Biotechnology - LSU AgCenter/media/system/d/b/8/2/... · 9 Cryoperservation: A New Industry for Aquatic Species Teence Tierschrr 10 Animal Biotechnology and the Future Robert A. Godke,

Louisiana Agriculture, Fall 2003 1

Fall 2003Vol. 46, No. 4

BiotechnologyIssue

BiotechnologyIssue

2 Louisiana Agriculture, Fall 2003

EDITORIAL BOARD:David J. Boethel (Chairman)Linda Foster BenedictBarbara Groves CornsJane HoneycuttTheresia LavergneRay McClainKenneth W. PaxtonWitoon PrinyawiwatkulT. Eugene ReaganJohn K. Saichuk

Published quarterly by the LouisianaAgricultural Experiment Station, LouisianaState University Agricultural Center,Baton Rouge, Louisiana. Subscriptions arefree. Send requests and any comments orquestions to:

Linda Foster Benedict, EditorLouisiana AgricultureP.O. Box 25100Baton Rouge, LA 70894-5100

phone (225) 578-2263fax (225) [email protected]

www.lsuagcenter.com

EDITOR: Linda Foster Benedict

DESIGNER: Barbara Groves Corns

PHOTO EDITOR: John Wozniak

The mention of a pesticide or use of a trade namefor any product is intended only as a report ofresearch and does not constitute an endorsementor recommendation by the Louisiana AgriculturalExperiment Station, nor does it imply that a men-tioned product is superior to other products of asimilar nature not mentioned. Uses of pesticidesdiscussed here have not necessarily been approvedby governmental regulatory agencies. Informationon approved uses normally appears on the manu-facturer’s label.

Material herein may be used by the press, radio andother media provided the meaning is not changed.Please give credit to the author and to the publica-tion for any material used.

OVERVIEW & PERSPECTIVE

n 1994, the summer issue of Louisiana Agriculture was dedicated to biotech-nology. Since then, many advances have been made in this rapidly changing fieldof research. This issue highlights new technologies to enhance crop and animalproductivity, to develop new uses for existing crops and animals, to add value totraditional production units and to answer basic research questions applicable toproduction system problems.

How has biotechnology changed the LSU AgCenter’s research efforts in thenine years since the 1994 issue? That issue began with the question, “What isbiotechnology?” Readers were provided with a glossary to help explain the newlanguage associated with recombinant DNA. Initial reports of weed and insect pestcontrol with transgenic cotton, soybean and rice were featured. Animal biotechnol-ogy articles about cloning, diagnostics and disease prevention were offered asexamples of how this technology might change the face of animal productionagriculture. Consumer acceptance of biotechnology was addressed.

This issue differs little despite impressive advances in the field. Public concernwith the safety of genetically modified plants and animals continues to present amajor challenge to the application of biotechnology to food consumed by thepublic. Genetically modified organisms (GMOs) that help reduce the use ofpesticides or that assist in the production of pharmaceutical compounds aregenerally more accepted than food products by the people of the United States.Scientists, public officials and researchers must continue to work together to ensurethe safety of GMOs and to educate the consumer about their safety and benefits.

The research content herein introduces readers to areas using biotechnology,ranging from increasing desired traits in cotton and disease resistance in rice andstrawberries, improving weed and insect resistance in a variety of crops, producingpharmaceutical chemicals in chicken eggs, and treating cancer with DNA vaccines.New technologies are being used to understand and disarm potential biologicalweapons and as an aid in the management of endangered Louisiana birds andmammals.

Daily, we, the public, are exposed through the media to news concerninggenetic-based technologies that offer promise as varied as that for the diagnosis andtreatment of diseases to the apprehension or exoneration of criminal suspects.AgCenter faculty are dedicated to improving the quality of life for Louisianians,and the safe use of biotechnology is just one of many tools they are using toaccomplish this mission. Genes and biotechnology are rapidly becoming entwinedinto the fabric of our daily lives, and this issue offers some of the innovative“threads” being pursued at the AgCenter.

Frederick M. Enright, Professor and Head,Department of Veterinary Science, LSUAgCenter, Baton Rouge, La.

I

Another Tool To Improve Life

LSU AgCenter

William B. Richardson, Chancellor

William H. Brown, Vice Chancellorand Director of Research

Paul D. Coreil, Vice Chancellorand Director of Extension

The Louisiana Agricultural Experiment Stationprovides equal opportunitiesin programs and employment.

Biotechnology

In LouisianaFrederick M. Enright


CONTRIBUTORS: Rick Bogren, JohnChaney, Mark Claesgens, Jane Honeycuttand Randy McClain


Volume 46, Number 4, Fall 2003

4 Gene Therapy for Cancer TreatmentFrederick M. Enright and Richard K. Cooper

6 Transforming Chickens to Lay ‘Golden’ EggsLinda Foster Benedict

8 New Vaccine Increases Broiler Breeder Chicken ProductionDaniel G. Satterlee

9 Cryoperservation: A New Industry for Aquatic SpeciesTerrence Tiersch

10 Animal Biotechnology and the FutureRobert A. Godke, Richard S. Denniston and Brett Reggio

14 Gene Wars: Biotechnology Can Help Control VirusesWilliam Todd, Laynette Spring, Jackie McManus, Brandye Smith and Richard K. Cooper

16 Slicing Years Off Rice Varietal ImprovementQi Ren Chu and Steven D. Linscombe

19 A Rice Field in a Petri Dish Randy McClain

20 Biotechnology and Control of Rice DiseasesM.C. “Chuck” Rush, Q. M. Shao, Shuli Zhang, A.K.M. Shahjahan, Kathy O’Reilly, Ding Shih,Donald Groth and Steven D. Linscombe

23 Clearfield 161 Has Rice Growers BuzzingRandy McClain

24 Clearfield Rice: It’s Not a GMOTimothy P. Croughan

27 Biotechnology for Herbicide, Disease Resistance in RiceJames H. Oard, Steven D. Linscombe and Donald Groth

29 Technology To Improve the Quality of Sweet Potato ‘Seed’Christopher A. Clark, Don R. LaBonte, Rodrigo A. Valverde, Mary W. Hoy,Pongtharin Lotrakul, Charalambos Kokkinos and Cecilia McGregor

32 Insect-resistant, Transgenic Soybeans: A New IPM ToolMatthew E. Baur, Bentley J. Fitzpatrick and David J. Boethel

34 Tracking Loopers with DNAMatthew E. Baur and David J. Boethel

35 Weed Management Made Easier with Herbicide-resistant CropsJames L. Griffin

38 Beyond Bollgard: Insect-resistant Cotton VarietiesB. Rogers Leonard, Stephen Micinski and Ralph Bagwell

40 Gene Mapping Fiber Traits in CottonGerald O. Myers and Muhanad Akash

41 Using Molecular Genetics in Natural Resource ManagementMichael Stine

42 Rescuing the Coast with BiotechnologyPrasanta K. Subudhi, Neil Parami, Alicia Ryan and Stephen A. Harrison

45 Biotechnology Improves Strawberry VarietiesCharles E. Johnson, Ding Shih and Joey Quebedeaux

46 Biotech Lab Opens for BusinessSvetlana Oard

47 Vaccines To Protect People from Germ WarfarePhilip H. Elzer and Sue D. Hagius

48 BEST Is Yet To ComeLinda Foster Benedict

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ON THE COVERThese are White Leghorn chickens at the LSUAgCenter’s Ben Hur Farm near Baton Rouge,La., who are normal except for one thing.When the hens lay eggs, the eggs containproinsulin, a precursor for making the druginsulin. Read about the new biotech companystarted because of AgCenter research on page6. Photo by John Wozniak.



enetic therapy is the introductionof a gene or group of genes into ananimal to either correct the result of anabnormal gene or to form a new productthat has a beneficial effect for theanimal or for those using productsderived from the animal.

Called a “DNA construct,” thenewly introduced material has a specificgenetic code to allow the desired gene tobe inserted into the animal’s own genes.The construct also has a promoterresponsible for forcing the gene for thenew product to be read, or expressed,and a specific genetic code to stop theexpression of the gene.

The DNA construct offers potentialin cancer research. Physicians have longsought treatments for cancer that destroycancer cells only. Most existing cancertreatments destroy not only cancer cellsbut also normal cells. The dreaded sideeffects of cancer chemotherapeuticdrugs—anemia, fever, weakness anddigestive upsets—are because of thedrugs’ destruction of normal cells.

Likewise, the DNA construct is ofinterest to veterinarians and animalscientists who have searched for a wayto sterilize pets, such as dogs and cats,without surgery. Surgical procedures,either castration in males or removal ofthe ovaries and uterus in females, areexpensive, time-consuming and involverisk to the animal.

In the past 10 years, scientists atthe LSU AgCenter have studied theeffectiveness of membrane-disruptingpeptides linked to ligands (smallmolecules that bind other molecules,frequently a hormone to its receptor) todestroy cancer cells selectively and tosterilize animals without surgery. Themembrane-disrupting, peptide-ligand

compounds are small proteins. Theyhave the unique characteristic of havingone side of the peptide molecule able tointeract and bind to fat (lipid in thecell’s membrane) with the other side ofthe molecule preferring to interact withwater (a major component of the fluidaround the outside of cells). Because ofthis “split personality,” these moleculesbind to lipids in the cell membrane andresult in membrane disruption. Whilesome cells are more susceptible todisruption than others (depends onthe types of lipids composing theirmembranes), in general, these mem-brane-disrupting peptides can destroyall types of cells. By themselves, theydo not demonstrate much selectivity inkilling cells.

Many cells in the body have uniqueproteins embedded in their cell mem-branes that recognize ligands. Theligands react with or join to membraneproteins called receptors much like akey (the ligand) fits into a lock (thereceptor). Thus, if scientists know aboutthe “lock,” the “key” can be found to fitit. The key may be used to direct drugs tospecific cells with the appropriate lock.

Prostatic, breast, testicular andovarian cancer cells (called carcinomas)frequently have receptors (locks) ontheir surfaces that bind to reproductivehormones (keys).Gonadotropin releasinghormone (GnRH) is asmall peptide in thebrain that binds tospecial cells in thepituitary gland calledgonadotropic cells.When GnRH binds tothem, these pituitarycells manufacture andsecrete two other largerprotein molecules calledluteinizing hormone(LH) and folliclestimulating hormone(FSH). The twohormones travel in thebloodstream from thepituitary and bind withcells in the testes of

males and ovaries of females and resultin sperm production and maturation inthe male and in egg production andmaturation in the female. Not only dothese ligands play an important role inreproduction, they are important in thelife cycle of selected cancer cells.

Because of their limited selectivitytoward the cells they destroy, LSUAgCenter scientists have combinedmembrane-disrupting peptides witheither the GnRH ligand or anotherligand, which is the 15 amino acidsegment of the beta chain of chorionicgonadotropin, referred to as beta CG.CG is an LH-like hormone made by theplacenta during pregnancy. Normal cellsand cancer cells with receptors for LHwill bind to CG because of this 15amino acid binding site.

These membrane-disruptingpeptides linked to either GnRH or tobeta CG have been studied for theirability to selectively destroy humanprostatic, breast and ovarian carcinomastransplanted into “nude” mice. These aremice with a genetic defect in theimmune system that prevents themfrom rejecting human tumors. If leftuntreated, these transplanted cells willgrow and spread (metastasize) andeventually kill these mice.

Frederick M. Enright, Professor and Head, andRichard K. Cooper, Professor, Department ofVeterinary Science, LSU AgCenter, Baton Rouge,La.

These “nude” mice are used in cancer research because theycan grow human tumors. The one on the right has beentreated with a beta CG hormone, which has made its tumordisappear.

Photo by William Hansel

G

Frederick M. Enright and Richard K. Cooperfor Cancer Treatment


This research has been conducted atthe Pennington Biomedical ResearchCenter in Baton Rouge, which is alsopart of the Louisiana State UniversitySystem. To date, these hormone-linkedpeptides have been successful inselectively destroying the primary(original tumor mass) and metastatictumor cells. More than 700 mice havebeen treated with these compounds.Unlike other cancer chemotherapeuticdrugs, these membrane-disruptingpeptides linked to ligands selectivelydestroy only the cancer cells and thegonadotropic cells in the pituitary ortesticular and ovarian cells having eitherGnRH or LH receptors. The onlyobserved side effects of thesecompounds were a loss of fertility inmales and females. Other vital organsin the treated mice remain normal.

In parallel studies conducted byLSU AgCenter scientists, the effects ofGnRH or beta CG-linked peptides onreproduction in normal female and malemice, rats, rabbits, fish, pigs and dogshave been studied. These studies havedemonstrated specificity in the rapiddestruction of gonadotropic cells in thepituitary gland by the GnRH-linkedpeptide or destruction of testicular cellsor ovarian cells by the beta CG-linked

peptide. That was the successfuloutcome of the research. However, asingle treatment of animals with thesepeptides resulted in only a temporaryloss of reproductive function.

For example, studies of micereceiving a single treatment showedthat for the first two weeks followingtreatment, the number of gonadotropiccells in the pituitary gland was reducedby 80 percent. By three to four weeksafter treatment, gonadotropic cells in thepituitary gland were returning. By fiveto six weeks post-treatment, the numberof gonadotropic cells was equal to thenumber of these cells in untreated mice.Thus, our hope of long-term sterilizationfollowing one injection of these peptideswas not realized.

Though repeated injections of thepeptides prolong the sterilization effect,this would be impractical for long-termsterilization of pets or livestock. Thereare several potential solutions to thisproblem. One is to package the peptidesfor slow release in the animal. We planto conduct studies using small osmoticpumps filled with the peptide. Thesepumps are implanted under the skinand will “trickle” out small amounts ofpeptides for extended periods. A secondsolution lay in introducing genes that

cause the animal itself to producethe peptides. This second approach isappealing because of the low cost ofgenetic constructs as compared to thecost of synthesizing peptides.

Data collection began on the effectsof these membrane-disrupting, peptide-ligand compounds on naturallyoccurring mammary gland adenocarci-nomas in dogs and cats. Richard Cooper,professor in the Department ofVeterinary Science, had previouslyinvented a unique vector with the abilityto efficiently insert stable, foreign DNAinto catfish. Using Cooper’s vector,DNA constructs of two differentpeptides (Phor11 and Phor14) linked toeither GnRH or to beta CG were made.By using the genes for the linkedpeptides, the animal itself could producethe drugs.

Initial studies to determine thesafety of the genetic constructs werecarried out in mice and dogs.Preliminary studies using DNAconstructs for membrane-disruptingpeptides and ligands have demonstratedthat the DNA constructs are safe andthat there is the same degree ofspecificity in the destruction of cellswith either LH or GnRH surfacereceptors as observed with the peptide-ligand compounds. Initial results infemale dogs suggest that the effectson the reproductive organs followingexposure to DNA constructs persist forat least 116 days. Additional studies areneeded to determine the length of thisextended sterilization.

Peptide therapy studies in dogs andcats with advanced mammary glandadenocarcinomas have begun. Both dogsand cats have demonstrated excellentresponses to the treatments. Followingintroduction of the genetic constructs,their tumors have undergone cellulardeath and have decreased dramaticallyin size. We are not claiming a cancercure but are optimistic about the abilityto effect these cancers. Additional workis necessary to learn to manipulate theconstructs and treatments dependingupon the situation. We need to deter-mine how long dog and cat cells willexpress the constructs. Prolongedexpression may be desirable in somecases while in others transientexpression would be preferable. Thebasic construct and the proteins beingexpressed may require modifications.Fine tuning the genetic constructs willrequire several additional years ofresearch.

Photos by Frederick M. Enright

These are tissue sections of breast cancer from a house cat before and after treatmentthrough gene therapy. The two views on the right are magnifications of the views on theleft. The top two pictures show the cancer cells (the little purple dots) invading the skin.The elongated feature in the top left photo is a hair follicle. The white and grayish spotsin the bottom two photos indicate the location of destroyed cancer cells following two one-week series of treatment with the gene constructs. AgCenter scientists are optimisticabout the ability of these gene constructs to treat breast cancer in cats and dogs.

Befo

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10 X 40 X


Transforming Chickens to Lay ‘Golden’ EggsEvery once in a while someone comes

along who can build a better mousetrap.And at the LSU AgCenter, that person isRichard Cooper, professor in the Depart-ment of Veterinary Science who’s come upwith a way to get chickens to lay eggscontaining human proteins.

These chickens are the reason a newbiotechnology company that will manufac-ture a precursor of insulin is located in anLSU AgCenter laboratory on the BatonRouge campus. Beginning in 2004, this com-pany, TransGenRx, appears likely to earnmillions of dollars in annual sales with apotential for spin-off companies that willproduce more pharmaceuticals at a fractionof the cost of conventional methods.

Cooper didn’t start his scientific careerthinking his claim to fame would be chickens.Fresh from earning a Ph.D. in medical micro-biology at the University of Georgia, hecame to the LSU AgCenter in 1990 to makechannel catfish more disease-resistant.

Laura Peak prepares bacterial cultures as part of insulin-related work for TransGenRx. She is one of Richard Cooper’s researchassociates. This photo ran on the front page of the Baton Rouge Advocate on Oct. 26, 2003.

Photo courtesy of Patrick Dennis at the Baton Rouge Advocate

He’d been fascinated by fish diseasessince his teen years in Jackson, Miss., whenhe managed to kill a thousand dollars’ worthof saltwater fish at his dad’s new pet store.

“I wanted to fix the problem and findout what caused it,” Cooper said.

That same curiosity led him to questionwhy it was so difficult to insert disease-resistant genes into catfish. At that time thetechnology guaranteed less than a 1 percentsuccess rate. The other 99 percent of thecatfish that had undergone the gene-alteringprocedure did not stay transgenic.

“I didn’t like the odds,” Cooper said.Over the next few years, he devel-

oped techniques that made for a betterequation. The LSU AgCenter team wasthe first to put in a gene for diseaseresistance in catfish. Now, 50 percent to80 percent of the offspring of geneticallyaltered catfish are disease-resistant.

Not to give away any patented secrets,but his techniques, in extremely simple terms,

Richard Cooper has come up with a way toget chickens to lay eggs containing humanproteins.

Phot

o by

John

Woz

niak



Gary Cadd, a former scientist with the AgCenter, isback on campus working for TransGenRx, the start-upbiotech company that has licensed technology fromthe AgCenter. TransGenRx (sponsored by theAgCenter) received $2.5 million in state funding tobuy equipment and set up laboratories in Wilson Hall.

involve three things. First, he shortened thesequence of the piece of DNA that’s in-serted into the organism by nearly 80 per-cent. This made transformation happenfaster.

Second, he inserted the foreign DNAinto the animal’s DNA sequence in a differ-ent manner than had ever been done before.

Third, he made sure the transporters ofthe foreign DNA, called “vectors,” self-destructed once they’d done their job. Thisis crucial for stability, allowing the desiredtraits to be passed on to the offspring.Otherwise, the foreign DNA could moveand get out of sequence, and the alterationwould be lost.

Meanwhile, a sequence of chance eventshappened in Cooper’s life that moved himfrom fish tanks to chicken coops.

He was at a crossroad about where togo with the transgenic technology used withthe fish, from a commercial viewpoint. Fed-eral law will not allow transgenic catfish tobe grown commercially because of concernsthat they will escape into the wild. The onlyway the business of growing transgenic,disease-resistant catfish could develop is ifthe catfish were sterile. No one has yetfound a way to make catfish sterile.

“Mother Nature has made this diffi-cult,” said Fred Enright, chair of the Depart-ment of Veterinary Science.

Cooper just happened to be in PaulaJacobi’s office when she received a phonecall from a biochemist-turned-businessman,Bill Fioretti of Dallas, Texas. Jacobi is incharge of intellectual properties for theLSU AgCenter and works with licensing agreements.

During the course of the conversation,Jacobi managed to connect Fioretti, the manwith the business sense, with Cooper, thescientist behind the technology. Together,they hatched a plan to try to make insulinwith transgenic chickens.

