" (Culpepper).
Lavendula vera Lavender Lamiaceae
Lavender oil is an insect repellent. It has been used as a rub for rheumatic complaints, aches and
pains. The dried blossoms are used to enhance teas. They make beautifully scented sachets which
will keep moths from damaging clothes.
Leonurus cardiaca Common Motherwort Lamiaceae
"There is no better herb to take melancholic vapours from the heart, and to strengthen it; it makes
mothers joyfull, and settles the womb, therefore it is called MOTHERWORT" Culpepper). Today,
it is used as a nerve tonic, and after childbirth.
Liatris spicata Blazing Star Asteraceae
A North American native, used by pioneers in treatment of venereal diseases and as a gargle.
Powdered leaves act as an insect repellent.

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Lobelia siphilitica Great Lobelia Lobeliaceae
Linnaeus gave this blue-flowered lobelia its name of 'siphilitica' because Peter Kalm, returning
from the New World in 1747, reported that the Indians used this plant as a cure for syphilis.
Lobelia tupa Devil's Tobacco Lobeliaceae
This plant was used by American and Chilean Indians as tobacco. The leaves contain lobeline,
which has a narcotic effect. It is used for treating bronchitis and spasmodic asthma, and the
resuscitation of newborn babies.
Marrubrium cylleneum Horehound Lamiaceae
This plant was listed by Hippocrates in 500 B.C., and has been used ever since for bronchial and
digestive complaints. It was known in ancient Egypt as "the seed of Horus". It is used as one of
the five bitter herbs for the Feast of Passover.
Matricaria chamomilla German Chamomile Asteraceae
This plant is the source of tisane, made from the leaves and used as a remedy for pain. The plant
possesses the same soothing qualities as Anthemis nobilis - Roman chamomile.
Melissa officinalis Lemon Balm Lamiaceae
This plant is a beloved of bees.
Mentha x gentilis Red mint Lamiaceae
Mentha requieni Creme-de-menthe
Mentha spicata Peppermint
These plants are used as peppermint flavoring in medicines and food, and also in creme-de-
menthe.
Mentha pulegium Pennyroyal Lamiaceae
"... a garland of pennyroyal worn about the head is of great force against swimming in the head"
(Gerard). Used as a medicinal, this plant prevents seasickness, and acts as an insecticide. It is
also used as a peppermint flavoring in medicines and food, and also in creme de menthe.
Mirabilis jalapa Four-o-clock Nyctaginaceae
The flower of this plant opens at four o'clock in the afternoon, and remains open all night. Its
roots contain the drug Jalap, which is used as a purgative medicine.
Monarda menthifolia Bee Balm Lamiaceae
The scented leaves of this plant are made into tea, called Oswego Tea.
Nepata cataria Common Catnip Lamiaceae
This aromatic plant has a curious fascination for cats. "It is much commended of some, if the juice
there-of be drunke with wine, to help those that are bruised by some fall, or some other accident"
(Parkinson).
Papaver somniferum Opium Poppy Papaveraceae
This plant was mentioned on Sumerian clay labels of 3500 B.C. It induces sleep and turbulent
dreams. The family name 'Papaver' is thought to come from the Celtic word 'pap', or porridge,
and refers to the custom of mixing poppy juice with gruel to put crying babies to sleep.
Plantago rubrifolia Plantain Plantaginaceae
In ancient days, this plant was used to relieve headaches, and to heal wounds. Alexander the Great
used this to cure his raging headaches. Today, it is used to relieve insect bites. One derivative,
annodine, soothes earaches and toothaches; the seeds are used for hemorrhoids and dysentery.

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Prunella vulgaris Common Self-heal Lamiaceae
Tea made from the leaves of this plant was used as cure-all for quinsy and stomach cramps.
Pulmonaria officinalis Blue Lungwort Boraginaceae
According to the Doctrine of Signatures, this plant was thought to be a cure for chest ailments,
because the leaves resemble lungs. In herbal medicine today, it is used to treat inflammation of the
bronchial tubes.
Rhamnus purshianus Cascara Rhamnaceae
The value of this tree lies in its bark, which yields cascara sagrada, a powerful purgative. It was
so named by missionaries. The translation is 'sacred bush'.
Rosa gallica Apothecary's Rose Rosaceae
The Romans believed the rose provided a cure against drunkenness, and floated rose petals in their
wine. A rose-scented pudding was given to sufferers of sore throats. All parts of the rose were
used in ancient remedies. Pliny lists more than thirty cures prepared with roses.
Ruta graveolens Common Rue Rutaceae
Ruta graveolens 'Variegata' Rue
"Herb of Grace" was used by the rich in nosegays to ward off evil airs as they walked in the
streets. Rue oil was used to arrest bleeding and calm intestinal spasms. "Rue maketh chaste: And
ere preserveth sight; Infuseth wit and putteth flies to flight" (Schola Salernitana).
Salix babylonica Weeping willow Salicaceae
Salix triandra French willow
This plant is the source of salicylic acid, the main ingredient of aspirin. It is used in the treatment
of diseases with rheumatic or gouty origin; diarrhea and dysentery.
Salvia officinalis Sage Lamiaceae
"To live for aye eat Sage in May". "Sage is singular good for the head and braine, quickeneth the
memorie and senses and restoreth health to those that hath the palsie" (Gerard)
Salvia sclarea Clary Sage Lamiaceae
In the Middle Ages, this plant was used for eye inflammation, hence the name 'Clear-Eye'. "Many
men when they have got the running of the reins (kidneys)...run to the bush of clary" (Culpepper).
Sambucus nigra European Elder Caprifoliaceae
This plant was often believed to have been inhabited by witches, so it was never used for
firewood, or to make a cradle. Its flowers can be used as an astringent in eye and skin lotions.
Elderberry jam is a good, natural laxative.
Sanguisorba minor Salad Burnet Rosaceae
"The leaves steeped in wine and drunken comfort the heart and make it merry..." (Gerard). This
plant was frequently called 'Toper's Plant'. Its leaves can be used in soups, salads and cool drinks
for a taste of cucumber.
Santolina chamaecyparissus Lavender Cotton Asteraceae
This plant was used in small quantities as a remedy against tapeworms; one which was not to be
given to children.
Saponaria officinalis Soapwort Caryophyllaceae
Medieval Arab physicians prescribed this plant for leprosy and various skin ailments. It was also
used to make a soapy lather to remove greasy spots from clothing, and to cure skin itches.

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Scrophularia nodosa Knotted Figwort Scrophulariaceae
A European native, related to the foxglove, used in folk medicine as an antidiabetic agent, in
dermatology and for reducing tumors.
Stachys byzantina Betony Lamiaceae
Anglo-Saxons considered this an important magical plant. A leaf in the purse gives protection
from witchcraft. Cure-all for violent blood "chilly need, "fear" and "angry snake bites".
Symp hytum asperum Rough Comfrey Boraginaceae
This plant was used as a tonic, and to suppress bleeding. It was also thought to hasten the healing
process.
Symphytum officinale Comfrey Boraginaceae
It has many names, including Knitbone and Consoleda, which indicate its usefulness in easing
sprains and helping to heal broken bones. Used as poultices or in a tisane. Comfrey is a
nutritional herb, rich in calcium, potassium, phosphorus, vitamins and trace elements.
Tanacetum vulgare Common Tansy Asteraceae
The leaves and flowers of this plant contain Tanacelum, which is used as a cure for intestinal
worms, and to prevent miscarriages. Charlemagne grew it in his garden in the 8th Century. Worn
in the shoe, this plant prevents ague.
Taxus baccata English Yew Taxaceae
This plant is very poisonous - not even safe to sleep under. It was used medicinally for disorders
of the spleen. It is mentioned by the third witch in Shakespeare's 'Hamlet'.
Vaccinium myrtillus Low Bilberry Ericaceae
The berries of this plant were listed by Abbess Hildegarde of Rupertsberg, in the 12th Century, as
being used as a medicinal drug. The leaves are hypoglycemic - they reduce blood sugar levels.
The fruits can be used as a decoction for diarrhea.
Verbascum densiflorum Mullein Scrophulariaceae
A decoction of these leaves is used to alleviate diarrhea. The Romans dipped the tall, flowering
stems in tallow and used them as torches.
Verbena officinalis Vervain Verbenaceae
In medieval times, this plant had a reputation as a potent healing aid. Its flowers were used to
restore failing vision.
Vinca major Periwinkle Apocynaceae
Astringent properties of these leaves have been recognized and used medicinally since medieval
times. This plant is used today in the treatment of leukemia.
Viola tricolor Heartsease Violaceae
This plant is a beloved of herbalists through the ages. According to the Doctrine of Signatures, its
heart-shaped leaves were thought to cure heart disease. It was once used as an ointment and
cathartic.

