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Ikuo Nakamura - One of the best experts on this subject based on the ideXlab platform.
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Transgenic Rice Expressing Wasabi Defensin Gene Exhibit Its Enhanced Resistance to Blast Fungus (Magnaporthe Grisea)
Rice Blast: Interaction with Rice and Control, 2020Co-Authors: Hiroyuki Kanzaki, Masahiro Nishihara, Saburo Yamamura, Shizuko Nirasawa, Hiromasa Saitoh, Kazumi Suzuki, Ryohei Terauchi, Ikuo NakamuraAbstract:A complementary DNA of defensin gene was isolated from Wasabi (Wasabia japonica) plant. This cDNA was cloned into PVX (Potato virus X) vector, and the vector inoculated to Nicotiana benthamiana plants to rapidly produce the defensin protein in planta. Purified Wasabi defensin showed strong antimicrobial activity toward Magnaporthe grisea. Thus, Wasabi defensin gene fused with a strong maize ubiquitin promoter was used for transformation of rice (Oryza sativa cv. Sasanishiki) by Agrobacterium tumefaciensmediated method. Transformants exhibited resistance to rice blast at various levels. Inheritance of blast resistance over the generations was confirmed. Two T3 lines (WT14–5 and WT43–5) stably expressing the defensin protein were challenged with blast fungus by using the press-injured spots inoculation method. The average size of disease lesions of the transgenic line WT43–5 was reduced to about half of that of non-transgenic plants. Transgenic rice plants producing Wasabi defensin are expected to possess a durable and wide-spectrum resistance (i.e. field resistance) against various rice blast races.
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retransformation of marker free potato for enhanced resistance against fungal pathogens by pyramiding chitinase and Wasabi defensin genes
Molecular Biotechnology, 2014Co-Authors: Raham Sher Khan, Valentine Otang Ntui, Nader Ahmed Darwish, Bushra Khattak, Kynet Kong, Kazuki Shimomae, Ikuo NakamuraAbstract:Multi-auto-transformation vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators and thus facilitating transgene stacking. In the study reported here, retransformation was carried out in marker-free transgenic potato CV. May Queen containing ChiC gene (isolated from Streptomyces griseus strain HUT 6037) with Wasabi defensin (WD) gene (isolated from Wasabia japonica) to pyramid the two disease resistant genes. Molecular analyses of the developed shoots confirmed the existence of both the genes of interest (ChiC and WD) in transgenic plants. Co-expression of the genes was confirmed by RT-PCR, northern blot, and western blot analyses. Disease resistance assay of in vitro plants showed that the transgenic lines co-expressing both the ChiC and WD genes had higher resistance against the fungal pathogens, Fusarium oxysporum (Fusarium wilt) and Alternaria solani (early blight) compared to the non-transformed control and the transgenic lines expressing either of the ChiC or WD genes. The disease resistance potential of the transgenic plants could be increased by transgene stacking or multiple transformations.
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increased resistance to fusarium wilt in transgenic tobacco lines co expressing chitinase and Wasabi defensin genes
Plant Pathology, 2011Co-Authors: Valentine Otang Ntui, Raham Sher Khan, Pejman Azadi, Dong Poh Chin, G Thirukkumaran, Ikuo NakamuraAbstract:Marker-free transgenic tobacco (Nicotiana tabacum) lines containing a chitinase (ChiC) gene isolated from Streptomyces griseus strain HUT 6037 were produced by Agrobacterium-mediated transformation. One marker-free transgenic line, TC-1, was retransformed with the Wasabi defensin (WD) gene, isolated from Wasabia japonica. Of the retransformed shoots, 37% co-expressed the ChiC/WD genes, as confirmed by western and northern analyses. Southern blot analysis showed that no chromosomal rearrangement was introduced between the first and the second transformation. Transgenic lines either expressing ChiC or WD, or co-expressing both genes were challenged with Fusarium oxysporum f.sp. nicotianae (Fon). Assessment of in vitro plant survival in the presence of Fon showed that transgenic lines co-expressing both genes had significantly enhanced protection against the fungus (infection indices 0·0–1.·2) compared with corresponding isogenic lines expressing either of the genes (infection indices 2·5–9·8). Whole-plant infection indices in transgenic lines were significantly related (r = 0·93, P < 0·01) to the extent of root colonization of the host, which ranged from 2·1% to 11·3% in lines co-expressing both genes, and from 16·8% to 37·7% in lines expressing just one of the genes (compared with 86·4% in non-transformed controls). Leaf extracts of transgenic lines also inhibited mycelial growth of Fon in vitro and caused hyphal abnormalities.
