Genome-wide association study of resistance to anthracnose in pepper (Capsicum chinense) germplasm.

BACKGROUND: Anthracnose is a fungal disease caused by Colletotrichum spp. that has a significant impact on worldwide pepper production. Colletotrichum scovillei is the most common pathogenic anthracnose-causing species in the Republic of Korea. RESULTS: The resistances of 197 pepper (Capsicum chinense) accessions deposited in Korea's National Agrobiodiversity Center were evaluated for their response against the virulent pathogens Colletotrichum acutatum isolate 'KSCa-1' and C. scovillei isolate 'Hana') in the field and in vitro methods for three consecutive years (2018 to 2020). The severity of the disease was recorded and compared between inoculation methods. Six phenotypically resistant pepper accessions were selected based on three years of disease data. All of the selected resistant pepper accessions outperformed the control resistant pepper in terms of resistance (PI 594,137). A genome-wide association study (GWAS) was carried out to identify single nucleotide polymorphisms (SNPs) associated with anthracnose resistance. An association analysis was performed using 53,518 SNPs and the disease score of the 2020 field and in vitro experiment results. Both field and in vitro experiments revealed 25 and 32 significantly associated SNPs, respectively. These SNPs were found on all chromosomes except Ch06 and Ch07 in the field experiment, whereas in the in vitro experiment they were found on all chromosomes except Ch04 and Ch11. CONCLUSION: In this study, six resistant C. chinense accessions were selected. Additionally, in this study, significantly associated SNPs were found in a gene that codes for a protein kinase receptor, such as serine/threonine-protein kinase, and other genes that are known to be involved in disease resistance. This may strengthen the role of these genes in the development of anthracnose resistance in Capsicum spp. As a result, the SNPs discovered to be strongly linked in this study can be used to identify a potential marker for selecting pepper material resistant to anthracnose, which will assist in the development of resistant varieties.

Pepper Mild Mottle Virus: An Infectious Pathogen in Pepper Production and a Potential Indicator of Domestic Water Quality.

Pepper (Capsicum spp.; Family: Solanaceae; 2n = 24) is an important crop cultivated worldwide for the consumption of its fresh and dried processed fruits. Pepper fruits are used as raw materials in a wide variety of industrial processes. As a multipurpose vegetable crop, there is a need to increase the yield. However, yield productivity of pepper is severely constrained by infectious plant pathogens, including viruses, bacteria, fungi, and oomycetes. The pepper mild mottle virus (PMMoV) is currently one of the most damaging pathogens associated with yield losses in pepper production worldwide. In addition to impacts on pepper productivity, PMMoV has been detected in domestic and aquatic water resources, as well as in the excreta of animals, including humans. Therefore, PMMoV has been suggested as a potential indicator of domestic water quality. These findings present additional concerns and trigger the need to control the infectious pathogen in crop production. This review provides an overview of the distribution, economic impacts, management, and genome sequence variation of some isolates of PMMoV. We also describe genetic resources available for crop breeding against PMMoV.

Assessment of respiratory and systemic toxicity of Benzalkonium chloride following a 14-day inhalation study in rats.

BACKGROUND: Although biocides at low concentrations have been used to control pests, they can be more harmful than industrial chemicals as humans are directly and frequently exposed to such biocides. Benzalkonium chloride (BAC or BKC) is a non-toxic substance used to control pests. Recently, BAC has been increasingly used as a component in humidifier disinfectants in Korea, raising a serious health concern. Moreover, it poses significant health hazards to workers handling the chemical because of direct exposure. In the present study, we aimed to evaluate the respiratory toxicity of BAC due to its inhalation at exposure concentrations of 0.8 (T1 group), 4 (T2 group) and 20 (T3 group) mg/m3. RESULTS: In our previous study on the acute inhalational toxicity of BAC, bleeding from the nasal cavity was observed in all the rats after exposure to 50 mg/m3 BAC. Therefore, in this study, 20 mg/m3 was set as the highest exposure concentration, followed by 4 and 0.8 mg/m3 as the medium and low concentrations for 6 h/day and 14 days, respectively. After exposure, recovery periods of 2 and 4 weeks were provided. Additionally, alveolar lavage fluid was analyzed in males of the BAC-exposed groups at the end of exposure and 2 weeks after exposure to evaluate oxidative damage. In the T3 group exposed to BAC, deep breathing, hoarseness, and nasal discharge were observed along with a decline in feed intake and body weight, and nasal discharge was also observed in the T1 and T2 groups. ROS/RNS, IL-1ß, IL-6, and MIP-2 levels decreased in a concentration-dependent manner in the bronchoalveolar lavage fluid. Histopathological examination showed cellular changes in the nasal cavity and the lungs of the TI, T2, and T3 groups. CONCLUSIONS: As a result, it was confirmed that the target organs in the respiratory system were the nasal cavity and the lungs. The adverse effects were evaluated as reversible responses to oxidative damage. Furthermore, the no observed adverse effect level was found to be less than 0.8 mg/m3 and the lowest benchmark dose was 0.0031 mg/m3. Accordingly, the derived no-effect level of BAC was calculated as 0.000062 mg/m3.

