Chlorophyll fluorescence assay for kanamycin resistance screening in transgenic plants

The applicability of a chlorophyll fluorescence assay for kanamycin (Km) resistance screening in transgenic tobacco (Nicotiana tabacum) and Arabidopsis thaliana plants was investigated. In wild-type leaves incubated in the presence of 200 mg/l Km, a decrease in maximum variable fluorescence ((Fv)m) and a significant increase in constant fluorescence (Fo) were observed. Using (Fv)m/Fo as a screening parameter, we were able to distinguish Km-treated samples from untreated samples within 4 days. This parameter was applied to Km resistance screening using tobacco plants transformed with the nptII gene via Agrobacterium. Among 74 shoots selected on medium containing 200 mg/l Km, 37 plants were scored as Km sensitive by the chlorophyll fluorescence assay. These 74 scorings proved to be accurate, as reconfirmed by (1) polymerase chain reaction amplification of the transgene, (2) enzymatic assay of neomycin phosphotransferase and (3) leaf disc assay. Using the chlorophyll fluorescence assay, we could also screen 3-week old Arabidopsis plants carrying the nptII gene. These results clearly demonstrate the reliability and efficiency of this nondestructive assay for Km resistance screening of transgenic plants. Received: 27 November 1995 / Revision received: 18 April 1997 / Accepted: 28 July 1997     

Fungal and bacterial disease resistance in transgenic plants expressing human lysozyme

The human lysozyme gene, which is assembled by the stepwise ligation of chemically synthesized oligonucleotides, was introduced into tobacco (Nicotiana tabacum cv `SR1') by the Agrobacterium-mediated method. The introduced human lysozyme gene was highly expressed under the control of the cauliflower mosaic virus 35S promoter, and the gene product accumulated in the transgenic tobacco plants. The transgenic tobacco plants showed enhanced resistance against the fungus Erysiphe cichoracearum – both conidia formation and mycelial growth were reduced, and the size of the colony was diminished. Microscopic observation revealed that the transgenic tobacco plants carried the resistant phenotype, analogous to that of the resistant cultivar `Kokubu' which had been selected by conventional breeding. Growth of the phytopathogenic bacterium Pseudomonas syringae pv. tabaci was also strongly retarded in the transgenic tobacco, and the chlorotic halo of the disease symptom was reduced to 17% of that observed in the wild-type tobacco. Thus, the introduction of a human lysozyme gene is an effective approach to protect crops against both fungal and bacterial diseases. Received: 9 September 1996 / Revision received: January 9 1997 / Accepted: 20 February 1997     

Genetic engineering of drought-resistant tobacco plants by introducing the trehalose phosphorylase (TP) gene from <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis>

This study generated transgenic tobacco plants expressing trehalose phosphorylase of Pleurotus sajor-caju (PsTP) constitutively under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Sixteen transgenic lines were selected by genomic Southern blot analysis for further study. Unlike yeast TPS1-transformed or Escherichia coli TPS1-transformed tobacco or potato, all of the PsTP transgenic tobacco lines showed normal growth phenotypes both in the culture tubes and soil mixture. The study measured the trehalose contents of PsTP-transformed tobacco plants as well as the wild type and empty vector-transformed control plants. Results showed that the PsTP transformant contained 6.3molg^–1 of plant tissues, while the wild type and the control plants had only minimal levels of trehalose. Because this study detected a significant amount of trehalose in PsTP transgenic tobacco plants, it decided to carry out a bioanalysis of the PsTP transgenic tobacco plants by drought treatment by not watering the plants for over 10days. A significant difference in drought resistance was observed from the second nonwatering day between the transgenic and the control tobacco plants. The transgenic tobacco plants had normal growth and did not wither, while the wild type and the only empty vector-transformed control plants withered severely. Among all the transgenic lines, line 10-4 showed the strongest resistance to drought stress. It did not wither even after 10days without watering. In addition, when the drought resistance of PsTP transgenic tobacco plants was tested using detached leaves, most transgenic plants, except one line, showed better capacity to retain water than the empty vector-transformed transgenic plant.     

