**3. Structures of flowers**

#### **3.1 Staminate flower**

Each staminate flower is composed of two stamens and 1–2 perianth pieces. *L. mirabile* has a single yellowish-white piece, and there are two deep red pieces in *L. leandri* (**Figure 2A**–**C**) [31, 32]. In both species, these pieces are fleshy cushion-like organs, formed of tanniniferous parenchyma with vascular supply, and covered with a unistratified epidermis with scattered sclereids. The epidermis lacks stomata and is covered by a thin cuticle (**Figure 2D**).

The stamens are composed of a short filament, and bitechae anthers. In the anther of *L. leandri* the anterior and posterior pollen sacs of each theca are about the same length, but the thecae are inserted at different heights in the connective tissue (**Figure 2E**). This displacement is the result of the adjustment that the thecae must undergo due to the presence of the upper piece of the perianth. In *L. mirabile* the anterior pollen sacs of each theca are 3/4 of the length of the posterior sacs (**Figure 2F**).

Each anther is tetrasporangiate (**Figure 2G**). The mature anther wall consists of the following layers (**Figure 2G**–**J**): unistratified epidermis with tanniniferous cytoplasm, without any stomata; bi-stratified endothecium with U-shaped fibrous thickening and tanniniferous cytoplasm; one middle layer, which in *L. mirabile* also has fibrous thickenings, forming a tri-stratified endothecium (**Figure 2G**). The tapetum is unistratified, and of secretory type (**Figure 2H**). No orbiculae are observed on the tapetal membrane and/or anther locule. The connective has a single vascular bundle; the surrounding parenchyma cells show the characteristic fibrous thickening of the endothecium, both on the dorsal and ventral sides of the connective. The development of the anther wall can be considered as being within the basic type proposed by Davis [39], although there are variations in the behavior of the middle layers, which develop thickening, constituting a pluristratified endothecium at maturity.

Primary sporogenous cells undergo several mitotic divisions, giving rise to uninucleate microspore mother cells. In meiosis I tetrahedral tetrads are formed by simultaneous cytokinesis, which remain surrounded by a callose wall. Mature pollen grains are released in a bicellular state, they are spheroidal, tricolpate-sincolpate, with a thin exine, and no protruding sculptures (**Figure 2K**).

#### **Figure 2.**

*Staminate flowers. L. leandri: A, D, F, H, J. L. mirabile: B, C, E, G, I, K. A and B: Mature inflorescence without scales, detail of secondary branches showing red perianth pieces and staminate flowers. C: Staminate flowers with dehiscent anthers. D: Stamens, anterior (left) and abaxial (right) views, showing offset in the insertion of thecae. E: Adaxial (left) and lateral (right) view showing different lengths of the pollen sacs. D and E: Arrows mark the lines of dehiscence. F: Longitudinal section of perianth piece with vascular bundles (vb). G: Transection of anther. H: Young pollen sac. I and J: Mature anther walls. K: Pollen grains. Abbreviations: cm: Microspore mother cells; ep: Epidermis; ml: Middle layers; en: endothecium; ta: Tapete. Scales: A–C: 1 cm; D, E: 0,2 mm; F: 0.1 mm; G, H: 0.2 mm; I, J: 20 μm; K: 10 μm.*

#### **3.2 Pistillate flower**

In both species of *Lophophytum* the pistillate flowers lack a perianth and are reduced to one pistil formed of a superior ovary, two styles and capitated stigmas (**Figure 3A**–**C**) [31–33]. The ovaries of *L. leandri* are arranged compactly in the axil of clavate bracts. These bracts have a thin basal portion and a capitated distal portion, which covers the top of the ovary (**Figure 3A**). *Lophophytum leandri* has

*Anatomy, Embryology and Life Cycle of* Lophophytum*, a Root-Holoparasitic Plant DOI: http://dx.doi.org/10.5772/intechopen.99981*

#### **Figure 3.**

*Pistillate flowers. L. leandri: A, B, E, G, I, K. L. mirabile: C, J, L, N, O. A–C: Scanning electron microscope of pistillate flowers, showing bract (br) and stigma positions. D, F, H, M: Successive stages of ovary and ovule development. D and E: Longitudinal section of ovary at the early stage, cup-shaped with a central placenta. F and G: Ovary with bilobed placenta. H and I: Ovary with two ategmic ovules in stages of megaspore mother cell. J: Megaspore mother cell. K: Metaphase I. L: Linear tetrad of megaspores. M–O: Tetranucleate embryo-sac in horizontal position. O: Details of four megasporic nuclei separated by a central vacuole. Note: All photographs were taken with the ovules in the same position (photo A); arrows indicate the direction of rotation of the megaspore mother cell/embryo-sac. Abbreviations: cp: Chalazal pole; mp: Micropylar pole; vb: Vascular bundles. Scales: A, C: 0,5 mm; 0,2 mm: B; 100 μm: E, G, I, N; 50 μm: J, K, L, O.*

two cavities in the apex of the ovary, on which two separate styles are inserted respectively (**Figure 3B**). *Lophophytum mirabile* lacks bracts, the ovary is cylindrical and is also compactly arranged, acquiring a well-defined hexagonal or square shape. The apex of the ovary has a single cavity where both styles are inserted (**Figure 3C**).

