Module II – Table of Contents › publications › bt_curriculum › bt_curic_mod… · Lesson Module II – Insect-Resistant Crops Using Bt Educators II Insect-Resistant Crops

iIowa State University Extension and ISU Office of Biotechnology

Module II – Table of Contents

II. Insect-Resistant Crops Using Bt Page

BACKGROUND INFORMATION FOR EDUCATORS

Bt corn ....................................................................................................... 55

Bt cotton .................................................................................................... 57

Bt potatoes ................................................................................................. 58

TEACHING RESOURCES

Laboratory lesson plan: Bt vs. the European Corn Borer ............................. 59

Laboratory lesson plan: Bt Detection – Corn Leaf Tissue Test ..................... 63

Internet ideas.............................................................................................. 65

Student handout: Learning More About European Corn Borers ...................... 67

Student handout: See For Yourself – Bt vs. European Corn Borers .................. 71

Student handout: See For Yourself – Bt Detection: Corn Leaf Tissue Test........ 75

Overhead transparency masters .................................................................. 79

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55Iowa State University Extension and ISU Office of Biotechnology

EducatorsLesson Module II – Insect-Resistant Crops Using Bt

II Insect-Resistant

Crops Using Bt

BACKGROUND INFORMATION

Bt Corn1

The European corn borer (ECB), Ostrinia nubilalis(Hübner), significantly affects production of field corn,popcorn, and sweet corn (Zea mays L.) Yield losses andcontrol expenditures associated with the ECB cost U.S.farmers more than 1 billion dollars annually.

The ECB is an introduced insect species that belongs tothe family Pyralidae in the order Lepidoptera. Itprobably arrived in North America during the early1900s in broom corn imported from Hungary and Italyfor the manufacture of brooms. First noticed nearBoston, Massachusetts, in 1917, the European cornborer also was found in 1921 in areas bordering LakeErie. It spread gradually from southern Michigan andnorthern Ohio. By the end of 1938, it had spread as farwest as the Wisconsin shore of Lake Michigan.

During its early history in the United States, the ECBproduced one generation per year. By the late 1930s,some of the ECB in eastern and north central stateswere able to produce two generations each year. Thistwo-generation ECB spread rapidly and soon becamedominant in the central Corn Belt. It reached Illinois in1939, Iowa in 1942, Nebraska in 1944, and SouthDakota in 1946.

Meanwhile, the single-generation ECB spread north-ward into northern Minnesota, North Dakota, and theCanadian provinces of Quebec, Manitoba, andSaskatchewan.

Later, three- and four-generation ECB appeared in thesouth along the Atlantic Coast and southwestward inMissouri, Arkansas, Kansas, Oklahoma, and the Gulfstates.

The insect has continued to spread throughout the corngrowing areas of the United States. In the years sincebeing discovered, the ECB has spread northward intoCanada, westward to the Rocky Mountains, andsouthward to Florida and New Mexico. It is nowpresent in all but the seven most western continentalstates.

How Corn Is Damaged by the European

Corn Borer1

A corn plant goes through a series of stages during itsgrowth and development. During these stages, theplant uses its resources for rapid growth and for generalplant maintenance. Because the plant’s ability towithstand stress varies during plant growth stages, thestage(s) at which it is attacked influences its ability todeal with injury from the ECB’s feeding. The corn plantis susceptible to ECB attack and injury after the 6th-leafstage through maturity of the grain.

During early stages of corn growth, there is between 5and 6 percent loss in grain yield for each larva on aplant. During ear development stages, the loss fromeach larva on a plant is about 2 to 4 percent. If cornplants experience prolonged moisture stress aftersignificant ECB feeding, the loss from each larva can beas high as 12 percent.

Damage and yield loss result from:

1. Leaf feeding (first generation);2. Midrib feeding (first and second generation);3. Stalk tunneling (first and second generation);4. Leaf sheath and collar feeding (second and third

generation); and5. Ear damage (second and third generation).

See illustrations of damage sites on p. 68 and p. 85.

ECB damage results in poor ear development, brokenstalks, and ears that fall off the plant before harvest.Most yield loss can be attributed to the impaired abilityof plants to produce normal amounts of grain due tothe effect of larval damage to leaf and stalk tissues.With strong autumn winds and dry weather, tunneling

A European corn borer (ECB) larva on a corn leaf. Keith Weller,ARS-USDA

56 Iowa State University Extension and ISU Office of Biotechnology

Educators Lesson Module II – Insect-Resistant Crops Using Bt

in the stalks and ear shanks can increase stalk andshank breakage, resulting in substantial loss of earsduring harvest.

Research has shown a close association betweensecond- and third-generation ECB infestation andincidence of stalk rot, caused by a fungus. This increasein stalk rot is directly related to ECB larvae boring intostalks and ear shanks, which provides an opening forthe fungus to enter. Losses due to weakened stalksincrease when corn harvest is delayed.

Loss of grain caused by direct feeding of the ECB onmature kernels is usually not important in field corn;however, in sweet corn and popcorn, losses can besignificant. Feeding on sweet corn is especiallyimportant to canners and gardeners.

How Bt Corn Helps Manage the

European Corn BorerFor a complete discussion of how the Bt gene affectsinsect digestion, see Lesson Module I of this curricu-lum. In brief, Bt (Bacillus thuringiensis) is a soilbacterium whose spores contain a crystalline (Cry)protein. When certain insects eat a plant part contain-ing the Cry protein, the protein breaks down andreleases a toxin, known as a delta-endotoxin. The toxinbinds to the insect’s intestinal lining, creating tiny holes.The damaged digestive system is paralyzed so the insectcannot digest its food. In a few days, the insect dies.Different types of Cry proteins affect different ordersof insects.

Current Status of Bt Corn Varieties2

The Environmental Protection Agency (EPA) regulates

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Table 3



insect-resistant crops. For detailed information aboutthe EPA’s regulatory authority, see the web site at http://www.epa.gov/pesticides/biopesticides/ or the publica-tion at http://www.biotech.iastate.edu/biotech_info_series/bio11.html.

The EPA issued a document titled Biopesticides Registra-tion Action Document for the Bacillus thuringiensis (Bt)Plant-Incorporated Protectants dated October 15, 2001.In this document, the EPA stated that Syngenta Seeds,Inc. (formerly Novartis Seeds, Inc.) and Mycogen Seedsc/o Dow AgroSciences had notified the EPA that theywould be phasing out their Event 176 corn productsbased on the Cry1Ab protein. The event expired onApril 1, 2001, for Syngenta Seeds and on June 30, 2001,for Mycogen Seeds. The companies must use existingseed of these products before or during the 2003growing season.

The Aventis company requested a voluntary cancella-tion of its Cry9C StarLink corn registration, and thecancellation became effective on February 20, 2001.StarLink was approved only for animal food use. Itsregistration was cancelled when the Cry9C protein wasfound in human food products during the fall of 2000.For more information about StarLink, see the web siteat http://www.extension.iastate.edu/Pages/grain/gmo/gmo.html.

