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                                   This unit was developed by

                      California Foundation for Agriculture in the Classroom
                                     2300 River Plaza Drive
                                  Sacramento, California 95833
                                   Telephone: (800) 700-AITC

                                       Mailing address:
                                    Post Office Box 15949
                                 Sacramento, California 95853

All or part of this educational unit may be reproduced for teacher and student classroom use.
          Permission for reproductions for other purposes must be obtained from the
                      California Foundation for Agriculture in the Classroom.

                                           1st Printing
                                         October 1997
                                  TABLE OF CONTENTS

ACKNOWLEDGMENTS ......................................................................................2
INTRODUCTION ..................................................................................................4


         UNIT OVERVIEW ......................................................................................5
         BEFORE YOU BEGIN ..............................................................................8


         LESSON 1: WHAT CAN WE DO?..........................................................13
         LESSON 2: THE MEETING ...................................................................21
         LESSON 3: THE PLASMID ...................................................................35
         LESSON 5: HOW DO GENES GET INTO PLANTS? ............................67
         LESSON 6: THE PRESENTATION .......................................................76


         WHERE DO GENES COME FROM? ......................................................81
         “CAREERS IN BIOTECHNOLOGY” STUDENT HANDOUT ..................82
         TEACHER RESOURCES AND REFERENCES ......................................83
         GLOSSARY ............................................................................................86

California Foundation for Agriculture in the Classroom 1997                                                        1

This unit was made possible through grants and support from Calgene, Inc., Monsanto Company,
Bio-Rad Laboratories, the California Farm Bureau Federation and the California Foundation for
Agriculture in the Classroom.

Calgene, Inc. is an agricultural biotechnology company which develops improved varieties of plants
and plant products for the fresh tomato, cottonseed and industrial and edible plant oils markets.

Monsanto Company is a global agricultural company which focuses on agricultural biotechnology and
the development and marketing of value-added food and fiber crops, as well as crop protection
products, performance materials, food ingredients and pharmaceuticals.

Bio-Rad Laboratories is a multinational manufacturer and supplier of products for life science
research and education.

The California Farm Bureau Federation actively represents, protects and advances the social and
economic interests of farm families and California communities by organizing productive agriculture
to provide group benefits and manage issues which affect its membership.

The California Foundation for Agriculture in the Classroom is dedicated to fostering a greater public
knowledge of the agricultural industry and seeks to enlighten students, educators and leaders in the
public and private sector about agriculture's vital, yet sometimes forgotten, role in American society
and the effect all citizens have on agriculture's well being.

We would like to thank the following people who helped create, write, revise and edit this unit. Their
comments and recommendations contributed significantly to the development of this unit. However,
their participation does not necessarily imply endorsement of all statements in the document.

Writers                               Field Testers                         Illustrators

Jean Kennedy                          Dorothy Reardon                       Patricia Houk
Science Teacher                       Science Teacher
Armijo High School                    Del Campo High School                 Maceo Montoya
Fairfield-Suisun Unified School       San Juan Unified School District
District                              Fair Oaks, CA
Fairfield, CA                                                               Layout, Typing and Design
                                      Francesca Lowe
Suzanne Weisker                       Science Teacher                       Margaret Anderson
Science Teacher                       Concord High School
Will C. Wood High School              Mount Diablo Unified                  Karin Bakotich
Vacaville Unified School District     School District
Vacaville, CA                         Concord, CA                           Sherri Hughes
                                      Kathleen McCarthy                     Tami Gutschall
Editor                                Science Teacher
                                      Concord High School
                                                                            Rae Lehutsky
Pamela Emery                          Mount Diablo Unified
Curriculum Specialist                 School District
                                      Concord, CA                           Cinamon Vann
California Foundation for
Agriculture in the Classroom
Sacramento, CA

2                                                   California Foundation for Agriculture in the Classroom 1997
Curriculum Advisory and Review Committee
David Anderson, Ph.D.                        David Hammond                            Maria Osborn
Director of Cotton Biotechnology             Educational Consultant                   Biology Teacher
J.G. Boswell Company                         Sacramento, CA                           Foothill High School
Corcoran, CA                                                                          Grant Joint Union High School District
                                             Carolyn Hayworth                         Sacramento, CA
Carol Bastiani                               Manager, Investor and Public Relations
Research Assistant                           Calgene, Inc.                            Frank Plescia
Department of Medical Biological Chemistry   Davis, CA                                Manager, Government Affairs
University of California                                                              Monsanto Company
Davis, CA                                    Catherine Houck, Ph.D.                   Roseville, CA
                                             Vice President, Product Development
Beth Brookhart                               and Variety Development                  Pam Schallock
Freelance Journalist                         Calgene, Inc.                            Fourth Grade Teacher
Bakersfield, CA                              Davis, CA                                Sandrini Elementary School
                                                                                      Panama-Buena Vista Union School District
Lucas Calpouzos, Ph.D.                       Lauren Hubbard                           Bakersfield, CA
Former Dean of Agriculture                   Graduate Student Researcher
California State University                  Plant Molecular Genetics                 Claudia Sellers
Chico, CA                                    University of California                 Graduate Student Researcher
                                             Berkeley, CA                             Department of Plant and Microbial Biology
Steve Clark                                                                           University of California
Science Teacher                              Gay Jividen                              Berkeley, CA
Monterey High School                         Senior Director of Research
Monterey Peninsula Unified School District   Cotton, Inc.                             Wynette Sills
Monterey, CA                                 Raleigh, NC                              Farmer
                                                                                      Pleasant Grove Farm
Norma Clawson                                Andy Kennedy                             Pleasant Grove, CA
Science Teacher                              Field Representative/Buyer
Cajon High School                            Colusa County Canning Company            Roger Sitkin
San Bernardino Unified School District       Williams, CA                             Farmer
Blue Jay, CA                                                                          Old Dog Ranch
                                             Jean Landeen                             Linden, CA
Constance Coman                              Agricultural Education Consultant
Biology Teacher                              California Department of Education       Barbara Soots
Winters High School                          Sacramento, CA                           Assistant Education Director
Winters Joint Unified School District                                                 Center for Engineering Plant Resistance
Winters, CA                                  Jeanne Layton                            Against Pathogens
                                             Research Biologist                       University of California
Lane Conn                                    Monsanto Company                         Davis, CA
Chairman                                     Chesterfield, MO
Stanford Human Genome Center                                                          Nancy Stevens
Stanford University                          Mark Linder                              Biology Teacher
Palo Alto, CA                                President                                San Rafael High School
                                             California Foundation for                San Rafael City High School District
Jenny Cuccinello                             Agriculture in the Classroom             San Rafael, CA
Science Teacher                              Sacramento, CA
Florin High School                                                                    KarenBeth Traiger
Elk Grove Unified School District            Ron Mardigian                            Science Resource Teacher
Sacramento, CA                               Product Manager                          Graystone Elementary School
                                             Bio-Rad Laboratories                     San Jose Unified School District
Judy Culbertson                              Hercules, CA                             San Jose, CA
Manager of Programs and Services
California Foundation for                    Martina McGloughlin                      Denise Van Horn
Agriculture in the Classroom                 Associate Director of Biotechnology      Fourth Grade Teacher
Sacramento, CA                               Biotechnology Division                   McSwain Elementary School
                                             University of California                 McSwain Union Elementary School District
Jerry Delsol                                 Davis, CA                                Atwater, CA
Agriscience Teacher
Woodland High School                         Craig McNamara                           John Vogt
Woodland Joint Unified School District       Farmer                                   Middle School Science Teacher
Woodland, CA                                 Sierra Orchards                          Creekside Middle School
                                             Winters, CA                              Cotati-Rohnert Park Unified School District
Jim Elam                                                                              Rohnert Park, CA
Animal Nutritionist                          Donna Mitten
Agricultural Technology, Incorporated        Genetic Engineering Consultant           Thea Wilkins, Ph.D.
Solvang, CA                                  Woodland, CA                             Associate Professor
                                                                                      Department of Agronomy & Range Science
Richard Engel                                Jeffrey O’Neal                           University of California
Project Coordinator                          Extension Coordinator                    Davis, CA
California Foundation for Agriculture        Biotechnology Program
in the Classroom                             University of California                 Mary Yale
Sacramento, CA                               Davis, CA                                Middle School Science Teacher
                                                                                      Grange Middle School
John Fedors                                  Janette Oaks                             Fairfield-Suisun Unified School District
Science Education Consultant                 Research Scientist                       Fairfield, CA
San Diego, CA                                Calgene, Inc.
                                             Davis, CA
Mary Jo Feeney, MS, RD, FADA
Director of Education
California Beef Council
Pleasanton, CA

California Foundation for Agriculture in the Classroom 1997                                                                         3

The Science Framework for California Public Schools emphasizes the need to make science
education more meaningful to students so they can apply what they learn in the classroom to their
daily lives. Since all students eat food and wear clothing, one natural connection between science
and the real world is agriculture. Advances in agricultural technology, especially in genetic
engineering, are continually making headlines and are an excellent way for educators to connect
current trends and issues in science to the lives of every student.

Agriculture is an enormous industry in the United States, especially in California. As more rural
areas become urbanized, more challenges exist to maintain and improve the quality of life on the
planet and feed the people of the world. It is extremely important to educate students about their
environment, agriculture and the current technologies and research that continue to make the Earth a
viable planet. Genetic engineering is a relatively new science that has blossomed over the last 40
years. Most genetic engineering research relates to agriculture and medicine. This unit will focus on
plant genetic engineering.

Genetic Engineering in Agriculture is a thematic high school unit that can be used at the end of a
genetics unit or an advanced unit on the cell. Throughout the unit, students are provided with the
scientific principles of genetic engineering and are encouraged to use their knowledge to think
critically, creatively and freely about the viability and ethics associated with genetic engineering and
agriculture. The introductory lessons present biotechnology terms as the students are presented
with a hypothetical scenario associated with two major environmental concerns—food and fiber
quality and the efficient use of resources. Students prepare interactive reports and perform DNA
transformation laboratory activities that teach genetic engineering concepts, such as how genetic
information is transferred from one organism into another through the use of plasmids and tissue
culture. The students then use their acquired knowledge to prepare a presentation to a hypothetical
research funding board to report their findings on one issue associated with the cotton industry—
gossypol quantities in cottonseed.

Genetic Engineering in Agriculture is one of many lesson plans provided by the California Foundation
for Agriculture in the Classroom. The Foundation appreciates the support from Calgene, Inc.,
Monsanto Company and Bio-Rad Laboratories for assisting in the funding and development of this
unit. Please contact the Foundation for assistance with furthering the integration of agriculture into
your curriculum. Comments on this unit and on other Agriculture in the Classroom resources are
always welcome and appreciated.

4                                                 California Foundation for Agriculture in the Classroom 1997
                                      UNIT OVERVIEW

This interdisciplinary thematic unit consists of six lessons on genetic engineering in agricultural crops
along with the background knowledge needed to complete the lessons. This unit is designed for
college preparatory biology or physiology students who have a basic understanding of genetics and
advanced knowledge of cell structures and functions. The lessons are designed to introduce
students to biotechnology and genetic engineering in a format that supports critical thinking and
problem-solving on current issues associated with these sciences.

As indicated in the first lesson, one agricultural challenge is presented to the students—the presence
of a chemical called gossypol in cottonseed. This chemical acts as a defense mechanism for the
plants, but can be potentially harmful to animals, including humans, if consumed in large quantities.
Through background information gathered from hypothetical, yet realistic, memos, meeting minutes
and newsletters, the students are asked to address the gossypol challenge. The lessons guide the
students to investigate gossypol using a genetic engineering approach and to test their procedures in
the laboratory, where they perform a hands-on transformation activity. As part of a research team,
the students are asked to present their findings to a research board requesting further funding of this

Before beginning this unit, your students should understand:

    •   The basic components of cells.
    •   The structures and functions of bacterial cell components, especially plasmids.
    •   The structure of DNA and the role of DNA in coding for traits.
    •   Cell transcription and translation.
    •   How the order of DNA base pairs determines amino acid selection and that the sequence of
        amino acids determines specific protein structure.
    •   Basic genetic principles.
    •   Sterile bacterial plate streaking techniques, including hands-on experience with this practice.

In its entirety, this unit will require approximately three weeks of class time plus several evenings of
student homework. It is possible, however, to perform only selected lessons from this six-lesson
unit. Long-range planning is required by the instructor so appropriate materials can be ordered and

California Foundation for Agriculture in the Classroom 1997                                                5
According to the 1990 Science Framework for California Public Schools, the students should
understand that:

    • All living things have genetic material in the forms of RNA and/or DNA (pp. 128-130).
    • Genetics is the study of heredity, the passing of traits from one generation to the next
      (pp. 128-130).
    • Genetic make-up is received from both parents, and is expressed as traits which can be
      predicted (pp. 128-130).
    • The genetic code instructs the production of enzymes and other proteins in cells (p. 130).
    • Genetic engineering is a biotechnological process where material from one organism is
      inserted into the genetic code of another organism (p. 136).
    • Genetic changes can be achieved through genetic engineering (p. 130).
    • Practical and ethical issues are important to consider in the field of genetic engineering
      (pp. 135-136).
    • Manipulative models can be used to enhance the understanding of complex ideas and
      technologies (pp. 153-155).

The 1988 Language Arts Framework states that students will:

    •   Read significantly meaningful literature that introduce new vocabulary and concepts (p. 29).

    •   Formulate and share ideas with others in small group work and discussion (p. 12).

    •   Participate in an oral language program that encourages a variety of writing, reading,
        speaking and listening activities (p. 4).

The 1988 California History-Social Studies Framework states that students will:

    •   Develop an appreciation of the many people who work to supply their daily needs
        (pp. 37-38).

    •   Acquire information by listening, observing and using community resources (pp. 40-42).

    •   Develop group interaction skills, such as a willingness to listen to the differing views of
        others, decision-making, compromising, resolving conflicts and leadership skills (p. 21).

6                                                 California Foundation for Agriculture in the Classroom 1997
The students should:

    • Understand that all genes are composed of the same basic compounds.
    • Understand that genetic engineers can bring bits of DNA together from many different
    • Realize the importance of sterile and safety techniques in a laboratory.
    • Discover that bacteria and viruses exist in nature and naturally inject pieces of DNA into other
    • Understand how bacterial DNA can be transformed, having different genotypes than they
      once had.
    • Analyze how genetic engineering has influenced and continues to influence agriculture.
    • Realize the complexity of advancing science technologies, including genetic engineering.
    • Understand the importance of having a strong science knowledge base.

It is assumed that students should be able to define and understand the following words which are
discussed in this unit. Definitions for many of these words are incorporated into the lessons. A
partial list of definitions is also provided in the glossary on pages 86-87.

• Agrobacterium                        • Gene Splicing                 • Recombinant DNA
• Amino Acids                          • Gene Therapy                  • Restriction Enzyme
• Antisense Sequence                   • Genes                         • RNA
• Base Pairing                         • Genetic Engineering           • Selectable Marker
• Biotechnology                        • Kanamycin                     • Terminator
• Chimeric Gene                        • Ligating Enzyme               • Tissue Culture
• Chromosomes                          • mRNA                          • Transcription
• Cleavage Site                        • Mutation                      • Transformation
• Cotyledon                            • Plasmid                       • Transgenic
• Crown Gall                           • Polygalacturonase             • Translation
• DNA                                  • Promoter                      • Vector
• Enzyme                               • Protein

California Foundation for Agriculture in the Classroom 1997                                          7
                              BEFORE YOU BEGIN

1. Review the lessons to gain understanding of the unit. Determine which lessons you will use and
   when you will teach them.