A new FGFR inhibitor disrupts the TGF-ß1-induced fibrotic process.

Pulmonary fibrosis (PF) is chronic and irreversible damage to the lung characterized by fibroblast activation and matrix deposition. Although recently approved novel anti-fibrotic agents can improve the lung function and survival of patients with PF, the overall outcomes remain poor. In this study, a novel imidazopurine compound, 3-(2-chloro-6-fluorobenzyl)-1,6,7-trimethyl-1H-imidazo[2,1-f]purine-2,4(3H,8H)-dione (IM-1918), markedly inhibited transforming growth factor (TGF)-ß-stimulated reporter activity and reduced the expression of representative fibrotic markers, such as connective tissue growth factor, fibronectin, collagen and α-smooth muscle actin, on human lung fibroblasts. However, IM-1918 neither decreased Smad-2 and Smad-3 nor affected p38MAPK and JNK. Instead, IM-1918 reduced Akt and extracellular signal-regulated kinase 1/2 phosphorylation increased by TGF-ß. Additionally, IM-1918 inhibited the phosphorylation of fibroblast growth factor receptors 1 and 3. In a bleomycin-induced murine lung fibrosis model, IM-1918 profoundly reduced fibrotic areas and decreased collagen and α-smooth muscle actin accumulation. These results suggest that IM-1918 can be applied to treat lung fibrosis.

Differentially expressed genes in iron-induced prion protein conversion.

The conversion of the cellular prion protein (PrPC) to the protease-resistant isoform is the key event in chronic neurodegenerative diseases, including transmissible spongiform encephalopathies (TSEs). Increased iron in prion-related disease has been observed due to the prion protein-ferritin complex. Additionally, the accumulation and conversion of recombinant PrP (rPrP) is specifically derived from Fe(III) but not Fe(II). Fe(III)-mediated PK-resistant PrP (PrPres) conversion occurs within a complex cellular environment rather than via direct contact between rPrP and Fe(III). In this study, differentially expressed genes correlated with prion degeneration by Fe(III) were identified using Affymetrix microarrays. Following Fe(III) treatment, 97 genes were differentially expressed, including 85 upregulated genes and 12 downregulated genes (≥1.5-fold change in expression). However, Fe(II) treatment produced moderate alterations in gene expression without inducing dramatic alterations in gene expression profiles. Moreover, functional grouping of identified genes indicated that the differentially regulated genes were highly associated with cell growth, cell maintenance, and intra- and extracellular transport. These findings showed that Fe(III) may influence the expression of genes involved in PrP folding by redox mechanisms. The identification of genes with altered expression patterns in neural cells may provide insights into PrP conversion mechanisms during the development and progression of prion-related diseases.

Development of Selectable Marker-Free Transgenic Rice Plants with Enhanced Seed Tocopherol Content through FLP/FRT-Mediated Spontaneous Auto-Excision.

Development of marker-free transgenic plants is a technical alternative for avoiding concerns about the safety of selectable marker genes used in genetically modified (GM) crops. Here, we describe the construction of a spontaneous self-excision binary vector using an oxidative stress-inducible modified FLP/FRT system and its successful application to produce marker-free transgenic rice plants with enhanced seed tocopherol content. To generate selectable marker-free transgenic rice plants, we constructed a binary vector using the hpt selectable marker gene and the rice codon-optimized FLP (mFLP) gene under the control of an oxidative stress-inducible promoter between two FRT sites, along with multiple cloning sites for convenient cloning of genes of interest. Using this pCMF binary vector with the NtTC gene, marker-free T1 transgenic rice plants expressing NtTC were produced by Agrobacterium-mediated stable transformation using hygromycin as a selective agent, followed by segregation of selectable marker genes. Furthermore, α-, γ-, and total tocopherol levels were significantly increased in seeds of the marker-free transgenic TC line compared with those of wild-type plants. Thus, this spontaneous auto-excision system, incorporating an oxidative stress-inducible mFLP/FRT system to eliminate the selectable marker gene, can be easily adopted and used to efficiently generate marker-free transgenic rice plants. Moreover, nutritional enhancement of rice seeds through elevation of tocopherol content coupled with this marker-free strategy may improve human health and public acceptance of GM rice.