“You can’t manipulate a chicken egg theway you can a fish egg,” Cooper said of thenew challenge.

But the change in animals provided himan opportunity to show his stuff. And Coo-per and the AgCenter have since patentedmore technology having to do with insertinggenes in chickens. Fioretti helped start a newcompany, now called TransGenRx, to takeadvantage of Cooper’s wizardry.

“Dr. Cooper solves puzzles,” Fiorettisaid of his new associate, who was found bycoincidence.

In the world of biotech start-up compa-nies, the race is on to produce humanproteins, such as insulin, in a cost-effectivemanner. So far, TransGenRx, which is ex-

Photos by John Wozniak

pected to bring hundreds of high-paying jobs to Baton Rouge, isahead of the competition.

“At least five other compa-nies are trying to do what weare,” Cooper said. “But we havea solid intellectual property base.”

At this writing, TransGenRxis expected to produce 30 kilo-grams of proinsulin in 2004, whichcould translate into $30 millionto $50 million in sales.

“This is an example of whatsupporting higher education cando for a state like Louisiana,” saidChancellor Bill Richardson. “TheLSU AgCenter plans to be theimpetus for more start-up com-panies.”

Meanwhile, Cooper has beenapproached by the U.S. Army toinvestigate the production of vac-cines to fight bioterrorism.Other products being looked at for futureventures include drugs to treat breastcancer, specialty drugs for rare diseasesthat drug companies heretofore have notbeen able to develop because of high costs,and diagnostic tests for cancer. Linda Foster Benedict

Richard Cooper, right, explains his science to Sen. Mary Landrieu (D-La.) during a recentvisit to AgCenter laboratories. Landrieu has pledged her assistance with the developmentof biotech businesses in Baton Rouge. Center is Barry Loder, TransGenRx CEO, talking toone of Landrieu’s aides.



oultry is Louisiana’s biggestanimal industry. In 2002, poultryexceeded $600 million in total value(gross farm income and value added).The production of broiler (meat-type)chickens accounts for more than 90percent of this value. Approximatelythree million broiler breeder eggs areset weekly to support the number ofchicks needed to produce the morethan 200 million broilers rearedannually in our state for humanconsumption. The poultry industryboasts of being the fifth largestemployer in the state.

The steady growth of Louisiana’spoultry industry mimics that of thenation and world. The U.S. poultryindustry is growing at a rate of about2 percent to 3 percent annually. In2002, about nine billion broilerchickens were produced in America,and our country’s production of thesemeat-type chickens constitutes onlyabout a quarter of broiler productionworldwide.

Continued growth in the broilerindustry is expected because of:

A predicted doubling of thehuman population within thenext 50 years.The likelihood that chickenmeat will remain a low-costsource of high-grade animalprotein.The favorable health imageof eating chicken products.The expectation that customeradaptability to new poultryproducts will remain positive.The lack of cultural impedi-ments to poultry consumption.

Because the broiler industry israpidly expanding and becauseselection for greater body weight inbroilers has been at the expense ofegg lay, the production of more eggsis becoming increasingly important.For example, to meet global demands

at current expansion rates, an extra 15million broiler breeders will be neededannually to produce 1.5 billion morebroilers for human consumption. Toaddress this need, LSU AgCenterresearchers have demonstrated thefeasibility of increasing egg productionin broiler breeder hens using chickeninhibin-based immunopharmaceuticals.

Inhibin is a peptide hormoneproduced in the ovary that acts as abrake on egg production. When aninhibin antigen is presented to the birdin the context of a foreign protein, theanimal’s immune system is fooled intoattacking inhibin. Similar toautoimmune diseases (for example,multiple sclerosis), the bird raisesantibodies to, and thereby inactivates,endogenous inhibin, which in essencereleases the brake on egg production.We have shown thatimmunoneutralization of inhibin byvaccination increases ovulation ratesin quail, chickens (both egg- and meat-type) and turkeys.

Over the past nine years, LSUAgCenter scientists have been workingin close cooperation with multiplecorporate sponsors to develop safe,inexpensive and effective inhibinvaccines. The scientific developmentof these patented vaccines (chickeninhibin-based antigens) has led to therecent discovery of a more practicalsynthetic vaccine. The vaccine iscomprised of a Multiple AntigenicPeptide (MAP) with a poly-lysinebackbone linked to four peptidescomprising the first 26 amino acidsof chicken alpha-inhibin. Whenappropriately administered to breederhens, it appears to be on track toproduce an increase of around 10percent in hen-day egg productionduring the industry standard 40 weeks oflay. This means that producers who usethe vaccine could experience an increasein egg lay of about two dozen eggs perhoused hen. This represents the singlelargest on-the-spot enhancement ofreproductive performance in the historyof the poultry industry. Indeed, it hastaken the breeders of egg-type chickensabout 20 years to achieve a lesser, about

7 percent, increase in egg lay bytraditional methods of geneticselection.

Depending on management, thevaccine increases the onset, magnitudeand duration of egg lay. Of equalimportance, regardless of how thelaying curve is affected, inhibinimmunoneutralization has shown nountoward effects on eggs produced byvaccinated hens or on the chicks thathatch from these eggs. The inter-vention does not alter hen body weightand livability; egg size and egg shellthickness; the fertility and hatchabilityof eggs; or livability of hatchlings toharvest age.

This remarkable story does notend with the hen. Active immuni-zation against inhibin holds significantpromise in enhancing the fertilizingcapacity of broiler breeder roosters aswell. In a preliminary study, this newvaccine was found to acceleratepuberty and increase fertility in agedmales.

With the continued emphasis onselection for body weight in yield-typebroiler breeders, overall egg fertility isdeclining at an alarming rate (about ahalf percent annually). This is mostlikely due to decreased gonadalfunction and copulation efficiency oflarge-bodied males. Marked declinesin fertility are now commonly seen inflocks with aged males (more than 36weeks old). This has caused producersto “spike” their old flocks with youngmales to increase fertility. Thispractice is expensive because addi-tional flocks of males have to bemaintained and risky from a bio-security standpoint because thestandard “all-in, all-out” practice usedin poultry is violated when youngoutsider males are introduced intoestablished flocks.

Inhibin immunoneutralizationof broiler breeders is a revolutionaryapproach that holds promise to changethe poultry industry forever in asimilar manner as bovine soma-totropin (BST) changed the dairyindustry.

New Vaccine Increases BroilerBreeder Chicken Production

Daniel G. Satterlee, Professor, Department ofAnimal Sciences, LSU AgCenter, Baton Rouge,La.

PDaniel G. Satterlee


he aquaculture industry is looking increasingly togenetic improvement for gains in production. But improvingthe genetics of aquatic species can take a long time. Withcatfish, for instance, a male typically spawns with only onefemale each season. Even if genetically superior males andfemales could be identified, the process of developing breed-ing stock and improved lines could take a decade or more.

Fish sperm were among the first cell types successfullymaintained through cryopreservation – the technique offreezing cells at extremely low temperatures so they remaingenetically stable and metabolically inert. This research,published 50 years ago, coincided with similar successes withdomestic livestock. In the intervening years, a multi-milliondollar global industry has developed for frozen livestocksperm while cryopreservation of aquatic species’ sperm is justnow on the verge of moving from the laboratory to the market.

The benefits of cryopreservation in aquaculture speciesinclude the following:

1. Cryopreservation can be used to improve hatcheryoperations by providing sperm on demand and simplifyingthe timing of induced spawning.

2. Frozen sperm can enhance efficient use of facilities andcreate new opportunities in the hatchery by eliminating theneed to maintain live males.

3. Valuable genetic lineages, such as endangered species,research models or improved farmed strains, can be protectedby storing frozen sperm. This could be critical for marinespecies such as shellfish, where valuable broodstocks mustbe stored in natural waters.

4. Sperm can be used in breeding programs to create new,improved lines and shape the genetic resources available foraquaculture operations. A dramatic example of this is in thedairy industry, which relies almost entirely upon cryo-preserved sperm to produce improvements in milk yields.

5. Cryopreserved sperm of aquatic species will likelybecome an entirely new industry within the coming decade.Large, highly valuable global markets for cryopreservedsperm of aquatic species are on the horizon.

Artificial insemination entails the collection of sperm andeggs so one male can fertilize eggs from several females andone female’s eggs can be fertilized by sperm from severalmales. This can lead to a matrix where a group of selectmales can be mated with a group of select females to developpopulations with distinctive traits. By having assayed theparents for genetic markers, the industry can develop brood-stocks with enhanced characteristics, such as growth rate,disease resistance or vigor. Such a process can also be usedto cross two different species (for instance, blue catfish andchannel catfish) to develop a new strain with hybrid vigor.

The process could also be used to develop triploid oystersby fertilizing diploid females with sperm harvested fromtetraploid males. The results would be triploid offspring whichwould be sterile, allowing them to convert energy to bodymass instead of reproduction. This could extend the harvestseason.

The dairy industry provides a business model fordeveloping an industry for cryopreserved sperm of fish andshellfish. In addition, this industry can provide equipment,protocols and facilities. We have worked for eight years withGenex Custom Collections, Inc. and the LSU AgCenter DairyImprovement Center to adopt for use with aquatic species theinfrastructure developed for dairy bulls. Other challengesinclude biosecurity concerns related to sample transfers,pricing structures and product quality control issues.

Another model for developing repositories for geneticresources comes from the newly formed National AnimalGermplasm Program (NAGP) of the U.S. Department ofAgriculture. The NAGP is patterned after the well-establishedUSDA National Plant Germplasm System. NAGP hascommittees for animals such as beef and dairy cattle, swine,goats and sheep, poultry and aquatic species. The AquaticSpecies Committee brings together members from univer-sities, industry and federal agencies. The LSU AgCenter isassisting the transition from cryopreservation research toapplication through work on protocol standardization, gametequality, sample labeling and database development to providea repository to protect genetic resources, including endangeredspecies, and to assist in developing existing and futureindustries for culturing aquatic species.

Photo by John Wozniak

Cryopreservation

Cryopreservation research in Terry Tiersch’s laboratory includeswork in the hatchery (above), laboratory and field.

T

Terrence R. Tiersch

A New Industry for Aquatic Species

Terrence R. Tiersch, Professor, Aquaculture Research Station, Baton Rouge,La.

AcknowledgmentsSupport was provided by the U.S. Department of Agriculture, theLouisiana and National Sea Grant College Programs, the NationalInstitutes of Health, the U.S. Fish and Wildlife Service and theLouisiana Catfish Promotion and Research Board. Other scientistsinvolved in the research include Mark Bates, John Buchanan, RexCaffey, Tyler Campbell, Michael Christensen, Daisy Dong, DonaldGlenn, Robert Lang, Carmen Paniagua-Chavez, German Poleo, KenRiley, Greg Roppolo and William Wayman.


ecent developments in cellbiology, molecular biology, immun-ology and genetic engineering havegiven new dimensions to research andapplication of biotechnology to farmanimals. Historically, artificial insem-ination, one of the early reproductivetechnologies, has provided excellentopportunities to expand the superiorgenetics of selected animals in plannedbreeding programs. With the develop-ment of applied aspects of embryotransfer technology (nonsurgicalcollection and transfer methods forcattle) in the mid 1970s, animal repro-duction again entered a new age oftechnical advancement. Although beefcattle prices, industry promotion andproducer interest enhanced the use ofthis technology in the late 1970s andearly 1980s, embryo transplantation ismore often used today by dairyproducers.

Embryo transfer methodologies inthe future will likely be conducted usingunique or laboratory-derived specializedembryos. In the years to come, theembryos for transfer will be producedwith frozen sperm from geneticallyvaluable males and oocytes (eggs)harvested from cows in the producer’sown herd, evaluated for gender andlikely tested for valuable genetic traitsbefore the embryos are transferred tothe recipient females. On the horizon,cloned embryos will be produced fromcells from valuable males and females,or even produced with foreign genesintroduced into the genetic makeup ofthe embryo before the cloningprocedure.

Selecting the Sex of EmbryosVarious studies have reported that

male embryos develop at a faster ratethan female embryos in the early stages

of embryonic development. The fasterdevelopment of male embryos has beendescribed for mouse, pig, cow andhuman embryos, and has been attributedto the presence of the Y chromosome inmale embryos. An alternative hypothesisis that before X chromosome inactiva-tion, the activity of two X chromosomessubsequently hinders the growth offemale embryos. Knowing that maleembryos generally grow faster, andthose are at a later stage of developmentthan female embryos, one could increasethe chances of producing a maleoffspring by selecting the mostdeveloped cattle embryos from anembryo collection to transfer torecipient females.

Microsurgical methods have beendeveloped to extract individual cells(called blastomeres) from early stageembryos. The cells remaining in thebiopsied embryo generally survive anddevelop into a viable offspring. Theefficiency of embryo production fromearly stage embryos, however, tends tolessen in farm animals because there arefewer cells remaining in the embryo.The individual cells removed from bothearly- and later-stage embryos can beused to determine the sex of eachembryo before transfer to a recipientfemale. Embryo sexing using the DNA(deoxyribonucleic acid) amplificationprocedure known as the polymerasechain reaction (PCR), with specificY-chromosome DNA probes, isremarkably accurate for sexing cattleembryos. This method has recently beenmade user-friendly and can be complet-ed within 2.5 to 6 hours after theembryos are harvested from donoranimals. Sexing later-stage embryos at6, 7 or 8 days of age before transplanta-tion is now available at most embryotransplant stations. There are at leasttwo commercial companies operatingin North America that distribute acomplete cattle embryo-sexing kit forin-field use. The capability of sexingembryos would give the producers theoption of selecting bull or heifer calvesfor market and reproductive manage-ment purposes.

Robert A. Godke, Professor; Richard S.Denniston, Assistant Professor; and Brett Reggio,Postdoctoral Fellow, Embryo BiotechnologyLaboratory, Reproductive Biology Center,Department of Animal Sciences, LSU AgCenter,Baton Rouge, La.

Animal Genetic TestingAfter removing individual cells

from the embryo (using microsurgery)just before transfer, genetic markers cannow be used to identify genes causinggenetic diseases in farm animals, forexample bovine leukocyte adhesiondeficiency, more commonly knownas BLAD, and economically importantquantitative trait loci (QTLs) can beidentified in the embryo.

Once a defective gene or a specificQTL is identified in the embryo, theproducer would have the option to eithertransfer or discard an embryo. Anembryo with a proper combination ofalleles for myostatin, thyroglobulin andcalcium-activated neural protease, forexample, could have an increasedmarket value because of its potential forenhanced growth rate, improvedmarbling and increased tenderness. Cellsof the embryo could also be tested toverify parentage or to enable selectionfor or against a phenotypic trait, such asred coat color in cattle. Selectingembryos with specific animal produc-tion traits (for example, high milkproduction or increased feed efficiency)would give the producer an advantage inefficiency over other producers notusing this molecular technology. Thispowerful new biotechnology tool hasthe potential to greatly enhance the live-stock industry in the years to come.

Monoclonal AntibodiesTo Detect Disease

Antibodies that aid in diagnosingand treating disease can now beproduced through animal biotechnology.Laboratory animals (for example,rabbits) are injected, usually two ormore times over several months, witha foreign protein and an adjuvant totrigger the animal to respond byproducing antibodies to the foreignprotein in their blood stream. After alaboratory cell fusion step, hybrid cellscalled hybridomas, under specificlaboratory culture conditions, willproduce antibodies to the originalforeign protein injected into the animal.

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Robert A. Godke, Richard S. Denniston and Brett Reggio

Animal Biotechnologyand the Future


These highly specific antibodies arereferred to as monoclonal antibodies.

These hybridoma cells areconsidered to be immortal and have thecapacity to produce large quantities ofantibodies under industrial conditions.Each antibody is uniquely specific for aselected protein. The specificity of themonoclonal antibodies makes theselaboratory-produced proteins useful inlive-animal diagnostic tests for variousinfectious agents and for immunologicaltreatment of infectious diseases in farmanimals.

The value of this new technologyfor producers will be in detectingdiseases in farm animals. Immuno-diagnostic kits, each with a specificmonoclonal antibody to a causativedisease agent, are now being used onfarms and ranches for rapid in-fieldidentification of specific diseases.These kits can help producers identifya disease before it ravages their herd.

Recombinant DNA ProductsUsing recombinant DNA technol-

ogy, a specific gene can be identified,removed from the cell nucleus with anenzyme, placed in a suitable vector andtransferred into a host microorganism,such as fast-growing bacteria. While inculture, these microorganisms canreproduce into a large populationcontaining many copies of the geneoriginally introduced into the micro-organism via the DNA-loaded vector.Multiple copies of the gene thensynthesize a highly specific peptideor protein product for commercial use.

Biotechnology companies nowproduce new recombinant DNA proteinsto treat various viral diseases in farmanimals. An example is a class ofnatural antiviral proteins called inter-ferons used to treat calf scours andvarious respiratory diseases, such asshipping fever in cattle.

In recent years, researchers haveisolated the genes for specific viralproteins by recombinant DNA technol-ogy. Once isolated, these genes can beused in conjunction with recombinantDNA technology to produce largequantities of the viral protein for use inanimal immunization programs. Themajor advantage of this approach overprevious vaccine production methods isthat the viruses are not present in thevaccine. This eliminates the potentialrisk of disease outbreaks in theproducer’s herd following immuniza-tion. Also, production costs for these

new vaccines are generally lower thanthe conventional manufactured vaccines.

Embryo CloningIn the early to mid 1980s,

procedures were developed to producegenetically identical twin offspring(clones) by bisecting or splittingindividual sheep, goat, swine, cattle andhorse embryos 5 to 8 days of age. A fineglass needle or a razor blade chip wasused to bisect the embryo. Thepregnancy rates (45 percent to 70percent) in cattle after the transfer ofhalf of a bisected embryo (known as“half” embryo or demi-embryo) aresimilar to those of intact embryos fromthe same donor female. Embryocollection, embryo microsurgery andtransfer of demi-embryos to recipientfemales can be completed in an hourwith this new reproductive technology.

Martha Gomez, an assistant professor in the LSU AgCenter’s Department of AnimalSciences, and her team at the Audubon Center for Research on Endangered Species(ACRES) in New Orleans, La., produced the world’s first clone of an African wildcat.This clone, named “Ditteaux,” was the center of attention at a recent pressconference. Gomez did her postdoctoral research with Earl Pope at ACRES andRobert Godke, Boyd Professor in animal sciences. The African wildcat is anendangered species. The AgCenter is a collaborator with ACRES on this project.

Photo by Linda Foster Benedict

For optimal success rates, each embryoshould not be bisected more than once.The technology has been continuallyrefined, and today methods are availableto bisect farm animal embryos with aglass microscope slide and a hand-heldrazor blade. This low-cost microsurgeryprocedure is simple, effective andrelatively easy to learn.

Embryo bisection offers thepotential of doubling the number ofviable embryo transplant offspringproduced from valuable donor females.For example, 100 good quality intactcattle embryos may result in 65transplant offspring born; whereas, 100similar quality embryos divided intohalves would yield 200 demi-embryos,which then may result in 130 split-embryo transplant calves born or a 130percent pregnancy rate from 100genetically superior embryos. Using this



procedure on beef cattle embryos hasbeen averaging 1 to 1.2 calves per donorembryo bisected. Twin calves producedby microsurgery will result in genet-ically identical offspring of the samesex. This would remove the concernfor bovine freemartinism, if both demi-embryos are transferred to the samerecipient female. A freemartin is aninfertile female calf born as co-twin toa male.

Research efforts are under way toimprove methods for freezing andstoring demi-embryos in liquid nitrogento subsequently produce twin calvesanytime during any calving season.With cryopreservation, the possibilityexists that one demi-embryo of the setcould be transferred to a recipientanimal and the remaining demi-embryoof the pair could be frozen for transferlater. If the offspring derived from thefirst “half” embryo was of the geneticquality the owner desired, the remainingdemi-embryo could then be thawed andtransferred to another recipient animalto produce the second twin offspring.The second demi-embryo (a clone) alsocould be marketed as a sexed embryoclone of established genetic quality.

