Ablation of Transgenic Pollen in the Model Perennial Grass Brachypodium sylvaticum

Interest in the use of perennial grasses, especially those utilizing C₄ metabolism, for biofuel production is increasing because of the low input cost and long-term land use benefits afforded by these plants relative to their annual counterparts. Efforts reliant on transgenic technology for the modification of C₄ grasses to improve plant tissue digestibility, stress tolerance and disease resistance must consider that most of these species utilize wind for transmission of pollen during sexual reproduction, a fact which represents a real potential for transgene escape to wild plant populations. To address this concern we are developing a transgene containment system for perennial grass species like switchgrass (Panicum virgatum). We are using the model perennial grass Brachypodium sylvaticum to evaluate the utility of novel transformation constructs to block transmission of transgenes via pollen. Using Agrobacterium-mediated transformation, we generated transgenic plants that express barnase under the control of four rice-derived pollen-specific promoters (PS1, PS2, PS3 and OsGEX2). Multiple independent transgenic lines for each construct have been evaluated by pollen staining and genetic segregation analyses. Alexander’s staining revealed that, relative to wildtype plants, >50% of the pollen collected from putative containment T0 lines was dead or severely deformed. Analysis of selfed T1 progeny showed that transgene heritability was 1:1, consistent with the expected segregation frequency for a male lethal gene, supporting the conclusion that successful ablation of transgenic pollen was achieved in these Brachypodium sylvaticum transgenic plants. Future work will focus on transferring these functionally validated transgene containment constructs into switchgrass.