Cytodifferentiation during callus initiation and somatic embryogenesis in Zea mays L. : [Cytodifferentiatie tijdens callusinitiatie en somatische embryogenese in Zea mays L.]

This thesis deals with cytomorphogenic aspects during various phases of regeneration in tissue cultures of Zeamays L. Regeneration through tissue culture has been shown in an increasing number of plant species and is applied on large scale in agriculture and horticulture. Nevertheless, several fundamental scientific data are lacking, which hampers further development of tissue culture, particularly where it concerns tissue culture of monocotyledoncus crops. For example, callus initiation and somatic embryogenesis are important processes during plant regeneration, which can be induced and controlled increasingly with success. However, basic knowledge with regard to these processes is often confined to dicotyledonous crops. In particular, information is lacking about structural changes at the cellular and subcellular level. Research on callus initiation and somatic embryogenesis in maize by structure analysis at the light and electron microscopical level therefore can lead to a better understanding of in vitro cultures, surely for maize, but possibly for monocotyledons in general.Chapter 1 gives a survey of the present information on callus initiation, callus development and somatic embryogenesis. At the end of this Chapter the aim of the thesis is described.In Chapter 2 callus initiation is described based on the results obtained from a light and electron microscopical study. A comparison is made between two inbred lines, A188 and A632, which differ in culture resonse. The initiation of callus in immature embryos has come about within three days after the onset of culture. The first day of culture is characterized by an increase in the number of cell organelles, changes in vacuolation and in nucleolar morphology. These ultrastructural changes take place in embryos of both genotypes and are not affected by 2,4D. After the first day of culture changes occur, dependent on genotype and culture conditions. In embryos of the A188 line a broad meristematic zone develops in the middle and basal scutellum under the influence of 2,4D. From this region somatic embryos and embryogenic compact callus arise. A second meristematic region is observed around the coleorhiza. In embryos of the A632 line callus formation occurs only around the coleorhiza. This callus tissue is non-embryogenic and not capable to regenerate. In the period of callus initiation a shock response can be discerned from a growth response. The shock response takes place in the whole scutellum of both genotypes during the first day of culture and is not affected by 2,4D. On the other hand, the growth response is a local event, after the first day of culture under the influence of 2,4D.In Chapter 3 the period of callus initiation is studied using autoradiography. Special attention was paid to the fraction of cells with DNA synthesizing activity and to the mitotic index in different scutellum regions. It appears that the shock response and the growth response also become manifest during the cell cycle. Moreover, the variation in culture response between the different regions of the scutellum is also expressed in the course of the cell cycle. We observed, that cells in the top of the scutellum terminate their DNA synthesizing and mitotic activity during the growth response, while cells in the middle and basis clearly retain these activities. The results further suggest that scutellum cells preferentially arrest in the G2 phase of the cell cycle during the first eight hours of culture.In the Chapters 4 and 5 the development of the embryoids via an embryogenic callus phase is described for the inbred line A188. Compact callus develops from the embryo when cultured on MS medium with 6% sucrose, whereas friable callus arises on N6 medium with 2% sucrose. Both callus types originate from the abaxial region of the scutellum. After a few days on N6 medium the scutellum is transformed into an almost translucent and soft callus tissue with a fragile appearance. This friable callus is composed of long aggregates of large vacuolated cells and embryogenic units, groups of small isodiametric cells. The cell aggregates are surrounded by large intercellular spaces, which gives the callus its fragile appearance. This friability is caused by cell dissociation between embryogenic cells as well as non-embryogenic cells, cell proliferation in the embryogenic unit and elongation and degeneration of the non-embryogenic cells. No vascular tissue is observed in friable callus. Transport of nutrients probably takes place through the intercellular spaces by diffusion. Compact callus is more organized; it contains vascular bundles around a centre of parenchyma cells, while intercellular spaces are lacking.Somatic embryogenesis in friable callus cultures starts with the transition of an unorganized embryogenic unit to an organized structure of embryogenic cells with a coordinated growth. The transition unit is regarded as an intermediate stage between the embryogenic unit and the somatic embryo. It is a globular structure with a distinct morphological difference between the apical cells and the basal cells that are connected with the callus. In this transition unit a polarity is established, which possibly leads to a gradient of hormones and nutrients. This polarity contributes to an important extend to the activation of somatic embryogenesis. The appearance of large numbers of microtubules in somatic embryos confirms the transition to a coordinated growth. The early somatic embryogenesis strongly resembles the zygotic embryo development with respect to shape and structure. The inability of the embryoid to develop beyond the late globular stage in the presence of 2,4D is probably due to the repressive effect of the auxin on the growth of the shoot apex.Chapter 6 shows the results of the study on the utility of isozymes as biochemical and cytochemical markers in tissue cultures of maize. The zymograms of glutamate dehydrogenase (GDH), peroxidase (PRX) and acid phosphatase (ACP) were analysed in different callus cultures. Variation was demonstrated in the isozyme patterns of GDH and PRX between embryogenic and non-embryogenic callus. Some isozyme bands appeared to correlate with the degree of organization, while the activity of other isozymes was dependent on the length of the culture period. When the same enzyme assays were carried out in situ on fresh callus samples, it appeared that only a clear discrimination in enzyme activity between embryogenic and non-embryogenic was possible with the GDH assay. Therefore, GDH is regarded as an useful biochemical and cytochemical marker in embryogenic callus of maize.In Chapter 7 it is demonstrated, that during pollen embryogenesis in maize, early microspores develop in multicellular pollen grains which consist of cells that differ in vacuolation. During the development repeated divisions especially take place in the vegetative cell. Although participation of the generative cell to the formation of the multicellular pollen grain can not be excluded, it is assumed, that in our experiment the multicellular pollen grain is mainly derived from the vegetative cell.In Chapter 8 the information, obtained from the Chapters 1 to 7, is summarized in the subjects callus initiation, callus development and somatic embryogenesis and provided with comments. At the end a hypothesis is formed, in which the different ways of regeneration are classed in one scheme. In this concept it is assumed that the regenerative capacity is genetically controlled and the expression of the genes becomes manifest under permissive conditions.