105
 APPENDIX 3
ENVIRONMENTAL AND PHYSIOLOGICAL ADAPTATIONS IN
APLINE PLANTS
Any ecologically organized section of a plant collection can be used to study adaptation. In this
case the UBC Alpine Garden collection, which is arranged by continental grouping, provides the
example. Students, working in groups of 4, are directed to select ten 'alpine' plants and their
adaptations. They should each record the name, family, origin and particulars of each adaptation.
When the task is completed, they should 'show and tell' their plants to another group. A short
competition to identify the greatest number of structural, environmental and physiological
adaptations in a group of plants within a fixed time may be a useful teaching tool.
The species chosen will depend on the plants that are showing the adaptations most effectively
at the time. This is the major constraint of using live collections either for environmental or
physiological study. Key features for alpine adaptations include:
low habit
adaptations for xeric conditions
compressed flowering time
relatively greater resource distribution towards
vegetative propagation.
perennial life form.
narrow leaves that reduce wind force.
thorns.
wax reflective properties.
easily rehydratable.
bright flowers
die-back
Several others can be identified.
Particular collections of plants allow the teaching of particular adaptive strategies without the
risks involved in a planned field mp. The Botanical Garden or any small local plant collection is
usually close at hand and can be used as the plants provide opportunity. Small display troughs and
greenhouse groupings can be used effectively to study adaptations.
NOTE: When teaching about any particular environment in a Garden setting, be sure that plants
really are naturally occurring in the environment. For example, in an alpine garden, a
naturally low-growing species may be included to provide some visual harmony. It
may not be an 'alpine'!

106
 Chapter 10

Microbial Ecology of the Oral Cavity

Barbara Dill
and
Meather Merilees

Department of Microbiology
University of British Columbia
Vancouver, British Columbia, Canada V6T 1W5

Note: This workshop was not included in this proceeding:

volume because it was not submitted.

107
108
 Chapter 11

Mapping Genes in C. elegans

Robert C . Johnsen
and
Denise V . Clark

Department of Biological Sciences,

Simon Fraser University,
Burnaby, B.C., Canada, V5A 1S6

Robert C. Johnsen received his B.Sc. in Biology from Simon

Fraser University in 1986. He is currently a Ph.D. student in
genetics at Simon Fraser University. His research interests include
genomic organization and the control of recombination.
Denise V. Clark received her B.Sc. in Biology from the University
of British Columbia in 1983. She is currently a Ph.D. student in
genetics at Simon Fraser University. Her research interests include
the study of developmentally essential genes and their organization
in C. elegans.

109
110
 NEMATODE GENETICS

In the mid 1960's after the breaking of the genetic code using viral and bacterial systems, many
molecular biologists began to turn their minds to an investigation of behaviour and the functioning
of the nervous system. About this time, Sydney Brenner in Cambridge, England began to look for
a suitable organism for genetic studies of this nature. In 1968, Brenner found an organism which
has a relatively simple nervous system (about 300 cells). It has only six pairs of chromosomes and
a short generation time (3 1/2 days at 20°C). It reproduces as a self-fertilizing hermaphrodite,
giving about 300 progeny. It can also be out-crossed to males. It is small (1 mm. long as an
adult) and transparent, thus enabling the morphological aspects of development to be observed
under the microscope using living material. In addition, strains can be maintained frozen in liquid
nitrogen. This organism is the free-living (non-parasitic) nematode Caenorhabditis elegans. By
1974, Brenner published a genetic map of C. elegans with 250 genes identified. Thus, this
hermaphroditic nematode was shown to be a suitable organism for genetic analysis. Since 1974,
several interesting mutations have been described and many lines of research have been followed
using C. elegans. This work has been reviewed by Riddle (1978).
This laboratory exercise will use C. elegans to illustrate how a new mutation in a gene of an
unknown location can be assigned to a specific chromosome. As well, the recombination distance
between the unknown and a known marker, will be calculated from F2 data. Unlike Drosophila,
crossing-over occurs in both the male and female germ lines in C. elegans.

NOTES ON THE BIOLOGY OF C. elegans

C. elegans is normally maintained as a self-fertilizing hermaphrodite, producing both eggs and

sperm. Spermatogenesis is complete before oogenesis. Developing oogonia move linearly along
each arm of a two-armed gonad to become mature oocytes. At maturity they pass through a bag of
sperm (spermatheca). It is here that fertilization takes place. Meiosis is completed after fertilization
and the egg-shell forms. Cleavage begins shortly after fertilization. Since C. elegans has a
transparent cuticle, it is possible to observe the developing eggs inside the mother. At about the
64-cell stage the "egg" is expelled to the outside through a mid-ventral opening, the vulva.
Life cycle

1. At 20°C, the average generation time is 3 1/2 days.

2. About 12-18h after an egg is laid, the shell ruptures and a first stage larva (L1) emerges.
3. The larva then goes through four larval molts L2, L3, L4 and adult. Essentially the stages
differ in appearance only in size. However there are two distinguishing features to notice:
a. Before the gonads are completely developed, the space of the vulva appears as a white
crescent shape ("moon"). This is especially noticeable in the L4 larvae, which are referred
to as "crescent stage." Although the vulva is ventral, the crescent appears to be on the side
of the worm (since the worm crawls on its side). Identification of this stage is important
for genetics, as it is a certain sign that no mating with a male has taken place yet
(virginity).

b. In the adult hermaphrodite developing eggs are visible.

4. An adult hermaphrodite will produce about 300 self-fertilized eggs over a period of 3 days at
20ºC.

111
Males
1. In order to perform crosses between different genotypes, males must be used.
2. As adults, males can be distinguished from adult hermaphrodites in that:
a. they are generally thinner (carry no eggs).
b. they have a copulatory tail apparatus (looks like a crochet hook). Larval males and
hermaphrodites are not easily distinguished.
3. During mating, the male sperm are passed to the hermaphrodite via the vulva.
4. Once the male sperm have entered the hermaphrodite, they are used for fertilization
(out-cross) in preference to the hermaphrodite's own sperm (self-cross).
Sex Determination
1. C. elegans has five autosomes (A) and one sex-chromosome (X).
2. Hermaphrodites, 5(AA),XX, produce 5A,X eggs and 5A,X sperm, while males,
5(AA),XO, produce 5A,X sperm and 5A,O sperm.
3. Thus self-cross progeny from hermaphrodites are all hermaphrodites. Consequently,
hermaphrodite strains can be maintained indefinitely (assuming enough food is available)
without mating.
4. Out-cross progeny, from hermaphrodite X male matings will be 50% males and
50% hermaphrodites. To maintain males, matings must be done at every generation.

NOTES ON GENERAL TECHNIQUES:

Culture Medium
1. C . elegans is grown on petri plates which have an agar base (nematode growth medium or
NGM). The agar is streaked with a strain of E. coli which does not grow too thickly
(OP50). The nematodes feed on the E. coli.
2. Occasionally, contaminating bacteria also grow and make visual observation of the worms
more difficult. Plates should therefore be kept covered when you are not manipulating
worms.
3. To avoid rapid evaporation, store plates upside down.
Handling Nematodes

1. Worms can be manipulated under a dissecting microscope at 12-25 X magnification, lighting

should be from below and should be diffuse.
2. Worms are transferred from one plate to another by lifting up a single one with a sharpened
wooden applicator stick, shifting the second plate into view, focusing on the E. coli and
gently resting the point of the stick on the agar (try to avoid poking holes into the agar).
Within a few seconds the worm will crawl off the stick onto the agar.
3. Be sure to use a FRESHLY sharpened stick whenever a different strain is handled (eggs
and small larvae may be on the used stick).
4. Eggs and larvae may also cling to adult worms. Therefore, when isolating males for a
cross, always transfer from the source plate to an intermediate plate first. After a few
minutes the males will have moved around enough to be free of clinging eggs and larvae,
and can be transferred to the mating plate.

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Maintaining a Hermaphrodite Strain

1. One or two young adult hermaphrodites are placed on a fresh plate.

2. After 3-4 days at about 20°C (7 days at 15ºC), some of the progeny worms are put onto
fresh plates.
3. This procedure is continued about every generation.
Mating Hermaphrodites and Males
1. Place at least 7 males on an intermediate plate.
2. Place about 4 young adult hermaphrodites onto a mating plate.
3. Transfer the isolated males to the mating plate. Keep this at about 20°C for 24h (males
won't mate at 15ºC).
4. 24h later, transfer single mated hermaphrodites to separate plates.
5. About 48h later start looking for progeny (remember, there may be some self-cross progeny).
Labelling of Plates

1. Use a permanent marking pen.

2. Label the bottom plate (in case plates are dropped and tops get mixed up).
3. Use small writing at the edge so that the writing does not obscure the microscope light.