Analysis of immune cell populations and cytokine profiles in murine splenocytes exposed to whole-body low-dose irradiation.

PURPOSE: In contrast to high-dose therapeutic irradiation, definitive research detailing the physiological effects of low-dose irradiation is limited. Notably, the immunological response elicited after low-dose irradiation remains controversial. MATERIALS AND METHODS: Female C57BL/6 mice were whole- body-irradiated with a single or three daily fractions up to a total dose of 0.1, 1, or 10 cGy. Blood and spleen were harvested 2, 7 and 14 days after irradiation. RESULTS: The splenic CD4(+) T cell subpopulations were temporarily increased at 2 days after single or fractionated irradiation, whereas the percentage of dendritic cells (DC) and macrophages was decreased. Whereas CD8(+) T cell populations were decreased in single-dose irradiated mice at day 7, early and sustained reduction of CD8(+) T cell numbers was observed in fractionated- dose-irradiated mice from day 2 until day 14. In addition, single-dose irradiation resulted in a Th1 cytokine expression profile, whereas fractionated-dose irradiation drove a Th2 shift. Additionally, increased expression of immune-related factors was observed at early time-points with single-dose irradiation, in contrast to the dose-independent induction following fractionated-dose irradiation. CONCLUSIONS: Our results demonstrate that low-dose irradiation modulates the immune response in mice, where the sensitivity and kinetics of the induced response vary according to the dosing method.

Quantitative proteomic analysis of single or fractionated radiation-induced proteins in human breast cancer MDA-MB-231 cells.

BACKGROUND: Radiotherapy is widely used to treat cancer alone or in combination with surgery, chemotherapy, and immunotherapy. However, damage to normal tissues and radioresistance of tumor cells are major obstacles to successful radiotherapy. Furthermore, the immune network around tumors appears to be connected to tumor progression and recurrence. METHODS: We investigated the cytosolic proteins produced by irradiated tumor cells by using a quantitative proteomic approach based on stable isotope labeling by amino acids in cell culture. MDA-MB-231 breast cancer cells were treated with a single or fractionated 10 Gray dose of (137)Cs γ-radiation, which was selected based on cell viability. RESULTS: Radiation-induced proteins were differentially expressed based on the fractionated times of radiation and were involved in multiple biological functions, including energy metabolism and cytoskeleton organization. We identified 46 proteins increased by at least 1.3-fold, and high ranks were determined for cathepsin D, gelsolin, arginino-succinate synthase 1, peroxiredoxin 5, and C-type mannose receptor 2. CONCLUSION: These results suggest that a number of tumor-derived factors upregulated by γ-radiation are promising targets for modulation of the immune response during radiation treatment.

Different strategies for producing naturally soluble form of common cytokine receptor γ chain.

The common cytokine receptor γ chain (γc) plays an essential role in regulating lymphoid homeostasis. In fact, alteration of this gene causes severe immunodeficiency in humans and animals. Although soluble γc (sγc) was identified in the late 1990s, much remains unknown about its production. This study describes various mechanisms underlying the generation of sγc isoforms in different species. Our data demonstrate that mouse γc and the avian ortholog γc-a did not generate sγc. Moreover, two mouse isoforms, CRA-a and mγc-b, encoded by transcripts lacking a transmembrane region by alternative splicing, did not yield sγc. However, in ducks, sγc was produced from a γc-b transcript lacking a transmembrane region by alternative splicing. In chickens, sγc was produced in normal cells and cell lines by proteolytic shedding of the γc-b isoform containing intron 5, which displayed a relatively high probability of proteolytic cleavage of the ectodomain. This shedding was suppressed by leupeptin, serine and cysteine protease inhibitor. Compared to the chicken ortholog γc-a, expression of γc-b mRNA was differentially regulated according to tissue type, developmental stage, and antigen stimulation. These data demonstrate several mechanisms for producing sγc and suggest a potential role for sγc in avian lymphoid homeostatic responses to environmental antigens.