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Stable integration and expression of Wasabi defensin gene in “Egusi” melon (Colocynthis citrullus L.) confers resistance to Fusarium wilt and Alternaria leaf spot
Plant Cell Reports, 2010Co-Authors: Valentine Otang Ntui, Raham Sher Khan, Gunaratnam Thirukkumaran, Pejman Azadi, Ikuo NakamuraAbstract:Production of “Egusi” melon ( Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer “Egusi” resistant to these diseases, cotyledonary explants of two “Egusi” genotypes, ‘Ejagham’ and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring Wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mgl^−l kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and ‘Ejagham’, respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype ‘Ejagham’ were transgenic. A DNA fragment corresponding to the Wasabi defensin gene or the selection marker npt II was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1–5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that Wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani , which causes Alternaria leaf spot and Fusarium oxysporum , which causes Fusarium wilt, as compared to that of untransformed plants.
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stable integration and expression of Wasabi defensin gene in egusi melon colocynthis citrullus l confers resistance to fusarium wilt and alternaria leaf spot
Plant Cell Reports, 2010Co-Authors: Valentine Otang Ntui, Raham Sher Khan, Gunaratnam Thirukkumaran, Pejman Azadi, Ikuo NakamuraAbstract:Production of “Egusi” melon (Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer “Egusi” resistant to these diseases, cotyledonary explants of two “Egusi” genotypes, ‘Ejagham’ and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring Wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mgl−l kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and ‘Ejagham’, respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype ‘Ejagham’ were transgenic. A DNA fragment corresponding to the Wasabi defensin gene or the selection marker nptII was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1–5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that Wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani, which causes Alternaria leaf spot and Fusarium oxysporum, which causes Fusarium wilt, as compared to that of untransformed plants.
Geoffrey P Savage - One of the best experts on this subject based on the ideXlab platform.
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Flavour components in the rhizome of soil-grown Wasabi.
2020Co-Authors: Tamanna Sultana, Geoffrey P Savage, N. G. Porter, David L. Mcneil, R.j. MartinAbstract:Wasabi, Japanese horseradish (Wasabia japonica (Miq) Matsum) is grown to prepare a green paste which is eaten with traditional Japanese dishes. The plant is grown as a perennial crop in Japan and is now also grown in New Zealand. The best quality Wasabi products are produced from the rhizomes (stems) although other parts of the plant such as petioles and leaves also possess some pungency and are also used as raw materials. The characteristic flavour of Wasabi comes from the volatile isothiocyanates (ITCs), which are evolved from glucosinolates by enzymatic hydrolysis when tissues are macerated. In this study the total isothiocyanate (ITC) and six different ITCs were measured in the rhizome of Wasabi grown at Lincoln under four different soil treatments. The level of total ITC ranged from 2425 to 2810 mg/kg fresh weight, which was significantly higher than the values reported in the literature for Wasabi grown in Japan (mean 1659 mg/ kg). Allyl isothiocyanate (AITC) was the main ITC and it contributed between 86 to 92% of the total ITC measured in the rhizomes. Overall, there were small changes in the individual ITCs as a result of the different treatments (control, lime, manure, lime and manure), however no correlation between ITC concentration and yield of plants was found. The total ITC contents quantified by GCMS were marginally higher than the total ITC’s measured by UV spectrometry after approximately one year storage of paraffin oil extract at 0 to -4oC. Comparison of isothiocyanate yield of Wasabi rhizome tissues grown either in soil or water Tamanna Sultana, G P Savage, D L McNeil and N G Porter Food group, Animal and Food Sciences Division, Lincoln University, Canterbury, New Zealand. New Zealand Institute for Crop & Food Science, Christchurch, New Zealand. Abstract The isothiocyanate (ITC) content of Wasabi, the Japanese horseradish (Wasabi japonica) was measured, by release from glucosinolates, in the rhizomes of plants grown in two traditional ways. Mature plants approximately 18 months old were harvested from two different commercial farms located in the south island of New Zealand. At one farm the plants were grown in raised soil beds while the plants in the other farm were grown in gravel irrigated by river water. Following harvest theThe isothiocyanate (ITC) content of Wasabi, the Japanese horseradish (Wasabi japonica) was measured, by release from glucosinolates, in the rhizomes of plants grown in two traditional ways. Mature plants approximately 18 months old were harvested from two different commercial farms located in the south island of New Zealand. At one farm the plants were grown in raised soil beds while the plants in the other farm were grown in gravel irrigated by river water. Following harvest the rhizomes from each growth medium were divided into five size groups based on the weight and length of the rhizomes. The different sized rhizomes were also subdivided into top, middle and bottom portions of the rhizomes and each portion was further subdivided into skin and cortex, and vascular and pith using a knife. The individual and total isothiocyanate content of each portion of the rhizomes were extracted using dichloromethane and measured using a GCFPD technique. The total isothiocyanate content of the rhizomes grown in soil increased (14.24 times) linearly from 15 to 100 g rhizome weight while the mean isothiocyanate content of the water grown Wasabi increased (11.13 times) non-linearly for similar sized rhizomes. Water grown rhizomes in the weight range 20 to 60 g gave significantly (P=0.030) higher total ITC (2.2-2.9 times) than similar sized soil grown Wasabi. Analysis of the tissues showed that the total and the individual isothiocyanates were found in significantly higher levels (73% and 64% respectively) in the skin and cortex compared to the vascular and pith tissues. Analysis of the isothiocyanate content of the different locations of the Wasabi rhizome showed that the lower portion of the rhizome contained significantly higher levels of both total and individual ITCs compared to the middle and top portions of the rhizome. Investigation of isothiocyanate yield of flowering and non-flowering tissues of Wasabi grown in a flooded system Tamanna Sultana, David L McNeil, Noel G Porter, G P Savage Food Group, Animal and Food Sciences Division, P O Box 84, Lincoln University, Canterbury, New Zealand. 2 Victorian Institute of Dryland Agriculture, Melbourne University, Horsham, Victoria, Australia. New Zealand Institute for Crop & Food Research Ltd, Christchurch, New Zealand.
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Shelf Life Studies of Three Wasabi Flavoured Sauces
Bangladesh Journal of Scientific and Industrial Research, 2009Co-Authors: Tamanna Sultana, Geoffrey P Savage, N. G. Porter, David L. Mcneil, SedcoleAbstract:Isothiocyanates (ITCs) contained in purees extracted from Wasabi ( Wasabia japonica (Miq) Matsum) can be used to manufacture a range of interesting spicy foods. In New Zealand, local manufacturers are showing interest in producing various forms of processed Wasabi based sauces. However, isothiocyanates have been shown to degrade quickly in some situations. Therefore, in this study, the stability of allyl ITC was investigated in three Wasabi flavoured products stored at four different temperatures (4, 10, 20 and 30°C) for 22 weeks. Two creamy (mayonnaise and tartare) sauces and a non-creamy sauce were prepared from an original recipe and flavoured with a known volume of "Wasabi oil". Two types of pouches (clear and metallic plastic) were used to store each product and allyl ITC content was measured in the stored sauces at two week intervals. The initial level of allyl ITC found in mayonnaise, tartare and smoky tomato sauces were 415.3, 411.4 and 144.7 mg/ kg respectively, prior to storage. Temperature showed a strong influence in reducing allyl ITC ( P =0.005 to
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Wasabi japanese horseradish
Bangladesh Journal of Scientific and Industrial Research, 2009Co-Authors: Tamanna Sultana, Geoffrey P SavageAbstract:Wasabi, Japanese Horseradish ( Wasabia japonica (Miq.) Matsum) is a perennial plant native to Japan. It has been cultivated in Japan for more than a thousand years and is now being grown in many countries as interest in Japanese cuisine expands. Wasabi can be grown in two main ways, either in flooded fields or in soil based mediums. The unique flavour of Wasabi comes from isothiocyanates (ITCs) which are evolved from precursor glucosinolates by the enzyme myrosinase when the tissue is disrupted. ITCs found in Wasabi are volatile, possess strong pungent smells and are toxic at high intakes. The overall flavour of Wasabi depends on individual ITC content. Allyl ITC is found in the highest concentration in all tissues, ranging from 86-92% of the total ITC content. Apart from flavouring sauces and foods Wasabi isothiocyanates have interesting anticancer effects. ITCs can also counter inflammatory conditions like asthma and anaphylaxis. ITCs have also been shown to inhibit platelet aggregation in the blood. Wasabi is a valuable crop that can be processed into a tasty condiment. Its production and consumption will increase as it becomes more appreciated in Western cuisine. Keywords : Wasabia japonica , Horseradish, Flavour compounds, Isothiocyanate (ITC), Glucosinolate, Allyl ITC, 3-butenyl ITC, 4-pentenyl ITC, 5-hexenyl ITC, 2-pheneylethyl ITC, Isopropyl ITC, Secbutyl ITC, Rhizome, Anticancer, Anti-asthmatic, Anti-inflammatory, Industrial application doi: 10.3329/bjsir.v43i4.2234 Bangladesh J. Sci. Ind. Res. 43(4) , 433-448, 2008
