Yoichiro Hoshino

Bialaphos stimulates shoot regeneration from hairy roots of snapdragon (Antirrhinum majus L.) transformed by Agrobacterium rhizogenes

Abstract Hairy roots of snapdragon (Antirrhinum ma-jus L.: Scrophulariaceae) induced by a wild-type strain of Agrobacterium rhizogenes were cultured on media containing various concentrations of a phosphinothricin-based herbicide, bialaphos, or plant growth regulators (PGRs). Adventitious shoot regeneration from hairy roots was observed with a low frequency (10%) on half-strength Murashige and Skoog medium. Addition of α-naphthalene-acetic acid in combination with 6-benzylaminopurine, thidiazuron, or zeatin to the medium had no effect on shoot regeneration from hairy roots. Although bialaphos at 0.9 mg l–1 or more was toxic to hairy roots, it significantly increased the shoot regeneration frequency up to 56% at 0.5 mg l–1. In contrast, non-transformed roots and leaves regenerated no shoots on media with or without bialaphos. Regenerated shoots detached from host roots readily developed roots on gellan-gum-solidified medium. Regenerated plants were successfully transferred to the greenhouse, but did not produce seed.

Adventitious shoot regeneration from cultured petal explants of carnation

Adventitious shoot regeneration was compared among leaf, stem and petal explants of carnation (Dianthus caryophyllus L.) cv. Scania on MS medium containing different concentrations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA). High frequency regeneration was obtained only from petal explants on the media containing 5 to 10 μM BA with or without 5 μM NAA. Among the cytokinins tested, N-2-chloro-4-pyridyl-N′-phenylurea and N-1,2,3-thiadiazol-5-yl-N′-N′-phenylurea were more effective than BA, kinetin, N6-2-isopentenyl adenine and zeatin on regeneration from petal explants. Although, high frequency shoot regeneration was obtained from all petal explants harvested from various developmental stages of buds, a significant decrease in regeneration capacity was observed in the explants obtained from fully-opened flowers. High frequency shoot regeneration was also obtained from the petal explants of cvs. Coral. Lena, Nora and White Sim, and an interspecific cultivar Eolo using the method developed in this study.

Highly efficient system of plant regeneration from protoplasts of grapevine (Vitis vinifera L.) through somatic embryogenesis by using embryogenic callus culture and activated charcoal

Abstract A simple protocol is described for high frequency plant regeneration from protoplasts isolated from leaf-derived embryogenic calli of grapevine ( Vitis vinifera L. cv. Koshusanjaku). The protoplasts successfully divided to form somatic embryos by culturing in gellan gum disc-method in which protoplasts were embedded in 2 g/l gellan gum-solidified Nitschs medium containing 2.0 mg/l NAA, 0.5 mg/l BA, 0.09 M sucrose and 0.3 M glucose at a density of 1 × 10 5 protoplasts/ml. For the continuous growth of the colonies without browning, it was essential to add 0.3% (w/v) AC in the liquid reservoir medium from the beginning of the culture. In this culture condition, protoplasts started to divide after 10 days of culture and grew into torpedo embryos 4 months after initiation of culture. The torpedo embryos thus obtained germinated normally by transferring onto 2 g/l gellan gum-solidified PGR-free Nitschs medium containing 30 g/l sucrose. The regenerated plants were successfully transferred to the greenhouse and showed normal morphology.

Intergeneric somatic hybrid plantlets between Dianthus barbatus and Gypsophila paniculata obtained by electrofusion.

Hypocotyl-derived protoplasts of Dianthus barbatus that had been pretreated with iodoacetamide were fused electrically with cell suspension culture-derived protoplasts of Gypsophila paniculata that could divide to form callus but could not regenerate shoots under the culture conditions used in this study. Electrofusion-derived calli which produced shoots were selected as putative somatic hybrids, and plantlets were subsequently regenerated from 2 of these selected calli. These plantlets, which in vitro produced flowers precociously, were identified as intergeneric somatic hybrids by nuclear ribosomal DNA analysis. Normal plants have not been established up to the present.

Plant regeneration from cell suspension-derived protoplasts of Saintpaulia ionantha Wendl

SummaryFriable calli were induced on leaf segments of Saintpaulia ionantha Wendl. on B5 medium containing 1 mg l−1 2,4-D and 2 g l−1 casein hydrolysate. Cell suspension cultures were readily established from these friable calli and protoplasts could be isolated from the cells with yields of 1–3×107/g f. wt.. By culturing in 0.1 % gellan gum-solidified B5 medium supplemented with 1 mg l−1 2,4-D and 0.1 M each of sucrose and mannitol at a density of 1×105/ml, the protoplasts divided within 6 days and formed macro-colonies after 2 months of culture. Shoot regeneration from protoplast-derived calli was obtained by sequential treatment of the calli with plant growth regulators: initially with 1 mg l−1 each of NAA and BA for 2 months followed by 0.01 mg l−1 NAA and 5 mg l−1 BA for 4 months. Regenerated plants were established after rooting of the shoots on half-strength MS medium, and successfully transferred to the greenhouse. The regenerated plants grew into flowering stage and showed the same phenotype as the parent plant.

Agrobacterium-mediated transformation of Saintpaulia ionantha Wendl.

Abstract Agrobacterium tumefaciens strain LBA4404 that has pTOK233 and other two strains that have pIG121Hm were co-cultivated with suspension cells of Saintpaulia ionantha Wendl. ‘Pink Veil’. These two plasmids contain an intron-gusA reporter gene, hygromycin phosphotransferase gene (hpt) and neomycin phosphotransferase II gene (nptII) as selective markers. After 48 h of co-cultivation, the cells inoculated with A. tumefaciens strain LBA4404 (pTOK233) showed the highest GUS activity. After 4 months of subculture on the selection medium supplemented with 50 mg l−1 hygromycin B, resistant calli appeared and adventitious shoots were regenerated 6 months after the transfer on B5 medium supplemented with 1 mg l−1 BA. The presence of the gusA gene in the genome DNA of regenerated plants was detected by PCR and genomic Southern blot analysis. Leaf lamina, another explant used for inoculation, rarely expressed transient GUS activity, and transgenic plants were not obtained.

Isolation of embryo sacs from Dianthus ovules by enzymatic treatments and microdissection

Abstract Mature ovules of Dianthus (Caryophyllaceae) were histologically observed by clearing and serial sectioning to characterize the cells of the embryo sac. The results show that the mature embryo sac was located deep inside the hemitropous ovule due to thick nucellar tissue at the micropylar region. For the isolation of the embryo sacs, ovules were collected from ovaries of flowers 1 day after anthesis, and treated with an enzyme solution for digesting cell walls on a gyratory shaker. After 12 h of enzyme treatment, these ovules were dissected using a glass needle under an inverted microscope to release the embryo sacs. The embryo sacs, characterized by their specific size, were successfully released by these successive treatments. The viability of the embryo sacs was more than 80% as assessed with fluorescein diacetate staining. Fluorescent staining with 4,6-diamidino-2-phenylindole revealed the nuclei of the egg apparatus in the isolated embryo sacs. The procedure for isolating embryo sacs established in this study will offer a new approach to further in vitro studies on fertilization in Dianthus.