Location of some of the C. elegans dpy genes:

LGI

LGII

dpy-18

dpy-11

LGX

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 NOTES ON THE GENETIC NOMENCLATURE OF C. elegans

1. C. elegans has six chromosomes (linkage groups):

LG's I, II, III, IV and V are the autosomes
LG X is the sex-chromosome.

2. A large number of genes have been identified and mapped. Among the genes that affect
the visible morphology of the worm, there are two major classes: Dpys and Uncs.
3. Mutations in dpy genes produce "dumpy", short fat worms. Mutations in unc genes produce
"uncoordinated worms that cannot move in the elegant sine wave of the wild-type (Wt).
4. 28 Dpy and 110 Unc genes have been mapped. They are named dpy-1, dpy-2, . . . and
unc-1, unc-2, . . . etc.
5. Mutant genotypes are written with small letters (underlined or in italics): e.g. dpy-1.
Mutant phenotypes are written with the first letter capitalized: e.g. Dpy.

GENETICS LABORATORY EXERCISE

Purpose: To map an unknown recessive Unc mutation of C. elegans (unc-?) to a particular

linkage group, and to do a two-factor cross to map its distance from a linked Dpy mutation.
General approach (see Experimental Procedures below)

1. Prepare a male carrying a recessive dpy-m marker as a heterozygote (dpy-m/+), by mating

Dpy-m hermaphrodites X Wt males.
2. Po cross: Mate Unc-? hermaphrodites X dpy-m/+ males
3. F1 x F1 cross: Select individual heterozygous F1 hermaphrodites onto separate plates
(using virgins) and allow these to produce self-cross F2 progeny.
4. F2 progeny: Score the phenotypes of the F2's to establish whether dpy-m and unc-?
linked.
5. Two Factor Cross: A two factor cross will be conducted to determine the recombination
distance of ync-? from the linked Dpy.
Theory

1. Diagram the crosses.

2. What F1 progeny do you expect from the Po cross? Genotypes? Phenotypes? Sexes?
Consider the 2 possibilities: either unc-? is on an autosome or unc-? is on the X
c hromosome.
3. From those F1 heterozygous hermaphrodites that received the dpy-m marker, what ratios of
F2 (i.e. Wt : Dpy : Unc : Dpy Unc) do you expect if dpy-m and unc-? are (a) linked and (b)
unlinked. If linked, assume 10% linkage.
Experimental Procedure:
1. Examine and practice handling C. elegans. Adjust microscope lighting (light must come
from below and be diffuse).
a. Compare different ages. A plate with wild-type hermaphrodites will be provided.
Keep also for 2.a. 1) below. Note: eggs, larvae, and adults. Try to identify crescent
stage L4 larvae, and transfer them to a fresh plate for practice.

b. Compare hermaphrodites and males. A plate with wild-type males and hermaphrodites
will be provided. Sort the two sexes onto fresh plates for practice.
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 c. Compare the phenotypes. A plate with a mixed hermaphrodite population will be
provided (i.e. with Dpy, Unc, and Wt worms). Sort the different phenotypes onto fresh
plates and see how they respond to gentle prodding on the head and the tail.
2. Your strains:
a. Each group will receive 3 homozygous hermaphrodite strains to be maintained
throughout the experiment:
1) wt from la. above.
2) unc-? this will be the same strain for all groups.
3) dpy-m this is a marker strain. Each group will receive a different dpy-m.
b. You will need these strains for the crosses below, and as reference phenotypes.
c. For each strain place 2-3 young adult hermaphrodites on a fresh plate. Remember to use
a FRESHLY sharpened stick for each strain. Repeat this every generation.
3. Mapping the unknown unc-?:
a. Prepare the dpy-m/+ males by mating Dpy-m hermaphrodites X Wt males.

1) Place at least 7 wild-type males onto an intermediate plate.

2) Place 4 Dpy-m hermaphrodites (L4 or young adult) onto another plate, the mating
plate.
3) Transfer the Wt males from the intermediate plate to the mating plate. Keep the
mating plate at 20°C (or room temperature).
4) 24h later, transfer the mated hermaphrodites to fresh plates (1 hermaphrodite per plate).
Keep the mating plate as well.
5) As the adult progeny emerge (2-3 days later), set up your Po cross.
b. Po cross:
1) From (3a) above screen the progeny. Self-cross progeny will be Dpy-m
hermaphrodites, while out-cross progeny will be phenotypically Wt males and
hermaphrodites.
2) Isolate at least 7 dpy-m/+ males onto an intermediate plate.
3) Place 4 Unc-? hermaphrodites (L4 to young adult) onto a mating plate.
4) Transfer 7 dpy-m/+ males from the intermediate plate to the mating plate.
5) 24h later, transfer the mated hermaphrodites to fresh plates (1 hermaphrodite/plate).
Keep the mating plate as well.
6) As the F1's mature (about 2 days later) set up your F1 self-cross. Also, inspect the
phenotypes of the F 1males. Is unc-? on LG X?

c. F1 x F1 self-cross:
1) Select 7 phenotypically Wt crescent stage F1 hermaphrodites onto individual plates.
If a F1 hermaphrodite is left too long on any one plate, the F2 progeny will be quite
crowded and asynchronous. This would make the F2 scoring very difficult. To
avoid this problem, the F1 hermaphrodites will be transferred to fresh plates
periodically, i.e sequential broods of F2's will be collected ("Brood A", and "Brood
B" etc.). To keep track of each individual F1 hermaphrodite (remember they do not
all have the same genotype), number these first 10 plates " 1A", "2A" . . . " 10A".
2) After 18-24h, transfer the F1 hermaphrodites to fresh plates (labelled
correspondingly "1B", "2B" etc.) to collect the B brood. Note whether there are
any progeny eggs on the A brood plates. 12h broods are advisable.

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 3) After 18-24h, remove the F1's, or transfer them once more for an 18h C brood if
there were no progeny on the A plates.

4) As soon as the F2's reach adulthood and you can recognize their phenotypes, score
them.
d. NOTE: It is important to brood the worms in order to:

1) have a small number of worms on any one plate

2) have a synchronous population

e. Score the F2 phenotypes.

1) Remove the adults as you score them.

2) As soon as enough F2's have matured so that you can tell which of the F1's carried
the dpy-m marker, continue scoring only from these F1's.

3) Continue scoring a plate until all the F2's have been removed.
4) Aim for scoring about a total of 300 F2's (from dpy-bearing F1 's). The data you
use should come only from plates that have had all their F2's scored. It is therefore
better to score the total progeny from a few F1's, rather than the early, partial
progeny from many F1's.

f. Record the data and have it available for collection and distribution to the class.
4. Two Factor Mapping:
From the compiled data, it should be apparent to which chromosome the Unc mutation
maps. By using the linked dpy-m unc ?, it will be possible to map the unc-? -dpy-m
distance. The procedure involves out-crossing the appropriate Dpy Unc to wild-type males
and picking heterozygotes in the F1 (these will be wild-type).
a. Determine to which chromosome unc-? maps and then obtain 5-6 young adult Dpy Uncs
from the appropriate group (i.e. that which shows linkage).
b. Place 10-12 wild-type males onto an intermediate plate for 5-10 minutes.
c. Place 5-6 dpy-m unc-? hermaphrodites on to a fresh mating plate.

d. Transfer the males from the intermediate plate to the mating plate. Keep the mating
plate at 20°C (or room temperature).

e. After 24 hrs, transfer the mated hermaphrodites to fresh plates (1/plate). Keep the
mating plate as well. You may transfer a few males to each plate if you wish.
f. When the F1's begin to develop, select wild-type L4 (virgin) hermaphrodites and
transfer them (1/plate) onto fresh plates (2 per individual in your group will suffice).
Remember, the presence of many males indicates out-crossing took place.
g. The hermaphrodites must be brooded at 12-18h intervals (five 12h broods is preferable).
h. Score the progeny on the brood plates to completion (usually 2-3 days). You do not
want to score the next generation.
i. Record the data and have it available for collection and distribution to the class.
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 QUESTIONS FOR CLASS
(Include these in your discussion)

1. Why do we not test to a dpy-m on the X-chromosome?

2. If hermaphrodites are true breeding, how do males arise and why are they maintainable?
3. Why do we not use the scoring data from the linkage determination, to determine the distance
between the two markers?

