We have used the tobacco thin cell layer 'in vitro' system to evaluate changes in polyamine titers as correlated with root differentiation and with variations in external pH during culture. We show that root differentiation in this system depends on both a rise in putrescine titers and a drop of pH, each of these two factors acting independently. With respect to polyamine titers, the most dramatic changes occur in the levels of putrescine liberated from perchloric acid-soluble conjugates. These titers increase from day 0 to day 7 of culture, reaching almost 2000nmolg -1 fresh weight. Inhibition of putrescine biosYnthesis prevents root initiation, while exogenous putrescine supply reverses this effect. We conclude that putrescine is a good marker for root differentiation.
Direct somatic embryos were differentiated on cotyledon transverse Thin Cell Layers (tTCLs) of Panax ginseng after 9 weeks in the Murashige and Skoog basal (MS) medium containing 2,4-D (5#M). When MS medium containing 2,4-9 (5#M) was used for seedling pretreatment and for tTCLs culture, somatic embryos were observed 2 weeks earlier, i.e. after 7 weeks of culture. On the tTCLs from seedlings pretreated with 2,4-D (5#M) combined with benzyladenine and zeatin at 0.1/zM (BZ), somatic embryos were observed after 6 weeks of culture and the percentage of embryogenesis was higher (62%) than when 2,4-D was used alone for pretreatment (40%). Similar results were also obtained from pretreatment with combinations of 2,4-D (5/zM) and thidiazuron (TDZ) (0.01, 0. I#M). When a combination of 2,4-D (5#M) and BZ (0.1#M) was used both for seedling pretreatment and for tTCLs culture, both somatic embryos and shoots were observed after only 3 weeks. As the concentration of BZ increased, the percentage of somatic embryogenesis decreased but the percentage of organogenesis increased. Similar responses were obtained with a combination of 2,4-9 (5#M) and TDZ (0.01#M). On the medium containing both NAA (0.3#M) and BZ (I#M), globular-and heart-stage embryos developed after 4 weeks of culture into cotyledonary-staged embryos which remained dormant after a short elongation of the embryo axis. The importance of seedling pretreatment by growth substances in enhancing somatic embryogenesis is reported.
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