Floral ontogeny and development of the model plant Nicotiana benthamiana

Nicotiana benthamiana is a model plant species widely used in molecular biology and biotechnology; however, it has historically lacked a detailed morphological framework for floral development. Here, we present a comprehensive characterization of N. benthamiana flower ontogeny, defining 14 floral stages from floral meristem initiation to anthesis. Using stereo- and scanning electron microscopy, we document the acropetal sequence of floral organ initiation and differentiation across four concentric whorls. Each stage is marked by distinct morphological features, starting with the domed floral meristem (Stage 1). Sepal primordia emerge first (Stage 2), followed by petal and stamen primordia (Stages 4 and 5), and two carpel primordia appear by Stage 6 to initiate gynoecium development. Through mid-development (Stages 6–8), petals and stamens expand within an enclosing calyx, while carpels remain unfused. By Stage 9, carpel fusion forms a single ovary with a differentiating stigma. Subsequent stages feature rapid elongation of petals and stamens, and partial calyx separation (Stages 11–12), allowing emergence of the corolla tube and style. Anthesis occurs at Stage 14, when the corolla lobes fully spread, exposing the mature reproductive organs. In parallel, we describe the coordinated development of male and female gametophytes, and further characterize post-anthesis fruit development, from fruit set through capsule maturation and dehiscence, thus completing the full reproductive cycle. This ontogenetic framework serves as a foundational reference for genetic and comparative development studies on flower organogenesis, reinforcing N. benthamiana as a versatile model system for Solanaceae research and a valuable tool for translational crop improvement. • Nicotiana benthamiana flower development is defined through 14 sequential stages • Floral ontogeny spans from the onset of the floral meristem to anthesis • Coordinated development of male and female gametophytes is studied • Fruit development is described from set to capsule maturation and dehiscence • N. benthamiana floral stages offers a framework for Solanaceae translational research