Use of the gene of antimicrobial peptide cecropin P1 for producing marker-free transgenic plants

The marker-free transgenic tobacco plants carrying a synthetic gene encoding the antimicrobial peptide cecropin P1 (cecP1) under the control of the cauliflower mosaic virus 35S RNA promoter were produced. The binary vector pBM, free of any selective genes of resistance to antibiotics or herbicides intended for selecting transgenic plants, was used for transformation. The transformants were screened on a nonselective medium by detecting cecropin P1 in plant cells according to the antibacterial activity of plant extracts and enzyme immunoassay. According to the two used methods, 2% of the analyzed regenerants were transformants. The resulting marker-free plants displayed a considerably increased resistance to microbial phytopathogens—the bacterium Erwinia carotovora and fungus Sclerotinia sclerotiorum. Thus, the gene cecP1 can be concurrently used as a target gene and a screening marker. The utility of cecP1 as a selective gene for direct selection of transformed plants is discussed.     

Improving freezing tolerance of transgenic tobacco expressing sucrose: sucrose 1-fructosyltransferase gene from Lactuca sativa

Sucrose: sucrose 1-fructosyltransferase (1-SST) cDNA from Lactuca sativa, coding the enzyme responsible for lower degree polymers fructan biosynthesis, was cloned by RT-PCR and RACE methods. The 1-SST cDNA under the control of CaMV 35S promoter was introduced into tobacco by Agrobacterium-mediated leaf disc transformation protocol. Fructan synthesis in vitro and carbohydrate analysis showed that sense transgenic tobacco plant displayed sucrose: sucrose 1-fructosyltransferse activity. After freezing stress, significant increases in electrolyte leakage and malondialdehyde were found in the wild type and anti-sense transgenic plants, while no apparent differences were observed in sense transgenic plants. Meanwhile, water soluble carbohydrate, fructan and fructose of sense transgenic plants remarkably increased, compared with those of wild type and anti-sense plants. No significant difference was detected in superoxide dismutase activity between transgenic and wild type plants. The above results demonstrated that the expression of 1-SST gene improved the freezing resistance of transgenic tobacco plants.     

Partial suppression of gene encoding proline dehydrogenase enhances plant tolerance to various abiotic stresses

The role of gene of proline dehydrogenase (PDH) in the maintenance of stress tolerance was investigated using the model transgenic plants of tobacco (Nicotiana tabacum L.) carrying an antisense suppressor of PDH gene (a fragment of Arabidopsis PDH gene under the control of cauliflower mosaic virus 35S promoter in antisense orientation) and notable for a low activity of PDH and elevated content of proline. The progeny of transgenic plants belonging to the 5th generation (T₅) with partially suppressed PDH activity was more resistant to various types of stress as compared with the control plants of tobacco, cv. Petit Havana SR-1 (SR1). The seedlings of transgenic lines cultured in Petri dishes on agar media supplemented with stress agents were resistant to high NaCl concentrations (200–300 mM) and water deficit simulated by an increased agar content in the medium (14 g/l) as compared to the control seedlings of cv. SR1. Juvenile plants of transgenic lines grown in pots filled with a mixture of vermiculite and perlite also manifested the higher resistance to water deficit and low temperatures (2°C and −2°C) than the control plants. Thus, the partial PDH suppression correlated with an increase in nonspecific resistance to different types of abiotic stress: salinity, water deficit, and low temperatures. Such transgenic lines of tobacco are promising genetic models for thorough investigation of molecular mechanisms of stress resistance in plants.     

Expression of giant silkmoth cecropin B genes in tobacco

Cecropin B is a small antibacterial peptide from the giant silkmothHyalophora cecropia. To reveal the potential of this peptide for engineering bacterial disease resistance into crops, several cecropin B gene constructs were made either for expression in the cytosol or for secretion. All constructs were cloned in a plant expression vector and introduced in tobacco viaAgrobacterium tumefaciens. A cDNA-derived cecropin B gene construct lacking the amino-terminal signal peptide was poorly expressed in transgenic plants at the mRNA level, whereas plants harbouring a full-length cDNA-derived construct containing the insect signal peptide, showed increased cecropin B-mRNA levels. Highest expression was found in plants harbouring a construct with a plant-gene-derived signal peptide. In none of the transgenic plants could the cecropin B peptide be detected. This is most likely caused by breakdown of the peptide by plant endogenous proteases, since a chemically synthesized cecropin B peptide was degraded within seconds in various plant cell extracts. This degradation could be prevented by the addition of specific protease inhibitors and by boiling the extract prior to adding the peptide. In addition, anionic detergents, in contrast to cationic, zwitter-ionic or non-ionic detergents, could prevent this degradation. Nevertheless, transgenic tobacco plants were evaluated for resistance toPseudomonas solanacearum, the causal agent of bacterial wilt of many crops, andP. syringae pv.tabaci, the causal agent of bacterial wildfire, which are highly susceptible to cecropin Bin vitro. No resistance was found. These experiments indicate that introduction and expression of cecropin B genes in tobacco does not result in detectable cecropin B protein levels and resistance to bacterial infections, most likely due to degradation of the protein by endogenous proteases.     

