Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen

Protoporphyrinogen oxidase (Protox), the penultimate step enzyme of the branch point for the biosynthetic pathway of Chl and hemes, is the target site of action of diphenyl ether (DPE) herbicides. However, Bacillus subtilis Protox is known to be resistant to the herbicides. In order to develop the herbicide-resistant plants, the transgenic rice plants were generated via expression of B. subtilis Protox gene under ubiquitin promoter targeted to the cytoplasm or to the plastid using Agrobacterium-mediated gene transformation. The integration and expression of the transgene were investigated at T0 generation by DNA and RNA blots. Most transgenic rice plants revealed one copy transgene insertion into the rice genome, but some with 3 copies. The expression levels of B. subtilis Protox mRNA appeared to correlate with the copy number. Furthermore, the plastidal transgenic lines exhibited much higher expression of the Protox mRNA than the cytoplasmic transgenic lines. The transgenic plants expressing the B. subtilis Protox gene at T0 generation were found to be resistant to oxyfluorfen when judged by cellular damage with respect to cellular leakage, Chl loss, and lipid peroxidation. The transgenic rice plants targeted to the plastid exhibited higher resistance to the herbicide than the transgenic plants targeted to the cytoplasm. In addition, possible resistance mechanisms in the transgenic plants to DPE herbicides are discussed.     

Transgenic rice plants expressing a trypsin inhibitor are resistant against rice stem borers, Chilo suppressalis

A synthetic gene, mwti1b, coding for a winged bean trypsin inhibitor WTI-1B, has been introduced and expressed in rice plants, Oryza sativa. Protein extracts from transgenic rice plants expressing the trypsin inhibitor inhibited the gut proteases of larvae of the serious insect pest, the rice stem borer, Chilo suppressalis (Lepidoptera: Pyralidae) in vitro. The growth of larvae reared on transgenic rice plants expressing WTI-1B at more than 1 ng/10 g total protein was significantly retarded compared to that on non-transgenic control plants.     

Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene

To analyze the physiological role of dehydroascorbate reductase (DHAR, EC 1.8.5.1) catalyzing the reduction of DHA to ascorbate in environmental stress adaptation, T1 transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants expressing a human DHAR gene in chloroplasts were biochemically characterized and tested for responses to various stresses. Fully expanded leaves of transgenic plants had about 2.29 times higher DHAR activity (units/g fresh wt) than non-transgenic (NT) plants. Interestingly, transgenic plants also showed a 1.43 times higher glutathione reductase activity than NT plants. As a result, the ratio of AsA/DHA was changed from 0.21 to 0.48, even though total ascorbate content was not significantly changed. When tobacco leaf discs were subjected to methyl viologen (MV) at 5 mumol/L and hydrogen peroxide (H2O2) at 200 mmol/L, transgenic plants showed about a 40% and 25% reduction in membrane damage relative to NT plants, respectively. Furthermore, transgenic seedlings showed enhanced tolerance to low temperature (15 degrees C) and NaCl (100 mmol/L) compared to NT plants. These results suggest that a human derived DHAR properly works for the protection against oxidative stress in plants.     

Transgenic tomato plants overexpressing chloroplastic monodehydroascorbate reductase are resistant to salt- and PEG-induced osmotic stress

RNA gel hybridization showed that the expression of monodehydroascorbate reductase (MDHAR) in the wild type (WT) tomato was decreased firstly and then increased under salt- and polyethylene glycol (PEG)-induced osmotic stress, and the maximum level was observed after treatment for 12 h. WT, sense transgenic and antisense transgenic tomato plants were used to analyze the antioxidative ability to cope with osmotic stresses. After salt stress, the fresh mass (FM) and height of sense transgenic lines were greater than those of antisense lines and WT plants. Under salt and PEG treatments, sense transgenic plants showed a lower level of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), a higher net photosynthetic rate (P _N), and the maximal photochemical efficiency of PSII (F_v/F_m) compared with WT and antisense transgenic plants. Moreover, sense lines maintained higher ascorbate peroxidase (APX) activity than WT and antisense plants under salt- and PEG-induced osmotic stress. These results indicate that chloroplastic MDHAR plays an important role in alleviating photoinhibition of PSII by elevating ascorbate (AsA) level under salt- and PEG-induced osmotic stress.     

