The effects of sucrose concentration, addition of ammonium nitrate, and exposure to N6-benzyl-adenine (BA) on multiplication potential with shoots derived from shoot cultures of 17- to 20-yr-old Douglas fir trees [Pseudotsuga menziesii (Mirb.) Franco] were compared. Each of these conditions, when compared independently, affected recurrent shoot multiplication and influenced shoot development, as measured by the abundance of shoot apices. Sucrose concentration was influential, the use of 25 g · liter−1 providing twice the multiplication obtained with 20 g · liter−1, and 14 × that obtained with the 30 g · liter−1 concentration routinely used (tree 11). Ammonium nitrate usage also improved multiplication, a 2.5 times improvement being obtained after incorporation of 100 mg · liter−1 NH4NO3 into the medium (tree 33). Shoot cultures were responsive but relatively sensitive to addition of BA, the best improvement in multiplication (5 times) being obtained with brief exposures to 3 mg · liter−1 BA (tree 11). Although shoot cultures were responsive to the conditions investigated, differences in shoot multiplication and development were not displayed for several weeks. It was not possible therefore to repeat all the treatments with more than one genotype; however, when this was possible a genotype-dependent variation in response was evident.
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Dunstan, D.I., Lashta, D.P., Kikcio, S.I. et al. Factors affecting recurrent shoot multiplication in in vitro cultures of 17- to 20-year-old douglas fir trees. In Vitro Cell Dev Biol - Plant 28, 33–38 (1992). https://doi.org/10.1007/BF02632190