FOR THE TECHNICIAN

Inquiries and stock requests should be directed to Mark Edgley, CGC, Division of Biological
Sciences, 110 Tucker Hall, University of Missouri, Columbia, MO, 65211. We recommend the
dpy's shown on the genetic map for markers and unc-42 for the unknown. You will also need the
wild-type N2 male stock.
Recipe for NGM (for 2L - we use 60mm petri plates):
6g NaCl
34g Agar (Sigma)
5g Bactopeptone
make up to 1.95L with distilled water
Autoclave and then add:
2 ml cholesterol (5 mg/ml in 95% EtOH)
2 ml 1M calcium chloride
2 ml 1M magnesium sulphate
50 ml 1M potassium phosphate pH 6
when set, streak with 1/2 ml liquid culture (10g tryptic soy broth/L) of OP50
(available from the CGC)

MISCELLANEOUS NOTES ON KEEPING WORMS

1. For long term maintenance of stocks, keep hermaphrodite strains at 15ºC with parafilm
to prevent desiccation. A dauer larva form survives for several months. Healthy, femle
worms can be recovered by placing a piece of the old agar on a fresh NGM plate and leaving it
for a few days.
2. Worms can be frozen in liquid nitrogen for indefinite lengths of time.
3. It is important to realize that generally only phenotypically wild-type males will
successfully mate

REFERENCES

Brenner, S. 1974. The genetics of Caenorhabditis elegans. Genetics 77: 71-94.

C. elegans Newsletter and Genetic Maps [available from the Caenorhabditis Genetics Center
(CGC), Division of Biological Sciences, 110 Tucker Hall, University of Missouri, Columbia,
MO, 65211].
Riddle, D. 1978. The genetics of development and behaviour in Caenorhabditis elegans. J.
Nematology 10: 1-16.
Wood, W.B. et. al., eds. 1988. The Nematode Caenorhabditis elegans. Cold Spring Harbor
Laboratory, New York.
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 Chapter 12

Trail-following in Snails:
A Behavior and Statistical Laboratory Exercise

Sandra V . Millen

Department of Zoology
6270 University Blvd.
University of British Columbia
Vancouver, British Columbia V6T 2A9

Sandra Millen is a lecturer in invertebrate zoology at the University

of British Columbia, Vancouver, B.C. Canada. For 15 years she
has been running labs in an introductory second year course and an
advanced third year course. The labs for the third year course have
a variety of themes on which the students run experiments, do the
appropriate statistics on their data, and write up a weekly lab report.
The snail lab is flexible enough to be modified for any year level,
and is one of Sandra's favorites, as her own research is on the
shell-less snails, the nudibranchs.

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 INTRODUCTION

Many species of snails have been shown to have the ability to make use of short distance
chemoreception to detect the trails laid by snails of their own species, and extract chemical
information from these trails. Snails may follow con-specific trails because they are gregarious
(Raftery 1982), wish to return home (Cook & Cook 1975), find a mate (Dinter 1974) or locate a
well-fed individual who may know a good food source (Pratt 1976). Snails rarely follow the trails
of other species unless they are predators and the trail is that of potential prey. However, some
snails do not distinguish between the trails of closely related species and their own species (Trott &
Dimock 1978). This ability of snails to detect and follow trails may be used in a variety of lab
exercises. The objective of the exercises listed here is both to teach the students about snails,
especially their locomotory and chemosensory abilities, and to generate data which can be
statistically tested. The statistical portion of the exercise can teach the need for a large sample size
and the normality of large variance in biological experiments as well as the tests themselves.
MATERIALS
One (or two) snail species that moves well (ie. fast) on a glass surface. Land snails can be used
if the surface is damp. Marine and pond snails need to be covered by water. A good marine snail is
Littorina sp. If starved animals are to be used, withhold food for one week prior to their use.
Aquarium tanks (10 gal.)
tracing paper
masking tape
pencils (2 colors)
calibrated map wheel for measuring the snails trail
Statistical Tests

These can be obtained from biological statistics texts.

Binomial test - for sample sizes under 25
Chi-square test - for sample sizes over 25.
F test - for homogeneity of variances
-
t test - requires homogenous variances
Mann-Whitney U test - for non-homogenous variances
(other non-parametric tests can be substituted)

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 METHODS
Tape tracing paper to the bottom of a clean aquarium tank. If the snail is aquatic, add just
enough water to cover the snail. Balance the aquarium between two stools or chairs, on a lab bench
if possible, so that the students can get under the aquarium to trace the snails movements. Place a
snail (marker) in the center of the aquarium and trace its path as it moves. The trail should be more
than 15 cm long and not go up the sides of the aquarium.
To obtain coincidence distances, start a second snail (tracker) facing onto the marker's trail.
Trace the trackers trail (t) with a second colored pencil for 15 cm after it contacts the markers trail
(m).
coincidence index = distance tracker follows marker x 100 %
15 cm

Examples:
Section A utilizes advanced statistics, section B, simple statistics. The A section can be done and
only the means compared (or means and variances) to simplify the statistical tests.
Section A: To see if snails can detect if the trail was laid by a fed or a starved snail, test fed (m)
with starved (t) vs. starved (m) with starved (t). To see if snails can distinguish sex pheromones in
the trail, test male (m) with male (t) vs. male (m) with female (t) (or vice versa). This test is only
useful if you can sex live animals (and they are not hermaphroditic). Some littorines have a red
penis behind the right tentacle which can be easily spotted. Most land snails, however, are
hermaphroditic. To see if snails can distinguish the trails of related snails, test species 1 (m) with
species 1 (t) vs. species 1 (m) with species 2 (t).
These experiments will give paired sets of coincidence indices. An F test will tell you if the
variances are homogenous or not. If they are use a t-test, if they are not, use a Mann-Whitney U
test, to see if there are statistically significant differences between the two sets of data. Because
variances tend to be rather high, you will need a large number of runs to get enough data to be
significant. Pooling class data is recommended.

Section B: These experiments can be used to quickly generate data which can be tested by much
simpler statistical tests: the binomial and chi-square tests. Use the binomial when the sample size is
under 25, the chi-square when it is larger.
Run a marker snail across a tank as far as you can, tracing the trail and marking its direction.
Start a tracker snail facing the trail and score it as follows:
+= turns the same direction as marker trail
- = turns in opposite direction

o = no response
r = retreat
Remove the tracker snail as soon as it has moved enough to be scored. The one long marker trail
can be used to score a number of tracker snails. Any of the three paired combinations of snails
suggested in Section A can be used as markers and trackers. Once the data has been obtained, first
test to see if trail following occurs ( + and - vs. o and r). If the answer is positive, indicating snails
do follow trails, pose the question, can they sense the direction of the trail? (+ vs -). Students
should work in pairs and each pair can do enough runs to obtain data for statistical testing.

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 REFERENCES CITED

Cook, Susan B. and Clayton B. Cook. 1975. Directionality in the trail-following response of the
pulmonate limpet Siphonaria alternata. Mar. Behav. Physiol. 3: 147- 155.
Dinter, Ingrid 1974. Pheromonal behavior in the marine snail Littorina littorea Linnaeus. The
Veliger 17(1): 37-39.
Pratt, David M. 1976. Intraspecific signalling of hunting success or failure in Urosalpinx cinerea
Say. Journ. Exp. Mar. Biol. Ecol. 21:7-9.
Raftery, Richard E. 1982. Littorina trail following: sexual preference, loss of polarized
information, and trail alterations. The Veliger 25(4): 378-382.
Trott, Thomas J. and Ronald V. Dimock Jr. 1978. Intraspecific trail following by the mud snail
Ilyanassa obsoleta. Mar. Behav. Physiol. 5: 91-101.

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 Chapter 13

Recording Action Potentials

From Cockroach Mechanoreceptors

Tom Linder

Department of Zoology
NJ - 15
University of Washington
Seattle, Washington 98195

Tom Linder received his Ph.D. in comparative physiology from the

University of Washington in 1971. His area of specialization is
cellular neurophysiology. After postdoctoral work, he began
teaching human physiology in 1975 at the University of
Washington, an enjoyable pursuit he continues today.