2. Order the appropriate supplies needed for the DNA transformation activities your students will
   perform. Lesson Four: The Research Starts requires the Bacterial Transformation Kit
   #166-0003-EDU from Bio-Rad Laboratories, plus other specialized equipment such as ultra-
   violet lights and incubators. Refer to the lesson on pages 51-54 and the resources on page 83
   for specific ordering information. Allow four to six weeks for the delivery of materials.

3. Obtain various references on general plant microbiology and genetic engineering. Several are
   described in the Teacher Resources and References section on pages 83-85.

4. Due to the complexity of the subject matter, many assumptions and generalizations have been
   made so your students can complete the activity without becoming overwhelmed. Several of the
   facts and generalizations are discussed below. Share this information with your students as you
   feel appropriate.

•   The gossypol content in cottonseed is an actual issue in agriculture today. Many attempts have
    been made to resolve this issue—from producing glandless cotton plants that do not contain
    gossypol to detoxifying the gossypol in the cottonseed. Unfortunately, to date, none of the
    methods have been successful at a commercial level.
•   In this unit, references are made to a gossypol “gene.” In actuality, there are numerous genes
    involved in gossypol production. Gossypol production is not a single gene trait.
•   Gossypol is actually a secondary metabolite (sesquiterpene), a chemical compound that deters
    chewing insects from eating the cotton plant. Gossypol is not a protein.
•   “Gossypol synthase” is the name of the gene which encodes for gossypol synthase enzyme
    production. This enzyme causes gossypol formation.
               Precursor                               Gossypol
                               (Gossypol Synthase)

•   The main objectives of this hypothetical research scenario are described in the flow chart below.
                       Understand the agricultural issues regarding gossypol.

                         Brainstorm ways to resolve the gossypol challenge.

                        Decide on a genetic engineering research approach.

    Perform numerous activities to insert the hypothetical gossypol gene into a bacterial plasmid.

Understand how the gossypol gene can be inserted into a cotton plant using bacteria and tissue culture.

Present a continued research proposal to find a regulator gene that will allow gossypol to exist in the
                      vegetable parts of the plant, but not in the cottonseed.

8                                                California Foundation for Agriculture in the Classroom 1997

While the sciences of genetics, genetic engineering and biotechnology are complicated, there are
many components that can easily be incorporated into the classroom. The following information can
help you better understand the subject matter and relay this information to your students.

What is biotechnology?

Biotechnology is a number of technologies which use biological organisms to produce useful
products, processes and services. Production can be carried out by intact organisms, such as yeast
or bacteria, or by natural substances, such as enzymes, from organisms. The use of yeast in bread-
making is a form of biotechnology. The use of bacteria and molds in cheese-making is another
example of simple biotechnology. In the 1970s, a new type of biotechnology began—genetic
engineering or recombinant DNA technology.

What is genetic engineering?

Genetic engineering is a process where genetic material (DNA) is taken from one organism and
inserted into the cells of another organism. Genetic engineering also can be the rearrangement of
gene location or the removal of genes. The “altered” organism then makes new substances or
performs new functions based on its new DNA. For example, the protein insulin, used in the
treatment of diabetes, now can be produced in large quantities by genetically engineered bacteria
and yeasts. Insulin was formerly extracted from pigs or cows. Some say the genetic engineering of
plants can make food more nutritious and plentiful, helping to feed the ever-rising world population.

What can genetic engineering do?

It can improve the ability of an organism to do something it already does. For example, an
adjustment in the amino acid balance in a particular corn variety improves the corn’s ability to be

It can suppress, or stop, an organism from doing something it already does. For example, the gene
that codes for the softening of tomatoes is “turned off” in a genetically engineered tomato variety so
the tomatoes do not soften as quickly.

It can make an organism do something new. For example, particular bacteria and yeasts have been
genetically engineered to produce chymosin, an enzyme used in cheese production.

What is a gene?

Genes are sequences of DNA which serve as blueprints for the production of proteins in all living
things. DNA is found in all cells, usually in the nuclei. In bacteria and viruses, which do not have
nuclei, the DNA floats within the cell. DNA is composed of six molecules: sugars, phosphates and
four bases. A gene produces a specific protein or has an assigned function.

California Foundation for Agriculture in the Classroom 1997                                              9
What is a protein?

Proteins are chains of amino acids that perform the necessary functions of living organisms. When
a gene is “expressed,” that means it is transcribed into mRNA which is used as a template for
translation into a protein. Some of these proteins perform specific functions themselves (such as
becoming insulin or muscle); others participate in the production of cell components (such as
becoming enzyme proteins that assist in making carbohydrates and fats); still others are regulatory
and modify gene expression.

What are some examples of genetically engineered products?

• Human growth hormone, normally produced in the human pituitary gland, can be made in
  bacteria to give to people who lack this hormone.

• Rabies vaccine can protect against the rabies virus.

• Oil-eating bacteria can clean up oil and gasoline spills efficiently.

• Healthier edible oils can be produced by genetically altered canola plants.

• Tomato plants can be altered to delay the onset of softening and rotting of fruit.

• Herbicide-resistant cotton can withstand the effects of sprays so weeds can be eradicated without
  harming the crop.

• Viral-resistant fruits and vegetables can resist viruses.

• Cheeses can be made using bacterial-produced rennet (an enzyme formerly taken from calves’

• Insecticidal proteins produced internally by plants can reduce the need for chemical pesticides.

How do we know that genetically engineered plant foods are safe?

Advanced technology, as well as standards and regulations set by food producers and governmental
agencies, have allowed the United States to maintain its safe food record. In fact, the United States
has the safest food supply in the world. The following information will help you better understand the
genetic engineering food safety guidelines.

Before any plant food developed through biotechnology is made available to the public, it undergoes
a safety evaluation. In 1992, the Food and Drug Administration (FDA) issued testing guidelines for
genetically engineered foods. The specific policies are under the title “Foods Derived From New
Plant Varieties.” There are other policies for products other than plants. The genetically engineered
plant food product guidelines are summarized as follows:

• Genetically modified plant foods shall be regulated exactly as traditionally produced foods.
• The food products will be judged on their individual safety, allergenicity, toxicity, etc., rather than
  on the methods used to produce them.
• Any new food additive produced via biotechnology will be evaluated for safety employing the
  same guidelines used for a traditional food additive (such as food coloring).

10                                                 California Foundation for Agriculture in the Classroom 1997
• Any food product that is found to contain material that could render it unsafe will not be allowed to
  enter commerce.
• If the introduced product contains an allergen, or if the production of the food has altered its
  nutritional value, the FDA may require informational labels.

As in the case for any food product, any genetically engineered plant food found to contain
substances not in keeping with the safety guidelines may be removed from the marketplace by the
FDA. The United Nation’s World Health Organization continues to debate the policies revolving
around genetically engineered food products.

How do we know if genetically-engineered plants are safe for the environment?

To insure that genetically engineered crop plants are safe, the United States Department of
Agriculture (USDA) oversees all field testing of genetically engineered products. Before a new crop
can move into commercial production, the USDA reviews the field-testing results. Field-testing
results and studies must demonstrate that plants altered using biotechnology react with ecosystems
in the same ways as do their traditionally produced plant counterparts.

What are some risks associated with genetic engineering?

As with any new technology, risks must be considered. Some criticism of genetic engineering
practices include the possibility that modifications in the genetic make-up of the plant could result in
some type of unknown toxin. The odds of that occurring in normal plant breeding and selection are
far greater than that occurring in genetic engineering. Genetic engineering involves only the
movement of specific genes with specific functions. In traditional plant breeding, crosses between
different varieties and wild relatives result in the transfer of many genes. The science of genetic
engineering is carefully monitored and the risks associated with any products and processes, such
as allergens and ecological impacts, are constantly addressed.

How can genetic engineering affect agriculture?

With increasing food needs around the world and the loss of farmland to urbanization, farmers must
constantly find ways to increase yields and lower production costs. As farmers continue to look for
renewable resources and safe ways to control pests and fertilize plants, genetic engineers continue
their research to assist agriculture.

• Pest-resistant plants are being developed through genetic engineering. For example,
  mungbeans, a staple in Asia, can now be commercially grown without the use of pesticides.
  Strawberries have also been genetically engineered to be resistant to root pests.

• Herbicide-tolerant cotton has been developed through genetic engineering. The herbicide
  bromoxynil is broken down by the cotton plant. This allows the cotton field to be sprayed with
  bromoxynil to kill weeds without affecting the cotton plant itself. This method of weed control
  greatly reduces the amount of herbicide used on cotton while increasing the yield of cotton per

• Genetic engineering is helping farmers diversify their crops. For example, ethanol produced from
  starches genetically added to potatoes can be used as a fuel, and genetically engineered plant
  oils in canola and soybean plants can be used to produce biodegradable plastics.

California Foundation for Agriculture in the Classroom 1997                                            11
What are the basic procedures for producing a genetically engineered plant product?

The actual procedures for producing a genetically engineered product are very complex. However,
most genetically engineered plant products are produced using the basic steps described below:

a) TRAIT IDENTIFICATION: Traits of organisms are identified.

b) GENE DISCOVERY: Genes for the desired traits are identified.

c) GENE CLONING: The desired gene is inserted into a bacterial cell and, as bacteria reproduce,
   the desired gene is also reproduced.

d) GENE VERIFICATION: Researchers study the copies of the gene using molecular techniques to
   verify that the replicated gene is precisely what is wanted.

e) GENE IMPLANTATION: Using a bacterium or other procedure, the desired DNA (gene) is
   transferred into the chromosomes of the host plant cells.

f)   CELL REGENERATION: Researchers select the plant cells that contain the new gene and
     regenerate whole plants from the selected plant cells.

g) THE NEW PLANT TESTING: Laboratory and field testing occur to verify the function and safety
   of the new plants.

h) SEED PRODUCTION: Seeds with the desired traits are produced using standards set for
   specific crop production.

12                                            California Foundation for Agriculture in the Classroom 1997
                     LESSON 1: WHAT CAN WE DO?
                                (An Introduction to Biotechnology)

The purpose of this activity is for students to realize that biotechnology, specifically genetic
engineering, is a tool that can be used to meet challenges that exist in agriculture.

• Biotechnology is the development of products using a biological process.
• Biotechnology is a science that affects every individual’s life.
• Many career opportunities exist in the field of biotechnology.
• One specialized application of biotechnology is genetic engineering.
• Many current challenges exist in the agricultural industry.
• Practical and ethical issues are important to consider in the field of genetic engineering.
• Agriculturalists, educators and industry representatives can work together to meet many of the
  challenges that exist in our society today.

For the class:

• Reference books on cotton, biotechnology and other pertinent topics (see pp. 83-85)
• Masking tape

For each group of two to four students:

• Here’s An Idea task list (p. 19)
• Butcher paper
• Markers

For each student:

• What Do You Think? memo (pp. 17-18)

California Foundation for Agriculture in the Classroom 1997                                        13
Teacher preparation ..................................................... 10 minutes
Class activity ................................................................ One or two 50-minute sessions
Homework .................................................................... Two nights

BIOTECHNOLOGY: If you split up the word, it is easy to understand. “Bio” stands for biology, living
things. “Tech” stands for technology, the tools and techniques used to study things. “Ology” means
the study of. Biotechnology, in simple terms, means the study of living things using various tools and
techniques. Here, “biotechnology” is the study of how technology is used to impact the function of
living things. Or rather, biotechnology is the development of products using technology in biological

This lesson presents facts about cotton and current agricultural challenges associated with it. In this
hypothetical yet realistic scenario, students learn about one real challenge that faces the cotton
industry—the high gossypol content in cottonseed. Using this example, the students work as
scientists to confront this challenge through research and development.

Gossypol is a naturally occurring chemical found in the seeds and plants of cotton. Located in the
pigment glands of cotton plants and cottonseed, it deters chewing insects and other animals from
eating the plants. The presence of gossypol in cultivated cotton has been an issue in the cotton
industry for over 40 years.

Cottonseed is used as a food source for both cattle and other animals. Mature cattle can eat small
amounts of cottonseed as part of their diet and be unaffected by the presence of gossypol. The
problem with the presence of gossypol in cottonseed, however, is that it is toxic to animals in high
quantities. For this reason, only limited quantities of cottonseed are permitted in cattle and chicken
feed and it is not a widespread ingredient in human food. The processing methods used to detoxify
the gossypol in the seed meal have been unacceptable because they decrease the nutritional value
of the meal and increase the cost of production.

One of the objectives of this unit is for students to realize the potential benefits and challenges of
producing cotton plants that contain gossypol in the leaves and stems, but not in their seeds.

The issue of gossypol content in various parts of the cultivated cotton plants is only one challenge of
thousands that are being researched today. This topic of study was chosen for many reasons, some
of which include its interesting history and current research possibilities. The issues addressed in
this unit are being addressed in the agriculture research community. To make this unit
understandable by high school students, however, many details have been left out of the research

14                                                           California Foundation for Agriculture in the Classroom 1997
1. Discuss the idea of this unit—to examine issues associated with agriculture and to investigate and
   propose resolutions to one or more agricultural challenges.
2. Divide the students into groups of two, three or four.
3. Distribute the What Do You Think? memo (pp. 17-18) written to Caitlin Noonan from Tom Davis.
4. Have the students read and discuss the memo and then complete the Here’s An Idea task list
   (p. 19), including an oral presentation of their ideas. Some possible ideas for the brainstorm list
    • Find wild strains of cotton that have gossypol in the vegetative parts of the plant, but not in the
      seed. Selectively breed those plants.
    • Find an economical way to detoxify the gossypol in the cottonseed through some type of
    • Find a way to make gossypol-free cotton plants resist chewing pests.
    • Genetically engineer a cotton plant to have gossypol in the plant, but not in the seeds.
    • Investigate the effects of heat on gossypol stability. Use this information in the processing of
      cottonseed to produce human food and silage.
5. As homework, have the students complete a response letter to Mr. Davis. In small groups, have
   the student share their response letters with one another.

There are many issues associated with agriculture that affect people and the environment. There
are also endless possibilities on how major challenges can be resolved through research and
investigation. The issue of gossypol content in cottonseed is one such issue.

• Have the students find out about other genetic engineering research projects currently under way
  by contacting biotechnology companies such as Calgene, Inc., Cotton, Inc., Genentech,
  Monsanto Company and the University of California Cooperative Extension.
• Throughout this unit, have the students learn more about the agricultural production of cotton.
• Invite a cotton grower or cotton ginning provider to your classroom to discuss the growing,
  harvesting and ginning of cotton.
• Have your students use the Internet to learn more about the agriculture of cotton and/or issues
  associated with genetic engineering.

California Foundation for Agriculture in the Classroom 1997                                           15
TO:       Caitlin Noonan
          Research Director
          Agri-Gene, Inc.

FROM:     Tom Davis
          Senior Research Investigator
          Cotton Research Associates

I recently attended the 20th Annual United States Department of Agriculture Symposium at the
University of California at Berkeley. It featured a lecture on specific issues in California
agriculture. One of the topics discussed was the gossypol content in cottonseed and cotton
plants and the issues associated with this naturally occurring chemical.