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comparison of flavour compounds in Wasabi and horseradish
Journal of Food Agriculture & Environment, 2003Co-Authors: Tamanna Sultana, Geoffrey P Savage, D L Mcneil, Noel G Porter, B ClarkAbstract:The Japanese horseradish (Wasabia japonica (Miq.) Matsum) and European horseradish (Amoracia rusticana) are aromatic herbs used as spices and condiments due to their characteristic flavour. The flavour of both comes from the liberation of volatile isothiocyanates (ITCs) by the hydrolysis of precursor glucosinolates. Seven ITCs were measured in this study in order to compare the flavour compounds of Wasabi rhizomes and horseradish roots. New Zealand grown horseradish contained 1900.7 mg total isothiocyanate/kg (on a fresh weight basis) while the level of total ITC in Wasabi was 2067.55 mg/kg. Allyl ITC (AITC) was the main ITC in both of the plants (1937.8 and 1658.1 mg/kg respectively in Wasabi and horseradish). 2-phenylethyl ITC (2-PEITC) was present as a major component after AITC (185.2 mg/ kg on a fresh weight basis) only in horseradish and therefore the radish-like flavour of 2-PEITC is likely to have a characteristic role in the overall flavour of horseradish. However, all minor ITCs (w-alkenyl ITCs, alkyl ITCs) occurred at higher levels in Wasabi rhizomes. The differences in ITC observed between Wasabi and horseradish could well explain the difference in taste between these two herbs.
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effects of fertilisation on the allyl isothiocyanate profile of above ground tissues of new zealand grown Wasabi
Journal of the Science of Food and Agriculture, 2002Co-Authors: Tamanna Sultana, Geoffrey P Savage, D L Mcneil, Noel G Porter, Richard J MartinAbstract:Wasabi (Wasabia japonica (Miq) Matsum) is a developing crop in New Zealand and is valued for its spicy taste and pungent smell. It is popular as a condiment for traditional and modern Japanese foods. However, the limited area suitable for Wasabi production in Japan has resulted in inability to meet increasing market demand. Isothiocyanates (ITCs) are sulphur compounds responsible for the unique flavour of Wasabi, with allyl isothiocyanate (AITC) being the main compound responsible for the pungency. In this study, AITC tissue concentration and yield were measured in three above-ground tissues of the Wasabi plant to investigate the effects of different fertiliser, manure and lime treatments. The highest tissue concentration of AITC was found in the rhizomes, ranging from 1564 to 3366 mg kg−1 (fresh weight basis), while the petioles and leaves contained 254–373 and 453–643 mg kg−1 respectively. Fertilisation with ammonium sulphate produced the highest-quality rhizomes (72% increase in AITC yield) and petioles (64% increase), but the best response in the leaves (51%) resulted from the manure treatment. Nitrogen fertiliser alone reduced the AITC yield by up to 15%. These results should help in formulating guidelines for production of high-quality Wasabi tops containing high levels of AITC. © 2002 Society of Chemical Industry
Valentine Otang Ntui - One of the best experts on this subject based on the ideXlab platform.
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retransformation of marker free potato for enhanced resistance against fungal pathogens by pyramiding chitinase and Wasabi defensin genes
Molecular Biotechnology, 2014Co-Authors: Raham Sher Khan, Valentine Otang Ntui, Nader Ahmed Darwish, Bushra Khattak, Kynet Kong, Kazuki Shimomae, Ikuo NakamuraAbstract:Multi-auto-transformation vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators and thus facilitating transgene stacking. In the study reported here, retransformation was carried out in marker-free transgenic potato CV. May Queen containing ChiC gene (isolated from Streptomyces griseus strain HUT 6037) with Wasabi defensin (WD) gene (isolated from Wasabia japonica) to pyramid the two disease resistant genes. Molecular analyses of the developed shoots confirmed the existence of both the genes of interest (ChiC and WD) in transgenic plants. Co-expression of the genes was confirmed by RT-PCR, northern blot, and western blot analyses. Disease resistance assay of in vitro plants showed that the transgenic lines co-expressing both the ChiC and WD genes had higher resistance against the fungal pathogens, Fusarium oxysporum (Fusarium wilt) and Alternaria solani (early blight) compared to the non-transformed control and the transgenic lines expressing either of the ChiC or WD genes. The disease resistance potential of the transgenic plants could be increased by transgene stacking or multiple transformations.