Transgenic Tobacco Plants Constitutively Overexpressing a Rice Thaumatin-like Protein (PR-5) Show Enhanced Resistance to Alternaria alternata

Overexpression of antifungal pathogenesis-related (PR) proteins in crop plants has the potential for enhancing resistance against fungal pathogens. Thaumatin-like proteins (TLPs) are one group (PR-5, permatins) of antifungal PR-proteins isolated from various plants. In the present study, a plasmid containing a cDNA of rice tlp (D34) under the control of the CaMV-35S promoter was introduced into tobacco plants through Agrobacterium-mediated transformation system. A considerable overproduction of TLP was observed in transformed tobacco plants by Western blot analysis. There was a large accumulation of tlp mRNA in transgenic plants as revealed by Northern blot analysis. Southern blot analysis of the DNA from transgenic tobacco plants confirmed the presence of the rice tlp gene in the genomic DNA of transgenic tobacco plants. Immunoblot analysis of intracellular and extracellular proteins of transgenic tobacco leaves using a Pinto bean TLP antibody demonstrated that the 23-kDa TLP was secreted into the extracellular matrix. T₂ progeny of regenerated plants transformed with TLP gene were tested for their disease reaction to Alternaria alternata, the brown spot pathogen. Transgenic tobacco plants expressing TLP at high levels showed enhanced tolerance to necrotization caused by the pathogen. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic transformation of Rangpur lime (Citrus limonia osbeck) with thebO (bacterio-opsin) genen and its initial evaluation forPhytophthora nicotianae resistance

Transgenic plants expressing the bacterio-opsin (bO) gene can spontaneously activate programmed cell death (ped) and may enhance broad-spectrum pathogen resistance by activating an intrinsic defense pathway in plant species such as tobacco and potato. In this work, we produced transgenic Rangpur lime plants with thebO gene, viaAgrobacterium tumefaciens-mediated transformation, and evaluated these plants forPhytophthora nicotianae resistance. Two transgenic lines were successfully regenerated and transformation was confirmed by GUS activity assay, PCR analysis, Southern, Northern and Western blot analyses, in addition to detecting the expressed bO protein by an immunological approach. Evaluation forPhytophthora nicotianae resistance was carried out by plant inoculations with the pathogen and quantification of the affected area. One of the two transgenic lines showed greater tolerance to the fungal pathogen as compared to the control, with significantly smaller stem lesions after pathogen challenge. This increase in pathogen tolerance is correlated with a significantly higher level of transgene expression in this line when compared with the other transgenic line. This is the first report of the introduction of a potentially important gene into Rangpur lime to provide novel pathogen tolerance.     

Light-grown plants of transgenic tobacco expressing an introduced oat phytochrome A gene under the control of a constitutive viral promoter exhibit persistent growth inhibition by far-red light

A comparison of the photoregulation of development has been made for etiolated and light-grown plants of wild-type (WT) tobacco (Nicotiana tabacun L.) and an isogenic transgenic line which expresses an introduced oat phytochrome gene (phyA) under the control of a constitutive viral promoter. Etiolated seedlings of both the WT and transgenic line showed irradiance-dependent inhibition of hypocotyl growth under continuous far-red (FR) light; transgenic seedlings showed a greater level of inhibition under a given fluence rate and this is considered to be the result of the heterologous phytochrome protein (PhyA) functioning in a compatible manner with the native etiolated phytochrome. Deetiolation of WT seedlings resulted in a loss of responsiveness to prolonged FR. Light-grown transgenic seedlings, however, continued to respond in an irradiance-dependent manner to prolonged FR and it is proposed that this is a specific function of the constitutive PhyA. Mature green plants of the WT and transgenic lines showed a qualitatively similar growth promotion to a brief end-of-day FR-treatment but this response was abolished in the transgenic plants under prolonged irradiation by this same FR source. Growth inhibition (McCormac et al. 1991, Planta 185, 162–170) and enhanced levels of nitrate-reductase activity under irradiance of low red:far-red ratio, as achieved by the FR-supplementation of white light, emphasised that the introduced PhyA was eliciting an aberrant mode of photoresponse compared with the normal phytochrome population of light-grown plants. Total levels of the oat-encoded phytochrome in the etiolated transgenic tobacco were shown to be influenced by the wavelength of continuous irradiation in a manner which was qualitatively similar to that seen for the native, etiolated tobacco phytochrome, and distinct from that seen in etiolated oat tissues. These results are discussed in terms of the proposal that the constitutive oat-PhyA pool in the transgenic plants leads to a persistence of a mode of response normally restricted to the situation in etiolated plants.Abbreviations FR far-red light - R red light - WL white light - WL + FR white light supplemented with FR - HIR high-irradiance response - PAR photosynthetically active radiation - Pr, Pfr R- and FR-absorbing forms of phytochrome - Ptot total phytochrome - phyA (PhyA) gene (encoded protein) for phytochrome - WT wild type This work was supported by an Agricultural and Food Research Council research grant to H.S. and A.M.; J.R. Cherry and R.D. Vierstra, (Department of Horticulture, University of Wisconsin-Madison, USA) are thanked for the provision of the transgenic tobacco line.     