Cotton is an important California commodity and is currently the leading commodity in Fresno
and Kings counties. It is believed that the first cultivation of cotton was in India. The American
Indians grew cotton in the early 1500s with true American cultivation beginning in 1621 when
the English settlers were provided cottonseed from the West Indies. The history of cotton
cultivation and processing techniques is fascinating and I will be happy to talk with you about
this further, if you so desire.

The purpose of this memo, however, is to discuss a possible research partnership in the area
of gossypol reduction in cottonseed. The presence of gossypol in cultivated cotton has been
an issue in the industry for more than 40 years. Here are some facts we already know about

      • Gossypol is a naturally occurring chemical found in the seeds and vegetative material
        of cotton. This chemical exists in the pigment glands of cotton plants and in the
        cottonseed. It deters chewing insects, such as caterpillars and other animals, from
        eating the plants.

      • Gossypol is a secondary metabolite produced from interactions between certain
        precursors and an enzyme called gossypol synthase.

      • Cottonseed is a food source for cattle and chickens. They can eat limited amounts of
        cottonseed as part of their diet and be unaffected by the presence of gossypol in the

      • Humans most often consume cottonseed in the form of oil. Many manufactured goods,
        including cookies and crackers, list cottonseed oil as an ingredient.

      • Cottonseed can be used as a filler in many foods. Since cottonseed is relatively
        tasteless, flavorings can be added to the seeds to produce items such as chocolate
        flavored chips and imitation nuts.

16                                             California Foundation for Agriculture in the Classroom 1997
     • It is the industry’s goal to increase the saleability of cottonseed so this by-product from
       cotton fiber production can be utilized more efficiently and effectively; therefore, it can
       benefit the cotton growers financially and potentially be a currently untapped food
       source for millions of people each year.

     • The problem with the presence of gossypol in cottonseed is that it is toxic in higher
       quantities. For this reason, only limited amounts of cottonseed are included in human
       foods and in cattle and chicken feed. Extensive studies have not been done on
       tolerance levels of gossypol; however, evidence shows that humans and cattle
       exposed to high levels of gossypol from cottonseed can sustain red blood cell scarring,
       liver damage and experience other medical problems.

     • Gossypol has some positive effects in animals. It is an anti-viral chemical, which
       means it reduces the propagation of some viruses, even in humans. Research
       indicates that it may also suppress the spread of cancer cells, such as those which
       result in leukemia.

     • As stated previously, gossypol is a natural pest deterrent. Plants containing high
       quantities of gossypol tend to resist chewing insect pests. Those with low levels of
       gossypol are not as successful in the field.

Here is our challenge. Cotton Research Associates would like to develop a cultivated cotton
plant that has high amounts of gossypol in its vegetative material, but little or no gossypol in its
seeds. To date, we have not been successful in achieving this goal, but are interested in
providing the funding for research to improve cotton production and the stability of the
cottonseed market.

I have been asked by your Board of Directors to investigate the feasibility of this task. I need
to gather data to present at its next meeting. At that time, the board will determine if funding
will be provided for such a project. I request your input on how to reduce or eliminate gossypol
in cottonseed. Please include your ideas on the following:

     —a definition of the work that you think should/could be done.
     —a brief explanation of how this work could be accomplished.
     —a list of pros and cons concerning each method you suggest.

I appreciate the time you are giving to this potential joint venture. I respect your opinions and
your continued support of scientific research in agricultural advances and look forward to
hearing from you soon.

California Foundation for Agriculture in the Classroom 1997                                       17
HERE’S AN IDEA                               Name(s)


     1) Brainstorm at least four potential ways gossypol levels in
        cottonseed can be reduced or gossypol could become less toxic to
        livestock and humans. During this brainstorming, you may use
        your textbook or other references to help you obtain ideas.

        Note: Think of scientific procedures and technologies used today that have resulted
        in healthier, more flavorful or more desirable foods. Examples include technologies
        used to develop leaner beef, low-fat cheeses, sweeter corn, tastier tomatoes and
        seedless grapes.

     2) Decide on two methods you think might be worth pursuing in regard
        to the gossypol issue. For each method, provide a brief description
        on how this could be achieved and the pros and cons for each

     3) On a large piece of butcher paper, prepare a visual aid that will
        help express your ideas. Prepare a three-minute explanation that
        describes your two favorite ideas.

     4) Present your ideas to the class.

     5) Listen to ideas presented by other groups.

     6) HOMEWORK—Assuming the role of Caitlin Noonan from
        Agri-Gene, Inc., write a reply to Mr. Davis discussing the various
        ideas presented by your class (research team). Be sure to discuss
        which method you suggest Cotton Research Associates pursue.
        The potential pros and cons of your recommendation should also
        be addressed.

18                                        California Foundation for Agriculture in the Classroom 1997
                                      COTTON PLANT

California Foundation for Agriculture in the Classroom 1997   19
                         LESSON 2: THE MEETING
              (The Decision for a Genetic Engineering Approach is Made)

The purpose of this activity is for students to learn how some research decisions are made and to
gain background information and begin preparatory work regarding the genetic engineering activity
they will perform.

•    Genetic engineering is one method used to alter
     the genetic make-up of an organism.
•    The genotype and the phenotype of an organism
     can be altered through genetic engineering.

For each group of four to six students:

•    The Meeting Task Sheet (pp. 32-33)
•    Access to a computer for word processing and newsletter layout
•    Sample public relations newsletters from a variety of companies

For each student:

•    Letter from Tom Davis (p. 25)
•    The Gossypol Meeting Transcription (pp. 26-31)

Since there are quite a few details mentioned in the meeting notes used in this activity, it is strongly
suggested that your students have an understanding of the following concepts and vocabulary prior
to this lesson:

•    the terms genotype and phenotype, and how the genotype of an organism affects its phenotype;
•    how DNA codes for protein;

20                                                California Foundation for Agriculture in the Classroom 1997
•   transcription and translation, and how they are associated with cell replication and protein
•   the structures of large chromosomes and plasmids in prokaryotes; and
•   the term gossypol and why it is a challenge in the cotton industry.

Teacher preparation .........................................20 minutes
Student activity .................................................Three to four 50-minute class periods, plus homework

The meeting notes are incorporated into this lesson for two reasons. One reason is to continue the
scenario with the students so the upcoming lessons have more meaning. Secondly, the meeting
minutes inform students, in a unique way, much of the background information needed to complete
the upcoming bacterial transformation activity. Based on the prior knowledge of your students, you
may need to explain some of the terminology and concepts. Provide the necessary supporting
activities so your students have comprehended the listed terms by the conclusion of this lesson.

• amino acid                   • gene                         • protein                         • transcription
• antisense                    • genome                       • selectable marker               • translation
• chimera                      • mRNA                         • sense                           • vector
• complimentary                • plasmid DNA                  • termination
  base pairs                                                    sequence
                               • promoter sequence

1. Discuss the previous activity, if necessary. Then, have each student individually read the letter
   from Tom Davis (p. 25) and the Gossypol Meeting Transcription (pp. 26-31).
2. Have a brief class discussion about the letter and transcription clarifying any facts that are
   unclear. If appropriate, make an overhead transparency of page 28 for your discussion.
3. Distribute The Meeting Task Sheet (pp. 32-33) to each student group of four to six students.
   Review the lesson, which requires the students to do research and develop a company public
   relations newsletter.
4. Allow sufficient time in class for students to develop draft ideas of the newsletter. Also, allow
   enough time for the students to proof each other’s work before turning in the final newsletter.
5. Upon completion, have the students share their newsletters with fellow classmates.

California Foundation for Agriculture in the Classroom 1997                                                        21
6. Individually, have the students write a one-page summary describing the most interesting
   concepts or facts they have learned. Clarify any misconceptions that have surfaced or plan
   appropriate questions or activities in upcoming lessons that will challenge the misconceptions.

Genetic engineering has been used to alter the genetic make-up of many agricultural products.
There are many challenges associated with the actual processes of genetic engineering as well as
ethical issues that must be considered when developing genetically engineered products.

• Coordinate time at your school computer lab to create the newsletter on the computer.
• Rather than a newsletter, have the students develop an editorial for a television show that
  discusses the information. Use video equipment, if available.
• Assign particular students the roles of the people in the meeting minutes. Have them orally
  present the information while the rest of the students listen.
• Turn the meeting transcription into a skit with appropriate characters and props.

• Invite a research scientist to your classroom to discuss how research ideas are formed and how
  funding sources for those ideas are obtained.
• Working with the English, social studies, journalism, mathematics and computer teachers at your
  school, expand this lesson into an extensive newsletter which may include editorials, polls,
  classified ads for appropriate jobs and statistical graphs.
• After completing this lesson, have the students discuss the profit or loss potential of such a

California Foundation for Agriculture in the Classroom 1997                                          23
                                    TOM DAVIS
                             Senior Research Investigator
                         COTTON RESEARCH ASSOCIATES
                                    123 Leaf Way
                             Cottonseed, Missouri 54321

                                   September 1, 1999

Caitlin Noonan
Research Director
Agri-Gene, Inc.
Cottontown, California 12345

Dear Caitlin,

       Thank you for your suggestion to have an informational meeting about how to
reduce the gossypol content in cottonseed. I have included the minutes from one
meeting we had on this topic. The group of people attending the meeting were very
informative and enlightened me as to the processes our company will be involved in as
we pursue this new venture.
       As you will see from the transcription, the Board of Directors decided that a
genetic engineering approach to the gossypol issue may best serve our purposes.
Thank you again, Caitlin, for your support on our project. I will be in touch with you as
things progress.


                                        Tom Davis
                                        Senior Research Investigator


cc: Alexandra Hoeppner
    Gordon Spicer

24                                         California Foundation for Agriculture in the Classroom 1997
                                         Sacramento, California

Meeting Attendees:

      Tom Davis, Senior Research Investigator, Cotton Research Associates
      Alexandra Hoeppner, Research Geneticist, Calgene, Inc.
      Gordon Spicer, Agronomist, University of California, Davis
      Assistants to Tom Davis, Cotton Research Associates

Tom:             Good morning. Thank you for attending our meeting. Our task today is to
                 investigate the feasibility of genetically engineering cotton plants to
                 contain cottonseed with little or no gossypol. There are several questions
                 we would like to have answered during this meeting.
             • We are aware that Calgene, Inc. created the Flavr Savr® tomato seeds.
               Why and how was this tomato developed? Can a similar protocol be
               used for our project?
             • I have heard the term “chimeric” used in relation to genetic engineering.
               What does this mean and will we be using a chimeric gene?
             • I understand there is a direct relationship between the DNA in a gene and
               protein production. Please explain this relationship to me in regards to
               gossypol and cotton.
                 I would like to turn this forum over to Alexandra Hoeppner, who will
                 discuss how the work at Calgene, Inc. on the Flavr-Savr® tomato seeds
                 was accomplished. The company performed research associated with
                 this tomato for ten years prior to its development. The genetically
                 engineered tomatoes are presently sold in selected stores throughout the
                 nation under the name MacGregor’s® tomatoes.
Alexandra:       Calgene developed the MacGregor’s® tomato in response to the tasteless
                 tomatoes usually available in the grocery stores during the winter months.
                 Normally, tomatoes consumed in the winter and spring months are picked
                 green so they can be stored and shipped without becoming too soft. The
                 tomatoes redden during shipping or in warehouses after being exposed to
                 ethylene gas, a gas naturally released by ripening fruit. The softening of
                 a tomato is associated with a single sense gene that causes the
                 development of a protein called “polygalacturonase” (PG). At Calgene,
                 we figured out how to place the backwards version of the PG gene, called
                 the “antisense PG” gene, into the tomato genome. The backwards
                 version is combined with the “sense PG” gene so replication and
                 expression of the softening PG gene cannot occur. Thus, the level of the
                 PG enzyme is reduced and the softening of the tomato is slowed. As a

California Foundation for Agriculture in the Classroom 1997                                25
             result, the tomatoes can be shipped vine ripened and have a more
             flavorful taste.
Tom:         How can you get the PG antisense strand to be expressed?
Alexandra:   In any organism, whether it is prokaryotic or eukaryotic, transcription of
             DNA to form mRNA must occur. In order for transcription to occur, an
             enzyme RNA polymerase must bind to a specific recognition site on the
             gene. This recognition site is called the promoter region. To reduce PG
             enzyme production, the PG gene is removed using restriction enzymes
             that cut DNA; then, the inverted gene, ASPG, is inserted next to the
             promoter. As a result, the ASPG gene instead of the PG gene is
             transcribed and translated. Thus, the polygalacturonase needed for fruit
             softening is not synthesized.
Tom:         How do you get the PG gene out of the genome and the ASPG gene into
             the genome? It seems difficult and complex.
Alexandra:   Developing the process was complex, but the process itself incorporates
             the natural actions of plants. To accomplish this task, we made use of a
             common reaction that occurs when a plant is injured. Frequently, when a
             plant gets a cut or another type of wound, it may get infected by a soil-
             borne bacterium called Agrobacterium. When the wound is infected by
             this bacterium, some of the bacterium’s DNA is transferred into the plant
             and a tumor is formed. This particular tumor is called a Crown Gall
             Tumor. We used this knowledge to our benefit.
             We also made use of a natural feature of many bacteria. Many bacteria
             contain genetic material called plasmids. Agrobacterium, like other
             bacteria such as E. coli, contain two types of genetic material, one larger
             chromosome and numerous smaller circular plasmids. Plasmids are
             relatively easy to remove from bacterial cells. Using specialized
             enzymes, called restriction enzymes, the PG gene is removed from the
             tomato genome, flipped backwards and then placed into a plasmid. The
             insertion of a gene is done with ligating enzymes. The backwards version
             of this gene is called the antisense PG gene or the ASPG gene. The
             plasmids containing the ASPG gene are put into living Agrobacteria.
             Then, when a wound is purposely made on a tomato leaf, the
             Agrobacteria containing the ASPG gene goes into the wound site and
             creates a callus—a scar—and transfers the ASPG gene into the plant.
             From this callus, tissue culture techniques are used to grow tomato plants
             from a single cell of the callus. Hence, the tomato plants grown from the
             callus have the new feature of the reduction of the non-softening PG
             enzyme because it binds with the ASPG gene that entered the cell
             through the plasmid. It’s a complex process, but is relatively easy now
             that we have the method down. (As Alexandra speaks, she is writing this
             schematic diagram on the board.)

