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During Apr. 1999, the lower branches of mature black walnut trees were removed and cut into sections 48 cm long and placed horizontally in plastic flats filled with perlite in a shaded polyethylene-covered greenhouse. Water was applied by drip emitters and care was taken to avoid overhead water contact with the stem sections. Within 2 months, elongating, green, leafy shoots were excised, brought into the laboratory, surface disinfested and placed in vitro onto agar-solidifed Long and Preece (LP) medium with 0.3 μM thidiazuron (TDZ), 0.5 μM indolebutyric acid (IBA), and either 0.1, 1.0, or 10.0 μM benzyladenine (BA). Explants were transferred to fresh medium after 1, 3, and 5 days in vitro and every 2 weeks thereafter. After 3 months in vitro, callus was excised and and explants were all placed on LP medium with 10 μM BA and 0.5 μM IBA for 4 weeks. They were then transferred to LP with 0.3 μM TDZ, 1.0 μM BA, and 0.5 μM IBA for 2 weeks. This 4-2-week alternation of media has continued for more than 6 months. After 4 months in vitro, shoot clusters were subdivided, and individual microshoots recultured. Of the original 260 explants, 30 survived and have been subdivided into 111 cultures. These explants have produced 132 axillary shoots that are also multiplying. Adult black walnut will acclimate and proliferate in vitro, but only with careful attention to detail and regular transfers to fresh medium.

313 In Vitro Shoot Multiplication of Adult Black Walnut (Juglans nigra L.)