Optimizing pollen germination and pollen viability estimates for Hydrangea macrophylla, Dichroa febrifuga, and their hybrids

The genus Hydrangea L. is comprised of some of the most popular ornamental plants in the world. The development of interspecific hybrids between Hydrangea species and within the family Hydrangeaceae would increase genetic diversity in the Hydrangea breeding pool and lead to unique combinations of desirable traits. Protocols to estimate Hydrangea pollen viability need to be optimized in order to perform basic studies on self- and cross-incompatibility, fertility of hybrids, formation of unreduced gametes, and pollen storage. Our goal is to quantify pollen production in Dichroa, Hydrangea, and their hybrids, determine the optimal media for pollen germination, and develop a series of correlations between staining and germinating that can be applied to stained pollen for a rapid, precise estimate of viability. Production of round versus oblong pollen grains was heavily influenced by species (F = 335.8, p < 0.001). Dichroa accessions produced an average of 86% round grains while Hydrangea accessions produced 65% round grains. Dichroa x Hydrangea hybrids averaged only 39% round grains. The optimal media composition for all accessions based on iterative test was 7.5% sucrose, 0.01% Ca(NO3)(2), and 0.025% H3BO3, and 10 mM 2-(N-morpholino)ethanesulfonic acid pH 5.5. Aniline blue-lactophenol staining overestimated viability by an average of 2.3x for Hydrangea and Dichroa, and an average of 4.1x for hybrids as compared to in vitro germination. The results from this experiment will provide more consistent results within and between laboratories and will easily fit into existing Hydrangea breeding programs.