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increased resistance to fusarium wilt in transgenic tobacco lines co expressing chitinase and Wasabi defensin genes
Plant Pathology, 2011Co-Authors: Valentine Otang Ntui, Raham Sher Khan, Pejman Azadi, Dong Poh Chin, G Thirukkumaran, Ikuo NakamuraAbstract:Marker-free transgenic tobacco (Nicotiana tabacum) lines containing a chitinase (ChiC) gene isolated from Streptomyces griseus strain HUT 6037 were produced by Agrobacterium-mediated transformation. One marker-free transgenic line, TC-1, was retransformed with the Wasabi defensin (WD) gene, isolated from Wasabia japonica. Of the retransformed shoots, 37% co-expressed the ChiC/WD genes, as confirmed by western and northern analyses. Southern blot analysis showed that no chromosomal rearrangement was introduced between the first and the second transformation. Transgenic lines either expressing ChiC or WD, or co-expressing both genes were challenged with Fusarium oxysporum f.sp. nicotianae (Fon). Assessment of in vitro plant survival in the presence of Fon showed that transgenic lines co-expressing both genes had significantly enhanced protection against the fungus (infection indices 0·0–1.·2) compared with corresponding isogenic lines expressing either of the genes (infection indices 2·5–9·8). Whole-plant infection indices in transgenic lines were significantly related (r = 0·93, P < 0·01) to the extent of root colonization of the host, which ranged from 2·1% to 11·3% in lines co-expressing both genes, and from 16·8% to 37·7% in lines expressing just one of the genes (compared with 86·4% in non-transformed controls). Leaf extracts of transgenic lines also inhibited mycelial growth of Fon in vitro and caused hyphal abnormalities.
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Stable integration and expression of Wasabi defensin gene in “Egusi” melon (Colocynthis citrullus L.) confers resistance to Fusarium wilt and Alternaria leaf spot
Plant Cell Reports, 2010Co-Authors: Valentine Otang Ntui, Raham Sher Khan, Gunaratnam Thirukkumaran, Pejman Azadi, Ikuo NakamuraAbstract:Production of “Egusi” melon ( Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer “Egusi” resistant to these diseases, cotyledonary explants of two “Egusi” genotypes, ‘Ejagham’ and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring Wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mgl^−l kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and ‘Ejagham’, respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype ‘Ejagham’ were transgenic. A DNA fragment corresponding to the Wasabi defensin gene or the selection marker npt II was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1–5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that Wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani , which causes Alternaria leaf spot and Fusarium oxysporum , which causes Fusarium wilt, as compared to that of untransformed plants.
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stable integration and expression of Wasabi defensin gene in egusi melon colocynthis citrullus l confers resistance to fusarium wilt and alternaria leaf spot
Plant Cell Reports, 2010Co-Authors: Valentine Otang Ntui, Raham Sher Khan, Gunaratnam Thirukkumaran, Pejman Azadi, Ikuo NakamuraAbstract:Production of “Egusi” melon (Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer “Egusi” resistant to these diseases, cotyledonary explants of two “Egusi” genotypes, ‘Ejagham’ and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring Wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mgl−l kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and ‘Ejagham’, respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype ‘Ejagham’ were transgenic. A DNA fragment corresponding to the Wasabi defensin gene or the selection marker nptII was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1–5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that Wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani, which causes Alternaria leaf spot and Fusarium oxysporum, which causes Fusarium wilt, as compared to that of untransformed plants.
Raham Sher Khan - One of the best experts on this subject based on the ideXlab platform.