A new method of cloning callednuclear transfer (separating andtransferring individual, undifferentiatedembryonic cells to enucleated oocytes)emerged in the mid to late 1980s.Multiple nuclear transfer-derivedoffspring from individual blastomeresfrom a single prehatched embryo havebeen produced in several farm animalspecies (sheep, cattle). Nuclear transfer-derived offspring have been producedfour generations from a single cattleembryo. Unexpectedly, some of thenuclear transplant calves have extendedgestation length, and there are reports ofabnormally large term offspring (calves,lambs), which often need assistance atbirth. The reason for these problems isnot clear, although laboratory cultureconditions have been implicated as apotential cause. Even though improve-ments in the nuclear transfer methodol-ogy are still needed, this approach has agreat deal of potential for seedstockprocedures in the future.

Somatic Cell CloningMore recently, there has been a

major breakthrough in the animalnuclear transfer procedure. With“Dolly,” the famous sheep, cells forcloning were harvested from themammary gland of a mature ewe. Thesemammary cells were incubated in the

laboratory to produce a larger popula-tion of similar type cells for the nucleartransfer procedure. The production ofcloned sheep in Scotland was importantbecause it was the first mammalproduced in the world from an adultdifferentiated body cell (somatic cell).

The cloning of adult sheep (Dollyand her sisters reported in 1997)stimulated interest in nuclear transfertechnology by the livestock industry. Toconstruct cloned embryos with this newapproach (Figure 1), take a somatic cellfrom a developing fetus or an adultanimal (male or female) and microsurg-ically transfer it to an unfertilizedoocyte from which the female nuclearDNA has been microsurgically removed

Figure 1. The basic scheme for somatic cell cloning. An individual somatic cell from adonor animal placed into an enucleated cow oocyte. The reconstructed oocyte is thenexposed to an electrofusion procedure to activate the cloning process. The nuclear DNAfrom the donor cell then directs the formation of a newly formed cloned embryo, which isthen transferred to a recipient female to be carried to term.

(called enucleation). The enucleatedoocyte with the newly introducedforeign somatic cell becomes activated(as though it had been naturallyfertilized) and the reprogrammednucleus directs embryonic cell develop-ment into a cloned embryo for subse-quent transfer to a recipient female.Once the donor somatic cell populationhas been prepared, hundreds of clonedembryos can be produced in thelaboratory on a weekly basis usingoocytes extracted from abattoir ovaries.

Use of this new biotechnology hastremendous potential. Somatic cellclones have been produced in mice,rabbits, cats, sheep, goats, swine,domestic beef and dairy cattle, exoticcattle and exotic sheep. Most recently,cloned mules, a cloned horse and clonedexotic cats have been produced. Cloningwould provide the cattle producer anopportunity to reproduce geneticallyvaluable seedstock animals, cloneanimals that have suffered a severeinjury such as a fractured leg and canno longer reproduce, or clone males thathad been prematurely castrated, such asa prize-winning show steer. Clonedcalves have now been produced fromfrozen adult cattle tissue stored in astandard deep freezer for years. It hasbeen proposed that sloughed off somaticcells from milk and semen could be usedto produce cloned farm animals. Ahealthy cloned calf has recently beenproduced from somatic cells extracted

This heifer calf, named “Ninja,” was thefirst somatic cell cloned calf produced atthe LSU AgCenter’s Embryo BiotechnologyLaboratory three years ago. Using this newcloning technology, the calf was producedfrom a small tissue sample taken from amature Brangus-based cow in the LSUReproductive Physiology breeding herd.

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from the milk (fresh colostrum) shortlyafter calving in a dairy cow.

Cloning technology would alsoprovide livestock producers with readyaccess to production-tested breedingstock, thus increasing the accuracy ofselection in their breeding herds. It hasbeen proposed that cloning F

1 terminal-

breed males to produce males for marketsteers might be the ultimate beefproduction management system. Withthis scenario, fewer cows would beneeded to produce annual replacementheifers, so more F

1 recipient females

could be available to produce the clonedF

1 males for use as steers. This scenario,

however, assumes that the new cloningmethodology becomes more efficientand economically feasible for cattleproducers.

Incorporating DNADNA is the genetic template stored

in a highly compacted nucleus and isneeded for the replication of living cells.This DNA material is stored in strandsas small nucleotide subunits (calledgenes) along the chromosomes thatreside in the nucleus or in the mitochon-dria residing in the cytoplasm of thecell. Individual genes are responsible forthe production of specific proteins in thecell. Some genes are expressed at higherlevels than others in cells at varioustimes, resulting in production of largeramounts of the corresponding protein atvarious stages of life.

Advances made during the lastdecade in molecular biology haveenabled scientists to identify and isolatespecific genes within the chromosomesof animal cells. In recent years, mucheffort has been directed toward incorpo-rating foreign DNA (genes) into thenuclear material of oocytes, termed genetransfer, and more recently into cells ofearly-stage embryos. The basic approachto gene insertion involves the transfer ofDNA via microinjection techniques intothe male pronuclei of recently sperm-activated ova. Following the fertilizationprocess, the resulting geneticallyengineered embryos are then transplant-ed into recipient females. The embryotransplant offspring would then have thepotential for gene expression (transgenicanimals with genetically alteredcapabilities) throughout various parts orall of their lives.

The first experiments usingmicroinjection of DNA into thepronucleus of a sperm-exposed oocyteintroduced hemoglobin genes andgrowth hormone genes of other

mammals(rabbits, humans)into mouse ova.Microinjectedgenes then insertinto the geneticmaterial duringthe fertilizationprocess, resultingin incorporationinto the nucleus ofthe resultingmouse embryos.These embryoswere transplantedinto mouserecipient females,which thenproduced live,transgenicoffspring. Asthese youngtransgenicoffspring started growing, copies oftheir inserted gene became activatedresulting in a foreign animal proteinbeing produced in the mouse.

The mice that had the insertedgrowth hormone gene produced elevatedlevels of growth hormone in their bodyresulting in larger animals than theirnontransgenic littermates. In addition,these introduced genes were ultimatelytransmitted to the offspring of the micethat had originally received the gene.Recent refinements in microinjectiontechniques have led to successfulintroduction of growth hormone genesinto sheep, pig and cow embryos,although the efficiency of geneincorporation was low and variable inexpression among the different animals.

This methodology does showpromise for genetic alteration (engineer-ing) of farm animals but more efficiencyis needed to be economically feasible.With the original gene injectionprocedure, it was difficult to control theamount of the foreign DNA incorpo-rated into the nuclear material of theresulting embryo and, furthermore,where the DNA was incorporated in thechromosomes. This generally results invariable levels of gene expression in theprogeny, with a portion of the animalsproducing low levels of the foreignprotein and others producing elevatedlevels of the protein. Researcherssubsequently have worked on develop-ing different methods for incorporatingDNA into embryonic and somatic cellsand on using targeted gene expression ofthe foreign gene in specific body tissues,such as the muscle.

One of the most obvious ways inwhich biotechnology can affect sheep,swine and beef production is byincreasing growth efficiency in marketanimals. Human growth hormone geneshave already been introduced into somefarm animals but the animals producedwith this incorporated gene generallydid not have a growth advantage overthose not receiving the gene. Effortshave been made to isolate the specificgrowth hormone gene of pigs and cattleand to use the gene appropriate for thatspecies to make transgenic animals.Alternative newer methods for DNAincorporation into ooctyes and embryos(sperm-mediated gene transfer,retrovirus vectors, electroporation,nuclear transfer) should improve theefficiency of this methodology.Although the current focus for trans-genic farm animal research is forbiomedical purposes, recent efforts havebeen directed toward the improvementof animal health, such as the lysostaphingene to reduce mastitis in dairy cattle.

The regulation of animal growthand development is a complex physio-logical process involving a multitude ofgenes in addition to the growth hormonegene. A recent example is the Callipagegene identified in sheep that canincrease the efficiency and the amountof muscle in growing lambs. Theinactivation of the myostatin gene inbeef cattle has been shown to dramatic-ally increase the amount of muscle inanimals compared with those withoutthe inactivated gene. It is anticipatedthat the other genes involved in growthand production efficiency will also be

LSU AgCenter scientists at the Embryo Biotechnology Laboratory(EBL) were part of the team that produced the world’s first clonedtransgenic goats in 1999, which produced pharmaceutical proteins intheir milk. Above are two recently cloned dairy goats produced at theEBL. They are transgenic and will have the ability to produce humanpharmaceutical proteins in their milk when they produce offspring.

Photo by Robert A. Godke


iruses cause lost productivity in allspecies of agricultural plants and animals.Viruses work by entering a cell andsubverting the essential functions ofthat host cell to replicate their own kind.Implicit in this strategy for survival areconsequences for their hosts, rangingfrom pain and suffering to possible death,depending upon the nature of the virus andthe particular host.

Because viruses are so small, theymust accomplish their entire life cycleusing a limited number of genes andstructural elements. Most viruses consistof only a nucleic acid core, either DNA orRNA, surrounded by at least one proteincoat, and sometimes, depending upon thetype of virus, a second overcoat consistingof a membrane with inserts of viralencoded proteins. These coats serve toprotect the viral nucleic acids as the virustravels from cell to cell within the host andduring transport between hosts. Some ofthe specific proteins on the viral surfaceare also designed as molecular keys thatenable the virus to enter the specific hostcell of its choosing.

Occasionally, virus infestationswreak havoc on a product and cause theimposition of regulatory limitationspreventing transport of the product fromthe farm. With animal agriculture,additional problems can and do arise.Because animals can serve as reservoirsfor the genetic mixing of genes fromdifferent viruses, the potential exists togenerate more virulent strains of virusescapable of infecting people and causingdisease. Clearly, there is a need to limitviral infections of our agriculturallyimportant species.

Vaccination LimitationHistorically and to date, most

attempts to control indigenous viruseshave depended upon stimulation of thehost immune system to recognize andinactivate the structural elements andprotein keys on the surface of each virusthrough vaccination strategies. Vacci-nation is often effective. But success

isolated and their role in growthregulation identified in the near future.Inserting multiple genes into an embryomay ultimately improve the growth andfeed efficiency in meat-producing farmanimals.

Transgenic pigs have been producedthat carry extra copies of the alpha-lactoalbumin gene to increase milkproduction during lactation. Once theseand other genes that enhance milksecretion are isolated, they could beused to make transgenic founder animalsto transmit these milk productioncapabilities to their offspring. Theultimate use of transgenic technologyfor producing farm animals in the futurewill likely be from using transgenicmales with genes of interest to transmitspecific traits to their offspring.

Gene FarmingGene farming (termed “pharming”)

refers to the concept of using farmanimals as biological factories tomanufacture commercially valuableproducts in their milk. It was firstreported that transgenic mice with genesincorporated in their mammary glandswere able to produce high levels ofhuman growth hormone in their milk.Since then, genes with site-directedpromoters have been produced that cansecrete human pharmaceutical peptidesand proteins in the milk of mice, rabbits,sheep, goats, swine and cattle. Thesetransgenic animals are then mated andwhen their offspring are produced, thefemales are allowed to nurse theiroffspring. The females are then milkedto obtain the human pharmaceuticalproteins from their milk. The humanproteins are then extracted from the milkand purified. Production of humanpharmaceutical products in this way hasbeen shown to be cheaper and moreefficient than by conventional industrialprocedures. This technology opens anew potential for the use of farmanimals.

This new biotechnology usingtransgenic farm animals to producebiopharmaceutical products in their milk(human anti-thrombin III, human serumalbumin and human blood clottingfactors VII, VIII and IX) has resulted inremarkable achievements in recentyears. The best example is anti-thrombinIII, a blood anticoagulant used for heartpatients and during surgery in humans,which will be the first of thesepharmaceutical products expected toreach the commercial market early in2004.

Gene Wars:Rapid Advances

Advances in assisted reproductivetechnologies during the last decade havebeen occurring at such a rapid rate thateven scientists themselves are amazed.The potential for use of these newbiotechnology procedures for animalproduction extend almost as far as onecan imagine. Tremendous progress hasbeen made in the development andapplication of these technologies.Although the availability and costeffectiveness of some of these newtechnologies still remain in question,there is little doubt about their potentialimpact on livestock production. Many ofthese applications will require moreresearch and in-field testing before theyreach the marketplace. It is obvious thatuse of these emerging technologies willrequire more intensive management bythe livestock producer. These newtechnologies, if economically practical,will provide the producers with theopportunity to change the geneticpotential of farm animals at a faster ratethan is possible by the conventionalmethods presently in use.

It is predicted that marker-assistedselection for both single and multiplegene traits will become a potent assistedreproductive technology for embryos,newborn offspring and young adultanimals. The challenge to the industrycomes in identifying those traits thatmerit the application of these newassisted reproductive technologies.

There is little doubt thatbiotechnology will contribute to theimprovement of human health andmedical treatments, improvedproduction of animal food sourcesand development of new commercialproducts. Biotechnology companiesare using these new methodologies toimprove animal production efficiency,increase animal disease resistance andto alter genetic traits in food animals.

In the future, biotechnologyproducts and new assisted reproductivetechnologies will likely become a largerpart of the livestock producer’s tools inembryo production and in the produc-tion of herd replacements. The researchapproach in our laboratory and that ofothers is to develop new assistedreproductive technologies that haveeconomic, agricultural and biomedicalapplications.

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and not present in unin-fected cells.These unique genetic signals can beexploited by altering the host cell so thatexpression of the viral signals can nolonger lead to the production of progenyvirus. Each virus, therefore, has thegenetic equivalent of an Achilles’ heel,either encoded in gene regulatorymechanisms or in unique methods of theviral nucleic acid replication.

Consider the common influenzatype viruses that cause so manyproblems for animals and humans. Allinfluenza viruses use single strands ofRNA as their genomes, and the singlestrands are in a form called negativeRNA, a complementary copy of themessenger RNA that host cells normallytranslate into gene products. At the earlystages of infection, the viral negativeRNA must first be converted tomessenger RNA so its encodedcommands can be understood bythe host cell. This conversion can beaccomplished only because the virusprovides the unique enzyme, an RNA-dependent polymerase, that allows thehost cell to convert the viral negativeRNA into a translatable form. The viruscarries this essential polymerase with itas it enters the host cell. Without thisenzyme, any form of negative RNAremains as nonsense to the host.

Unique functions of pathogens,such as this, provide opportunities fordisruption without causing undue harmto the host. The unique RNA polymerasecan be exploited. For example, the hostcells of plants and animals can beengineered to produce small amounts ofnegative RNA encoding a product thatwould shut the cell down upon infectionwith the virus. Without the virus, thenegative RNA could never be tran-scribed, so uninfected cells could neverbe harmed. The consequence of beinginfected with a negative RNA viruswould be to shut down the few initiallyinfected cells; the virus would beprevented from producing progeny andcausing disease.

is typically limited to a particular strain ofvirus. Furthermore, viruses have evolvedalong with their hosts and have developedways to escape the immune response byaltering the protein keys to confuse hostimmunity. Vaccinations, though useful,are often short-term solutions of limitedeffectiveness.

In addition to vaccines, some progresshas been made to design drugs specificenough to interfere with unique viralfunctions without causing too many sideeffects for the host. However, because of theprice of these drugs, agricultural applicationsare not realistic.

Despite limitations, there has neverbeen a better time to be optimistic aboutour ability to overcome these pesky viralinfections. Because of recent advances inmolecular genetics and biotechnology,strategies to prevent viral infections arewithin our future. Instead of basingprevention on the proteins of viruses,opportunities now exist to modify geneticinteractions between viruses and host cells,to the detriment of the virus.

Gene wars between viruses and hostcells have always existed at every level—from viruses that infect bacteria to humans.The innate genetic ability to inhibit mostviruses is what prevents each species frombeing inundated with all but the select fewviruses that have figured how to workaround the genetic mechanisms of inhibitionused by that particular host. With advancesin biotechnology, it is now possible to assistgenetics to overcome the remainingrecalcitrant viruses.

Biotechnology to the RescueEach type of virus has unique signals

used to control the host cell of its choosing.The trick is to modify the genetics of thehost in ways that will prevent the targetedclass of virus from fulfilling its quest toproduce more of its own kind. Once avirus enters a cell, it exists as nucleic acid,separate from the protective protein coat,and is now entirely dependent upon the cell.We believe that each class of virus encodesgenetic signals that are unique to the virus

To reach this goal, we haveengineered plasmids to express bothnegative RNA encoding genes to shutdown cells, along with marker genes,and we have expressed these plasmidswithin cells cultured in the laboratory.When the molecular dynamics aresufficiently understood at the culturedcell level, such constructs could beengineered into farm animals. Theneed for such protection is significantto agriculture and of potential impor-tance to human health. For example,it is the genetic mixing of human andbird forms of influenza in pigs thatgenerates new forms of humaninfluenza. Limiting influenza virusinfections of domesticated pigs andbirds would decrease opportunitiesfor the influenza viruses to recombineinto more virulent forms that couldinfect humans. We believe thatgenetic strategies to modify host cellsof agriculturally important plants andanimals for resistance to specificpathogens will contribute to the futureprofitability of agriculture and willimprove human health.

At the LSU AgCenter weenvision a time when viral infectionsof plants and animals will terminate atthe beginning of the infectious cyclebefore production of the viral progenyessential to cause disease. Themodified plants and animals will begenetically incapable of beinginfected by specific viral pathogens.As agricultural scientists we lookforward to being part of the researchprograms that will be responsible forlimiting viral infections of our plantand animal commodity products.

Biotechnology Can Help Control VirusesWilliam Todd, Laynette Spring, Jackie McManus, Brandye Smith and Richard K. Cooper

William Todd, Professor; Laynette Spring,Jackie McManus and Brandye Smith, allResearch Associates; and Richard K. Cooper,Professor, Department of Veterinary Science,LSU AgCenter, Baton Rouge, La.


The mixture of sucrose and micronutrients in the Petri dishenhance the chances of haploids spontaneously doubling.In a few weeks, these cells begin to grow into tiny plants.

Anthers collected from rice plants are put in a nutrient mixture.

Slicing Years OffRice Varietal Improvement

Qi Ren Chu and Steven D. Linscombe

Photos by Qi Ren Chu


evelopment of new varieties atthe LSU AgCenter’s Rice ResearchStation in Crowley, La., has been oftremendous benefit to the U.S. riceindustry. In 2002, about 63 percent ofthe rice acreage in Louisiana, Arkansas,Mississippi, Texas and Missouri wasseeded to LSU AgCenter-releasedvarieties. Research on varietalimprovement and release of new ricevarieties to farmers have significantlyincreased Louisiana’s statewide yields.For example, in 1990, average yieldswere about 5,100 pounds per acre. Butin 2001, that average had jumped tomore than 5,900.

Most of the varieties developedhave been created through conventionalbreeding. In conventional breeding, thepollen grain of the male parent and theegg of the female parent each containhalf of the chromosomes of a normalrice plant; the pollen grain and egg aresaid to be haploid. When these cellsunite, the fertilized cell becomes diploidbecause it contains twice the number ofchromosomes, the full complement ofchromosomes of the normal plant.

The first population of plantsfollowing a cross is uniform or homo-geneous; however, each individualcontains genes from each parent and,because the parents were different, theseplants are considered heterozygous.When these plants are self-fertilized,as is the typical rice crop, the resultingpopulation of plants is extremelyvariable, or heterogeneous.

At this point in conventionalbreeding, the plant breeder begins toselect for desirable characteristics from

Once the plants reach a certain level of maturity, they are readyfor transfer to the greenhouse. As many as 16 plants can developfrom 100 anthers. This is considered success.

The plants begin to take shape in less than three months after theanthers are placed in the culture media.

