Trait improvement of turfgrass through genetic engineering is important to the turfgrass industry and the environment. However, the possibility of transgene escape to wild and non-transformed species raises commercial and ecological concerns. The purpose of this project is to determine the usefulness of genetically engineered male sterility in turfgrass for producing stable, environmentally safe transgenic perennials with enhanced agronomic performance. We have generated male-sterile, herbicide-resistant transgenic creeping bentgrass and demonstrated that transgenes are inherited by and co-segregate in the T1 progeny. We have also demonstrated, under greenhouse conditions, that the male sterility engineered in the transgenic plants is complete and effective in preventing gene flow. In this project, we will evaluate the inheritance of both male sterility and the herbicide resistance by the T2 progeny, and carry out field trail studies using T2 plants to test whether male sterility will provide effective control of transgene escape compared to open pollination and the use of spatial and temporal management systems to minimize gene flow in transgenic turfgrasses. If the effectiveness of male sterility in controlling transgene escape is confirmed in this field study, it will not only produce male-sterile transgenic creeping bentgrass with herbicide resistance as a potential commercial product, but also confirm the feasibility of using the same strategy in other perennial grass species, producing environmentally safe transgenic plants with enhanced agronomic performance. OBJECTIVES: Our opportunity is to develop the basis for the establishment of a system for the production of environmentally safe, transgenic turfgrass with improved traits using genetically engineered male sterility. Gene constructs containing a rice tapetum-specific promoter driving cytotoxic ribonuclease gene barnase or the antisense of a rice fertility gene rts, linked with a rice ubiquitin promoter driving an herbicide resistance gene bar have been introduced into creeping bentgrass (Agrostis stolonifera), cv. Penn-A-4. While cell-specific expression of a cytotoxic molecule or the antisense of a male fertility gene blocked pollen development in the transgenic plants, causing male sterility, the expression of the bar gene resulted in herbicide (bialaphos) resistance in the transgenic T0 plants. The male-sterile, herbicide-resistant transgenic T0 plants were then cross-pollinated with non-transgenic wild-type plants. The seeds from that cross were collected and planted to produce T1 plants. Both the male sterility causing element and bar gene were found to be inherited by, and co-segregate in the progeny and function at sufficient efficiencies. The male-sterile, herbicide-resistant transgenic T1 plants were cross-pollinated again with non-transgenic wild-type plants for seeds to produce T2 plants. Our phase I objective is to conduct field trail studies using T2 plants to test whether male sterility will provide effective control of transgene escape and to evaluate the inheritance of both male sterility and the herbicide resistance by the T2 progeny. If we prove that male sterility and herbicide resistance can be continuously transmitted, co-segregate and stably expressed in the T2 progeny, and that under field conditions, male sterility is complete and effective in controlling intra- and inter-specific gene flow, this result will not only lead to a commercial product, which can also serve as foundational germplasm for the introduction of additional value-added traits, but also provide the basis for commercialization of this technology with other target turf species in phase II. APPROACH: We have obtained T2 seeds from the male-sterile transgenic creeping bentgrass (Agrostis stolonifera) plants (cv. Penn-A-4) that were cross-pollinated with pollen from non-transformed wild-type plants. We have planted the T2 seeds obtained from cross-pollination to generate T2 plants. During our phase I research, we will conduct herbicide spray experiments on the seedlings of T2 plants to confirm the normal Mendelian segregation and stable expression of transgenes in T2 progeny. We will conduct molecular characterization (PCR, Southern and Northern) on all the T2 plants to determine the transmission and segregation of transgenes in the T2 plants. We will vernalize the T2 plants to ensure that they eventually flower. We will examine the pollen viability in the T2 plants harboring male sterility genes and determine the inheritance of the engineered male sterility in the progeny. We will conduct cross-pollination of the male-sterile T2 plants with the pollen from non-transformed wild-type plants to produce T3 seeds. We will conduct field trail studies to test, under field conditions, the completeness and effectiveness of the male sterility engineered in turfgrass in preventing transgene escape through pollen. The field trail studies will be modeled after our previous work (Wipff and Fricker, Diversity 16:36-39, 2000; Int Turfgrass Soc Res J 9:224-242, 2001) using the male-sterile, herbicide-resistant T2 plants
