Transgenic Miscanthus lutarioriparius that co-expresses the Cry 2Aa# and Bar genes

2019 ◽  
Vol 99 (6) ◽  
pp. 841-851
Author(s):  
Yanan Xia ◽  
Jun Xu ◽  
Junyi Duan ◽  
Qingbo Liu ◽  
Hongmei Huang ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transgenic Plants ◽  
Herbicide Tolerance ◽  
Transformation Method ◽  
Energy Crop ◽  
Embryogenic Calli ◽  
Transformation Protocol ◽  
Agrobacterium Mediated Transformation ◽  
Domestic Species ◽  
Miscanthus Lutarioriparius

Miscanthus lutarioriparius, a domestic species of China, belongs to the genus of perennial rhizomatous Miscanthus and is a promising energy crop for biomass production. To establish an Agrobacterium tumefaciens transformation protocol and obtain transgenic plants co-expressing the Cry 2Aa# and Bar genes, an Agrobacterium-mediated transformation method for M. lutarioriparius was developed in this study. To inhibit tissue browning, optimized callus induction and regeneration media were developed. Embryogenic calli were efficiently induced from immature inflorescences on Murashige & Skoog (MS) medium containing 0.2 g L−1 citric acid. Agrobacterium tumefaciens EHA105 carrying pC3300 with the Cry 2Aa# and Bar genes were introduced into embryogenic calli. Six transgenic lines were selected by Basta screening and confirmed by genomic PCR. The herbicide tolerance of M. lutarioriparius transgenic plants was verified by spraying with glufosinate and expression of the Cry 2Aa# protein by ELISA. Therefore, an Agrobacterium-mediated transformation protocol for M. lutarioriparius was established and the co-expression of herbicide tolerance and Cry 2Aa# was successfully introduced with the system.

scholarly journals Optimization of the parameters of Agrobacterium-mediated transformation of bar-shaped millet

2020 ◽  
pp. 32-43
Author(s):  
A.T. Zhumabek ◽  
◽  
Y.M. Ramankulov ◽  
S.A. Manabayeva ◽  
◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Optical Density ◽  
Panicum Virgatum ◽  
Virulence Gene ◽  
Transformation Method ◽  
Bacterial Suspension ◽  
Embryogenic Calli ◽  
Agrobacterium Mediated Transformation ◽  
Agrobacterial Transformation ◽  
Panicum Virgatum L

An efficient Agrobacterium-mediated transformation method has been developed and optimized for switchgrass (Panicum virgatum L.). The aim of this study is to optimize the conditions for agrobacterial transformation of upland switchgrass cultivars Alamo, Forestburg, Pathfinder, Shawnee, and Trailblazer. For genetic transformation, the authors have used embryogenic calli of switchgrass cultivars. As a result of the study the effect of the virulence gene inducer, surfactant, optical density of bacterial suspension and co-cultivation time, it has been revealed that the presence of acetosyringone with silwet-L77 in the medium, bacterial suspension at an optical density of 0.6 and 8 days of co-cultivation with bacteria is optimal for Agrobacterium– mediated transformation of embryogenic calli of switchgrass. The results obtained can serve as a basis for the creation of transgenic plants of switchgrass.

scholarly journals Evaluasi varietas, sumber eksplan dan strain Agrobacterium terhadap keberhasilan transformasi tebu dengan gen P5CS Evaluation of varieties, explant sources, and Agrobacterium strains for successful sugarcane transformation using P5CS gene

2016 ◽  
Vol 83 (1) ◽  
Author(s):  
Hayati MINARSIH ◽  
Dwi SUBIYARTI ◽  
Imron RIYADI ◽  
Soekarno Mismana PUTRA ◽  
Laksmi AMBARSARI
Keyword(s):  
Drought Stress ◽  
Agrobacterium Tumefaciens ◽  
Somatic Embryos ◽  
Saccharum Officinarum ◽  
Transformation Method ◽  
Embryogenic Calli ◽  
Temporary Immersion System ◽  
Agrobacterium Strain ◽  
Solid Media ◽  
P5cs Gene

