Pollen characteristics of intergeneric crossings in the family of the Ranunculaceae

Ranunculus asiaticus L. and Anemone coronaria L. (Ranunculaceae) are interesting cut flowers. There is a large variation in leaves, flower shape and flower colour within these species. Intergeneric hybridization might therefore result in the creation of new phenotypes with original combinations of these forms and colours. Spontaneous intergeneric crossings between Ranunculus asiaticus and Anemone coronaria are not reported and little research effort is given to controlled crossings. Crossing barriers occur frequently when intergeneric crosses are attempted. A pre-fertilization barrier for these species is linked to a non corresponding flowering time. Therefore a study of pollen viability and pollen storage was undertaken. Plants were grown in the greenhouse from September till May and pollen characteristics were studied. The average pollen diameter was for Ranunculus and Anemone respectively 0.028 mm and 0.031 mm. Pollen can be binucleate or trinucleate. Binucleate pollen grows easier than trinucleate pollen in culture conditions. Both genera used in our experiment had binucleate pollen. A frequently used method to assess pollen viability is in vitro germination. Sixteen germination media were tested for three Anemone and two Ranunculus cultivars. Germination rates as well as pollen tube lengths were measured. For Anemone a general pollen germination medium, containing 100 mg/l H3BO3, 700 mg/l Ca/(NO3)2.4H2O, 200 mg/l MgSO4.7H2O, 100 mg/l KNO3, 100 g/l sugar, 150 g/l PEG 6000 and 0.5 g/l MES (pH 6.0), resulted in the highest germination rate (22.6 % ± 7.3 %) and the longest pollen tube (132 µm ± 12 µm) for the three cultivars studied. The two cultivars of Ranunculus reacted different to the pollen germination media. One of the cultivars had the highest germination rate on the same medium as Anemone (23 % ± 3 %), the other cultivar preferred a medium with higher sugar content (200 g/l) and lower boron content (10 mg/l H3BO3) (pH 5.8) (22.7 % ± 3 %). The length of the pollen tube growth was for Ranunculus the best in 62 mg/l H3BO3, 236 mg/l Ca(NO3)2.4H2O and 100 g/l sugar (pH 5.8) (717 µm ± 183 µm). The growth of the pollen tube in vivo was also assured by an aniline blue staining method in order to study the occurrence of prezygotic barriers. In most cases of the crossings between Ranunculus and Anemone (and vice versa) the pollen tube did not reach the ovules. In order to overcome this barrier, in vivo as well as in vitro techniques will be further optimised. Pollen viability during storage was studied in the following temperature conditions: 4°C, -20°C, -80°C and a direct immersion in liquid nitrogen (-196°C). Viability was tested 0h, 24h, 2 weeks, one month, 3 months and 6 months after the start of the treatment with fluorescein diacetate (FDA). Crossability barriers imposed by different flowering periods of the parents might be effectively overcome through pollen storage. As the pollen storage protocol was not working properly (6 months of storage resulted in a drastic lose of viability (< 20%)), further research to synchronise the flowering period is needed.