26                                        California Foundation for Agriculture in the Classroom 1997
California Foundation for Agriculture in the Classroom 1997   27
Tom:          It seems that this new tomato plant genome is composed of DNA from
              several different sources—the original plant and Agrobacterium.
Alexandra:    Yes, it is. This is called a “chimera.” Chimera means “from several
              sources.” In this case, the genome is from the original tomato plant as
              well as the Agrobacterium, which contains the antisense PG gene cloned
              from a different tomato plant.
Tom:          I heard that you grew the Agrobacterium in a solution that contained the
              antibiotic Kanamycin. Why was this done and what effects will this
              antibiotic have on the person who eats the tomato? Can they become
              resistant to this antibiotic by eating your tomatoes?
Alexandra:    It is common practice to use what is called a selectable marker when
              transferring genetic material. Let me explain. . . When the plasmid is
              made, it contains the promoter sequence, the ASPG gene, the selectable
              marker and a terminating sequence—a sequence on the DNA which stops
              the formation of mRNA. When the mRNA is made during transcription,
              both the ASPG gene and the selectable marker gene are transcribed at
              the same time. The selectable marker for our tomatoes is the resistance
              to the antibiotic Kanamycin. This antibiotic resistance serves several
              purposes. It serves as a control so only the bacteria we want will survive.
              You see, after the plasmid is placed back into the bacterium, the
              bacterium is grown on a special medium plate which contains the
              antibiotic. It will kill all of the bacteria except those that have been
              transformed—those that have the softening gene removed and the
              Kanamycin resistance gene. Secondly, it is easy to see antibiotic
              resistance in bacteria—the bacteria either grow or don’t. It is much more
              time-consuming to see if the softening occurs or doesn’t occur.
              Therefore, when a new gene is inserted, a gene for antibiotic resistance is
              also included. Sometimes two selectable markers are used.
                         CHIMERIC GENE CONSTRUCT DIAGRAM

       Promoter                 Gene                  Selectable                  Terminator
       Sequence              of Interest               Marker                     Sequence

              And . . . to answer your questions about humans becoming resistant to
              the antibiotic during the process—this is, frankly, not possible. When
              people eat the tomatoes, they digest the DNA as well. The DNA is not
              being inserted into the human cells. The antibiotic resistance is part of
              the plant genome and is only recognized by plants.
Tom:          What about the question of feasibility concerning the use of this
              procedure for reducing the gossypol content in cottonseed? Gordon, can
              you address this?
Gordon:       I believe that it is theoretically possible to reduce the gossypol in
              cottonseed without reducing the amount of gossypol in the adult plant.

28                                         California Foundation for Agriculture in the Classroom 1997
                 This question intrigued me and I asked one of my graduate students to
                 begin researching this question. She found out that it is possible to
                 produce a gossypol-free plant. This has been done by breeding cotton
                 plants that are homozygous recessive for glandless cotton. These plants
                 will produce only plants that do not make gossypol. They are not grown
                 commercially at present because they do not resist chewing insects;
                 therefore, they are not as productive and may require more use of
                 chemical pesticides. These homozygous recessive plants, however, will
                 suit our research purposes since they produce no gossypol in their
                 genomes. I would suggest that we start by determining if a glandless
                 plant can make gossypol when a genetically engineered plasmid
                 containing the gossypol gene is inserted.

                 If this works, this is half our battle. We still need to somehow transform
                 the plant so that gossypol is made in the adult plant only, not in the seed.
                 During our research, we found some reference to wild strains of cotton
                 that have a gene that regulates the production of gossypol synthase so
                 that gossypol is made only in an adult plant, but not the embryonic part or
                 seed. If we can isolate this gene, we can create glandless plants that can
                 be infected with a transformed bacterium that contains both a gossypol
                 gene and a regulator gene. This way gossypol will be made only in the
                 adult plant. This is where the real research and funding will be needed.
                 (Gordon draws this schematic on the board as he speaks.)

                                       Produce glandless gossypol plants
                                               from wild strains

                                           Isolate gossypol gene and
                                         gossypol regulator genes from
                                                  cotton plants

                                          Insert gossypol and gossypol
                                              regulator genes into
                                                 glandless cotton

                                         Use tissue culture techniques to
                                          produce cotton plants which
                                          have gossypol in the plants,
                                               but not in the seeds.

Alexandra:       Do you foresee using the “antisense” method of reducing the gossypol
                 content or will you try to just control gossypol production?

California Foundation for Agriculture in the Classroom 1997                                29
Gordon:   Good question . . . At this point, I think it would be best to insert the
          gossypol gene and then regulate it with a promoter sequence rather than
          incorporate the antisense–mirror image of the gene. Another thing, really
          quick if I may . . . we are calling this gene the “gossypol gene” for
          simplicity. In actuality, it is a group of genes that produce an enzyme
          called gossypol synthase. This gossypol synthase is the protein that
          causes gossypol production. We’re after the genes that create this
          enzyme. But for ease, let’s continue to call the group of genes needed
          the “gossypol gene.”
Tom:      Do you have suggestions about how we can do some short transformation
          experiments to see what might happen?
Gordon:   Well, in fact, I do! Not only do I have a graduate student who has isolated
          this gossypol gene, we think we have a selectable marker that can be
          used as well. Last summer, a high school student, Jorge Villalobos, took
          a five-week summer research internship with us. Jorge not only mapped
          the gene adjacent to the gossypol gene, but in a serendipitous event,
          determined that this adjacent gene becomes luminescent when grown in a
          medium that contains a special sugar called “arabinose.” It would make
          an easy second marker gene for you. You could immediately know if
          transformation has occurred correctly just by looking at the bacteria grown
          in the media containing arabinose. Since it glows beautifully under a UV
          light, we named this plasmid the PGLO™ plasmid.
          The research that you are proposing will be quite involved. I propose that
          we try to attach the gossypol gene to the glow marker, put these genes
          into a plasmid and then insert the plasmid into E. coli or Agrobacterium. If
          this is possible, we should be able to use tissue culture techniques to
          grow a glandless cotton plant that produces gossypol. This is step one.
          Step two will be to find a regulator gene that controls gossypol production.
          Step three will be to insert the regulator gene into this plasmid that we just
          created. This will take quite a bit of research and will require a major
          funding source.
Tom:      That sounds great! It gives us a great place to start. I would like to
          publish an account of all of this information in our PR newsletter. This will
          keep the farmers abreast of what is happening in research and will inform
          our staff and stockholders of potential products. Hmmm. . . I wonder what
          environmental impact studies we will have to do as this progresses! Let’s
          keep this in mind.
          Thank you everyone, for participating in this meeting. There has been an
          extensive amount of information presented here. My committee and I will
          assimilate it and ask further questions as they arise.

30                                     California Foundation for Agriculture in the Classroom 1997
THE MEETING TASK SHEET                                        Names

Assume the role of the communications department for Cotton Research Associates.
You and your colleagues, under Tom Davis’ supervision, have been asked to create
one of the monthly public relations newsletters for company members and the public.
The goal of this newsletter is to highlight one aspect of research Cotton Research
Associates is involved in and to be forthright in discussing issues and challenges that
this particular research project addresses.

Your newsletter should have a minimum of six articles, plus any other information you
would like to add (pictures, upcoming events open to the public, etc.). Determine how
the newsletter tasks will be divided among your group members. Each member of your
group is responsible for the following:

     • gathering background information for all articles;
     • writing one article;
     • proofing all articles for technical accuracy, grammar and spelling;
     • participating in the layout, design and typing or illustrating of the newsletter.

                                POSSIBLE NEWS ARTICLE TOPICS

      • What is going to be Cotton Research Associates’ new genetic engineering
        venture in relation to the gossypol content in cottonseed?
      • Why is the gossypol content in cottonseed an issue in agriculture?
      • What is a transformed bacterial cell?
      • What is MacGregor’s® tomato and how was it developed? How is this
        knowledge going to be applied to the cotton industry?
      • What considerations need to be taken into account when placing a gene into a
      • What are some ethical issues that surround genetic engineering and how
        should the public learn about and address these issues?
      • Discuss the profit or loss potential of producing genetically engineered cotton.
      • What environmental impacts must Cotton Research Associates consider?

California Foundation for Agriculture in the Classroom 1997                                31
The following vocabulary should be included in your articles: Agrobacterium, amino
acid, biotechnology, chimera, chromosome, enzyme, restriction enzyme, gene, genetic
engineering, mRNA, plasmid, protein, promoter sequence, selectable marker,
terminating sequence, transcription, translation and vector.


_____ 1)   Review the meeting minutes and discuss with your colleagues any areas
           that confuse you. Assist your colleagues in understanding the key points.

_____ 2)   Skim over newsletter samples you have at home and those provided by
           your teacher. As a team, discuss format and content ideas you particularly
           like or dislike.

_____ 3)   Create an overall format for your newsletter, including a title.

_____ 4)   Gather the information needed to write the articles and discuss as a group
           the contents of each article.

_____ 5)   Write the draft articles individually.

_____ 6)   Review the work of your colleagues for technical accuracy, grammar and

_____ 7)   Review the articles to make sure all required vocabulary words are

_____ 8)   Prepare the final version of the newsletter, making sure you follow any
           rules and guidelines provided by your instructor regarding size, content and

_____ 9)   Share your newsletter with your classmates.

32                                          California Foundation for Agriculture in the Classroom 1997
                           LESSON 3: THE PLASMID
                              (How to Insert a Gene into a Plasmid)

The purpose of this activity is for students to gain a greater understanding of how DNA from one
organism can be inserted into DNA of another organism. The focus is on the insertion of the
gossypol gene and the PGLO™ and antibiotic resistance selectable markers into a plasmid.

• A chimeric gene is created by bringing bits of DNA
  together from various sources.
• Each chimeric gene has certain elements, including a
  promoter sequence, a gene of interest, a selectable
  marker and a terminator sequence.
• DNA codes for proteins.
• Restriction and ligating enzymes are used to “cut” and
  “glue” DNA material, respectively.

For each student:

• Letter to Tom Davis (p. 39)
• Inserting DNA Into a Plasmid news article (p. 40)

For the teacher:

• Inserting DNA Into a Plasmid news article (p. 40)
• DNA Insertion Into a Plasmid answer key (pp. 49-50)

For each partnership:

• Gossypol DNA handout duplicated onto white paper (p. 41)
• Plasmid DNA handout duplicated onto colored paper (p. 42)
• Restriction Enzyme Reference Page (p. 43)
• DNA Insertion Into a Plasmid activity sheet (pp. 44-47)

California Foundation for Agriculture in the Classroom 1997                                        33
Teacher preparation ..................................................... 15 minutes
Student activity ...............................................................One 50-minute session, plus a 15-minute
                                                                                summary and review

Students should have a basic understanding of the functions of DNA and RNA including the
processes of transcription and translation. Transcription is the process in which RNA polymerase
synthesizes a messenger RNA chain by reading the code of a DNA sense strand. Translation is the
process in which the messenger RNA (mRNA) is decoded to produce amino acids. The amino acids
link together in specific sequences directed by the mRNA to form specialized proteins. These
proteins are then used somewhere in the cell.

A chimeric gene construct generally consists of a promoter sequence, the gene of interest, a
selectable marker and a terminator sequence. The promoter sequence indicates the place in which
DNA transcription for protein synthesis should begin. The promoter can also dictate where and
when the gene of interest is expressed. The gene of interest is the gene that one wishes to transfer
to another organism. Examples include virus resistance, color or added nutrition. Since it often is
difficult to determine whether a specific gene, such as increased nutrition, is transferred, a selectable
marker is used. A selectable marker is an easily identifiable gene, such as antibiotic resistance or
phosphorescense, that is attached to the desired gene so that it is obvious whether or not the genetic
transformation has occurred. The terminator sequence is a section of DNA that stops the mRNA
translation for protein synthesis.

                                  CHIMERIC GENE CONSTRUCT DIAGRAM

       Promoter                           Gene                          Selectable                    Terminator
       Sequence                        of Interest                       Marker                       Sequence

Chimeric gene constructs are created by bringing bits of DNA together from various sources. These
sources may include DNA from bacteria, viruses, plants or animals. A list of genetically engineered
crops and the source of the gene inserted is located on page 81. Genetic engineers isolate desired
genes using restriction enzymes. Restriction enzymes identify specific sequences of DNA bases
and make a cut at these specific sites on a DNA molecule. Ligating enzymes “glue” bits of DNA
strands together.

By cutting the DNA molecule into small pieces using restriction enzymes, scientists are able to
discover and study genes. There are four restriction enzymes commonly used in genetic
engineering. Each type differs in the type of cut it makes. Over 175 different restriction enzymes are
known and are characterized with respect to their cleavage sites and the “sticky ends” that are
available for attachment. Sticky ends are the single strands of DNA left after a restriction enzyme
separates the base pairs. Some examples are described below. The Bam HI and Sac I restriction
enzymes are used to cut the polygalaturonase genes from tomato genomes. This method is used by
Calgene, Inc. to produce the MacGregor’s® tomato—a tomato that resists softening.

34                                                         California Foundation for Agriculture in the Classroom 1997
        Restriction Enzyme                 Recognition Site            Cleavage Site
               EcoR I                        GAATTC                      G / AATTC
               Hind III                      AAGCTT                      A / AGCTT
               Bam HI                        GGATCC                      G / GATCC
                 Sac I                       GTCGAC                      G / TCGAC
                                                                      / = cleavage site

This activity shows the students how restriction and
ligating enzymes work. It is important for the students
to realize that where a gene inserts itself into another
genome is dependent on biochemical and
environmental factors as well as the availability of
sticky ends. Where a chimeric gene construct
chooses to attach itself can affect other processes of
the organism.

A typical gene insertion challenge is that scientists
want a gene to insert itself in a location that will not
affect other plant genes. One such example has
happened in the cotton plant. Researchers were able
to isolate a gene that could increase fiber strength.
The problem was that it always inserted itself in the
middle of a gene sequence that caused cotton boll
formation. The cotton boll is where the fiber grows.
Thus, when the gene for fiber strength was inserted
the mechanism for cotton boll formation was
interrupted and no cotton bolls would form. In
conclusion, the fiber strength gene could not be
expressed. Since this study, other fiber strength
genes have been identified and have been
successfully inserted into certain cotton varieties.

1. Read aloud the memo to Tom Davis from Alexandra Hoeppner (p. 39). Discuss how
   communication and cooperation are very important components of scientific research.

2. Distribute the Inserting DNA Into a Plasmid article (p. 40) to the students. Have them read it
   individually and discuss, in small groups, five key points of the article.

3. Have the students individually create a sequencing map which shows the steps that must occur
   for DNA insertion into a plasmid. Have them save their ideas and amend them at the conclusion
   of this activity.

4. Have the students pair up and complete the DNA Insertion Into a Plasmid Paper Model activity
   (pp. 44-47).

5. Discuss the results and challenges of this activity. Include a discussion of student challenges as
   well as actual research challenges.

California Foundation for Agriculture in the Classroom 1997                                         35
6. Have the students refer to their pre-activity sequencing maps. With their newly gained
   knowledge from this activity, have them amend or rewrite the sequence of events necessary to
   insert DNA into a plasmid.

Restriction and ligating enzymes are used to cut and paste pieces of DNA into genomes.
Restriction and ligating enzymes are specific and affect DNA molecules in specific ways.

•    Assign the article on page 40 as a homework reading assignment.

•    In preparation for upcoming activities, have the students practice pouring agar plates, plating
     bacteria and using sterile techniques.
•    Have student groups research various aspects of cotton production—its life cycle, the ginning
     process, the varieties of cotton, the economic impact of cotton on society, etc.

36                                                California Foundation for Agriculture in the Classroom 1997

TO:                  Tom Davis

FROM:                Alexandra Hoeppner

RE:                  Gossypol Gene Insertion into an E. Coli Plasmid

Thank you for including me as a researcher in your project. Enclosed is a reading
selection that discusses how DNA is added to a plasmid. I think it explains what we will
be doing. Remember, we will cut out the gossypol gene from a cotton genome and
then put it into E. Coli plasmids. This plasmid example depicts most of the preliminary
work that we will be testing.