Expression of Cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.     

Heterologous expression of chloroplast‐localized geranylgeranyl pyrophosphate synthase confers fast plant growth,early flowering and increased seed yield

Geranylgeranyl pyrophosphate synthase (GGPS) is a key enzyme for a structurally diverse class of isoprenoid biosynthetic metabolites including gibberellins, carotenoids, chlorophylls and rubber. We expressed a chloroplast‐targeted GGPS isolated from sunflower (Helianthus annuus) under control of the cauliflower mosaic virus 35S promoter in tobacco (Nicotiana tabacum). The resulting transgenic tobacco plants expressing heterologous GGPS showed remarkably enhanced growth (an increase in shoot and root biomass and height), early flowering, increased number of seed pods and greater seed yield compared with that of GUS‐transgenic lines (control) or wild‐type plants. The gibberellin levels in HaGGPS‐transgenic plants were higher than those in control plants, indicating that the observed phenotype may result from increased gibberellin content. However, in HaGGPS‐transformant tobacco plants, we did not observe the phenotypic defects such as reduced chlorophyll content and greater petiole and stalk length, which were previously reported for transgenic plants expressing gibberellin biosynthetic genes. Fast plant growth was also observed in HaGGPS‐expressing Arabidopsis and dandelion plants. The results of this study suggest that GGPS expression in crop plants may yield desirable agronomic traits, including enhanced growth of shoots and roots, early flowering, greater numbers of seed pods and/or higher seed yield. This research has potential applications for fast production of plant biomass that provides commercially valuable biomaterials or bioenergy.     

Enhanced quantitative resistance to Leptosphaeria maculans conferred by expression of a novel antimicrobial peptide in canola (Brassica napus L.)

The novel antimicrobial peptide MiAMP1, originally isolated from the seeds of Macadamia integrifolia, was constitutively expressed in transgenic tobacco and canola plants to test its effect on disease resistance. Analysis of plants transformed with 35S-MiAMP1 construct by northern and western blot analyses demonstrated the presence of MiAMP1 mRNA and the mature peptide in the transgenic plants. The MiAMP1 purified from the leaves of transgenic plants was biologically active with the same in vitro antifungal activity as native MiAMP1 purified from the seeds of macadamia. The effect of MiAMP1 expression on the economically important canola pathogen Leptosphaeria maculans (causal agent of blackleg disease) was evaluated in comparison with an untransformed control line and an azygous segregant derived from one of the transgenic lines. Lesion development on the cotyledons of the inoculated canola seedlings was significantly reduced in the T₂ progeny of seven independently transformed transgenic lines. These results suggested that, transgenic canola expressing MiAMP1 may be useful for the management of blackleg disease.     

Differential Expression of Endogenous Ferritin Genes and Iron Homeostasis Alteration in Transgenic Tobacco Overexpressing Soybean Ferritin Gene

For studying the effects of endogenous ferritin gene expressions (NtFer1, GenBank accession number ay083924; and NtFer2, GenBank accession number ay141105) on the iron homeostasis in transgenic tobacco (Nicotiana tabacum L.) plants expressing soybean (Glycine max Merr) ferritin gene (SoyFer1, GenBank accession number m64337), the transgenic tobacco has been produced by placing soybean ferritin cDNA cassette under the control of the CaMV 35S promoter. The exogenous gene expression was examined by both Northern- and Western-blot analyses. Comparison of endogenous ferritin gene expressions between nontransformant and transgenic tobacco plants showed that the expression of NtFer1 was increased in the leaves of transgenic tobacco plants, whereas the NtFer2 expression was unchanged. The iron concentration in the leaves of transgenic tobacco plants was about 1.5-folds higher than that in nontransformant. Enhanced growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weights significantly greater than those in the nontransformant. These results demonstrated that exogenous ferritin expression induced increased expression of at least one of the endogenous ferritin genes in transgenic tobacco plants by enhancing the ferric chelate reductase activity and iron transport ability of the root, and improved the rate of photosynthesis.     

