Homologues of the flavonoid 3′,5′-hydroxylase (F3′5′H) gene, a key gene determining flower color, were obtained from a Verbena hybrida (verbena) cultivar Temari Violet, verbena cultivar Tapien Pink, and Clitoria ternatea (butterfly pea). The expression of the Temari and butterfly pea homologues in yeast confirmed that they encoded F3′5′H. The two genes under the control of an enhanced cauliflower mosaic virus 35S promoter were introduced into verbena Temari Sakura. Some of the transgenic verbena plants had elevated delphinidin contents and flower color altered toward violet. Interestingly, the butterfly pea F3′5′H gene yielded more delphinidin and gave clearer flower color change than the verbena Temari gene in the transgenic verbena plants. The results indicate that the choice of the gene source should be considered to obtain strong phenotypic changes, even if the genes encode the same enzymatic activity. We also cloned some flavonoid biosynthetic genes from verbenas. The potential usefulness of verbena in the phytomonitoring of environmental pollutants is also discussed.
Post-transcriptional gene silencing, such as antisense suppression, sense suppression (or cosuppression), and RNAi, is often used to down-regulate a target gene in transgenic plants. Novel flower color is industrially important; furthermore, flower color is a convenient tool to monitor the stability of such silencing. Previously, we obtained white torenia through sense suppression of chalcone synthase or dihydroflavonol 4-reductase (Suzuki et al. 2000). However, their phenotypes were not stable. In this study, we suppressed the anthocyanidin synthase gene using three methods in transgenic torenia. About half of the transgenic torenia plants gave white flowers by RNAi suppression of the gene, while antisense and sense suppression yielded a few and no white flowers, respectively. The white flower color obtained by RNAi has been stable for three years in a greenhouse. This study shows the usefulness of RNAi to suppress a target gene.
Nierembergia sp., a popular floricultural species, only has violet and white flower color and lacks pink to red. To elucidate the reason, we analyzed its flavonoids. Its major anthocyanin was determined to be delphinidin 3-O-(6-O-(4-O-(4-O-(6-O-caffeoyl-β-D-glucopyranosyl)-p-coumaroyl)-α-L-rhamnosyl)-β-D-glucopyranosyl)-5-O-β-D-glucopyranoside. The petals rarely contained cyanidin and pelargonidin, and they contained more flavonols than anthocyanins. We also characterized the biosynthetic pathway by cloning the cDNAs encoding enzymes involved in the flavonoid biosynthesis pathway: chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), dihydroflavonol 4-reductase (DFR), flavonol synthase (FLS), and UDP-rhamnose: anthocyanidin 3-glucoside rhamnosyltransferase (3RT). Northern blot analysis revealed that the expressions of CHS, F3′5′H, DFR, and 3RT genes were coordinately regulated in parallel with anthocyanin accumulation in the petals, indicating that anthocyanin biosynthesis is transcriptionally regulated; on the other hand, the transcripts of the F3′H gene were rarely detected. Antisense suppression of the F3′5′H gene decreased the amount of F3′5′H transcripts and that of delphinidin. It was noteworthy that the color of the transgenic flower changed from violet to white rather than to reddish, which was the expectation.
To produce white-flowered gentian plants, we attempted to suppress the chalcone synthase (chs) gene by Agrobacterium-mediated transformation. A binary vector, pSMABcCHS, harboring antisense cDNA of chs isolated from the gentian Gentiana triflora cv. Maciry under control of the CaMV35S promoter was transformed into an interspecific hybrid gentian (cv. Albireo; G. scabra×G. triflora). The vector also contained the bar gene as a selectable marker. Three out of seventeen transgenic plants showed completely white flowers with 10 to 25% reduced anthocyanin content compared with the wild-type. Molecular analyses confirmed integration of the foreign genes and suppression of chs mRNA accumulation in their petals. Application of commercial herbicide including bialaphos showed that the transformants were strongly resistant. Segregation of the white-flowered and herbicide-resistant traits was tested using a T1 progeny obtained from crossing with a blue-flowered parental line. The results clearly showed that these two traits are inherited dominantly as linked traits in the T1 progeny, suggesting that these transgenic plants are useful resources for production of white-flowered gentians. These results also demonstrated for the first time the inheritance of foreign genes and genetic modification of flower color in transgenic gentian plants.