The gossypol gene has already been mapped. We now need to determine which
restriction enzyme we can use to insert the gene into the plasmid genome. We are
working it out on paper prior to trying it in the lab. I hope you can join us in this
preliminary work. By participating in placing the plasmid into the bacterial cell, you will
gain understanding of the steps in this process which will help you prepare for your
presentation to your board.

I have attached an idea that I have used in the past. It shows how DNA is inserted into
a plasmid on the molecular level. I have used this idea during presentations, and it
may be something you can use with your Board of Directors. I hope it helps to clarify
our process.


    INSERTING DNA INTO A PLASMID                                    Prokaryotic cells contain one large chromosome.
   (A Genetic Engineering Technique Using Plants and Bacteria)   This chromosome contains most of the DNA.
by Tanisha Bradley
                                                                 Sometimes prokaryotic cells also have several small
                                                                 circular pieces of DNA called plasmids. These
                                                                 plasmids contain genes which code for proteins that
                                                                 are beneficial to the survival of the cell.
                                                                    The first plasmids discovered contained genes for
                                                                 antibiotic resistance. Geneticists believe that plasmids
                                                                 contain these genes because they neutralize the action
                                                                 of an antibiotic on the bacterial cell. To counteract the
                                                                 effects of antibiotics which kill the bacteria, large
                                                                 quantities of the enzyme are required. More copies of
                                                                 the antibiotic-resistant gene that produces the enzyme

California Foundation for Agriculture in the Classroom 1997                                                            37
can be carried on several plasmids than can be                  Plasmids have been used in the process of gene
incorporated into one large chromosome.                       splicing in a variety of study areas, including
  Geneticists take advantage of prokaryotic plasmids          medicine and agriculture.
by incorporating the DNA of a desired gene into the              To perform the necessary procedure that will place
genome of the plasmid. When the plasmid replicates,           a piece of DNA into a plasmid, researchers use a
the desired gene is also replicated. This way the             restriction enzyme.     A restriction enzyme is a
information in the gene is passed from one generation         specialized enzyme that cuts the DNA at a site where
to the next as the bacterial cell divides. More               the base pairs are arranged in a specific order. For
importantly, as the DNA in the plasmid is transcribed         example, the restriction enzyme Bam HI cuts DNA
into mRNA, the desired DNA from the implanted gene            between the two Gs in the sequence GGATCC.
also gets transcribed into mRNA so that translation                            G GATCC
can occur. Translation is when the mRNA is decoded                              C CTAGG
to produce amino acids which attach together to make              You may notice that the DNA is palindromic,
the desired proteins.                                         which means the base pairs read the same each way,
                                                              backward and forward. Since the structure of DNA is
                                                              the same in all organisms, the same enzymes can be
                                                              used in both prokaryotic and eukaryotic cells.
                                                                 When geneticists want to insert a gene into another
                                                              organism, they cut out the desired DNA from an
                                                              organism using restriction enzymes. Using the same
                                                              restriction enzyme, plasmids from a bacterial cell are
                                                              cut in one spot to open it up. When DNA is cut, or
                                                              spliced, this leaves the two open ends chemically
                                                              active. These chemically active ends are called
                                                              “sticky ends.” Because of DNA’s complimentary
                                                              base-pairing rules, a sticky end will readily
                                                              recombine with another piece of DNA with
                                                              complimentary bases in order to chemically bond and
                                                              once again become stable. When the new DNA is
                                                              placed in with the cut plasmid, ligating enzymes are
                                                              used to seal the new connection. It is possible for
                                                              plasmids to recombine with themselves or with other
                                                              compatible sticky ends to get a genome arrangement
                                                              other than the one desired.
                                                                After the plasmid has been inserted into a
                                                              bacterium, the scientist grows the bacterium on an
                                                              agar plate to create the colony of bacteria with the
                                                              new genotype. A selectable marker is used to identify
                                                              the cells that have been transformed in the desired

38                                                      California Foundation for Agriculture in the Classroom 1997
                                      GOSSYPOL DNA
             1                            2                          3                  4
 A                     G     G      GGGGG            C        A              T   G   ••••••••   C
 G                     C     A      GGGGG            T        A              T   G   ••••••••   C
 A                     T     T      GGGGG            A        A              T   G   ••••••••   C
 A                     T     C      GGGGG            G        T              A   G   ••••••••   C
 A                     T     G      GGGGG            C        T              A   G   ••••••••   C
 G                     C     C      GGGGG            G        T   ••••••••   A   A   ••••••••   T
 G                     C     G      GGGGG            C        T   ••••••••   A   A   ••••••••   T
 G                     C     G      GGGGG            C        A   ••••••••   T   A   ••••••••   T
 C                     G     G      GGGGG            C        A   ••••••••   T   T   ••••••••   A
 A                     T     G      GGGGG            C        A   ••••••••   T   T   ••••••••   A
 A                     T     A      GGGGG            T        C   ••••••••   G   T              A
 A                     T     A      GGGGG            T        C   ••••••••   G   T              A
 T                     A     A      GGGGG            T        C   ••••••••   G   C              G
 T                     A     A      GGGGG            T        C   ••••••••   G   G              C
 T                     A     A      GGGGG            T        T   ••••••••   A   C              G
 A                     T     T      GGGGG            A        T   ••••••••   A   C              G
 G                     C     T      GGGGG            A        A   ••••••••   T   A              T
 G                     C     T      GGGGG            A        G   ••••••••   C   A              T
 C                     G     C      GGGGG            G        G   ••••••••   C   A              T
 C                     G     C      GGGGG            G        G   ••••••••   C   G              C
 C                     G     C      GGGGG            G        G   ••••••••   C   G              C
 A    GGGGG            T     T      GGGGG            A        G   ••••••••   C   C              G
 A    GGGGG            T     A      GGGGG            T        A   ••••••••   T   C              G
 A    GGGGG            T     G      GGGGG            C        A   ••••••••   T   T              A
 A    GGGGG            T     G                       C        A   ••••••••   T   T              A
 A    GGGGG            T     G                       C        T   ••••••••   A   T              A

         GGG         Glow Gene                                     •••••     Gossypol Gene

California Foundation for Agriculture in the Classroom 1997                                     81
                    PLASMID DNA
      1              2                      3                           4
 A          T   A          T    C      ]]]]            G     A                   T
 C          G   A   ]]]]   T    C      ]]]]            G     A                   T
 G          C   T   ]]]]   A    G      ]]]]            C     A                   T
 G          C   T   ]]]]   A    A      ]]]]            T     C                   G
 G          C   T   ]]]]   A    G                      C     C                   G
 T   ««««   A   C   ]]]]   G    A                      T     T                   A
 C   ««««   G   C   ]]]]   G    A                      T     G                   C
 A   ««««   T   G   ]]]]   C    T                      A     G                   C
 A   ««««   T   C   ]]]]   G    C                      G     A                   T
 C   ««««   G   A   ]]]]   T    G      zzzz            C     G                   C
 G   ««««   C   G   ]]]]   C    G      zzzz            C     G                   C
 G          C   C   ]]]]   G    A      zzzz            T     G                   C
 G          C   T   ]]]]   A    T      zzzz            A     C                   G
 G          C   C   ]]]]   G    A      zzzz            T     T                   A
 C          G   G   ]]]]   C    T      zzzz            A     T                   A
 A          T   A   ]]]]   T    A                      T     A                   T
 A          T   G   ]]]]   C    G                      C     G                   C
 G          C   A   ]]]]   T    C                      G     T                   A
 G          C   T   ]]]]   A    C                      G     T                   A
 C          G   C   ]]]]   G    A                      T     A                   T
 C          G   C   ]]]]   G    A                      T     A                   T
 A          T   C   ]]]]   G    A                      T     A                   T
 A          T   G   ]]]]   C    C                      G     C                   G
 G          C   T   ]]]]   A    C                      G     C                   G
 G          C   A   ]]]]   T    G                      C     G                   C
 G          C   T   ]]]]   A    A                      T     C                   G

82                         California Foundation for Agriculture in the Classroom 1997
           Promoter Sequence                                  Ampicillin Gene
Terminator Sequence                     ]]]                                 zzz

     C              G                                         C           G
     C              G                                         T           A
     T              A                                         C           G
     G              C               Ava II                    G           C       Sac II
     G              C                                         A           T
                                                              G           C

     T              A                                         G           C
     T              A                                         G           C
     C              G                                         A           T
     G              C            Hind III                     T           A       Bam HI
     A              T                                         C           G
     A              T                                         C           G

                                                              G           C
     G              C                                         A           T
     G              C                                         A           T
     C              G              Hpa II                     T           A       EcoR I
     C              G                                         T           A
                                                              C           G

California Foundation for Agriculture in the Classroom 1997                                83
                         (Paper Model Student Activity)

The purpose of this activity is for you to build a model that will help you
understand the workings and complexities of restriction and ligating
enzymes as they pertain to inserting a piece of DNA into plasmid DNA.

At the conclusion of this activity, you should be quite familiar with restriction
and ligating enzymes, sticky ends, DNA structure and chimeric gene

For each partnership:

• Plasmid DNA handout (colored paper)
• Gossypol DNA handout (white paper)
• Restriction Enzyme Reference Page
• Tape
• Scissors


1.   Carefully observe the gossypol DNA and the plasmid DNA. Using the
     keys, find the areas needed to produce the desired chimeric gene and
     then answer Question #1.

2.   Observe the available restriction enzymes on the attached page.
     Remember . . . restriction enzymes cut the DNA. Answer Question #2.

84                                     California Foundation for Agriculture in the Classroom 1997
 3.    Using scissors, cut the plasmid DNA and attach each strip to form one
       long strand of DNA. Attach in the order that the lanes are numbered

 4.    Attach the loose end of #4 to the loose end of #1 to form a circular

 5.    Cut and attach the plant DNA in a similar manner as you did the
       plasmid DNA, but do not attach to form a circle. This represents the
       piece of the eukaryotic gossypol DNA that will be placed into the

 6.    From the restriction enzyme page, locate the area on the plasmid where
       each restriction enzyme will cut. Pencil in where the cut would be.
       Repeat for the plant DNA. Complete the chart under Question #3 and
       answer Question #4.

 7.    Using scissors, cut the plasmid in the same manner that the chosen
       restriction enzyme would cut it so that it opens up into a straight line.

 8.    Cut the plant DNA at the site of the chosen restriction enzyme.

 9.    Piece together the sticky ends of the plasmid DNA to attach to the
       sticky end of the plant DNA. All of the pieces should form a circle.

10.    Using tape (DNA ligase), attach the plant DNA to the plasmid DNA at
       the sticky end sites. You should end up with a larger circular plasmid
       than found in #4.

11.    Double check to make sure that all of the needed parts of the plasmid
       DNA are present for the desired protein to be made. Answer Questions
       #5 through #8 and complete the Conclusion Statement as homework.

 California Foundation for Agriculture in the Classroom 1997                       85
IV. QUESTIONS       (Write your answers on a separate sheet of paper)

1.   Explain what areas of the plasmid DNA and the plant DNA will be
     needed to create the desired chimeric gene.
2.   List the sticky ends formed by:
        a) Hpa II
        b) Ava II
        c) EcoR I
3.   Copy and complete the chart below:

      Restriction      Should            Why? Give your reasons why the
       Enzyme           use?             enzyme should or should not be used.
                     (State yes or no)

        Ava II

        Sac II

        Hpa II

       Bam HI

        EcoR I

       Hind III

4.   Which restriction enzyme is the best to use and why?
5.   Why was the plasmid DNA taped in a circle while the plant DNA was
6.   If an analogy can be made about the scissors representing the
     restriction enzyme, what would the tape represent?
7.   Does the plasmid you just made contain all of the components needed
     to make protein synthesis occur? Explain your answer.
8.   Many diabetics use human insulin that is made from the bacterial cell
     E. coli. How can a eukaryotic gene be placed in a prokaryotic cell?

86                                           California Foundation for Agriculture in the Classroom 1997

Write a summary statement for Mr. Davis that he can use as a handout for
his presentation to the Board of Directors.

Special thanks to John Fedors of San Diego, California, for his contributions to
this lesson.

California Foundation for Agriculture in the Classroom 1997                    87
                                            (Answer Key)

1.   Explain what areas of the plasmid DNA and the plant DNA will be needed to create the
     desired chimeric gene.

     The glow gene and gossypol gene need to be cut from the plant DNA strand (requiring
     two cuts). The plasmid gene will need to open up after the promoter and before the
     terminator sequence (requiring one cut). The ampicillin gene on the plasmid DNA
     must not be cut.

2.   List the sticky ends formed by:

        a) Hpa II       There will be a two G-C sticky ends.

        b) Ava II       There will be a one G-T-C sticky end and one C-A-G sticky end.

        c) EcoR I       There will be a one A-A-T-T sticky end and one T-T-A-A sticky end.

3.   Copy and complete the chart below:

        Restriction     Should use?         Why? Give your reasons why the enzyme should or
         Enzyme         (State yes or no)   should not be used.
          Ava II              No            Cuts plasmid DNA, but not plant DNA.

          Sac II              No            Cuts plasmid DNA, but not plant DNA. Cuts in the
                                            middle of the ampicillin gene.

          Hpa II             Yes            Cuts out the glow and gossypol genes in the
                                            plant. Opens the plasmid in one spot and does
                                            not interfere with the promoter sequence,
                                            ampicillin gene or terminator sequence.

         Bam HI               No            Cuts plant DNA, but not plasmid DNA. Will
                                            interfere with the glow gene.

          EcoR I              No            Does not cut either plant or plasmid DNA.

          Hind III            No            Does not cut either plant or plasmid DNA.

4.   Which restriction enzyme is the best to use and why?

     Hpa II. This restriction enzyme cuts the plasmid after the promoter sequence, but
     before the terminator sequence without affecting the ampicillin gene. It also cuts the
     glow and gossypol genes from the plant DNA so they can be removed as a unit to be
     placed into the plasmid.

88                                               California Foundation for Agriculture in the Classroom 1997
California Foundation for Agriculture in the Classroom 1997   89
5.   Why was the plasmid DNA taped in a circle while the plant DNA was not?

     A plasmid is a circular piece of DNA. The plant DNA is a paper model sequence from
     a very large model of a cotton chromosome. After the plant DNA has been placed into
     the plasmid DNA, the entire sequence will be circular.

6.   If an analogy can be made about the scissors representing the restriction enzyme, what
     would the tape represent?

     The tape would be the “glue” that fuses the sticky ends of the DNA together after they
     find complimentary base-pair matches. The tape represents the enzyme DNA ligase.

7.   Does the plasmid you just made contain all of the components needed to make protein
     synthesis occur? Explain your answer.

     Yes. It contains the promoter sequence, the gene of choice, the selectable markers
     and the terminator sequence. For protein synthesis to begin, the promoter sequence
     must be present to start the process. The terminator sequence must be present to
     end the process.

8.   Many diabetics use human insulin that is made from the bacterial cell E. coli. How can a
     eukaryotic gene be placed in a prokaryotic cell?

     After the desired gene has been identified and mapped, the correct restriction enzyme
     needs to be identified so that the placement of this sequence can be cut from the
     eukaryotic cell and placed into the plasmid. Because prokaryotic and eukaryotic DNA
     are the same chemically, the same restriction enzyme is used to cut both strands of
     DNA. The gene of choice needs to be cut in two places so that it can be removed from
     the DNA strand. The plasmid needs to be cut in one spot so insertion can occur. It
     should be cut after the promoter sequence and before the terminator sequence so
     that the placement of the gene of choice allows for successful protein synthesis.

