Limonium spp. are known to have sexual and apomixis (asexual reproduction through seeds) reproductive modes. Here, we present dissection protocol developed for ovules of Limonium spp. using differential interference contrast (DIC) microscopy. This protocol permits better handling of ovules and offers certain advantages over earlier techniques particularly in larger ovules. This method also enables observation of meiosis and embryo sac development in intact ovules, and the readily detection of events distinguishing sexual and apomictic development.
[Background] To describe the events that occur during ovule development it is necessary to cytologically examine ovules. This study can involve microscopic observation of paraffin- or resin-embedded, sectioned material, or cleared organs. The first cytological investigations into ovule and embryo sac development in sexual and apomictic Limonium species were published in the pioneer works of D’Amato (1940; 1949). In these works, flowers were fixed using the Karpechenko’s method, embedded in paraffin, sectioned and stained with Heidenhain’s iron haematoxylin, which stains chromatin and chromosomes in the cell nuclei. Flower buds sectioning using these methods can result in preparations with poor quality, due to partial disruption structural integrity of individual cells. A more facile alternative is clearing formalin:acetic acid:ethyl alcohol fixed organs and staining with pure Mayer’s hemalum (Wallis, 1957; Stelly et al., 1984). This technique requires much less time and labor, particularly for species which usually only form a small ovule within the ovary, which is the case of Limonium spp. However, in both small and large ovules chloral hydrate worked better than methyl salicylate as a clearing solution, because in this latter fluid ovules become quite fragile and difficult to handle during experiments. Our approach with an enzymatic digestion of ovules helps to reveal the central mass of tissue, the nucellus and the two integuments it covers, particularly in large ovules. Examples of meiotic and ameiotic ovules and embryo sacs cleared in chloral hydrate were observed under differential interference contrast optics.
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