A staining protocol for identifying secondary compounds in Myrtaceae

Abstract

Premise of the study: Here we propose a staining protocol using toluidine blue (TBO) and ruthenium red to reliably identify secondary compounds in the leaves of some species of Myrtaceae. •

Methods and results: Leaves of 10 species representing 10 different genera of Myrtaceae were processed and stained using five different combinations of ruthenium red and TBO. Optimal staining conditions were determined as 1 min of ruthenium red (0.05% aqueous) and 45 s of TBO (0.1% aqueous). Secondary compounds clearly identified under this treatment include mucilage in the mesophyll, polyphenols in the cuticle, lignin in fibers and xylem, tannins and carboxylated polysaccharides in the epidermis, and pectic substances in the primary cell walls. •

Conclusions: Potential applications of this protocol include systematic, phytochemical, and ecological investigations in Myrtaceae. It might be applicable to other plant families rich in secondary compounds and could be used as a preliminary screening method for extraction of these elements.

Keywords: Myrtaceae; anatomy; ruthenium red; staining; toluidine blue.

Fig. 1.

Transverse light micrographs (LM) of leaves of some species of Myrtaceae stained with the staining treatment T5. (A) Acmena smithii: Clear highlight of lignified cells (blue) and mucilage (red). (B) Eugenia reinwardtiana: Carboxylated polysaccharides in epidermis. (C) Syzygium australe: Phloem cells stained red and xylem stained blue-green. (D) Gossia floribunda: Mesophyll cells containing dark stained pigments, probably tannins. (E) Myrceugenia parvifolia: Pectic substances in primary cell walls observed in spongy parenchyma (red). (F) Waterhousea floribunda: Cuticle with polyphenols stained blue and fibers with lignin stained blue-purple. (G) Luma apiculata: Red staining of pectic substances and mucilage. (H) Ugni molinae: Mucilage in spongy parenchyma and clear difference between polyphenols in xylem (lignin) and phloem. Scale bars = 100 µm.

Fig. 2.

Transverse light micrographs (LM) of leaves of Acmena smithii, showing comparisons between treatments T2 (A, B), T3 (C, D), T4 (E, F), and T5 (G, H). Treatment T1 showed similar results to T5. (A, B) Polyphenols highlighted in the cuticle (blue) and mucilage (red); however, the lignified elements in the midrib are not differentiated. (C, D) Secondary compounds are not differentiated for masking of TBO. (E, F) Secondary compounds are not differentiated for masking of ruthenium red. (G, H) Secondary compounds are clearly visible with treatment T5, e.g., polyphenols in the cuticle, xylem, and fibers (lignin, blue); mucilage and pectic substances in the mesophyll (red); tannins in the phloem (red); and carboxylated polysaccharides in the epidermis (pink). Scale bars = 100 μm.

Fig. 3.

Transverse light micrographs (LM) of leaves of Eugenia reinwardtiana, showing comparisons between treatments T2 (A, B), T3 (C, D), T4 (E, F), and T5 (G, H). Treatment T1 showed similar results to T5. (A, B) Only polyphenols in the cuticle (blue) are differentiated as the red staining is predominant. (C, D) Secondary compounds are not differentiated for masking of TBO, except for the cuticle. (E, F) Secondary compounds are not differentiated for masking of ruthenium red. (G, H) Secondary compounds are clearly visible in treatment T5, and the xylem and phloem are clearly differentiated, e.g., polyphenols in the cuticle, xylem, and fibers (lignin, blue); mucilage and pectic substances in the mesophyll (red); tannins in the phloem (red); and carboxylated polysaccharides in the epidermis (pink). Red tannins in the epidermis are observable with all the treatments. Scale bars = 100 µm.