Qi Ren Chu, Associate Professor, Rice ResearchStation, Crowley, La., and Steven D. Linscombe,Professor and Director of the Southwest Region,LSU AgCenter, Crowley, La.

which is produced a new generation ofplants. Each succeeding generation isidentified by the letter F (filial, refers tosons and daughters) and a number. Forexample, the first generation is the F

1and the sixth is the F

6 generation. By the

F6 generation, through continuous self-

fertilization and selection by thebreeder, lines within the population willbecome uniform or homogeneous. Bythe time these lines move through yieldtesting, at least three more years of self-fertilization and selecting will haveoccurred. About nine generations willhave been produced in the process.

Several years ago, LSU AgCenterscientists introduced doubled haploidtechnology into the breeding program.This technology reduces by three tofour years the time required to produceuniform lines. It involves makinghaploid plants from heterozygous F

1diploid plants and then doubling thechromosomes to produce a new diploidplant referred to as doubled haploids. Akey characteristic of the doubled haploidis its homozygosity. In conventionalbreeding, reaching this point would haverequired at least six generations of self-fertilization and selection by the breederto achieve this state of uniformity.

Some of the steps in doubledhaploid technology are illustrated inFigures 1 through 4. Callus induction isthe formation of undifferentiated tissue,tissue consisting of unspecialized cells,which will later be induced to differ-entiate into leaves, roots and other plantorgans. The process of plantregeneration includes those proceduresrequired to produce differentiated

tissues and new plants. Once plants havebeen devel-oped, they are placed in thelater stages of conventional varietydevelopment such as progeny rowevaluation, preliminary yield testingand advanced yield testing.

U.S. commercial long-grainvarieties initially showed little responseto doubled haploid techniques whencompared to Asian rice, which are allshort- or medium-grain. Systematictesting of long-grain crosses to dozensof culture media enabled us to developculture media (designated as Chu basicmedium) suitable for callus inductionand plant regeneration of the U.S. long-grain crosses. Furthermore, doubledhaploid plants with high regenerationability were identified and are now usedas bridging parents to consistentlyproduce thousands of doubled haploidplants annually. In the beginning, only 2percent of attempts resulted in a doubledhaploid plant. That figure has risen to 16percent.

Numerous doubled haploid lineswith various new plant types have beencreated, selected and advanced. Theselines recombine the genes fromcommercial cultivars such as Cocodrie,Cypress, Wells, Francis and someChinese varieties. Many new linesexhibit compact plant types, erect andV-shaped leaves, and a moderately thick

D


leaf blade in contrast to Cypress andCocodrie and more tillers with a thickercanopy that differs from Wells andFrancis. Panicle sizes of the new linesare in between Cocodrie and Francis.All these lines have good seedling vigor,desirable maturity, high milling qualityand high yield potential.

After the doubled haploid plants mature in the greenhouse, theyare transferred to field test plots.

Qi Ren Chu, a rice breeder at the Rice Research Station inCrowley, La., addresses the audience gathered for the annual fieldday June 26, 2003.

Superior breeding lines developedso far have widened the geneticdiversity of current cultivars. They notonly carry genes for various grain yieldcomponents but also carry genes thataffect ideal grain quality and diseaseresistance. The uniformity and geneticstability of these lines facilitate the plant

breeder’s ability to select for desirabletraits.

Experiments indicated recom-bination between doubled haploid linesproduced a wide range of superiorhomozygous (uniform) progeny thatcarry desirable long-grain traits. It hasbeen demonstrated repeatedly thatsufficient quantities of long-graindoubled haploid plants can be producedfor breeding purposes. The averageyields of three of these entries are:LA2091—8,729 pounds per acre,LA2085—8,165 pounds per acre andLA2088—8,052 pounds per acre. Theaverage yields of the high-yieldingvarieties were: Francis—8,638,Cocodrie—7,783 and Cheniere 7,526pounds per acre. Seed increases of theseentries are under way.

Doubled haploid breeding researchconducted at the Rice Station hasresulted in the accumulation of a largenumber of promising doubled haploidlines. Most of these lines were derivedfrom crosses made by the anther cultureproject with desirable bridging parents.Evaluation of these lines indicatesacceptable traits for yield, plant height,seedling vigor, maturity date andmilling. Most of these lines have shownyield potential similar to or better thanthe dominant commercial cultivarsCocodrie and Francis. Multi-locationand multi-year testing of these lines isunder way to confirm any commercialvalue. New varieties are expected tocome from efforts of this project withinin the next few years.

These paper caps keep the pollen trapped, allowing for mating ofthe rice varieties.

Photo by Qi Ren Chu

Photo by Qi Ren Chu

Photo by Linda Foster Benedict


LSU AgCenter rice breeder Qi RenChu grows the equivalent of thousands ofacres of rice in his lab – a room the size ofyour kitchen.

“Instead of growing plants in a field,we grow five million pollen (grains) in aPetri dish,” explains Chu, straining to helpa layman understand how he coaxes greenrice plants to sprout in what starts out asa dish full of pollen swimming in a specialchemical soup.

“The process has been established. Itis legitimate. It is not a question of whetherwe can do it. It is a question of what scaleis possible and what is the marketability ofwhat you create,” says Chu, a native ofChina recognized as one of his country’stop rice breeders.

Chu cut his teeth as a research assis-tant and associate plant breeder at a toprice lab in Shanghai, China, starting in the1970s. He earned a Ph.D. in genetics fromLSU in 1988 and later rose to full profes-sor and director of the Agro Biotechnol-ogy Center at the Shanghai Academy ofAgricultural Sciences back home.

Chu signed on as a full-time ricebreeder at the LSU AgCenter’s Rice Re-search Station in Crowley, La., in 1995.His specialty is anther culture, the processof growing complete rice plants from justthe male half of the rice plant’s internalreproductive hardware.

The technique, first perfected in corndecades ago, requires a researcher toextract anthers from the head of the riceplant and make them grow in a Petri dish.Each anther contains 1,000 or more grainsof pollen, the male part of a rice plant. Thepollen is considered a “haploid,” meaningit has 12 chromosomes or exactly half ofa rice plant’s normal 24-chromosomemake-up.

When growing in soil, a rice plantneeds both pollen and its ovule or egg (theplant’s female half, also with 12 chromo-somes) to create the next generation ofseed. The chromosomes are the seat ofthe rice plant’s genes, which govern ev-erything from how quickly a rice plant willmature, how hearty it is against diseaseand how tall it will grow.

But Chu bypasses the normal fertiliza-tion method and instead uses a methodknown as a “doubled haploid” to grow acomplete but uniform series of plants – all(or at least most) of which have the samecharacteristics.

The idea is to speed up efforts to breednew types of rice that mesh the attributes ofone variety of rice with the favored aspectsof another. For instance, the goal might beto create a new rice variety that maturesearly (like Rice “A”) but also one that hasstrong resistance to disease (like Rice “B”).

Using the doubled haploid method letsscientists breed rice more quickly, bypassingat least some of the confusion that comeswhen trying to select plants with a nearlyuniform set of characteristics in the field.

In doubled haploids, the rice pollen isplaced in a Petri dish suspended in a mixtureof sucrose and other micronutrients thatenhance the chances of the 12 male chromo-somes spontaneously doubling to give you acomplete plant.

In conventional rice breeding, it cantake up to 10 generations of growing plantsin the field after an artificial hybridization or“cross” is made to perfect a 99.99 percentpure variety. Crossing “Rice A” with “RiceB” in the field isn’t an exact science, espe-cially when juggling the thousands of genes ina rice plant.

Plants in the field might appear identicalat first, but neighboring plants from thesame cross can have different degrees ofdisease resistance, for example, dependingon whether Plant A’s traits or Plant B’s endup as dominant.

“In conventional breeding, you makethe initial cross, grow out the F

1 plants, grow

the F2 (second generation), select a large

number of plants, grow the F3 (third genera-

tion), look to see which plants are uniform,”Chu said. “With doubled haploids, we gofrom anther (or pollen) in a Petri dish toplants in the greenhouse within two and ahalf months. The pollen regenerates intoentire plants.”

Chu says an 8 percent success rate(eight anthers growing up to be plants out of100 anthers in a dish) when breeding U.S.long-grain rice varieties is acceptable and

A Rice Field in a Petri Dish‘Doubled Haploids’ Speed Breeding

gives researchers enough healthy plantsfor further testing.

Steve Linscombe, director of theSouthwest Region and a rice breeder, saidthe doubled haploid method is anothertool in the rice breeder’s arsenal.

Linscombe said he expects one ormore of Chu’s creations to be released bythe LSU AgCenter in the next two to fiveyears.

At this point, conventional rice breed-ing methods are still the research station’sNo. 1 focus. And breeders use a winternursery in Puerto Rico, which allows formultiple rice crops every year, to speedthe development of new varieties. Use ofthe winter nursery has cut conventionalrice breeding time frames to roughly eightyears – down from about 12 years not solong ago.

But Linscombe said Chu’s approachhas a definite place and could result in newvarieties being released in as few as sixyears from Petri dish to farm.

Chu said he’s optimistic about sev-eral varieties in the doubled haploidpipeline.

“We have dozens of lines with excel-lent yield potential. Sooner or later wewill release one,” Chu predicts.

Linscombe cautions that it still takesa lot of field work to fully test any newrice variety.

“You can’t fully analyze yields on anindividual plant basis in the greenhouse,”Linscombe said. “Field conditions, with alltheir variables, are difficult to mimic in thegreenhouse. Disease in a real field is awhole new ballgame.”

That’s why even varieties that start inChu’s lab still have to weather severalyears of analysis outdoors, including prog-eny row testing, preliminary yield tests,advanced yield trials and three years ofmulti-state testing. “We not only work inthe lab, we put a lot of time and effort inthe field as well,” Chu said.

Chu’s research project, like manyothers at the Rice Station, is supported byLouisiana rice farmers through check-offcontributions under the direction of theLouisiana Rice Research Board. Randy McClain


iseases are a major constraintto rice production in Louisiana and theother Gulf of Mexico rice-producingstates. They cause millions of dollars indirect losses and losses related to the useof control measures. The most damagingdiseases are sheath blight and rice blast,caused by fungal pathogens, andbacterial panicle blight and sheath rot.Several fungicides are available to helpcontrol sheath blight and blast, but theyare expensive and require superiormanagement to obtain economiccontrol. No pesticides are availablefor controlling bacterial panicle blight.

Biotechnology-related research isbecoming increasingly important in thedevelopment of measures to supplementpesticides and conventional breedingefforts for disease control. In a coor-dinated effort involving the ricepathology laboratory in the Departmentof Plant Pathology and Crop Physiology,the Department of Biological Sciences,the School of Veterinary Medicine andthe Rice Research Station, research isbeing conducted in the laboratory andfield to develop new technologies fordisease control.

Improving ConventionalBreeding Through‘Clonal Variation’

Sheath blight is the leading cause ofdisease loss in Louisiana rice. No sourceof complete resistance has been

identified throughout the world forRhizoctonia solani, the cause of thisdisease, but some rice varieties havepartial resistance. For many years wehave been locating sources of partialresistance and incorporating them intoour breeding efforts.

In our search for better sources ofpartial resistance, we have exploited atechnology called clonal variation. Clonalvariation comes from the increased rate ofmutations found in plants regeneratedthrough tissue culture. Tissue culture isthe process of creating new plants fromcells in a test tube.

Using tissue culture technology wehave developed a new source of high-level partial resistance from a suscep-tible variety. Two lines having this newresistance were registered as the “elitelines” LSBR-5 and LSBR-33. Theselines have been used for several years,along with other natural, high-levelsources of partial resistance identifiedin our program, to generate thousandsof breeding lines with sheath blightresistance.

Each year new lines are screened fordisease resistance, yielding ability, grainshape, and quality and cooking character-istics. The best materials are advanced tothe rice breeding program at the RiceResearch Station in Crowley, La.

TransformationUsing Foreign Genes

Transformation is a technologywhere foreign genes, or genes fromother plants, animals and microorgan-isms, can be transferred to a crop plantto introduce a new trait. We areinterested in transferring “PR” genes,or genes involved in plant diseaseresistance, from other plants to rice.The genes are cloned from the sourceorganism and put into plasmid DNA,which is a type of bacterial DNA thatcan be transferred into the target plant.The procedure we use involves coatingsmall particles of gold with plasmidDNA containing the foreign gene andshooting them with an air-pressuredevice into cultured rice cells.

Biotechnologyand Control of Rice DiseasesM.C. “Chuck” Rush, Q. M. Shao, Shuli Zhang, A.K.M. Shahjahan,Kathy O’Reilly, Ding Shih, Donald Groth and Steven D. Linscombe

Our laboratory began working withrice transformation in the early 1990s.We first transformed rice with the genefor resistance to the antibiotic hygro-mycin (hpt gene). Since then we havetransformed many varieties with a genefor resistance to Liberty herbicide,including the commercial varietiesDrew and Cypress (Figure 1).

The PR genes with which we areworking include a thionin gene frombarley, a chitinase gene from soybeanand a beta glucanase gene from tobacco.All of these genes are involved indefense against diseases in the plantfrom which they were derived. We havetransformed rice with the thionin geneand found in preliminary greenhousetests that it greatly increased resistanceto the bacterial panicle blight and sheathrot disease.

We also used a process calledcotransformation to transform riceplants with different combinations ofthe chitinase and beta glucanase genes,which plants use to defend againstfungal pathogens, along with the genefor resistance to Liberty herbicide.These plants show a high level ofresistance to sheath blight in greenhouseand field tests (Figures 2, 3 and 4). Theyalso show resistance to Libertyherbicide. We still have to cross thismaterial with nontransformed plants to

M.C. “Chuck” Rush, Professor; Q.M. Shao,Postdoctoral Researcher; Shuli Zhang, GraduateAssistant; and A.K.M. Shahjahan, PostdoctoralResearcher (now Assistant Professor of Biology atBaton Rouge Community College), Department ofPlant Pathology and Crop Physiology, LSUAgCenter, Baton Rouge, La.; Kathy O’Reilly,Associate Professor, LSU School of VeterinaryMedicine, Baton Rouge, La.; Ding Shih,Associate Professor, Department of BiologicalSciences, LSU, Baton Rouge, La.; Donald Groth,Professor, and Steven D. Linscombe, Professorand Director, Southwest Region, LSU AgCenter,Rice Research Station, Crowley, La.

Figure 1. Rice transformed for resistance toLiberty herbicide. These rows were sprayedwith Liberty herbicide. Note the dead rowof nontransgenic rice.

Photos by M.C. “Chuck” RushD


varieties. For commercial use oftransformed plants, single transform-ation events must be registered by thefederal government in a process similarto that used for new pesticides. Theseregistered transformation events canthen be used to develop new varietiesby conventional breeding. The originaltransformant would not be released as avariety.

We used our Liberty-resistant transformants inextensive testing to determinehow many generations ofcrossing and backcrossing itwould take to transfer thetransgene from a singletransformation event into acommercial variety. Four tofive backcrosses over two anda half years were required torecover stable transformed lineswith the characteristics of thevariety. These characteristicsincluded appearance, height,grain shape and quality, time-to-maturity and yield potential.These transformed lines areavailable to the breedingprogram at the Rice ResearchStation.

Other research withtransformed plants showedthat when many transformationevents (individual transformedplants with the same transgene)were crossed, insertion was notcompletely random. Thissuggests that certain chromo-some sites were more likely tohave the transgene inserted. Byidentifying these sites and thefactors controlling insertion, wemay be able to dictate insertionsites when developing trans-genic plants. This would allowus to avoid the transformationproblems associated withinsertion in unfavorablelocations and benefit frominsertion into pre-selectedsites that favor gene expression.

Using Biotechnologywith Pesticides

Using Cypress ricetransformed with the bar gene,which is the gene that createsresistance to Liberty herbicide,we have conducted field testsfor several years to determine ifLiberty herbicide could be usedas a pesticide to control rice

diseases. Liberty herbicide is anantibiotic as well as a herbicide. Astransgenic rice varieties with Libertyresistance become available to ricegrowers, it is important to know theeffects of Liberty on rice diseases aswell as weeds. Also, there is potentialto greatly increase the use of Liberty inrice.

We determined the rates andtimings for use of Liberty as a fungicidein the field. In comparative field testsconducted for three years, Libertyapplied at the correct rate and timingprovided significantly better control ofsheath blight than Moncut fungicide andwas equal to or better than Quadrisfungicide. These are the fungicides nowused to control sheath blight inLouisiana rice.

Our research demonstrates thatLiberty used as a fungicide on trans-genic rice will have dramatic effects ondiseases. In preliminary field studiesLiberty also appeared to reduce bacterialpanicle blight. In laboratory tests, thecompound was highly inhibitory to thesheath blight, blast and bacterial panicleblight pathogens, as well as severalother rice pathogens common inLouisiana rice. The compound has thepotential to be a broad spectrumpesticide for controlling both weeds anddiseases in rice.

Rice Panicle Blight PathogenRice produced in the U.S. Gulf

Coast area has a long history of lossto panicle blighting. The damage inLouisiana was severe in 1995 and 1998,years with record high temperatures.Yield losses in some fields wereestimated as high as 40 percent.

In 1996, the cause of this panicleblighting was identified by ourlaboratory as the seedborne bacterialplant pathogen Burkholderia glumae.Further studies have implicated B.gladioli, B. plantarii and B. cepacia asother potential causal agents of panicleblighting in rice. The relationship ofthese plant pathogens to bacterialpanicle blighting and sheath rot inrice must be determined to developprocedures for identifying commercialseed infected with the causal bacteria.

Rice crops planted with infectedseeds will suffer severe losses duringsummers with unusually high nighttimetemperatures. The symptoms ofbacterial panicle blight include seedlingblight, sheath rot and panicle blight(Figure 5). Research in this project isdirected toward developing a method for

Figure 4. Resistant lesion on Taipei 309 rice afterpoint inoculation (inside white circle). Resistance wasconferred by transforming this plant with thechitinase and glucanase genes from soybean andtobacco, respectively.

Figure 3. Sheath blight developing on nontransgenicTaipei 309 rice after point inoculation.

Figure 2. Sheath blight developing on the susceptiblerice variety Cocodrie after point inoculation withRhizoctonia solani.

determine whether the sheath blightresistance is stable and if diseaseresistance is linked to Liberty resistance.

Several years ago, we transformedTaipei 309 and Nipponbare rice with thegene for resistance to Liberty herbicide.These plants were selected because theywere easy to use in tissue culture andgave stable resistance. They are not U.S.

Photos by M.C. “Chuck” Rush


identifying infected certified seed withhigh levels of the causal bacterialpathogens. Damage can be avoided bynot planting seed with high levels ofbacterial infection or by treatinginfected seed with antibacterialcompounds. This will greatly reducethe potential for loss by producers inLouisiana and other Southern rice-producing states.

The goals of our research are toclarify the causal relationships of theBurkholderia species associated withblighted rice, to develop procedures forextracting pathogenic bacteria frominfected seeds, to develop monoclonaland polyclonal antibodies specific for B.glumae and B. gladioli and to developan enzyme-linked immunosorbant assay(ELISA) system for identifying thebacterial pathogens from rice seeds. Thesensitivity and specificity of the assayare being determined using an existingcollection of rice-field isolates of B.glumae, B. gladioli, B. plantarii and B.cepacia. Development of this tech-nology will help increase rice yieldsand minimize some of the economicuncertainty related to rice production.

AcknowledgmentThis research was supported in part bythe Louisiana Rice Research Board anda Louisiana Board of Regents’ grant.

(Left) Panicles to be inoculated werecovered with an open wax paper tubeto protect other panicles on the plant.

(Left) Resistant transgenic Lafitte plantafter inoculation with B. glumae. Notethat the panicle remained healthy.

(Above) Nontransgenic Lafitte plant inoculated with B. glumae showing typical bacterialpanicle blight symptoms.

Figure 5. Severe bacterial panicle blightsymptoms on rice.


Photos by Chuck Rush


Rice farmer Danny Koch noticed something special as hepiloted a combine the size of a small house through his 82-acre fieldof Clearfield 161 rice this summer.

“This field was tremendously infested with red rice last year,”said Koch, who farms just north of Eunice. “Look at it this year.There’s not a stalk in here. It’s amazing.”

