Teoría Crítica de la Comunicación

Reproductive development begins in wheat after the transition of the apical meristem to reproductive growth and continues through a series of definable processes until anthesis, many of which occur whilst the immature spike is enclosed within the culm (Vahamidis et al., 2014). A number of staging scales

58 for wheat reproductive development have been suggested, many with the aim for assisting the timing of agronomic interventions such as fertiliser and growth regulator applications (Waddington et al., 1983; Vahamidis et al., 2014). Much of this male reproductive development takes place before GS51, when the spike first becomes externally visible. Therefore, in order to

investigate the effect of any treatment upon these processes or collect material at specific developmental stages, a means of accurately linking distinct external morphological features to anther development stages whilst it is enclosed within the culm is required.

Male reproductive development begins in wheat with the specification of stamen primordia on the apical meristem and reaches completion upon dehiscence when the pollen is released from the anther, some remaining inside the floret and falling upon its own stigma (De Vries, 1971). The nuclear division of wheat pollen cells proceeds through meiosis of a sporophytic pollen mother cell, the release of unicellular microspores from tetrads and their subsequent mitosis to form mature pollen cells (Goss, 1968) as occurs in other species. El- Ghazaly and Jansen (1986) and Mizelle et al., (1989) used transmission electron microscopy (TEM) to investigate the changes that occur within wheat anthers during pollen development in order to characterise the effect of a chemical hybridising agent. They divided wheat anther development into 7 distinct stages: 1) precallose, 2) central callose – prophase I meiosis, 3) dyad and tetrad, 4) young, free microspores, 5) vacuolated microspore, 6) vacuolated pollen grain, 7) near-mature, tri-nuclear pollen grain. Saini et al., (1984) also described 7 stages of wheat anther development (Fig. 3.2.) but described the mitosis of vegetative and generative nuclei as two distinct events prior to pollen maturation.

59 Table 3.1. Description of wheat anther and pollen development. Wheat anther and pollen development stages, beginning at the formation of sporogenic Pollen Mother Cells (PMCs), gametogenesis via meiosis and mitosis and concomitant development of anther and tapetum cell layers. Pollen development is complete with the formation of trinuclear cells with fully formed outer layer and accumulated starch reserves. Adapted from Saini et al., (1984).

During the pre-meiosis stage, large sporogenous cells are present surrounded by four cell layers, the innermost being comprised of rectangular, single nuclear tapetum cells with heavily staining cytoplasm. Mizelle et al., (1989) define the beginning of the meiosis stage by the expansion of the

microsporangium, creating a locule into which the secretion of callose causes a distinctive star shape to be formed. As meiosis progresses and dyads followed by tetrads are formed inside callose envelopes, tapetum cells become binuclear, their plastids become dense and the primary wall expands as pro-Ubisch bodies become evident within the cytosol and on the plasma membrane surface (El-Ghazaly and Jensen, 1986; Mizelle et al., 1989).

Stage of microspore

development Feature of anther anatomy

Pre-meiosis Anther wall consists of epidermis, endothecium, middle layer and a

tapetum surrounding PMC

Meiosis Each PMC enclosed within a callose wall

Young microspores

Callose walls are broken down. Microspores aporate, thin-walled with large nuclei align along the periphery of the anther lumen. Degeneration of tapetum commences

Vacuolated microspores

Microspores irregularly shaped and in contact with the tapetum. Wall and pore formation continues. Pro-Ubisch bodies from on inner walls of degenerating tapetum cells. Anther diameter increases

Mitosis I Microspore nucleus divides to form vegetative and generative nuclei.

Pollen begins to accumulate starch

Mitosis II Generative nucleus divides to form two ovoid sperm nuclei. Tapetal cells

degenerated. Pollen becomes spherical

Mature Pollen Only two outer layers of anther wall remain. Pollen consists of an outer 3-

60 Upon release of young microspores, the tapetum primary wall completely degrades and a pore forms within the microspore orientated towards the tapetum, and Ubisch bodies assume their distinctive spiked shape as

sporopollenin is deposited upon them. The nucleus of the young microspore migrates from the centre of the cell to the periphery and the cell becomes vacuolated (Mizelle et al., 1989). The rapid expansion of the microspores during vacuolation appears to compress the tapetum layer and Mizelle et al., (1989) define this as the beginning of tapetum degeneration. Mitosis I occurs after the vacuolated pollen grain stage (Table 3.1.), resulting in a vegetative and generative cell. Tapetum degeneration continues throughout this stage with tapetal cytosol precipitating into the locule and large lipid deposits becoming evident. Throughout this period sporopollenin deposition on the microspore continues leading to a visible increase in exine thickness (El- Ghazaly and Jensen, 1986; Mizelle et al., 1989).

Unlike Saini et al., (1984), Mizelle et al., (1989) group Mitosis II and mature pollen stages together. At the near-mature tri-nuclear pollen grain stage the generative cell divides to form two sperm cells whilst the vegetative cell migrates to the pore (Goss, 1968). The cytoplasm now proliferates into the central vacuole and becomes rich in starch-storing plastids which stain heavily. At this stage the tapetum layer is in an advanced stage of degeneration, however, although some debris such as plastids and

endoplasmic reticulum fragments can still be recognised (Mizelle et al., 1989), it is not clear whether these components retain any function at this point in pollen development.

Interestingly, this description of anther development in wheat indicates that the tapetum layer is degenerating but still present during pollen mitosis II. In rice the tapetum layer is generally described as disintegrating immediately prior to pollen mitosis (Li et al., 2006a; Huang et al., 2009) and in Arabidopsis degeneration starts much earlier, during meiosis, and is complete before the end of mitosis (Parish and Li, 2010).This suggests that despite common processes occurring during anther development, there are important

61 differences between species. The secretory function of the tapetum and correct timing of its PCD is vital for wheat fertility and is vulnerable to abiotic stress (see section 1.5.) and it is therefore necessary to ensure that any anther development scale incorporates accurate descriptions of the changes that occur in tapetum cells.

3.1.3. Existing non-destructive reproductive development staging