After 2 weeks in the dark, leafy shoots were initiated on subcultured callus tissue of triploid quaking aspen on Wolter's medium without auxin and with 6-benzylaminopurine at 0.05, 0.10., 0.15, or 0.20 milligram per liter substituted for kinetin. Shoots transferred to Wolter's medium under light (323 milliphots) grew large roots and were isolated as plantlets.
Trees were produced from firm white callus tissue of triploid quaking aspen (Populus tremuloides Michx.), initiated on a modified Wolter and Skoog defined medium and subcultured monthly for two years. When subcultured to medium without auxin, kinetin or supplements, but containing 0.15 mg/liter BAP (6‐benzylaminopurine), multiple stunted shoots appeared on most inocula. However, at 0.05 mg/liter BAP, only a few vigorous shoots per piece were initiated, but seven rooted on the callus: two in the dark with BAP and five in 200 ft‐c of light with 0.04 mg/liter 2,4‐D. After proliferation of the roots on the medium surface, four shoots elongated and were planted in semi‐sterilized soil, then were given 3100 ft‐c of light for rapid growth into trees. Both light sources were on for 16 hr/day. Two trees were also grown from stunted shoots excised from the callus and rooted in soil.
Whole Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seed embryos were placed on defined medium containing 0.05 or 0.1 mg/ℓ BAP (benzylaminopurine). One vigorous shoot usually grew from the tip of each swollen cotyledon on the 0.05 BAP medium, but small multiple shoots were produced on the 0.1 BAP medium. After transfer of embryos or excised cotyledons to medium without hormones, the vigorous shoots developed normally but the small multiple shoots did not. However, when cotyledons were grown on medium containing auxin and cytokinin, callus was produced that was isolated and subcultured monthly to fresh medium. Shoots were produced from subcultured cotyledon callus in 11 of 35 cultures, for a frequency of 31%. Two of 30–50 excised shoots rooted after treatment with 10 mg/ℓ IBA (indolebutyric acid). A few shoots were also produced from subcultured needle callus and from stem explant callus from young seedling material.
Liquid‐grown callus was used to study the nutritional requirements for rooting of a triploid form of normally diploid quaking aspen (Populus tremuloides Michx.). In modified Wolter's liquid medium, tan rough‐surfaced spheres of callus grew rapidly when supplied with a high concentration of 2,4‐D (0.5 mg/liter), but light‐yellow uniformly smooth and firm spheres grew more slowly with a low level of 2,4‐D (0.04 mg/liter) plus kinetin. When tissue was grown uncut in liquid for 1, 2, or 3 months, then subcultured to a low‐2,4‐D agar medium, rooting increased primarily with the age of the source tissue rather than the initial explant size. The surface of the youngest tissue source was almost smooth, but free columnar proliferations extended out from the surface for two or three cells in 2‐month‐old tissue and for three to six cells in the 3‐month‐old tissue. The relationship of increased rooting with increased surface cell‐proliferation of older tissue was not determined anatomically.
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