2.4. Pollen germination and viability tests
In vitro pollen germination was assessed by using hanging drop technique as suggested by Stanley and Linskens (1974).
The liquid germination medium contained 150 g l−1polyethylene glycol (PEG 4000), 4.88 g l−1N-morpholinoethanosulfonic acid in potassium hydroxide buffer (MES-KOH, pH 6.4), 200 mg l−1MgSO4, 100 mg l−1 KNO3, 100 mg l−1H3BO3, 700 mg l−1Ca(NO3)2and 200 g l−1sucrose at pH 5.5 (modified Vivian-Smith et al., 1992).
A minimum of 100 pollen grains per slide were randomly counted under a light microscope (Leica DM 1000) at 20× magnification.
Pollen was adjudged as having germinated when the length of the pollen tube was equal to, or exceeded the pollen diameter.