A correlative approach using light microscope and electron microscope was carried out in this study to assess floret characteristics and microspore developmental stages in an ornamental sunflower cv. Prado Red. The relationship between microspore stage with anther culture response on various culture media was also investigated. The results revealed a decrease in the percentages of mid-to late- uninucleate microspores from 39.33% of the outer unopened disk floret whorl 1 to 15.33% of the inner floret whorl 3. Disk floret length and anther size were positively correlated with the microspore stage. The callus induction was significantly affected by culture medium but not disk floret whorls and the interaction between them, while the percentage of embryogenic calli was significantly affected by culture media and floret whorls but not the interaction between them. Among the four tested media, MS medium supplemented with 2 mg l^-1 α-napthalene acetic acid (NAA), 1 mg l^-1 N6-benzylaminopurine (BAP) and 100 ml l^-1 coconut water produced the highest callus induction (60.44%) and embryogenic callus production (20.45%). Hence larger disk florets with higher percentages of mid-to late-uninucleate microspores responded well and produced embryogenic callus in the optimum culture medium. © 2018, Khon Kaen University,Research and Technology Transfer Affairs Division. All rights reserved.