The chloroplast genome of higher plants contains eleven ndh genes encoding homologs of mitochondrial complex I subunits. The enigmatic occurrence of the respiratory complex in chloroplasts suggests the possibility that NAD (P) H dehydrogenase catalyzes cyclic electron flow around photosystem I and/or chlororespiratory electron flow, as it does in cyanobacteria. Establishment of a chloroplast transformation technique in tobacco facilitated the reverse genetic approach. Although some conclusions are still matters of controversy, it is sure that the gene product, NDH, functions to donate electrons to plastoquinone. Although NDH is dispensable under mild growth conditions, ndhB disruptant is more sensitive to high light stress, suggesting that NDH functions in minor compensation for the electron flow to cope with changes in environmental conditions. Here, we review the physiological function of NDH in chloroplasts, including important subjects that are still unclear.

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Somatic embryogenesis in carrot (Daucus carota L.) is strongly inhibited by certain factors that accumulate in the medium of cultures with high embryogenic carrot cell density. We previously identified p-hydroxybenzyl alcohol as one of these inhibitory factors. In this study, we performed a detailed analysis of the effects of p-hydroxybenzyl alcohol on somatic embryogenesis. p-Hydroxybenzyl alcohol strongly inhibited the formation of somatic embryos by suppressing cell division. The inhibitory effect on the development of globular embryos was stronger than that on the development of heart-shaped and torpedo-shaped embryos. In contrast, p-hydroxybenzyl alcohol had no effect on the proliferation of both undifferentiated embryogenic cells or non-embryogenic cells. These results indicate that p-hydroxybenzyl alcohol inhibits the division of cells in a way that is specific to the early stage of somatic embryogenesis and that plays an important role in the formation of somatic embryos.

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Moricandia arvensis (L.) DC. ha bseen identified as a C3-C4 intermediate species based on its low CO₂ compensation point, low photorespiratory activity and partial degree of Kranz anatomy. However, M. arvensis does not have key enzymes for C4 photosynthesis such as phosphoenolpyruvate carboxylase (PEPC). We introduced maize C4 PEPC cDNA under the control of the cauliflower mosaic virus 35S promoter into M. arvensis by Agrobacterium-mediated gene transfer. Most of the transgenic plants accumulated large amounts of the transcripts of the introduced PEPC gene, although the amount of the PEPC protein was rather small. The amounts of the maize PEPC protein in the transgenic plants were positively correlated with the activity of PEPC. Elevated PEPC activity, twice as high as that of the wild-type control, was observed in some transgenic plants.

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Our final goal is to breed dwarf cultivars of foxglove (Digitalis purpurea L.) using Agrobacterium rhizogenes. For this aim, we examined an effective method for hairy root induction, the phytohormone condition for regeneration from the root and traits of the regenerants. In the case of using a filter paper soaked with 1/2MS liquid medium for co-cultivation of leaf pieces with the bacteria, 30-40 hairy roots per 100 pieces were expected to be induced. Higher effect was given for regeneration from the hairy root through callus in zeatin addition than with BA, kinetin and no phytohormone added. In potted plants, numerical values of plant height, number of leaves, spike length, number of florets and leaf size were lower in a strain of transformants than in non-transformants, and both types of the plants exhibited similar plant forms and leaf morphologies. These traits provide novel possibilities for the breeding of dwarf cultivars of foxglove.

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Genetically transformed plants of virus-resistant genotype of Gossypium hirsutum L. CIM-443 were generated by using Agrobacterium and particle bombardment method. An insecticidal cry1Ab gene derived from Bacillus thuringiensis was introduced in the plants. Inoculated tissues were selected on MS medium containing kanamycin. The resultant plants showed protection against Lepidopteran insect Helicoverpa armigera. Molecular analysis showed the presence of cry1Ab gene in the genome of transformed plant. Immunological analysis of the transgenic plants indicated expression of Cry1Ab protein upto 100-120ng per mg of fresh leaf weight.

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An experimental system of somatic embryogenesis at a high frequency has been established in Asparagus officinalis L. The best medium for asparagus somatic embryogenesis was MS medium. Sucrose was found to be the best carbon source. Optimum combination of phytohormones was 0.5mgl^-1 2, 4-D with 1.0mgl^-1 BA. More than 80% of the culture cells developed into somatic embryos when MS medium supplemented with 0.5mgl^-1 2, 4-D, 1.0mgl^-1 BA and 20gl-1 sucrose was used. Interestingly, heart-shaped and mature embryos with two cotyledons, which are the typical structures of dicotyledonous embryos, were observed in this system although asparagus is a monocotyledonous species. Complete plants were regenerated from the mature embryos with one or two cotyledons at a frequency over 50%. On the basis of these results, multiple pathways of somatic embryogenesis in asparagus were proposed.

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The hydraulic properties of tissue-cultured soybean (Glycine max [L.] Merr.) embryos grown on trehalose-containing culture media were investigated. Water potentials of culture media ranged from -0.13 to -0.65MPa when sucrose and/or trehalose concentrations were altered. The wall extensibility was greatly larger than hydraulic conductance, and the size of the effective turgor was much smaller than that of the growth-induced water potential, indicating that hydraulic conductance and the growth-induced water potential were predominantly regulating growth of soybean embryos under tissue culture conditions. The hydraulic conductance of soybean embryos grown on trehalose-containing media became smaller than that in sucrose-containing media. When both sucrose and trehalose were added to the culture media, the hydraulic conductance became an intermediate between only sucrose-containing and only trehalose-containing treatments. It is suggested that trehalose might reduce hydraulic conductance in soybean embryos, resulting in growth retardation of soybean embryos.