We successfully produced dwarf potted gentian using wild-type Agrobacterium rhizogenes A4 strain (ATCC43057) harboring an agropine type plasmid (pRiA4). A number of hairy roots were induced by direct inoculation with A. rhizogenes to the stem and leaf tissues in vitro. Adventitious shoots were regenerated from the hairy roots on Murashige and Skoog medium containing 10 mg l−1 N-phenyl-N′-(1,2,3-thiadiazol-5-yl) urea, 0.1 or 1.0 mg l−1 1-naphthaleneacetic acid, and 3 g l−1 gellan gum. Regenerated plants were then cultured in vitro for at least six months and tested for the absence of viable A. rhizogenes within their tissues. After receiving authorization from the Ministry of Agriculture, Forestry and Fisheries, Japan, the plants were acclimated and grown in a greenhouse and/or field. Opine synthesis and rolC gene expression were analyzed to demonstrate successful introduction and expression of the transferred DNA. In total, 122 lines of variant types of dwarf plants with blue, white, and pink flowers were obtained, which might be useful for molecular breeding of a series of dwarf potted gentian cultivars.
We reported previously that the genetically modified rice lines expressing OASA1D, a feedback-insensitive anthranilate synthase (AS) α-subunit gene of rice, showed remarkable increase of free tryptophan (Trp) in calli. In the present study, we investigated transformation of &ldqou;Kusahonami” (Oryza sativa L.), a rice variety for animal feed with this gene. In order to avoid the co-integration of vector backbone sequences and taking into account the fact that OASA1D gene exert resistance to 5-methyltryptophan (5MT), gene cassette vector consisting of a promoter, OASA1D cDNA and terminator was constructed and introduced directly to rice calli by use of potassium titanate whisker-supersonic method. Transgenicity of regenerated plants and the copy number of the transgene in the genome were analyzed by Southern blot. Fertile transgenic plants carrying low copy number of transgene and producing high level of free Trp in leaves and seeds were obtained. Genetic stability of the transgene has been demonstrated. This cultivar is a common variety for animal feed, these transgenic plants may be useful to establish an actual variety that is nutritionally improved.
Loading of cytosolic nitrite into the chloroplast in nitrate assimilation of rice was modified by introducing a gene for chloroplastic nitrite transport from cucumber. Rice (Oryza sativa L. japonica cv. Nipponbare) was transformed with a cDNA (CsNitr1-L) that encodes a nitrite transporter of cucumber under the control of the CaMV35S promoter. CsNitr1-L transgenic rice grown by hydroponics with nitrate supplied as the sole nitrogen source grew as well as plants grown with ammonia. In contrast the growth of the untransformed or mock-transformed control rice was retarded on a nitrate-containing medium by about 60% compared to plants grown with ammonia. Nitrite was not beneficial to the growth of the untransformed or mock-transformed control rice but sustained that of CsNitr1-L transgenic rice. Low steady-state concentration of nitrite in leaves as well as delayed non-photochemical quenching of chlorophyll fluorescence in CsNitr1-L transgenic rice seems indicative of higher nitrite use in chloroplasts.