Current Bt corn registrations are set to automaticallyexpire on October 15, 2008. These corn registrationsare in Table 3 on the previous page.

Bt Cotton3

The Bt protein targets Lepidopteran insects, includingpink bollworm (Pectinophora gossypiella [Saunders]),tobacco budworm (Heliothis virescens [ Fabricius]) andto a lesser extent the bollworm (Heliothis zea [Boddie]).Another major pest of cotton, the boll weevil, is notcontrolled by the Bt protein but has been the target ofan eradication program in the U.S.

Before the boll weevil eradication program, the chemi-cal insecticides used against the boll weevil controlledmost other insects on cotton, including bollworm andbudworm. The eradication program used malathionagainst the boll weevil. Malathion eradicated bollweevils and controlled many other secondary pests.However, malathion only injures some insects and killsbeneficial predatory insects, but does not kill bollwormsand budworms. Therefore, when farmers switchedfrom conventional pesticides to malathion, there was arapid increase of bollworms and budworms.

How Cotton Is Damaged by Bollworms

and BudwormsThe cotton bollworm is the larva or caterpillar of thecorn earworm moth. While this pest prefers corn as itshost plant, it will infest cotton, peanut, sorghum,soybean, tobacco, tomato, and several other cultivatedplants. The tobacco budworm is a close relative of thebollworm and prefers tobacco over cotton.

On cotton, the larvae of these insects require about twoto three weeks to grow and mature. While they grow,they eat. What they eat depends on the stage of thecotton plant when they hatch. For example, up to fourgenerations of these insects occur each year in NorthCarolina. The early generations feed on the tenderupper parts of the plant where new leaves are expand-ing. Later generations move into the blooms, buds, andbolls. A boll is the round seed pod in which the cottondevelops. As seen in the photo above, the insectsburrow into the immature bolls, hollowing them outand giving easy access to plant diseases.

How Bt Cotton Helps Manage the

Bollworms and BudwormsAs shown in Table 1, Lesson Module I, different types ofCry proteins produced by the soil bacterium Bacillusthuringiensis affect different insects. At present, theonly Bt cotton product on the market produces theCry1Ac protein. This protein interferes with thedigestive tracts of the pink bollworm, the tobaccobudworm, and the cotton bollworm.

Current Status of Bt CottonThe EPA has determined that the Cry1Ac productregistration will automatically expire on midnightSeptember 30, 2006, except for the external, unsprayedrefuge option that will expire September 30, 2004.

Cotton bollworm on an immature cotton boll. ARS-USDA



The current Bt cotton registration is as follows:

Crop Name: Cry1Ac Bt CottonPesticide Name: Bacillus thuringiensis kurstaki Delta-Endotoxin as Produced by the Cry1Ac Gene and ItsControlling Sequences as Expressed in CottonTrade and Other Names: BollGard®

Uses: Full commercial use in cotton with geographiclimitations due to weedy relativesTarget Pest(s): Cotton bollworm, tobacco budworm,and pink bollwormRegistrant: Monsanto Company

700 Chesterfield Parkway NorthSt. Louis, Missouri 63198

Bt Potatoes4

The Colorado potato beetle, Leptinotarsa decemlineata(Say), is a major potato pest. It was first recognized as apotato pest in Colorado in 1859 after settlers intro-duced potato plants into the insect’s native range of theeastern Rocky Mountains. The native host for thisinsect is a relative of potato, buffalo bur (Solanumrostratum). It took the beetles about 30 years to adaptto eating potato plants, after which they migrated eastfeeding on potato plants grown in farms and gardensthroughout the Great Plains and Ohio River Valley.

The Colorado potato beetle expanded its range east-ward approximately 85 miles per year, reaching the EastCoast by 1874. The insect is now found in Europe andhas spread eastward to Turkey.

How Potatoes Are Damaged by the

Colorado Potato BeetleThe adult Colorado potato beetle overwinters in the soileither in the potato field itself or in the borders of afield. The following spring, adults begin to emerge atabout the same time potatoes emerge. Adults feed for ashort time in the spring, then begin to mate and layclusters of 10-30 yellow eggs on the underside of theleaf. Females typically lay 350 or more eggs duringtheir life span that can last several weeks.

The Colorado potato beetle has few natural enemies,and those enemies that do feed on the eggs, larvae,pupae, or adults have little impact on the number ofColorado potato beetles. Larvae and adults both feedon the leaves of potato plants and will completelydefoliate a plant if left untreated.

A Colorado potato beetle. Scott Bauer, ARS-USDA

How Bt Potatoes Help Manage the

Colorado Potato BeetleNewLeaf® potato varieties were marketed undercontract with NatureMark, a subsidiary of Monsanto(http://www.naturemark.com). These geneticallyengineered plants contain a bacterial gene from Bacillusthuringiensis var. tenebrionis that produces a protein thatis toxic only to beetles. A limited number of geneticallyengineered varieties have been available for commercialuse. NewLeaf potatoes produced the protein toxin inall green tissues and beetle mortality was essentially100%.

Starting with the 2001 growing season, Monsantodecided to no longer market its NatureMark potatoesdue to the reluctance of some major processors to buythe genetically enginered potatoes in response toconcern about lack of consumer acceptance.

Current Status of Bt PotatoesThe EPA has determined that the Bt potato productdoes not have an expiring registration. The current Btpotato registration is as follows:

Crop Name: Cry3A Bt PotatoPesticide Name: Bacillus thuringiensis Cry3A Delta-Endotoxin and the Genetic Material Necessary for itsProduction in PotatoTrade and Other Names: NewLeaf®

Uses: Full Commercial Use in PotatoesTarget Pest(s): Colorado Potato BeetleRegistrant: Monsanto Company

700 Chesterfield Parkway NorthSt. Louis, Missouri 63198



Credit Notes1Excerpted from or based on European Corn BorerEcology and Management, NCR-327, Charles E. Mason,Marlin E. Rice, Dennis D. Calvin, John W. Van Duyn,William B. Showers, William D. Hutchison, John F.Witkowski, Randall A. Higgins, David W. Onstad, andGalen P. Dively. Iowa State University Extension,publishing state, July 1996. Excerpts viewable on theInternet at http://www.ent.iastate.edu/pest/cornborer/.Used with permission.

2Based on information contained in BiopesticidesRegistration Action Document – Bacillus thuringiensis (Bt)Plant-Incorporated Protectants, U.S. EnvironmentalProtection Agency, Office of Pesticide Programs,Biopesticides and Pollution Prevention Division,October 15, 2001, p. I1-I2. Available in pdf form athttp://www.epa.gov/pesticides/biopesticides/pips/bt_brad.htm. Used with permission.

3Based on information contained in: Biopesticides Registration Action Document – Bacillusthuringiensis (Bt) Plant-Incorporated Protectants, U.S.Environmental Protection Agency, Office of PesticidePrograms, Biopesticides and Pollution Prevention

Division, October 15, 2001, p. I19, I21. Available in pdfform at http://www.epa.gov/pesticides/biopesticides/pips/bt_brad.htm. Used with permission.

