Development of Improved Turfgrass with Herbicide Resistance and

Chapter 19

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Development of Improved Turfgrass with Herbicide Resistance and Enhanced Disease Resistance through Transformation F. C . Belanger, C . Laramore, S. Bonos,W. A. Meyer, and P. R. Day Rutgers University, 59 Dudley Road, New Brunswick, N J 08901

Genetic engineering of creeping bentgrass has the potential to provide alternative pest management approaches for golf courses. Bentgrass transformation is very efficient so transgenic plants can readily be produced for evaluation of promising genes. The effectiveness of genetically engineered herbicide resistance in creeping bentgrass has been demonstrated in multiple field tests. This trait is now ready to be incorporated into a commercial cultivar. This would allow the substitution of a nonselective herbicide with low environmental impact for herbicides with greater soil longevity and higher chance to contaminate groundwater. Transgenic bentgrass plants containing several potential disease resistance genes are currently being field tested. If any of these genes proves effective this would result in a reduction in the fungicide use required to maintain bentgrass. There is great interest in improving the pest management options for creeping bentgrass through a combination of biotechnology and plant breeding. Two major categories of pests encountered on golf courses are weeds and diseases. There is significant potential for genetic engineering to have an impact on both of these problems. Our goal is to provide golf course managers with herbicide resistant cultivars for more effective weed control, and disease resistant cultivars which can be maintained in a more environmentally sound and cost-effective manner. Bentgrass Transformation We have a highly efficient transformation system for creeping bentgrass using particle bombardment (i). We use embryogénie callus as our target tissue. Our efficiency is such that we routinely obtain multiple independent transformed plants from each bombardment event. We currently have many independently transformed lines containing a herbicide resistance gene and potential disease resistance genes. These plants are being field tested. The development of an improved cultivar of creeping bentgrass using genetic engineering will be more complex than the production of a transformed plant. Rather it will require the integration of the biotechnology program with the breeding

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Clark and Kenna; Fate and Management of Turfgrass Chemicals ACS Symposium Series; American Chemical Society: Washington, DC, 1999.

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program. Creeping bentgrass is a primarily self-incompatible cross-pollinating species. Cultivars are a population of genotypes produced by intercrossing several superior genotypes. Both plant fertility and stable expression of a transgene in the progeny will therefore be required for commercial application of genetic engineering in bentgrass. Following field evaluation of the original transgenic plants, those displaying the best turf qualities will be crossed with the most advanced bentgrass germplasm from the Rutgers breeding program. It will require at least two or more cycles of recurrent phenotypic selection and evaluation before commercial varieties can be developed. Weed Control An approach to weed control that has proved successful in a number of crop species is the engineering of resistance to nonselective herbicides, such as glyphosate (Roundup) or glufosinate (Finale). Resistance genes for both of these herbicides are known. Crop species, such as corn, soybean, and canola, engineered to express one or the other of these genes are commercially available. Fields of such engineered crops can thus be sprayed with the appropriate herbicide resulting in broad spectrum weed control and no damage to the crop. Both of these herbicides are very effective and considered to be environmentally benign. Both are rapidly degraded after application by soil bacteria and both are non-toxic to mammals (2-5). Because of the benefits of low environmental impact and broad spectrum effectiveness, genetic engineering of crops for resistance to these herbicides has received considerable attention. This approach to weed control in bentgrass has tremendous potential. Bentgrass is grown as a single species and currently there is no effective method of controlling Poa annua which is a serious weed problem. It is thus an ideal candidate for the genetic engineering approach. The ability to spray a bentgrass green planted with herbicide-resistant transgenic plants with one of the nonselective herbicides would offer a convenient and effective method of weed control. This approach obviously requires the routine use of an herbicide. Because of the positive attributes of both glyphosate and bialaphos, there is little concern that their use will negatively affect the environment. An additional benefit of the use of herbicide resistant bentgrass for control of Poa annua would be the reduction in the amount of insecticide used to control the annual bluegrass weevil (Listronotus maculicollis). The Rutgers turfgrass biotechnology program has produced herbicide-resistant creeping bentgrass by transformation with the bar gene, which confers resistance to glufosinate (4-5). These transformed plants were field tested and shown to be resistant at I X and 3X the normal application rate (6). Additionally, the transformed plants were shown to be fertile and the resistance gene was transmitted to the progeny as would be expected for a nuclear encoded gene (6). Both the effectiveness of the herbicide resistance gene and the fertility of the original transformants was thus established. These results illustrate the potential of genetic engineering in providing a new approach to weed control in bentgrass. For this approach to be commercially useful, transformed plants expressing either the glyphosate or glufosinate resistance genes would need to be incorporated into a breeding program for development of a cultivar with overall good turfgrass qualities, in addition to the herbicide resistance trait.


