Abstract: In this study, investigation of some anther structures in Sanguisorba minor Scop. subsp. muricata was aimed. Therefore, anthers were squashed by modified glycerine-gelatine method and additionally different histochemical stainings also were tested. The result illustrated that in different parts of anther, such as marginal, connection tissue, starch and protein granules accumulated. Furthermore, during maturation these deposits were detected in pollen grains also. In addition, a very interesting feature, balloon-like structures filled by proteins, were observed in anther cavity. In the light of these findings, their importance on pollen development was discussed.
INTRODUCTION
Many morphological and embryological studies have been performed so far and then the different features of anthers have been revealed (Bhojvani and Soh, 2001; Lersten, 2004). For instance, in family of Rosaceae, anthers are monosporangiate and dinocotyledonous type, as is commonly found in other Angiospermae families (Johri, 1984). Additionally, some cytogenetical faults during pollen development have also been determined (Rivero-Guerro, 2008; Duarte-Silva et al., 2010). And pollen morphology studies were mostly done (Hebda and Chinnappa, 1990; Hesse et al., 2009). However, from literature, there is still a significant gap in anther structure or function in some genus, as Sanguisorba. Whereas, to more learn about the relationship between anther structures and pollen development, it is necessary to conduct several studies in cytochemical, histochemical and physiological fields on anthers of different plants, as possible (Ying-Qiang et al., 2004; Lindstrom et al., 1999; Hansson and El-Ghazaly 2000; El-Ghazaly et al., 2001). Therefore, it is likely to bring to light on reproductive success of pollens (El-Ghazaly et al., 2001). Consequently, the aim of this study is to examine anther anatomy and its importance on pollen development in Sanguisorba minor.
MATERIALS AND METHODS
Materials used were freshly collected from Balkan Campus, University of Trakya in 2009-2010. As for method followed partially by Md. Islam and et al. (2004) study. Fresh anthers were dissected in mixture of CaNO3 0.03% and Boric acid 0.001% on which a glass slide with the aid of dissecting needles. After few minutes, two drop of Calberla solution, a fuchsine pollen stain (glycerol 16% v/v., ethyl alcohol 33% v/v. and basic fuchsine 0.02% v/v) and examined microscopically for anther and pollens at 100-200-400 magnifications. For histochemical tests, anther were dissected, as described above, but preparats were separately stained with lughole for starch and protein localisation, Sudan III for lipids, Fehling for total carbonhydrates and Toluidin Blue for phenolic compounds and acidic polyanions and then mounted with pure glycerin-gelatin mixture (Jensen, 1962; Jafari and Ghanbarian, 2007). All investigations were done under Prior light microscope and photographs were taken with Olympus BH-2 Photomicroscope.
RESULTS
From all squash preparations, in anthers dense viscous substances were seen (Fig. 1). Furthermore balloon-like and bulging enormous sizes of structures also exist. According to histochemical preparations, dense starch accumulation in anthers wall tissues observed (Fig. 1a). In addition, bulging or balloon-like structures with protein granules were found (Fig. 1b). Large balloon-like structures were with protein granules and filled up entirely anther cavity. This structure firstly covered by a membrane, but afterwards, is without the membrane so that these granules transfer to pollen grains. During squashing preparations, the balloon-like structures burst and ejected theirs protein granules as viscose substance. Furthermore, it is detected that pollen grains to have starch granules (Fig. 1c).
| Fig. 1: | During anther squashing yellowish-brown secretion (arrows), a. in anther walls starch accumulation as a line (arrows), a. Balloon-like structures within protein granules, c. Pollens bearing starch. Bars: (1,1a). 100 μ, (b). 10 μ, (c). 50 μ |
DISCUSSION
This study is especially based on some anther structures and its importance on pollen maturation in Sanguisorba minor. From results, in vessel of connection tissue and anther margins, lughole reacted positive with deeply purple-black. Therefore, especially presence of starch granules in connection tissue and anther margins related to vessel tissue, suggested logical that these granules could originate from other organs. As for complex balloon-like structures bearing protein granules, were connected with each other and vessel tissue, thus widened enormously in anther cavity. In authors opinion, starch and protein granules are related with pollen maturation. Because it was determined mature Sangusiorba pollens to bear starch grains. Similar results were indicated in a previous report (Tian et al., 1998). Regarding protein granules, they will be possibly used in architectural plan of pollen coat as pollenkit, the most common viscous fluid material (Chehregani et al., 2004; Sharma et al., 2009). Present idea was supported by a recent study in which pollenkit was classified in several types depending on its viscosity and heterogeneity, except balloon-like structures (Pacini and Hesse, 2005). These are new for both Rosaceae family and Sangusiorba genus.
From literatures, only some study have been foundon pollen morphology and taxonomy in Sanguisorba and/or Rosaceae family (Latif, 2004; Chung et al., 2010) but not anther tissues. However, the functions of anther in all stage of pollen development were emphasised and pollen abortion were, therefore, dependent on anther defects (Loukides et al., 1995; Izhar and Frankel, 1971; Sanders et al., 1998). Similarly, De Halac and Harte (1995), in their own ultrastructural and histochemical study, proved precisely that the deviation from fertile pollen development was correlated with abnormalities of the tapetum and outer cell layers of anther wall. In authors view, fertilisation success of Sanguisorba pollens is also closely related to both tapetum and these balloon-like structures and any defects on the tapetum and/or balloon-like structures caused sterile pollen formation.