A Cryopreservation Strategy for Brassicaceae Pollen in Hybrid Breeding

In Brassicaceae cross-breeding, asynchronous flowering and geographic separation often cause pollen shortages that severely constrain hybridization. Although pollen cryopreservation offers an effective solution, Brassicaceae pollen is typically short-lived due to its tricellular structure, thin exine, and high desiccation sensitivity, necessitating optimized cryopreservation protocols. In this study, we optimized a pollen cryopreservation protocol for three representatives Brassicaceae species: Brassica rapa L. (Chinese cabbage), Brassica oleracea L. (cabbage), and Barbarea vulgaris R. Br. (European rockcress). An in vitro pollen germination system was optimized to reliably assess pollen viability before and after cryopreservation. Key parameters including pollen collection time, drying duration, freezing procedure, and thawing conditions were systematically evaluated. The optimal protocol comprised: pollen collection at 8:00–10:00, drying at 28 °C and 2% relative humidity for 1 h, precooling at −20 °C for 30 min, storage at −80 °C, and thawing under running tap water (ca. 25 °C). Following 30-day cryopreservation, pollen maintained high germination rates (75.19% for Brassica rapa L., 71.18% for Brassica oleracea L., 80.33% for Barbarea vulgaris) and produced comparable silique development, seed quality, and seed germination rates to those of fresh pollen following pollination. This study established a reliable and efficient cryopreservation system for Brassicaceae pollen that effectively overcomes asynchronous flowering and geographic barriers in hybridization, thereby improving breeding efficiency and facilitating germplasm innovation for Brassicaceae crops.