DNA synthesis, cell division and specific cytodifferentiation in cultured pea root cortical explants

Abstract

Pea root segments cut 10–11 mm behind the tip of germinating seedlings were prepared by removal of the central cylinders with a tissue punch. These cortical explants were cultured aseptically on nutrient medium containing auxin with and without added cytokinin. In the absence of kinetin, the cortical cells enlarged and separated but failed to show DNA synthesis, mitosis, cell division or subsequent cytodifferentiation. In the presence of 1 ppm kinetin, cortical nuclei showed 3H-thymidine incorporation beginning between 24 and 32 hr; mitoses began about 48 hr, reaching a maximum of 6% at 60 hr. From an initial number of 8000 cells per segment, the cell count increased to 37,000 by day 7 and 140,000 by day 21. At the outset all mitoses were tetraploid; with time the proportion of tetraploid mitotic cells decreased and an octaploid population increased. A frequency of less than 10% diploid mitoses was observed after day 5. Only 25% of the cortical cells showed initial labeling. Beginning on day 7 tracheary elements differentiated from cortical derivatives. By day 14 about 25% and by day 21 about 35% of the total cell population had formed tracheary elements. As a system for analysis in biochemical and cytological terms, pea cortical explants represent an excellent system for the study of cytodifferentiation.