A unique tool to advancing thedevelopment of new turfgrass varieties.

by ARTHUR P. WEBER

A more modern means of micro engineering turfgrass plantsinvolves the use of a gene gun. Selected DNA is fired into livingplant cells using an explosive charge to insert the DNA into thehost chromosomes. The cells are regenerated into whole plants,hopefully carrying the new gene.

TRANSGENICTURFGRASSES

and older of the technologies uses thebacterial species Agrobacterium tuma-taciens to carry the gene of interestinto the host plant. Agrobacterium, amicroorganism that causes plant dis-ease (Le., galls) and has been knownsince the turn of the 20th century,possesses its own genetic engineeringsystem. In nature, the bacterium sendsits own genes into the infested host andinserts them into plant chromosomes.Researchers take advantage of thismeans of transforming plants by infect-ing them with laboratory-developedAgrobacterium mutants whose dis-ease-carrying genes were replaced withspecifically chosen DNA. In effect, thebacterium then acts as a microengineer,doing all the work.

The second, more modem means ofmicro engineering uses a gene gun thatwas developed, in part, to allow for the

will kill corn-borer pests thattry to eat theplant.

The derivedbenefits have notoccurred withoutenvironmentalconcerns. In thewake of thesegenetic advancesare reports thatcom engineeredto carry the Bttoxin might harmbutterflies orother non-targetinsects andwildlife. Contro-versy relating tohuman andanimal healthover the longterm derives fromconcerns thatplants with genesfrom viral patho-gens mightcombine withother viruses to create new viral strains.It is thought by some groups that thetransgenic plants could create newallergies or exacerbate existing ones,such as the recent claims of increasedallergic reactions to genetically alteredsoybeans.

Notwithstanding, breakthroughs ingenetics have made it possible toimprove crop plants and farm produc-tivity in ways conventional breederscould only dream about. The possibili-ties are endless. With conventionalbreeding, it can take seven or moreyears to produce a new plant that maybe only marginally superior to its pre-decessors. Genetic modification allowsresearchers to insert a wide array ofnew genes into a plant and makeimprovements more efficiently.

Two main methods prevail forgenetic engineering in plants: The first

OURfundamental understandingof the biological sciences standsready to explode, and the bene-

fits to be derived loom large. Forgolfers, biotechnology promises toaccelerate the improvement of turfgrassspecies using genetic engineering tech-niquesY) Genetic Modification (GM),Le. the splicing of genes from one orga-nism to another unrelated organism tocombine traits that would otherwisebe highly unlikely to occur together,is a natural succession to the earlierrealization and success of the USGAGreen Section Research Program, agoal of which is to "develop turfgrasseswith enhanced stress tolerance andreduced supplemental water require-ments, pesticide use and costs."

Among the most desirable charac-teristics of such GM turfgrasses wouldbe: (2)

1. Ability to survive high and lowtemperature extremes.

2. Reduced need for pesticides byincreasing resistance to disease, insects,nematode, and weed encroachments.

3. Tolerance of intensive traffic.4. Reduced requirements for mow-

ing, irrigation, and fertilization.5. Tolerance of non-potable water.6. Stability of inherited charac-

teristics.7. Tolerance of acid, alkaline, or

saline soils.8. Tolerance of smog and other

pollutants.9. Increased shade tolerance.Transgenic biotechnology, although

still in its infancy, has already becomea significant commercial reality. Genestaken from other organisms can bespliced into food plant DNA, e.g. com,soybeans, and canola. Herbicide toler-ance to products such as Roundup(glyphosate) has been conferred usinggenetic engineering so weeds can becontrolled without harming the crops.Com seed, by carrying a gene derivedfrom the bacterium Bacillus thurni-giensis (Bt) produces the Bt toxin that

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Traditional breeding methods can take ten years and beyond to successfully bring a new turfgrass variety to market.Genetic engineering techniques can produce significant improvements beyond a traditional breeding program.

transformation of plants that cannot beinfected with Agrobacterium. In usingthe gene gun, selected DNA, coatedonto gold or platinum microparticles, isfired into living plant cells, either cellcultures or embryos, using an explosivecharge. The cells are punctured by themicrobullets, and the DNA enters thenucleus and then inserts into the hostchromosomes.

The cells, those infected by Agro-bacterium or shot by the biolistic gun,are regenerated into whole plants,which then carry the new gene or genesof interest. These plants are tested,cloned, and ultimately can provide theseed for a new plant variety.

One of the new and exciting experi-mental approaches for discovering thefunction of genes is DNA microarrays.From them, extensive databases ofquantitative information can be ob-tained about the degree to which genesrespond to pathogens, pests, drought,cold, salt, growth regulators, herbi-cides, and other agricultural chemicals.These gene expression databases willprovide novel insights into the genes

32 USGA GREEN SECfION RECORD

that control complex responses, andthey will create an opportunity toassign functional information to genesof otherwise unknown function. Thisinformation will ultimately help con-ventional plant breeding programs be-come more efficient in developing newvarieties.

Toward these ends, the USGA GreenSection Turfgrass and EnvironmentalResearch Program, among other con-ventional and biotechnological turf-grass breeding methods, has committed$835,000 over the next five years to thefollowing university studies alreadyunderway:

"A Multigene-Transfer Strategy toImprove Disease and EnvironmentalStress Resistance in Creeping Bent-grass," Michigan State University,Mariam R. Sticklen, Start Date 1998, 3years, total funding $75,000.

