Embryo induction and plant regeneration from cultured anthers and pistils of orchardgrass (Dactylis glomerata)

Direct embryogenesis was observed from Dactylis glomerata L. [orchardgrass (2N=4X=28)] anthers incubated at 25 C for 6 weeks on Schenk and Hildebrandt (SH) medium containing 3% sucrose and 30 pM dicamba [SH-30 (3,6 dichloro-o-anisic acid)]. Subsequent experi-ments showed that SH-30 supplemented with up to 5.0 g/1 casein hydro- lysate did not affect and 0.1 to 5.0 mg/1 benzylaminopurine or kinetin hindered the embryogenic response. Furthermore, anther orientation had no significant effect on the embryogenic response and no embryos were obtained from culture of isolated microspores. However, SH-30 containing 94 sucrose promoted embryogenesis and an anther density of 10/ml initiated more embryos per anther than the 100 anthers/ml treatment. Anthers initiated embryo-like structures from within micro- spores after culture on SH-30 for 6 weeks at 4 C. In another experi-ment, anthers were exposed to 0, 3, or 6 week cold pretreatments (4 C) and then cultured at 25 C on SH medium containing 0, 10, 20, or 30 pM dicamba and 9% sucrose. Embryo initiation was affected by an interaction of cold pretreatment and dicamba concentration. Stereomicroscopy showed that the 6 week cold pretreated anthers initi-ated indirect embryos from callus masses derived from microspores. Five of six plants regenerated from the 6 week cold pretreated anthers consisted of cells with 28 chromosomes. However, 1 plant had 14, 28, and up to 112 chromosomes and 1 to 8 nucleoli per nucleus. Direct embryogenesis was observed from ovary and style regions of unpollinated pistils cultured on SH-30 for 3 to 4 weeks at 25 C. After 5 and 6 weeks culture, however, embryos disorganized and produced calli which proliferated into numerous secondary embryos. An upright pistil orientation initiated a significantly greater embryo- genic response compared to those pistils cultured flat on the medium. All examined plants regenerated from pistil culture possessed the somatic chromosome number of 28 except for 1 mixoploid with 14, 28, and 56 chromosomes. Culture of excised unpollinated ovules failed to initiate any embryos when cultured on SH-30 with or without kinetin concentrations up to 5.0 mg/1. However, 1 of 60 ovules excised from pistils cultured for 3 weeks on SH-30 and then recultured on SH-30 initiated callus and embryos. Histology of pistils cultured for 3 weeks showed possible cell divisions within the embryo sac.