Transgenic plants that produce anti-insect substances are vital in improving crop yields and in reducing the environmental risks of chemical insecticides. Enhancin is a metalloprotease produced in occlusion bodies of the Trichoplusia ni granulovirus (TnGV). It is a key substance that enhances infection of the nucleopolyhedrovirus in lepidopteran insects. Rice (Oryza sativa L. cv. Nipponbare) protoplasts were cotransformed with pREXΦVEF and pLTHyg, which respectively bear the chimeric enhancin gene and the hygromycin-resistance gene. Hygromycin-resistant regeneration plants were examined by genomic polymerase chain reaction and genomic Southern and northern blotting analyses to confirm the presence and expression of the enhancin gene. Fourteen transgenic plant lines harboring the enhancin gene were obtained, and stable inheritance and expression of the enhancin gene were confirmed in the second, third, and fourth plant generations. Feeding Spodoptera exigua larvae leaves of enhancin-expressing rice plants in the presence of S. exigua nucleopolyhedrovirus occlusion bodies enhanced infection of the virus. Further, the development of Pseudaletia separata, S. exigua, and S. litura, none of which are host insects of TnGV, was inhibited when these larvae were fed enhancin-expressing rice leaves. This indicates that expression of the enhancin gene confers resistance to lepidopteran insect pests in rice.
Overexpression of an oat thionin gene (Asthi1) confers enhanced disease resistance of rice (a cultivar “Chiyohonami”) against seed-transmitted phytopathogenic bacteria. The isolated paddy field test for evaluation of agronomic traits using T4 transgenic rice plants which are homozygous with a high level of Asthi1 protein has shown that transgenic rice possesses, in addition to enhanced anti-bacterial resistance, slight different characteristics compared with the original cultivar such as slight decrease in plant height, grain yield and simultaneity in heading behavior in addition to 2 days earlier in heading date. To address whether this difference was caused by ectopic high level expression of the transgene, T4 plants were back-crossed with wild-type and selfed, and the progenies were analyzed. Almost all characteristics observed in the field were found to be genetically segregated. While only a limited number of plants were used, the present findings indicate that the altered characteristics found in transgenic plants were not attributable to integration of Asthi1 transgene or its high expression itself, but instead to possible spontaneous mutations which may have occurred during in vitro culture for Agrobacterium-mediated transformation.
To engineer insect-resistant Solanum plants, a peroxidase C2 gene (prxC2) from horseradish was introduced into Solanum integrifolium Poir. cv. Hiranasu. We produced 77 transgenic Hiranasu plants. Peroxidase expression was confirmed at the transcriptional and translational levels by northern blot analysis and by measuring peroxidase activity, respectively. Feeding test results show that transformant no. 180 is more resistant to corn earworm (Heliothis armigera) and common cutworm (Spodoptera litura) than the wild-type plants. We also found a correlation between insect resistance and lignin content in the transgenic plants. In particular, the lignin content of transformant no. 180 was 30% higher than that of wild-type plants. These results further confirm that peroxidase is functionally expressed in transgenic plants and suggest that the increased lignin content is a basis for the insect resistance in transgenic Hiranasu plants.
With the purpose of enhancing environmental stress tolerance of sweet potato (Ipomoea batatas, cv. Kokei 14), we transformed this plant with spermidine synthase genes derived from Cucurbita ficifolia (FSPD1). The FSPD1-transgenic plants showed twice as high spermidine content as the wild type (WT) counterpart in both leaves and storage roots. One of the most characteristic features of the transgenic plants was the increase in the number of storage roots formed under both non-stress and stressful environments. Salt and drought stresses suppressed storage root growth, but the transgenic plants were less affected producing significantly larger mass of storage roots and starches than WT plants under either stress. The transgenic plants also showed increased tolerance to chilling- and heat-mediated damage to photosynthesis compared to the WT plants. Thus, sweet potato was made more tolerant to environmental stresses through introduction of the FSPD1 genes. This improved tolerance may involve enhanced oxidative stress tolerance at least partially because the transgenic plants were more tolerant to paraquat, a potent oxidative stress inducer, than the WT plants. From these results, the FSPD1 gene is considered useful for gene transfer technology aiming at improving environmental stress tolerance of sweet potato.