This study focuses molecular solu-tions to the biotic (i.e., pest) problemsand abiotic (Le., heat, humidity, etc.)problems associated in the manage-ment of creeping bentgrass turf. Aseries of available genes has been

inserted into existing creeping bent-grass varieties to ascertain if resistanceto various stresses can be improved.Early results indicate that various genes(e.g., elm chitinase, proteinase inhibi-tor, glufosinate resistance, and manni-tol dehydrogenase) can be successfullyinserted into the bentgrass genome.However, with the exception of theglufosinate resistance gene, improvedstress tolerance has been somewhatlimited. The genes can be found in thetransformed bentgrass plants but donot significantly improve disease resis-tance or increase the amount of man-nitol to help with drought and salttolerance.

"Hybrid Bermudagrass Improvementby Genetic Transformation," NorthCarolina State University, Rongda Qu,Start Date 1998, 3 years, total funding$75,000.

The research is developing a geneticengineering protocol for hybrid ber-mudagrass varieties. Bermudagrass hasbeen more difficult to work with dueto problems producing viable plantembryos, and eventually healthy plants,

A gene resistant to the herbicide glyphosate was successfully inserted into the planton the right. Plants without the inserted gene react with the results on the left whentreated with glyphosate.

from tissue culture callus. Once areproducible technique is developed,research efforts will focus on insertinggenes that confer nematode resistanceinto the bermudagrass clones.

"Bermudagrass Cold Hardiness:Characterization of Plants for FreezeTolerance and Character of Low-Tem-perature Induced Genes," OklahomaState University, Charles M. Taliaferro,Start Date 1998, 5 years, total funding$125,000.

This research will reduce the risk offreeze injury to bermudagrass grown intemperate regions. The project is accu-rately assessing the freeze tolerance ofbermudagrass cultivars, isolating genesresponsible for enhanced freeze toler-ance, and enhancing knowledge of thefundamental mechanisms associatedwith cold tolerance. Substantial prog-ress toward isolating the characterizingcold regulated proteins responsible forimproved freeze tolerance in bermuda-grass was achieved. Measured geneactivity increases of 75 to 100 percentin crown and root tissues occurredafter 24 hours of exposure to coldtemperatures.

"A Turfgrass Genome Project: Inte-gration of Cynodon Chromosomeswith Molecular Maps of Cereals,"University of Georgia, Andrew H.Paterson, Start Date 1999,5 years, totalfunding $125,000.

The research project is producing thefirst primary molecular map for thechromosomes of bermudagrass. Thisinformation will be compared withmolecular maps of the major cerealcrops in order to gain access to thewealth of genetic information pro-duced by scientists around the world.The map will be useful for investigatingmany aspects of turfgrass populationbiology and genetics, and provide amolecular conduit for turf improve-ment. Significant progress on charac-terizing DNA from bermudagrass anddeveloping molecular markers wasaccomplished during the last year. Thefocus in the future will turn to full-scale genetic mapping and identifyingquantitative traitloci (QTLs) of imp or-tant turfgrass characteristics.

"Development of Improved Bent-grass Cultivars with Herbicide Resis-tance, Enhanced Disease Resistance,and Abiotic Stress Tolerance throughBiotechnology," Rutgers University/Cook College, Faith Belanger, StartDate 1998, 5 years, total funding$250,000.

This project will help conserve golfcourse natural resources while provid-

ing quality playing surfaces by improv-ing creeping bentgrass through genetictransformation. The work has concen-trated on important bentgrass varietiesand selections developed for golfgreens in the Northeast. New bentgrasscultivars with improved stress toleranceand disease resistance are under devel-opment through a combination ofmolecular and conventional plantbreeding efforts. The effectiveness ofgenetically engineered herbicide resis-tance in creeping bentgrass wasdemonstrated in several field tests, andthe trait is now incorporated into a newcultivar. There are 50 new transgeniclines of creeping bentgrass expressingone of five potential disease resistancegenes.

"Transformation of Bermudagrassfor Improved Fungal Resistance,"Oklahoma State University, Michael P.Anderson, Start Date 1998, 5 years,total funding $125,000.

The long-term goal of this project isto improve bermudagrass resistance tospring dead spot using gene transfor-mation. The disease is active in the falland early spring when temperatures arecool and moisture is plentiful. A genetictransformation system was developedfor a forage-type bermudgrass becauseit had previously demonstrated superiorgrowth and plant regeneration poten-tial in tissue culture. Efforts to identifyan anti-fungal protein antagonistic tospring dead spot are making progress.

Despite some of the environmentaland health concerns, research effortsshould be supported to ascertain howturfgrass improvement efforts coulduse the new genetic engineering tech-niques. Propelled by the skillful appli-cation of genetic modification tech-niques, the dramatic improvement ofgolf course turfgrasses, as part of acomprehensive plant revolution, is nowwell underway. Ultimately, it will be acombination of new genetic modifica-tion tools with existing conventionalplant improvement techniques thatwill provide turfgrass varieties with en-hanced stress tolerance and. reducedsupplemental water requirements,pesticide use, and costs.

References(l)Sticklen, M. B., & Kenna, M. P., 1998,Turfgrasses Biotechnology, Ann ArborPress, Chelsea, Michigan.(2)Kenna,M. P., & Snow, J. T., 1999, USGATurfgrass & Environmental ResearchProgram.

ARTHUR P. WEBER, a semi-retiredchemical and nuclear engineer, has beenan active member of the USGA GreenSection Committee since 1984. A longtimeGreen Committee chairman, he was theprincipal author behind the Old WestburyGolf and Country Club (NY) Code ofEnvironmental Conduct, a leading set ofprinciples for golf course maintenance.

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