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126
 INTRODUCTION

Action potentials can be recorded with both intracellular and extracellular electrodes. With
intracellular electrodes the tiny tip of a micropipette pierces the plasma membrane, allowing the
actual electrical potential difference across the membrane to be recorded. At rest, a steady
membrane potential of about -70 mv is recorded. As an action potential passes by the point of the
recording, the membrane depolarizes to about +50 mv and then about one millisecond later returns
to the resting level.
Often, however, a neurophysiologist does not need to know the actual changes in the membrane
potential, but only when an action potential occurs. In this case, an extracellular recording is
usually adequate. Electrodes are placed outside a neuron to record the electrical potential (voltage)
changes occurring in the extracellular fluid. The technique works because ionic current flowing
across membranes during depolarizations simultaneously causes ionic current to flow in the
extracellular fluid, producing electrical potential changes.
The electrical potential changes detected by extracellular electrodes are much smaller than those
recorded with an intracellular electrode. Also, the time course of the potential changes is not the
same, but distorted.
Although analyzing extracellular potential changes in detail is complex, a few simple principles
help understand the recording. Electrical potentials are always recorded between two locations,
since it is the difference in electrical potential that is significant. Usually the electrode connected to
the positive input of the voltage recording device (the "positive electrode") is placed just outside the
neuron. The negative electrode is then placed either outside the same neuron at a distance or else
anywhere in the fluid surrounding the neuron. Often recordings pick up less interference if the
negative electrode is connected to ground. At rest, no electrical potential is recorded -- both
electrodes are in the extracellular fluid. If only the positive electrode is close to the neuron, the
voltage measuring device detects a positive electrical potential as an action potential moves towards
the positive electrode; conversely, an action potential travelling away from the positive electrode
causes a negative electrical potential change. Thus, if an action potential conducts towards and
then past the electrode, the electrical potential will first be positive and then negative. If both the
positive and negative electrodes are near the neuron, the recording is more complex, consisting of a
summation of the potential changes detected at the two electrodes.
Sensory (afferent) axons in the leg of a cockroach offer an excellent opportunity for observing
action potentials and for studying important concepts in sensory physiology. Most of the largest
sensory neurons detect movements of the spines ("bristles") on the leg. The long portion of the leg
closest to the body is termed the femur. The next portion is the tibia.
As Figure 1 shows, each spine on the femur or tibia is suspended on a flexible membrane
within a stiff socket. The sensory innervation is not in the spine itself, but at the flexible
membrane at the base of the spine. The sensory structure is called a campanifom sensillum. It
consists mainly of a tiny dome of cuticle with the dendrite of a single sensory neuron attached to
its inner surface. The dendrite passes through a canal in the wall of the spine to the cell body at the
base of the spine. The axon projects all the way to the central nervous system via a leg nerve that
contains the axons of many sensory receptors, as well as axons of motor neurons innervating the
muscles.

127
Figure 1. Sensory innervation of femur or tibia spine.

When the spine is pushed from its resting position, the dendrite becomes depolarized due to
mechanical forces which are not fully understood. The depolarization produces action potentials at
a frequency reflectingthe degree of depolarization.

PROCEDURE

Cut off the hind leg of an adult cockroach. Cockroaches are easier to handle if cooled in a
refrigerator first. The wound will heal, so place the animal back in the container. Make sure the
two insect pins on the holder are clean. Use fine sandpaper to clean if necessary. Gently impale
the femur on one of the insect pins on the holder and the tibia on the other. Position the holder
under a dissecting microscope so that you can see the leg with its spines.
Connect the cable from the holder to the preamplifier and oscilloscope. Adjust the gain to give a
deflection of the oscilloscopebeam of one centimeter for each 0.2 mv. The preamplifier should be
recording frequencies between roughly 50 and 5000 HZ. The oscilloscope should be recording
AC. Observe the action potentials. What is their amplitude and form?

Make a careful drawing of the leg and the position of the large spines that you can see on the femur
and tibia.

128
Use a small probe to touch various parts of the leg and observe the response. Why doesn't
touching each spine give an action potential of the same height?

What types of movements of a spine give the largest response? Test as many spines on the leg as
you can and mark the direction of their maximal sensitivity with an arrow on the diagram you
drew.

Can you verify that only one sensory receptor appears to be associated with each spine?

Do receptors differ in their response to a steady stimulus? A receptor that continues to respond is
called tonic; a receptor that responds only briefly is called phasic. Indicate any observations of this
type on your diagram.

Test some fine hairs, as opposed to stiff spines. Any response? Tap the holder. What receptors
might produce this response? Blow on the preparation. What receptors are responsible? The
terminal segments on the leg are called the tarsi, including the one at the tip with claws. Test
responses to moving the tarsi.

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 CONSTRUCTION OF THE HOLDER

To make the holder you will need a number 15 cork, two 00 insect pins, some fine sandpaper,
needle-nose pliers, solder and a soldering iron, and a shielded cable suitable for connecting the
holder to your voltage measuring device. Usually this would be a preamplifier, which in turn
would be connected to an oscilloscope. The electronic apparatus used for the traditional frog
sciatic nerve experiment is ideal. However, just about any voltage measuring device capable of
recording brief voltage changes of about 0.1 mv should work. The negative input to the voltage
recording device can be grounded.

Figure 2. Holder.

Sand any paint or varnish off the insect pins. Grasp one pin about one centimeter from its tip
with the needle-nosed pliers and force the pin obliquely into the side of the cork until about 5 mm
protrudes from the top of the cork (see Figure 2). Insert the second pin in the same fashion
approximately one centimeter away. Use the needle-nose pliers to bend the final 2 mm of the tips
of the needles so that they point vertically. Finally, solder the wires in the cable to the pins. One
pin can be ground if your apparatus is of this type. If your cable is relatively stiff, it would be a
good idea to solder several inches of a more flexible cable to the end and attach the more flexible
cable to the pins. The holder will be sturdier if the junction of the pin and the cable is covered by a
thick layer of epoxy glue. Also, a few drops of any type of black wax applied to the top of the
cork will make the spines more visible under the dissecting microscope.

REFERENCES

Chapman, K. M. 1965. Campaniform sensilla on the tactile spins of the legs of the cockroach.
J. Exp. Biol. 42:191-203.
French, A. S. and E. J. Saunders. 1981. The mechanosensory apparatus of the femoral tactile
spine of the cockroach, Periplaneta americana. Cell Tissue Res. 219:53-68.

Note: This experiment was developed in collaboration with John Palka

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 Chapter 14

Plant Hormones: Bioassay for Gibberellin

Sandra Lyn Biroc

Department of Molecular, Cellular and Developmental Biology

University of Colorado
Boulder, CO 80309-0347

Sandra Biroc received her B.A. in Biology from San Fernando

Valley State College, California, in 1970 and her Ph.D. in Cell and
Developmental Biology from The Johns Hopkins University,
Maryland, 1975. She has taught courses at U.C. Davis and Cal.
State Univ. Sacramento. She is presently lab coordinator in the
Molecular, Cellular and Developmental Biology Department at the
University of Colorado, Boulder, where she is in charge of the
upper division lab courses in Cell Biology and Developmental
Biology. In each course there are about 200 students that are
separated into multiple sections of lab. She is deeply committed to
providing quality laboratory instruction to undergraduates. Her
most recent publication is a student manual of lab exercises for
Junior and Senior level developmental biology students.

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132
 INTRODUCTION

The cereal grain seed germinates by first imbibing water then releasing hormones to
begin digesting the protein and starch stored in the endosperm. Gibberellic acid is a
hormone that is released by the embryo, travels to the aleuronelayer and interacts with the
genome of those cells to cause production of alpha- amylase. The enzyme travels to the
endosperm which is broken down to simpler molecules such as sugars and amino acids.
This bioassay uses wheat seeds with the embryo (wheat germ) removed. The hormone is
added at various concentrations and allowed to incubate. After two days, the solution is
assayed for the presence of reducing sugars with the Benedicts test. The color is compared
to known concentrations of glucose to estimate amount of sugar produced. This is a highly
enjoyable lab exercise because the results are clear cut, it is easy to do and the results are
pretty colors. It is a very inexpensive exercise to prepare.

PRINCIPLES

Hormone Action

A hormone is a chemical substance that is produced in one place in an organism, is

released into the body fluids, and has its effect on a target tissue in a place remote from
the cells that produce it (Marx 1984). Hormones are in a sense chemical messengers that
carry information from one type of cell to another. Not all cells are capable of responding
to a given hormone, and not all sensitive cells respond the same way.
Animal Hormones

Animal hormones fall roughly into two categories: peptide hormones and steroid
hormones. A peptide hormone interacts with its target cell by binding to a specific
receptor located on the external cell membrane. The binding of hormone to receptor sets
off a series of events that lead ultimately to the response. Cyclic AMP has been termed
the "second messenger" because it can mimic the effect of some hormones (Sutherland,
1972). Other substances can mimic the effect, such as Ca2+ and polyphosphoinositides
(see Marx, 1984 and Berridge, 1985)) and are also sometimes called "second messengers."
The steroid hormones act by entering a cell (steroids are lipid soluble), binding to a
receptor, either cytoplasmic or nuclear, and interacting with the chromatin to turn on a gene
or set of genes. This phenomenon can be visualized by analyzing the puffing pattern of
chromosomes from Drosophila salivary glands (Ashburner, et al., 1974).
Amplification

Both classes of hormones show amplification of the signal. For instance, for each
molecule of insulin (a peptide hormone), there might be 10 molecules of adenyl cyclase
activated, 100 molecules of cAMP, and 1000 molecules of kinase activated. And for
estrogen (a steroid hormone) there might be a set of 10 genes turned on, which make 100
RNA molecules, which make 1000 protein molecules. This amplification process makes it
possible to elicit a high cellular response from only a few hormone molecules.

