Plant regeneration from embryogenic cultures initiated from mature loblolly pine zygotic embryos

Mature zygotic embryos of eight (open-pollinated) families of loblolly pine (Pinus taeda L.) were cultured on eight different basal salt formulations, each supplemented with 36.2 muM 2,4-dichlorophenoxyacetic acid, 17.8 muM 6 -benzyladenine, 18.6 muM kinetin, 500 mg l(-1) casein hydrolysate, and 500 mg l(-1) L-glutamine for 9 wk; embryogenic tissue was formed on cotyledons, hypocotyls, and radicles of mature zygotic embryos. Callus was subcultured on the callus, proliferation, medium, the same as the induction medium, ba t with one-fifth concentration of auxin and cytokinin for 9 wk. On this med ium a white to translucent, glossy, mucilaginous embryogenic callus contain ing embryogenic suspensor masses (ESMs) was obtained. The highest frequency of explants forming embryogenic tissue, 17%, occurred on a modified Murash ige and Skoog salts basal medium containing the concentration of KNO3, Ca(N O3)(2)-4H(2)O, NH4NO3, KCI, ZnSO4-7H(2)O(2), and MnSO4-H2O, 720, 1900, 00 2 50, 25.8, and 25.35 mg l(-1), respectively. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus prolifer ation medium. Liquid cultures containing ESMs were transferred to medium co ntaining abscisic acid, polyethylene glycols, and activated charcoal for st imulating ther production of cotyledonary somatic embryos. Mature somatic e mbryos germinated for 4-12 wk on medium containing indole-butyric acid, gib berellic acid, 6-benzyladenine, activated charcoal, and reduced sucrose con centration (15 g l(-1)). Two hundred and ninety-one regenerated plantlets w ere transferred to a perlite:peatmoss:vermiculite (1:1:1) mixture, then the plants were transplanted to soil in the earth, and 73 plantlets survived i n the field.