Prion diseases as transmissible zoonotic diseases.

Prion diseases, also called transmissible spongiform encephalopathies (TSEs), lead to neurological dysfunction in animals and are fatal. Infectious prion proteins are causative agents of many mammalian TSEs, including scrapie (in sheep), chronic wasting disease (in deer and elk), bovine spongiform encephalopathy (BSE; in cattle), and Creutzfeldt-Jakob disease (CJD; in humans). BSE, better known as mad cow disease, is among the many recently discovered zoonotic diseases. BSE cases were first reported in the United Kingdom in 1986. Variant CJD (vCJD) is a disease that was first detected in 1996, which affects humans and is linked to the BSE epidemic in cattle. vCJD is presumed to be caused by consumption of contaminated meat and other food products derived from affected cattle. The BSE epidemic peaked in 1992 and decreased thereafter; this decline is continuing sharply owing to intensive surveillance and screening programs in the Western world. However, there are still new outbreaks and/or progression of prion diseases, including atypical BSE, and iatrogenic CJD and vCJD via organ transplantation and blood transfusion. This paper summarizes studies on prions, particularly on prion molecular mechanisms, BSE, vCJD, and diagnostic procedures. Risk perception and communication policies of the European Union for the prevention of prion diseases are also addressed to provide recommendations for appropriate government policies in Korea.

Effect of orally administered Lactobacillus brevis HY7401 in a food allergy mouse model.

We had found that orally administered Lactobacillus species were effective immune modulators in ovalbumin (OVA)-sensitized mice. To validate these findings, we investigated the effects of orally administered Lactobacillus brevis HY7401 in OVA-T cell receptor transgenic mice. This strain showed a tendency to induce Th1 cytokines and inhibit Th2 cytokines. All assayed isotypes of OVA-specific antibody were effectively reduced. Systemic anaphylaxis was also relatively reduced with the probiotic administration. These results reveal that L. brevis HY7401 might be useful to promote anti-allergic processes through oral administration.

Immunomodulatory and anti-allergic effects of orally administered Lactobacillus species in ovalbumin-sensitized mice.

We investigated the effects of orally administered probiotic bacteria (Lactobacillus species) as allergic immune modulators in ovalbumin (OVA)-sensitized mice. BALB/c mice were intraperitoneally injected with OVA twice at a 2-week interval for allergy sensitization. The mice were then orally administered Lactobacillus casei YIT9029 (L1), L. casei HY7201 (L2), L. brevis HY7401 (L3), or L. plantarum HY20301 (L4) every 2 days for 3 weeks. Total IgE levels significantly decreased in sera of L3-administered mice but increased in the other groups. OVA-specific IgE levels decreased slightly in sera of mice administered L1, L3, and L4 but increased significantly in L2-administered mice. In passive cutaneous anaphylaxis (PCA) using sera from administered mice, only the L3-administered group showed reaction inhibition. High expression of TLR-2 with interferon (IFN)-gamma stimulation on peripheral blood mononuclear cells occurred in L3- or L4-administered mice. Th1 cytokines, including IFN-gamma and interleukin (IL)- 12, increased in splenocytes of L3-administered mice; however, IL-4 decreased in L1- and L4-administered groups; IL-5 decreased in all experimental groups. IL-6 decreased in the L3-administered group; and IL-10 decreased in L1-, L2-, and L3-administered groups. L3 induced antiallergic effects by increasing Th1 cytokines, decreasing Th2 cytokines, and inhibiting the PCA reaction, whereas L2 administration increased allergic effects.

Development of a chloroplast DNA marker for monitoring of transgene introgression in Brassica napus L.

Chloroplast molecular markers can provide useful information for high-resolution analysis of inter- and intra-specific variation in Brassicaceae and for differentiation between its species. Combining data generated from nuclear and chloroplast markers enables the study of seed and pollen movement, and assists in the assessment of gene-flow from genetically modified (GM) plants through hybridization studies. To develop chloroplast DNA markers for monitoring of transgene introgression in Brassica napus L., we searched for sequence variations in the chloroplast (cp) genome, and developed a simple cpDNA marker that is reliable, time-saving, and easily discriminates among 4 species (B. napus, B. rapa, Raphanus sativus, and Sinapis alba) based on PCR-product length polymorphism. This marker will be useful to identify maternal lineages and to estimate transgene movement of GM canola.