In the storage roots of sweetpotato (Ipomoea batatas (L.) Lam. cv. Kokei 14), 10 to 20% of the starch is essentially unbranched linear amylose and the other major component is branched amylopectin. The starch branching enzymes, which are responsible for production of amylopectin to form α-1,6-linkages in the glucan can be divided into two classes, class A (e.g. potato and maize SBEII, pea SBEI) and class B (e.g. potato and maize SBEI, pea SBEII). On the bases of the registered cDNA of sweetpotato SBEII (IbSBEII) encoding class A branching enzyme, we constructed double-stranded RNA (dsRNA) interference vectors and introduced them into sweetpotato genome via Agrobacterium-mediated gene transformation. We obtained eight independent transgenic plants by using two kinds of RNA interference (RNAi) constructs, encoding GBSSI 1st intron-spliced RNA or a GUS fragment-spliced RNA, respectively. All transgenic plants were confirmed not to express IbSBEII by RT-PCR and to have the starch with a higher amylose content than the non-transgenic control (up to 25% compared to 10% in the control). Both constructs induced the same level of silencing of IbSBEII in all transgenic plants. The morphological characters showed no significant differences between the transgenic and control plants. Starch yield of transgenic tubers was slightly lower than that of non-transgenic tubers. The starch granules of the transgenic plants were similar to those of typical sweetpotato starchs in shape and the distribution in granule size, but slightly different in grain structure.
Interferon-α (IFN-α) is a principle cytokine that plays a key regulatory role in mammalian immune systems. The recombinant DNA of IFN-α is composed of the signal sequence region for rice 10 kDa prolamin cDNA, the amino-terminal region of β-glucuronidase DNA, and the mature polypeptide region of human IFN-αDNA, and is expected to produce a biologically active form of IFN-α. This chimerical DNA for coding IFN-α under the control of a cauliflower mosaic virus 35S promoter and a 5′-intron of rice cytosolic superoxide dismutase gene (sodcC1), was transformed into dwarf rice by an Agrobacterium-mediated system. Three lines of transgenic rice plant expressing various levels of IFN-α polypeptide were finally generated. The expression level of the recombinant polypeptide in each line was analyzed by an IFN-α antiviral activity assay and enzyme immunoassay. Higher expression of IFN-α was achieved in developing seed endosperm in two of transgenic rice lines. The replacement of the native signal peptide of IFN-α with the prolamin signal peptide was effective for transporting the IFN-α polypeptide into the ER-derived protein body of developing seed endosperm. These results suggest that rice can be used to produce many biologically active mammalian proteins that accumulate in target organelles such as protein bodies.
Two genes for acetoacetyl-CoA reductase (phbB) and poly-3-hydroxybutyrate (PHB) synthase (phbC), from Ralstonia eutropha were transformed into rice and Tamarix to change their fiber characteristics by producing PHB in these plants. Expression of the genes was detected by RT-PCR. The enzyme activity of phbB was confirmed by measuring NADPH-dependent acetyl-CoA consumption. Western blot analysis was used to detect the protein product of phbC. PHB accumulated in the transformed rice to an average level of 3 mg g−1 dry weight. Similar levels were detected in the transformed Tamarix. Wood and plastic combination (WPC) boards were prepared from the transformed rice and Tamarix. Differential scanning calorimetry (DSC) analysis of rice WPC board measured a melting point (Tm) that was distinct from those of boards made of 100% PHB or PHB-blended cellulose. A unique DSC peak was present for the sample of transformed rice. A measure of pressure deformation showed a higher compression resistance in the sample made of transformed rice and the PHB-blended sample. Analysis of thermal extension showed enhanced stabilities for the PHB-blended sample and the samples made of transformed rice and Tamarix. It was also shown that transgenic blending of PHB prevented moisture absorption in samples made from rice and Tamarix. These results indicated that the accumulation of the plastic in the plants results in improved characteristics in the sample boards.
