2018 ASHS Annual Conference

Phloem-limited diseases, such as citrus greening (Hunaglongbing; HLB), are becoming increasingly pervasive, threatening the existence of crops around the world. Studies of phloem diseases are complicated by the inaccessibility of the phloem tissue. Phloem cells are located buried inside the plant body and are interspersed with other cell types. In addition, phloem cells are amongst the smallest cells in a plant kingdom and make up a small percent of the total cell population within a plant. Together these properties create a complex research challenge. Protoplasts should provide an alternative approach to the study of phloem cells in isolation, especially HLB studies, where the causing agent Candidatus Liberibacter asiaticus remains unculturable. However, crucial to this hypothesis is the ability to distinguish sieve elements (SE) and companion cells (CC) within a population of protoplasts. For this purpose, we aimed at developing a system to allow the distinction of SE and CC from the remaining cells. We present evidence that using a combination of Neutral Red (acidic compartments), Mito Tracker Green (mitochondria), Hoechst 3342 (nucleus) and chloroplast autofluorescence, allows for the identification of SE and CC protoplasts from citrus leaf tissue. Isolated SE and CC offer an additional approach to advance studies on HLB.