Melon is an important fruit in the Japanese food market. Accordingly, to be a leading variety in Japan, the fruit must meet demands from growers, markets, and consumers. Melon breeders have made great efforts to incorporate such demands into breeding programs. We have developed methods for improving two important traits, shelf-life and fruit size, by genetic engineering. First, we isolated and characterized genes related to these characteristics and subsequently used the genes to develop methods for altering shelf-life and fruit size. We isolated two ethylene receptor genes, Cm-ERS1 and Cm-ETR1, which we expected to be related to shelf-life, and developed methods for altering the ethylene sensitivity of the plants. We also isolated a gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, Cm-HMGR, which we expected to be related to fruit enlargement, and developed methods for altering fruit size. These two methods will provide basic technologies for the molecular breeding of melon.
Modification of diet by means of genetic engineering methods shuld be an effective way to overcome problems derived from micronutrient deficiency. Here, we review the current status of knowledge of the micronutrient contents in native foods and matters of concern for the genetic engineering of their nutritional contents, and summarize the results of experiments in the development of high iron crops by ferritin gene transformation. This means that by increasing the storage capacity of iron in plants the potential activities of the iron uptake related mechanism can be induced and thereby create high iron content crops. Finally, we also discuss the possibility of storing more iron in ferritin and the expanded application of ferritin to further breeding other than iron storage.
The sulfur atom is an essential nutrient for living organisms because it plays a central role in protein folding, enzyme catalysis and maintenance of the redox status of cells. Microorganisms and plants can synthesize organic sulfur compounds, including cysteine, methionine and glutathione, from inorganic sulfur compounds such as sulfates. In contrast, animals utilize organic sulfur compounds that are mainly synthesized by plants. In the last decade, many genes whose products are involved in sulfate assimilation have been isolated from higher plants, and it has been revealed that there exist multiple isoforms for each step. The different properties of isoforms has been examined for sulfate transporters, which serve at the first step of sulfate assimilation, and for O-acetylserine(thiol)lyases, which catalyze the incorporation of sulfide into cysteine. Currently, however, it is not clear why plants developed multiple forms with similar catalytic properties. Mechanisms of regulation of the sulfate assimilation pathway have also attracted much attention, since metabolites such as cysteine and glutathione affect related enzymatic activity or corresponding gene expression. The precursor of cysteine, O-acetylserine, is a key compound in maintaining the balance between sulfate and nitrate assimilation in higher plants. In addition to nutrition, sulfate assimilation is important for conferring tolerance against environmental stresses including heavy metals. Genetic engineering of components of the sulfate assimilation pathway is a useful approach to generate useful plants for agriculture and phytoremediation.
A suspension culture of tobacco cells was transformed with a gene encoding barley lectin to obtain an efficient production system of lectin. Lectin excreting transgenic tobacco was constructed by transformation with barley lectin deleted of the C-terminal vacuole targeting peptide (CTPP). 2, 4-Dichlorophenoxyacetic acid (2, 4-D) was examined for its effect on the excretion of lectin at different concentrations from 0 to 5mgl-1. 2, 4-D addition increased excretion efficiency, defined as the amount of excreted lectin per cell, but reduced the growth rate. Microscopic observations showed loosening of the cell wall, which is assumed to be one of the causes of the enhanced excretion. Decrease in extracellular lectin was observed in spite of the growth of cells, which was confirmed to be caused by a degradation process in the culture medium. These results suggest that an efficient production process of lectin would include a process of simultaneously removing culture medium with high lectin content before degradation occurs and maintaining the cells at high excretion efficiency.
We have previously isolated a cDNA clone, which is similar in sequence to the Tat binding protein (TBP) of human immunodeficiency virus 1, from the anthers of Brassica rapa and designated it BrTBP. In the present study we isolated a genomic clone of BrTBP from B. rapa and found several sequence motifs conserved in various pollen-expressed genes. The 1.7-kb promoter region of BrTBP was fused to the GUS gene and introduced into tobacco and Arabidopsis thaliana. GUS expression was observed exclusively in mature pollen after anthesis in transgenic tobacco, while it was observed in tricellular pollen starting two days before anthesis in A. thaliana. Such expression patterns at the late stage of pollen development have not been reported in other pollen-specific promoters. These results indicate that the BrTBP promoter region used in this study has a unique activation pattern in tobacco and A. thaliana.
Cd-tolerant (CCd1) cells of carrot (Daucus carota) were selected from wild (CW) cells under 100μM Cd. In the CW and CCd1 cells, the maximum activity of superoxide dismutase was found 1 day and 4 days after Cd treatment, respectively. The activity of ascorbate peroxidase in the CW and CCd1 cells decreased by 80-160 and 160μM Cd, respectively. The activity of glutathione reductase in the CW cells decreased drastically by Cd; whereas, the activity in the CCd1 cells increased. The content of ascorbate in the CW cells decreased remarkably when the Cd treatment was applied, while that in the CCd1 cells also decreased at 4 days but then recovered at 8 days after the Cd treatment. We speculate that Cd tolerance in the CCd1 cells is due to the high activity of glutathione reductase and maintenance of the content of ascorbate.