During the development of pistillate flowers, the pistil primordium is initiated from hemispheric meristems on the surface of secondary rachis of a young inflorescence (**Figure 3D** and **E**). In *L. leandri*, the bracts develop first, and then ovarian primordia are formed in their axils. In *L. mirabile* only ovarian primordia are formed. In both species the meristem acquires a cup-shaped form. In the center of the pistil a sub-spherical protrusion corresponding to the placenta is developed. Until the closure of the ovary, the ovarian cavity is unique and almost undetectable; the placenta is located at the base of the ovary and is perfectly distinctive from the tissue of the carpels.

The placenta grows occupying the whole cavity of the single locule, the upper end acquires a sharpened shape and it is united postgenitally to the top of the carpels; therefore the mature ovarian cavity is divided into two locules (**Figure 3F** and **G**). The placenta is laterally enlarged giving rise to two-lobed projections in each locule, which are 90° curved towards the base of the ovary, resulting in two ovule primordia. This primordium of ovules occupies the entire cavity of the locules.

Two ategmic ovules are inserted on the upper portion of a central column placenta. The two locules are almost completely obstructed by the ovules (**Figures 3I** and **4A, B**). As the ovules develop, they are reduced to the nucellus and lack of integuments, but the female gametophyte is developed inside (**Figure 2J–O**). The term micropyle is not applicable in its usual sense, therefore it has been designated as a "micropylar pole" at the apex of the nucellus, which is where the megaspore mother cell develops. Vascular supply is absent in the placenta and the ovule, so the chalaza and the funiculus cannot be defined. Therefore, the basal portion is called the "chalazal pole" where the nucellus is attached to the placenta (**Figure 3D**, **E**, **J**, **L**, **N**, **O**).

The mature ovary wall consists of several layers of parenchyma and two zones are recognized: the outer layers are tanniniferous and the internal layers have starch grains (**Figures 3I**, **N** and **4B**). Different types of sclereids have been differentiated between the two zones: both species present a brachysclereid ring in the apical portion of the ovary (**Figure 4C**). In *L. mirabile* four clusters of macrosclereids are also formed at the base of the ovary, which alternate with the vascular bundles. Vascular bundles show scarce development; elements of the xylem vessels have ingrowths.

The styles are solid and are formed of elongated parenchymal cells with dense tanniniferous cytoplasmic contents. Cells of the center of style are smaller but are not differentiated into transmission tissue. The stigmas are capitate and have depressions on their surfaces, where adhered pollen grains can be observed.

Megasporogenesis [31–33]: In both ovules, a conspicuous cell develops below the nucellar epidermis and acquires archesporial features, this cell gives rise to the megaspores mother cell (MMC) (**Figure 3J**). The MMC meiosis I and II happen normally, resulting in four identical nuclei, which are arranged a linear tetrad or "T" shaped (**Figure 3K**, **L**). These four nuclei migrate in pairs to opposite poles of the cell, all of which participate in the formation of a tetrasporic type of embryo-sac (**Figure 3M**–**O**). The polarity of the embryo-sac is determined by the displacement of the nuclei and the presence of a central large vacuole. At this stage of the tetranucleated coenocyte the embryo-sac is in a horizontal position relative to the main axis of the gynoecium, this apparent shift is due to the growth of the ovule by increased cell division on the dorsal side of the nucellus.

Megagametogenesis: During the migration of the two pairs of nuclei to opposite poles, the four-nucleate megagametophyte extends and becomes "J" shaped (**Figure 4A**, **D**). Each pair of nuclei undergoes a mitotic division creating an eight-nucleate embryo-sac (ES). Four nuclei remain near to the chalazal pole and the other four move towards the micropylar pole, separated by vacuoles. Three nuclei remain at each end of the ES, and the fourth nucleus moves towards the center of the central cell, where the cytoplasm is gradually increased in density (**Figure 4E**). The three nuclei at the micropylar end of the embryo-sac are compacted and reduced; cytokinesis occurs at this pole and creates three antipodes (**Figure 4F**). At a later stage, when the triad of nuclei at the chalazal pole is

*Anatomy, Embryology and Life Cycle of* Lophophytum*, a Root-Holoparasitic Plant DOI: http://dx.doi.org/10.5772/intechopen.99981*

#### **Figure 4.**

*Embryo sac (ES) development in L. leandri; A and B: Scheme and light microscopy photo of ovary, showing position of ovules and embryo sacs. C: Detail of sclereids. D: Tetranucleate ES. E: Tetrasporic, 8-nuclei ES (arrow indicates the direction of rotation of ES). F: Antipodes (an) and part of the central cell (pn). G: Eggcell (ec) and the pair of synergids with filiform apparatus (sn). Abbreviations: cp: Chalazal pole; Ilo: Inner layer of ovary; mp: Micropylar pole; olo: Outer layer of ovary; ov: Ovule; sc: Sclereids; st: Stigma. Scales: B, D, E: 100 μm; C, F, G: 10 μm.*

organized as the egg-apparatus, the antipodal cells disappear. At the chalazal pole of the ovule, the typical egg-apparatus is developed, composed of an egg-cell in a central position and two adjacent synergids-cells, all of which determine an inverted embryo-sac (**Figure 4E** and **G**). The synergids-cells are smaller than the egg-cell; their vacuole being oriented towards the center of the megagametophyte, and a prominent filiform apparatus is developed. This tetrasporic, 8-nuclei embryo-sac follows an Adoxa type organization (**Figure 4F**).
