The endocarp is made up of one or two layers of small cells that cover the inside of the fruit’s cavity (Figure 10.5c).
At the center of the carpellary leaf are found specialized cells, the papillae. The papillae are intensely stained by Schiff reagent (Figure 10.5d). In agreement with the previous observations (De Lanessan, 1886), the papillae are also intensely stained by Nile Red (Figure 10.6), indicating the presence of high concentration of storage lipids. Some proteinaceous material, stained greenish-blue by Naphthol Blue Black, can be seen in the papillae and in the central cavity in the immediate vicinity of the apical ends of the papillae (Figures 10.5c and d).
FIGURE 10.6 (See color insert following page 136.) Visualization of lipid storage in the papillae of a mature vanilla bean after Nile Red staining (imaging with confocal microscope Zeiss 510 Meta, laser 488 nm and 405 nm, yellow: Nile Red staining, blue: autofluorescence of walls and papillae).
Each side of the pod bears a placenta composed of four to five layers of parenchyma cells and covered by an epidermis. The placenta is divided into two longitudinal placental laminae-bearing funicles, made up of three or four layers of cells (Figure 10.5b), to which seeds are attached. On cross-sectioning, each pair of placental laminae appears as finger-shaped lobes bent inside the central cavity (Figure 10.5d). The cells of the epidermis of the placental laminae contain numerous lipid storage vesicles, as in the papillae.
At maturity, the cavity of the fruit contains numerous small seeds (0.2 μm on average) that are oblong in shape with a dark-colored integument. Each seed is attached to a long, narrow funicle. The seeds are held in mucilage that was found to be essentially polysaccharidic in nature (Odoux and Brillouet, 2009); remnants of this mucilage stained in light pink are shown in Figure 10.5d around the holes previously occupied by the seeds.
Vanilla Bean Ontogenesis: From the Flower to the Mature Pod
The vanilla flowers, in groups of 10 or 15, form small bunches at the leaf axil. White, greenish, or pale yellow in color, they have the typical structure of orchid flowers, the most evolved of all the flowers in the plant kingdom. The perianth of these flowers is made up of three sepals and three petals. The lowest petal of the flower, the lip, is usually large, and is spurred. Under the perianth is a very long ovary, which ends with a short pedicel attaching the flower to the inflorescence axis (Figure 10.2). Before pollination, the vanilla ovary is far from being fully developed. After pollination—natural pollinators are not very well known (Lubinsky et al., 2006)—the perianth withers and falls off (Figure 10.2), while the wall of the inferior ovary progressively evolves to form the fruit’s pericarp (capsule), and the ovules inside the cavity of the ovary develop into seeds. The first sign of the ovary developing into a fruit is a considerable and rapid increase in its size. From an anatomical and histocytological viewpoint, the most spectacular change concerns the inner part of the ovary. After fertilization, a polarized elongation of the endocarp cells toward the cavity of the ovary can be observed. These cells will develop into secretory trichomes, the papillae. At 9 or 14 days after pollination, the papillae remain undifferentiated (Figures 10.5a and b). At this stage, the upper part of the central cavity of the pollinated ovary contains a tissue composed of degenerative cells deeply stained in pink by Schiff reagent (Figure 10.5b). This tissue could correspond to the tissues termed “transmitting tissues” by Arber (1937), a parenchyma that provides a nutrient substrate, which aids the pollen tube to grow through the style and inside the ovary cavity (Figures 10.5a and b).
The differentiation zone of the papillae is not continuous; it is located at the center of the carpel leaf, in the pericarp zone situated under the three triangularly arranged vascular bundles (Figure 10.5b). Two months after pollination, the papilla cells begin their elongation and differentiation. At this stage, their length can reach 20 μm (Figure 10.5c). At maturity, the papillae are around 200 μm in length (Fig ure 10.5d).
Under ultraviolet light, the papillae autofluoresce in white (Figure 10.7a). The cell wall of the mature papillae, which thickens in an uneven manner, undergoes lysis in its distal extremity, facilitating the secretion of its content into the cavity of the capsule (Figure 10.5d).
FIGURE 10.7 Fresh cross sections (100 μm) of mature vanilla bean (8 months after pollination) observed with epifluorescence microscope (Leica DM6000, filter A: 340–380 nm excitation, 425–800 emission). (a) a general view of placenta and papillae; (b) magnification of funicle, seeds, and “matrix.” en, Endocarp; fu, funicle; pl, placenta; vb, vascular bundle.
A white fluorescent substance (Figures 10.7b and 10.8) can also be observed, which surrounds the seeds and partially fills the bean cavity. This substance—called the “matrix” by French (2005)—does not appear to be of cellular origin (i.e., the extremity of funicles), but rather appears to be amorphous and different from the polysaccharidic mucilage.
FIGURE 10.8 Longitudinal view of a mature bean opened in one of the three corners (in the papillae area) with a razor blade and observed with stereomicroscope Zeiss Lumar V12 [white fluorescence of walls (in the mesocarp), papillae and “matrix” = autofluorescence through UV excitation].
The placentas (especially the placental lamina extremity and funicles) also autofluoresce (golden yellow fluorescence, Figures 10.7a and b).
In the mesocarp, white fluorescent globules can be observed in the cells, which may correspond to polyphenols. The lignified tissues of the vascular bundles (xylem and sclerenchyma fibers) emit a bluish autofluorescence linked to the lignin contained in their walls (Figure 10.7a).
Ovule Ontogenesis: From the Ovule to the Seed
In vanilla plants, there is a considerable interval between microsporogenesis, which produces the pollen, and macrosporogenesis, which results in the embryo sac. Surprisingly, the differentiation of the ovules at the top of the placentas mainly occurs after pollination. At two days after pollination, the ovules remain undifferentiated (Figure 10.9a). The placental laminae branch out into a large number of funicles that constitute the placentas (Figure 10.9a). They are made up of four layers of cells that gradually vacuolate from the base (the placental lamina side) to the top. The end of each placenta contains a pool of meristematic cells (actively dividing cells that have dense cytoplasm with a centrally positioned nucleus) (Figure 10.9a). It is this mer-istematic end that assures the growth of the placenta, and later the shift of the ovules into the terminal position. This is clear at 15 days after pollination (Figure 10.9b). The outer integument and the inner integument of the ovule differentiate almost simultaneously with the individualization of the spore mother cell, which will undergo meiosis (Figure 10.9b). The outer integument is made up of four layers of cells and up to eight at its base. This characteristic differs from the other orchids, whose inner integument is made up of a single layer of cells. The inner integument and the outer integument of the ovule are not fused and there is a gap between the two.
The nucellus shows a considerable development in relation to other orchids; it persists in the form of several cells at the base of the seeds when they are disseminated.
Most commonly in orchids, endosperm development is limited to 10 nuclei on an average (up to a maximum of 12). This endosperm is rapidly digested, from the first divisions of the zygote. Unlike the endosperm, the nucellus persists until the embryo development ends, particularly at the chalaza, when the seed is mature. The zygote is surrounded by a thickened outer cell wall and has a central star-shaped nucleus with a single nucleolus stained in black by Naphthol Blue Black (Figure 10.9c). The embryo, which ceases to develop very early on (just after the globular stage but before the torpedo stage), accumulates lipid and protein reserves in the form of aleurone grains that are intensely stained black by Naphthol Blue Black (Figure 10.9d).