90                                           California Foundation for Agriculture in the Classroom 1997
                     TRANSFORMING DNA
                               (A Bacterial Transformation Activity)

The purpose of this laboratory activity is for students to have firsthand experience transferring DNA
from a plasmid into a bacterial cell. Specialized laboratory techniques will be taught and practiced.

• The genome of an organism can be changed by adding or removing DNA.
• Genetic transformation involves the insertion of a gene into an organism in order to change the
  organism’s traits.
• Genetic transformation is used in many areas of biotechnology, including agriculture and
• Selectable markers are used so successful DNA transformation can easily be identified.
• Examples of selectable markers include antibiotic resistance and bioluminescence.
• Specialized laboratory techniques, such as heat shock, sterile techniques, bacterial plate
  streaking and quantitative measuring, are important components of biotechnology.

For the teacher and class:

• Biotechnology Explorer-Bacterial Transformation Kit #166-0003-EDU from Bio-Rad Laboratories
  (see page 83 for ordering information)
• Microwave oven
• 37° C incubator
• Temperature-controlled water bath
• Celsius thermometer
• 1-liter flask
• 250 ml flask
• 500 ml graduated cylinder
• Distilled water
• Crushed ice

California Foundation for Agriculture in the Classroom 1997                                         91
• 10% bleach solution in squirt bottles
• Mini centrifuge

For each team:

                Supplied With Kit                                                 Not Included in Kit
• 2 microtubes: one for (-DNA) and one for (+DNA)                     • Recombinant Memo by Alexandra
                                                                        Hoeppner (p. 55)
• Stock bacteria in a Petri dish
                                                                      • The Research Starts activity sheet
• Plasmid
                                                                        (pp. 56-63)
• 4 Petri dishes:
                                                                      • Permanent marker
  1 with LB agar only; 2 with LB agar and ampicillin
  (amp); 1 with LB agar, ampicillin (amp) and                         • UV lamp
  arabinose (ara)
                                                                      • Digital watch with second-hand
• Sterile transformation buffer
                                                                      • 42° C water bath
• LB broth
                                                                      • 500 ml graduated cylinder
• Pipettes, sterile
                                                                      • Distilled water
• 3 inoculation loops
                                                                      • Styrofoam cup with crushed ice
• Microtube racks
                                                                      • 10% bleach solution in squirt bottle
                                                                      • Paper towels
                                                                      • Waste container with 10% bleach solution
                                                                      • Safety goggles, gloves and lab apron
                                                                      • Celsius thermometer

Teacher preparation ....................................................... 3 hours (see page 7 of the Biotechnology
                                                                                Explorer, Bacterial Transformation manual)
Student activity ............................................................... Four 50-minute sessions

Your students will perform a genetic transformation laboratory activity. Genetic transformation is
used in many areas of biotechnology, including agriculture. Some examples include the insertion of
genes into plants to make them more frost or pest resistant, drought tolerant or more flavorful.

This activity was adapted with permission from Bio-Rad’s Biotechnology Explorer, Bacterial
Transformation Kit (#166-0003-EDU). The kit includes background information and lab preparation

92                                                         California Foundation for Agriculture in the Classroom 1997
instructions as well as a student manual. For your thorough understanding, it is highly
recommended that you review and follow the instructions provided with the kit. You may also
choose to provide supplemental information and use student questions that are provided in the kit

In this activity, the gossypol scenario will continue as students act as researchers to insert the
gossypol gene into glandless cotton plants. This activity will not teach laboratory techniques only, but
also will allow your students to get a flavor of what true research entails. The selectable markers of
antibiotic resistance and bioluminescence will help easily identify the transformed bacterial cells.
The antibiotic resistance gene is required so nontransformed cells do not survive—the
bioluminescence gene is added for a colorful experience.

Approximately three hours of preparation time will be needed for a well-equipped laboratory.
Review and follow the teacher preparation guidelines provided in the Biotechnology Explorer-
Bacterial Transformation Kit (#166-0003-EDU) from Bio-Rad Laboratories.


1. Attach The Research Starts: Transforming DNA activity (pp. 56-63) to the Recombinant Memo
   (p. 55) and distribute the packets to the students. Review the memo and discuss what the
   students will be doing.
2. Discuss the purpose of the lab, laboratory protocol and safety instructions.
3. Have the students complete the pre-lab activity questions (p. 57).


1. This is the actual day of the experiment. All supplies should be prepared, organized and
   available to the students.
2. Have the students complete the lab and activity sheet designated Day 2.
3. Assign appropriate questions for homework.

California Foundation for Agriculture in the Classroom 1997                                           93

Have the students complete the activities designated for Day 3 on their activity sheets, including
making appropriate data tables and answering the questions. Assign the conclusion statement
writing assignment as homework.


Discuss the laboratory activity, clarifying any confusing points and appropriate applications to the
gossypol scenario.

•    Genetic transformation activities, using sterile techniques, allow scientists to transfer genetic
     material from one organism to another. Bacteria are often used for transformations because of
     their size and fast replication rates.

•    Adapt this lesson to other transformation kits available to educators.
•    Perform other laboratory activities, such as bacterial plate streaking, preparing agar plates and
     DNA fingerprinting.

•    Complete the Transformation Efficiency* Extension Activity provided on pages 64-65.
•    Using the Internet and other sources, have the students research how and why bacterial
     transformation activities are used in agricultural, medical and pharmaceutical research.

94                                                California Foundation for Agriculture in the Classroom 1997
*   This procedure was adapted, with permission, from Bio-Rad’s Biotechnology Explorer, Bacterial Transformation laboratory activity
    (#166-0003-EDU). For further inquiries contact Bio-Rad at 1-800-4BIORAD or

California Foundation for Agriculture in the Classroom 1997                                                                            95

TO:          Tom Davis

FROM:        Alexandra Hoeppner

RE:          The Recombinant

Thank you for the check that your association sent us for preliminary work. We
followed up on the suggestion from Ms. Noonan and have found and created a chimera
containing the gossypol gene, the glow gene and the antibiotic resistance gene. This
sequence has already been inserted into a plasmid. The new sequence is shown in
the diagram below.

                                   Gossypol                 Antibiotic
 Promoter       Glow Gene           Gene                 Resistance Gene          Terminator

We will attempt to insert this chimera into an E. coli bacterial cell next week as
scheduled. I am pleased that you will assist us in the laboratory. By participating, you
will have a better understanding of the protocols used in this research. Once we are
successful at inserting this chimera into the plasmid, we will need to insert the plasmid
into the cotton plant and determine growth rate success.

If the procedure is successful, we will work on the gossypol regulator gene. This is
where the sponsorship from your group will become essential. I appreciate our
partnership as we work closely together to obtain funding for this important endeavor.

I have enclosed the protocol that we will be using next week. We are looking forward
to seeing you again.

96                                         California Foundation for Agriculture in the Classroom 1997
THE RESEARCH STARTS                                           Name


You are about to complete a genetic transformation activity. Genetic
transformation means that one or more genes are inserted into an
organism’s DNA to change the organism’s traits. In this experiment, we are
supposing that you are attempting to insert the gossypol gene into an E. coli
bacteria. You will know if this transformation is successful because the
selectable markers of ampicillin resistance and bioluminescent glow are
attached to this gossypol gene. You can detect the transformed bacteria by
locating the bacteria that survive (have ampicillin resistance) and glow (have
the glow gene that causes glowing when the sugar arabinose is present)
when plated on an agar plate containing ampicillin and arabinose. Those
that survive also, hypothetically, carry the desired trait of the gossypol gene.

Special Note

In actuality, the identification of genes and the creation of the chimeric gene
constructs in the plasmids require extensive laboratory work and research.
For the sake of time and complexity, we are assuming this preliminary work
has already been done by other researchers.

California Foundation for Agriculture in the Classroom 1997                   97

1. What genes are being placed into the transformed plasmid?
2. What organism is to receive this plasmid?
3. List at least three reasons why a bacterium was chosen for this experiment.
4. What are the two selectable markers used in this procedure? Why were these
   markers selected?
5. What safety concerns must you use when doing this lab and why?
6. Copy and complete the chart below to predict what you expect to find after your
   experiment is complete. Also, write your hypothesis in an “if . . . then” format.


                              LB (-DNA)   LB/AMP (-DNA)    LB/AMP (+DNA)        LB/AMP/ARA (+DNA)
      Will plate show
      transformed cells?
      (+ for yes, - for no)

      Estimated number
      of colonies

7. What do the labels (+DNA) and (-DNA) represent?
8. Explain what LB, AMP and ARA represent.
9. On which of the four plates listed in #7 do you expect to see growth?
10. Which of the four plates are considered to be control plates and why?
11. What does it mean if growth appears on a plate?
12. Step #7 of the laboratory procedure is only completed for the (+DNA) tube. Why?

98                                             California Foundation for Agriculture in the Classroom 1997

Materials needed for each team:                               Other materials needed:

• Microtubes: one for (-DNA) and one for (+DNA)               • UV lamp
• Stock bacteria in a Petri dish                              • Microwave oven
• Plasmid                                                     • 37° C incubator
• 4 Petri dishes:                                             • 42° C water bath with thermometer
  1 with LB agar only; 2 with LB agar and                     • Waste container with 10% bleach solution
  ampicillin (amp); 1 with LB agar, ampicillin
  (amp) and Arabinose (ara)                                   • Safety goggles, rubber gloves and lab apron
• Sterile transformation buffer                               • Fischer or Bunsen burner
• LB broth                                                    • 10% bleach solution in spray bottle
• Pipettes, sterile
• 3 inoculation loops
• Microtube racks
• Second-hand watch or timer
• 500 ml graduated cylinder
• Distilled water
• Styrofoam cup with crushed ice
• Paper towels


1.    Put on safety clothing as instructed. Wearing rubber gloves and goggles, sterilize
      your counter top using paper towels and the 10% bleach solution.

2.    Pick up all materials required for your team. Note where community items are

3.    Using the permanent marker, label the top
      and side of one microtube (+DNA) and the
      second (-DNA). On both tubes, write your lab
      group number and the date. Note: One lab
      group in your team will work with the (-DNA)
      tube and one will work with the (+DNA) tube.
      Be sure to label the top of the vial as the ink
      may rub off the side when placed in the water
      bath. Place the microtubes in the microtube

4.    Using one sterile pipette and sterile techniques, transfer 250 µl of the calcium
      chloride (CaCl2) solution into each of the microtubes from #3. Store on ice to chill

California Foundation for Agriculture in the Classroom 1997                                                99
      the calcium chloride. Dispose of the pipette in the designated waste container. A
      sketch of the pipette with the volume marking is shown.

5.    Observe your stock plate by holding it under the UV light. Copy and complete the
      data chart below. Using a sterile loop, collect a single average-sized colony of
      stock bacteria and transfer it to the microtube labeled (+DNA). Immerse the loop
      into the CaCl2 and swirl the loop so that all of the bacteria are transferred into the
      microtube. Stir until the bacteria are evenly mixed with no clumps observed.
      Dispose of the loop in the waste container. Place the tube into the ice bath.


      Observe and describe the colonies on the starter plate using the following chart.

                        Size of                                                         Visible
                    colonies (estimate                         Distribution of       appearance
                    in mm using a ruler on
       Number of    the outside of the Petri
                                               Color of         colonies on          when viewed
        colonies             dish)             colonies          the plate           with UV light

                    Largest ___

                    Smallest ___

                    Majority ___

100                                              California Foundation for Agriculture in the Classroom 1997
6.    Using another sterile loop, repeat #5 using the (-DNA) tube. Dispose of the loop
      in the designated waste container.

7.    Immerse a third sterile loop into the stock plasmid solution. Pick up some of the
      plasmid solution in the loop. It should look like a film in the ring. Gently stir the
      plasmids in the loop into the tube labeled (+DNA). Cap the tube and replace in
      the ice. DO NOT ADD PLASMID TO THE (-DNA) TUBE. Close the cap of the (-
      DNA) tube and place it in the ice. Make sure both tubes are capped and in the

8.    Check the time and incubate on ice for 10 minutes.

9.    While the tubes are incubating, label the bottom of your four Petri dishes as

California Foundation for Agriculture in the Classroom 1997                               101
10.   After your ten minutes are complete, heat shock the bacterial cells. To do this,
      place the rack of tubes into the 42°C water bath for exactly 50 seconds. Make sure
      that the bottoms of the tubes sit in the warm water bath. When the 50 seconds are
      expired, carry the ice container to the warm water bath, remove the rack of tubes
      and immediately place them back on the ice, making sure that the bottom of the
      tubes are in the ice. This timing is critical for successful transformation. Be
      accurate with your time! Incubate on the ice for two minutes.

11. After the two-minute ice
    incubation, remove the rack
    and tubes from the ice and
    place them on the bench top.
    Open the (+DNA) tube and,
    using a sterile pipette, add 250
    µl of LB broth. Close the tube.
    Dispose of the pipette in the
    designated waste container.
    Incubate at room temperature
    for 10 minutes.

12. Repeat #11 using the (-DNA) tube. Incubate at room temperature for 10 minutes.

13. After 10 minutes, tap both tubes to mix the bacteria with the LB broth. Use a
    sterile pipette and transfer 100 µl from the (+DNA) tube onto the LB/amp plate.
    Discard the pipette.

14. Using a sterile loop, gently spread the liquid from #13 evenly around the agar in
    the Petri dish. Be careful that you do not gouge the agar. DO NOT LIFT OFF

15. Repeat #13 and #14 using a new pipette and loop for each of the other plates. Be
    sure you transfer the (+DNA) to the (+DNA) plate and the (-DNA) to the (-DNA)
    plates. Discard the used pipettes and loops in the designated waste container
    immediately after each transfer.

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16. Allow the liquid to dry for a short time. Turn the Petri
    dishes upside down and stack them. Tape the Petri
    dishes together and incubate at 37°C for 24 hours.
    If necessary, the Petri dishes can be incubated at
    room temperature for three days.



1.    Carefully remove the tape from the Petri dishes and observe the bacterial growth
      by placing each Petri dish under the UV light. DO NOT OPEN THE PETRI DISH
      AND AGAR. Record your observations.
2.    Count all of the colonies on each Petri dish. A colony will appear as a small circle
      of bacteria on the agar. Complete the chart named Data Chart #2.


      Observe and record the data from the four plates on Day 2:

                                                  Qualitative    Quantitative Observations*
              Plate             Sketch           Observations*     (count the number of colonies)

       LB / amp

       LB / amp / ara


       LB / amp

California Foundation for Agriculture in the Classroom 1997                                         103
      *Note:   Qualitative observations are characteristics such as color, shape and general appearance.
               Quantitative observations are observations you can measure such as amount and size.

3.    After discussing your results with your team, complete the questions and
      conclusions of this activity.


1.    What was the purpose of using four types of agar plate(s)?

2.    Which plate(s) were considered to be control plate(s)? What purpose did each

3     Which plate showed the most growth and why? Which showed the least growth
      and why?

4.    By simply looking at your Petri dish, how do you know if the gossypol gene was
      placed into the E. coli?

5.    Compare your observations of the starter plate and the transformed plate. How
      was the phenotype of the transformed bacteria different from the phenotype of the
      original bacteria?