Abstract Genetic transformation can be used as an alter-native to develop sugarcane (Saccharum officinarum L.) tolerant to drought stress. P5CS gene has a role in biosynthesis of proline, an amino acid that accumulated under drought stress conditions. Transfer of a P5CS gene construct into plant cells in conjunction with regeneration of transgenic plantlets may develop sugarcane tolerant to drought stress. The aim of this research was to obtain an optimal transformation method which includes a suitable strain of Agrobacterium tumefaciens, and the best sugarcane explant and variety. The results showed that transfer of P5CS gene has been successfully carried out on sugarcane explants from solid media-derived calli, embryogenic calli and somatic embryos derived from temporary immersion system (TIS) culture. Whilst Agrobacterium strain LBA4404 was indicated as the most effective transformation vector. The regeneration of Kidang Kencana variety transformants from calli and somatic embryos was better than those of PS 881 and PS 891. The best performance of transformants based on the source of explants obtained from somatic embryos from TIS culture. Moreover, a succesfull Agrobacterium mediated transformation on sugarcane was indicated by transient expression of Gus gene and the ability of the transformants grew in a selection medium containing 50 ppm of kanamycin.Abstrak Transformasi genetik dapat digunakan sebagai upaya untuk merakit tebu (Saccharum officinarum L.) toleran terhadap cekaman kekeringan. Gen P5CS diketahui  berperan  dalam  biosintesis  prolin,  yaitu asam amino yang umumnya terakumulasi ketika tanaman mengalami cekaman kekeringan. Transfor-masi gen P5CS dan regenerasi transgeniknya mungkin dapat menghasilkan tanaman tebu trans-genik yang toleran terhadap cekaman kekeringan. Tujuan penelitian ini adalah untuk mendapatkan metode transformasi yang optimum yang mencakup strain Agrobacterium tumefaciens yang sesuai, sumber eksplan dan varietas tebu terbaik sebagai target transformasi. Hasil penelitian menunjukkan bahwa transformasi gen P5CS telah berhasil dilakukan ke eksplan tebu baik yang berupa kalus asal media padat maupun kalus embriogenik dan embrio somatik asal kultur sistem perendaman sesaat (SPS). Sementara itu strain A. tumefaciens LBA4404 menunjukkan hasil yang paling efektif sebagai vektor transformasi. Pertumbuhan transforman baik pada kalus maupun embrio somatik pada varietas Kidang Kencana terlihat paling baik dibandingkan dengan varietas PS 881 dan PS 891. Sumber eksplan yang paling efektif adalah embrio somatik yang diperoleh dari  kultur SPS. Keberhasilan transformasi tebu me-lalui Agrobacterium ditunjukkan oleh ekspresi transien dari gen GUS dan kemampuan dari trans-forman untuk tumbuh di media yang mengandung    50 ppm kanamisin.

scholarly journals Agrobacterium-mediated in planta genetic transformation of winter wheat (Triticum aestivum L.)

2018 ◽  
Vol 22 ◽  
pp. 293-298
Author(s):  
S. I. Mykhalska ◽  
A. G. Komisarenko ◽  
V. M. Kurchii ◽  
O. M. Tishchenko
Keyword(s):  
Triticum Aestivum ◽  
Transgenic Plants ◽  
Genetic Transformation ◽  
Winter Wheat ◽  
Natural Frequency ◽  
Triticum Aestivum L ◽  
Transformation Method ◽  
In Planta ◽  
Agrobacterium Mediated Transformation ◽  
Pollination Methods

Aim. To optimize the agrobacterium-mediated method of winter wheat transformation (Triticum aestivum L.); to select the conditions and period of inoculation to effectively transfer the genes during pollination. Methods. Agrobacterium-mediated in planta genetic transformation of winter wheat (Triticum aestivum L.) during pollination. Results. The conditions for agrobacterium-mediated transformation method of winter wheat during natural (frequency pollination was 1 %) and non-natural (frequency pollination was 4 %) pollination were defined. Conclusions. The possibility of integrating transgenes into the genome of winter wheat plants by the method of Agrobacterium-mediated transformation in planta in the process of forced and natural pollination is demonstrated. It is found that the transformation efficiency to a large extent depends on the plant genotype and the method of carrying out the transformation procedure. The selection of transgenic plants under water deficit conditions allowed to identify the plants with functional transgene. The signs of functioning transgene have been remaining in the next generation of genetically modified winter wheat. Keywords: Triticum aestivum L., Agrobacterium-mediated transformation in planta, transgenic plants, seeds.

scholarly journals Agrobacterium-mediated genetic transformation of Chinese chestnut (Castanea mollissima Blume)

2019 ◽  
Vol 140 (1) ◽  
pp. 95-103 ◽  
Author(s):  
Zhi-lin Sun ◽  
Xiao Li ◽  
Wan Zhou ◽  
Jun-di Yan ◽  
Yue-rong Gao ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Economic Value ◽  
Target Material ◽  
Normal Growth ◽  
Transformation Method ◽  
Transformation System ◽  
Embryogenic Calli ◽  
Chinese Chestnut ◽  
Castanea Mollissima

