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At monthly intervals for 1 year, one branch was removed from the lower crown of three 30-year-old trees of black walnut ( Juglans nigra  L.). The basal 1.3 m of each branch was cut into four 32-cm-long segments that were placed horizontally in shallow plastic trays filled with perlite and watered daily with tap water. Branch segments cut early in the dormant season (29 Sept., 31 Oct., or 1 Dec.) or shortly after flushing (6 June) produced few, if any, epicormic sprouts. Approximately half the branch segments cut on 3 Jan. or 3 Feb. produced one sprout that elongated slowly. Most branch segments cut in the late dormant season (2 Mar., 30 Mar., 3 May) or growing season (5 July, 4 Aug., 6 Sept.) produced one or two sprouts &gt;20 mm long. To prepare explants for in vitro culture, the terminal 2.5 cm was harvested when sprouts exceeded 3.0 cm, trimmed of all leaves, and disinfested. Explants were placed vertically in liquid Long &amp; Preece (LP) medium supplemented with 3% sucrose, 0.3 μM TDZ, 0.05 μM IBA, and 1 μM BA. When shoots began to elongate (4 to 6 weeks), they were then placed horizontally on agar-solidified LP medium with liquid LP overlays to induce axillary shoot proliferation. Advantages of forcing epicormic sprouts on large branch segments are: 1) they can be a source of in vitro explant material for 6 to 7 months a year, 2) aseptic cultures can be easily obtained, 3) shoots from the base of branches may show more juvenility than shoots forced from branch tips, 4) softwood shoot wilting is not a problem as with forcing shoots from branch tips, 5) the procedure does not require preparing and changing forcing solutions, and 6) branch segments should have more stored food than dormant branch tips for forcing softwood growth.

Epicormic Sprouts from Branch Segments of Mature Juglans nigra L. as an Explant Source for in Vitro Culture