Prion protein conversion induced by trivalent iron in vesicular trafficking.

Iron dyshomeostasis has been observed in prion diseases; however, little is known regarding the contribution of the oxidation state of iron to prion protein (PrP) conversion. In this study, PrP(C)-deficient HpL3-4 cells were exposed to divalent [Fe(II)] or trivalent [Fe(III)] iron, followed by exogenous recombinant PrP (rPrP) treatment. We then analyzed the accumulation of internalized rPrP and its biochemical properties, including its resistance to both proteinase K (PK) digestion and detergent solubility. Fe(III), but not Fe(II), induced the accumulation of internalized rPrP, which was partially converted to detergent-insoluble and PK-resistant PrP (PrP(res)). The Fe(III)-induced PrP(res) generation required an intact cell structure, and it was hindered by U18666A, an inhibitor of vesicular trafficking, but not by NH4Cl, an inhibitor of endolysosomal acidification. These observations implicated that the Fe(III)-mediated PrP(res) conversion likely occurs during endosomal vesicular trafficking rather than in the acidic environment of lysosomes.

Protective immunity conferred by the C-terminal fragment of recombinant Pasteurella multocida toxin.

Pasteurella multocida serogroup D, producing P. multocida toxin (PMT), is a causative pathogen of progressive atrophic rhinitis (PAR) in swine. To evaluate the protective immunity and vaccination efficacy of the truncated form of PMT, a C-terminal form of recombinant PMT (designated PMT2.3; amino acid residues 505 to 1285 of PMT) was expressed in an Escherichia coli expression system, and the humoral and cellular immune responses to PMT2.3 were investigated. PMT2.3 vaccination in mice led to high levels of the anti-PMT antibody with a high neutralizing antibody titer. PMT2.3 also induced a cellular immune response to PMT, as demonstrated by the lymphocyte proliferation assay. Furthermore, strong protection against a homologous challenge with P. multocida was also observed in mice vaccinated with PMT2.3. In PMT2.3 vaccination in swine, high levels of serum antibody titers were observed in offspring from sows vaccinated with PMT2.3. Offspring from sows vaccinated with PMT2.3 or toxoid showed a good growth performance as depicted by mean body weight at the time of sacrifice, as well as in average daily gain in the postweaning period. Low levels of pathological lesions in turbinate atrophy and pneumonia were also observed in these offspring. Therefore, we consider PMT2.3--in the truncated and nontoxic recombinant PMT form--to be an attractive candidate for a subunit vaccine against PAR induced by P. multocida infection.

Effects of a single nucleotide polymorphism in the chicken NK-lysin gene on antimicrobial activity and cytotoxicity of cancer cells.

NK-lysin is an effector protein of the innate immune system and an important component of host protection. We isolated a SNP in the NK-lysin coding sequence among different chicken breeds. This A to G substitution at the position 271 nucleotide in the ORF results in an Asn (N) to Asp (D) amino acid alteration. We synthesized two 30-aa peptides (N29N and N29D) to compare the biological activity of the helix 2-loop-helix 3 region of NK-lysin resulting from the polymorphic gene. Both peptides were found to be cytotoxic in bacteria and tumor cell cultures at micromolar concentrations. The N29N peptide, however, exhibited greater antibacterial and anticancer activity than the N29D peptide. Circular dichroism spectroscopy of the two peptides in negatively charged single unilamellar vesicles showed spectra typical of α-helical peptides. The helical profile of N29D was reduced substantially compared with that of N29N. However, no structural change was observed in neutral vesicles. ζ-Potential measurements of liposomes incubated with increasing peptide concentrations allowed surface charge neutralization with a negatively charged lipid, but not with a zwitterionic lipid. This result suggests that a difference in electrostatic interaction between lipid membranes and the helical peptides results from the polymorphic gene and is subsequently an important factor in cell lytic activity of variant NK-lysin peptides.

Stability of orally administered immunoglobulin in the gastrointestinal tract.

