Identification of sieve elements and companion cell protoplasts by a combination of brightfield and fluorescence microscopy

PREMISE OF THE STUDY: Phloem‐limited diseases are becoming increasingly pervasive, threatening the existence of crops worldwide. Studies of phloem diseases are complicated by the inaccessibility of the phloem tissue. Phloem cells are located deep inside the plant body, are interspersed with other cell types, are among the smallest cells in the plant kingdom, and make up a small percentage of the total cell population in a plant. These properties make phloem cells difficult to investigate. METHODS: We used leaf midrib protoplasts and a combination of organelle‐specific dyes including Neutral Red (acidic compartments), MitoTracker Green (mitochondria), Hoechst 3342 (nucleus), and chloroplast autofluorescence. We examined the protoplasts under light and fluorescence microscopy. RESULTS: When observed using brightfield and fluorescence microscopy, sieve element protoplasts were distinguished by the lack of vacuole and a nucleus, and by containing various mitochondria. Companion cells showed a dense cytoplasm and various small vacuoles. They also revealed their characteristic large nucleus and abundant mitochondria. DISCUSSION: We present evidence that a combination of organelle‐specific dyes and autofluorescence allows for the identification of sieve elements and companion cell protoplasts from citrus leaf tissue.