Credit the emergence of Clearfield 161, a new rice varietyreleased by the LSU AgCenter’s Rice Research Station in Crowley,for Koch’s and other farmers’ clean fields.

Used in combination with BASF’s NewPath herbicide, theClearfield rice system has shown 99 percent or more control of redrice weeds in fields throughout the U.S. rice belt this year.

Koch put the pencil to his Clearfield 161 harvest data and cameup with this: He averaged just over 42 barrels of rice per acre andexperienced excellent milling quality. Some other farmers in south-west Louisiana reported even higher yields with Clearfield 161,which was available for commercial use for the first time in 2003.

“I would have made nothing in this field if it hadn’t been forClearfield 161,” Koch said.

Clearfield 161 got its start when Tim Croughan, an AgCenterscientist at the Rice Station, discovered that by bathing particularrice seeds in ethyl-methane sulfonate (Clearfield 161 comes fromthe seed of Cypress, another AgCenter rice variety with strongyields and top-notch milling characteristics), he could create micro-scopic changes in the rice plant’s genetics and increase herbicideresistance.

The end result was that Clearfield 161 became so resistant tothe effects of imazethapyr, the active ingredient in the NewPathherbicide, that farmers can spray the red rice weeds in their fieldswith NewPath and not harm the good rice growing nearby.

Before Clearfield technology, any herbicide that killed red riceweeds also killed the good rice growing next to it. Red rice is atroublesome weed problem in southwest Louisiana . It competes forthe same nutrients and hurts both yields and milling quality of thegood rice harvested from infested fields.

Clearfield 161 is not a genetically modified crop (GMO)because no gene is inserted into the rice plant. Instead, chemicalmanipulation is used to encourage mutations in the rice plant’sDNA similar to changes that occur naturally anyway.

“Using ethyl-methane sulfonate just makes it more likely formutations to occur,” said Croughan. The trick is to foster the rightmutations that affect the plant’s herbicide resistance.

Top officials with Horizon Ag, a Memphis-based firm thatmarkets Clearfield seed nationally, say they expect to see a big jumpin the number of acres planted with the new variety in 2004.

“We expect at least 75,000 acres to be planted in Louisiana in2004, up from 30,000 acres this year,” said Randy Ouzts, Horizon’sgeneral manager. Nationally, he projects at least 375,000 acres tobe planted in 2004, almost double this year’s total.

“Clearfield 161’s milling quality is fine, very good. I haven’theard one complaint,” said Bill Dore, president of the Supreme RiceMill in Crowley.

Steve Linscombe, director of the LSU AgCenter’s SouthwestRegion and a rice breeder, agrees. He said Clearfield 161 willresurrect some poor-performing fields that were economic losersin years past because of severe red rice problems.

“All the results on yields and grain quality look very good,”Linscombe said after reviewing recent LSU AgCenter test data on

Clearfield 161 Has Rice Growers BuzzingClearfield 161. “In some fields this year, farmers had the highestyields they’ve ever had when you consider all their past red riceproblems and the losses that can cause.”

Tommy Ellett, co-owner of Angelina Plantation in ConcordiaParish, participated in a test with AgCenter rice specialist JohnnySaichuk. Side-by-side fields were planted under similar conditions –one field with Clearfield 161 and the other in the popular Cocodrierice variety. Both fields historically had severe red rice problems.

In mid-summer, Ellett could see the difference at a glance.“We’ve got 99 percent red rice control (on the 161 field). It’s likedaylight and dark between the two fields.”

At harvest, the Clearfield 161 test plot yielded 52.9 barrels ofrice per acre, slightly better than the neighboring Cocodrie field.

One word of caution: Clearfield 161 seed costs more thanmany conventional rice varieties, so it may not be a farmer’s bestchoice in every instance. But proponents say using it could put more

cash in a producer’s pocket depending on how severe red rice wasin a particular field and to what extent the weeds reduced the yield,grade or the milling quality of the good rice.

Many farmers who used Clearfield 161 this year planted theirfields at seeding rates ranging from 80 pounds to as much as 100pounds an acre. That may be too much. AgCenter experts familiarwith the way the Clearfield 161 rice plant grows (it tillers very welljust like its parent variety, Cypress) say farmers will probably be ableto reduce seeding rates significantly next year without hurtingproductivity. Lower seeding rates mean lower costs.

One problem to watch for with Clearfield 161 is that the newrice variety is susceptible to sheath blight, a rice disease thatflourishes in wet growing conditions.

Linscombe said growers who opt for Clearfield 161 shouldinclude an appropriate fungicide in their production programs.

LSU AgCenter rice research, including development of theClearfield rice technology, is supported in part by the Louisiana RiceResearch Board and by check-off funds contributed by rice farmersacross the state. Randy McClain

Louisiana rice farmers report that they are pleased with Clearfield161, the latest version of the herbicide-resistant rice that wasdeveloped at the LSU AgCenter. The variety allows farmers tocontrol red rice, which has been their No.1 weed enemy.

Photo by Mark Claesgens



ice farmers throughout the worldface a unique weed problem. A weedyrelative of cultivated rice, red rice, caninvade and severely infest rice fields,both lowering yields and reducing theselling price of the harvested grain.Most of Louisiana’s rice acreage isinfested, at least to some extent, withthis weed. Because of its close geneticrelationship to commercial rice, red ricehas proved difficult to control. Noherbicide yet developed can adequatelycontrol red rice without also injuring orkilling conventional rice.

Since sufficiently selectiveherbicides were not forthcoming, analternative approach to red rice control

Timothy P. Croughan, Professor, Rice ResearchStation, Crowley, La.

R was explored. Rather than continuingto search for a new herbicide with thedesired specificity, this alternativeinvolved trying to change the rice plantinstead. The goal was a plant that wouldthrive despite being sprayed with analready existing herbicide known to killred rice.

Genetic engineering is one wayto alter a rice plant’s sensitivity toherbicides. This involves adding a genefor herbicide resistance from anotherorganism. The resulting plant is termeda genetically modified organism(GMO), and any subsequent varietiesdeveloped from this plant that possessthe introduced trait are GMOs as well.

A different approach is to searchfor an individual rice plant that hasundergone a slight alteration in itsnatural inventory of genetic information,

resulting in the development ofresistance to the herbicide. While thereis no assurance that such plants can everbe found, the odds can be improvedsomewhat by using techniques thatincrease the rate of genetic changesabove the rates of mutations thatnaturally occur in all living things. Theresulting herbicide-resistant rice plantwould contain a slightly altered but stillnatural complement of genetic infor-mation. It would not be a GMO, since itcontains no inserted gene from anotherorganism.

A genetically engineered plantmight be produced fairly rapidly inthe laboratory; however, before a newvariety developed from that plant couldbe released to farmers, approximately$10 million must be spent on testing togain government approvals.

Timothy P. Croughan

Clearfield Rice: It’s Not a GMOClearfield Rice: It’s Not a GMO


of such crops is not an issue. OnceCanada’s approval is obtained, the ricecan be freely exported to all foreignmarkets.

Natural genetic change was usedto develop Clearfield rice, which isresistant to the chemical group ofherbicides called imidazolinones. Theseherbicides are new and have significantadvantages. The imidazolinone herbi-cides target a biological mechanismspecific to plants. This target, termedthe AHAS enzyme, is involved in theproduction of the amino acids leucine,isoleucine and valine. Plants require thecontinued production of these aminoacids to survive. Imidazolinones workas herbicides because they block theAHAS enzyme, preventing the produc-tion of the amino acids. Without theseamino acids, the weeds whither and die.

The AHAS enzyme is one of theapproximately 50,000 enzyme systemsin rice, and there are roughly 650building blocks (amino acids) in the riceAHAS enzyme. There was no certainty

that herbicide-resistant forms of AHAScould be found in rice. Essentially, thesearch for rice with resistance toimidazolinone herbicides involvedlooking for a needle in a haystack, ifsuch a “needle” existed in the first place.

Environmentally FriendlyAlthough imidazolinones are toxic

to weeds, they do not affect animals,insects or people, which lack the AHASenzyme that the herbicide disrupts.Thus, imidazolinones are environment-ally friendly herbicides.

While not toxic to animals, imidaz-olinones are so potent to weeds that ittakes only 1 to 2 ounces per acre tocontrol nearly all rice field weeds, andthe herbicides are particularly effectiveon red rice. By comparison, many riceherbicides now used are applied at ratesof several pounds per acre, and they donot control red rice. Replacing theselarger volume herbicides with theimidazolinone herbicides will result ina reduction in herbicide release into the

GMO FoodsPerhaps more important is the

significant resistance to GMO foods inparts of the world. The United States hasgenerally been accepting of GMO foods,but major U.S. food companies typicallyfunction internationally as well. Agrowing number of companies in theU.S. and overseas are banning GMOingredients in their products to assureconsumer acceptance worldwide. Thistrend continues, not so much becauseof scientific concerns on the part of thecompanies regarding GMO ingredients,but as a practical approach to aninternational marketplace that includesconsumers who have personal doubtsabout GMO food.

In contrast, only Canada requiresapproval before crops changed througha more natural means can be exported tothat country. Because more than 1,000varieties of a number of crops havealready been developed through thistechnique and grown worldwide overthe last 50 years, consumer acceptance

Photo by Mark ClaesgensMid-day thunderstorms roll toward rice fields farmed by Charles Reiners of Branch, La. He ran his combine until the lastpossible moment, when a wall of rain pummeled the field. A friend to the growing season, precipitation is a foe to harvest.


environment, and the imidazolinoneherbicides are less toxic to begin with.

After more than a decade ofsearching through some 1 billion riceseeds and plants, an individual plantwith an AHAS enzyme resistant toimidazolinone herbicides was found.This single survivor was transferred tothe greenhouse for further work. Theseeds it produced were planted, andthe resulting seedlings sprayed withherbicide. They also were resistant,proving that the parental plant wasindeed herbicide-resistant and not an“escape,” a plant missed while spraying.It also proved that its progeny inheritedthe resistance trait.

A program for breeding the traitinto higher-yielding varieties wasthen initiated, and conventional plantbreeding techniques were used totransfer the trait into established andpromising new rice varieties. Seed

Photo by Randy McClain

A combine stands at rest after cutting through a field of Clearfield 161 rice this pastAugust. The field is near Rayne, La., and farmed by Danny Koch, who praised the

yields of 161 and the red rice control achieved from using the variety.

collected from these crosses was plantedin the field, comprising more than 2,000unique rows. As the rice approachedharvest, rice breeders were invited tovisit the field and select seed fromplants they felt showed promise. Thefirst two U.S. varieties of Clearfield riceoriginated from this field and werereleased from the LSU AgCenter’s RiceResearch Station as CL 121 and CL 141.

Meanwhile, the search continuedfor additional herbicide-resistant riceplants, with the hope of finding a higherresistance level. The first plantdiscovered had sufficient resistancefor commercial use, but sometimesexhibited injury symptoms from theherbicide application. The plantsrecovered and yields were not affected,but a higher resistance level wouldavoid the temporary discoloration andslowing of growth sometimes observed.

During the subsequent five-yearperiod, another billion seeds and

seedlings were tested before a secondherbicide-resistant plant was discovered.Further testing of progeny from thisplant indicated that it was considerablymore herbicide-resistant than the firstplant discovered. It exhibited almost noinjury symptoms, even when subjectedto excessively high rates of the mostpotent imidazolinone herbicides tested.It was also high-yielding and producedexcellent quality grain. An increase ofthe seeds tracing back to this discoveredplant was conducted to develop the ricevariety named CL 161 for release togrowers. Within a year CL 161essentially replaced CL 121 and CL 141,and the acreage planted to Clearfieldrice in 2003 increased by nearlythreefold over the previous year. Thenumber of acres planted with thisnaturally herbicide-resistant rice isexpected to continue to increase forseveral years.



ice is one of Louisiana’s leadingagricultural commodities, with nearly532,000 acres planted in 2002,producing an average yield of 5,772pounds per acre. The 2002 gross farmincome reached $122.8 million, andvalue added in marketing, processingand transportation increased that amountto $159.6 million. In spite of theseimpressive figures, Louisiana rankslowest of U.S. rice-producing states forgrain yield, in part because weeds anddisease can substantially lower netreturns to producers. Geneticallyengineered or transgenic rice has beendeveloped in the LSU AgCenter thatshows the potential to avert weed anddisease problems, which will benefitthe Louisiana rice industry.

Development of LibertyHerbicide-resistant Rice

Various weeds in Louisiana ricefields, such as the noxious weed redrice, can cause significant losses ingrain yield and quality. To help developeffective weed management tools, wehave established an efficient method tointroduce genes for herbicide resistanceand other traits into elite Louisiana ricevarieties and lines. From this initialwork, we produced and tested nineindependent transgenic lines derivedfrom the variety Cocodrie that containeda gene originally constructed in thelaboratory by Bayer Crop Science, Inc.This synthetic gene conferred highlevels of resistance to the broad-spectrum Liberty herbicide in transgenicCocodrie during greenhouse and fieldtrials (Figure 1).

The transgenic lines were crossedonce to normal Cocodrie and thenadvanced to the next generation. Nineselected families were resistant at fieldapplication rates of Liberty at the 3- to4-leaf stage in a yield trial at the LSUAgCenter’s Rice Research Station inCrowley, La. Two transgenic linesyielded more than 7,000 pounds peracre, and one line, RGL1513, produced7,992 pounds per acre. It was the top-

James H. Oard, Steven D. Linscombe and Donald Groth

yielding long-grain entry in the trial(Table 1). RGL1513 produced 954pounds per acre more than Cypress, 656pounds per acre more than Cocodrie and370 pounds per acre more than Wells.Maturity and height of RGL1513 were

Figure 1. Transgenic Cocodrie after application of Liberty herbicide in the greenhouse.Normal Cocodrie plants in the right tray have yellow leaves one week after application ofLiberty herbicide. In the left tray, transgenic Cocodrie plants containing a gene resistant toLiberty appear normal and healthy after herbicide treatment.

James H. Oard, Professor, Department ofAgronomy, LSU AgCenter, Baton Rouge, La.;Steven D. Linscombe, Professor, Rice ResearchStation, and Director, Southwest Region, LSUAgCenter, Crowley, La., and Donald Groth,Professor, Rice Research Station, Crowley, La.

Photo by James H. Oard

Line/Variety Maturitya Height Yield(in.) (lb./A)

RGL1513 81 41 7992LL185 78 40 7946RGL1496 79 42 7822LL624 83 39 7736LL001 83 37 7429

Cocodrie 79 38 7336Cypress 80 39 7042Wells 81 41 7622

Table 1. Maturity, height and grain yieldof transgenic Liberty resistant lines andnon-transgenic Cocodrie, Cypress andWells, Rice Research Station, 2001.

a Days from planting to heading

identical to that of Wells and one to twodays later in maturity and 2 to 3 inchestaller than Cocodrie and Cypress. Thisresearch demonstrates that use ofherbicide-resistance genes in transgenicLouisiana rice varieties can contribute toeffective, long-term weed managementstrategies.

Development of RiceResistant to Sheath Blight

Sheath blight, caused by the fungusRhizoctonia solani, is the primary foliardisease of rice in Louisiana. All com-mercial Louisiana rice varieties are

R

for Herbicide,Disease Resistance in RiceBiotechnology


susceptible to thisdisease, which cancut rice yields inhalf. Recentadvances havebeen made throughtraditionalbreeding methods.But progress isslow because highlevels of resistanceare controlled bymany genes thatmust be combinedwith severaldesirable factorsfor high grain yieldand quality.

To addressthis challenge, wetransferred thechitinase genefrom rice and theglucanase genefrom Arabidopsisthaliana, a fast-growing plantuseful in research,to the sheathblight-susceptiblevariety Cocodrie.Chitinase and glucanase proteins arestrong antifungal agents that in previousstudies with tobacco have reducedinfection by Rhizoctonia solani. Aftergene transfer, more that 100 plants weregrown in the greenhouse and inoculatedwith the sheath blight fungus. Six plantsshowed higher levels of resistance thannontransgenic Cocodrie. One exampleis shown in Figure 2.

From this material, 52 lines wereadvanced and inoculated with R. solaniat the Rice Station in 2001. Eight lines

After testing in the laboratory, scientists tested the transgeniclines derived from Cocodrie in the field at the Rice ResearchStation in Crowley, La.

Figure 2. Response of normal and transgenic Cocodrie to infection in the greenhouse by the sheath blight fungusRhizoctonia solani. Transgenic Cocodrie on the left shows light infection three weeks after exposure, and normalCocodrie on the right shows extensive damage to leaves and stems.

Photos by James H. Oard

exhibited greater resistance than normalCypress and Cocodrie, and the engineer-ed chitinase protein was detected inleaves of transgenic lines, whereas nocorresponding protein was found innormal Cocodrie.

In 2002, seeds from individualplants of the resistant lines were plantedat the Rice Station and inoculated withthe sheath blight fungus. Three linesfrom independent events showedincreased resistance when compared tonon-transformed Cocodrie. Height and

maturity, always a major hurdle toovercome when using traditionalbreeding methods, were essentiallyidentical in the lines compared tonormal Cocodrie.

Future work will involve additionalmolecular and field analysis of thetransgenic lines. This researchdemonstrates the potential of genet-ically engineered rice to enhancedisease resistance thereby increasinggrain yields and economic returns forthe Louisiana rice industry.


weet potatoes are grown commercially by beddingwhole potatoes in the field and transplanting cuttings from thesprouts produced in the beds to the production field. Thus, it isone of many crops, including potatoes, sugarcane andstrawberries, grown by vegetative propagation.

Commonly, varieties of these vegetatively propagatedcrops decline because disease-causing pathogens, includingviruses and bacteria, gradually accumulate in the plantingstock over years of production. An added problem in sweetpotato production is the high frequency of mutations, whichcan cause defects such as changing the flesh color from thenormal orange to yellow or white (Figure 1). The frequencyof mutation in sweet potatoes is thought to be greater than inother crops because the part of the plant used for propagation,the storage root, does not have “eyes” as does a potato tuber,but must instead produce buds from vegetative cells. Thisprocess is thought to favor mutation.

In modern production, the rate of decline of varieties isslowed by programs that carefully produce foundation seedfrom stock free of viruses and mutations. In Louisiana,foundation seed is produced at the Sweet Potato ResearchStation at Chase and sold to growers to use in crop production.

Virus-tested PlantsVirus-tested sweet potato plants can be produced by a

plant tissue culture process known as meristem-tip culture,in which the smallest possible piece (less than 0.5 mm) isremoved from the growing tip of a selected plant. Thismeristem tip is regenerated in tissue culture into a new plant(Figure 2), which then must be thoroughly tested to be certainthat all viruses have been eliminated.

This involves a time-consuming, laborious method ofgrafting the tissue-culture derived plant at least three times toa seedling of an indicator plant, the Brazilian morning glory.This plant develops obvious symptoms when infected withknown sweet potato viruses. In addition, because mutationscan occur during meristem-tip culture, each virus-tested plantmust be grown in the field to be certain that it remains true tothe original characteristics of the particular variety.

Once the mericlone (the plant derived from a singlemeristem) has passed each of these tests, it is maintained intissue culture by cutting the stem into segments containing atleast one node on which is a preformed bud. A new plant cangrow from this bud with minimal risk of mutation. Since thisonly minimizes without entirely eliminating the risk ofmutation, this tissue culture stock must be periodicallyretested to be certain it is true to type.

The tools of molecular biology offer promise forimproving the process of producing sweet potato foundationseed in several ways. The most important is in improving theefficiency, accuracy and reliability of testing for the presenceof viruses, other pathogens and mutations, not only during theinitial process of generating the stock tissue cultures, but also

during the subsequent process of producing foundation seedand even farmers’ seed in the field.

Virus DetectionTwo groups of viruses found in Louisiana sweet potatoes

are a current focus of research: the aphid-transmitted poty-viruses and the whitefly-transmitted geminiviruses. It ispossible to isolate each of these viruses in other host plantsmore suitable for virus purification and characterization.

