Histochemical Examination of Shoot Initiation in Cultured Cotyledon Explants of Radiata Pine

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Abstract
Histochemical events underlying the process of shoot initiation were studied in cotyledonary explants of P. radiata cultured in the presence of N6-benzyladenine (BA). The initial explant was mitotically active and showed intense staining for nucleic acids and for cytoplasmic as well as nuclear basic proteins. The cells of initial explants were rich in reserves such as lipids, proteins, and starch and also showed uniform localization of activity of various enzymes. The cotyledons cultured without BA showed a rapid decline in their reserves. The localization of nucleic acids, proteins, and enzyme activity was random, and the intensity of staining decreased in these cells. In constrast, in the cotyledons cultured in the presence of BA, the shootforming layers in contact with the medium showed increased staining for DNA, RNA, and cytoplasmic and nuclear proteins. Starch reserves declined in most cells of the explant, but the epidermal and subepidermal layers maintained abundant starch grains, which disappeared only as the meristematic tissue was formed. Lipase activity was confined to the shoot-forming layers during the initial stages of shoot formation but during the later stages was detected in cells underlying the meristematic tissue. Increased staining intensities for acid phosphatases, ATPase, succinate dehydrogenase and peroxidase were found in shoot-forming regions of the cotyledons. These findings agree with the hypothesis that the initiation of organized development in vitro involves a shift in metabolism that precedes and is coincident with the process.