Embryogenesis in Zea mays L. : a structural approach to maize caryopsis development in vivo and in vitro

In this thesis the embryological development of the maize plant ( <u>Zea</u><u>mays</u> L.) is described. The investigations aim at analysing the development of polarity, the initiation of meristems, the differentiation of tissues within the embryo and the interaction between the embryo and the extra-embryonal tissues of the growing caryopsis. The (ultra) structural aspects of the ontogenesis of both the embryo and endosperm form the main topic.Caryopses which developed in vivo were used; besides that, tissue culture techniques were applied to pollinate in vitro, to culture excised immature embryos, to regenerate somatic embryos and to initiate callus formation. The structural analysis was mainly based upon transmission electron microscopy, scanning electron microscopy and light microscopy with the belonging (immuno)cytochemical and morphometrical techniques. The gathered information contributes to the knowledge of embryogenesis in vivo and in vitro and to the practical insight in how to influence embryogenesis. The comparison of inbred lines and the comparison of in vivo and in vitro embryo development in particular resulted in a better understanding of the embryogenesis of maize.Comparable investigations mainly date from the first half of this century and information on the level of organelles lacks for the greater part in those reports. More recently, sub-microscopical investigations on the in vivo embryogenesis of maize have been performed incidentally. The interaction of embryo and endosperm, however, has not been considered intensively. The latter aspect has been stressed in the present investigations. In the various chapters of this thesis one will find subsequently a treatise of the structure of the ovule and embryo sac (megagametophyte) in the progamic phase, of the fertilization, the early embryogenesis in vivo and in vitro, the interaction between the young embryo and the endosperm, and finally the developmental history of the endosperm and the fruit wall (pericarp).The general introduction reviews the literature, summarizes the aims of the study and of the various chapters, and describes the morphology and anatomy of the inflorescences of maize, its pistillate spikelets and the pistil itself.Chapter 1 presents the results of an ultrastructural study in which two inbred lines, A188 and BMS, are compared. The distribution of organelles in the cells of the megagametophyte has been determined qualitatively and quantitatively.Before fertilization the synergids exhibit a conspicuous distribution of organelles such as mitochondria which might be correlated to the transmembranal transport of nutrients in the filiform apparatus. With reference to the location of the cytoplasm in the egg cell, the cytoplasm in the zygote moves towards the antipodal side of the cell. A change in polarity is thus created and it will be maintained in the developing proembryo. In the central cell of the megagametophyte the cytoplasm changes place, too, because of fertilization. The structure and the number of organelles point to a high metabolic capacity which permits the fast synthesis of cytoplasm and the frequent occurrence of cell divisions. Differences between both inbred lines are expressed in, amongst others, the sizes of organelles and sometimes in their functions such as the accumulation of starch in plastids. Concerning the structure of the ovule, the development of the inner integument and the micropylar part of the nucellus appears different in the two lines. In strain BMS the pollen tube penetrates the integument and the nucellus without curving whereas in A188 no straight growth was detected.Chapter 2 comprises a light microscopical investigation of the morphogenesis of the proembryo from 1 up to 7 days after pollination (DAP). The cellular polarity of the zygote results in an unequal first cell division at about 36 hours after pollination. The twocelled proembryo is spherical and this shape is maintained up to 3 DAP. In this period several small apical cells and somewhat larger basal cells are formed. A pattern in cell division was not observed. Hereafter, the increase in length is mainly caused by cell division and directed elongation in the parts below the apex. Because of the stretching of the proembryo, its apex, which will form the embryo proper, is directed towards the future germinal face of the caryopsis. This phenomenon can be explained by the bending of the suspensor which is caused by an intensive development of the endosperm near the base of the suspensor. The data mentioned point towards the importance of polarity, to the function of the suspensor which pushes the embryo apex into the endosperm and to the morphogenetic influence of the endosperm.Chapter 3 gives a compilation of data concerning the induction of bilateral symmetry in the embryo, the development of a single cotyledon, and the formation of the embryo axis. The morphogenesis and cytological differentiation of the young embryo are analysed in order to follow the initiation and development of the apical meristems from the proembryo stage (5 to 6 DAP) up to the moment the shoot and root have been formed (about 13 DAP). The morphology of the embryo is determined by SEM, its histogenesis with LM and its cytological development with TEM. The original radial symmetry of the proembryo changes into a bilateral symmetry. This is caused or influenced by the excentric position of the embryo apex in the endosperm which on its turn influences the embryo as an exogenous factor. The development of the shoot meristem is preceded by the formation and enlargement of the scutellum and is localized in the protoderm at the future germinal face of the embryo. The shoot meristem is oriented asymmetrically because a second cotyledon is not developed. One or two days after the appearance of the shoot meristem, the root meristem is established at the inside of the embryo proper and in line with the suspensor. Initially it is discernable as a group of cells with few vacuoles and much cytoplasm. The new root-shoot axis deviates from the original length axis of the proembryo but during further development a sideward outgrowth of shoot and coleoptile is impeded mechanically by the pericarp and further development of tissues in the root-shoot axis of the embryo.Chapter 4 presents the investigations on in vitro pollination