Effects of a wide range of concentrations of four salts (NaCl, KCl, MgCl₂, CaCl₂) and different pHs (pH 4.5, 5.0, 5.7, 6.2) on colony formation, were investigated in protoplast culture of mangrove, Bruguiera sexangula, suspension cells. The results were compared to those obtained with protoplasts of tobacco BY-2 cells and leaf protoplasts of Populus alba. A 96-multiwell culture plate method was used and only 250 to 2500 protoplasts in 55 μl medium were sufficient to test the effect of each treatment. The effects of four salts were always found to inhibit colony formation of Populus and tobacco protoplasts at pH 5.7. In contrast, MgCl₂ and CaCl₂ stimulated further development of B. sexangula protoplasts, while KCl had an inhibitory effect. The highest number of colony formation was obtained at pH 5.7 without salt additions. However, at lower pHs, some stimulatory effects were found at low concentrations of all four salts tested. Depending on the pHs, different patterns of stimulation and inhibition of four salts were obtained in B. sexangula protoplasts.

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Phytic acid (myo-inositol hexakisphosphate) is the storage form of phosphorus in plant seeds. Reduction of phytic acid levels in seeds leads to both an increase in the availability of phosphorus and a reduction in the environmental load. Myo-inositol-1-phosphate (Ins(3)P₁) synthase (EC 5.5.1.4) catalyzes the first step in phytic acid biosynthesis. We attempted to reduce the phytic acid content of seeds through manipulation of the expression of the Ins(3)P₁ synthase gene. A rice Ins(3)P₁ synthase gene, RINO1, was transformed into rice plants in the antisense or sense orientation under the control of the CaMV 35S promoter or the RINO1 promoter. Some T₁ seeds that transformed both with RINO1 promoter::antisense RINO1 gene and with 35S promoter::sense RINO1 gene increased the amount of inorganic phosphates, as compared to those of non-transgenic plants. This result suggested a molar-equivalent decrease in phytic acid. We also examined RINO1 promoter activity using a β-glucuronidase (GUS) reporter gene. Finally, we discuss the strategy of molecular breeding to reduce phytic acid levels in seeds.

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The UDP-galactose: anthocyanin 3-O-galactosyltransferase (ACGaT) cDNA was cloned from cell-suspension cultures of Aralia cordata Thunb. (Araliaceae). The cDNA was isolated from a cDNA library using a galactosyltransferase cDNA from Vigna mungo. The cDNA contained an open reading frame encoding 452 amino acids with a calculated molecular mass of 50.0 kDa. The deduced amino acid sequence of ACGaT cDNA included a plant secondary product glycosyltransferase signature sequence and was similar to the flavonoid 3-O-glycosyltransferases from petunia and grape. The enzymatic activity of the recombinant ACGaT expressed in Escherichia coli was detected toward anthocyanins and flavonols as an aglycon and UDP-galactose as a sugar donor. The reaction product for quercetin and UDP-galactose catalyzed by the recombinant ACGaT was identified as quercetin 3-O-β-galactoside using nuclear magnetic resonance spectroscopy.

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In vitro somatic embryogenesis of Castanopsis cuspidata var. sieboldii was achieved from immature embryo-derived callus and the morphological course of events during embryo development was evaluated by scanning electron microscopy technique (SEM). Embryogenic callus could be initiated from immature embryos and optimum induction of callus was observed on Murashige and Skoog (MS) medium supplemented with 10 μM 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 1 μM benzyl‐aminopurine (BAP). Somatic embryo development was occurred on half-strength basal MS medium supplemented with 2 μM BAP, 0.25 μM naphthaleneacetic acid (NAA). Somatic embryo maturation was accomplished using 1 μM zeatin, 0.25 μM indolebutyric acid (IBA). SEM observations indicated development of an extracellular material at an early stage of differentiation and a course of differentiation leading to development of somatic embryos.

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In order to understand cell-cell interactions involved in xylem differentiation, we studied intercellular molecules in an in vitro Zinnia xylogenic culture system, where single mesophyll cells transdifferentiate into tracheary elements (TEs) and xylem parenchyma cells. We found that UV-absorbing substances accumulated predominantly in xylogenesis-inducing medium and kept increasing even after the TEs died. This accumulation was inhibited by L-α-aminooxy-β-phenylpropionic acid (AOPP), an inhibitor of phenylalanine ammonia-lyase, and also by brefeldin A, an inhibitor of vesicle transport. These results indicated that living non-TE cells, probably xylem parenchyma cells, secrete some kinds of phenylpropanoids via a vesicle transport system. Further experiment showed that inhibition of brassinosteroid biosynthesis by uniconazole suppressed TE differentiation, but not the secretion of UV-absorbing substances into the medium, implying that differentiation of xylem parenchyma cells might not be strongly affected by the depletion of endogenous brassinosteroids.

