Use of aniline blue stain to observing pollen tubes development in different Manihot Mill. species

The lack of a reliable test to evaluate pollen viability in species of the genus Manihot is one of the primary limitations for cassava genetic improvement. Thus, the objective of the present study was to apply aniline blue stain to observing pollen tubes development in different Manihot Mill. species, in order to estimate the crossability, pollen germination time, pollen tube size and pollen longevity. Pollen grains from 2 accessions of the cultivated species M. esculenta spp. esculenta and 4 accessions of 3 wild species (M. esculenta ssp. flabellifolia (Pohl) Cif, M. anomala Pohl, and M. irwinii D. J. Roger and Appan) were used. To visualize the pollen tubes, pollinated stigmas was deposited on a glass slide with 3 drops of aniline blue stain and transferred to an optical fluorescence microscope, where they were visualized and photographed using the 10x ocular lens. The pollen crossability rate was estimated by the percentage of germinated pollen, with well-developed pollen tubes in relation to the total number of pollen grains counted per stigma. The experimental design was entirely random with 3 repetitions, each repetition consisting of 100 pollen grains. In addition to the estimation of crossability, the germination time after pollination, pollen tube size, and pollen longevity were also determined to the accession FLA 029V-01. Significant difference was observed between the crossings. Pollen tubes developed after 45 min of contact between the pollen and the stigma. The average pollen tube size 24 h after pollination was 2.9 mm. The studies examining pollen longevity showed that germination did not occur when the pollen grains were evaluated 24 h after anthesis. Thus, the use of aniline blue stain can be considered an effective method for observing pollen tubes development and studying the pollen morphology, longevity, viability and Manihot crossability.   Key words: Cassava, genetic resources, pre-breeding.