Hypocotyl sections of Rhaphiolepis umbellata (Thunb.) Makino were transformed by Agrobacterium tumefaciens bearing a binary vector pIG121-AtNiR, which contains cDNA of the nitrite reductase gene from Arabidopsis thaliana (Atni) under the control of a modified cauliflower mosaic virus 35S promoter and chimeric hygromycin phosphotransferase gene (hph). A 4% of the hypocotyl explants transfected with Agrobacterium formed hygromycin resistant adventitious shoots, and most of them rooted upon root induction treatment. The presence and expression of the introduced transgene in putative transgenic plants (12 months after transfection) were respectively confirmed by polymerase chain reaction (PCR) analysis using primers specific to Atni and by western blot analysis using anti NiR antibody. A total of 37 transgenic plant lines were obtained. Plants 33 months after the transfection were fumigated with 200±50 ppb 15NO2 under the natural light for one week in the fumigation chamber in a confined glasshouse. The amount of total nitrogen derived from NO2 (reflecting uptake of NO2) and that of Kjeldahl nitrogen derived from NO2 (reflecting assimilation of NO2) were determined using a mass spectrometer. One of the 9 transgenic plants tested was 1.6–2.0 times higher both in the uptake and assimilation of NO2 than non-transformed wild-type ones.
Hydrogen sulfide is a major environmental pollutant, highly toxic to living organisms at high concentrations. Even at low concentrations, it causes an unpleasant odor from wetlands, especially from wastewater. Plants can utilize hydrogen sulfide as a sulfur source to synthesize cysteine, which then serves as the principal substrate for synthesis of other sulfur containing compounds including glutathione and methionine. It was thus feasible to use aquatic plants, which possess high potential for sulfur assimilation, to remove hydrogen sulfide from the wetland. To this end, we have generated transgenic rice plants over-expressing cysteine synthase, a key enzyme in the sulfur assimilation pathway, and evaluated their capacity for sulfur uptake on hydrogen sulfide treatment. The obtained transgenic plants exhibited 3-fold elevated cysteine synthase activity, and incorporated more hydrogen sulfide into cysteine and glutathione than their wild type counterparts upon exposure to a high level of hydrogen sulfide. These observations suggest that over-expression of cysteine synthase in aquatic plants is a viable approach to remove hydrogen sulfide from polluted environments.
Azadirachta excelsa belongs to the family Meliaceae and is one of the most important silviculture trees in the tropics. In this study, we established a somatic embryogenesis system and succeeded in construction of transgenic lines expressing genes for Bar, GUS and GFP employing the Agrobacterium-mediated transformation technique. Somatic embryos were obtained after approximately two months of culture on Murashige-Skoog (MS) medium containing 5 μM 6-benzyladenine. Plantlets were then regenerated by transferring somatic embryos to modified 1/2 MS medium without phytohormones. We initially attempted genetic transformation using two different A. tumefaciens strains, and found only the strain LBA4404 to be active in infection to explants. The strain EHA101 was totally inactive, suggesting that a specific interaction between Agrobacterium sp. and host plant might be critical for gene transfer. The possibility is discussed of applying the methodology developed in this work to the practical propagation of tropical trees.
The evaluation of genetically modified (GM) plants under special screened greenhouse conditions is the first step of assessing the risks of pollen dispersal from GM plants. To obtain fundamental information on the effective use of special screened greenhouses, we examined the pollen grain sizes of the major plants used for transgenic studies in Japan to estimate the effective fine-mesh size for covering the openings of special screened greenhouses to reduce pollen dispersal. Second, we examined the potential of small insects as possible pollen vectors. Finally, we investigated the relationship between fine-mesh size and air temperature in a covered space using a model of a special screened greenhouse. Although using smaller mesh sizes for covering the openings was clearly more effective in reducing pollen dispersal, the smaller mesh raised the air temperature substantially and resulted in poorer growing conditions for plants. We discuss this dilemma and suggest conclusions based on our research.
Soil microbial community analysis is one of the most important elements in the environmental risk assessment of transgenic plants. Recent technical advances in this area now enable us to assess the impact of plant genotypes on soil microbial communities with rapid, simple and less biased molecular techniques than the previously used conventional microbiological methodologies. We review the use of these modern molecular techniques from the aspect of environmental assessments of transgenic plants.