# Morphological, Anatomical, and Phytochemical Studies of _[PERSON]_ [PERSON]. [PERSON]
## 2 Results and Discussion  
### Macro- and Microscopy Analysis  
_C. acaulis_ cyspelae (Figure 1B) are oblong, with 4-6 mm length and approx. 1 mm width. They are brown and are covered with silver hairs. They possess pappus with a length from 15 to 17 mm, which consists of 5-8 hairs stuck together at the base. The thousand seed weight without the calyx fluff is 3.60 \(\pm\) 0.08 g.  
As shown by the microscopic imaging, the mature cyspela (Figure 2A) was mostly occupied by a large embryo (Figure 2B-D), which was covered by the pericarp overlain by the testa (Figure 2E). In transverse section, the shape of the cyspela was mainly elliptical (Figure 2B). The embryo cells (cells of both the epidermis and parenchyma) were filled with storage materials in the form of approximately globular structures (Figure 2F).  
Figure 2: Structure of the _Carlina acaulis_ cyspela. (**A**). Morphology of the cyspela in scanning electron microscopy (SEM), bar 1 mm. (**B**). Transverse section of the cyspela; embryo (**E**) in SEM, bar 1 mm. (**C**). Cross section through the cyspela; embryo (**E**) in SEM, bar 500 \(\upmu\)m. (**D**). A part of cross section through the cyspela; two cotyledons are well visible (**C**), bar 100 \(\upmu\)m. (**E**). A part of transverse section through the cyspela; pericarp and testa (Pt), cotyledon (**C**), bar 100 \(\upmu\)m. (**F**). Parenchyma cells of the cotyledon; note the cytoplasm filled with storage material, nucleus (N), bar 10 \(\upmu\)m.  
The NBB staining revealed that these structures were protein bodies (Figure 3A,B). The results of PAS reaction confirmed the presence of carbohydrates located mostly in the cell walls, and no starch grains were detected in the embryo cells (Figure 3C). The Sudan Black B staining gave a negative result, which indicated the absence of lipid bodies (Figure 3D). In the plant material treated with the osmium tetroxide solution (Figure 4A,B), some embryo cells had osmiophilic granules in the cytoplasm, suggesting their lipid character.  
We did not detect starch grains in the embryo cells of _Carlina acaulis_; however, as shown by [PERSON] [19], the occurrence of starch grains in cotyledons depends on the species. In other species from the Asteraceae family starch was not found as storage material in embryos, e.g., in sunflower cultivars [15] and _Trichocline catharinensis_[20].  
Protein reserves are an important storage of nitrogenous compounds, which are essential during germination and development of young seedlings. They are a common seed storage material [21]. In plants, proteins are selectively accumulated and packaged in specialized vacuoles referred to as protein bodies [22; 23]. As reported by [PERSON] and [PERSON] [21], protein bodies are located in the entire embryo or endosperm or they are restricted to a specialized layer. Here, we show that protein bodies in _Carlina acaulis_ are found in the entire embryo and are dominant organelles in embryo cells.  
Studies have reported the presence of protein bodies in the embryo cells in the other members of the Asteraceae family as well [15; 20].  
Figure 3: Cytochemistry of _Carlina acaulis_ embryo cells. (**A,B**). NBB staining, bar 50 \(\upmu\)m and bar 10 \(\upmu\)m. (**C**). PAS reaction’ note the presence of polysaccharides only in the cell walls, bar 50 \(\upmu\)m. (**D**). Sudan Black B staining, bar 10 \(\upmu\)m.  
Using chemical methods, we showed that _Carlina acaulis_ cypselae contained oils (fatty acids); however, Sudan Black B staining gave a negative result, which indicated the absence of lipid bodies. In contrast, lipid bodies were a common cell feature of embryo cells in other Asteraceae species [15; 20]. Thus, this problem deserves future analysis to be conducted using transmission electron microscopy.  
[PERSON] et al. [20] found that _Trichocline catharinensis_ seeds were orthodox seeds. Additionally, our anatomical and histochemical observations indicate that _Carlina acaulis_ seeds belong to this type.