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Shikonin production on the stem of shoot cultures of Lithospermum erythrorhizon was controlled by use of various culture media and light irradiation. Microscopic analysis of the shoot cultures revealed that the red pigment formation was only observed on the stem surface and the stem hairs of shoots cultured in the dark. Cross section of the shoot stem which formed red pigment in the dark was morphologically similar to that of shoots cultured under illumination. Red pigment accumulation was strictly localized in the outer surface of epidermal cells. The localization of these pigments was similarly observed in root tissues generated from the cultured shoots as well as field-grown roots. Northern blot analysis indicated that LEDI-2 gene, which is one of the candidates for the regulatory element of shikonin biosynthesis and specifically expressed in the root system of the intact plants, was also expressed in the stem of shoot cultures when producing shikonin.

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Lily, Lilium longiflorum has been used to study meiosis for advantages of the synchronous development among sporogenous cells within an anther. In order to identify genes expressed at meiosis, we carried out random sequencing of a cDNA library which represented the mRNAs from microsporocytes of L. longiflorum at the zygotene stage of meiotic prophase. To avoid redundancy of cDNA clones and to obtain novel sequence information efficiently, we exploited a simple and versatile “self-hybridization” strategy. Single-run DNA sequence data were obtained from 418 cDNA clones. Deduced amino acid sequences of 171 cDNA clones, which comprised 41% of the selected cDNA clones, showed significant similarity to amino acid sequences registered in the database. These included cDNA sequences for previously identified meiosis-associated genes. However, 59% of the selected clones showed no significant homology to known gene products.

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A novel cDNA corresponding to a gene designated PnMADS1 and encoding a MADS-box protein was isolated from the short-day plant Pharbitis nil (Japanese morning glory) cv. Violet. Phylogenetic analysis of the amino acid sequence encoded by PnMADS1 indicated that PnMADS1 does not belong to any of the major sub-groups of MADS-box proteins. The protein exhibited some similarity to proteins in the so-called ‘orphan group’ of unclassified MADS-box proteins that are mainly expressed in vegetative organs. The most similar MADS-box genes were AGL24 and StMADS16 (53.2% and 52.3% identity at the deduced amino-acid level, respectively). We obtained evidence that PnMADS1 mRNA accumulates in both vegetative and floral meristems but not in any other vegetative organs. Our results indicate that PnMADS1 is a novel MADS-box protein in terms of both structure and pattern of expression.

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LIM18, originally identified as a lily gene specifically expressed in meiosis I of pollen mother cells, encodes a protein which is highly homologous to heat shock protein 70. The anti-LIM18 antibody raised against recombinant LIM18 protein recognized at least three proteins (68, 69 and 70kDa). However, two dimensional PAGE analysis revealed at least six different proteins recognized by the antibody, suggesting the existence of multiple HSP70-like proteins in lily microsporocytes. Immuno-fluorescence microscopy of anther tissue revealed that LIM18 immunoreactive materials are localized both in the cytosol and in the nucleus of microsporocytes. A cell fractionation experiment also suggested that LIM18 or its immunologically-related molecules is present in the nucleus. Analysis of transgenic tobacco BY-2 cells expressing LIM 18-GFP fusion protein indicated the nuclear translocation of LIM18 molecule. These results suggest that LIM18 is a meiosis-associated HSP70-like protein that may be involved in nuclear and/or chromosome events during meiosis I of microsporogenesis.

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In the detached cotyledons of carrot, Daucus carota L., somatic embryogenesis was induced by 2-week treatment with sucrose at 0.29-0.73M both in the epidermal cells (type-1) and the callus-like cells generated at the cut end of the cotyledons (type-2). Type-2 embryos were produced predominantly, after the treatment with 0.29-0.44M sucrose and type-1 embryos after treatment with 0.58-0.73M sucrose. Sucrose and glucose were more effective than mannitol and sorbitol for the induction of somatic embryogenesis. Furthermore, both type-1 and type-2 embryos were induced by sucrose and glucose, but only type-2 embryos by mannitol and sorbitol. These results suggest that sucrose does not work as a mere osmoticum.

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Zygotic embryos of Cryptomeria japonica were collected from 20 clones and cultured on half-concentration of Murashige and Skoog medium containing 10μM2, 4-dichlorophenoxyacetic acid to examine the differences in embryogenic tissue (ET) initiation among seed families. ETs were obtained in all the seed families tested, with an overall initiation rate of 45.6% (689 from 1512 explants). However, initiation rates and maintenance rates differed considerably among seed families. They varied from 7.5% to 78.5% and from 1.2% to 27.8%, respectively.

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To establish a transformation system and contribute to breeding of dwarf cultivars in Vaccaria pyramidata using Agrobacterium rhizogenes, regeneration from a hairy root and traits of the regenerant were examined. The hairy roots were induced and actively grew on phytohormone-free 1/2MS media solidified with 2% gellan gum. Shoots regenerated from the root through callus on the 1/2MS media supplemented with 1.0-2.0mgl^-1 of zeatin and with 0.5mgl^-1 of IBA and 1.0mgl^-1 of zeatin. Plant height of a potted transformant was lower than those of non-transformants, and leaves of the transformant curled. Differences in morphologies and sizes of flowers in both types of the plants were little, although the starting date of flowering was 44 days later in the transformant than non-transformants. The results throw a new light on genetic engineering and the breeding of dwarf cultivars in Vaccaria.

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A cDNA encoding putative chloroplast ω6 fatty acid desaturase was isolated from a cDNA library of marine Chlamydomonas sp. strain W-80. The mRNA level of this gene under various conditions of stress was examined by northern blotting analysis, and the transcript level was increased under a cold-stressed (4°C) condition.

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