Abstract Chinese chestnut (Castanea mollissima) is an important germplasm resource for the breeding of Castanea species worldwide with vital ecological and economic value. Biotechnology overcomes the limitations of traditional breeding and accelerates germplasm improvement. However, a genetic transformation system for Chinese chestnut has not yet been established. In this study, a stable and efficient Agrobacterium-mediated genetic transformation method for Chinese chestnut is described. Embryogenic calli of C. mollissima cv. ‘Yanshanhongli’ were used as the target material. The sensitivity of embryogenic calli to kanamycin was determined, whereby the proliferation of non-transformed calli was completely inhibited at 180 mg/L. Antibiotic inhibition results for Chinese chestnut embryogenic calli showed that 50 mg/L cefotaxime and 500 μM timentin completely inhibited the growth of Agrobacterium tumefaciens but did not affect the normal growth of Chinese chestnut embryogenic calli. When embryogenic calli were co-cultured for 2 days with Agrobacterium tumefaciens strain AGL1 harboring the PBI121-EGFP plasmid, an embryogenic callus transformation efficiency of 4.55% was obtained, and two transgenic chimera were acquired. This Agrobacterium-mediated transformation system for Chinese chestnut provides a fundamental platform for genetic improvement of core germplasm and for further verification of gene function.

scholarly journals Regeneration of oil palm plantlets introduced by P5CS gene using Agrobacterium-mediated transformation

2019 ◽  
Vol 87 (2) ◽  
Author(s):  
Asmini Budiani ◽  
Imam Bagus Nugroho ◽  
Hayati Minarsih ◽  
Imron Riyadi
Keyword(s):  
Escherichia Coli ◽  
Agrobacterium Tumefaciens ◽  
Genetic Engineering ◽  
Oil Palm ◽  
Elaeis Guineensis ◽  
Sybr Green ◽  
Gus Assay ◽  
Embryogenic Calli ◽  
Agrobacterium Mediated Transformation ◽  
P5cs Gene

AbstrakCekaman kekeringan dapat mempengaruhi produktivitas tanaman perkebunan. Rekayasa genetika merupakan salah satu cara untuk meningkatkan produktivitas tanaman perkebunan penting seperti kelapa sawit. Tujuan dari penelitian ini adalah melakukan perekayasaan kelapa sawit melalui introduksi gen P5CS dengan transformasi berbasis Agrobacterium untuk meningkatkan ketahanan tanaman terhadap cekaman kekeringan. Pada penelitian ini perakitan kelapa sawit transgenik yang tahan terhadap cekaman kekeringan dilakukan melalui transformasi gen P5CS (Δ1-pyrroline-5-carboxylate synthetase) ke dalam kalus embriogenik (embryogenic calli – EC) menggunakan Agrobacterium. Plasmid pBI_P5CS yang membawa gen P5CS ditransfer dari Escherichia coli XL1 Blue ke Agrobacterium tumefaciens AGL1 melalui konjugasi. Selanjutnya klon Agrobacterium yang membawa plasmid pBI_P5CS digunakan untuk menginfeksi kalus embriogenik kelapa sawit dengan perlakuan 100 ppm asetosiringon. Kalus transforman diregenerasi pada media de Fossard (DF) yang ditambahkan 50 ppm kanamisin dan 250 ppm sefotaksim. Kalus transforman diseleksi melalui uji GUS dan metode PCR menggunakan primer NPTII dan P5CS1. Uji GUS dilakukan untuk menyeleksi kalus transforman yang ditunjukkan dengan reaksi positif pembentukan warna biru pada kalus yang berhasil ditransformasi dengan konstruk pBI_P5CS. Pengujian dengan menggunakan PCR memberikan hasil positif dengan adanya profil pita PCR pada visualisasi menggunakan pewarnaan SYBR Green, yang menunjukkan amplikon berukuran ~ 0,7 kb untuk gen NPTII dan ~ 0,4 kb untuk gen P5CS pada elektroforesis dengan gel agarosa. Hasil dari penelitian ini adalah diperolehnya kalus transforman terseleksi yang telah diregenerasi dan tumbuh menjadi planlet.[Kata kunci: cekaman kekeringan, Elaeis guineensis Jacq., rekayasa genetika, planlet]Abstract      Environmental abiotic stressors particularly drought has detrimental effects upon the productivity of estate crops. Increasing the crop tolerance towards drought stress through genetic engineering is one of the strategies employed to maintain steady productivity of valuable crop, i.e. oil palm. The aim of this study was to engineer oil palm with a better tolerance towards drought by introducing P5CS (Δ1-pyrroline-5-carboxylate synthetase) gene via Agrobacterium–mediated transformation into embryogenic calli (EC). The pBI_P5CS plasmid harboring P5CS gene was transferred from Escherichia coli XL1 Blue to Agrobacterium tumefaciens AGL1 by conjugation. The positive clone of transformed Agrobacterium was then used to infect oil palm EC by the addition of 100 ppm acetosyringone. The transformed ECs were regenerated in the de Fossard (DF) media supplemented by 50 ppm kanamycin and 250 ppm cefotaxime followed by GUS assay and PCR-based screening using NPTII and P5CS1 primers. The positive EC clones were confirmed by GUS assay, which produced blue coloration on positive transformed oil palm EC. A positive result of PCR screenings was depicted by PCR products in SYBR Green staining gel agarose electrophoresis with the expected band size of ~ 0.7 kb for the NPTII gene and ~ 0.4 kb for the P5CS gene. This study has successfully selected and regenerated pBI_P5CS transformed oil palm embryogenic calli into plantlets.[Keywords: drought tolerance, Elaeis guineensis Jacq., genetic engineering, plantlets]

