3. Results3.1. Relationship between flower bud morphology and microspore developmental stageCytological study shows five different developmental stages of the pollen grains: tetrad, mid-uninucleate, late-uninucleate, early-binucleate and maturity. Results showed that microspore developmental stages of baby primrose were closely related with flower bud length and morphology (Fig. 1). The youngest anthers (from buds <3.0 mm in length) mainly contained tetrad microspores (58%). Anthers from 3.0 to 4.0 mm flower buds, exhibited a predominance of mid-uninucleate microspores (56%). Among microspores obtained from anthers of 4.0–5.0 mm flower buds, 45% were in the late-uninucleate stage, with cells having a condensed nucleus shifted to the pollen wall, and 19% were in the early-binucleate stage, clearly containing with two nuclei. Anthers from the buds in early flowering (>5.0 mm) contained 71% mature pollen grains. In most plant species, microspores at the late-uninucleate to the early-binucleate stage are the most responsive to induction (Ferrie et al., 1995, Seguí-Simarro and Nuez, 2008 and Germanà, 2011). Based on the results, flower buds just prior to blooming, which had notable swelling, lengths of 4.0–5.0 mm, and petals beginning to emerge from the calyx, were selected for culturing to induce callus formation.
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