Cultures were initiated from immature seeds of three species of magnolia: sweetbay magnolia (Magnolia virginiana L.), fraser magnolia (M. fraseri Walt.) and yellow cucumbertree [M. acuminata var. cordata (Michx.) Sarg.]. Immature seeds were bisected longitudinally and cultured on a solidified conditioning medium containing 2 mg 2,4-D/liter, 0.25 mg BA/liter, 40 g sucrose/liter, and 1 g casein hydrolysate/liter. Cultures were maintained in the dark at 22C and transferred to fresh medium at monthly intervals. Within 2 months of culture, somatic embryos or proembryogenic masses proliferated from one end of the endosperm mass. Somatic embryos and proembryogenic masses of each species were cultured on a hormone-free version of the conditioning medium to complete maturation and then transferred to the same hormone-free medium, minus casein hydrolysate, to initiate germination. Germinants were transferred to a hormone-free plantlet development medium for conversion. Plantlets of all three species survived transfer to soil mix and continued to grow. Chemical names used: (2,4 -dichlorophenoxy) acetic acid (2,4-D), N- (phenylmethyl)-1H-purin-6-amine (BA).
A protocol to achieve efficient plant regeneration from juvenile black locust (Robinia pseudoacacia L.) explants is described. Direct adventitious shoots were induced from cotyledon explants on woody plant medium containing 22.2 μm BA and 0.4 μm 2,4-D. Shoots developed and new shoots were induced when the explants were transferred to medium without growth regulators. The effect of dark incubation on shoot regeneration from cotyledons indicated that 15 days of darkness resulted in a high regeneration frequency (91.7%). Adventitious shoot formation also was induced from sections of in vitro-derived leaves cultured in darkness on Murashige and Skoog medium supplemented with 4.4 μm BA and 24.6 μm IBA. A shoot regeneration frequency of 89% was obtained when explants were subcultured on a medium containing 4.4 μm BA and 0.5 μm IAA. Shoots were rooted on Schenk and Hildebrandt medium with or without IBA. Plantlets were acclimatized and grown in the greenhouse. Chemical names used: N -(phenylmethyl)-1H -purin-6-amine (BA); 2,4-dichlorophenoxyacetic acid (2,4-D); indole-3-acetic acid (IAA); indole-3-butyric acid (IBA).
Bigleaf magnolia (Magnolia macrophylla Michx.) cultures were initiated from immature seeds on an induction medium containing 9.0 μm 2,4-D, 1.1μm BA, and 1 g casein hydrolysate/liter. After 2 months on induction medium, one culture produced adventive embryos. Clumps of embryos transferred to liquid induction medium proliferated as nodules, which grew in diameter, but failed to produce embryos while maintained in induction medium. Nodules transferred to basal medium produced clumps of somatic embryos, which continued to produce repetitive embryos with monthly transfer to fresh basal medium. Individual embryos transferred to basal medium lacking casein hydrolysate germinated and leaves expanded. Plantlets derived from these embryos were transferred to potting mix and acclimatized to greenhouse conditions. Chemical names used: (2,4-dichlorophenoxy)acetic acid (2,4-D); N -(phenylmethyl)-lH-purin-6-amine (BA).
Low conversion rates of somatic embryos and poor early growth of somatic embryo-derived plantlets of some forest trees may be related as much to prolonged maintenance in vitro as to basic developmental problems with the embryos. We tested ex vitro conversion as an alternative method for producing the rare North American pyramid magnolia (Magnolia pyramidata Bartram) plantlets from somatic embryos. Tissue cultures were initiated from immature seed explants of pyramid magnolia. Immature seeds collected from each of three trees formed proembryogenic masses (PEMs) following 7 to 10 weeks of continuous culture on semisolid medium containing 9.0 μm 2,4-D, 1.1 μm BA, and 1 g casein hydrolysate/liter. PEMs transferred to semisolid medium without plant growth regulators produced somatic embryos that germinated following transfer to the same medium without casein hydrolysate. Conversion frequency to plantlets was higher and plantlets were more vigorous when germinants were transferred directly to potting mix and grown in a humidifying chamber instead of being maintained in plantlet development medium in test tubes. Chemical names used: 2,4-dichlorophenoxyacetic acid (2,4-D); N-(phenylmethyl)-1H-purine-6-amine (BA).