FIGURE 7.

Characterization of the different developmental pathways of C. sativa microspores and pollen grains cultured under in vitro conditions. The different developmental pathways are described as follows: (A,A′) An enriched population of vacuolate microspores and young bi-cellular pollen grains just after in vitro culture stablishment: Arrows point to dead microspores, which does not exhibit any fluorescence. (B,B′) Gametophytic pathway developed by a pollen grain culminated with the emission of a prominent pollinic tube. (C,C′) Illustration of both, gametophytic and sporophytic pathways developed by microspores after a week-long cold-shock bud pretreatment. (D,D′) Microspore under sporophytic development showing five nuclei as a result of successive divisions promoted by cold-shock bud pretreatment. (E,E′) Pollen-derived embryogenic structure generated after cold-shock bud pretreatment and three weeks of in vitro culture. (F) First C. sativa microspore-derived embryos: Illustration of a secondary embryogenesis event which resulted in the development of a globular embryo on a heart-shaped microspore-derived embryo: arrow points to the globular embryo. (F′) Protoderm development (arrow) in the embryogenic structure, which showed a well-organized and regular cellular structure (inset in F′). (A–E) Differential Interference Contrast (DIC) microscope images. (A′) Fluorescent microscope image after FDA staining. (B′–E′) Fluorescent microscope images after DAPI staining. (F) Stereomicroscope image. (F′) Bright-field microscope image. Scale bars (A–E,A′–E′): 10 μm. Scale bar (F): 1 mm. Scale bars (F′) and (inset in F′): 50 μm. v, vacuoles; pt, pollinic tube; vn, vegetative nucleus; s, spermatids; gn, generative nucleus; n, sporophytic nucleus.