Each of the most important potyviruses that infectLouisiana sweet potatoes has been purified and used to

Figure 1. A sweet potato root with a section that has mutatedfrom the normal orange flesh to light yellow flesh.

Christopher A. Clark, Professor, Plant Pathology and Crop Physiology; DonR. LaBonte, Professor, Horticulture; Rodrigo A. Valverde, Professor; MaryW. Hoy, Research Associate; Pongtharin Lotrakul, former Graduate Student;and Charalambos Kokkinos, Graduate Student, Plant Pathology & CropPhysiology; and Cecilia McGregor, Graduate Student, Horticulture, LSUAgCenter, Baton Rouge, La.

Figure 2. Tissue culture plantlets of Beauregard sweet potato.

Photo by Don R. LaBonteS

Christopher A. Clark, Don R. LaBonte, Rodrigo A. Valverde, Mary W. Hoy,Pongtharin Lotrakul, Charalambos Kokkinos and Cecilia McGregor

Photo by Christopher A. Clark


prepare antisera in cooperation with colleagues at theInternational Potato Center in Lima, Peru. These antisera haveproved useful in serological assays to identify the virusestransmitted into the Brazilian morningglory indicator plants.

Unfortunately, sweet potato plants contain unusuallylarge concentrations of a number of substances that interferewith serological assays, including latex, polyphenols andpolysaccharides. Thus, this type of test has not proved reliablein detecting these viruses directly from sweet potato. In thecase of geminiviruses, detection using serological approacheshas not been successful in most crops.

An alternative to using serology, which detects primarilythe protein coat associated with viruses, is to detect thenucleic acid component of the viruses. A variety of methodscan be used for this purpose.

PCR-based AssayOne method developed at the LSU AgCenter to detect

the sweet potato geminiviruses is a polymerase chain reaction(PCR)-based assay. First, the sequence of nucleotide basesthat make up a portion of the DNA of this virus was deter-mined. This sequence was then used to design short segmentsof DNA that could be used as primers in the synthesis of aDNA product using the virus DNA as a template. Thus, ifvirus DNA is present in an extract from a plant, when the PCRtest is run using these primers, a DNA product is producedthat can be identified using gel electrophoresis.

This method has the advantage of both speed and sensitiv-ity. But, although it is useful for confirming the presence of

geminiviruses in a few samples, it is too expensive for usein cases requiring large numbers of samples be tested.

Hybridization AssayResearch is underway to determine if molecular hybri-

dization assays can be developed that could screen largenumbers of samples less expensively for sweet potatogeminiviruses. Hybridization assays involve extracting DNAfrom plant samples, placing the extracts on spots on sheets ofnitrocellulose membrane and then adding a specific probeconsisting of virus-specific DNA that is labeled withchemiluminescent marker. If virus DNA is present in thespot on the membrane, the probe will bind to it, and then themarker will produce a chemical reaction that can be observed(Figure 3).

Photo by John ChaneyThese are newly harvested sweet potatoes from the farm of Carl Ducote of Bunkie, La. They are theBeauregard variety, which was developed at the LSU AgCenter and is the country’s most popular variety.

Figure 3. A hybridization assayinvolves extracting DNA fromplant samples, placing theextracts on spots on sheets ofnitrocellulose membrane andthen adding a specific probeconsisting of virus-specific DNAlabeled with chemiluminescentmarker. If virus DNA is present,the probe will bind to it, andthe marker will produce a spot.


Real-time PCRA different approach is being tested for the potyviruses.

Real-time PCR is an adaptation of the basic PCR techniquethat increases the specificity and allows relative quantificationof the target nucleic acid. In addition to using specific primersas in simple PCR, a fluorogenic probe complementary to theDNA between the primers is used. As the PCR reactionamplifies the target DNA, the fluorescent signal is releasedfrom the probe and the fluorescence generated can bemeasured as the reaction proceeds (Figure 4).

In preliminary experiments, promising results have beenobtained for detection as well as quantification of one each ofthe potyviruses and geminiviruses directly from infectedleaves. Future investigations will explore whether real-timePCR can be used to detect viruses in the tissue culture plantsand obviate the need for graft indexing and also to quantifyviruses in different breeding lines of sweet potatoes todetermine if they have virus resistance.

Mutation DetectionDramatic color changes in root “seed”

stock are easy to spot and eliminate;however, during meristem-tip culture androutine tissue culture multiplication, it canbe difficult to detect more subtle changesthat can affect root shape and yield. This isof particular concern because of the smallnumber of tissue culture plants used togenerate the tens of thousands of plantsrequired for planting acres of land forgrower foundation seed. A mutation in oneof these initial increase plants would bemultiplied greatly and go virtuallyundetected until the crop is harvested.

Using DNA fingerprintingtechnology, we have been able to detectvariation in some mutant versus true-to-type plant fingerprint profiles. Unfor-tunately, some plants with varying degreesof mutations differ little in fingerprintprofiles. The problem is that just oneprobe, akin to one used to detect a specificvirus, is not available. Mutations likelyoccur in many different genes, hence ourinterest in using a new technique calledDNA microarrays to examine the entireplant genome.

Mary Hoy, research associate, is one of the scientists working to improve the quality ofsweet potato seed.


Essentially, DNA microarrays contain tens of thousandsof probes for detecting thousands of active plant genessimultaneously. The detection process is similar to the onedescribed above for viruses. In this case, however, we extractplant RNA, which contains a class of RNA called messengerRNA or RNA transcripts. These RNA transcripts are used inthe plant cell as a template to generate the actual protein fora given gene. Comparing DNA microarrays for true-to-typeplants and mutant plants should show us genes inactivated (nomessenger RNA) because of a mutation. This is an expensiveand laborious technique but may ensure our ability to maintainand propagate true-to-type plants of a variety.

Resistance to Viruses and Other PathogensDisease resistance has been used to control many

important sweet potato diseases caused by bacteria, fungi andnematodes. It is the most economical means of disease controlfrom a farmer’s perspective because it can reduce reliance onchemicals for disease control. However, it can take manyyears to identify a suitable source of resistance for anyparticular disease. Using time-consuming screening methods,it can require many more years to incorporate resistance intoa desirable variety.

Breeding for resistance appears even more daunting forsweet potato viruses. Symptoms often do not correlate witheffects viruses have on their host plants. There is a complexof viruses potentially involved. And efforts to date haveidentified few sources of virus resistance in sweetpotatogermplasm.

Biotechnology offers several tools that might be appliedto these problems. As indicated, real-time PCR is beinginvestigated as a means of quantifying viruses in plants thatcould be used to identify whether breeding lines are able toresist viruses by suppressing replication of the virus. Futureresearch will be directed at determining if molecular markerscan be used to identify resistant lines without the necessity oflabor-intensive screening methods.

Figure 4. Each curve in this Real-time PCR represents a differentunknown sample. From the geometric phase of each curve, ascientist is not only able to detect the presence of a virus, butalso calculate the absolute or relative amount of virus present.


he integrated pest management(IPM) approach to insect controlinvolves multiple tactics. Host plantresistance is one. Pest-resistant plantscan reduce pest population growth, thenumber of pesticide applications andthe environmental impact of pesticides.Though some research has beenconducted on breeding insect resistanceinto soybean plants, most soybeanbreeding programs focus on increasingyield. Transgenic technology, however,allows for the rapid development ofinsect-resistant plants that also havegood agronomic characteristics.

The IPM approach is based onthe use of economic thresholds. Theeconomic threshold is the pest popu-lation level at which controls areinitiated to prevent the loss in yield

Insect-resistant, TransgenicSoybeans: A New IPM ToolMatthew E. Baur, Bentley J. Fitzpatrick and David J. Boethel

Matthew E. Baur, Assistant Professor; Bentley J.Fitzpatrick, Research Associate; and David J.Boethel, LSU AgCenter Associate Vice Chancellorand Professor, Department of Entomology, BatonRouge, La.

caused by pest damage to the crop thatwould be equal to the cost of controllingthat pest using chemical intervention.Pest populations are maintained at alevel below the economic threshold bymonitoring and the as-needed use ofinsecticides. By employing economicthresholds, the producer and environ-mental costs are reduced by eliminatingunnecessary insecticide applications.Use of economic thresholds also limitsthe exposure of the pest population tocontrol measures, thereby reducingthe possibility of pesticide resistance.

In recent years, researchers in-volved in developing IPM strategieshave studied preventive treatments.This differs from the strategy involvingeconomic thresholds and as-neededapplications of insecticides, but stillcan be viewed as falling within the IPMconcept. Preventive treatments can beespecially useful in geographic areasthat have historical, annual problemswith certain insect pests. The transgenictechnology in which proteins withinsecticidal properties are engineeredinto plants might be viewed as apreventive measure. As with other

Figure 1. Velvetbean caterpillar

Photo by Matthew E. Baur

preventive treatments, this technologycould be useful in areas of Louisianathat historically experience highpopulations of target pests.

The two target insects for insect-resistant, transgenic soybeans inLouisiana are the velvetbean caterpillar(Figure 1) and the soybean looper (onpage 34). These pests feed on the leavesof the soybean plant and can severelylimit yield. Velvetbean caterpillarpopulations can reach damaging levelsrapidly. Many producers in areas wherevelvetbean caterpillar is a significantproblem, primarily in the southern partof the state, apply a preventive treatmentof Dimilin when plants are in fullbloom. In the northeastern part of thestate, soybean looper can be a signi-ficant problem, especially where soy-beans are grown close to cotton. Fewpreventive measures are accessible forthe control of this pest, and this insecthas been difficult to control using manyof the historically recommendedinsecticides, such as pyrethroids andcarbamates. Both of these insects can bemanaged using early-maturing soybeanvarieties planted early in the season.However, this may not be an optionbecause of weather.

In 1998, LSU AgCenter entomol-ogists conducted the first field trialsever to evaluate the effectiveness oftransgenic soybean lines developedby Monsanto Co. that contained theinsecticidal protein from Bacillusthuringiensis (Bt). That year, andin subsequent years, we have seenexceptional control in the field (Figure2) and the laboratory of velvetbeancaterpillar and soybean looper usingthese transgenic soybean lines. Inaddition, there was no direct impacton insect predators often found in thesefields. Trials in recent years havecontinued to examine the effectivenessagainst insects but have begun to focuson the agronomic properties ofexperimental lines.

Bt soybeans could be beneficial toproducers, but several issues must beaddressed before adoption of this newtechnology:

T


How would the introduction ofanother Bt crop affect resistancemanagement in pests that attackseveral Bt crops? Several insect pestsin Louisiana attack multiple Bt crops.The cotton bollworm (also known as thecorn earworm) attacks corn, cotton andsoybean, and soybean looper attackssoybean and cotton. Resistancemanagement plans for Bt crops alreadyrestrict the acreage of Bt corn that canbe planted in cotton-growing regions.Bt soybeans could fall under similarrestrictions.

What additional cost will beincurred by the producer for thistechnology? The increase in cost forgenetically engineered seed is referredto as the technology fee. For instance,soybean producers pay a technology feeof $10 to $15 for seed with the RoundupReady technology. Cotton producersalso are charged more for Bt cotton seedthan for conventional seed. But, it is lessexpensive to control insects in soybeansthan in cotton, so it is unlikely that atechnology fee as large as that chargedfor Bt cotton would be assessed tosoybean producers.

What is the cost-to-benefit ratio?Currently, the technology only offerscontrol of lepidopteran pests (moths)and would have to be integrated withmanagement of other major soybeanpests such as the threecornered alfalfahopper and the stink bug complex.Frequently, these pests occur simultan-eously with caterpillar defoliators andcan be controlled with the sameinsecticides. This is especially truefor the velvetbean caterpillar but notalways the case for the soybean looperbecause of the looper’s resistance tocertain classes of insecticides thatwill still control the other members ofthe late-season insect pest complex onsoybeans.

How will the presence of a secondtransgenic technology affect theexport of soybeans to the worldmarket? Many European markets limitthe importation of products producedfrom transgenic plants. This limits theability of Louisiana soybean producersto export their product to these markets,because Louisiana producers make useof the Roundup Ready technology.Roundup Ready soybeans are transgenicand at harvest these seed are not keptseparate from conventional seed.Therefore, markets averse to trans-genic plant products would alreadydiscriminate against soybeansproduced in this state. The addition

Figure 2. Field at the LSU AgCenter’s St. Gabriel Research Station illustrating theeffectiveness of the insect-resistant, transgenic soybeans compared to nontransgenicsoybeans under severe velvetbean caterpillar pressure.


of insect-resistant transgenic soybeanwould not significantly alter thatpicture.

The development of Bt transgenicsoybeans offers an additional tool to beused in soybean IPM programs.However, the above questions will haveto be answered and further studies

conducted to evaluate the experimentallines’ agronomic competitiveness beforethe product will appeal to producers.This technology will complementexisting soybean IPM programs andhas the potential for enhancing thesustainability and profitability ofsoybean production in Louisiana.


Soybean looper larva

Two insect pest species that would be targets for insect-resistant, transgenic soybean varieties—velvetbean caterpillarand soybean looper—originate south of the Tropic of Cancer andmigrate into Louisiana.

The soybean looper is difficult to control with insecticidesbecause it has developed resistance to several products used forinsect control on soybeans. Researchers are puzzled at how thesoybean looper has developed and maintained resistance toorganophosphate, carbamate and pyrethroid insecticides whenexposure within Louisiana soybeans to these insecticides hasbeen low.

In the region where the soybean looper originates, it com-pletes development on vegetable and ornamental crops thatreceive many insecticidal applications. This continued exposureto insecticidal pressure may have selected individuals with higherlevels of tolerance to these products and may have contributedsignificantly to control problems in Louisiana soybeans.

LSU AgCenter scientists have focused efforts on identifyingsoybean looper populations in areas south of the Tropic ofCancer (south Texas, south Florida and the Caribbean) todetermine the susceptibility of these populations to insecticides.If we find populations in these geographic areas that providemigrants to Louisiana with high levels of tolerance or resistance,this would explain why populations within Louisiana are difficultto control.

We used DNA fingerprinting to determine the geneticsimilarity among populations, and we assessed susceptibility tofour insecticides: Condor (Bacillus thuringiensis or Bt), Larvin(thiodicarb), Tracer (spinosad) and Denim (emamectin ben-zoate). Three of the four insecticides (Bt, thiodicarb and spinosad)were chosen because they are recommended for soybean loopercontrol in soybeans in Louisiana, and emamectin benzoate was

Matthew E. Baur, Assistant Professor, and David J. Boethel, LSUAgCenter Associate Vice Chancellor and Professor, Department ofEntomology, LSU AgCenter, Baton Rouge, La.

included because it is not registered for use on soybeans inLouisiana. We did not include pyrethroid or organophosphorousinsecticides in our screening panel because soybean looperpopulations are not susceptible to these products. Therefore,these are not recommended for control.

Populations in Puerto Rico were the most resistant to theinsecticides; populations from Texas were intermediate, and thosefrom Florida were the most susceptible. In three of the four yearsstudied, populations from Texas and Florida appeared to providemost of the migrants to Louisiana, but evidence from the last yearof the study (2000) indicated some migrated from Puerto Rico.

These studies demonstrate that soybean looper populations,separated by large geographic distances, manage to exchangegenetic information. There is widespread interbreeding amongpopulations. The results from these studies also point to thepossibility of rapid resistance development in the soybean looperpopulations. This information could affect the way in which theinsect-resistant, transgenic soybean varieties may be used.

To assess the threat of resistance in soybean looper popula-tions adequately, scientists still need to discover the mode ofinheritance of resistance traits, and, if possible, the mechanismsof resistance. As more sophisticated molecular biological tech-niques become available, researchers will be better able toaddress many of these questions about insecticide resistancedevelopment. Although only DNA fingerprinting was used here,these molecular techniques have been important in determininginsecticide resistance mechanisms.

Tracking Loopers with DNA


Matthew E. Baur and David J. Boethel

Tracking Loopers with DNA


n traveling through the major crop-producing areas of Louisiana in the late1980s and early 1990s, it was commonto see fields infested with many grassand broadleaf weeds. In some cases,it was difficult even to distinguishthe crop. At that time, particularly insoybeans, the herbicides were so narrowin their weed control spectrum that twoor three herbicides applied togethermight be needed to control all weeds infields. But because commodity priceswere low and herbicide costs were high,it was just not economical to control allweeds present in fields. Consequently,

weeds thrived and caused significantyield loss.

Traveling through those same areasof the state in 2003 presents a starklydifferent picture. Fields are cleanerthan they have ever been. This positivechange can be attributed directly toadvancements in weed managementtechnologies through the developmentof herbicide-resistant crops. These newcrops, even though developed for themost part by private companies, wereevaluated extensively by LSU AgCenterweed scientists. These weed controltechnologies include the following.

Roundup ReadySoybeans, Corn, Cotton

Roundup and other glyphosate-containing products are nonselective,foliar-applied herbicides that controlmany annual and perennial weeds.Roundup was initially evaluated inthe South for preplant weed control in

Photo by John Chaney

James L. Griffin, Lee F. Mason LSU AlumniAssociation Professor, Department of Agronomyand Environmental Management, LSU AgCenter,Baton Rouge, La.

Use of herbicide-resistant soybeans has made a remarkable differencein the late-summer Louisiana landscape. No longer do you see weedy

soybean fields, a common sight 10 years ago.

James L. Griffin

I



reduced tillage systems, but the roleexpanded with the development ofherbicide-resistant crops. The glypho-sate-resistance (Roundup Ready) genefrom Monsanto was introduced in theUnited States in soybeans in 1996 andin cotton in 1997. In 2003, about 90percent of the soybean, 40 percent ofthe corn and 85 percent of the cottonacreage in Louisiana was planted withRoundup Ready varieties.

Liberty Link CornGlufosinate (trade name Liberty) is

a nonselective, foliar-applied herbicidenot only effective on grasses but also onmany hard-to-control broadleaf weeds.In general, Liberty is less active ongrasses when compared with glyphosatebut is more effective than glyphosate onsome broadleaf weeds. As with glypho-sate, Liberty was initially evaluated asa preplant herbicide in reduced tillagesystems, and both herbicides have littlesoil residual activity. Glufosinate-resistant (Liberty Link) corn wasmarketed in the South in 2003, but inLouisiana less than 5 percent of the cornacreage was planted with Liberty Linkvarieties. Rice lines have been devel-oped in Louisiana with resistance toglufosinate, and research is underwayto evaluate weed control programs.However, the technology is still in theearly stage of development.

BXN CottonWeed control technology using the

BXN system in cotton was first evalu-ated in 1990. Cotton varieties with theBXN trait, unlike susceptible weeds, areable to tolerate Buctril herbicide throughproduction of nitrilase enzyme, whichmetabolizes bromoxynil, the activeingredient in Buctril, to an inactiveform. This technology offers cottongrowers the flexibility to control manybroadleaf weeds as a foliar applicationwithout fear of crop injury. In 2003, lessthan 5 percent of the cotton acreage inLouisiana was planted with BXNvarieties.

Clearfield Corn, RiceIn Clearfield corn, Lightning

herbicide (a premix of imazethapyr,the active ingredient in NewPath andPursuit, and imazapyr, the activeingredient in Arsenal) can be usedto manage weeds. Lightning’s weedspectrum is not as broad as Roundup or

Liberty, but it does provide some soilresidual weed control. In 2003, lessthan 5 percent of the corn acreage inLouisiana was planted with Clearfieldvarieties.

By exposing rice seed to chemicalmutating agents and treating seedlingswith herbicide, researchers at the RiceResearch Station in Crowley, La., havedeveloped rice varieties tolerant toNewPath herbicide, which contains theactive ingredient imazethapyr. See thearticles about Clearfield rice on pages

23 through 26. The Clearfield tech-nology from BASF has providedgrowers with the ability to selectivelycontrol red rice in domestic rice—abreakthrough in the management of thismajor pest problem. In 2003, around 10percent of the rice acreage in Louisianawas planted with Clearfield varieties.Unlike Roundup Ready, Liberty Linkand BXN technologies, the Clearfieldtechnology is not considered a GMO(genetically modified organism).