6.    Why was an antibiotic gene used in this transformation, since it has nothing to do
      with creating a gossypol-producing cotton plant from a gossypol-deficient cotton

7.    For the transformed bacteria to glow, what two factors must be available in the
      environment? What would happen to a bacterium with this gene, without the
      correct environment? Explain your answer.

8.    Explain how this laboratory activity shows that the following sequence does or
      does not hold true.

                                           DNA         RNA          Protein        Trait

9.    Does this laboratory project complete the project for Cotton Research Associates?
      If so, explain. If not, what must be done after this transformation has occurred?


Write a one page explanation of the experiment that you just completed. Be sure to
explain the variables and controls. Explain what the results were and how you know
whether or not a transformation occurred.

104                                                   California Foundation for Agriculture in the Classroom 1997
*   This procedure was adapted, with permission, from Bio-Rad’s Biotechnology Explorer, Bacterial Transformation laboratory activity
    (#166-0003-EDU). For further inquiries contact Bio-Rad at 1-800-4BIORAD or

California Foundation for Agriculture in the Classroom 1997                                                                        105
                     EXTENSION ACTIVITY

It is important for genetic engineering businesses to know the number of cells that have
been successfully transformed. Knowing the number of transformed cells is critical in
determining the success of this type of procedure.

To calculate the transformation efficiency, you will need to use the following formula:

Transformation Efficiency = Number of cells growing on the LB/amp/ara plate
                                Amount of DNA spread on the agar plate

To calculate this, you will need to complete the following information below:

1. Check your data chart for the number of glowing colonies on your LB/amp/ara plate.
   It can be assumed that each of these colonies originated from a single cell. By
   counting the glowing colonies, you know how many cells were transformed.

                                                    Total Number of cells =

2. To calculate the amount of DNA spread on the agar plates, you will need to
   determine the amount of plasmid that was placed into the microtube. Use the
   formula below to calculate this information.

           DNA (µg) = concentration of DNA (µg/µl) X volume of DNA (µl)

      The concentration of the DNA was 0.03 µg/µl. The loop held 10 µl of plasmid.

                       Total amount of DNA (µg) used in this experiment =

3. You now need to determine the ratio of DNA used. The liquid in your microtube
   after all the ingredients were added consisted of CaCl2, LB broth and bacteria
   containing the plasmid. You now need to calculate the ratio of the bacteria that was
   spread to that in the microtube. To do this, determine from your procedure the
   amount of DNA spread on the LB/amp/ara plate as well as the total amount of fluid
   in the microtube. (Hint: This will include the CaCl2, LB broth and the amount of
   plasmid.) Once that has been determined, use the formula to calculate the ratio of
   DNA used.
          Ratio of DNA used = Volume spread on the LB/amp/ara plate
                               Total sample volume in test tube

106                                        California Foundation for Agriculture in the Classroom 1997
                                                                Ratio of DNA used =

4. The amount of DNA spread on the LB/amp/ara plate now needs to be calculated to
   determine the number of micrograms of DNA spread. To do this, use the following

     Plasmid DNA spread (µg) = Total amount of DNA used (µg) x ratio of DNA used

    (Hint: The total amount of DNA used can be found in #2, while the ratio of DNA
           can be found in #3.)

                                                         Plasmid DNA spread (µg) =

5. Transformation efficiency can now be calculated. To do this, use the following

     Transformation Efficiency =            Total number of cells growing on the agar plate
                                           Amount of plasmid DNA spread on the agar plate

    (Hint: The numbers for this equation can be found from #3 and #4 above.)

                                                         Transformation efficiency =

Note: This should be a large number. Scientists usually convert numbers into
      scientific notation. To do this, write the first numeral to appear between 1 and
      10. Count the number of places you moved the decimal to do this. The number
      of places you needed to move the decimal will be the number of places to the
      upper right of the ten. For example, the number 5400 will be expressed as 5.4 x
      103. Express your transformation efficiency in scientific notation.

                                                         Transformation efficiency =

6. Most researchers find the transformation efficiency to be between 5 x 103 and
   5 x 104. How does your transformation efficiency compare to this figure?

California Foundation for Agriculture in the Classroom 1997                                   107
*   This procedure was adapted, with permission, from Bio-Rad’s Biotechnology Explorer, Bacterial Transformation laboratory activity
    (#166-0003-EDU). For further inquiries, contact Bio-Rad at 1-800-4BIORAD or

108                                                              California Foundation for Agriculture in the Classroom 1997
               (Using Bacteria to Insert Chimeric Genes into Plant Genomes)

The purpose of this activity is to sequentially show students how chimeric genes enter plants using
natural bacteria. Students will discover that naturally occurring bacteria can be used to produce
genetically transformed plants.

• Agrobacterium and E. coli are bacteria that naturally exist in the environment.
• Tissue culture is a method of asexual reproduction that allows genetically identical organisms to
  be propagated from one cell.
• Co-cultivation is a process by which two known organisms grow together—one example is
  Agrobacterium and plant cells.
• Genetic material can be transferred from one organism into another through the use of
  specialized bacteria.

For each partnership:
• Follow-Up Research Memo (p. 70)
• Agrobacterium Summary Sheet (p. 71)
• Tissue Culture Sequencing Activity (pp. 72-73)
• Glue stick
• Scissors
• Ruler
• 12” x 18” paper
• Markers
• Tissue Culture In Plants reading assignment—optional (p. 69)
• Photographs of tissue culture methods in the laboratory (available in many textbooks)

Teacher preparation ......................................... 10 minutes

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Student activity ................................................. One 40-minute session
After this lesson, the students should have a basic understanding of how to produce genetically
identical plants using natural processes and tissue culture. Although the processes are quite
complex, the students will understand that once the system for replication of genetically engineered
plants is refined for a particular situation, laboratory technicians are able to complete the work with
adequate training. It is appropriate to talk with students about the various career opportunities in the
area of biotechnology. In fact, encouraging students to participate in laboratory internships could be
beneficial to both the students and the biotechnology industry.

1. Explain the concept of tissue culture and its use as a biotechnological tool. If appropriate, have
   the students read and complete the Tissue Culture in Plants lesson (p. 69) at home prior to the
   sequencing activity they do in class.
2. Distribute the Follow-Up Research memo (p. 70) and Agrobacterium Summary Sheet (p. 71) to
   the students. Have the students read and discuss the material. Be sure to discuss the
   relationship between the transformation activity and tissue culture—transformation precedes
   tissue culture.
3. Distribute the Tissue Culture Sequencing Activity Sheet (pp. 72-73) to pairs of students. Have
   the students complete the pre-activity questions, the sequencing activity and the follow-up
   questions. Discuss the results. A Tissue Culture Sequencing Activity answer key is provided on
   page 75.

• Genetically identical plants can be produced using tissue culture techniques.

• Assign a tissue culture textbook reading assignment to students.
• Have the students perform actual tissue culture activities.


• Visit a biotechnology company which uses tissue culture as a propagation technique.
• Investigate how hydroponic set-ups are used in tissue culture.
• Infect tobacco plants with Agrobacterium and observe gall formation.

110                                                         California Foundation for Agriculture in the Classroom 1997
• Observe galls in your environment—oak galls are common in California.

California Foundation for Agriculture in the Classroom 1997               111
TISSUE CULTURE IN PLANTS                                      Name ______________________

Read the following information and answer the questions.

Tissue culture includes methods of asexual propagation. Such methods allow
researchers to produce numerous genetically identical plants from one cell. This
method can involve the placement of sterilized terminal shoots, flower or leaf buds onto
a sterile agar gel or nutrient medium. The medium must contain proper nutrients and
growth-regulating chemicals. Most media contain auxin, the hormone responsible for
root formation, and cytokinin, the hormone responsible for shoot formation.

Tissue culture procedures must be performed in a sterile environment in order to
prevent infection. The work is done in a laminar flow hood that is equipped with an
alcohol lamp and dissecting instruments sterilized in 95% ethyl alcohol. Before placing
plant tissue into the hood, the plant is treated with a solution of 10% bleach and 70%
ethyl alcohol or hydrogen peroxide for 10 to 20 minutes. After washing, a few cells are
removed and plated onto the agar gel. A callus (ball of undifferentiated cells) will begin
to grow. The callus is the scar tissue of the plant. This callus can then eventually
develop into a new plant.

Tissue culture can be used to propagate valuable
genetic lines or to regenerate tissue. Many plants
have the unique ability to regenerate via tissue
culture. This is called totipotency—the ability of a
single cell to regenerate into a complete organism.
Tissue culture is one of many laboratory techniques
used by many plant biotechnology companies.


1. Is tissue culture a method of sexual propagation or asexual propagation? What
   does this mean?
2. For successful growth, what must be present in the growing medium and why?
3. Why do you suppose tissue culture needs to be performed in a sterile environment?
4. How are the new plants produced from tissue culture related to the “parent” plant?
5. Explain how the parent tissue is disinfected.
6. What is a callus?
7. Give some examples of how you think tissue culture could be used in agriculture.
8. Write one question or concern you have about tissue culture.

112                                        California Foundation for Agriculture in the Classroom 1997

TO:              Tom Davis

FROM:            Alexandra Hoeppner

RE:              Follow-up Research

I hope that it was beneficial for you to work with us last month. The first part
of our project was successful—getting the gossypol gene and glow gene
successfully into the plasmid. We have found, however, that Agrobacterium
is easier to work with than E. coli. The transformation procedures work
similarly in both bacteria. I am including a short blurb about Agrobacterium
that was written for another project.

We now need to determine if this newly transformed bacteria can be placed
successfully into the cotton plant and still function appropriately. If we are
successful at creating a glandless cotton plant that is genetically engineered
to produce gossypol, we will be ready to move forward with funding
proposals which will allow us to do more research on the gossypol regulator

I have included some information about what we need to do next. Your
artists will find it useful as they sketch out a storyboard for your Board of
Directors. Good luck with your presentation. I’m rooting for your success!

California Foundation for Agriculture in the Classroom 1997                     113
                                          (Week 3)

Editorial Note:    This brief description is one of an eight-part series of summaries intended
                   to provide all staff members of Agri-Gene an overview of biotechnological
                   concepts. It corresponds with our once-a-month lunch lecture series on
                   specific topics.

Agrobacterium is a bacterium that naturally lives in the soil and causes a plant disease
called crown gall. Agrobacterium causes this disease by putting some of its plasmid DNA
into the plant’s DNA and tells the plant to grow the gall. The gall supplies the bacterium with
food to continue growing and dividing, thus spreading the disease. When a susceptible
plant is wounded by poor cultivation practices or animals, Agrobacterium can transfer its
DNA into the plant’s DNA. In the laboratory, wounds are purposely caused by cutting the

The mechanisms of Agrobacterium and crown gall disease are used in tissue culture.
Currently, we are trying to insert a gossypol gene and a glow gene into the genome of a
glandless cotton plant. In other words, we are taking a plant that does not have the ability to
produce gossypol and trying to give it the ability to make gossypol. If this works, we will
seek funding from Cotton Research Associates for further research to find a regulator gene
that will allow gossypol to be made only in the leaf and stem portions of the plant, but not in
the cottonseed. The success of this venture will be discussed at an upcoming brown bag
lunch lecture.

When genetic engineers use Agrobacterium, they remove the DNA between the genes
which causes a gall to grow and replace it with new desired DNA sequences and introduce
a selectable marker, such as an antibiotic resistance gene.

The selectable marker, such as an antibiotic resistance gene, gives a bacterium the ability
to grow in an antibiotic that would normally kill it. Approximately two out of 1,000 plants
produced through tissue culture will be transformed plants. These “transformed” plants
contain the desired trait, such as the gossypol producing gene, and the selectable marker,
such as the antibiotic resistance gene.

To get a gene into a plant, a genetic engineer needs to use both tissue culture techniques
and the natural DNA capabilities of Agrobacterium. After a seed germinates and the
cotyledon leaves form, the cotyledon leaves are cut into pieces and placed onto an agar
feeder plate that conditions the cotyledons overnight. The cotyledons are then placed into
an Agrobacterium solution for about five minutes. The Agrobacterium used has its DNA
that causes crown gall growth removed, and it contains the chimeric gene that scientists
want to incorporate into the plant DNA. The cotyledons are then removed from this solution
and placed back on to the feeder plate for two days. After about five days, the cotyledons
start to form offshoots and some callus (scar tissue) forms. The offshoots are transplanted
onto an agar plate. As they mature, they are transferred onto a final agar plate that
contains rooting solution and antibiotic. The antibiotic selects for only those plants which

114                                          California Foundation for Agriculture in the Classroom 1997
have been transformed. Finally, the transformed plant is planted into soil for further growth
and more tests.

California Foundation for Agriculture in the Classroom 1997                                115
                                   (Student Activity Sheet)


The purpose of this activity is for you to visualize the process a laboratory technician performs
to create numerous plants made of genetically identical DNA. This sequencing activity you are
about to perform illustrates the replication of glandless cotton plants that have the gossypol
producing gene inserted into their cells.


1.     What is a crown gall?
2.     Why are cotyledons used in this experiment?
3.     Why is an antibiotic used in the nutrient solution?
4.     Is it possible for Agrobacterium to be put in a solution and be absorbed through the
       roots? Explain.
5.     Why are new plants containing a chimeric gene developed using tissue culture
6.     What is the chimeric gene your group has developed?


• Tissue Culture Sequencing Activity Sheet
• Scissors
• Glue
• Ruler
• 12” x 18” paper
• Markers


1.    Review the Follow-Up Research Memo and the Agrobacterium Summary Sheet.
2.    Study the pictures on the attached Tissue Culture Sequencing Activity Sheet.
3.    Using scissors, cut out the boxes and determine the correct sequence—start to finish.
4.    Place the boxes, in order, on the paper provided, leaving space for a caption that explains
      each picture.
5.    Number each picture and glue it on the paper. Write a caption for each picture explaining
      the sequential steps of this tissue culture procedure.

116                                            California Foundation for Agriculture in the Classroom 1997
          Agar plate with rooting solution                              Clipped Cotyledon

       Transformed             Not Transformed
      (roots) Healthy       (no roots) grows poorly
                     Cotyledons                                        Cotyledon offshoots

      Overnight conditioning of Cotyledons                                Co-Cultivation

                                                          Cotyledon with growth hormone, Agrobacterium
                                                                       Kanamycin, nutrients
              Transplanted offshoots                                 Seed with nutrient agar

                    Feeder plate                              Offshoots transferred to a pot with soil

California Foundation for Agriculture in the Classroom 1997                                              117
                                             (Answer Key)

         Agar plate with rooting solution                             Clipped Cotyledon

       Transformed          Not Transformed
      (roots) Healthy    (no roots) grows poorly
                   Cotyledons                                        Cotyledon offshoots

      Overnight conditioning of Cotyledons                              Co-Cultivation

                                                     Cotyledon with growth hormone, Agrobacterium
                                                                  Kanamycin, nutrients
             Transplanted offshoots                                Seed with nutrient agar

118                                              California Foundation for Agriculture in the Classroom 1997
                    Feeder plate                              Offshoots transferred to a pot with soil

California Foundation for Agriculture in the Classroom 1997                                              119
                  LESSON 6: THE PRESENTATION
                 (Presenting Your Research and Seeking Further Funding)

The purpose of this lesson is to encourage the students to think about the biotechnology activities
they have performed and to present their findings to the class (the Board of Directors of Cotton
Research Associates).

