Identification of Citrus Species Based on Pollen Morphology: A Comparative Study Using Light and Scanning Electron Microscopy

Citrus is an important cultivated genus within the family Rutaceae. Pollen morphological traits are valuable for interspecific classification and studies of reproductive biology. However, the pollen morphology of Citrus species in Pakistan has not been comprehensively evaluated using both qualitative and quantitative approaches. This study investigated pollen characteristics of ten Citrus species from Pakistan using light microscopy (LM) and scanning electron microscopy (SEM). The species examined included Citrus sinensis L., Citrus aurantium L., Citrus aurantifolia, Citrus limonum, Citrus limmetta, Citrus madurensis, Citrus medica L., Citrus paradisi, Citrus limon L., and Citrus maxima. Fresh floral specimens were collected from different locations in Peshawar, Khyber Pakhtunkhwa, Pakistan. For each species, pollen grains were obtained from five individual plants, and from each plant, three flowers were sampled. For each flower, pollen was extracted from three anthers. For each pollen parameter, we measured three pollens in order to avoid statistical errors and obtain accurate data. Qualitative traits, including pollen shape, pollen size, polarity, aperture type, and exine ornamentation, as well as quantitative traits including polar diameter, equatorial diameter, colpi length and width, exine thickness, and mesocolpi length, were measured and compared. Pollen grains were mostly prolate-spheroidal, oblate-spheroidal, and sub-oblate in shape. Aperture types ranged from tetracolporate to pentacolporate. Pollen size varied from small to medium, with predominantly isopolar polarity across species. SEM observations revealed perforate and perforate-reticulate exine sculpturing. Polar diameter ranged from 22.55 to 34.0 μm, while equatorial diameter ranged from 22.25 to 31.83 μm. Colpi length was highest in Citrus madurensis (3.38 μm) and lowest in Citrus maxima (5.4 μm), whereas colpi width was highest in Citrus maxima (5.4 μm) and lowest in Citrus limmetta (2.5 μm). The observed qualitative and quantitative pollen characters provide important diagnostic features for accurate identification of Citrus species. These findings highlight the taxonomic significance of pollen morphology in distinguishing species within the genus Citrus.