“Bollworm Complex” and “Budworms” in Insect andRelated Pests of Field Crops, AG-271, North CarolinaCooperative Extension Service, North Carolina StateUniversity. Viewable on the Internet at http://ipm.ncsu.edu/AG271/cotton/bollworms.html and http://ipm.ncsu.edu/AG271/tobacco/budworms.html. Used withpermission.

4Based on information contained in:Colorado Potato Beetle, David Ragsdale and Edward B.Radcliffe, University of Minnesota Extension Service.Available online at http://www.vegedge.umn.edu/vegpest/cpb.htm. Used with permission.

Biopesticides Registration Action Document – Bacillusthuringiensis (Bt) Plant-Incorporated Protectants, U.S.Environmental Protection Agency, Office of PesticidePrograms, Biopesticides and Pollution PreventionDivision, October 15, 2001, p. I22. Available in pdfform at http://www.epa.gov/pesticides/biopesticides/pips/bt_brad.htm. Used with permission.

II IIII

Insect-Resistant

Crops Using Bt

TEACHING RESOURCES

Laboratory Lesson Plan:

Bt vs. the European Corn Borer

Science Content

• Students will observe and gather data on the effectsof the European corn borer (ECB) on traditionaland Bt corn.

• Students will apply the knowledge and lab data tosolve common problems in today’s agriculture.

IIScience Education Standards

Science as Inquiry, Content Standard A, AbilitiesNecessary to do Scientific Inquiry

– Design and conduct scientific investigations(p. 175)

– Use technology and mathematics to improveinvestigations and communications (p. 175)

– Formulate and revise scientific explanations andmodels using logic and evidence (p. 175)

Life Science, Content Standard C– The cell (p. 184)– Molecular basis of heredity (p. 185)– Interdependence of organisms (p. 186)

Source: National Science Education Standards, ©National Academy ofSciences, 1996. Used with permission. Page numbers refer to theseventh printing, November 1999 – also available on the Internet athttp://books.nap.edu/html/nses/pdf/index.html.

Science Process Skills

• Observing• Relating• Inferring• Applying



Life Skills

• Science processing• Making decisions• Critical thinking• Problem solving

Time

Preparation: Two hours, beginning three weeks beforethe day of the lab activity

Doing the activity: 40 minutes

Materials

Before doing this activity, students should read thepublication, Insect-Resistant Crops Through GeneticEngineering, Biotechnology Information Series, NCR#553, available free to Iowa teachers from the ISUOffice of Biotechnology (contact information below) oron the Internet at http://www.biotech.iastate.edu/publications/biotech_info_series/bio9.html.

• Bt corn seeds and regular corn seeds*• Potting soil• Sand or aquarium rock• Container for plants to grow [4-6 inch peat/plastic

starter pots or large (14"w x 24"l x 11"h) con-tainer]

• European corn borer (ECB) eggs*• Petri dishes (100 mm x 20 mm are best)• Light source for growing plants• Marking pen• Fine-tipped watercolor paintbrush or toothpicks

*Available to Iowa teachers through Iowa StateUniversity’s Office of Biotechnology, phone toll-free inIowa 800-643-9504 or e-mail [email protected].

Optional: Make copies of student handouts II-a and II-b on p. 67-74 or use the overhead transparency mastersII-a through II-f on p. 79-89.

Lesson Plan

This lab needs some advance planning. You will haveto coordinate the arrival times of the corn seed and ECBeggs. Plan time for shipping and preparation. Whenthe corn reaches the 2-3 leaf stage, you will need toorder the ECB egg masses from the ISU Office ofBiotechnology. You need to let the corn grow to thisstage because young corn plants have a natural resis-tance for a short time. The office will request the eggmasses from the USDA-ARS Corn Insect Research Unit,

who will send them to you.

Planting Your Corn – 10-14 Days Before Activity

You or your students should grow approximately 15plants of Bt and non-Bt corn. You will need to plantyour corn 10-14 days before the lab activity. Remem-ber that the corn will grow wide and tall. There areseveral ways to grow your corn. A large inexpensiveplastic container (14"w x 24"l x 11"h), such as onesmade for clothes storage, works well. A container ofthis size could be used to grow 12-15 plants. Place 1.5inches of sand or aquarium rock at the bottom of thecontainer. Over the sand and/or aquarium rock, add 2-3 inches of potting soil. A maximum of three rows withfive plants is enough for the lab activity. An alternativeis to grow each plant in 4-6 inch individual pots ofplastic or peat. It will take the corn 5-7 days to germi-nate, depending on the temperature. Grow the cornuntil it has 2-3 leaves, then order your ECB eggs.

Finding Out About Bt Corn

During the 10-14 days that the corn is growing,teachers may want students to use the library and/orInternet to research Bt corn.

Preparing and Incubating ECB Eggs

It is important to keep the ECB eggs in high humidity.If you want to keep the eggs dormant for up to 7 days,place them in a plastic bag with a damp paper toweland store them in the refrigerator.

If you receive egg masses sent to you on wax paper,scrape the back side of the wax paper (the side withoutthe eggs) on the sharp edge of a table. Turn the waxpaper over (upside down) and snap the paper. The eggmasses will pop off. Make sure you have an areacleared to collect the egg masses because they will flyeverywhere. Collect the egg masses in the containersyou intend to use for incubation. If you receiveindividual egg masses, they will be ready for incubationwhen they arrive. Make sure the corn has 3 leavesbefore incubating the ECB eggs.

Hatching ECB Eggs – 3-5 Days Before Activity

A. Using Petri DishesFor best results, you should incubate the egg masses at80ºF (27ºC) until the eggs reach what is commonlycalled the blackhead stage. Eggs close to hatching havedistinct black centers, which are the black heads of thelarvae that are visible through the translucent eggshells.A standard incubator set at the correct temperatureworks. If an incubator is not available, use a styrofoamcooler with a work light suspended over it. Adjust thelight’s height until the temperature reads 27ºC. Keep a



moist paper towel over the container of eggs masses forhumidity. DO NOT let the egg masses sit in water for along period. Incubate the eggs until black heads appearin the mass.

When the black heads appear, you and your studentscan observe the larvae of the ECB using a microscopeunder scan or low power objectives or a dissectingmicroscope. You should be able to see the larvae movein the eggs. At this time, the eggs are only about a dayfrom hatching. It is critical that the ECB eggs be usedwhen they get to the blackhead stage of their develop-ment. One egg mass yields about 10-20 larvae.

B. Using a Glass JarAn alternative to using petri dishes is to hatch the ECBlarvae in a jar. Place water in a glass jar and swirl untilthe sides are wet. Remove the excess water from the jar.Using a damp watercolor paintbrush, place the eggmasses inside the jar on the sides and let dry. Place adamp paper towel in the jar and seal with the lid.Incubate at 80ºF (27ºC) for approximately 3-5 days oruntil the eggs hatch. You can drop corn leaf cuttingsinto the jar and observe the ECB larvae’s activity. Makesure you mark and keep track of the two varieties ofleaves (Bt and non-Bt). Notching one variety of cornleaf can aid in identification.

