This report describes the successful plant regeneration via somatic embryogenesis from immature zygotic embryos of Cryptomeria japonica D. Don. For the induction of embryogenic tissue, we determined that the optimal medium contained N6-benzyladenine and 2,4-dichlorophenoxyacetic acid. Immature zygotic embryos that were collected at the end of June yielded embryogenic tissue at the highest frequency. Embryogenic tissues that had proliferated in liquid medium included small and loosely packed cells and elongating or elongated cells. We used ten cell lines to determine the optimal medium for the development of somatic embryos. Induced somatic embryos germinated with synchronous sprouting of cotyledons, hypocotyls and roots. Gibberellin A3 in the germination medium had a positive effect on both the elongation of hypocotyls and the survival of seedlings. The frequencies of induction and germination of somatic embryos differed among the cell lines examined. Most of the seedlings grew normally. This system of somatic embryogenesis required 4-5 months for the regeneration of C. japonica plantlets from immature zygotic embryos.

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We report an improved transformation system for Lombardy poplar (Populus nigra var. italica). A new binary vector, pBF2, with 11 unique restriction enzyme sites and the normal neomycin phosphotransferase II (NPTII) gene was constructed for the transformation of Lombardy poplar. Genetically transformed adventitious shoots were directly regenerated after cocultivation of stem segments with Agrobacterium tumefaciens EHA105 that harbored a binary vector with genes for the NPTII and enhanced green fluorescent protein. Successful transformation was confirmed by fluorescence microscopy, immunoblotting and Southern blotting analyses, and resistance to kanamycin and geneticin. This transformation system requires less time than our previous method for the regeneration of transgenic shoots. When explants were incubated on a smedium containing dithiothreitol, the transformation frequency increased to approximately 20%.

Genetically transformed lombardy poplar (Populus nigra L. var. italica Koehne) plants were regenerated by co-cultivation of stem segments with Agrobacterium tumefaciens strain LBA4404 that harbored a binary vector (pBI121) which included genes for [5-glucuronidase (GUS) and neomycin phosphotransferase. Successful transformation was confirmed by the ability of stem segments to produce calli in the presence of kanamycin, histochemical and fluorometric assays of GUS activity in plant tissues, and Southern blot analysis.