• Biotechnology, specifically genetic engineering, is a complicated subject both scientifically and
• Collaborative efforts between industry, educators, agriculturalists and the government are
  advantageous and crucial in today’s technological society.
• In today’s technological society, it is important for all people to have an understanding of science
  and to have problem-solving and critical thinking skills.

For each team:

• Presentation Task Sheet (p. 79)
• Butcher paper
• Markers
• Supplies to prepare visual aides

Teacher preparation ............................................... 10 minutes
Student presentation preparation ........................... One or two 50-minute sessions, plus homework
Student presentations and class discussion ......... Two 50-minute sessions


120                                                California Foundation for Agriculture in the Classroom 1997
Throughout this unit, you have provided students with opportunities and tools to learn about the
science of biotechnology, specifically genetic engineering. It is important that you provide time for
your students to process what they experienced and share their knowledge with others.
Many issues associated with genetic engineering are constantly arising in the news. Through their
experiences in the lab, your students will be better prepared to think critically about such issues. It is
important to remind students to think critically, considering the author(s) or source(s) of their
information, before making decisions of their own.

1. Divide students into groups of three to four. Provide a Presentation Task Sheet to each group
   and discuss the purpose of the presentation they are to prepare and the procedures they are to
2. Provide enough time in class for your students to prepare the drafts of their presentation script
   and visual aids. The final products should be created at home with time in class to finalize group
   presentation plans.
3. Have each student group present reports to the class. Discuss appropriate concerns and ask
   appropriate questions. Assess students on the thoughtfulness of the topics and on the quality of
   their presentations.
4. Conclude this unit by having the students write an evaluation of the unit . . . what they liked, what
   they did not like, what they learned, what challenges they had, what opinions they now have on
   the subject of genetic engineering in plants, etc.

• Assign one group to prepare a presentation which would not support further funding of the
• Have each student or student group prepare a written funding proposal to Cotton Research
  Associates. Provide specific grant-writing guidelines for the students to follow.
• Develop a role-play where students act as different members of a Cotton Research Associates
  funding meeting. Some students could prepare a presentation, other students could support the
  funding, others could oppose funding and some students can act as the voting members of the
• Invite parents to the oral presentations.
• Have the students apply what they learned to another situation, such as producing sweeter
  strawberries or drought tolerant lettuce.

• Have the students write a follow-up proposal to the one they just completed. What research
  would they like to pursue next?

California Foundation for Agriculture in the Classroom 1997                                           121
• Invite various people into class to discuss their professions—research scientists, fund
  development coordinators, city board members, biotechnological industry representatives, etc.
            Discuss farmers’ concerns and appreciation of genetically engineered crops.

122                                            California Foundation for Agriculture in the Classroom 1997
                       PRESENTATION TASK SHEET


As Cotton Research Associates representatives, you have now gathered the information you need
for your presentation to the Board of Directors of Cotton Research Associates. The purpose of this
presentation is to gather support for the funding needed to complete the development of a new strain
of cotton that still resists pests, but no longer has gossypol in the seed. Your group will develop and
present an oral presentation to the Board of Directors.


        1) Discuss with your teammates the importance of developing a strain of cotton that does not
           have gossypol in its seed.
        2) Review the process by which a chimeric gene can be inserted into a cotton genome.
           Remember to discuss the functions of Agrobacterium, tissue culture, selectable markers,
           promoter and terminator sequences and restriction and ligating enzymes.
        3) Discuss how the desired phenotype is expressed after protein synthesis.
        4) Discuss bioethical issues pertaining to this particular process. What groups may support
           this project and why? What groups may oppose this project and why?
        5) Prepare your presentation to the Board of Directors requesting financial support of this
           project. The presentation should focus on the information reviewed and discussed in
           steps 1–5 above and clearly state how further funding will be used. Use the following
           checklist to determine the completeness of your presentation.
                   Does your presentation include information discussed in #1 through #4 above?
                   Have you explained the research already performed and what research still needs
                   to be funded before this project is successful?
                   Does your presentation clearly state how further funding will be used?
                   Is your presentation organized in a sequential fashion clearly expressing your
                   Are your visual aids eye catching, easy to follow, attractive to potential viewers and
                   Is the name of your project, with the author’s names, clearly visible?
                   Is the scientific information accurate?
                   Did you proof for grammar and spelling errors on all written work?
                   Have all members of your team contributed in an equal manner to the completion
                   of the project?
                   Do all group members have a part in the oral presentation?
                   Did you practice your oral presentation?
                          Do all members of your group agree that the presentation, preparation and
                                         display are complete?

California Foundation for Agriculture in the Classroom 1997                                           123
                  WHERE DO GENES COME FROM?
This chart lists a variety of agricultural crops that have been genetically engineered. It also provides
you with information on the sources of the genetic materials, the names of the genes and
descriptions of how the new traits are expressed.

      Crop           Source of Genes             Name of Gene                          New Trait

Tomato               Tomato                Antisense enzyme                To soften slowly to allow it to
                                                                           remain on the vine longer

                     Virus                 Coat protein                    Virus resistance

                     Bacteria              Sugar                           Extra sweet

                     Bacteria              Vitamin A, B-carotene           Extra nutrition

Canola               Various plants        Enzymes for oil                 Lower content of saturated oils

                     Various plants        Synthesis                       Special oil compositions for
                                                                           producing shampoo, synthetic
                                                                           lubricants and shortenings

Squash,              Virus                 Coat protein                    Virus resistance

Potato               Bacteria              Starch                          Increased nutrition

Soybean,             Legumes and nuts      Storage proteins                Increased protein in the plant
Sunflower,                                                                 by-products so they can be
Canola                                                                     used for nutritious animal feed

Chrysanthemum        Bacteria              Antisense pigment genes         Pure white petal color

                     Plants                Ripening genes                  Increased size and firmness

Papaya               Plants                Ripening genes                  Increased flavor and firmness

                     Virus                 Coat protein                    Virus resistance

124                                                California Foundation for Agriculture in the Classroom 1997
                           CAREERS IN BIOTECHNOLOGY

A career in biotechnology offers an individual a wide range of career choices. A strong background
in science is recommended. The diagram below shows the connection between biotechnology and
the sciences.

                                   A MOSAIC OF BIOTECH FIELDS AND CAREERS


                                   Ecologist                    Biochemistry
                                                                                    Genetics            Evoluntionary
                                                                                                          Genetics            Anthropologist
                                                                                  Animal Scientist

                                                          Biochemical Genetics                                              Biologist
   Genetic Counselor

     Medicine             Medical Genetics                                                   General Genetics

    Pathologist         Pharmacologist

             Molecular                                             Genetics                                              Systematics

                                                                                                                  Behavioral Genetics
                                               Horticulturist                     Botanist


                                                                               Ecosystems                  Development
                       Euthenics               Geographer
                                                                                Ecologist                    Biologist

California Foundation for Agriculture in the Classroom 1997                                                                              125

Ag Access: Agricultural Book Source, P. O. Box 2208, Davis, CA 95617; (916) 756-7177.
      Request a catalog of agricultural books.

Agriscience—Fundamentals and Applications textbook, Elmer L. Cooper, Delmar Publishers,
       Inc., 1990. A general high school agriscience textbook written in an easy-to-read format.

“Agricultural Biotechnology: A World of Opportunity” video, National FFA Center,
       5632 Mt. Vernon Memorial Highway, Alexandra, VA 22309-0160;
       (703) 360-3600. This video describes the various career opportunities available in
       agricultural biotechnology.

American Society for Microbiology, ASM Finance Department, 1325 Massachusetts Avenue, NW,
      Washington, DC 20005-4171; Fax: (202) 942-9347. Members of this organization receive
      newsletters and journals. Contact the association for membership information.

An Introduction to Biotechnology: A Junior High Unit, Monsanto Fund and National Science
       Foundation Mathematics and Science Education Center, 8001 National Bridge Road,
       246 Benton Hall, St. Louis, MO 63121; (314) 553-5552. Educational lessons designed to
       teach about biotechnology, including genetic engineering.

Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547; (800) 424-6723. The
      company provides many biotechnological classroom kits, including the DNA transformation
      kit recommended for use with this unit. Request a free catalog.

“Biotechnology—Careers for the 21st Century” video, National Association of Biology Teachers,
       11250 Roger Bacon Drive #19, Reston, VA 22090; (703) 471-1134. This video shares
       interviews of people involved in the many careers associated with biotechnology.

Biotechnology Education Program, University of California, Davis, CA 95616; (916) 752-3260.
       Request information specific to your needs, or a packet of general information on

Biotechnology: Microbes and the Environment pamphlet, Steve Nitt, Center for Science
       Information, 1990. Provides information on how microbes can be used as a biotechnology

Blazing a Genetic Trail pamphlet, Howard Hughes Medical Institute, 6701 Rockledge Drive, Bethesda,
       Maryland 20817. A pamphlet on human genetics and medicine.

“Building a Better Tomato” article, Ricki Lewis, High Technology, May 1996. A highly detailed
       discussion of tomato biotechnology.

Calgene, Inc., Public Affairs, 1910 Fifth Street, Davis, CA 95616; (916) 753-6313;
      Fax: (916) 753-1510. A genetic engineering company that has press packets available on
      some of their products, including plant oils, tomatoes and cotton.

126                                            California Foundation for Agriculture in the Classroom 1997
California Foundation for Agriculture in the Classroom, P.O. Box 15949, Sacramento, CA
       95853; (800) 700-AITC. Provides low-cost educational resources and lessons which
       incorporate agriculture into existing curricula. Request a Teacher Resource Guide and ask
       to be put on the mailing list.

California Science Teachers Association, 3550 Watt Avenue, #120, Sacramento, CA 95821-
       2666; (916) 979-7004. This association provides newsletters and journals to California
       science educators about ideas, issues and trends in science education.

Carolina Biological Supply Company, 2700 York Road, Burlington, NC 27215; (800) 334-5551,
       ext. 5310. This company has a wide variety of science supplies, including DNA
       fingerprinting and transformation kits. Request a catalog.

“Cotton’s Journey From Seed To You” kit, The Alaca Company, P.O. Box 55, Tranquility, CA
      93668; Fax: (209) 698-5190. Contains booklet, teaching guide, student manual, video,
      cotton samples and other materials to teach students the journey of cotton from the farm to
      the home. Approximate cost is $70.

“Food Biotechnology” video, Dr. Christine Bruhn, Center for Consumer Research, University of
      California, Davis, CA 95616; (916) 752-2774. A video and worksheet which educates
      consumers about food biotechnology and the current issues associated with it.

Genetics Heads for the Supermarket pamphlet, World Book Encyclopedia, 1560 Sherman
       Avenue, Suite 1111, Evanston, Illinois 60201, Publ #5285. An easy-to-understand reading
       on food biotechnology.

Industrial Biotechnology Association, 1625 K Street NW, Suite 1100 Washington, DC 20006;
       (202) 857-0244. A variety of basic and detailed information on biotechnology, including
       genetic engineering.

Logal Science and Math Software, 125 Cambridgepark Drive, Cambridge, MA 02140;
       (800) 564-2587. A variety of CDs and other software programs are available. The Molecular
       Biology Explorer 3.0 CD ROM is a good supplemental tool for studying molecular biology.
       Request a catalog.

National Cotton Council of America, P.O. Box 12285, Memphis, TN 38182; (901) 274-9030.
       Several resources are available on United States cotton production.

Plant Molecular Biology, A Practical Approach textbook, C.H. Shaw, IRL Press Limited, 1988.
       Provides practical, simple information on plant molecular biology.

Recombinant DNA: A Short Course textbook, J. Watson, J. Tooze and D. Kurtz, Scientific
     American Books, W. H. Freeman and Company, New York, 1983. Provides illustrations and
     graphics of recombinant DNA technology.

Science Framework for California Public Schools, K-12, 1990. Bureau of Publications, Sales
      Unit. California Department of Education, P.O. Box 271, Sacramento, CA 95812-0271;
      (916) 445-1260. The state guidelines for teaching science. Other subject matter frameworks
      are also available.

The Agricultural Dictionary book, Ray V. Herren, and Roy L. Donahue, Delmar Publishers, Inc.,
      1991. Provides definitions of agricultural terms.

California Foundation for Agriculture in the Classroom 1997                                      127
The Cartoon Guide to Genetics booklet, Larry Genick, Harper Collins Publishers, 1991. Can be
      used to add humor to your genetics lessons.

University of California Cooperative Extension, Refer to your local telephone book in the
       government or county section for address and phone number. Practical and research
       publications are available on agriculture. One such newsletter is called the California Cotton

128                                             California Foundation for Agriculture in the Classroom 1997

AGROBACTERIUM—A pathogenic bacterium that causes crown gall disease. Used in genetic
   engineering to put chimeric genes into plants.

AMINO ACID—The building blocks of proteins. Codons code for amino acids.

BIOTECHNOLOGY—A number of technologies involving living organisms used to produce useful
    products, processes and services.

CHIMERIC GENE—A gene that contains DNA from at least two different sources.

CHROMOSOME—DNA strands responsible for the determination and transmission of hereditary

CLEAVAGE SITE—The site on a DNA strand that restriction enzymes recognize and then cut.

CODON—A triplet of bases within a molecule of DNA or mRNA that codes for a particular amino

COTYLEDON—Often the first “leaves” on a new plant; part of a seed that provides nutrients and
    protection to a plant embryo.

E. COLI—A bacterium naturally found in the environment in a variety of strains. Selected strains are
     used in genetic engineering.

ENZYME—A protein that functions as a biological catalyst.

GENE SPLICING—A procedure by which one DNA molecule is attached to another DNA molecule.

GENES—Sections of DNA that code for a specific trait.

GENETIC ENGINEERING—Alteration of an organism by inserting genes from another organism into
    its genome.

LIGATING ENZYME—Enzymes used to fuse or “glue” DNA strands together.

mRNA—Messenger RNA; codes for the production of amino acids.

MUTATION—A random change in the genetic material.

PLASMID—Circular DNA molecules found in bacteria that are not part of a chromosome.

PROTEIN—Chains of amino acids that perform the necessary functions of living organisms.

PROMOTER SEQUENCE—A sequence of bases within a DNA strand that initiates transcription by
   RNA polymerase.

California Foundation for Agriculture in the Classroom 1997                                      129
RECOMBINANT DNA—A DNA strand comprised of pieces of DNA from two or more different
    sources spliced together.

RESTRICTION ENZYME—An enzyme that cuts a DNA molecule at a specific nucleotide sequence.

SELECTABLE MARKER—An identifiable gene used to determine whether or not a genetic
    transformation has taken place.

TERMINATOR SEQUENCE—A sequence of bases on a DNA molecule that stops protein synthesis.

TISSUE CULTURE—A method of asexual propagation which allows genetically identical plants to
    be propagated from one cell.

TRANSCRIPTION—The process by which the genetic information contained in DNA is converted to
    a molecule of RNA.

TRANSLATION—The process by which genetic information encoded in mRNA in the form of
    codons is converted into a sequence of amino acids that form a protein chain.

130                                         California Foundation for Agriculture in the Classroom 1997

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