C. Using Live PlantsThe egg masses can be applied to the underside of thethird or later corn leaves or in the whorl of the plant.The whorl is the tube-like growing point near the top ofthe plant’s stem/stalk from which new leaves spiral out.You can place the egg masses on corn leaves by using afine-tipped watercolor paintbrush or toothpick. Toplace the egg masses on the underside of the leaf or inthe whorl, pick up a blackheaded egg mass with thebrush or toothpick and touch it to the underside of theleaf or in the whorl.

The Day Before the Activity

Two weeks ago, you started growing two varieties ofcorn, Bt and non-Bt, that are used in this investigation.

Three to five days ago, you began incubating ECB eggmasses.

Now that the corn has grown enough and the ECB eggmasses show black heads, you are ready to proceed withthe lab.

Ask students to read the publication Insect-ResistantCrops Through Genetic Engineering, BiotechnologyInformation Series, NCR #553. Other options are to

ask students to read handout II-a found on p. 67, or usethe overhead transparencies on p. 79-89 to providebackground for the activity.

Tell students: “You are going to repeat the procedure thatresearchers first used to test corn for resistance to theEuropean corn borer (ECB). The ECB causes billions ofdollars worth of damage each year to the corn grown in theUnited States. Control of ECB without the use of insecti-cides has long been a goal of the corn industry. In this lab,you will investigate a variety of transgenic corn thatcontains a gene from the bacteria Bacillus thuringiensis(Bt). The Bt protein is lethal to the ECB when consumed.During the time you have been investigating the transgeniccorn variety, you have been doing some library or Internetresearch (as directed by the teacher). With the research anddata from the lab, you will make predictions about thenumbers of actively feeding larvae and/or percent of leafdamage you will see on Bt and non-Bt corn.”

If doing the petri dish protocol, ask students to recordtheir predictions about the percentage of leaf damagethey will see on the first and second days after thelarvae hatch on the leaves. In the petri dish protocol,nearly all larvae will be dead by the second day.

If doing the live plant protocol, ask students to countthe number of larvae on their plants as soon as theyhatch. Ask students to record their predictions aboutthe percentage of leaf damage and numbers of activelyfeeding larvae that they will see on their corn plants ondays 1, 2, 3, and 4.

Students may record their predictions on the optionaldata sheets on p. 73-74.

Doing the Activity

PART I - Petri Dish Protocol

1. Use a marking pen to write “non-Bt” on the lefthalf of the petri dish and “Bt” on the right half.

2. Place a piece of filter paper in the petri dish.

3. Add 2-3 drops of water to the filter paper.

4. Cut a 1-1 1/2" piece of leaf off each variety of corn.Make sure you keep track of each variety. Re-searchers mark one leaf with a notch to keep track.

5. Place the non-Bt corn leaf on the left side of theplate and the Bt corn on the right side.



6. Place the blackheaded egg masses on the leavesusing a fine-tipped watercolor paintbrush ortoothpick.

7. Cover the top of the petri dish with a damp papertowel and place the lid over the dish. During thecourse of the investigation, keep the paper toweldamp so the humidity will be high. Re-wet thepaper towel when necessary.

8. Incubate at 27ºC (80ºF) until the eggs hatch,which will be about 1-3 days.

9. Observe the ECB behavior and eating activity foreach corn variety.

10. Replace the leaf pieces with fresh cuttings as theold leaf segments dry and/or are consumed.

PART II - Live Plant Protocol

At the same time you infest your leaf cuttings, you caninfest some of the growing plants. Do not infest allyour plants. You will need more cuttings for your petridish part of the investigation. Do not leave youruninfested plants close to the infested ones because thelarvae can move from one plant to another and all theplants may become infested.

1. Using a wet fine-tipped watercolor paintbrush ortoothpick, pick up a blackheaded egg mass andplace it on the underside of the leaf or in the whorl.Place 2-3 egg masses on each leaf of the corn plant.

2. Keep track of the corn varieties and record yourobservations daily.

Reflect and ApplyThese follow-up activity questions appear on optionalstudent handout II-b on p. 72.

Petri Dish Protocol

1. How did the level of leaf damage you observed onday 1 for the non-Bt leaf cuttings compare to thelevel of damage you observed on day 1 for the Btleaf cuttings?

Even on day 1, students probably will observe moreleaf damage on the non-Bt corn leaf cuttings than onthe Bt corn leaf cuttings.

2. How did the level of leaf damage you observed onday 2 for the non-Bt leaf cuttings compare to the

level of damage you observed on day 2 for the Btleaf cuttings?

By day 2, students will observe extensive damage onthe non-Bt leaf, while the Bt leaf will remain with verylittle damage.

Live Plant Protocol

1. How many larvae were on your plants at thebeginning of this experiment?

Student answers will vary.

2. When corn is infected by ECB this early in itsgrowth, assume that there will be a 5% grain lossfor each larva you find on a single plant. Calculatehow much grain loss you could have expected foreach of your plants.

Student answers will vary.

3. How did the level of leaf damage and number ofactively feeding borers you observed on each dayfor your non-Bt corn plant compare to the levels ofdamage and borers you observed for your Bt cornplant?

The level of leaf damage for the non-Bt corn plantshould have steadily increased until by day 4, the non-Bt corn leaves should be nearly consumed. Thenumber of actively feeding borers should haveremained nearly steady.

For the Bt corn plant, the level of damage probablystabilized by day 2 as the number of living borerssteadily decreased until very few, if any, were alive byday 4.

Credit Note

This activity was developed by Mike Zeller for theOffice of Biotechnology, Iowa State University, and isused with permission.



Laboratory Lesson Plan:

Bt Detection – Corn Leaf Tissue

Test

This activity can be done as part of the preceeding “Bt vs.European Corn Borer” lab activity that begins on p. 59 orcan stand alone as a separate activity. Iowa teachers canorder the Bt and non-Bt corn seed and up to six QuickStix™

test sets per class free of charge by contacting the ISUOffice of Biotechnology, phone toll-free 800-643-9504, ore-mail [email protected]. Additional QuickStix test setsmay be ordered directly from EnviroLogix (see address atend of this lesson plan).

Science Content

• Students will observe and gather data on the effectsof the European corn borer (ECB) on traditionaland Bt corn.

• Students will apply the knowledge and lab data tosolve common problems in today’s agriculture.

Science Education Standards

Science as Inquiry, Content Standard A, AbilitiesNecessary to do Scientific Inquiry

– Design and conduct scientific investigations(p. 175)

– Use technology and mathematics to improveinvestigations and communications (p. 175)

– Formulate and revise scientific explanations andmodels using logic and evidence (p. 175)

Life Science, Content Standard C– The cell (p. 184)– Molecular basis of heredity (p. 185)– Interdependence of organisms (p. 186)

Source: National Science Education Standards, ©National Academy ofSciences, 1996. Used with permission. Page numbers refer to theseventh printing, November 1999 – also available on the Internet athttp://books.nap.edu/html/nses/pdf/index.html.

