A simple and efficient method for onion pollen preservation: Germination, dehydration, storage conditions, and seed production

The preservation of viable pollen is essential to overcome the problems related to the asynchronous flowering of the parental lines in onion hybrid breeding programs. The aim of this study was to establish a simple, inex-pensive, and easily reproducible protocol for medium-term onion pollen storage. First, the conditions for assessing the in vitro pollen germination were optimized. The liquid medium favored the counting of germination of pollen grains in comparison to the solid medium. The addition of 75 mg/l Ca(NO3)2. 4H2O to the medium did not improve pollen germination, while that of 150 mg/l Ca(NO3)2. 4H2O inhibited pollen germination. The highest germination percentage was achieved by incubation at 30-35 degrees C in the dark. Second, fresh or dehydrated pollen (maintained in a desiccator with silica gel at 25 degrees C for 18 h) was stored at 4, -20, and -80 degrees C for two years to study pollen preservation. In addition, the viability and germination capacity of stored pollen were periodi-cally evaluated at 0, 15 and 30 days; 2 and 6 months; and 1 and 2 years. Pollen viability was best retained at low relative humidity and temperatures below zero. Dehydration was essential for pollen preservation at -20 and -80 degrees C. The results showed that dehydrated pollen stored at -20 degrees C could be used, with guarantees, for polli-nation throughout the flowering season. However, the highest viability and in vitro germination percentages after two years of storage (29 and 32%, respectively) were achieved with dehydrated pollen stored at -80 degrees C. Finally, the capacity of stored pollen to produce seeds was confirmed in crosses with male sterile lines. In this way, dehydrated pollen stored at -80 degrees C for two years produced an average of 47.9 seeds/100 flowers, representing 43% of the seed in the control crosses. This is the first report in onion research of seed production after polli-nation with preserved pollen at -80 degrees C for two years.

Este trabajo ha sido financiado por el Ministerio de Economía y Competitividad INIA-FEDER (RTA2015-00042-C02-01), por el Convenio EEAD-CSIC con el CITA-Aragón para la «Producción de dobles haploides de cebolla y desarrollo de un protocolo de conservación de polen», y por el Gobierno de Aragón (Grupos A11-20R y A08-20R).

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