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Plant hormones
The plant hormones fall into five main categories: the auxins, the cytokinins, the
gibberellins, growth inhibitors, and ethylene. Only one, gibberellic acid, will be studied.
Auxins are plant hormones produced by shoot meristems which help cause stem
elongation in the cells just beneath the meristem. One example of an auxin is indole acetic
acid (IAA). Cytokinins seem to be made in the roots and travel upward in the plant. The
ratio of cytokinin to auxin determines whether a tissue grown in culture will be root tissue
or shoot tissue; high cytokinin favors shoots, high auxin favors roots. The gibberellins
promote stem elongation in intact plants. Abscisic acid and other growth inhibitors are
responsible for dormancy and the release of fruit and leaves, Ethylene is a gas and is best
known for its role in the ripening of fruit. Some chemical formulas for these plant
hormones are shown in Figure 1.

Ethylene Abscisic acid Indoleacetic acid

(an auxin)

Zeatin (a cytokinin) Gibberellic acid

Figure 1. Some plant hormones.

134
 The traditional bioassay for gibberellic acid makes use of its ability to induce starch
breakdown in the endosperm of a barley seed (Fig. 2) whose embryo has been removed.
In nature, a seed germinates by first imbibing water, which induces the embryo to release
gibberellic acid. The aleurone layer (surrounding the endosperm) responds to the
hormone stimulation by producing specific RNA molecules that code for the
starch-digesting enzyme, alpha-amylase. Embryo-less half barley grains will merely
imbibe water for several days in culture unless supplied with an exogenous source of
gibberellic acid (Coombe et al, 1967). After only two days in the presence of the hormone,
the alpha-amylase produced by the aleurone has digested enough endosperm starch (a large
polymer of glucose residues) to be detected by a simple chemical test. Benedict's reagent
(bluish, copper solution) turns yellow, orange, or red when heated in the presence of the
free aldehyde of reducing sugars (such as glucose monomers):

+ Benedicts reagent ---------> Copper oxide

Cu2+ in alkaline Cu 2 O
citrate complex yellow or red
GLUCOSE GLUCOSE
(closed) (open)

This entire process can be inhibited by Actinomycin D, which blocks RNA synthesis,
suggesting that gibberellin somehow causes specific genes to be turned on (see review by
Galston and Davies, 1970).

Seed coat

Aleurone
Endosperm
Embryo

Figure 2. Longitudinal section of a barley grain.

Dose Response Curve

In any experiment where a drug or hormone is being used, it is important to determine
the optimal dosage to use. A dose response curve is constructed by varying the
concentration of drug or hormone and observing the degree of response the system gives
(Fig. 3).

135
 Biological
response

10-6 10-5 10-4 10-3

Concentration of hormone (moles per liter)

Figure 3. Dose response curve. Varying concentrations of hormone are tested and the
response observed. The data are plotted with concentration on the abscissa and response
on the ordinate.

PROCEDURE

Starch breakdown in embryo-less wheat half-seeds induced by gibberellic acid. The dose
response curve.
Session #1
1. Select 10 wheat seeds and place them on a sheet of dental wax. Using a sharp razor
blade, cut each seed in half so that you have a half containing the embryo and a half that
does not. Discard the half containing the embryo. Slice the remaining half down the
middle and place both "quarters" into a 100 ml beaker.
2. Prepare three more batches of 10 seeds and place them in a second, third and fourth
beaker.
3. Surface sterilize the embryo-less half seeds by treating as follows:
a) Add 10 ml of half-strength Clorox. Swirl, then allow to sit 10 minutes. Aspirate
off the liquid using a sterile pasteur pipette.
b) Add 10 ml sterile 0.01 N HCl. Swirl. Aspirate.
c) Add 10 ml sterile water. Swirl. Aspirate.
d) Add 10 ml sterile water. Swirl. Aspirate.
e) Sterilize a spatula by wiping it with 70% ethanol. Use this spatula to scoop the
seeds into each of four sterile, 50 mm petri plates.
4. Add 10 ml of sterile acetate buffer to each plate.

136
 Prepare two serial 100-fold dilutions of the hormone. Work sterilely.
a) Pipette 1 ml of acetate buffer into each of two sterile test tubes. Label these A
(1:100) and B (1:10,000).
b) Remove 10 µl of hormone from the 1 mM stock solution and add it to the first
tube. Swirl to mix.
c) Remove 10 µl from A and add it to B. Swirl to mix.

6. Add the hormone dilutions to the petri plates.

a) Add 10 µ1 of 1 mM gibberellic solution to one plate for a concentration of 1000
nanomolar.
b) Add 10 µl of "A" dilution to a second plate for a concentration of 10 nanomolar
c) Add 10 µ1 of "B" dilution to a third plate for a concentration of 0.1 nanomolar.
d) Make no additions to the fourth (control) plate.
7. Label four test tubes as:
day 0, 1000 nM
day 0, 10 nM
day 0, 0.1 nM
day 0, control
Remove 1 ml from each plate and pipette into the waiting test tubes. Store these tubes in
the freezer. These are the "zero-time controls."
8. Tape the plates together, four high, label the tape clearly and place these in the box your
TA has set aside for your section. The box will remain at room temperature for two
days and then will be placed in the refrigerator until the section meets again.

Session #2

[The lab instructor will place your plates in the refrigerator after two days of incubation to
slow down any bacterial or fungal growth. Ideally, these should be assayed after two days
of incubation.]
1. Examine the three plates for evidence of softening of the endosperm (starchy) tissue. Jot
down the observations in your notebook.
2. Prepare to test the supernatant liquid for the presence of reducing sugars.
a) Retrieve the zero-time samples from the freezer. Thaw.
b) Remove 1 ml from each petri plate and place the liquid in separate 9 ml test tubes.
Label these with a permanent (not water soluble) marking pen.
3. Prepare a standard curve of glucose
a) Set out 5 test tubes, label these 1,2,3,4 and 5.
b) Add 0.9 ml acetate buffer to tubes 2, 3 , 4 and 5.
c) Add 1 ml stock dextrose to tube #l.
d) Add 0.1 ml stock dextrose to tube #2. Swirl to mix.
e) Add 0.1 ml from tube #2 to tube #3. Swirl to mix.
f ) Repeat step "e" until all five dilutions are made.

137
4. The color reaction
a) Prepare a boiling water bath using a 250 ml beaker filled one third full of water
and a few boiling chips.
b) Add 2 drops of Benedict's solution to all thirteen test tubes. Swirl to mix. Place
the tubes in the water bath for 2-3 minutes.
c) Remove the tubes, swirl, and jot down the observations in your notebook.

Tube # Sample Color Observed

10% dextrose
1% dextrose
0.1 % dextrose
0.01% dextrose
0.001 % dextrose
Control plate, day 0
1000 nanomolar gibberellin, day 0
10 nanomolar gibberellin, day 0
0.1 nanomolar gibberellin, day 0
Control plate, day 2
1000 nanomolar gibberellin, day 2
10 nanomolar gibberellin, day 2
0.1 nanomolar gibberellin, day 2

VOCABULARY

hormone, target tissue, responding cell, peptide hormone, steroid hormone, receptor,
second messenger, amplification of the signal, auxin, cytokinin, gibberellin, aleurone
layer, alpha-amylase, reducing sugar.

QUESTIONS

1. Animal hormones fall into two major classes. What are they? What is the difference in
their mode of action within the cell?
2. Explain what is meant by a "second messenger" and give an example.
3. Hormones occur in very low concentrations and yet can elicit very dramatic cellular
responses. Give an example of signal amplification.
4. Name two plant hormones other than gibberellic acid and describe what each does.
5. In a natural seed, what is the source of gibberellic acid? Why did you have to supply
gibberellic acid to the experimental seeds?

6. Explain what is meant by a "reducing sugar." Chemically, explain why the control plate
did not show any reducing sugars present, but the treated plate did. Where does the
sugar come from?

138
7. Plant hormones fall into five categories (according to some sources).
a) Name three of the five. At least name one major hormone from three of the five
categories.
b) What is the name of the plant hormone that you added to the petri plates of
embryo-less wheat half-seeds?
8. A wheat seed undergoes a series of cellular changes in response to imbibing water. In
the natural situation (not a laboratory experiment) what is the source of the hormone?
What tissue does it act on? What molecule does that tissue make in response to the
hormone? What does that molecule do to the starch?
source of hormone
responding tissue
molecule produced
action on starch

DIRECTIONS TO THE PREPARATOR

Biologicals; Barley, wheat or oat seeds. Purchase from a local seed store.
Equipment: test tubes, sterile 60 mm petri plates, alcohol lamp, spatula, razor blade, dental
wax, pipettes, boiling water bath, heating plates, microliter capillary pipettes (10 µ1) or
automatic pipettor set for 10 µ1 and sterile tips, permanent marking pen.
Solutions: Clorox, 0.01N HCl (autoclaved), acetate buffer (autoclaved), distilled water
(autoclaved), gibberellic acid (1 mM, sterile), Benedict's reagent, 70% alcohol for
sterilizing.
Benedict's reagent;
1. Add 173 gm Na citrate and 100 gm Na2C03 to 800 ml d-H 2 O. Warm and stir to
dissolve.
2. Cool and filter.
3. Add d-H 2 0 to 850 ml.
4. Dissolve 17.3 gm CuSO 4 in 100 ml d-H2O. Add SLOWLY with stirring to 1
liter.
5. Add d-H 2 O to 1 liter.
Gibberellic acid solution: (1 mM)
35 mg GA3 (Sigma) plus 10 ml 95% ethanol. Dissolves completely in 5 minutes.
Add 90 ml d-water. No cloudiness evident. Sterilize by filtration. If refrigerated
promptly, can be stored for years.
Acetate buffer:
M.W. gm/liter molarity
sodium acetate 86 0.172 2 mM
calcium chloride 111 2.2 20 mM
Add salts to 800 ml d-water, adjust pH to 4.2 with 0.1 N HCl; bring volume to 1 liter;
autoclave 20 minutes. cool. store at room temp.