scholarly journals Evaluasi varietas, sumber eksplan dan strain Agrobacterium terhadap keberhasilan transformasi tebu dengan gen P5CS Evaluation of varieties, explant sources, and Agrobacterium strains for successful sugarcane transformation using P5CS gene

2016 ◽  
Vol 83 (1) ◽  
Author(s):  
Hayati MINARSIH ◽  
Dwi SUBIYARTI ◽  
Imron RIYADI ◽  
Soekarno Mismana PUTRA ◽  
Laksmi AMBARSARI
Keyword(s):  
Drought Stress ◽  
Agrobacterium Tumefaciens ◽  
Somatic Embryos ◽  
Saccharum Officinarum ◽  
Transformation Method ◽  
Embryogenic Calli ◽  
Temporary Immersion System ◽  
Agrobacterium Strain ◽  
Solid Media ◽  
P5cs Gene

Abstract Genetic transformation can be used as an alter-native to develop sugarcane (Saccharum officinarum L.) tolerant to drought stress. P5CS gene has a role in biosynthesis of proline, an amino acid that accumulated under drought stress conditions. Transfer of a P5CS gene construct into plant cells in conjunction with regeneration of transgenic plantlets may develop sugarcane tolerant to drought stress. The aim of this research was to obtain an optimal transformation method which includes a suitable strain of Agrobacterium tumefaciens, and the best sugarcane explant and variety. The results showed that transfer of P5CS gene has been successfully carried out on sugarcane explants from solid media-derived calli, embryogenic calli and somatic embryos derived from temporary immersion system (TIS) culture. Whilst Agrobacterium strain LBA4404 was indicated as the most effective transformation vector. The regeneration of Kidang Kencana variety transformants from calli and somatic embryos was better than those of PS 881 and PS 891. The best performance of transformants based on the source of explants obtained from somatic embryos from TIS culture. Moreover, a succesfull Agrobacterium mediated transformation on sugarcane was indicated by transient expression of Gus gene and the ability of the transformants grew in a selection medium containing 50 ppm of kanamycin.Abstrak Transformasi genetik dapat digunakan sebagai upaya untuk merakit tebu (Saccharum officinarum L.) toleran terhadap cekaman kekeringan. Gen P5CS diketahui  berperan  dalam  biosintesis  prolin,  yaitu asam amino yang umumnya terakumulasi ketika tanaman mengalami cekaman kekeringan. Transfor-masi gen P5CS dan regenerasi transgeniknya mungkin dapat menghasilkan tanaman tebu trans-genik yang toleran terhadap cekaman kekeringan. Tujuan penelitian ini adalah untuk mendapatkan metode transformasi yang optimum yang mencakup strain Agrobacterium tumefaciens yang sesuai, sumber eksplan dan varietas tebu terbaik sebagai target transformasi. Hasil penelitian menunjukkan bahwa transformasi gen P5CS telah berhasil dilakukan ke eksplan tebu baik yang berupa kalus asal media padat maupun kalus embriogenik dan embrio somatik asal kultur sistem perendaman sesaat (SPS). Sementara itu strain A. tumefaciens LBA4404 menunjukkan hasil yang paling efektif sebagai vektor transformasi. Pertumbuhan transforman baik pada kalus maupun embrio somatik pada varietas Kidang Kencana terlihat paling baik dibandingkan dengan varietas PS 881 dan PS 891. Sumber eksplan yang paling efektif adalah embrio somatik yang diperoleh dari  kultur SPS. Keberhasilan transformasi tebu me-lalui Agrobacterium ditunjukkan oleh ekspresi transien dari gen GUS dan kemampuan dari trans-forman untuk tumbuh di media yang mengandung    50 ppm kanamisin.

T-DNA integration patterns in transgenic plants mediated by Agrobacterium tumefaciens

2011 ◽  
Vol 33 (12) ◽  
pp. 1327-1334 ◽  
Author(s):  
Lin YANG ◽  
Feng-Ling FU ◽  
Wan-Chen LI
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transgenic Plants ◽  
Dna Integration
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