Science Process Skills

• Observing• Relating• Inferring

Life Skills

• Science processing

• Making decisions• Critical thinking• Problem solving

Time

Preparation: Two hours, beginning three weeks beforethe day of the lab activity

Doing the activity: 40 minutes

Materials

Materials Per Class

2 Bt corn plants labeled only with a reference number2 non-Bt corn plants labeled only with a referencenumberSix QuickStix test sets*

Materials for Each Group of Four

1 QuickStix strip*1 disposable 1.5 ml tube with punch caps*1 tissue pestle*10 drops (350 ul) of extraction buffer*1 disposable pipette*1 pair of scissors

*Available to Iowa teachers through Iowa StateUniversity’s Office of Biotechnology

Optional: Make copies of student handout II-c onp. 75-77.

Lesson Plan

Planting Your Corn – 10-14 Days Before Activity

You or your students should obtain approximately 15plants of Bt and non-Bt corn.You will need to plant yourcorn 10-14 days before thelab activity. Remember thatthe corn will grow wide andtall. There are several waysto grow your corn. A largeinexpensive plastic con-tainer (14"w x 24"l x 11"h), such as ones made forclothes storage, works well. Place 1.5 inches of sand oraquarium rock at the bottom of the container. Over thesand and/or aquarium rock, add 2-3 inches of pottingsoil. A container of this size could be used to grow 12-15 plants. A maximum of three rows with five plants is

Growing Corn



enough for the lab activity. An alternative is to groweach plant in 4 to 6 inch individual pots of plastic orpeat. It will take the corn 5-7 days to germinate,depending on the temperature. The leaves of the cornplant should be large enough to cover the opening of a1.5 ml tube. Make sure to test the leaf tissue beforeexposing the corn plant to the European corn borereggs if you are doing this activity as part of the “Bt vs.European Corn Borer” activity.

How the Test Works

Corn that has been genetically engineered with a Btgene will produce Bt protein in the leaf tissue. TheQuickStix kit detects the proteins produced by thecrystalline proteins Cry1Ab and Cry1Ac. To conductthe test, a leaf tissue sample is taken from the plant andpulverized with a pestle. Then the proteins that may bepresent in the ground-up leaf tissue are solubilized in anextraction buffer where they can be detected by usingan immunochromatographic test. This test usesantibodies on the strips to detect the presence of Btprotein in the leaf. A control line will appear on eachstrip. If Bt protein is present in the leaf, the reactionproduces a second line under the control line.

Each Envirologix QuickStix test strip has an absorbentpad at each end. The protective tape with the arrowindicates the end of the strip to insert into the extrac-tion tube. The extraction buffer with the dissolvedproteins (if present) will travel up the membrane stripand be absorbed in the larger pad at the top of the strip.The portion of the strip between the protective tape andthe absorbent pad at the top of the strip is used to viewthe reactions as described in the “Interpreting theResults” section of these instructions.

Doing the Activity

Pre-Lab

The instructor should set out an equal number of Btand non-Bt corn plants. Number the plants for refer-ence only. Do not indicate which corn plants are Bt andnon-Bt. Several groups should test identical plants toconfirm results. Have the students observe the plantsand record a hypothesis about which plants they believeto be genetically engineered.

Extraction Procedure

Be very careful to prevent sample-to-sample cross-contamination with plant tissue, fluids, or disposables.Repeat the protocol for each sample to be tested, using anew tube and pestle for each.

1. Label a 1.5 ml tube with the reference number ofthe plant you are testing.

2. Using a scissors, cut off a 1 1/2" (~4 cm) by 1/2"(~1.5 cm) piece of leaf from each plant to be tested.

3. Sandwich a section of the leaf between the cap andthe body of the 1.5 ml tube.

4. Snap down the cap ofthe tube so that itpunches out a circularpiece of the leaf. Insome cases, the capdoes not completelypunch the leaf. Withthe cap still down, tearexcess leaf tissue fromaround the cap withyour fingers.

5. Repeat step 4 a second time with the sametube to collect a total of two circular pieces of tissuefrom the same plant.

6. Push the leaf punches down into the taperedbottom of the tube with the pestle.

7. Insert the pestle into the tube and grind the tissueby rotating the pestle against the sides of the tubewith a twisting motion for 20-30 seconds. Makesure the leaf tissue is well ground before proceed-ing to step 8.

8. Using a plastic pipette,carefully draw up theentire contents of thetube marked “Extrac-tion Buffer” and add itto the tube containingthe ground-up leaftissue. Try to avoidexcessive bubbles asthe extraction buffer isdrawn and added tothe tissue tube.

9. Repeat the grindingprocess to mix thetissue with the extraction buffer (5-10 seconds).

10. Place the EnviroLogix QuickStix strip into theextraction solution, being sure to insert the endindicated by the arrows on the protective tape.

Step 4

Step 8



11. After inserting the stripinto the extractionsolution tube, you willnotice liquid travelingup the membrane striptoward the absorbentpad at the top of thestrip.

Interpreting the Results

12. Soon after the membrane strip is completely wet, acontrol line will appear on the membrane strip justbelow the top absorbent pad. This line indicatesthat the strip is functioning correctly.

13. Allow the strip todevelop for 10 minutesbefore making a finalinterpretation of theresults. (You may wantto ask your students torecord their results onthe data table providedwith the lab materials.)

14. The development of asecond line below thecontrol line is a positiveresult that indicates that Bt protein is present in thetissue. A positive result means that the leaf tissuewas taken from Bt corn. The absence of the secondline indicates a negative result for the Bt protein,meaning the leaf tissue sample was taken fromnon-Bt corn.

Reflect and Apply

These questions on are on student handout II-c onp. 76.

1. Compare the results of your QuickStix tests to thepredictions you made before you started. Why doyou think your predictions agreed or disagreedwith the test results?

Probably few, if any, students will have made fourcorrect predictions. Their answers for why they did ordid not make the correct predictions should all focuson the fact that Bt corn plants look the same as non-Btcorn plants. Correct predictions only could have beenmade by chance.

2. What uses can you think of for a Bt detection testlike the one you used for this activity?

Because Bt and non-Bt corn plants are indistinguish-able visually, this type of Bt test can be used to verify ifBt corn was grown by a farmer. The test also can helpscientists determine which plants have the Bt gene.

Internet Ideas

Bt Corn & European Corn Borer: Long-Term SuccessThrough Resistance Managementhttp://www.extension.umn.edu/distribution/cropsystems/ DC7055.html©1997 Regents of the University of Minnesota. Allrights reserved. Also available in print as North CentralRegional Extension Publication NCR 602, ExtensionDistribution Center, Iowa State University, 119 Printingand Publication Building, Ames, IA 50011-3171 Ph:515-294-5247.

