Micropropagation of eastern white pine (Pinusstrobus L.)

Abstract

Cotyledons, epicotyls, and hypocotyls from embryos and "seedlings" of Pinusstrobus formed shoots following exposure to cytokinin on a Schenk and Hildebrandt medium. Benzyladenine and 2-isopentenyladenine were both effective at inducing shoot formation but benzyladenine was 5–10 times more potent. There was no synergistic interaction between these two cytokinins. Benzyladenine concentrations of 0.5–1.0 mg L−1 gave consistently good results with cotyledons. A 2-week cytokinin exposure produced uniformly good results and longer exposures caused callusing and vitrification. Pretreating seeds with hydrogen peroxide for 3 days stimulated caulogenesis. Addition of phenolics to medium containing benzyladenine also stimulated shoot formation and di-substituted phenolics were more effective than p-hydroxybenzoic acid. A short liquid pulse with high concentrations of benzyladenine induced caulogenesis but was not as effective as the application of cytokinin through agar-solidified culture medium. Cotyledons excised from embryos in cultured megagametophytes or from cultured embryos were more caulogenic than cotyledons from imbibed seeds. Best caulogenesis occurred after 1 week of direct embryo culture or culture of the embryo inside the megagametophyte. The caulogenic capacity of cotyledons from cultured embryos declined after 1 week but they retained competence to form shoots for 1 month. Epicotyls and hypocotyl sections from 1-week-old cultured embryos were also caulogenic but roots were not. Epicotyls responded best at benzyladenine levels less than 1 mg L−1, but hypocotyls required higher concentrations. Caulogenesis from hypocotyls was polar in that most shoots formed at the upper end of the apical hypocotyl half. The basal hypocotyl half was less caulogenic and produced callus. Roots only produced callus. Charcoal inhibited shoot induction when it was included with benzyladenine. Charcoal also inhibited rhizogenesis when included in the final rooting medium. Shoot elongation was promoted by charcoal when it was given during the second subculture on basal medium for 1 month. Shoot elongation occurred on full-strength basal medium. Reduced concentrations of minerals and organics failed to enhance shoot elongation. Approximately 80% of microshoots rooted invitro on quarter-strength basal medium without any hormone application. An auxin pulse treatment had little effect on rooting. Application of rooting powders containing indolebutyric acid increased the number of roots formed but had little effect on rooting frequency.