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retransformation of marker free potato for enhanced resistance against fungal pathogens by pyramiding chitinase and Wasabi defensin genes
Molecular Biotechnology, 2014Co-Authors: Raham Sher Khan, Valentine Otang Ntui, Nader Ahmed Darwish, Bushra Khattak, Kynet Kong, Kazuki Shimomae, Ikuo NakamuraAbstract:Multi-auto-transformation vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators and thus facilitating transgene stacking. In the study reported here, retransformation was carried out in marker-free transgenic potato CV. May Queen containing ChiC gene (isolated from Streptomyces griseus strain HUT 6037) with Wasabi defensin (WD) gene (isolated from Wasabia japonica) to pyramid the two disease resistant genes. Molecular analyses of the developed shoots confirmed the existence of both the genes of interest (ChiC and WD) in transgenic plants. Co-expression of the genes was confirmed by RT-PCR, northern blot, and western blot analyses. Disease resistance assay of in vitro plants showed that the transgenic lines co-expressing both the ChiC and WD genes had higher resistance against the fungal pathogens, Fusarium oxysporum (Fusarium wilt) and Alternaria solani (early blight) compared to the non-transformed control and the transgenic lines expressing either of the ChiC or WD genes. The disease resistance potential of the transgenic plants could be increased by transgene stacking or multiple transformations.
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increased resistance to fusarium wilt in transgenic tobacco lines co expressing chitinase and Wasabi defensin genes
Plant Pathology, 2011Co-Authors: Valentine Otang Ntui, Raham Sher Khan, Pejman Azadi, Dong Poh Chin, G Thirukkumaran, Ikuo NakamuraAbstract:Marker-free transgenic tobacco (Nicotiana tabacum) lines containing a chitinase (ChiC) gene isolated from Streptomyces griseus strain HUT 6037 were produced by Agrobacterium-mediated transformation. One marker-free transgenic line, TC-1, was retransformed with the Wasabi defensin (WD) gene, isolated from Wasabia japonica. Of the retransformed shoots, 37% co-expressed the ChiC/WD genes, as confirmed by western and northern analyses. Southern blot analysis showed that no chromosomal rearrangement was introduced between the first and the second transformation. Transgenic lines either expressing ChiC or WD, or co-expressing both genes were challenged with Fusarium oxysporum f.sp. nicotianae (Fon). Assessment of in vitro plant survival in the presence of Fon showed that transgenic lines co-expressing both genes had significantly enhanced protection against the fungus (infection indices 0·0–1.·2) compared with corresponding isogenic lines expressing either of the genes (infection indices 2·5–9·8). Whole-plant infection indices in transgenic lines were significantly related (r = 0·93, P < 0·01) to the extent of root colonization of the host, which ranged from 2·1% to 11·3% in lines co-expressing both genes, and from 16·8% to 37·7% in lines expressing just one of the genes (compared with 86·4% in non-transformed controls). Leaf extracts of transgenic lines also inhibited mycelial growth of Fon in vitro and caused hyphal abnormalities.
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Stable integration and expression of Wasabi defensin gene in “Egusi” melon (Colocynthis citrullus L.) confers resistance to Fusarium wilt and Alternaria leaf spot
Plant Cell Reports, 2010Co-Authors: Valentine Otang Ntui, Raham Sher Khan, Gunaratnam Thirukkumaran, Pejman Azadi, Ikuo NakamuraAbstract:Production of “Egusi” melon ( Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer “Egusi” resistant to these diseases, cotyledonary explants of two “Egusi” genotypes, ‘Ejagham’ and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring Wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mgl^−l kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and ‘Ejagham’, respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype ‘Ejagham’ were transgenic. A DNA fragment corresponding to the Wasabi defensin gene or the selection marker npt II was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1–5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that Wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani , which causes Alternaria leaf spot and Fusarium oxysporum , which causes Fusarium wilt, as compared to that of untransformed plants.
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stable integration and expression of Wasabi defensin gene in egusi melon colocynthis citrullus l confers resistance to fusarium wilt and alternaria leaf spot
Plant Cell Reports, 2010Co-Authors: Valentine Otang Ntui, Raham Sher Khan, Gunaratnam Thirukkumaran, Pejman Azadi, Ikuo NakamuraAbstract:Production of “Egusi” melon (Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer “Egusi” resistant to these diseases, cotyledonary explants of two “Egusi” genotypes, ‘Ejagham’ and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring Wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mgl−l kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and ‘Ejagham’, respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype ‘Ejagham’ were transgenic. A DNA fragment corresponding to the Wasabi defensin gene or the selection marker nptII was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1–5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that Wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani, which causes Alternaria leaf spot and Fusarium oxysporum, which causes Fusarium wilt, as compared to that of untransformed plants.