铁皮石斛DoSMT2基因的功能分析

为了揭示铁皮石斛(Dendrobium officinale)甾醇C-24甲基转移酶2基因(DoSMT2)在甾醇代谢过程的功能,该研究通过根癌农杆菌介导法将来源于铁皮石斛的DoSMT2基因转化烟草(Nicotiana tabacum),并采用qRT-PCR技术检测DoSMT2基因在转基因烟草叶片中的表达,采用气相色谱质谱法分析菜油甾醇和谷甾醇的含量。结果显示:(1)成功获得DoSMT2基因的开放阅读框(1 119 bp),并成功构建正义植物表达载体质粒pCXSN-DoSMT2,经农杆菌介导的烟草叶盘转化法转化烟草并鉴定,获得4株阳性转基因烟草植株。(2)Southern blot结果表明,4株转基因烟草植株都有1条杂交信号带,而非转基因烟草植株没有,说明外源DoSMT2基因都以单拷贝整合到4株转基因烟草基因组中。(3)qRT-PCR检测显示,非转基因烟草未检测到外源DoSMT2基因的表达,4株转基因烟草都能检测到DoSMT2基因的表达,且表达水平差异极显著,各株系表达量高低依次为P3P1P2(P4)。(4)气相色谱质谱分析显示,转DoSMT2基因烟草叶片的菜油甾醇含量均极显著低于非转基因烟草叶片,而谷甾醇含量均极显著高于非转基因烟草叶片。研究表明,DoSMT2具有催化24-亚甲基胆甾烯醇转化形成24-亚乙基胆甾烯醇活性。     

Overexpression of glutamate decarboxylase in transgenic tobacco plants confers resistance to the northern root-knot nematode

Previous research suggests that the endogenous synthesis of gamma-aminobutyrate (GABA), a naturally occurring inhibitory neurotransmitter, serves as a plant defense mechanism against invertebrate pests. Here, we tested the hypothesis that elevated GABA levels in engineered tobacco confer resistance to the northern root nematode (Meloidogyne hapla). This nematode species was chosen because of its sedentary nature and economic importance in Canada. We derived nine phenotypically normal, homozygous lines of transgenic tobacco (Nicotiana tabacum L.), which contain one or two copies of a full-length, chimeric tobacco glutamate decarboxylase (GAD) cDNA or a mutant version that lacks the autoinhibitory calmodulin-binding domain, under the control of a chimeric octopine synthase/mannopine synthase promoter. Regardless of experimental protocol, uninfected transgenic lines consistently contained higher GABA concentrations than wild-type controls. Growth chamber trials revealed that 9–12 weeks after inoculation of tobacco transplants with the northern root-knot nematode, mature plants of five lines possessed significantly fewer egg masses on the root surface when the data were expressed on both root and root fresh weight bases. Therefore, it can be concluded that constitutive transgenic expression of GAD conferred resistance against the root-knot nematode in phenotypically normal tobacco plants, probably via a GABA-based mechanism.     

Multiple resistance to sulfonylureas and imidazolinones conferred by an acetohydroxyacid synthase gene with separate mutations for selective resistance

Summary The acetohydroxyacid synthase (AHAS) gene from the Arabidopsis thaliana mutant line GH90 carrying the imidazolinone resistance allele imr1 was cloned. Expression of the AHAS gene under the control of the CaMV 35S promoter in transgenic tobacco resulted in selective imidazolinone resistance, confirming that the single base-pair change found near the 3 end of the coding region of this gene is responsible for imidazolinone resistance. A chimeric AHAS gene containing both the imr1 mutation and the csr1 mutation, responsible for selective resistance to sulfonylurea herbicides, was constructed. It conferred on transgenic tobacco plants resistance to both sulfonylurea and imidazolinone herbicides. The data illustrate that a multiple-resistance phenotype can be achieved in an AHAS gene through combinations of separate mutations, each of which individually confers resistance to only one class of herbicides.