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Disease Resistance Another major pest problem encountered on golf courses is disease control. Bentgrass is highly susceptible to a large number of fungal diseases and generally requires considerable fungicide use (7). Golf courses throughout the country are under pressure to reduce their inputs of fungicides. A number of alternative approaches to disease control are being investigated, such as management strategies, biocontrol, and cultivar improvement through breeding. It is likely that such an integrated pest management (IPM) strategy to disease (8) will ultimately provide the best control. We are hopeful that genetic engineering may also contribute to an IPM approach. We are specifically investigating the potential of genetic engineering in providing some disease control. The production of transgenic creeping bentgrass cultivars with enhanced disease resistance could help in reducing dependence on chemicals with potentially adverse environmental impacts. Unlike herbicide resistance, where two extremely effective resistance genes are known which function in virtually any plant species, disease resistance genes with such broad applicability are not yet known. There are, however, a number of genes which have shown promising results in some species. We are working with some of these to see if they may be useful in creeping bentgrass. We consider it wise to work with several genes at the same time since we do not know which, if any, of the genes will be most effective. Our transformation system is efficient, so we can readily obtain transgenic plants containing the genes of interest. Plants containing transgenes giving the best effect will be chosen for continuing in the breeding program. As new beneficial genes are identified in other species, we plan to incorporate them into our program. We have a number of independent transgenic lines expressing some potential disease resistance genes and we are currently at the stage of evaluating their effectiveness. We do not expect any one gene to produce complete disease resistance or stress tolerance. Because the current use of fungicides is so high, however, genes which can confer measurable improvements in disease resistance will be extremely valuable in turfgrass maintenance. Bacterio-opsin. Bacterio-opsin is a proton pump protein from the bacterium Halobacterium halobium. Mittler et al. (9) reported that expression of bacterio-opsin in tobacco protected the plants from viral and bacterial pathogens. Transgenic plants expressing bacterio-opsin were able to block the replication of tobacco mosaic virus and thus exhibited fewer symptoms of infection (9). Transgenic plants also prevented disease symptoms and growth of the bacterial pathogen Pseudomonas syringae pv tabaci (9). The mechanism of bacterio-opsin induced pathogen resistance may be through activation of the plant defenses since pathogenesis-related proteins are constitutively expressed in the bacterio-opsin expressing transgenic tobacco (9). Expression of bacterio-opsin in potato resulted in dramatic resistance to the US1 isolate of the fungal pathogen which causes late blight disease, Phytophthora infestans (10). The plants were not, however, resistant to a more aggressive isolate (US8) of the pathogen (10). The degree of resistance conferred by a particular transgene is likely to vary depending on the strain or race of a pathogen. However, since bacterio-opsin expression can confer resistance to pathogens as diverse as fungi, viruses, and bacteria it is a good candidate gene to confer broad spectrum resistance to turfgrass diseases.



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Pokeweed Antiviral Protein. The pokeweed antiviral protein (PAP) is a ribosome-inactivating protein from the plant Phytolacca americana. PAP expression in transgenic tobacco confers broad spectrum resistance to several plant viruses (11). Like bacterio-opsin, the mechanism of PAP-induced viral resistance may also be through activation of the plant defenses, since pathogenesis-related proteins are induced (12). Expression of the wild type form of PAP in transgenic tobacco was toxic to the plants (11). A C-terminal deletion of the PAP coding sequence has reduced plant toxicity when expressed in transgenic plants, yet maintains the antiviral activity (13). Expression of the C-terminal deletion in tobacco also shows protection against the fungal pathogen Rhizoctonia solani (12). PAP, thus is another good candidate gene for inducing broad spectrum pathogen resistance in turf. Glucose Oxidase. Glucose oxidase is an active oxygen species-generating enzyme from the fungus Aspergillus niger (14). It acts on the substrates glucose and oxygen yielding gluconic acid and hydrogen peroxide. Wu et al. (15) found that expression of glucose oxidase in potato resulted in resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora and the fungal pathogen Phytophthora infestons. The mechanism of action of glucose oxidase may be two-fold. Hydrogen peroxide, a product of glucose oxidase, is itself toxic to many microbial pathogens. In fact, glucose oxidase was found to be the active agent from the biocontrol fungus Talaromycesflavus(16). Hydrogen peroxide may thus directly inhibit invading organisms. Hydrogen peroxide also activates the plant defenses, inducing systemic acquired resistance (17) and expression of pathogenesis-related proteins (18). Glucose oxidase is thus another good candidate gene for inducing broad spectrum pathogen resistance in turfgrass Field Test. In the summer of 1997 we established a field trial of some of our bentgrass transformants expressing the genes described above. In order to evaluate the plants under their normal use conditions they were maintained as mowed spaced plants. Our preliminary data obtained in the fall of 1997, based on natural dollar spot infection in the field, was promising. A number of transgenic lines showed significantly less disease than the controls. We are currently pursuing more rigorous field evaluations based on inoculation with the pathogen. Any transgenic lines exhibiting enhanced disease resistance, relative to the controls, will be selected for the breeding program.