Crop Genetics: Crop Genetic Engineeringhttp://croptechnology.unl.edu/©University of Nebraska, 2000, 2001, 2002. This sitehas lessons on backcross breeding, plant tissue culture,DNA and DNA extraction, gene cloning, European cornborer and Bacillus thuringiensis, gene design, andtransformation.

A section especially for high school(http://croptechnology.unl.edu/highschool/index.html)has lessons about DNA and DNA extraction, genecloning, gene design, transformation, and plantbreeding.

A nutrition section of the site (http:/croptechnology.unl.edu/nutrition/ ) contains lessons about basicbiotechnology, allergenicity, and nutrient content ofgenetically engineered foods.

In addition to the lessons, a number of animations maybe viewed online. The animations may be downloadedafter completing an online form describing the intendeduse. The animations include the following and more:

• the life cycle of the European corn borer and howBt affects the ECB

• gene constructs (Bt gene)• three gene coding regions and how they are used to

produce a Bt gene• map of the ECB generations in the U.S.• map of ECB spread from east to west in the U.S.• a general overview of the crop genetic engineering

process

Results

Bt Non-

Bt

Step 11



• backcross breeding of transgenic lines• bacterial transformation• gene gun• gene cloning• DNA structure• constructing a gene library• making a recombinant plasmid• the differences between conventional plant

breeding and plant breeding using biotechnology

European Corn Borer Home Page of the Iowa StateUniversity Department of Entomologyhttp://www.ent.iastate.edu/pest/cornborer/

The Microbial World: Bacillus thuringiensishttp://helios.bto.ed.ac.uk/bto/microbes/bt.htmProduced by Jim Deacon, Institute of Cell andMolecular Biology, and Biology Teaching Organization,University of Edinburgh, UK. 2001.

Credit Note

This laboratory activity was developed by Mike Zeller,ISU Biotechnology Outreach Education Coordinator, inconsultation with EnviroLogix, Inc., and is used withtheir permission The activity uses the QuickStix™ StripKit manufactured by EnviroLogix, Inc.

QuickStix™ is a trademark of EnviroLogix, Inc. Forordering information, mail or phone:

EnviroLogix500 Riverside Industrial ParkwayPortland, ME 04103-1418 USAPhone: (207) 797-0300FAX: (207) 797-7533E-mail: [email protected]: http://www.envirologix.com

67

Learning more about . . .

Iowa State University Extension and ISU Office of Biotechnology

Student Handout

European Corn Borers

The European Corn Borer1

The European corn borer (ECB), whose scientific nameis Ostrinia nubilalis, is an insect that significantly affectsproduction of field corn, popcorn, and sweet corn.Overall, yield losses and money spent trying to controlthe ECB cost farmers in the United States more than 1billion dollars annually.

As its name suggests, the ECB is not native to the UnitedStates. It is an introduced insect species that belongs tothe family Pyralidae in the order Lepidoptera. It prob-ably arrived in North America during the early 1900s inbroom corn imported from Hungary and Italy for themanufacture of brooms. First noticed near Boston,Massachusetts, in 1917, the European corn borer alsowas found by 1921 in areas bordering Lake Erie. Itspread gradually from southern Michigan and northernOhio. By the end of 1938, it had spread as far west as theWisconsin shore of Lake Michigan.

During its early history in the United States, the ECBproduced one generation per year. By the late 1930s,some of the ECB in eastern and north central states wereable to produce two generations each year. This two-generation ECB spread rapidly and soon becamedominant in the central Corn Belt. It reached Illinois in1939, Iowa in 1942, Nebraska in 1944, and SouthDakota in 1946.

Meanwhile, the single-generation ECB spread north-ward into northern Minnesota, North Dakota, and theCanadian provinces of Quebec, Manitoba, andSaskatchewan.

Later, three- and four-generation ECB appeared in thesouth along the Atlantic Coast and southwestward inMissouri, Arkansas, Kansas, Oklahoma, and the Gulfstates.

The insect has continued to spread throughout the corngrowing areas of the United States. In the years sincebeing discovered, the ECB has spread northward intoCanada, westward to the Rocky Mountains, andsouthward to Florida and New Mexico. It is nowpresent in all but the seven most western continentalstates.

The Life Cycle of European Corn Borer1

The ECB goes through four stages of development: egg,larva (borer), pupa, and adult moth. In the northernstates, its larvae spend the winter inside corn stalkresidue that remains after harvest. In spring, the larvaebecome pupas. When the adult moths emerge fromthe pupas, they lay eggs on the underside of the leavesof growing corn plants. The larvae developing in theeggs form black heads shortly before they emerge. Asthey grow, the larvae feed on the leaves and stalks ofthe growing corn plants, creating holes. Eventually, thelarvae chew holes in the stalk and enter, where theycontinue to feed and develop into a pupa.

In the continental United States, only the seven unshaded westernstates have escaped the European corn borer.

II-aLesson Module II – Insect-Resistant Crops Using Bt

A European corn borer larva on a corn leaf. Keith Weller ARS-USDA

68

Student Handout


Lesson Module II – Insect-Resistant Crops Using Bt

Adult moths emerge from the stalk, and the cyclebegins again. In the north central part of the U.S., theECB can complete one or two generations beforewinter. In the southern U.S., up to four generations canbe completed each year.

How European Corn Borer Damage

Affects Corn1

For field corn grown for grain, yield losses from theECB are primarily caused by the added stress to theplant’s normal growth and maintenance activities. Acorn plant being fed on by corn borers must divertsome of its energy away from normal growth anddevelopment to deal with the damage caused by theborers. Because the corn plant’s ability to withstandstress varies during plant growth stages, the stage(s)when it is attacked influences its ability to deal withinjury. The corn plant is susceptible to ECB attack andinjury after the 6th-leaf stage through grain maturity.

During early stages of corn growth, there is between 5and 6 percent loss in grain yield for each larva on aplant. During ear development stages, the loss fromeach larva is about 2 to 4 percent. However, if cornplants experience prolonged moisture stress aftersignificant ECB tunneling, the loss from each larva canbe as high as 12 percent.

Damage and yield loss result from:

1. Leaf feeding (first generation);2. Midrib feeding (first and second generation);3. Stalk tunneling (first and second generation);4. Leaf sheath and collar feeding (second and third

generation); and 5. Ear damage (second and third generation).

ECB damage results in poor ear development, brokenstalks, and ears that drop from the plant before harvest.Most yield loss can be attributed to the impaired abilityof plants to produce normal amounts of grain due tothe effects of larval damage in leaf and stalk tissues.During dry and windy autumn weather, tunneling inthe stalks and ear shanks can increase stalk and shankbreakage, resulting in substantial ear loss before harvest.

Research has shown a close association betweensecond- and third-generation ECB infestation andincidence of stalk rot, a fungal disease. This increasein stalk rot is directly related to ECB larvae boring intostalks and ear shanks, giving the fungus easy entry intothe stalk. Losses due to weakening of the stalk fromtunneling and stalk rot increase when corn harvest isdelayed. Early harvest will reduce losses caused bystalk rot and the ECB.