Expression of carrot ECP (DcECP) genes encoding late-embryogenesis abundant proteins is embryo-specific and ABA-inducible. The expression is regulated by C-ABI3, a carrot homologue of Arabidopsis ABI3. To understand the molecular mechanisms controlling ABA-inducible and embryo-specific gene expression, we compared and analyzed the promoter regions of some ECP genes. The accumulation of DcECPs mRNA in response to ABA was not inhibited by an inhibitor of protein synthesis, cycloheximide. These results indicate that the ABA-inducible expression of the DcECPs did not require de novo protein synthesis. Sequence comparison among some ECP gene promoters revealed that the promoters contained several conserved motifs including ABRE (ABA responsive element)-like ACGT core motifs, and an Sph box (CATGCATG), which has been identified as a motif mediating gene activation of maize antocyanin regulatory gene C1. To investigate the promoter activity of DcECP31 promoter region, we carried out a deletion analysis with a transient assay system using protoplasts of embryogenic cells or with a transformation system using embryogenic cells of carrot. We found that the -250bp upstream region of the DcECP31 promoter is sufficient for embryo-specific and ABA-inducible promoter activity. Following deletion analysis of DcECP40 promoter, we clarified that the distal (-670∼-390) and proximal regions (-140∼-50), are essential for the ABA-inducible expression.
Expression of carrot embryogenic cell specific protein genes (DcECPs) that are late-embryogenesis abundant proteins is ABA-inducible and embryo-specific. The expression of some DcECPs is controlled by an embryo-specific transcription factor C-ABI3, a carrot homologue of Arabidopsis ABI3. In order to understand the molecular mechanisms regulating ABA-inducible and embryo-specific expression of DcECP genes, we carried out a detailed analysis of DcECP31 promoter with deletion analysis, base-substitution mutagenesis and electrophoretic mobility shift assay (EMSA). We identified two important elements in the promoter of DcECP31, motif X (CACACGTGGG) and motif Y (CACACGTATC), which are sufficient for the embryo-specific and ABA-inducible promoter activity. By precise EMSA analysis, it was demonstrated that a nuclear protein which has sequence specific-binding ability binds to the flanking ACGT core motifs.
The effect of various growth regulators, especially of Thidiazuron (TDZ), on seed germination and subsequent differentiation capabilities of hypocotyl, cotyledon, leaf, internode, root and petiole explants was studied in the Indian mulberry, Morus indica cultivars K2 and DD. Of the various hormones tried, thidiazuron at a concentration of 5μM, gave the highest percentage of adventitious bud formation in both the hypocotyl and cotyledon explants obtained from the seedlings germinated on 0.5μM TDZ. The young tissue was more responsive and showed up to 50% regeneration from hypocotyl and 70% from cotyledon explants in M. indica cv. K2 when cultured for 10 days on TDZ containing medium. The leaf explants produced adventitious buds after 30 days of culture on 2.5μM TDZ. The 7-day old leaf explants taken from in vitro cultured axillary buds were more efficient in terms of regeneration percentage than the leaf explants derived from the seedlings or from the plants growing in the experimental field. The regenerated shoots were sub-cultured on to Shoot Elongation Medium (SEM: MS+0.5mgl-1 BAP+0.5mgl-1 GA3+2mgl-1 AgNO3) for further growth. Healthy and vigorously growing roots were readily obtained on 1mgl-1 NAA+0.1% activated charcoal (RIM: Root Induction Medium) within three weeks of culture at a rooting percentage of 70-76%. Successfully acclimatized plantlets (95-100%), transferred to the experimental plots, are now well established in the field.
We investigated the induction of β-glucuronidase (GUS) activity in transgenic Nicotiana plumbaginifolia harboring palg2b promoter-uidA chimeric gene after light irradiation. GUS activity was increased distinctly in leaves of transgenic plants by white light irradiation and was slightly increased in transgenic green calli by blue light irradiation. The present results indicate that the g2b promoter might be useful to control the expression of a foreign gene in transgenic plants.
To investigate the use of the DLRA system in plants, expression of firefly luciferase (F-luc) and Renilla luciferase (R-luc) reporter genes in plant cells introduced by microprojectile bombardment was examined. Although the relative activity of F-luc and R-luc varied among types of plant cells, the assay system is highly sensitive and can be applicable to various plant cells.
An efficient method for regeneration of Ipomoea aquatica was established. Cotyledons with petioles produced adventitious shoots near the excised margins situated only on the distal side, which rooted within one week in a 1/2 MS solid medium without phytohormones. The frequency of adventitious shoot formation depended on the developmental stage of the seedlings. Upon transfer to hydroponics in a greenhouse, all plantlets grew to maturation.