scholarly journals Reconstruction and validation of three different binary vectors suitable for generation of genetically engineered Helicoverpa protected crops

2019 ◽  
Vol 79 (01) ◽  
Author(s):  
Nayana Hazarika ◽  
Rashmi Rekha Boruah ◽  
Pratap Jyoti Handique ◽  
Sumita Acharjee ◽  
Bidyut Kumar Sarmah
Keyword(s):  
Transgenic Crops ