Cytochemical investigations on multiple bud formation in tissue cultures of Pinus coulteri

Cytochemical events associated with formation of multiple shoots on cultured embryonic explants of Pinus coulteri were studied. The first step in the developmental pattern leading to multiple adventitious bud formation was the establishment of organogenetic domains defined by increased RNA, DNA, and basic nuclear proteins. The succeeding step was the accumulation of reserves such as lipids, proteins, and especially starch. This was followed by utilization of reserves characterized by high lipase and amylase activity in the domains. However, some of the subjacent callus cells also exhibited high lipase and amylase activity, indicating the mobilization of reserves for organogenesis. As meristemoids formed, there was an increase in enzyme activities concerned with energy production and utilization. Differentiating shoot primordia and developing needles showed high activities of acid phosphatase, glucose-6-phosphatase, and succinate dehydrogenase. Intense localization of reducing substances (chiefly ascorbic acid) and increased ATPase activity suggested that there was a considerable utilization of energy associated with elevated metabolism in the meristemoids and developing buds. Peroxidase was strongly localized in regions of growth and differentiation, especially developing vascular strands. It is concluded that these cytochemical events reflect distinctive changes in cellular metabolism associated with the intitiation and development of multiple shoots on the cultured explants of P. coulteri.