The Azolla-Anabaena azollae association permits the study of a symbiotic relationship between a blue-green alga and a green plant under laboratory conditions. Previous studies on the physiology of the symbiotic association were not well defined and were limited in scope. Various aspects of mineral nutrition, temperature, and light intensity on the growth of the organism have been reported (22). We are not aware of any studies on the metabolic functions of N2 fixation, respiration, and photosynthesis in the individual organisms or their interaction in the symbiotic association.This manuscript is a report of initial studies on the characterization of the Azolla-Anabaena azollae symbiotic relationship. We describe the morphology, a method of freeing the fronds of the symbiotic alga, and isolation procedures devised to fractionate Azolla-Anabaena azollae for metabolic studies. The companion publication deals with aspects of acetylene reduction (nitrogenase activity) (25).

A new procedure is reported for high-yield isolation of guard cell protoplasts from Viia faba L. Delayed Light emission and P700 content plus absorption and fluorescence emission spectra of these protoplast extracts are reported. It is concluded that both photosystems are present. The presence of photosystem II 12Step 2. The leaf pieces were infitrated under reduced pressure with enzymic digestion medium consisting of 150 ml 0.3 M mannitol, 10 mm sodium ascorbate, 10 mm CaCl2, 4% (w/v) Cellulysin (pH 5.5). Incubation was for 1 h at 30 C in a 1-liter flask in a shaker bath (60 5-cm excursions/min).Step 3. One hundred fifty ml 0.7 M mannitol was added and incubation was continued for 1 h.Step 4. Digestion was interrupted by pouring the material over a 295-,um screen (Nitex) and washing with 0.7 M mannitol containing 10 mm sodium ascorbate. Epidermal strips (coming from both surfaces) and vein nets retained on the screen were transferred to 0.7 M mannitol containing 10 mM sodium ascorbate. Then the epidermal strips were manually separated from the vein nets and transferred to a separate container. The strips had few adhering mesophyll cells, but epidermal cells were intact.In some experiments, mesophyll cells which passed through the screen were carried through steps similar to the remainder of the isolation procedure and used as controls.Step 5. Digestion was continued for I h as in Step 2 except (a) mannitol was increased to 0.7 M, (b) volume was reduced to 100 ml, and (c) shaker-bath speed was reduced to 30 excursions/min.Step 6. The strips were collected and washed as in Step 4. If microscopic examination showed contaminating cells, Steps 5 and 6 were repeated.Step 7. Digestion was continued until guard cell protoplasts were released (about 3 h). These protoplasts were isolated by passage through a cascade of screens (nominally 295, 166, and 20 pm) and collected by centrifugation (lSOOg, 5 min). The protoplasts were washed three times with 0.7 M mannitol containing 10 mM sodium ascorbate and examined for purity.Comments. Isolation of guard-cell protoplasts has been reported (25,32, 43

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