Domesticated pennycress is a predominantly self-pollinated crop

Domesticated pennycress (Thlaspi arvense L.) is an emerging winter annual oilseed crop developed from field pennycress to deliver commercial agronomic performance, low-carbon feedstock for biofuel production, and value-added animal feed. Effective breeding, seed production, and stewardship of this newly domesticated crop requires a clear understanding of its potential for pollen-mediated outcrossing. Although prior evidence suggests that field pennycress is predominantly self-pollinated, direct experimental evidence remains limited. Here, we integrate laboratory, greenhouse, and field experiments to rigorously evaluate pollen viability, pollen movement, and outcrossing potential in domesticated pennycress. Pollen viability declined rapidly with increasing temperature and environmental exposure: pollen stored at 4C reached 50% viability after 12 h, whereas pollen held at 37C reached the same threshold after 2.5 h. Exposure to outdoor conditions with natural light and wind reduced viability to 50% within 1.6 h. Under greenhouse conditions, no cross-pollination was detected among plants flowering in close proximity within an agitated pollen-containment covering. Detectable cross-pollination occurred primarily when emasculated flowers were directly exposed to donor pollen, demonstrating that intact flowers strongly favor self-fertilization. Field trials using a trackable knockout gene edit provided no detectable evidence of pollen movement under field conditions across two locations, even at short distances from donor plots. Together, these results provide strong, multi-environment evidence that domesticated pennycress is predominantly self-pollinated and that outcrossing is rare under practical greenhouse and field production conditions.