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Pod storage protein (PSP) accumulates in both developing pods and wounded leaves of common bean (Phaseolus vulgaris). Since jasmonic acid (JA) is known to be involved in wound responses, we examined hormonal regulation of PSP expression. Methyl jasmonate (MeJA)-induced expression of PSP was blocked by both salicylic acid (SA) and ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). The PSP promoter fused to β-glucuronidase (GUS) coding region was introduced into Arabidoposis thaliana using Agrobacterium infection. No MeJA-inducible GUS expression was found in the transformants. The chimeric gene was then introduced into stems of common bean using particle bombardment. Exogenously applied MeJA enhanced GUS activity, and this was prevented by both SA and ACC. Deletion analysis of the promoter indicated that the region between positions-747 and-555 included cis-regulatory elements for JA induction and ethylene suppression, and that cis-elements for SA-suppression were located in the region downstream of position-86.

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Six turnip cultivars (Brassica rapa var. rapifera) exhibited shoot regeneration ability of 0-44.0% from their hypocotyl sections. Shoot regeneration from hypocotyl sections of 5 turnip cultivars was markedly enhanced by adding AgNO₃ into a shoot regeneration medium. Transgenic turnip plants were obtained by the Agrobacterium-mediated transformation procedure incorporating AgNO₃ in the shoot regeneration medium. Transformation efficiencies (percentage of stable transformants per total sections infected) were 1.0% and 0.5% for ‘Hinonakabu’ and ‘Honbenidaimarukabu’.

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Cryopreservation of Diospyros at-150°C followed by a prefreezing treatment using dormant winter buds was investigated. Twig sections bearing one bud each were first partly dehydrated at room temperature for 3 h. Then they were subjected to a successive prefreezing at-5, -10, -15, -20 and-30°C at 24 h intervals, and finally preserved at-150°C in a deep freezer. Thawing in the air at 25°C gave the highest rate (about 70%) of in vitro shoot formation from cryopreserved buds. Shoot regeneration by bud grafting was not successful. Dormant buds appear to be a suitable material for long-term storage of some Diospyros species.

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The RPN10 subunit is a component of the 19S regulatory complex that is essential for the ubiquitin-dependent proteolytic activity of the 26S proteasome (EC 3.4.99.46). To address the functional role of RPN10, we examined phenotypic changes that occurred when the rpn10 gene was overexpressed in rice plants (Oryza sativa L cv Nipponbare). These transgenic plants showed enhanced resistance to canavanine, suggesting that the overexpression of the rpn10 gene accelerates the degradation of abnormal proteins containing canavanine instead of arginine.

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Brugmansia versicolor Lagerheim of the family Solanaceae was propagated through shoot tip culture and shoots were cold-stored in vitro at 5, 10 or 15°C under light or dark condition. All the shoots died after cold storage for 6 months at 5°C irrespective of light condition. When shoots were stored for 12 months at 15°C under light illumination, the best 100% survival rate was obtained. The plants regenerated from shoots stored for 6 or 12 months retained the ability to accumulate scopolamine as much as the control plants which were raised from shoots maintained under normal culture condition without cold-storage. These findings show that slow growth storage of in vitro B. versicolor shoots at 15°C can be used as a germplasm conservation system for short-or medium-term duration without deterioration of the ability to accumulate the secondary metabolites.

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In radish (Raphanus sativus L.) cotyledons, γ-glutamyltransferase (GGT, EC 2.3.2.2) activity was detected in both fractions extracted with grinding buffer alone (low ionic strength buffer) and the same buffer supplemented with 1 M NaCl (high ionic strength buffer). The former was referred to as soluble GGT and the latter, as bound GGT. Bound GGT was not solubilized by 1% Triton X-100. The activities of both soluble and bound GGTs were inhibited by acivicin, an inhibitor of mammalian and Escherichia coli GGTs. The activity of GGT extracted with the high ionic strength buffer disappeared when protoplasts were prepared, indicating the localization of bound GGT to cell walls. These results strongly indicate the occurrence of two forms of GGTs, soluble and bound ones. The two forms of GGTs were also found in Arabidopsis thaliana, cabbage, pea, soybean, maize and rice among the eight plants tested, in addition to radish.

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