A soybean field at the LSU AgCenter’s Dean Lee Research Station


Herbicide Resistance:Advantages, Concerns

Development of herbicide-resistantcrops has offered weed managementoptions with economical advantages toLouisiana growers. Availability of thesenew technologies has enabled soybean,corn, cotton and rice growers to manageproblem weeds that have limitedproduction more effectively. Weeds mayno longer be the most limiting factor tocrop yield potential in Louisiana.

These technologies have resulted ina shift toward reduced tillage systems.Of the soybean, corn, cotton and riceacreage in the state, around 50 percentis managed using some form of reducedtillage or minimum tillage. Research inthe state has clearly shown a reductionin soil and herbicide loss from fieldswhere reduced tillage programs havebeen implemented. Additionally, theavailability of herbicide-resistanttechnologies has resulted in a shifttoward dependence on use of post-

emergence foliar-applied herbicidesthat allow growers to customize weedcontrol programs on a field-by-fieldbasis. This integrated approach to weedmanagement has reduced the cost peracre and the potential for carryoverproblems associated with use of somesoil-applied herbicides.

Along with the benefits of thetechnologies, however, come potentialproblems. Availability of RoundupReady technology in many crops grownin rotation with one another and thecontinued use of glyphosate can increasethe potential for development of weedsresistant to the herbicide. In Tennesseeand Arkansas, a glyphosate-resistantmarestail has been identified, and, inother states, ryegrass resistant toglyphosate has been identified. In time,weeds will develop resistance to a herb-icide when used year after year. InLouisiana there are no documented casesof weeds resistant to glyphosate so far,but researchers have observed a shift inweed populations toward those lesssensitive to glyphosate where theherbicide has been used for several years.

The most effective means tohelp avoid development of herbicideresistant-weeds is to alternate herbicideuse. For example, a grower might use aRoundup Ready variety for a couple ofyears and then change to a Liberty Linkor Clearfield variety. This can bedifficult to do when considering allfactors, but using herbicides withdifferent modes of action ina rotation program will help prevent orslow the development of herbicideresistance.

Photo by John Chaney

AcknowledgmentPersonnel involved in research to evaluateherbicide-resistant crops include P. RoyVidrine, Donnie K. Miller, Bill J. Williams,Eric P. Webster, Dearl A. Sanders, Reed P.Lencse, Timothy P. Croughan, Steven D.Linscombe, Ronald E. Strahan, Steve T.Kelly, Daniel B. Reynolds, David L. Jordan,Richard W. Costello, Joseph H. Pankey,Chris B. Corkern, Jeffrey M. Ellis, Patrick A.Clay, David Y. Lanclos, D. Alan Peters,Jason A. Bond, Lee M. Prochaska, Kristie J.Pellerin, Jeffrey A. Masson, Wei Zhang,Sujatha Sankula, Andrew J. Lanie, MichaelP. Braverman and Stephen H. Crawford.The Louisiana Soybean and Grain Researchand Promotion Board, Cotton Incorporatedand the Rice Research Board have providedfunds to support weed control research.


he first caterpillar-resistanttransgenic cotton varieties (Bollgard)were approved by the U.S.Environmental Protection Agency(EPA) in 1996. The Bollgard technologyhas successfully reduced the frequencyof sprays for caterpillar pests by abouthalf. The excessive costs of controllingpests with foliar insecticides and the riskof economical yield losses allowedBollgard to become widely acceptedby producers. In 2002, more than 75percent of Louisiana’s total cottonacreage was planted to Bollgardvarieties.

Bollgard varieties produce theCry1Ac protein from Bacillus thur-ingiensis. This protein is toxic to thelarval stages of many lepidopteran(moth) pests of cotton, but the primarytargets in cotton include the tobaccobudworm and the pink bollworm.Bollgard demonstrates only limitedactivity against other pests such asarmyworms, soybean looper and

bollworm. If these pests are present athigh densities or populations persist foran extended period, supplementalinsecticide applications are justifiedto prevent yield loss.

Recent advances in geneticengineering technologies within theagrochemical industries have produced asecond generation of caterpillar-resistantcotton germplasm. These cotton linescontain two separate B. thuringiensisproteins and have improved the targetspectrum of caterpillar pests.Monsanto’s new genetically engineeredcotton (Bollgard II) was derived byincorporating the Cry2Ab protein fromB. thuringiensis into commerciallyavailable Bollgard cotton varieties.Levels of Cry1Ac protein expression inBollgard II are similar to the levels ofCry1Ac expressed in Bollgard; there-fore, tobacco budworm control is alsosimilar.

Dow AgroSciences has employed asimilar strategy and developed anothermultiple protein product(WideStrike) with efficacyagainst a wide range ofcaterpillar pests. The Wide-Strike cotton lines expressCry1Ac and Cry1F proteinsfrom B. thuringiensisstrains.

AssessingPerformance

LSU AgCenterentomologists have had theopportunity to evaluate theperformance of the newtransgenic cottons againstthe common insect pests inLouisiana. Experimentalcotton lines containing theBollgard II technologieshave been tested in fieldand laboratory trials since1998. Evaluation ofWideStrike technologybegan in 1999. Theobjectives of these studieshave been to document theefficacy of these productsagainst a spectrum of

B. Rogers Leonard, Professor, Macon RidgeResearch Station, Winnsboro, La.; StephenMicinski, Associate Professor, Red River ResearchStation, Bossier City, La.; Ralph Bagwell,Associate Professor, Scott Research, Extensionand Education Center, Winnsboro, La.

caterpillar pests and to determine theiroptimum role in an overall cotton insectpest management system.

Field trials of Bollgard II andWideStrike demonstrated satisfactorycontrol of mixed Heliothine (tobaccobudworm and bollworm) populationsand late-season foliage feeding insectsincluding soybean looper and beetarmyworm. A summary of 11 testsindicated Bollgard II had the lowestaverage number of Heliothine-infestedfruiting forms and associated damagecompared to that in conventional cottonand Bollgard cotton (Table 1). BollgardII also demonstrated satisfactory controlof foliage-feeding insects compared toconventional non-transgenic andBollgard cottons (Figure 1). In a similarseries of trials, WideStrike demonstratedsuccessful control of Heliothines andfoliage-feeding insect pests (Table 2,Figure 2).

In addition to Bollgard andWideStrike, Syngenta Corporation is

Boll damage from tobacco budworm andbollworm has been uncommon in thesecond generation transgenic cottons.

Photo by B. Rogers Leonard

T

B. Rogers Leonard, Stephen Micinski and Ralph Bagwell

Table 2. Mean seasonal percentage of fruiting formsinfested with bollworm/tobacco budworm larvaeand associated damage for selected cottongenotypes and insecticide spray regimes.

Genotype Treatment Percent Percentlarvae damage

Conventional Nonsprayed 5.7 18.6Conventional Sprayed 3.0 8.6WideStrike Nonsprayed 0.5 2.1

Summary of four trials in Louisiana during 2001-2002.

Table 1. Mean seasonal percentage of fruiting formsinfested with bollworm/tobacco budworm larvaeand associated damage for selected cottongenotypes and insecticide spray regimes.

Genotype Treatment Percent Percentlarvae damage

Conventional Nonsprayed 5.1 16.5Conventional Sprayed 1.9 8.1Bollgard Nonsprayed 0.8 3.3Bollgard II Nonsprayed <0.1 1.5

Summary of 11 trials in Louisiana during 1999-2002.

Beyond BollgardInsect-resistant Cotton Varieties


A complex of bug pests including stink bugs (in both photos) will become more frequentproblems in Bollgard II and WideStrike cottons.

Figure 1. Efficacy of Bollgard II cottongenotypes against foliage feeding pests.

Figure 2. Efficacy of WideStrike cottongenotypes against foliage feeding pests.

developing a vegetative insecticidalprotein (VipCot) technology. Althoughfewer trials have evaluated theperformance of the VipCot trait,the preliminary data indicate a targetefficacy spectrum similar to that of theother technologies. As transgenictechnology continues to evolve in thefuture, LSU AgCenter scientists willevaluate additional insecticidal plantproteins.

Insect ManagementStrategies

Bollgard II and WideStrike willfurther reduce the need for foliar spraysof insecticides against caterpillar pestsof cotton; however, reduction inspraying inadvertently gives rise tomore secondary cotton pests that canlead to significant yield losses. Severalsecondary pests of cotton, including acomplex of tarnished plant bugs andstink bugs, will increase their pest statusas the need for insecticides is furtherreduced in Bollgard II and WideStrikecotton fields. Recently, LSU AgCenterscientists have intensified efforts todevelop cost-effective solutions formanaging the tarnished plant bug andstink bug complex. Using Bollgard IIor WideStrike as the primary strategyagainst caterpillar pests, researchprojects focus on emerging pestproblems. Sampling procedures,establishing action thresholds fortreatment initiation, efficacy trialsfor foliar insecticides and studying theinteractions of multiple pest problemsare examples of cotton IPM researchthat will improve the successfulimplementation of novel transgenicproducts.

Conclusions andRecommendations

The multiple protein products,Bollgard II and WideStrike, havedemonstrated satisfactory control ofcaterpillar pests including tobaccobudworm, bollworm, soybean looperand beet armyworm. These technologieswill further reduce and, in someinstances, eliminate a requirement ofsupplemental control of caterpillar pestswith foliar applications of insecticides incotton. Neither of these two products isactive against noncaterpillar pests,which will likely increase the occur-rence of tarnished plant bug and stinkbug problems.

Photos by B. Rogers Leonard

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otton is the most important textile fiber crop and theworld’s second-most important oil-seed crop after soybeans.Cotton is grown commercially in the temperate and tropicalregions of more than 50 countries. In the United States, cottonis a major agricultural crop and was grown on more than 12.2million acres in 2002. In Louisiana in 2002, cotton wasproduced on about 490,000 acres, which was well belowthe 50-year average of about 680,000 acres.

Although traditional plant improvement efforts havesuccessfully modified the crop to meet the needs of bothproducers and consumers, genetic engineering has been usedto address several important pest problems such as weeds andlepidopterous (caterpillar) pests. Future improvements incotton will depend upon the concerted application of tradi-tional plant breeding, genetic engineering and moleculargenetic tools to increase yield and fiber quality. Yield remainsthe single most important criterion for the development ofnew varieties in modern cotton improvement programs, yetmodern spinning technologies require that similar efforts beplaced upon improving fiber quality.

Fiber quality is evaluated by a combination of traits:micronaire (fiber maturity), length (longer fiber can bespun into finer yarn), strength, elongation (elasticity) anduniformity. Genetic linkage maps are essential to locate thegenes involved in the expression of these traits. This caneasily be done for simple heritable traits based on one gene,but it is also possible for complex traits based on more genes(quantitative trait loci or QTL).

QTL involved in the expression of the five standard fiberquality traits have been identified by means of amplifiedfragment length polymorphisms (AFLP) markers. This isthe marker system of choice because of the low amount ofpolymorphism (variation) detectable by other DNA markertechnologies.

The initial step to identifying QTL for fiber quality traitsinvolved the construction of a genetic linkage map in uplandcotton. Upland cotton is the type grown in most of the UnitedStates, and the only type grown in Louisiana. In 2001, this wasdone using AFLP technology from a cross between a high

Gene Mapping Fiber Traits in CottonGerald O. Myers and Muhanad Akash

Gerald O. Myers, Associate Professor, and Muhanad Akash,Graduate Student, Department of Agronomy and EnvironmentalManagement, LSU AgCenter, Baton Rouge, La.

fiber quality line, PeeDee 2165 and Paymaster 54, ahistorically important high-yielding cotton variety.

In 2002, these upland cotton plants were grown at theDean Lee Research Station in Alexandria, La., and the BenHur Research Farm in Baton Rouge, La., to collect fiber foranalysis. The fiber was analyzed using both interval analysisand composite interval analysis methods to detect QTL forthe five fiber quality traits and to place them on the previouslygenerated AFLP linkage map. Multiple QTL methods wereused to eliminate any biased estimates of effects of size andlocation that can be introduced by using single QTL methods.

The genetic linkage map generated contained 143 AFLPmarkers assigned to 13 major and 15 minor linkage groupsand covered 37.7 percent of the cotton genome. Linkageanalysis between these markers and the studied fiber qualitytraits indicated the following, in brief:

One QTL for fiber elongation explained approximately50 percent of the variation.

Five QTL for fiber length were detected via intervalanalysis and seven via composite interval analysis. Only oneof these QTL was detected by both methods and had a majorimpact on fiber length.

Two marker intervals were detected as being significantby both interval mapping and composite interval mapping,with one explaining about 18 percent and the other about 53percent of the variation in fiber uniformity.

Eleven QTL for fiber strength were detected, five byinterval mapping and six by composite interval mapping. Ofthese, three QTL for fiber strength were detected by bothmethods and explained from 14 percent to 31.4 percent of theobserved variation. This number of QTL for fiber strength wasin close agreement with previous studies.

QTL detected for micronaire numbered three byinterval mapping and six by composite interval mapping. Acomparison of the two mapping methods discovered two QTLcommon to both. One of these had a relatively minor effect,explaining about 9 percent of the variation, whereas the otherhad a more pronounced effect, explaining about 29 percent ofthe variation observed.

The results from these experiments confirm thequantitative nature of inheritance for these five majorfiber quality traits. As a consequence, the improve-ment of cotton for these characteristics cannot beeasily transferred from one variety to another andwill take time. The detection of a few QTL that hada relatively larger effect on fiber quality traits makesthese prime candidates for further investigation,primarily through the development of more detailedgenetic linkage maps. If these QTL can then belocated physically, then marker-assisted selection toeffect more rapid advances in cotton fiber quality willbe possible.


C

Cotton farmers have been pleased with somewhat higher prices for theircrop this season, compared with the past few years.

Photo by Mark Claesgens


n the past decade there have been major advances inmolecular genetics research. A wide variety of DNA-basedmarkers have been developed. These include randomamplified polymorphic DNAs (RAPD), amplified fragmentpolymorphisms (AFLP) and DNA microsatellites. Thesemarkers are used to map genes, study population geneticsand produce DNA fingerprints. The advantage of these newtools is that the number of genetic markers is potentiallyunlimited for many species. In addition, thesebiotechnology-based tools can be applied to a wide rangeof topics and species.

Longleaf PineOne molecular genetics project involves mapping genes

in longleaf pine. This species of pine has many desirablequalities, including excellent wood quality, insect anddisease resistance, and is associated with fire-managedecosystems in the southeastern United States. Longleaf pine,however, has an extended juvenile phase, called the grassstage, during which height growth does not occur. Theunpredictable nature of the grass stage contributes to limiteduse of longleaf pine in forests managed intensively.

The increased use of other pine species and a reductionin the frequency of fires have reduced the area of longleafpine forests in the Southeast and have affected other species,such as the red-cockaded woodpecker and the gophertortoise, both of which are dependent upon longleaf pineforests.

In conjunction with U.S. Department of AgricultureForest Service scientists in Mississippi, LSU AgCenterresearchers are mapping genes that regulate early heightgrowth in longleaf pine. To date, we have mapped 11 genesusing RAPD markers and are using this information in abreeding program. The end result should be that with theuse of DNA markers, we may be able to breed longleaf pinetrees without the grass stage in about half the time thatwould be required in a traditional breeding program. Thesuccessful integration of genetic mapping with the breedingprogram may result in increased use of longleaf pine inindustrial forests. This work is continuing, and we haveexpanded our research to include several other tree andwildlife species.

Louisiana Black BearThe Louisiana black bear is a federally listed

endangered species that survives in several smallpopulations in Louisiana. LSU AgCenter researchers usedDNA microsatellites to estimate abundance of the bears indifferent locations of the state. The traditional method is tocatch the bears, tag them and then see how often a differentbear or a previously captured bear is caught. With the ratioof previously caught to newly caught bears, it is possible toestimate the number of bears in an area. In addition to beingexpensive, this technique is potentially hazardous to boththe bears and the researchers.

Michael Stine, Associate Professor, School of Renewable NaturalResources, LSU AgCenter, Baton Rouge, La.

Instead of capturing the bears, we now put out trapsin bear habitat that snag hair samples from passing bears.Then, using the polymerase chain reaction to analyze theDNA in the hair follicles, we can identify each bear caughtin the hair snare. By determining a recapture rate, we areable to provide accurate population size estimates. Becauseof the increased efficiency of the DNA-based techniques,many more bears were sampled than would be possible withtraditional methods, and this has provided the best estimatesof population sizes to date.

With these techniques, it will be possible to monitorgenetic diversity within and among populations and tominimize the chances of inbreeding in the new populationwhile, at the same time, following the genetic health of thecurrent populations.

Eastern CottonwoodEastern cottonwood is the fastest growing tree in the

eastern United States and is increasingly being used inshort-rotation, high-intensity forestry operations. Inaddition to their fast growth rate, cottonwood trees areeasily propagated by using rooted cuttings. This allows thebest ones to be rapidly cloned. LSU AgCenter researchershave used AFLP markers to estimate the amount of geneticdiversity in southeastern populations and to assist in aneastern cottonwood breeding program coordinated byMississippi State University. Because AFLP markers areso highly variable, scientists can easily identify eachindividual in our breeding program by its DNA fingerprint.This allows us to improve the efficiency of breeding byincreasing the accuracy of the data and to assure proprietaryrights by producing a DNA fingerprint for each clone.

Henslow’s SparrowHenslow’s sparrow is a small bird commonly found in

prairie and pine habitats in Louisiana. In recent decades,this species has suffered a significant decline in population.One problem in studying Henslow’s sparrows is that it isimpossible to accurately distinguish between the two sexesin the field. The inability to determine the sex of the birdsmakes monitoring the health of breeding populationsdifficult and imprecise. However, by using DNA markers todetermine the sexes of captured birds, the researcher needsonly to gather a small blood sample and can determine thesex of the bird in the laboratory.

These diverse research projects have all benefited fromthe application of molecular genetics. Basically, once theDNA is extracted from the leaves, blood or hair, themethods of analysis are the same and can be applied to suchdiverse topics as breeding trees and managing endangeredwildlife species.

IMichael Stine


Using Biotechnologyfor Coastal RestorationPrasanta K. Subudhi, Neil Parami, Alicia Ryan and Stephen A. Harrison

Using Biotechnologyfor Coastal Restoration



isappearance of Louisiana coastat the rate of 30 square miles a year isno trifling matter. Louisiana’s wetland,which provides food and habitat tonumerous plants and animals, is oneof the most productive ecosystems onearth. Both natural and human activitieshave seriously disturbed this ecosystemto such an extent that the native plantsare no longer able to adapt to thischanging environment.

While the use of native vegetationis well accepted as a cost-effectivemeasure to prevent coastal land loss,the concept of genetically improvingwetland plants for the changing marshenvironments is quite novel. LSUAgCenter scientists are focusing ondevelopment of genetically superiorplants in native plant species forunstable coastal areas. A significantcomponent of this activity involvesuse of DNA technology throughcharacterization and combining ofuseful genes in important coastal plantspecies. Following are a few activitiesinvolving application of this new

Prasanta K. Subudhi, Assistant Professor; NeilParami and Alicia Ryan, Graduate Students; andStephen A. Harrison, Professor, Department ofAgronomy, LSU AgCenter, Baton Rouge, La.

molecular tool to help conserve andexploit the existing genetic resourcesfor coastal restoration efforts.

DNA in Coastal PlantsSmooth cord grass, the dominant

plant species of the Louisiana marshes,was our first target. We collected 126smooth cord grass accessions from 11parishes in south Louisiana andevaluated for both vegetative andreproductive traits; several superioraccessions were selected for detailedevaluation. Forty selected accessionswere also DNA fingerprinted to gatherfirst-hand knowledge about thedistribution of genetic diversity amongthe native accessions. A DNAfingerprint is a banding pattern uniqueto an individual organism.

