Protoplasts as a Means of Studying Chloroplast Development in Vitro

Protoplasts obtained enzymically from etiolated primary leaves of oat were illuminated in vitro, and the process of etioplast chloroplast transformation followed. Chloroplast development proceeded up to 6 hours of incubation in the light (20 C). During this period, complete photosynthetic light and dark reactions were constituted, in addition to prolamellar body-degrading protease activity.In parallel, electron microscopic investigations showed a pronounced decrease in prolamellar body area from 89.5 square micrometers at 0 hours to 40.6 square micrometers at 6 hours, whereas the length of the thylakoid membranes (prothylakoids, thylakoids) increased from 21 micrometers at 0 hour to about 293 micrometers at 6 hours. This was accompanied by the formation of grana (bi- and polythylakoids).A comparison of plastid structure and function, developed within isolated protoplasts and those of organelles greened in intact leaves up to 6 hours, showed that there was only a slight lag in development of plastids illuminated in vitro to those illuminated in vivo. However, times of in vitro illumination longer than 6 hours resulted in signs of deterioration and the lack of further development, except for photosystem I activities.