139
 REFERENCES

Alberts B. et al. (1983). Molecular Biology of the Cell. Garland. New York, pp. 510-516.
Altman, J. (1988). Ins and outs of cell signalling. Nature 331:119-120.
Ashburner, M. Chihara, C., Meltzer, P., and G. Richards. (1974). Temporal control of puffing
activity in polytene chromosomes. Cold Spring Harbor Symp. Quant. Biol. 38:655-662.
Berridge, M.J. (1985). The molecular basis of communication within the cell. Sci. Am. Oct.
pp 142-152.
Biroc, S. L. (1986). Developmental Biology; A Laboratory Manual with Readings. Macmillan.
New York.
Conn, E.E. and P.K. Stumpf (1972). Outlines of Biochemistry. John Wiley & Sons. New York.
Coombe, B.G., Cohen, D. and L.G. Paleg (1967). Barley endosperm bioassay for gibberellins.
I. Parameters of the response system. Plant Physiol. 42:105-112.
Curtis, H. (1979). Biology. 3d ed. Worth Publishers. New York. pp, 491-507.
Darnell, Lodish Baltimore (1986). Molecular Cell Biology. Scientific American Books. Freeman.
New York.
Galston, A.W. and Davies, P.J. (1970). Control mechanisms in plant development.
Prentice-Hall, Inc. Englewood Cliffs, New Jersey.
Higgins, T.J.V., Zwar, J.A. and J.V. Jacobsen (1976). Nature 260:166.
Jacobsen, J.V. and L.R. Beach (1985). Control of transcription of a-amylase and rRNA genes in
barley aleurone protoplasts by gibberellin and abscisic acid. Nature 3 16:275-277.
Lang, A. (1957). The effect of gibberellin upon flower formation. Proc. Natl. Acad. Sci.
43:709-7 17.
Marx, J.L. (1984). A new view of receptor action. Science 224:27 1.
Mozer, T.J. (1980). Control of Protein Synthesis-in barley aleurone layers by the plant hormones
gibberellic acid and abscisic acid. Cell 20:479-485.
Wittwer, S.H., Bukovac, M.J., Sell, H.M. and L.E. Weller (1957). Some effects of gibberellin
on flowering and fruit setting. Plant Physiol. 32:39-41.

140
 Chapter 15

The Genetics of Eye Color in

Drosophila melanogaster

Carol Pollock

Biology Program
University of British Columbia
Vancouver, British Columbia V6T 2B1

Carol Pollock is a lecturer in the Biology Laboratory program at the

University of British Columbia.

Note to Instructors:
This exercise has been set up as requiring six weeks. It can be done completely by
students in which case short periods of time are required each week for six weeks.
Alternatively, the crosses could be set up for the students and they could begin the
exercise further along e.g. at Week 3. The two-week chromatography exercise can
be put in anywhere between Week 1 and Week 5. It is assumed that students doing
this exercise will have had some experience with basic Mendelian genetics. If not,
then the exercise can be modified to include basic principles of meiosis, Mendelian
genetics and gene/enzyme relationships.
Schedule:
Week 1: set up parental crosses
Week 2: remove parents
Week 3: chromatography of eye pigments
F1 self cross
Week 4: remove F1 parents
analyze chromatograms
Week 6: analyze F2 data
Week 7: report due

141
142
 INTRODUCTION

The purpose of this exercise is to investigate the inheritance of various eye colors in mutant
strains of Drosophila. We will also use these mutants to demonstrate the relationship between the
information carried by the DNA molecule (genotype) and the characteristics of that organism
(phenotype) as well as to demonstrate the way that gene products interact to produce phenotypic
characteristics.

PIGMENT PRODUCING PATHWAYS

There are two separate biochemical pathways leading to the production of eye pigments in
Drosophila. One produces the brown pigments, ommochromes, and the other produces red
pigments, pterins. See Figure 1. In addition to pigment production, the pigment must also be
bound to a granule in the pigment cell of the eye. Failure of this binding process, for example by a
mutation, results in the lack of pigment in the eye regardless of the pigments produced.
A. Ommochrome pathway (brown pigments):

enz a enz b enz c enz d

tryptophan -----------> Ia -----------> Ib -----------> Ic -----------> xanthommatin

where enz a, enz b, etc. are the enzymes and Ia, Ib, etc.
are the intermediates of the pathway.
pigment
B. Pterin pathway (red pigments): binding
enzyme
DROS

enz 1 enz 4
purine riboside ---------------- > I1 ---------------- > SEP -----------
pigment
in eye
ISOX
other pathways
KEY:
DROS = drosopterin
SEP = sepiapterin
XAN = xanthopterin
ISOX = isoxanthopterin
where enz 1, enz 2, etc. are the enzymes and I1 is an intermediate in the pathway.
Figure 1. Pigment production pathways in Drosophila melanogaster. (simplified)

143
 WEEK 1
Drosophila crosses
Each pair of students will be given a specific cross to follow through three generations: the
parental generation (P), the first generation of offspring (F1) and the second generation of
offspring (F2). For our crosses Drosophila are grown on a cornmeal medium to which dextrose,
agar, yeast extract and a mold inhibitor have been added.
Phenotypes
Examine the flies on demonstration and be sure you can accurately identify each phenotype.
Use the space below for any notes you wish to make on these phenotypes. Each strain differs
from the wild-type in eye color only. Note: there is a "wild" type for all traits; it is the phenotype
that was first observed in wild populations of the organism.
Stocks to be used in crosses:

wild type (+)

scarlet (st)
brown (bw)
white (w)
sepia (se)
Distinguishing the Sex of Adult Flies

In order to set up crosses it is necessary to mate males and females. Therefore it is very
important to be able to distinguish the sexes. Also, some genes are sex-linked and show different
patterns of inheritance in males and females. Therefore, in determining the mode of inheritance of
a genetic trait, it is important to distinguish the sex of the flies and to record the number of flies of
each sex in each phenotype.
Adult males and females can easily be distinguished in a number of ways using the dissecting
microscope (see Figure 2).
1. The abdomen of the female is longer (seven segments) and pointed at the end with several dark
transverse stripes. The male abdomen has only five segments with two narrow dark stripes and a
heavily pigmented tip.
2. The males have a sex comb of about 10 black bristles on the forelegs; females do not have a
sex comb.
3. Males have obvious external genitalia on the ventral surface; females do not.
Crosses
The following crosses are to be set up:
Cross 1A: brown-eyed female x wild-type male
Cross 1B: wild-type female x brown-eyed male
Cross 2A: scarlet-eyed female x wild-type male
Cross 2B: wild-type female x scarlet-eyed male

144
 Cross 3A: brown-eyed female x scarlet-eyed male
Cross 3B: scarlet-eyed female x brown-eyed male

Cross 4A: white-eyed female x wild-type male

Cross 4B: wild-type female x white-eyed male
Cross 5A: sepia-eyed female x wild-type male
Cross 5B: wild-type female x sepia-eyed male
Cross 6A: white-eyed female x sepia-eyed male
Cross 6B: sepia-eyed female x white-eyed male
Note: crosses A and B are reciprocal crosses, i.e. the strains involved in the crosses are the
same but the phenotype of the female and male parents is reversed.

Figure 2. Distinguishing characteristics of male and female Drosophila.

Procedure

1. Etherize your parental flies for 2-3 min. CAUTION: Ether is flammable. Keep away from all
open flames.
2. Place 5 females and 5 males for each cross in a fresh vial.
3. Indicate your names and cross number on each vial.
4. Leave the vials on their side until the flies revive.
5. Your vials will be incubated at 25ºC and returned to you next week.
145
 WEEK 2

Drosophila crosses - Removal of Parents

In your culture vials the adults are the original parents. Note the presence of the other stages of
the life cycle (see Figure 3).
Procedure

1. Etherize your flies - do not worry about over-etherizing.

2. Discard your parental flies into the morgue (a soap solution which will kill the flies).
3. Your vials will be incubated at 25ºC and returned to you next week.