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transgenic potatoes expressing Wasabi defensin peptide confer partial resistance to gray mold botrytis cinerea
Plant Biotechnology, 2006Co-Authors: Raham Sher Khan, Masahiro Nishihara, Saburo Yamamura, Ikuo NakamuraAbstract:Five potato (Solanum tuberosum L.) cultivars were transformed with Agrobacterium tumefaciens strain EHA101 harboring Wasabi defensin gene (isolated from Wasabia japonica L.) in a binary plasmid vector, pEKH1. The infected tuber explants co-cultivated for 3 days resulted in higher transformation efficiency (7-50% higher) for all cultivars than 2 days co-cultivation. PCR analysis showed an amplified fragment of Wasabi defensin gene and the selectable marker, nptII gene in the genomic DNA of all clones rooted on MS medium supplemented with 100 mgl � 1 kanamycin, suggesting their transgenic nature. Southern blot analysis confirmed that transgenic plants integrated 1-6 copies of Wasabi defensin gene into their genome. Expression of Wasabi defensin protein was confirmed in the leaf extracts of independent transgenic clones by Western blot analysis. Antifungal assay of detached leaves from non-transformed control and transgenic plants indicated that transgenic plants were partially resistant to the fungal pathogen, Botrytis cinerea (gray mold).
Tetsuro Ogawa - One of the best experts on this subject based on the ideXlab platform.
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Wasabi leaf extracts attenuate adipocyte hypertrophy through pparγ and ampk
Bioscience Biotechnology and Biochemistry, 2016Co-Authors: Yasuo Oowatari, Tetsuro Ogawa, Takuya Katsube, Kiyohisa Iinuma, Hisae YoshitomiAbstract:Hypertrophy of adipocytes in obese adipose tissues causes metabolic abnormality by adipocytokine dysregulation, which promotes type 2 diabetes mellitus, hypertension, and dyslipidemia. We investigated the effects of Wasabi (Wasabia japonica Matsum) leaf extracts on metabolic abnormalities in SHRSP.Z-Leprfa/IzmDmcr rats (SHRSP/ZF), which are a model of metabolic syndrome. Male SHRSP/ZF rats aged 7 weeks were divided into two groups: control and Wasabi leaf extract (WLE) groups, which received water or oral treatment with 4 g/kg/day WLE for 6 weeks. WLE improved the body weight gain and high blood pressure in SHRSP/ZF rats, and the plasma triglyceride levels were significantly lower in the WLE group. Adipocyte hypertrophy was markedly prevented in adipose tissue. The expression of PPARγ and subsequent downstream genes was suppressed in the WLE group adipose tissues. Our data suggest that WLE inhibits adipose hypertrophy by suppressing PPARγ expression in adipose tissue and stimulating the AMPK activity by i...
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anti obesity effects of hot water extract from Wasabi Wasabia japonica matsum leaves in mice fed high fat diets
Nutrition Research and Practice, 2013Co-Authors: Masayuki Yamasaki, Tetsuro Ogawa, Takuya Katsube, Yukikazu Yamasaki, Li Wang, Kuninori ShiwakuAbstract:The anti-obesity effects of a hot water extract from Wasabi (Wasabia japonica Matsum.) leaves (WLE), without its specific pungent constituents, such as allyl-isothiocyanate, were investigated in high fat-diet induced mice. C57J/BL mice were fed a high-fat diet (control group) or a high-fat diet supplemented with 5% WLE (WLE group). Physical parameters and blood profiles were determined. Gene expression associated with lipid metabolism in liver and white adipose tissue were analyzed. After 120 days of feeding, significantly lower body weight gain, liver weight and epididymal white adipose tissue weight was observed in the WLE group compared to the control group. In liver gene expression within the WLE group, PPARα was significantly enhanced and SREBP-1c was significantly suppressed. Subsequent downstream genes controlled by these regulators were significantly suppressed. In epididymal white adipose tissue of the WLE group, expression of leptin, PPARγ, and C/EBPα were significantly suppressed and adiponectin was significantly enhanced. Acox, related to fatty acid oxidization in adipocytes, was also enhanced. Our results demonstrate that the WLE dietary supplement induces mild suppression of obesity in a high-fat diet induced mice, possibly due to suppression of lipid accumulation in liver and white adipose tissue.