II-a

ear damage

stalk tunneling

First and second generation damage sites

midribfeeding

leaf feeding

whorl

Second and third generation damage sites

leaf sheathfeeding

ear shankbreakage

collarfeeding


Student HandoutII-a


… and justice for allThe U.S. Department of Agriculture (USDA) prohibits discrimination in allits programs and activities on the basis of race, color, national origin,gender, religion, age, disability, political beliefs, sexual orientation, andmarital or family status. (Not all prohibited bases apply to all programs.)Many materials can be made available in alternative formats for ADAclients. To file a complaint of discrimination, write USDA, Office of CivilRights, Room 326-W, Whitten Building, 14th and Independence Avenue,SW, Washington, DC 20250-9410 or call 202-720-5964.

Issued in furtherance of Cooperative Extension work, Acts of May 8 andJune 30, 1914 in cooperation with the U.S. Department of Agriculture.Stanley R. Johnson, director, Cooperative Extension Service, Iowa StateUniversity of Science and Technology, Ames, Iowa.

Credit Notes

1Excerpted from or based on European Corn BorerEcology and Management, NCR-327, Charles E. Mason,Marlin E. Rice, Dennis D. Calvin, John W. Van Duyn,William B. Showers, William D. Hutchison, John F.Witkowski, Randall A. Higgins, David W. Onstad, andGalen P. Dively. Iowa State University Extension,publishing state, July 1996. Excerpts viewable on theInternet at http://www.ent.iastate.edu/pest/cornborer/.Used with permission.

Learn the Language

European corn borer

An insect of the Lepidopteran order with moth,egg, larval, and pupa stages that feed on corn andother plants

Collar

The light-colored band near the bottom of a cornleaf

Ear

The woody, tubular cob to which corn kernels areattached

Generation

One complete life cycle from pupa, to moth, to egg,to larva, to pupa again

Larvae

The immature worm or caterpillar stage ofEuropean corn borers

Leaf sheath

The bottom part of the corn leaf that wraps aroundthe stalk

Midrib

The central vein of a leaf

Pupa

The cocoon stage of an insect that occurs betweenthe larval and moth stages

Stalk

The stem of the corn plant

Stalk rot

A fungal disease that causes premature decay ofcorn stalks before the plant reaches maturity

Whorl

The tightly coiled leaves at the top of a corn stalkthat unroll as the plant grows

Iowa State University Extension and ISU Office of Biotechnology 71

See for yourself . . .

Student HandoutLesson Module II – Insect-Resistant Crops Using Bt II-b

Bt vs. European Corn Borers

Bt vs. European Corn Borers

Two weeks ago, you started growing the two varieties ofcorn, Bt and non-Bt, to use in this activity.

Three to five days ago, you began incubating ECB eggmasses.

Now that the corn has grown enough and the ECB eggmasses show black heads, you are ready to proceed withthe lab.

While investigating Bt corn, your teacher may haveasked you to do some library or Internet research abouthow the Bt protein controls certain insects. Recordyour predictions about the results of this experiment onthe petri dish data sheet and/or the live plant protocoldata sheet, as directed by your teacher.

Doing the Activity

PART I - Petri Dish Protocol

1. Use a marking pen to write non-Bt on the left halfof the petri dish and Bt on the right half.

2. Place a piece of filter paper in the petri dish.

3. Add 2-3 drops of water to the filter paper.

4. Cut a 1-1 1/2" piece of leaf off each variety of corn.Make sure you keep track of each variety.Researchers mark one leaf with a notch to keeptrack.

5. Place the non-Bt corn leaf on the left side of theplate and the Bt corn on the right side.

6. Place 2-3 blackheaded egg masses on each leafusing a fine-tipped watercolor paintbrush or atoothpick.

7. Cover the top of the petri dish with a damp papertowel and place the lid over the dish. During thecourse of the investigation, keep the paper towel

damp so the humidity will be high. Re-wet thepaper towel when necessary.

8. Incubate at 27ºC (80ºF) until the eggs hatch,which will be about 1-3 days.

9. Observe the ECB behavior and eating activity foreach corn variety. Record your observations aboutleaf damage daily on the petri dish data sheet.

10. Replace the leaf pieces with fresh cuttings of thesame variety as the old leaf segments dry and/or areconsumed.

PART II - Live Plant Protocol

At the same time you infest your leaf cuttings, you caninfest some of the growing plants. Do not infest allyour plants. You will need more cuttings for your petridish part of the investigation. Do not leave youruninfested plants close to the infested ones because thelarvae can move from one plant to another and all theplants may become infested.

Record your predictions on the live plant data sheetabout the percentage of leaf damage and numbers offeeding larvae that you will observe each day.

1. Using a wet, fine-tipped watercolor paintbrush or atoothpick, pick up a blackheaded egg mass and

A European corn borer larva on acorn leaf. Keith Weller, ARS-USDA

A European corn borer moth.ARS-USDA

Iowa State University Extension and ISU Office of Biotechnology72

Student Handout

place it a leaf. Place 2-3 egg masses on each leaf ofthe corn plant. At the beginning of the experimentwhen the egg masses hatch, count the number oflarvae on each of your plants.

2. Record your observations about the percentage ofleaf damage and the number of feeding larvae dailyon the live plant data sheet.

Reflect and Apply

Petri Dish Protocol



Credit Note

This activity was developed by Mike Zeller for theOffice of Biotechnology, Iowa State University, and isused with permission.



II-bLesson Module II – Insect-Resistant Crops Using Bt

Live Plant Protocol

1. How many larvae were on your plants at thebeginning of this experiment?

2. When corn is infected by ECB this early in itsgrowth, assume that there will be a 5% grain lossfor each larva you find on a single plant. Calculatethe percentage of grain loss you could haveexpected for each of your plants.

3. How did the level of leaf damage and number ofactively feeding borers you observed on each dayfor your non-Bt corn plant compare to the levels ofdamage and borers you observed for your Bt cornplant?


Student Handout II-b

Petri Dish Protocol Data Sheet

Name ________________________________________ Group ___________________________________

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tnalPtB tnalPtB-noN

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tnalPtB tnalPtB-noN

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2yaD


Iowa State University Extension and ISU Office of Biotechnology74

Student Handout II-b

snoitciderP

faeLfoegatnecreP

egamaDeavraLgnideeFfo.oN

tB tB-noN tB tB-noN

1yaD

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Live Plant Protocol Data Sheet

Name ________________________________________ Group ___________________________________



See for yourself . . .

Student Handout

Bt Detection – Corn Leaf Tissue Test

II-cLesson Module II – Insect-Resistant Crops Using Bt

Bt Detection – Corn Leaf

Tissue Test

Corn that has been genetically engineered with a Btgene will express Bt protein in the leaf tissue. TheQuickStix™ kit detects the endotoxins produced by thecrystalline proteins Cry1Ab and Cry1Ac. To conductthe test, you will use a pestle to grind up a leaf tissuesample from the corn plants. Then the proteins thatmay be present in the ground-up leaf tissue aresolubilized in an extraction buffer where they can bedetected by using a test strip. This assay uses antibod-ies on the strips to detect the presence of Bt protein inthe leaf. A control line will appear on each strip. If Btprotein is present in the leaf, the reaction produces asecond line under the control line. If no Bt protein ispresent in the leaf, no second line appears.

