Preferential freezing avoidance localised in anthers and embryo sacs in wintering Daphne kamtschatica var. jezoensis flower buds visualised by magnetic resonance imaging.

To explore diversity in cold hardiness mechanisms, high resolution magnetic resonance imaging (MRI) was used to visualise freezing behaviours in wintering Daphne kamtschatica var. jezoensis flower buds, which have naked florets and no bud scales. MRI images showed that anthers remained stably supercooled to the range from -14 to -21°C or lower while most other tissues froze by -7°C. Freezing of some anthers detected in MRI images between -14 and -21°C corresponded with numerous low temperature exotherms and also with the 'all-or-nothing' type of anther injuries. In ovules/pistils, only embryo sacs remained supercooled at -7°C or lower, but slowly dehydrated during further cooling. Cryomicroscopic observation revealed ice formation in the cavities of calyx tubes and pistils but detected no ice in embryo sacs or in anthers. The distribution of ice nucleation activity in floral tissues corroborated the tissue freezing behaviours. Filaments likely work as the ice blocking barrier that prevents ice intrusion from extracellularly frozen calyx tubes to connecting unfrozen anthers. Unique freezing behaviours were demonstrated in Daphne flower buds: preferential freezing avoidance in male and female gametophytes and their surrounding tissues (by stable supercooling in anthers and by supercooling with slow dehydration in embryo sacs) while the remaining tissues tolerate extracellular freezing.