and the development of embryos which are cultured under various experimental conditions. The pollination of excised pistillate spikelets resulted in the development of 24% of the ovaries. Next it was determined at which age and developmental stage excised immature embryos were still able to germinate and to grow to maturity on nutrient media without growth regulators. It was found that both apical meristems have to be formed in order to establish an endogenous regulation for further root and shoot development. Embryos should have an age of at least 9 DAP and a size of about 1.5 mm.Regeneration phenomena have been studied in immature embryos. The presence of 2,4 D appeared a prerequisite for the induction of regeneration. In order to initiate regeneration, the germinal face of the embryo had to be placed onto the nutrient medium. Various tissues were formed such as chlorenchyma, collenchyma, vascular and callus tissues and more complex structures such as adventitious shoots, roots and somatic embryos. Finally attempts have been made to initiate embryogenesis in suspension cultures. Cell suspensions were prepared from callus tissues which were generated by cultured embryos. A suspension of living cells was established. The cells, however, were formed in root meristem-like cell conglomerates and did not divide at all.Chapter 5 presents an ultrastructural study of the interaction between the embryo and the endosperm from 3 to 11 DAP. Pathways of nutrient flow towards the embryo are characterized structurally by the positions and numbers of various organelles. Nutrients enter the ovule through the placento-chalazal region. They are taken up by the endosperm in a layer of cells with transfer characteristics, and move to various regions in the inner endosperm such as the region of the embryo suspensor. The nutrient flow is characterized structurally by the positions of organelles such as mitochondria, dictyosomes, ER and osmiophilic droplets. A second pathway of nutrient flow, which runs from the degenerating nucellus towards the endosperm, is deduced from the positions of mitochondria in the outer endosperm cells. They were found especially adjacent to the cell membranes bordering the nucellus.From early developmental stages onwards (3 to 5 DAP) the endosperm cells in the basal region of the suspensor exhibit a large number of ordered ER profiles. Products, synthesized by the ER, are likely to be ingested by the suspensor of the embryo seeing the accumulation of osmiophilic material in invaginations of the cell membranes bordering the suspensor cells. Energy is supplied locally by mitochondria which are found here in particular. Near to the enlarging embryo proper endosperm cells degenerate continuously and the degraded products are also taken up by the embryo.The in vivo development of the extra-embryonal parts of the caryopsis is described in Chapter 6. Transmission electron micro~ scopy, LM and immunocytochemical techniques are used to visualize cell differentiation in the pericarp, integument, nucellus and endosperm from 3 to 2 2 DAP. The arrangements of the microtubular cytoskeleton in these tissues are determined and the accumulation of organelles in the various layers of the endosperm is quantified. Microtubules were observed in three main configurations: a. Spindle tubules which function in the movement of the chromosomes; b. cytoplasmic microtubules which run throughout the cytoplasm and which are likely to have a function in the motility and movement of organelles and in stabilizing the cytoplasm; and c. cytoplasmic microtubules which run in the cortical cytoplasm. The function of the cortical microtubules is related to cytomorphogenesis.In the pericarp three zones differentiate. The development of cell shapes appears to be related to the organization of the cytoskeleton. The cell differentiation in each zone of the pericarp coincided with a certain arrangement of the cortical microtubules. Mesocarp cells exhibited preferentially criss- cross microtubules. These cells were not cylindrical, had thin cell walls and disappeared almost completely, including the cell walls. Endocarp and exocarp cells elongated and got thick cell walls. The differentiation coincided with parallel arrangements of microtubules. Most cells had degenerated at 22 DAP but their cell walls remained. In the cells of the integument, which is a transient tissue, most microtubules are arranged normal to the length axis of the cylindrical cells. Nucellus cells were mostly isodiametrical but the outer cells were flattened. Depending on the type of the cell shape either criss-cross or parallel arrangements of microtubules were observed. At 17 DAP only cell walls of the epidermis and its cuticle were observed. The endosperm development is characterized by the formation of the aleurone layer, some sub-aleurone layers and the inner endosperm. The cells of the various layers have different shapes and a different patterning of microtubules. Bundles of cortical microtubules were found in parallel arrays along the anticlinal cell walls of the aleurone cells. Criss-cross patterns were found in isodiametrical cells of the sub-aleurone layers and parallel configurations in cells which had a length axis.From these results it is concluded that when cortical microtubules are found in parallel arrangements in growing cells, elongation is about perpendicular to the microtubules. When cortical microtubules are arranged in criss-cross textures, isodiametrical patterns of growth are found or are to be expected. Although the development of cell shapes can be predicted it has as yet not been found which factor determines the microtubular configuration.The distribution, the sizes and numbers of organelles within the various cell layers of the endosperm, were determined. Clear differences were found in the three layers with respect to variations in the accumulation of storage products such as starch in the plastids, proteins in the two types of protein bodies and of lipids in the spherosomes.Finally in Chapter 7 the data of the foregoing chapters are summarized and related to each other. In a model of differentiation endogenous and exogenous factors which influence the development of the embryo shape continuously are postulated. Some developmental stages are mentioned in particular and the influence of transport and the accumulation of nutrients, of polarity, genetical variability and experimental parameters are emphasized.