Growth Regulator Effects, Metabolite Changes, and Respiration during Shoot Initiation in Cultured Cotyledon Explants of Pinus radiata

Adventitious shoot initiation in cotyledon explants from 6-7-day-old germinated seed of Pinus radiata requires exogenous cytokinin. This requirement was examined in terms of both concentration and duration. Treatments resulting in an insufficient cytokinin supply, particularly during the first 3 days in culture, dramatically reduced organogenesis. Other phytohormones and some other growth-active substances also reduced this cytokinin-induced shoot formation and promoted callus formation. Changes in metabolite pool sizes and in respiratory activity were monitored over the first 21 days in culture, during which meristematic tissue formation occurred along the entire length of the cotyledon. Lipids and free sugars were depleted ca. 10-fold and sixfold, respectively, and there was a steady decline in free amino-N levels. However, the protein-N pool remained relatively large. While there was a continued increase in cotyledon fresh weight, respiration decreased initially and then increased slightly during later stages of meristematic tissue formation.