An optimization method for measuring the stomata in cassava (Manihot esculenta Crantz) under multiple abiotic stresses.

As a gateway for gas exchange, pores regulate the transport of air and water in carbon assimilation, respiration, and transpiration to quickly adapt to environmental changes. Therefore, the study of stomatal movement characteristics of plants is helpful to strengthen the understanding of the mechanism of plant response to multi-environmental stress, and can improve the function of plant resistance to stresses. The stomatal movement of Arabidopsis leaves was observed by staining the stomata with rhodamine 6G, but this method has not been reported in other plant leaf stomata studies. Taking cassava as an example, the correlation between cassava stomatal movement and cassava response to stress was observed by using and improving the staining method. Rhodamine 6G is a biological stain widely used in cell biology and molecular biology. It was found that 1 μM rhodamine 6G could stain the stomata of cassava without affecting stomatal movement (n = 109, p < 0.05). In addition, we proposed that stomata fixed with 4% concentration of formaldehyde after staining were closest to the stomatal morphology of cassava epidermis, so as to observe stomatal movement under different environmental stresses more accurately. Previous methods of measuring stomatal pore size by autofluorescence of cell wall needs to fix the cells for 6 h, but Rhodamine staining can only be observed in 2 min, which greatly improves the experimental efficiency. Compared with the traditional exfoliation method (e.g., Arabidopsis), this method can reduce the damage of the leaves and observe the stomata of the whole leaves more completely, so that the experimental results are more complete. In addition, the method enables continuous leaf processing and observation. Using this method, we further compared four different cassava varieties (i.e., KU50, SC16, SC8, and SC205) and found that there are differences in stomatal density (SD) among cassava varieties, and the difference in the SD directly affects the stress resistance of cassava (n = 107, p < 0.001). This finding has important implications for studying the mechanism of plant response to environmental stress through stomata.