Takeshi Mohri

Agrobacterium tumefaciens-mediated transformation of Japanese white birch (Betula platyphylla var. japonica)

Abstract Genetically transformed Japanese white birch ( Betula platyphylla var. japonica ) plants were regenerated after co-cultivation of leaf discs with Agrobacterium tumefaciens strain LBA4404 that harbored a binary vector (pBI121) which included genes for β -glucuronidase (GUS) and neomycin phosphotransferase. The efficiency of Agrobacterium tumefaciens -mediated transformation of Japanese white birch was markedly increased by the inclusion of acetosyringone in the co-cultivation medium. Successful transformation was confirmed by the ability of leaf discs to produce calli in the presence of kanamycin, by histochemical and fluorometric assays of GUS activity in plant tissues, and by Southern blot analysis. With this transformation system, about 4 months were required for regeneration of the transgenic birch plants from leaf discs. The frequency of transformation was approximately 18%.

Agrobacterium-mediated transformation of lombardy poplar (Populus nigra L. var.italica Koehne) using stem segments

Genetically transformed lombardy poplar (Populus nigra L. var.italica Koehne) plants were regenerated by co-cultivation of stem segments withAgrobacterium tumefaciens strain LBA4404 that harbored a binary vector (pBI121) which included genes for β-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.

An improved transformation system for Lombardy poplar (Populus nigra var. italica)

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%.

Robinia pseudoacacia inner-bark lectin promoter expresses GUS also predominantly in phloem of transgenic tobacco

Summary Robinia pseudoacacia L., a leguminous deciduous tree, accumulates lectins in the inner bark (phloem) and seeds. The lectin gene Rplec2 is expressed predominantly in the inner bark. To examine the expression pattern directed by the 5′-upstream region (from -793 bp to +26 bp relative to the transcription start site) of Rplec2 in heterologous plants, a transcriptional fusion of this region with the β-glucuronidase (GUS) reporter gene was introduced into tobacco plants. The resulting transformants showed higher GUS activity in the stems than in the leaves and roots. Histochemical assay of GUS activity demonstrated that the expression of the GUS gene was confined to internal and external phloem, parenchyma cells adjacent to the phloem, and xylem parenchyma cells in the stems. In petioles, the GUS gene was expressed in phloem and nearby parenchyma cells, but not in leaf mesophyll cells. GUS activity was also detected in the phloem of the root. In addition, sequence analysis of the 5′-upstream region of Rplec2 revealed three conserved motifs shared with several previously reported phloem-specific promoters. These results indicate that the 5′-upstream region of Rplec2 functions as a promoter that directs preferential expression of the GUS gene to the phloem of transgenic tobacco plants.

A Biolistic Approach for the Transfer and Expression of a Luciferase Gene in Paulownia (Paulownia fortunei)

Genetic engineering has the potential to allow the selective improvement of a single trait in forest trees without the loss of any of the desired traits of the parental line. Microprojectile-mediated DNA transfer in trees, first demonstrated in poplar (McCown et al., 1991), offers an alternative to gene transfer with Agrobacterium vectors. Stable transformation has been demonstrated in poplar (McCown et aL, 1991) and in spruce (Ellis et aL, 1993). Paulownia (Paulownia fortunei) is a fast-growing species of hardwood tree that is native to East Asia and it is of great commercial value. However, to our knowledge, no studies of the transformation of paulownia have been documented. We have explored microprojectile-mediated DNA transfer in paulownia. In this report, we describe the simple microprojectile-mediated DNA transfer and the transient expression of a gene for luciferase in paulownia.