Transgenic tobacco plants expressing a rice cysteine synthase gene are tolerant to toxic levels of cadmium

Plants tolerate heavy metals through sequestration with cysteine-rich peptides, phytochelatins. In this reaction, the rate limiting step is considered to be the supply of cysteine, which is synthesized by cysteine synthase (CS, EC 4.2.99.8) from hydrogen sulfide andO-acetylserine. In this study, we transformed tobacco (Nicotiana tabacum) plants withRCS1, a cytosolic cysteine synthase gene of rice (Oryza sativa), and examined their sensitivity to cadmium. The transgenic plants had up to 3-fold higher activity of cysteine synthase than wild-type plants. Upon exposure to cadmium, they exhibited obvious tolerance with much greater growth than wild-type plants. The level of phytochelatins in shoots was higher in transgenic than in wild-type plants after cadmium treatment, suggesting that cadmium was actively trapped by phytochelatins. However, the cadmium concentration per g fresh weight of whole transgenic plants was 20 percnt; lower than that of wild-type plants, suggesting cadmium to be either actively excreted or diluted by fast growth. Genetic analysis of progenies clearly showed segregation of cadmium tolerance, indicating that the trait resulted from the introduced gene. These results suggest that introduction of a cysteine synthase gene into tobacco plants resulted not only in high level production of sulfur-containing compounds that detoxify cadmium, but also in active elimination of cadmium toxicity from plant bodies.     

Transgenic fertile japonica rice plants expressing a modified cryIA(b) gene resistant to yellow stem borer

The japonica rice variety Taipei 309 was cotransformed by particle bombardment of immature embryo-derived embryogenic calli with a modified δ-endotoxin gene cryIA(b) of Bacillus thuringiensis (Bt) under the control of the rice Actin1 promoter, and the hygromycin resistance gene, hph driven by the CaMV35S promoter. Selected transgenic rice plants showed enhanced insecticidal activity against yellow stem borer (Scirpophaga incertulas), with mortality rates reaching up to 100% in a bioassay with cut stems. Introduction and expression of the Actin1 promoter-Bt gene into rice provides japonica rice germplasm resistant to insect attack. Received: 21 March 1997 / Revision received: 23 June 1997 / Accepted: 5 July 1997     

Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses

Transgenic tobacco plants expressing the coat protein (CP) gene of tobacco mosaic virus were tested for resistance against infection by five other tobamoviruses sharing 45-82% homology in CP amino acid sequence with the CP of tobacco mosaic virus. The transgenic plants (CP+) showed significant delays in systemic disease development after inoculation with tomato mosaic virus or tobacco mild green mosaic virus compared to the control (CP-) plants, but showed no resistance against infection by ribgrass mosaic virus. On a transgenic local lesion host, the CP+ plants showed greatly reduced numbers of necrotic lesions compared to the CP- plants after inoculation with tomato mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, and Odontoglossum ringspot virus but not ribgrass mosaic virus. The implications of these results are discussed in relation to the possible mechanism(s) of CP-mediated protection.     

Transgenic rice plants ectopically expressing AtBAK1 are semi-dwarfed and hypersensitive to 24-epibrassinolide

Brassinosteroids (BRs) are endogenous plant hormones essential for plant growth and development. Brassinosteroid insensitive1 (BRI1)-assocaiated receptor kinase (BAK1) is one of the key components in the BR signal transduction pathway due to its direct association with the BR receptor, BRI1. Although BRI1 and its orthologs have been identified from both dicotyledonous and monocotyledonous plants, less is known about BAK1 and its orthologs in higher plants other than Arabidopsis. This article provides the first piece of evidence that AtBAK1 can greatly affect growth and development of rice plants when ectopically expressed, suggesting that rice may share similar BR perception mechanism via BRI1/BAK1 complex. Interestingly, transgenic rice plants displayed semi-dwarfism and shortened primary roots. Physiological analysis and cell morphology assay demonstrated that the observed phenotypes in transgenic plants were presumably caused by hypersensitivity to endogenous levels of BRs, different from BR insensitive and deficient rice mutants. Consistently, several known BR inducible genes were also upregulated in transgenic rice plants, further suggesting that BAK1 was able to affect BR signaling in rice. On the other hand, the transgenic plants generated by overproducing AtBAK1 may potentially have agricultural applications because the dwarfed phenotype is generally resistant to lodging, while the fertility remains unaffected.     

