A new method for determination of the photosynthetic pathway in grasses

An easy and inexpensive method of determining the photosynthetic pathway in grasses using a dye widely used in microscopy. To evaluate the efficiency of a new histochemical test for determination of the photosynthetic pathway in grasses (Poacea). Leaves of 58 grass species were sectioned transversally, and the sections treated with a 2% sodium hypochlorite solution to clarify the tissue. After discoloration, sections were washed with distilled water and double-stained with astra blue and safranin (1% each in 50% ethanol) for 1 min. Sections were then mounted between microscopy glass slides and coverslips using water. Grass species showing red staining of the bundle sheath cells were considered C4, and species with translucent bundle sheath were considered C3. The results of the histochemical test were then compared with results from carbon isotope composition analysis and the relevant scientific literature. Observations from the histochemical test were congruent with results from δ(13)C isotope composition analysis, and with data previously presented in the scientific literature. The proposed histochemical test proved efficient for characterization of the photosynthetic pathway in the tested grasses; however, the method should be further tested in a greater number of grass species, encompassing, preferably, all Poacea subfamilies. Future studies may elucidate if the proposed method can effectively be used in other botanical families. Furthermore, additional investigations may determine whether the phenolic compounds indicated by the histochemical test are exclusive to the bundle sheath of C4 grasses and if possible relations exist between these phenolic compounds and the C4 photosynthetic pathway in grasses.