Several accessions with distinctgenetic patterns have been selectedand crossed in a breeding program todevelop genetically superior populationsfor coastal vegetation activities. Thegenetic variability revealed through ouranalysis will enable identification and

LSU AgCenter researchers grow smooth cord grass, at left and above, in research ponds at the Ben HurResearch Farm south of campus. The piece of grass above is laden with anthers, the male part of the flower.


incorporation of useful genes indesirable combinations usingbiotechnology.

In a related project, 43 survivingplants from brown marsh areas weresampled and subjected to DNA analysisto determine their genetic composition.Preliminary analysis indicates that theseplants are quite different from the onlyreleased variety, Vermilion, in theirgenetic makeup. These plants will be asource of genes for breeding improvedvarieties of smooth cord grass.

Little is known about theperformance and genetic purity of theseed-produced progeny of the clonallypropagated Vermilion. Although clonalpropagation is required to maintaingenetic purity and provide quality

D


assurance, it is also necessary to reducethe cost of coastal vegetation projects.Our research, using DNA markers, willdetermine if the seed-propagated plantsmaintain both genetic integrity andperformance comparable to Vermilion.

Sea oats, once abundant on theLouisiana coast, help build sand dunes.A collection of sea oat germplasm fromseveral Gulf and Atlantic states fromTexas to Virginia has been assembledand is being analyzed. Seed yield is lowin southern sea oats compared tonorthern accessions and has been amajor limitation for use in beach nour-ishment projects. Genetic shuffling ofDNA from these two sources can lead todevelopment of sea oats that can adaptto low elevation Louisiana beaches.

Designing Native PlantsDNA technology has demonstrated

its usefulness as a selection tool inapplied crop improvement. Techniqueshave been developed to identify DNAthat controls a specific attribute.Previously, breeders used visual or fieldscreening to select their dream plants.Now, DNA markers make it possible tomuch more accurately choose superiorplants, speeding the overall process ofgenetic improvement. This process issometimes referred to as marker-assistedselection.

LSU AgCenter scientists are alsomaking progress in developingtechnology for aerial seeding for largemarsh plantings. Unfortunately, smoothcord grass does not produce many seeds,and the seeds have poor storability. Tomake the aerial seeding of smooth cordgrass a viable proposition, a series oftechnologies including DNA techniquesare being employed to improve the seedyield and storability. As the projectadvances, we intend to target traits likevegetative vigor and extend them to seaoats. Our research will also expand toother coastal plant species includingSpartina patens, bitter panicum andmangroves, to name a few that haveproven potential in maintaining thecoastal ecosystem.

Adequate vegetative cover of thecoastal marshes through the introductionof genetically diverse plant materials isneeded to prevent disintegration.Discovery of the natural assemblage ofgenes, their role and reconfiguration innative plant species to improve theiradaptation, survival, propagation andproductivity will continue to be an LSUAgCenter research goal.


As part of the research to develop new varieties of smooth cord grass, which is thedominant species of the Louisiana marshes, scientists collect the anthers by cutting them.Prasanta Subhudi, one of the LSU AgCenter scientists involved in coastal research, appliespollen from one variety to another using a paint brush. The scientists have collected 126smooth cord grass accessions from 11 parishes in south Louisiana.


trawberries are one of the mostpopular fruit crops grown in the world.Per capita consumption of freshstrawberries in the United States hasincreased in the past 10 years and ispredicted to continue to rise in theforeseeable future. Most U.S. commer-cial production of strawberries is inCalifornia where the arid climate andlow disease pressure make idealgrowing conditions.

Most of Louisiana’s commercialstrawberry production is in Livingstonand Tangipahoa parishes. Louisianagrowers set transplants in Octoberand begin harvesting in late February.Harvesting continues through earlyMay. Two of the major hazards tostrawberry production in Louisiana arediseases that attack the plants and thecold winter weather that freezes thetender young fruit. Farmers routinelyprotect the crop from freezing with rowcovers and sprinkling systems. Diseases,however, are much harder to controlbecause of Louisiana’s heavy rainfalland warm weather, an ideal growingcondition for pathogenic fungi thatattack both plants and fruit.

Growers in Louisiana mustregularly spray fungicides to maintainhealthy plants and marketable fruit.Fungicide sprays are applied weekly andsometimes twice weekly during harvest.A grower often will apply fungicides 15times or more times during a growingseason.

Because of all the spraying, diseasecontrol is a major expense in strawberryproduction for Louisiana growers. Amore effective and less expensivemethod would be to develop strawberryvarieties resistant to prevalent diseases.Traditional breeding methods involvemaking crosses and growing a largenumber of seedlings to select individualplants with desirable traits. Thestrawberry has a complex geneticsystem that requires a breeder to screentens of thousands of seedlings to retrievethe desirable combination. This long-range commitment often takes 10 ormore years to develop a new strawberryvariety.

A serious strawberry disease inLouisiana and in the Gulf Coast region

is anthracnose crown rot. This fungaldisease can lead to heavy crop lossesand even total crop loss under certainweather conditions. The primary fungalspecies that cause this disease areColletotrichum fragariae andColletotrichum acutatum. Currently,there is no fungicide registered for useon strawberry that reliably controls theanthracnose disease. The developmentof strawberry cultivars resistant to theanthracnose disease should provide ameans for reliable, long-term diseasecontrol and offer economical benefitsby reducing or even eliminating theneed for costly spraying.

In the mid 1990s, LSU AgCenterscientists launched a project to developstrawberry lines resistant to anthracnosedisease. In this project, scientists usedthe soil bacterium Agrobacteriumtumefaciens to genetically transformChandler strawberry plants into astronger, more disease-resistant variety.This bacterium has the ability to transferDNA to the host plant. When used as agene vector, the Agrobacterium isaltered in the laboratory to containdesirable genes.

The genes of interest are forproduction of two enzymes thatenhance the defense mechanisms ofthe strawberry plant. Specifically, theAgrobacterium was altered to contain agene from snap bean for the productionof the enzyme chitinase and a gene fromtobacco for production of the enzymeglucanase. These enzymes are foundnaturally in strawberry plants in variousconcentrations. When transferred intothe strawberry from the Agrobacterium,these new genes could potentiallyincrease the production of the naturallyoccurring defense agents in thestrawberry.

Leaf strips of Chandler strawberryplants were inoculated with transformedAgrobacterium tumefaciens and thencultured on a regeneration mediumcontaining antibiotics to control growthof Agrobacterium and to select out thenontransformed cells. Plants regeneratedthrough this method were tested for thepresence of the new genes by biochem-ical analysis. Transformed plants weretransferred to peat pellets in a transition

chamber and remained for 21 daysbefore being transferred to a green-house. Transgenic lines were evaluatedunder field conditions for three years.Fruit size, shape and overall qualitywere evaluated along with commercialvarieties. Plants from nine transgeniclines were inoculated under controlledconditions with a virulent isolate ofColletrotrichum, which is the causalagent of anthracnose. After evaluation,disease severity ratings indicated thatthe transgenic lines vary in theirresistance to this prevalent pathogen.Several transgenic lines exhibited moreresistance than the nontransgeniccontrol.

The transgenic strawberry plants inthis study varied considerably in fruitcharacteristics and disease resistance.The primary purpose of these experi-ments was to evaluate and developmethods for transferring genes tostrawberry plants. One of the severalchallenges to the plant breeder in thisprocess is the uncontrollable morphol-ogical and physiological changes thattake place during the tissue cultureprocedure. The methods developedwill enable scientists to produce largenumbers of plants from which to choosethe desired horticultural characteristics.This research has established basicmethods from which scientists canintegrate other useful genes into thestrawberry genome.

Strawberry plants resistant toprevalent diseases would enable growersto use fewer pesticides in production.This reduces the cost of production andlessens the impact of pesticides on theenvironment. Evidence from these andother experiments indicate a goodpossibility of developing a newstrawberry variety for Louisianagrowers.

Charlie Johnson, Professor, Department ofHorticulture, LSU AgCenter, Baton Rouge, La.;Ding Shih Professor, Department of BiologicalSciences, LSU AgCenter, Baton Rouge, La.; andJoey Quebedeaux , Research Associate, HammondResearch Station, Hammond, La.

SCharles E. Johnson, Ding Shih and Joey Quebedeaux


Biotechnology


A primary goal of the LSU AgCenter’s Biotechnology Labora-tory (ABL) is to develop new commercial products and establishbiotechnology as one of Louisiana’s future leading industries. TheABL is a multidisciplinary research group that includes scientistsfrom the departments of Agronomy, Biological Sciences, Entomol-ogy, Human Ecology, Plant Pathology and Crop Physiology, Veteri-nary Sciences and the Pennington Biomedical Research Center.Both plant and animal scientific disciplines are represented.

ABL’s mission is to create superior agronomic crops andanimals and to foster economic partnerships among academic units,business and government agencies creating long-term economic andbusiness opportunities to benefit Louisiana through the use ofagricultural biotechnology.

Agro-biotechnology has vast potential for application in suchareas as disease-resistant and high-yield crops and animals, greenplastics and biomass-based fuels. Newly emerging areas include

How ABL WorksABL has formal and informal relationships with a variety of

organizations to enhance biotechnology research and development.Success in modern biotechnology requires high-tech equipment andcombined efforts of scientists from different disciplines to design andcomplete necessary work for identifying and developing new prod-ucts. The intellectual foundation of the ABL is based on scientificexpertise covering a wide range of biological and agriculturalsciences. Using our intellectual foundation and potential of the ABL,we assist science, business and government in Louisiana by combin-ing and moving biotechnology from the research lab to commercialproducts and developing beneficial partnerships among appropriateparties. ABL, which unites specialists in different areas, manages thismultistage process by establishing economic partnerships amongacademia, business and state agencies.

ABL has state-of-the-art laboratory facilities where scientistsconduct research programs in the related areas of genetic transfor-mation technologies, molecular animal and plant breeding (includingrice and sugarcane transformation, mice and catfish transforma-tion), targeted treatment of cancer, human nutrition and obesity,and related areas. Current projects include:

Developing crops resistant to pathogenic bacteria andfungi. We screen numerous natural and synthetic antimicrobialpeptides as potential inhibitors of pathogenic bacteria and fungiimportant for Louisiana. Three peptides have been identified withhigh in vitro activity against sheath blight, and they may be able toserve as potent agents against this devastating disease of rice. Aftertesting, potential antimicrobial peptides will be introduced into rice,sugarcane, strawberries and other Louisiana crops.

Production of valuable commercial products in sugar-cane. We are developing a technique for economically effectiveprotein production in sugarcane. Sugarcane has potential to be a“factory” for low-cost production of valuable commercial productssuch as biological polymers, pharmaceutical compounds (antibodiesand vaccines), industrial enzymes and nanomaterials. Plant-derivedmedicines represent a novel opportunity to develop new treat-ments produced from renewable raw materials that can be pro-duced in vast quantities quickly and as needed. These medicines willbe safe and effective, but also more affordable than existing pharma-ceutical products derived from bacteria, animals or animal cellfactories requiring a large investment in manufacturing capacity.

Partnership with biotech businesses. We are helping aprivate company, TransGenRx, by providing laboratory space. Seethe article on pages 6 and 7. Providing space and equipment plays acritical role in establishing new biotech businesses. In exchange,TransGenRx licenses LSU AgCenter technology.

More information about the ABL is available at its Web site:www/lsuagcenter.com/biotechlab.

Svetlana Oard, Assistant Professor, Veterinary Science, LSU AgCenter,Baton Rouge, La.

Svetlana Oard, left, is manager of the AgCenter’s BiotechnologyLaboratory, also known as ABL. Tamara Chouljenko is a researchassociate in veterinary science who works in the ABL.


plant- and animal-made pharmaceuticals and nanotechnology. Forexample, green plastics (biological polymers derived from grain oragricultural biomass) could substitute for petroleum-derived poly-mers. Biomass-based fuels derived from crops could complementtraditional gas and oil sources. Animal- and plant-made pharmaceu-ticals could significantly reduce prices of important drugs.

The research area of nanotechnology opens new possibilitiesby exploiting extraordinary properties of biological molecules andcell processes. Nanotechnology is based on protein engineering andproduction to create new materials in miniature for industries.Examples include biomaterials interfacing with living tissues toachieve natural tissue architectures for cellular implants and, there-fore, functional replacement of tissues and organs. Other examplesinclude drug-loaded nanoparticles that could deliver drugs intotargeted cells, nanosensors that could monitor living systems, andbiologically-based assembly of electronic materials. By 2015, themarket for nanotechnology is expected to exceed $1 trillion.


Svetlana Oard

Biotech Lab Opens for Business


he bacterial genus Brucellaincludes six recognized species. Theyare characterized by the animals thatthey preferentially infect. Three of thesebacteria were classified by the Centersfor Disease Control as “agents of massdestruction” after the Sept. 11, 2001,tragic events in this country. They areB. abortus, B. melitensis and B. suis.

Brucellae cause abortion andinfertility in wild and domestic animals.Several of the brucellae can be trans-mitted from animals to humans. There isno approved vaccine for human use, andmost of the animal vaccines are virulentto humans. Thus, we need to find a safeand efficacious vaccine that can be usedin humans.

With the concentration of livestock,lack of genetic diversity, increased farmsizes, importation of animals andincreased international travel, agri-culture around the world could bevulnerable to a terrorism attack throughthe use of brucellae. Brucellae arehighly infectious, can be easily aero-solized and are stable during production.Because of their sensitivity to directsunlight, these bacteria can be destroyedin the environment over time. Sincethere are no human vaccines againstbrucellosis, most, if not all populations,have little or no natural immunity to thisorganism.

Human infection can be caused byingestion of Brucella-infected raw milkproducts, exposure to infected animalsand aerosolization of the organism.Brucellosis in humans is characterizedby a cyclical fever that starts two tothree weeks after exposure. Nightsweats, headaches, backaches andgeneral malaise are symptoms associ-ated with acute infection. Chronicbrucellosis can lead to arthritis,dementia and even death.

Of all of the brucellae, B. melitensisbest fits the criteria of a biological andagricultural weapon because little isneeded to infect humans. Low dosesinfect most mammals. These brucellaeaccount for tremendous economic lossin agriculture worldwide. This microbewas found in biological warheads during

the Gulf War, and the U.S. Army hashad an active vaccine developmentprogram since that time. So far theseefforts have produced no efficaciousvaccines for military or civilians againstbrucellosis.

Spurred by the lack of knowledgeof the molecular biology of the genusBrucella, which is necessary for thedevelopment of vaccines, LSUAgCenter scientists were part of aproject to sequence the genome of B.

More than 500 proteins have beenidentified in B. melitensis using thegenomic sequence, and experimentsstudying the up and down regulation ofthese proteins under different conditionsare under way. We have compared theproteome of three Brucella strains,including strain 16M, grown underexperimental conditions; and we havefound many differences in theirproteomes. We are now looking at theproteome expressed in goats infectedwith strain 16M versus the proteinsexpressed in the laboratory-growncultures. We predict that we will finddifferent numbers or levels of proteinsneeded for survival in the host.

Using this information, proteins ofinterest can be modified or deleted fromvirulent strains. One such protein isOMP25, a major structural outermembrane protein, which we havestudied extensively. Genetic mutantslacking OMP25 were made in virulentstrain 16M. By studying the proteomesof these mutants, the up and downregulation of other proteins can bedetermined. Comparing these differ-ences can lead to knowledge aboutpathogenicity, virulence and potentialdiagnostic tools. The OMP25 mutantsshow promise as potential animalvaccines.

Beyond these direct scientificobservations, results of genomic andproteomic studies may be used in thedevelopment of rapid detectionmethodology, finger printing, newdiagnostics and the development ofmore potent animal and potential humanvaccines. To date there is no safe humanvaccine available against B. melitensisinfection, and it is our hope to find onein the near future.

Philip H. Elzer, Associate Professor, and Sue D.Hagius, Research Associate, Department ofVeterinary Science, LSU AgCenter, Baton Rouge,La.

Agriculture around theworld could be vulnerableto a terrorism attackthrough the use of thebacteria brucellae.

melitensis. A genome is the entirecomplement of genetic material presentin each cell of an organism. In cooper-ation with scientists at the University ofScranton in Pennsylvania, the AgCenterrole was to work with the B. melitensisstrain 16M. We have been doing this forthe past eight years using the goat as amodel system. Strain 16M causesdisease in goats and humans.

The genome of B. melitensis strain16M was sequenced. Furtherinvestigation has led to the discoveryof genes that influence the ability of thebacteria to establish infections and causedisease. This was the first Brucellaspecies sequenced and published in thescientific literature. It was a template forthe sequencing of two other Brucellaspecies: B. suis and B. abortus.

With the sequence completed andthe knowledge interpreted, the next stepin the process was to examine theBrucella proteome. The genomicsequence allows for the comprehensiveand rapid analysis of the organism’sproteome. The proteome is defined asthe entire set of proteins.

T

Philip H. Elzer and Sue D. Hagius

Vaccines To Protect Peoplefrom Germ Warfare

Vaccines To Protect Peoplefrom Germ Warfare


Non-profit Org.U.S. Postage

PAIDPermit No. 733Baton Rouge, LA

LSU AgCenterP.O. Box 25100Baton Rouge, LA 70894-5100

Inside:Research on dogs and cats has

found a promising treatment forcancer. The scientists have received apatent for their find. Page 4

Creating superior breeding animalsin the aquaculture world is withinreach. The secret is“cryopreservation.”

Page 9

A disease-resistant rice plantwould make Louisiana rice farmershappy. LSU AgCenter scientists aregetting closer through biotechnology.

Page 20

Biotechnology helps track Louisianablack bears. Page 41

If you think the biotechnology research in this edition of themagazine is pretty amazing, wait until you see what the LSUAgCenter will be doing in the next few years.

That’s the premise behind Biotechnology Education for Stu-dents and Teachers – BEST – a program designed to help assure aflow of fresh talent into research.

One arm of BEST reaches into the state’s high schools and pullsout top-notch science teachers and their students for a biotechnol-ogy-intense, six-week summer session on campus.

“Our goal with this program is to improve the level of scienceeducation in the state and expose students to science and biotech-nology research,” said Richard Tulley, BEST director. “We hopesome of these young people will choose biotechnology research asa career.”

This past summer six pairs of students and teachers from acrossthe state worked under the tutelage of many of the scientistsfeatured in this issue of Louisiana Agriculture.

Another part of the BEST program tries to make a differenceat the undergraduate level. Each spring, LSU undergraduate stu-dents can apply for a $5,000 stipend to do a biotechnology researchproject over the course of two semesters. Each student works witha professor who oversees the project.

This year three students were awarded these grants. Onestudent is examining how resistant starch reduces body fat. Anotheris working with frozen cattle oocytes. And the third is developing aprotocol for growing chicken cells.

Then there are the three BEST graduate assistantships, each forthree years, and the four postdoctorates, one year each, subject torenewal.

BEST Is Yet To Come

One of the graduate students awarded a graduate assistantship,Allison Landry, works with Robert Godke, Boyd Professor in AnimalSciences. Her research involves developing new methods of nucleartransfer that she hopes will increase birth rates in cattle and sheep.

The final part of the program is a new three-hour credit course,taught by Tulley, which provides an overview of biotechnology.

BEST came about in 2001, through a $2.5 million donation fromthe Gordon A. Cain Foundation. Cain was a businessman inHouston whose father had been a Louisiana county agent.

For more information about the program, you may contactTulley at (225) 578-7879 or [email protected]. He hasalready begun recruiting high school science teachers for the 2004summer program. Each teacher will receive a $6,000 stipend, andeach student will receive a $3,000 stipend. Linda Foster Benedict

Documents

Biotechnology - LSU AgCenter/media/system/d/b/8/2/... · 9 Cryoperservation: A New Industry for Aquatic Species Teence Tierschrr 10 Animal Biotechnology and the Future Robert A. Godke,