Transgenic Arabidopsis plants expressing Agrobacterium tumefaciens VirD2 protein are less susceptible to Agrobacterium transformation

Agrobacterium tumefaciens causes crown gall disease on many plant species and can result in considerable economic losses. Here we report a new strategy to control crown gall disease by over-expressing Agrobacterium tumefaciens VirD2 protein in plants. Transgenic Arabidopsis plants over-expressing virD2 from constitutive or wound-inducible promoters are less susceptible to Agrobacterium -mediated transformation. Additionally, the transient introduction of an A. tumefaciens virD2 gene in tobacco BY-2 cells reduces subsequent Agrobacterium -mediated transformation.     

Transgenic sweetpotato plants expressing an <Emphasis Type="Italic">LOS</Emphasis>5 gene are tolerant to salt stress

Transgenic sweetpotato (cv. Lizixiang) plants exhibiting enhanced salt tolerance were developed using LOW OSMOTIC STRESS 5 (LOS5) with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbors the pCAMBIA1300 binary vector with the LOS5 and hygromycin phosphotransferase II (hptII) genes. Selection culture was conducted using 25 mg l⁻¹ hygromycin. A total of 26 plants were produced from the inoculated 200 cell aggregates of Lizixiang via somatic embryogenesis. PCR analysis showed that 23 of the 26 regenerated plants were transgenic plants. All of the transgenic plants exhibited higher salt tolerance compared to the untransformed control plants by in vitro assay for salt tolerance with 86 mM NaCl. When plants were exposed to 86 mM NaCl, 16 transgenic plants had significantly higher levels of superoxide dismutase (SOD), proline, and abscisic acid (ABA) and significantly lower malonaldehyde (MDA) contents than those in untransformed control plants. Salt tolerance of these 16 plants was further evaluated with Hoagland solution containing 86 mM NaCl in a greenhouse. Four of the sixteen had significantly better growth and rooting ability than the remaining 12 plants and control plants. Stable integration of the LOS5 gene into the genome of the 4 salt-tolerant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated LOS5 gene ranged from 1 to 3. High level of LOS5 gene expression in the 4 salt-tolerant transgenic plants was demonstrated by real-time quantitative PCR analysis. This study provides an important approach for improving salt tolerance of sweetpotato.     

Transgenic tobacco plants expressing the Arabidopsis thaliana nitrilase II enzyme

Nitrilase (E.C. 3.5.5.1) cloned from Arabidopsis thaliana converts indole-3-acetonitrile to the plant growth hormone, indole-3-acetic acid in vitro. To probe the capacity of this enzyme under physiological conditions in vivo, the cDNA PM255, encoding nitrilase II, was stably integrated into the genome of Nicotiana tabacum by direct protoplast transformation under the control of the CaMV-35S promotor. The regenerated plants appeared phenotypically normal. Nitrilase II was expressed, based on the occurrence of its mRNA and polypeptide. The enzyme was catalytically active, when extracted from leaf tissue of transgenic plants (specific activity: 25 fkat mg^?1 protein with indole3-acetonitrile as substrate). This level of activity was lower than that found in A. thaliana, and this was deemed essential for the in vivo analysis. Leaf tissue from the transgenic plants converted 1-[¹³C]-indole-3-acetonitrile to 1-[¹³C]-indole-3-acetic acid in vivo as determined by HPLC/ GC-MS analysis. Untransformed tobacco was unable to catalyze this reaction. When transgenic seeds were grown on medium in the absence of indole-3-acetonitrile, germination and seedling growth appeared normal. In the presence of micromolar levels of exogenous indole-3-acetonitrile, a strong auxin-overproducing phenotype developed resulting in increased lateral root formation (at 10 µM indole-3-acetonitrile) or stunted shoot growth, excessive lateral root initiation, inhibition of root out-growth and callus formation at the root/shoot interface (at 100 µM indole-3-acetonitrile). Collectively, these data prove the ability of nitrilase II to convert low micromolar levels of indole-3-acetonitrile to indole-3-acetic acid in vivo, even when expressed at subphysiological levels thereby conferring a high-auxin phenotype upon transgenic plants. Thus, the A. thaliana nitrilase activity, which exceeds that of the transgenic plants, would be sufficient to meet the requirements for auxin biosynthesis in vivo.     

Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut

The corn smut fungus, Ustilago maydis, is a global pathogen responsible for extensive agricultural losses. Control of corn smut using traditional breeding has met with limited success because natural resistance to U. maydis is organ specific and involves numerous maize genes. Here, we present a transgenic approach by constitutively expressing the Totivirus antifungal protein KP4, in maize. Transgenic maize plants expressed high levels of KP4 with no apparent negative impact on plant development and displayed robust resistance to U. maydis challenges to both the stem and ear tissues in the greenhouse. More broadly, these results demonstrate that a high level of organ independent fungal resistance can be afforded by transgenic expression of this family of antifungal proteins.     

Transgenic Arabidopsis expressing osmolyte glycine betaine synthesizing enzymes from halophilic methanogen promote tolerance to drought and salt stress

Gene duplication events exert key functions on gene innovations during the evolution of the eukaryotic genomes. A large portion of the total gene content in plants arose from tandem duplications events, which often result in paralog genes with high sequence identity. Ubiquitin ligases or E3 enzymes are components of the ubiquitin proteasome system that function during the transfer of the ubiquitin molecule to the substrate. In plants, several E3s have expanded in their genomes as multigene families. To gain insight into the consequences of gene duplications on the expansion and diversification of E3s, we examined the evolutionary basis of a cluster of six genes, duplC-ATLs, which arose from segmental and tandem duplication events in Brassicaceae. The assessment of the expression suggested two patterns that are supported by lineage. While retention of expression domains was observed, an apparent absence or reduction of expression was also inferred. We found that two duplC-ATL genes underwent pseudogenization and that, in one case, gene expression is probably regained. Our findings provide insights into the evolution of gene families in plants, defining key events on the expansion of the Arabidopsis Tóxicos en Levadura family of E3 ligases.     

Transgenic maize plants expressing a fungal phytase gene

Maize seeds are the major ingredient of commercial pig and poultry feed. Phosphorus in maize seeds exists predominantly in the form of phytate. Phytate phosphorus is not available to monogastric animals and phosphate supplementation is required for optimal animal growth. Undigested phytate in animal manure is considered a major source of phosphorus pollution to the environment from agricultural production. Microbial phytase produced by fermentation as a feed additive is widely used to manage the nutritional and environmental problems caused by phytate, but the approach is associated with production costs for the enzyme and requirement of special cares in feed processing and diet formulation. An alternative approach would be to produce plant seeds that contain high phytase activities. We have over-expressed Aspergillus niger phyA2 gene in maize seeds using a construct driven by the maize embryo-specific globulin-1 promoter. Low-copy-number transgenic lines with simple integration patterns were identified. Western-blot analysis showed that the maize-expressed phytase protein was smaller than that expressed in yeast, apparently due to different glycosylation. Phytase activity in transgenic maize seeds reached approximately 2,200 units per kg seed, about a 50-fold increase compared to non-transgenic maize seeds. The phytase expression was stable across four generations. The transgenic seeds germinated normally. Our results show that the phytase expression lines can be used for development of new maize hybrids to improve phosphorus availability and reduce the impact of animal production on the environment.     

Transgenic crop plants expressing synthetic cry9Aa gene are protected against insect damage

A synthetic gene sequence of cry9Aa was made to achieve high expression levels in a plant cell. Tobacco, potato, cauliflower and turnip rape plants were transformed with this synthetic gene driven by the double 35S promoter using Agrobacterium tumefaciens LBA4404. The presence and expression of the synthetic cry9Aa gene was evaluated in Southern, Northern and Western analysis and with insect bioassays. The expression of the gene in tobacco plants reached a level of 5 pg of mRNA per 1 μg of total RNA and 0.3% of soluble protein or 1.4 μg of Cry9Aa protein per 1 g of leaf material. The expression level in the other species was three to ten times lower. Tobacco plants were also transformed with a truncated native cry9Aa gene construct and with a translational fusion construct of the truncated native cry9Aa and the uidA (GUS) gene sequence. The constructs were transformed in tobacco plants under the control of the same promoter as the synthetic cry9Aa. The expression level of the native cry9Aa gene constructs ranged from 0.03 to 1 pg of cry9Aa mRNA per 1 μg of total RNA. The protein was undetectable in Western analysis. In comparison to the native constructs the expression level of the synthetic cry9Aa gene was five to ten times higher at the mRNA level and at least 50 times higher at the translational level. Bioassays against Plutella xylostella performed with transgenic cauliflower showed high insecticidal activity of the plants expressing the synthetic cry9Aa gene.     

Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens

 The rice chitinase gene (RCC2), classified as class I chitinase, was introduced into the somatic embryos of grapevine (Vitis vinifera L. cv. Neo Muscut) by Agrobacterium infection. After co-cultivation with Agrobacterium, somatic embryos were transferred onto Murashige and Skoog hormone-free medium supplemented with 50 mg/l kanamycin. Transformed secondary or tertiary embryos were selected, and then more than 20 transgenic plantlets were recovered. Two transformants showed enhanced resistance against powdery mildew caused by Uncinula necator. Few disease symptoms were observed on leaves of these transformants compared with those of the non-transformant, although browning and necrotic symptoms, which seemed to constitute a hypersensitive reaction, were observed. Scanning electron microscopic observation revealed that conidial germination, mycelial growth and conidial formation were suppressed on the leaf surface of the transformant. The transgenic grapevines obtained also exhibited slight resistance against Elisinoe ampelina inducing anthracnose, resulting in a reduction in disease lesions. The relationship between the expression of the foreign chitinase gene and the disease resistance is discussed. Received: 5 April 1999 / Revision received: 13 September 1999 / Accepted: 6 October 1999     

Characterization of sunlight-grown transgenic rice plants expressing Arabidopsis phytochrome A

Transgenic rice (Oryza sativa) overexpressing Arabidopsis phytochrome A (phyA) was cultivated up to the T₃ generation in paddy to elucidate the role of phyA in determining the plant architecture and the productivity of sunlight-grown rice plants. PhyA is light-labile and controls plant growth in response to the far-red light-dependent high-irradiance response as well as the very low fluence response. The Arabidopsis phyA gene linked to the rice rbcS promoter was transformed into embryogenic rice calli, and the calli were regenerated to whole plants. Compared to wild-type seedlings, the rbcS::PHYA transgenic seedlings contained more phyA when grown in the dark, and at least 10-fold more phyA when exposed to white light. When grown in paddy, the phyA transgenic plants in general exhibited reduced plant height (dwarfing), larger grain size, higher chlorophyll content, smaller tiller number, and low grain fertility compared to wild-type plants. The heading stage was not significantly changed. However, it is likely that a certain level of phyA is a prerequisite for induction of such changes. It is suggested that phyA overproduction in rice could be a useful tool to improve rice grain productivity by the larger grain size that increases grain yield and the dwarfing that tolerates lodging-associated damage.     

Transgenic tobacco plants expressing the bacterial mc gene resist virus infection

A bacterial rnc gene coding for a double-stranded RNA-dependent RNase III endoribonuclease and a mutant, rnc70, were expressed in tobacco plants. The RNase III protein produced in the transgenic plants was the same size as the bacterial protein. Expression of the wild-type gene could cause stunting in some plant lines, but not in others. Expression of the mutant protein did not affect normal growth and development of the transgenic plants. Transgenic plants of the R1 and R2 generations, expressing the wild type, as well as a mutant protein, were resistant to infection by three disparate RNA plant viruses with a divided genome but not against two viruses with a single-stranded RNA genome. Introduction of the rnc gene in crop plants may provide resistance to economically important virus diseases.     

Transgenic rice plants expressing a novel antifreeze glycopeptide possess resistance to cold and disease

Freezing injury and disease are both restrictive factors in crop production. In order to improve the tolerance ability to these stresses, a better way is to carry out genetic engineering by transferring dualfunctional genes. A predicted rice antifreeze glycopeptide gene was purposefully selected from rice blast-induced cDNA library. Northern blot demonstrated that the gene is expressed not only in blast-infected rice leaves, but also in low temperature-treated rice. In addition, the expressed protein in Escherichia coli exhibits strong antifreeze activities. The gene was overexpressed in rice plants transformed via Agrobacterium tumefacient EHA105. Overall 112 T0 transformants were obtained in this research. Cold tolerance and disease resistance of T1 transformants were, respectively, investigated. The results showed that plants containing overexpressed transgene can withstand -1 degrees C for 24 h without severe chilling injury after thawed, and that disease symptoms of the parallel transformants are highly reduced in response to blast infection, when compared with controls. The relationship of the gene and several pathogenesis-related protein genes to be chosen was analyzed and discussed. All these results confirmed the dual role of the cloned gene, and implied that genetic engineering using this kind of gene is a promising method to reduce biotic and abiotic stresses.