Regenerable embryogenic suspension cultures of soybean (Glycine max [L.] Merrill, cv. Jack) were transformed by microprojectile bombardment with a β-glucuronidase (gus) gene and a hygromycin phosphotransferase (hpt) gene under control of the cauliflower mosaic virus (CaMV) 35S promoter on different plasmids mixed together. Many independent transgenic clones were obtained by selection for hygromycin resistance. GUS activity was detected in 45% of the transgenic clones showing that cotransformation occurred at high frequency. Stable integration of both the gus reporter gene and the hpt selectable marker were further confirmed by polymerase chain reaction (PCR) amplification of both genes using double primer sets together in the same reaction. Southern blot hybridization analysis also showed the presence of the foreign genes in genomic DNA. The frequency of embryo development was increased and the time span required for embryo development was reduced by the use of a liquid medium to induce embryo development. An average of 3645 GUS-positive spots and ten transgenic clones were produced per bombardment by this procedure giving a 0.27% ratio of stable to transient expression. The procedure described here can be used for modification of agronomic traits and for study of gene regulation in soybean.

View full abstract

Liquid media suitable for the induction and micropropagation of protocorm-like bodies (PLBs) in Vandofinetia Nara ‘Yumika Pink’ were developed by modifying MS basal medium. The best medium obtained for PLBs induction was one-fourth strength MS medium containing 30g/l sucrose and for PLBs micropropagation was one-fourth strength MS medium containing 10g/l sucrose in which FeEDTA was replaced by 6.95mg/l FeSO₄7H₂O. Usefulness of the latter medium was confirmed in other monopodial orchids such as Renanetia, Darwinara, Ascocentrum and Ascofinetia. After transfer onto 2g/l Hyponex (N:P:K=10:30:20) medium containing 2g/l proteose peptone and 20g/l sucrose for 2 months, the PLBs (both those regenerating and those not regenerating plantlets) were subcultured into MS medium containing 30g/l sucrose at 2 months intervals for 4 months. More than 10, 000 plantlets ready for transplanting to pots were obtained efficiently from one floral bud within a year for each orchid cultivar. These results suggest that the methodology could be used on a commercial scale for micropropagation of a wide range of monopodial orchids.

View full abstract

Adventitious shoots and roots were directly formed from young flower bud explants of Agapanthus africanus on Murashige and Skoog (MS) medium containing 30g·L^-1 sucrose, 2g·L^-1 gellan gum, and kinetin or thidiazuron at 1mg·L^-1 in combination with 1mg·L^-1 NAA. Friable calli with a high rate of plant regeneration were also induced by culturing the explants on medium containing NAA, picloram or 2, 4-D at concentrations varying from 1 to 10mg·L^-1, especially at low concentrations of picloram or 2, 4-D. Shoots were readily induced from these calli when transferred onto plant growth regulator-free or 1mg·L^-1 zeatin-containing MS medium. Nodular compact calli were also formed with friable calli on medium supplemented with 2, 4-D or picloram. The compact calli induced with 1mg·L^-1 picloram developed into somatic embryos. In both shoot and embryo regeneration systems, complete plantlets were obtained when they were transferred onto a medium containing no plant growth regulator.

View full abstract

Fine cell suspension cultures with plant regeneration ability were established from leaf-like structures which were produced on friable green calli originated from an immature embryo of rose (R. hybrida cv. Gelb Dagmer Hastrap×R. chinensis var. mutabilis). The leaf-like structures were initially cultured in liquid MS medium containing 2mg/l 2, 4-D for inducing calli, which were then transferred to the medium containing a reduced concentration of 2, 4-D (0.01mg/l) for establishing rapidly proliferating fine cells. The cells thus obtained showed the ability to regenerate embryo-like structures on gellan gum-solidified MS medium containing 1mg/l BA. The embryo-like structures developed into normal plantlets through adventitious shoot formation on MS medium containing 0.1mg/l TDZ. The plantlets were successfully established on the soil.

View full abstract

Hairy root cultures of Trichosanthes kirilowii var. japonica (Cucurbitaceae) were established by direct inoculation of Agrobacterium rhizogenes ATCC15834 on sterile seedlings. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis of intracellular proteins showed that karasurins, ribosome-inactivating proteins (RIPs) in root tubers of T. kirilowii var. japonica, were not accumulated in the hairy roots. A major basic protein produced by the hairy roots was tentatively identified as a class III chitinase based on N-terminal amino acid sequence homology comparisons. In spite of the apparent lack of karasurins, the basic protein fraction from the hairy roots exhibited ribosome-inactivating activity in the rabbit reticulocyte system, suggesting the presence of RIPs in the hairy roots.

