2018 ASHS Annual Conference

In vitro pollen germination and viability assays are commonly used to assess pollen quality. Two of the most frequently used germination methods are the hanging drop technique and the use of agar plates. Different authors have reported a significant effect of pollen density on pollen germination. Therefore, the pollen concentration must be standardized between different samples when performing germination tests. A fast, reliable and possibly non-destructive method is needed, since pollen grains can start germination as soon as five minutes after being mixed with germination media.

Stored pollen (-20 °C) from five apple cultivars (‘Gala’, ‘Granny Smith’, ‘Honeycrisp’, ‘Red Delicious’ and ‘Rome Beauty’) were used for this experiment. A stock solution for each cultivar was created by suspending 0.5 grams of pollen in 20 ml pollen germination media (10 g L^-1 sucrose, 40 mg L^-1 boric acid). A serial dilution with three replicates for each cultivar stock was created (0, 25, 50, 75, 100 %). The absorbance (optical density) between 400 and 700 nm (1 nm intervals, scan speed 600 nm min^-1) was measured using a spectrophotometer (Cary 60 UV-Vis, Agilent Technologies). A hemocytometer was used to count the number of pollen grains of the dilutions.

The cultivar specific regression models showed a coefficient of determination of 0.98, 0.98, 0.96, 0.98, 0.98 for ‘Gala’, ‘Granny Smith’, ‘Honeycrisp’, ‘Red Delicious’ and ‘Rome Beauty’, respectively. The R-squared value decreased slightly when using a general regression model that includes all five cultivars (R² = 0.89). The decreases could be explained by variations in pollen grain size between cultivars. Every tested wavelength was suitable to estimate the optical density of pollen solutions.

There are two applications for the described method. Optical Density (OD_{400 - 700}) can be used to adjust pollen concentration in suspension. Using a spectrophotometer is a fast, reliable and non-destructive approach that can be integrated with in vitro germination and viability assays. Furthermore, this method would allow for estimation of the pollen yield of different pollinizers. A standard method for quantifying pollen yield is the use of a hemocytometer. However, this method can be time consuming when screening large populations. Cultivar specific OD models are necessary when the variation in pollen grain size becomes too large.