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suppressive effect of hot water extract of Wasabi Wasabia japonica matsum leaves on the differentiation of 3t3 l1 preadipocytes
Food Chemistry, 2010Co-Authors: Tetsuro Ogawa, Takuya Katsube, Hiromasa Tabata, Yukari Ohta, Yukikazu Yamasaki, Masayuki Yamasaki, Kuninori ShiwakuAbstract:This study investigated the effect of hot water extract of Wasabi (Wasabia japonica Matsum.) leaves (WLE), without its specific pungent constituents, such as allyl isothiocyanate, on the differentiation of 3T3-L1 preadipocytes. WLE suppressed the increase in glycerol-3-phosphate dehydrogenase (GPDH) activity and triglyceride (TG) accumulation, markers of adipogenesis, in a dose-dependent manner. Quantitative real time RT-PCR results showed that WLE significantly reduced the mRNA expression levels of peroxisome proliferator-activated receptor (PPAR) γ and CCAAT/enhancer-binding protein (C/EBP) α, both key adipogenic transcription factors, as subsequently were the mRNA expression levels of their target genes, such as adipocyte fatty acid binding protein 2 (aP2). Western blot analysis results showed that the protein expression levels of both PPARγ and C/EBPα were also inhibited by WLE. Thus, WLE suppressed the differentiation of 3T3-L1 preadipocytes, and the suppressive effect was mediated, in part, through the altered regulation of PPARγ, C/EBPα, and other specific genes, such as aP2. These results suggest that WLE may prevent obesity and insulin resistance by inhibiting the differentiation of preadipocytes.
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anti influenza virus activity of extract of japanese Wasabi leaves discarded in summer
Journal of the Science of Food and Agriculture, 2008Co-Authors: Kyo Mochida, Tetsuro OgawaAbstract:BACKGROUND: Japanese Wasabi (Wasabia japonica) is now habitually used as a spice in some kinds of Japanese foods, and its pungent taste and flavor are preferred. Generally, rhizomes and winter leaves are used as a spice and for processed foods such as pickled Wasabi. Since the leaf area of summer leaves is far greater than that of winter leaves, they are not used for food, and are discarded. Thus, we need to develop an effective use for summer leaves. We investigated anti-influenza virus activity in these summer leaves as a new function. RESULTS: Seventy percent ethanol extracts of leaves harvested in July exhibited a high replication inhibition rate (98% or higher) in the type A strain (AH1N1, A/shimane/48/2002), its subtype (AH3AB, A/shimane/122/2002), and type B strain (B/shimane/2/2002). The extracts of summer leaves exhibited the same anti-influenza virus activity as winter leaves, and showed a stronger activity than stems, roots, and rhizomes. CONCLUSION: A potent anti-influenza virus activity was discovered in summer leaves of Japanese Wasabi. The ethanol extracts inhibited influenza virus replication regardless of the hemagglutinin antigen type. Therefore, such extracts are expected to be a promising source of a novel anti-influenza virus agent. Copyright © 2008 Society of Chemical Industry
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Anti‐influenza virus activity of extract of Japanese Wasabi leaves discarded in summer
Journal of the Science of Food and Agriculture, 2008Co-Authors: Kyo Mochida, Tetsuro OgawaAbstract:BACKGROUND: Japanese Wasabi (Wasabia japonica) is now habitually used as a spice in some kinds of Japanese foods, and its pungent taste and flavor are preferred. Generally, rhizomes and winter leaves are used as a spice and for processed foods such as pickled Wasabi. Since the leaf area of summer leaves is far greater than that of winter leaves, they are not used for food, and are discarded. Thus, we need to develop an effective use for summer leaves. We investigated anti-influenza virus activity in these summer leaves as a new function. RESULTS: Seventy percent ethanol extracts of leaves harvested in July exhibited a high replication inhibition rate (98% or higher) in the type A strain (AH1N1, A/shimane/48/2002), its subtype (AH3AB, A/shimane/122/2002), and type B strain (B/shimane/2/2002). The extracts of summer leaves exhibited the same anti-influenza virus activity as winter leaves, and showed a stronger activity than stems, roots, and rhizomes. CONCLUSION: A potent anti-influenza virus activity was discovered in summer leaves of Japanese Wasabi. The ethanol extracts inhibited influenza virus replication regardless of the hemagglutinin antigen type. Therefore, such extracts are expected to be a promising source of a novel anti-influenza virus agent. Copyright © 2008 Society of Chemical Industry
