Nuclear changes associated with cellular dedifferentiation in pea root cortical cells cultured in vitro

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Abstract
Root cortical tissues explanted from seedling roots of the garden pea, Pisum sativum L., cv. Little Marvel cultured in sterile medium containing indoleacetic acid and kinetin were fixed, sectioned and studied with the electron microscope. Nuclear changes over 60 h of culture were examined during the events of dedifferentiation of mature parenchyma cells into subdivided newly meristematic derivatives. Table 1 summarizes the events which are evident in 7 population classes designated I—VII. The initially small, round nucleus with a single, unvacuolated nucleolus and highly condensed and disperse chromatin showed marked volume increase, nucleolar enlargement of almost 20-fold, development of diffuse and then clumped chromatin and then subdivision of the nucleoli into a number (5–10) of small nucleoli immediately preceding cell division. Mitoses involved cells with diplochromosomes and daughter nuclei with doubled chromosome number, either 4n or 8n. Subdivided cortical cells showed an increased ratio of nucleus to cytoplasm although the nuclei were reduced in volume to that of the original nuclear population. Nucleoli in subdivided cells were small and unvacuolated and chromatin changed from an initially condensed state through a diffuse and disperse condition to a condensed form. Cortical cells divided at least twice in rapid succession before initiating new events leading to redifferentiation. Nuclear changes in dedifferentiation in pea were comparable in many respects to the similar process studied in animal systems including eye lens regeneration, limb regeneration and activation of division in blood lymphocytes. The cytological changes in nuclear structure could be correlated with documented changes in DNA, RNA and protein levels in these systems. By 60 h cells presumed to be ready for redifferentiation and cell specialization were observed in the subdivided population but structural evidence for commitment to a new course of cytodifferentiation was not obtained.