THE EXPLORATION OF STOMATAL DEVELOPMENT IN BARLEY USING THE TILLMORE COLLECTION

Stomata are small pores on the leaf surface that regulate photosynthetic gas exchange and transpiration water loss. Stomatal distribution, morphology, and movement are key traits for water use efficiency and the adaptation of plants to different environmental conditions. These traits are not easy to evaluate because of the current phenotyping techniques that are laborintensive and time-consuming and therefore the understanding of the molecular mechanism and the genetic control of stomatal development is advancing in model species such as Arabidopsis and B. distachyon, however it remains poorly understood in crops1. In addition, there is a scarce availability of phenotypic and genotypic variation related to stomatal traits in barley and other cereal crops, making it even more difficult to conduct genetic studies on stomatal development. We performed a phenotypic screening on 3600 barley TILLING mutant lines from the TILLMore collection2 grown in seed trays for up to 21 days and acquiring images from the basal portion of the third leaf of each line using an optical microscope. Images were then analyzed for stomatal density and distribution using ImageJ. We found a wide range of phenotypic variation for stomatal density, size and morphology showing a normal distribution of values. From the first 1000 lines, we selected a panel of 16 lines showing strong differences in stomatal density and size, and we proceeded to grow plants in pots in replicates acquiring images from the third, fifth, and flag leaf to confirm the phenotypic variation. Among the 16 lines, three lines showed a significative higher stomatal density compared to the control plants. Since the distribution and frequency of stomata are synchronized with cell growth and division, we also measured the length and width of the leaf and of portion observed with the microscope to correlate stomatal density and distribution to leaf dimensions. Furthermore, we developed a quick protocol for image acquisition of the epidermis with a single-cell resolution, allowing us to count both stomata and epidermis cells and thus to calculate the stomatal index, which is relatively constant and independent from leaf dimension. Determining the genetic basis of stomatal development is of fundamental importance to improve crop adaptation to environmental changes and could be addressed evaluating TILLING mutants.