Summary In summary, we feel that biotechnology, in combination with conventional breeding, has considerable potential for improving pest management options for creeping bentgrass. From experiences with other crops and from the field tests of transgenic bentgrass, genetically engineered herbicide resistance is likely to be a successful pest management strategy for weed control. This would allow the substitution of a nonselective herbicide with low environmental impact for herbicides with greater soil longevity and higher chance to contaminate groundwater. Biotechnology may also contribute to disease control. If successful this would result in a reduction in the fungicide use required to maintain bentgrass. We currently have many plants transformed with candidate genes for disease resistance.


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We will be evaluating the efficacy of the genes and incorporating the best plants into a breeding program for cultivar improvement. Acknowledgments This work was supported by the United States Golf Assoiation.


Literature Cited 1. Klein, T.M.; Fromm,M.;Weissinger, Α.; Tomes, D.; Schaaf, S.; Sletten, M.; Sanford, J.C. Proc.Natl.Acad. Sci. USA 1988, 85, 4305-4309. 2. Franz, J.E. In The Herbicide Glyphosate; Grossman, E. and Atkinson, D., Eds.; Discovery, development and chemistry of glyphosate. Butterworths: London, 1985. pp 3-17. 3. Kishore, G . M . ; Shah, D . M . Ann. Rev. Biochem. 1988, 57, 627-663. 4. Hartman, C.; Lee, L.; Day, P.; Turner, N . Bio/technology, 1994, 12, 919-923. 5. Lee, L.; Laramore, C.; Day, P.; Turner, N. Crop Sci. 1996, 36, 401-406. 6. Lee, L.; Laramore, C.; Hartman, C.L.; Yang, L.; Funk, C.R.; Grande, J.; Murphy, J.A.; Johnston, S.A.; Majek, B.A.; Turner, N.E.; Day, P.R. International Turfgrass Society Research Journal 1997, 8:337-344. 7. Vargas, J.M. Jr. Management of Turfgrass Diseases, second edition. C R C Press: Boca Raton, F L , 1994. 8. Schumann, G.L.; Vittum, P.J.; Elliott, M.L.; Cobb, P.P. IPM Handbook for Golf Courses. Ann Arbor Press, Inc.: Chelsea, MI, 1998; pp 123-149. 9. Mittler, R.; Shulaev, V.; Lam, E. Plant Cell 1995, 7, 29-42. 10. Abad, M.S.; Hakimi, S.M.; Kaniewski, W.; Rommens, C.M.T.; Shulaev, V.; Lam, E.; Shah, D . M . Molecular Plant Microbe Interactions 1997, 10, 635-645. 11. Lodge, J.K.; Kaniewski, W.K.; Turner, N.E. Proc. Natl. Acad. Sci. USA 1993, 90, 7089-7093. 12. Zoubenko, O.; Uckun, F.; Hur, Y.; Chet, I.; Turner, N. Nature Biotechnology 1997, 15, 992-996. 13. Hur, Y . ; Hwang, D-J.; Zoubenko, O.; Coetzer, C.; Uckun, F . M . ; Tumer, N.E. Proc.Natl.Acad. Sci. USA 1995, 92, 8448-8452. 14. Frederick, K.R.; Tung, J.; Emerick, R.S.; Masiarz, F.R.; Chamberlain, S.H.; Vasavada, Α.; Rosenberg, S.; Chakraborty, S.; Schopter, L . M . ; Massey, V .J.Biol. Chem. 1990, 265, 3793-3802. 15. Wu, G.; Shortt, B.J.; Lawrence, E.B.; Levine, E.B.; Fitzsimmons, K.C.; Shah, D . M . Plant Cell 1995, 7, 1357-1368. 16. Kim, K . K . ; Fravel, D.R.; Papavizas, G.C. Phytopathology 1988, 78, 488-492. 17. Chen, Z.; Silva, H.; Klessig, D.F. Science 1993, 162, 1883-1886. 18. Klessig, D.F.; Malamy, J. PlantMol.Biol. 1994, 26, 1439-1458.


Development of Improved Turfgrass with Herbicide Resistance and

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