Doing the Activity

Materials Per Class

2 Bt corn plants labeled only with a reference number2 non-Bt corn plants labeled only with a referencenumberQuickStix test sets

Materials for Each Group of Four

1 QuickStix Strip1 disposable 1.5 ml tube with punch caps1 tissue pestle10 drops (350 ul) of extraction buffer1 disposable pipette1 pair of scissors

Predictions

Carefully observe the four plants that your class will betesting. Write the four reference numbers for the plantson the four lines below the test strip diagrams at thebottom of this page. With a pencil, draw a second linebelow the thick black control line for the referenceplants you think will contain the Bt protein.

Procedure

Be very careful to prevent sample-to-sample cross-contamination with plant tissue, fluids, or disposables.Repeat the protocol for each sample to be tested, usinga new tube and pestle for each.

1. Label a 1.5 ml tube with the reference number ofthe plant you have been assigned by your teacher.

2. Using a scissors, cut off a 1 1/2 inch (~4 cm) by 1/2inch (~1.5 cm) piece of leaf from each plant to betested.

ELI

X Q

UIC

KS

TIX

ELI

X Q

UIC

KS

TIX

ELI

X Q

UIC

KS

TIX

ELI

X Q

UIC

KS

TIX

Reference no. Reference no. Reference no.Reference no.

←←←←←Controlline

76

Student Handout



3. Sandwich a section ofthe leaf between thecap and the body ofthe 1.5 ml tube.

4. Snap down the capof the tube so that itpunches out a circularpiece of the leaf. If the cap does not completelypunch the leaf, with the cap still down, tear excessleaf tissue from around the cap with your fingers.

5. Repeat step 4 a second time with the same tube tocollect a total of two circular pieces of tissue fromthe same plant.

6. Push the leaf punches down into the taperedbottom of the tube with the pestle.

7. Insert the pestle into the tube and grind the tissueby rotating the pestle against the sides of the tubewith a twistingmotion for 20-30seconds. Make surethe leaf tissue is wellground beforeproceeding to step 8.

8. Using a plasticpipette, carefullydraw up the contentsof the tube marked“Extraction Buffer”and add it to thetube containing theground-up leaftissue. Try to avoidexcessive bubbles asthe extraction buffer is drawn and added to thetissue tube.

9. Repeat the grinding process of step 7 to mix thetissue with the extraction buffer (5-10 seconds).

10. Place the EnviroLogix QuickStix strip into theextraction solution, being sure to insert the endindicated by the arrowson the protective tape.

11. After inserting the stripinto the extractionsolution tube, you will

Step 8

Step 4

Step 11

notice liquid traveling up the membrane striptoward the absorbent pad at the top of the strip.

Interpreting the Results

12. Soon after the membrane strip is completely wet, acontrol line will appear on the membrane strip justbelow the top absorbent pad. This line indicatesthat the strip is functioning correctly.

13. Allow the strip to develop for 10 minutes beforemaking a final interpretation of the results. Yourteacher may ask you to record your results on thedata table provided with the lab materials.

14. The development of a second line below thecontrol line is a positive result that indicates that Btendotoxin (protein) is present in the tissue. Apositive result means that the leaf tissue was takenfrom Bt corn. The absence of the second lineindicates a negative result for the Bt endotoxin,meaning the leaf tissue sample was taken fromnon-Bt corn.

Reflect and Apply

1. Compare the results of your QuickStix tests to thepredictions you made before you started. Why doyou think your predictions agreed or disagreedwith the test results?

2. What uses can you think of for a Bt detection testlike the one you used for this activity?

77

Student Handout


Credit Note

This laboratory activity was developed by Mike Zeller,ISU Biotechnology Outreach Education Coordinator, inconsultation with EnviroLogix, Inc., and is used withtheir permission. The activity uses the QuickStix™ StripKit manufactured by EnviroLogix, Inc.

QuickStix™ is a trademark of EnviroLogix, Inc.





Lesson Module II – Insect-Resistant Crops Using Bt Overhead Master II-a

About the

European Corn Borer (ECB)

The larvae of European corn borerfeed on corn, sorghum, cotton, andvegetables.

One-generation ECB was first noticedin the U.S. in 1917 near Boston, Mass.

By the 1930’s, two-generation ECBappeared in the eastern and northcentral U.S.

Later, three- and four-generation ECBappeared in the southern U.S.


Lesson Module II – Insect-Resistant Crops Using Bt Overhead Master II-b

Current U.S. Distribution

European Corn Borer

The European corn borer is nowpresent in all the continental states,except Washington, Oregon,California, Idaho, Nevada, Utah, andArizona.


Lesson Module II – Insect-Resistant Crops Using Bt Overhead Master II-c

Corn Damage from

European Corn Borer

Corn plants are susceptible after the6th-leaf stage.

Damage and yield loss result fromeach of the corn borer generations.

• Leaf feeding – 1st

• Midrib feeding – 1st and 2nd

• Stalk tunneling – 1st and 2nd

• Leaf sheath and collar feeding –2nd and 3rd

• Ear damage – 2nd and 3rd


Lesson Module II – Insect-Resistant Crops Using Bt Overhead Master II-d

Damage Sites

First and secondgeneration

Second and thirdgeneration

leaf sheath feeding

leaf feeding

midribfeeding

stalk tunneling

whorl

ear shankbreakage

collarfeeding

eardamage



Excerpted from or based on European Corn Borer Ecology and Management, NCR-327,Charles E. Mason, Marlin E. Rice, Dennis D. Calvin, John W. Van Duyn, William B.Showers, William D. Hutchison, John F. Witkowski, Randall A. Higgins, David W.Onstad, and Galen P. Dively. Iowa State University Extension, publishing state, July1996. Excerpts viewable on the Internet at http://www.ent.iastate.edu/pest/cornborer/.Used with permission.

Overhead Master II-e

Damage Impacts from

European Corn Borer (ECB)

Corn’s ability to withstand stressvaries during plant growth stages.

A corn plant’s stage at the time ofECB attack influences yield loss.

• For early stages, expect 5 - 6% yieldloss for each larva on a plant.

• For ear development stages, expect2 - 4% loss for each larva.

• For prolonged moisture stress aftersignificant ECB tunneling, expect upto 12% loss for each larva.



How Bt Helps Control

ECB Damage

Bacillus thuringiensis is a rod-shapedsoil bacterium.

Its spores contain a crystalline(Cry) protein, such as Cry1Ab orCry1F.

In certain insects like European cornborers, the Cry protein releases intothe gut a protein called delta-endotoxin.

Delta-endotoxin creates tiny holes inthe borer’s intestinal lining, whichstops digestion.

The borer dies in a few days.

Bt

Bt

Overhead Master II-f

Documents

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