View full abstract

Localized cell death and activation of phenylpropanoid metabolism in suspension-cultured rice cells are induced by factors secreted by blast fungus. Microscopic observation revealed that the death of cells at the periphery of calli appeared within 1h after addition of a TCA-precipitable fraction (TCAp) of the fungus culture filtrate. The number of dead cells continued to increase for up to 24h, followed by general browning of the calli after about 48h. However, the culture still contained uninjured cells within the callus clumps, and there was no significant difference in culture viability from non-treated callus. Phenylalanine ammonialyase (PAL) and peroxidase (POX) activities were induced by TCAp and reached a maximum at 6h and 1-2 days after treatment, respectively. No cultivar-race specificity was found in PAL activity of cultured cells induced by TCAp. Phenolic compounds were found in callus tissue treated with TCAp. Among these phenolic compounds, accumulation of p-coumaric acid was remarkable. Fractions of fungal culture filtrate obtained by C₁₈ reverse-phase chromatography were assayed for the ability to induce cell browning, PAL activity in cultured cells. Among the individual fractions, that which had the most potent activity to induce browning stimulated PAL activity.

View full abstract

Somatic embryos and adventitious shoots were initiated from leaf explants of Primula cuneifolia var. hakusanensis on the media containing various cytokinins. The best treatment for induction of somatic embryos was on the medium containing 1.0mg/l of TDZ or 5.0-10.0mg/l of zeatin. Kinetin had no effect on the regeneration from leaf explants. Somatic embryos and adventitious shoots grew to plantlets on LS-hormone-free medium. After acclimatization in the growth chamber, the regenerated plantlets flowered and set seeds.

View full abstract

Three amino acid residues (histidine-217, aspartic acid-219, and histidine-274) in Hyoscyamus niger hyoscyamine 6β-hydroxylase are strictly conserved among 2-oxoglutarate-dependent dioxygenases and other structurally related enzymes. These residues were investigated by chemical modification and site-directed mutagenesis. The hydroxylase was expressed in E. coli as a fusion protein to maltose-binding protein. Modification of histidine residues by diethyl pyrocarbonate inactivated the recombinant wild-type hydrox-ylase. Inactivation was prevented most effectively by the presence of 2-oxoglutarate. Mutation of histidine-217 to glutamine, histidine-274 to glutamine, or aspartic acid-219 to either histidine or asparagine inactivated the hydroxylase, whereas substitution of loosely conserved histidine-66 with glutamine did not decrease the catalytic activity of the enzyme. These results suggest that histidine-217, aspartic acid-219, and histidine-274 play important roles in the hydroxylase function, and may be the ligands to the active-site iron.

View full abstract

Callus and adventitious root cultures of Cornus capitata were established and the production of tannins and related compounds was determined in various culture conditions (media, plant growth regulators, light, etc.). Calli produced mainly procyanidins such as (+)-catechin and procyanidin B-3 on Murashige and Skoog (MS) and Woody Plant (WP) media. The maximum content of (+)-catechin (0.1%, as dry weight) was observed in the calli cultured on WP medium supplemented with 0.1mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.1mg/l benzyladenine (BA), or 2.0mg/l naphthaleneacetic acid (NAA) and 0.1mg/l BA in the light. In contrast, adventitious roots cultured in MS liquid medium supplemented with 3.0mg/l indoleacetic acid (IAA) produced a high level of gallotannins such as 1, 2, 3, 6-tetra-O-galloyl-β-D-glucose and 1, 2, 3, 4, 6-penta-O-galloyl-β-D-glucose.

View full abstract

The adventitious root cultures were induced from leaf segments of Rubia tinctorum L. (madder) in MS medium supplemented with 15μM indoleacetic acid and 0.5μM kinetin. The effects of CuCl₂ and glutathione on phytochelatins (class III methallothioneins) induction and the production of anthraquinone pigments were studied. Addition of Cu ions to MS medium induced phytochelatins and affected the production of anthraquinone pigments. The addition of glutathione which did not induce phytochelatins by itself, augmented the content of phytochelatins induced by Cu ions at an early stage of culture. The addition of glutathione alone resulted in a marked increase in production of anthraquinone pigments, particularly lucidin-3-O-primeveroside.

View full abstract