Agrobacterium-mediated production of transgenic plants of Muscari armeniacum Leichtl. ex Bak.

Abstract

A system for the production of transgenic plants has been developed for the Liliaceous ornamental plant Muscari armeniacum Leichtl. ex Bak via Agrobacterium-mediated transformation of embryogenic cultures. Leaf-derived embryogenic cultures were co-cultivated with each of three A. tumefaciens strains, all of which harbored the binary vector carrying the neomycin phosphotransferase II (nptII), hygromycin phosphotransferase (hpt) and intron-containing β-glucuronidase (gus-intron) genes in the T-DNA region. Following co-cultivation, the embryogenic cultures were cultured on a medium containing 500 mg l -1 cefotaxime for 1 week followed by a medium containing 75 mg l -1 hygromycin in addition to cefotaxime. After 4-5 weeks, several hygromycin-resistant (Hyg r ) cell clusters were produced from the co-cultivated embryogenic cultures. The highest efficiency of production of Hyg r cell clusters was obtained when embryogenic cultures were inoculated with A. tumefaciens EHA101/pIG121Hm in the presence of 100 µM acetosyringone (AS) and 0.1% (v/v) of a surfactant (Tween20) followed by co-cultivation in the presence of 100 µM AS. Hyg r embryogenic cultures developed into complete plants via somatic embryogenesis, and most of them were verified to be transgenic by GUS histochemical assay and polymerase chain reaction analysis. Southern blot analysis revealed the integration of one to five copies of the transgene into the genome of transgenic plants, but most of them had one or two copies.

An optimized Agrobacterium -mediated transformation procedure for Phaseolus acutifolius A. Gray

“…In these cases, an improvement in the transient expression levels will not necessarily result in better stable transformation (Shen et al 1993;Ishida et al 1996;Maximova et al 1998). However, most studies have shown that conditions leading to enhanced transient expression do result in a higher number of transformed plants (Cao et al 1998;Kondo et al 2000;Niu et al 2000;Zhang et al 2000;Choi et al 2001;Trifonova et al 2001;Suzuki and Nakano 2002).…”

Production of transgenic lily plants by Agrobacterium -mediated transformation

“…Southern blot analysis has been the usual means to verify the transgenic nature and to determine the copy number of transgenes in plant genomes (Watad et al 1998;Suzuki et al 2001;Suzuki and Nakano 2002). As this technique requires relatively large amounts of genomic DNA, it is difficult to obtain a clear signal if the genome size of the subject plant is large.…”

“…Suzuki et al 2001;Suzuki and Nakano 2002) or regeneration medium (e.g. Watad et al 1998) was used for selecting transformed lines.…”

“…In the past few years, successful application of Agrobacterium-mediated transformation has been reported for some Liliaceous species, including Asparagus officinalis (Kisaka and Kameya 1998), Allium sativum (Kondo et al 2000), Allium cepa (Eady et al 2000), Agapanthus praecox (Suzuki et al 2001), and Muscari armeniacum (Suzuki and Nakano 2002), suggesting that transformation with this method could be applicable to other Liliaceous species. However, in the genus Lilium, Agrobacterium-mediated production of transgenic plants has not yet been reported, although transient expression of the gus reporter gene following cocultivation with Agrobacterium has been demonstrated for an interspecific hybrid cv.…”

Heterologous expression of the flavonoid 3?,5?-hydroxylase gene of Vinca major alters flower color in transgenic Petunia hybrida

“…Southern hybridization was performed according to Suzuki and Nakano (2002). V. major genomic DNA (50 mg) was digested with either HindIII, XbaI, ScaI or SacI.…”