WEEK 3

Chromatography of Pterin Pigments

We will use paper chromatography to separate the pterin pigments which will then be identified
next week on the basis of their fluorescence in ultraviolet light. The ommochrome pigments will
not appear on the chromatograms.
Materials
filter paper, pencil, ruler, glass rod, forceps, beaker with solvent (2:1 n-propanol: 1%
NH4OH)
strains of Drosophila: wild-type (+)
sepia (se)
white (w)
brown (bw)
scarlet (st)
Procedure: Work in pairs.

1. Obtain a piece of filter paper 15 cm x 23 cm and, with a pencil, lightly draw a line 1.5 cm from
the longer edge. This will be the lower edge. Be careful not to get fingerprints on the filter
paper.
2. With a pencil, initial the upper right hand comer.
3 . With a forceps, place one white (w) fly on the pencil line 3 cm from the left edge of the paper
(see Figure 4). Holding the fly in place, use a glass rod to crush the head onto the pencil line.
Discard the body of the fly. Label this spot in pencil below the line - see Figure 4. Repeat
with four more white flies. Make sure all the heads are crushed in exactly the same spot.
4. Wash the glass rod and dry it with a paper towel. Place one brown fly 3 cm from the white
flies. Crush the head. Label this spot in pencil below the line - see Figure 4. Repeat with four
more brown flies. Make sure all the heads are crushed in exactly the same spot.

146
 (look on walls
of vial)

LARVA (1)
-
4 .

-..
. ...

-after 2nd molt in culture medi .. . 2)

. . .
.
-up to 4.5 mm long
1 day -after 1st molt

Figure 3. Life cycle of Drosophila melanogaster.

A.B.

w bw se st +

Figure 4. A chromatogram spotted and labelled.

147
5. Repeat step 4 with sepia (se), scarlet (st) and wild-type (+) flies.
6. Allow the spots to dry.
7 . Roll your filter paper into a cylinder so that the spots are on the inside. Staple the cylinder so
that the edges are neither touching nor overlapping. (Figure 5).

Figure 5: A chromatogram folded and stapled, ready for running in solvent.

8. Place this cylinder in a beaker which has approximately 1 cm of solvent in it (the solvent must
not come higher than the pencil line). Cover the beaker with foil and let sit for 2 hours 20
minutes or until the solvent front is 1 cm from the top of the paper.
9. Remove the cylinder from the beaker and place your chromatogram where indicated by your
T.A. until next week.

Drosophila crosses - F1 self Cross

1. Etherize your flies for 2-3 minutes.

2. Separate into males and females.
3. Note the phenotype of each sex and record in the space below.
F1 females:

F1 males:

Do not discard your flies!

4. Check your results with your T.A.
5. Select five males and five females and place in a fresh vial.
6. Label the vial with your name and the number of your parental cross.

148
7. Leave the vial on its side until the flies revive.
8. Your vials will be incubated at 25ºC and returned to you next week.
9. Discard any leftover F1 flies into the morgue.

WEEK 4

Drosophila crosses - Removal of F1 Parents

1. Etherize your flies in your F1 self vial.
2. Remove all flies from this vial.
3. Discard flies into morgue.
4. Your flies will be returned to you in two weeks.

Analysis of Chromatograms
Introduction

The pathway for the production of the red pigments, the pterins, in Drosophila is outlined in
Figure 1B. Pigments such as drosopterin (DROS), sepiapterin (SEP), xanthopterin (XAN) and
isoxanthopterin, (ISOX) can be identified on the basis of their fluorescence in ultraviolet light.
When ultraviolet light is absorbed by organic compounds, such as pigments, some of the absorbed
energy can be emitted as heat or as light of a longer wavelength, i.e., in the visible range. When
the separated pigments are observed in ultraviolet light, each pigment fluoresces a characteristic
color. This color, as well as its relative position on the chromatogram, can be used to identify the
pigment. None of the brown pigments (ommochromes) will appear on the chromatograrn.
Results

1. Examine your chromatogram in ultraviolet light. CAUTION - Ultraviolet light can be

dangerous. Do not look directly at the source,

2. Compare your results with the sample chromatogram of wild-type pigments on the next page
(Figure 6) and complete Table 1. The pigments may not appear as separate spots but they may
blend into each other. Use the relative distance each pigment has moved from the original
position of the sample to help in your identification.

149
 Solvent Front Color

SEP yellow-green

XAN green-blue

ISOX violet-blue

DROS orange
original position of sample

Figure 6. Sample chromatogram of wild-type Drosophila eye pigments

Table 1. Results of chromatographic separation of Drosophila eye pigments.

KEY:
- = absent
+ = small amount
++ = moderate amount
+++ = large amount

Strain DROS ISOX XAN SEP

+
se
W

bw
st

150
Analysis of results

If a mutation in the DNA occurs such that one enzyme in the biochemical pathway is not
functional, then the intermediate acted on by that enzyme could accumulate. For example, in the
pathway
enz 1 enz 2 enz 3 enz 4
... ----------> I1----------> I2 ----------> I3 ---------- >I4
if enz 3 is not functional, then I2 could accumulate. If there is a branch in the pathway e.g.

enz 3 I3
enz 1 enz 2
------------> I1 ------------> I2

and the non-functional enzyme occurs after the branch, e.g. enz 3, then I2 could accumulate and/or
extra I4 could be produced (the extra I4 doesn't necessarily accumulate - why?).

Compare the pigments present in each of the mutant strains with the pigments found in the wild
type and by the presence or absence or amount of each and try to determine which enzyme in the
pigment pathways (see Figure 1) could be non-functional. Remember the brown (ommochrome)
pigments do not appear on the chromatogram, therefore you must also consider the phenotype i.e.
the actual eye color of each strain. Xanthommatin is the only ommochrome bound to granules,
therefore if a mutant has no brown pigments assume enz d (see Figure 1A) is non-functional.
Remember if one enzyme in the pterin pathway is blocked (excluding the first one), you should
see an increase in at least one of the intermediates or products of the pathway. Also keep in mind
that the pigments must be bound to the granules in the eye for any color to be observed.
Complete the following table:
Strain Defective enzyme Reasons
W

bw
se
st

151
 WEEK 6
Drosophila crosses - Analysis of F2
Results

1. Etherize your flies.

2. Separate into males and females.
3. Determine the phenotypes of each sex and the number of each phenotype. Record your results
here and on the blackboard.

PHENOTYPE(S) . . NUMBER
females

males

4. Using the data tables provided, record the results of all crosses.

Analysis of Results

For each cross 1 - 6 you must use the results provided to:
1. Decide on the mode of inheritance of each phenotype i.e. how many genes are involved in each
cross, how many alleles of each gene and which alleles are dominant, recessive, linked, etc.
You must provide the reasons for your decision. Use Analysis Table 1 for this part of your
assignment.

2. Do a Chi-Square test: χ2 = Σ(obs - exp)2

exp

to confirm your suggested mode of inheritance. Show all your work, including the calculation
of expected values. If sex-linkage is involved you must do separate Chi-Square tests for males
and for females in each of the reciprocal crosses.
3. Relate the results of each cross to the enzyme defective in each of the parents, the F1 and all the
F2 phenotypes observed. If sex-linkage is not involved, you may do either cross A or B. Use
Analysis Table 2 for this part of your assignment.
Remember: Explain all your reasoning and show all your work. Be sure to explain any
abbreviations or symbols used. Your assignment is due in one week.

152
 (6 copies of this table are needed)
Data Table: Cross:
Cross A Cross B
Phenotype Phenotype
a. Parental cross
P female

P male

b. F1 Progeny
F1females

F1 males

c. F2 Progeny (from
F1 self) include
numbers of each
phenotype
F2 females

F2 males

d. Examine the F1 and F2 data. If sex does not appear to be a factor i.e. results for the F 1 and F2
of the reciprocal crosses are similar, then add all the numbers for each F2 phenotype i.e. F2
females cross A + F2 males cross A + F2 females cross B and F2 males cross B. Calculate the
F2 phenotypic ratio on the basis of the combined data.
phenotypes:
number observed:
ratio observed:

If sex does appear to be a factor i.e. the results for the F1 and F2 of the reciprocal crosses are
not similar, then do not combine your data. Calculate the phenotypic ratio separately for F 2
males and F2 females and for crosses A and B.

153
Analysis Table This table has been condensed)

Analysis Table 2. (6 copies of this table are needed. Also note that this table has been condensed.)

Cross:
 REFERENCES

Hadorn, E. 1962. Fractionating the Fruit Fly. Sci. Am. Vol. 206, pp 101-110.
Phillips, J. P. and Forrest, H. S. 1980. Ommochromes and Pteridines. The Generics and Biology
of Drosophila. M . Ashburner and T.R.F. Wright, Ed., Academic Press, N.Y. Vol. 2d, pp 541-
623.

155