Germination and dormancy of single tomato seeds : a study using non-invasive molecular and biophysical techniques

Formation , germination and dormancy of seeds are important steps in the life cycle of higher plants. The seed is the generative dispersal unit, which enables plants to spread and survive through periods or seasons of less favourable conditions. In agriculture tomato is an important crop and seed companies go through big efforts to deliver uniformly germinating seed batches. Uniform germination of a seed lot does not often come naturally. Seed to seed variation in timing of germination and also dormancy cause non-uniform germination of seed batches. This variation and dormancy of tomato seeds is the subject of the experimental work presented in this thesis. Several molecular and biophysical techniques have been used to expand our knowledge of tomato seed physiology.The firefly luciferase-luciferin system has been used in two distinct techniques to study single tomato seeds. A reporter gene construct consisting of a CaMV 35S promoter and the luciferase gene was introduced in tomato by Agrobacterium mediated transformation (Chapter 2). Transgenic seeds were obtained and imbibed in 0.1 mM luciferin solutions. The expression of the luciferase gene was linked with photon emission from the seeds during germination. Luciferase was expressed in a developmental pattern during germination in all germinating seeds. Luciferase expression increased during germination. Although the expression pattern of luciferase was intrinsically linked with the completion of germination, the luciferase activity of a single seed could not be used as a prediction of the time point of visible germination or of the germination rate of a single seed. This was due to the combination of both a time component and an intrinsic variation in the level of expression.Both primarily and secondarily dormant tomato seeds did not show luciferase activity. This enabled us to distinguish, non destructively, dormant from germinating tomato seeds prior to radicle protrusion and, hence, separation of those seeds for future experiments.Luciferase was also used to visualize distribution of ATP in sections of tomato seeds during dormancy and germination (Chapter 3). It was shown that not the overall ATP level or concentration of a seed was related to germination or dormancy per se , but merely the localised increase of ATP levels in the radicle. Dormant tomato seeds did not show an increase in the level of ATP in the radicle.Germination of seeds starts with the uptake of water and finishes by water uptake by the radicle at the initiation of seedling growth. Water uptake by tomato seeds was studied with the use of NMR-imaging (Chapter 4) . Water uptake resulted in an uneven distribution of water over the seed tissues. The endosperm had higher water content during germination. Radicle protrusion was accompanied by an uptake of extra water, thereby stretching the endosperm outward which resulted in rupture of the endosperm cap, which marked the end of germination. In contrast with the commonly adopted model in which seeds take up extra water only after germination, tomato seeds showed this extra water uptake prior to germination.Linker histones play an important role in the regulation of gene expression by remodelling DNA architecture. Distinct linker histones are thereby under control of different developmental processes in plants. With this in mind we have studied the expression of two different linker histones in tomato, which were originally believed to be under control of either GA or ABA, by the use of reverse-transciptase PCR. ABA and GA are antagonists in the regulation of seed germination and this makes both linker histones excellent candidates to play a role in the regulation of germination and dormancy of tomato seeds (Chapter 5). It was shown that the two different linker histones were differentially expressed in seeds, in relation with dormancy or germination. The linker histones also appeared not to be necessarily under direct control of either GA or ABA. A model is presented in which dormancy and germination are controlled by the linker histones, which, on their turn, are under direct control of phytochrome signal transduction. Expression of the histones may be stimulated or accompanied by ABA or GA.