Oral administration of immunoglobulin in the colostrum or egg yolk has been considered an effective tool for preventing enterobacterial infection via passive immunization. During this process, the transmission and residence of the active immunoglobulin are the most important conditions for successful protection. We investigated the stability of encapsulated colostrum and egg yolk immunoglobulin for the effective transmission of immunoglobulin in the gastrointestinal (GI) tract. First, we measured GI transit time. Contrast media passed through and reached the stomach within 10 min, the small intestine within 3.5 h, and the cecum within 5 h. Both the encapsulated colostrum containing anti-hepatitis A virus (HAV) antibody (IgG) and egg yolk with anti-rotavirus antibody (IgY) showed lower antibody activity than the non-encapsulated colostrum did in the stomach after administration; however, significantly higher antibody activities were observed in the encapsulated groups than in the non-encapsulated groups in the small intestine 3.5 h after the administration. In the large intestine, the antibody activities of the encapsulated groups were maintained or slightly increased in a time-dependent manner; however, the titers of each non-capsulated control were as low as the negative controls. Therefore, this encapsulation is considered a useful tool for the delivery of active antibody through the GI tract.

Sequence variations of the bovine prion protein gene (PRNP) in native Korean Hanwoo cattle.

Bovine spongiform encephalopathy (BSE) is one of the fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs) caused by infectious prion proteins. Genetic variations correlated with susceptibility or resistance to TSE in humans and sheep have not been reported for bovine strains including those from Holstein, Jersey, and Japanese Black cattle. Here, we investigated bovine prion protein gene (PRNP) variations in Hanwoo cattle [Bos (B.) taurus coreanae], a native breed in Korea. We identified mutations and polymorphisms in the coding region of PRNP, determined their frequency, and evaluated their significance. We identified four synonymous polymorphisms and two non-synonymous mutations in PRNP, but found no novel polymorphisms. The sequence and number of octapeptide repeats were completely conserved, and the haplotype frequency of the coding region was similar to that of other B. taurus strains. When we examined the 23-bp and 12-bp insertion/deletion (indel) polymorphisms in the non-coding region of PRNP, Hanwoo cattle had a lower deletion allele and 23-bp del/12-bp del haplotype frequency than healthy and BSE-affected animals of other strains. Thus, Hanwoo are seemingly less susceptible to BSE than other strains due to the 23-bp and 12-bp indel polymorphisms.

Antigenicity of partial fragments of recombinant Pasteurella multocida toxin.

Pasteurella multocida serogroup D strain, which produces P. multocida toxin (PMT), is a widespread and harmful pathogen of respiratory diseases such as pneumonia and progressive atrophic rhinitis (PAR) in swine. Vaccination has been considered the most desirable and effective approach for controlling the diseases caused by toxigenic P. multocida. To investigate the antigenicity and immunogenicity of partial fragments of recombinant PMT, recombinant proteins of the N-terminal (PMT-A), middle (PMT-B), Cterminal (PMT-C), and middle-C-terminal (PMT2.3) regions of PMT were successfully produced in an Escherichia coli expression system. The molecular masses of PMT-A, PMT-B, PMT-C, and PMT2.3 were ca. 53, 55, 35, and 84 kDa, respectively, purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. All the recombinant proteins except for PMT-A showed immune responses to antisera obtained from a swine showing symptoms of PAR. Moreover, high titers of PMT-specific antibodies were raised from mice immunized with each of the recombinant proteins; however, the immunoreactivities of the antibodies to authentic PMT and heat-inactivated whole bacteria were different, respectively. In the protection study, the highest protection against homologous challenge was shown in the case of PMT2.3; relatively poor protections occurred for the other PMT fragments.

Auto-excision of selectable marker genes from transgenic tobacco via a stress inducible FLP/FRT site-specific recombination system.

Antibiotic resistance marker genes are powerful selection tools for use in plant transformation processes. However, once transformation is accomplished, the presence of these resistance genes is no longer necessary and can even be undesirable. We herein describe the successful excision of antibiotic resistance genes from transgenic plants via the use of an oxidative stress-inducible FLP gene. FLP encodes a recombinase that can eliminate FLP and hpt selection genes flanked by two FRT sites. During a transformation procedure in tobacco, transformants were obtained by selection on hygromycin media. Regenerants of the initial transformants were screened for selective marker excision in hydrogen peroxide supplemented media and both the FLP and hpt genes were found to have been eliminated. About 13-41% of regenerated shoots on hydrogen peroxide media were marker-free. This auto-excision system, mediated by the oxidative stress-inducible FLP/FRT system to eliminate a selectable marker gene can be very readily adopted and used to efficiently generate marker-free transgenic plants.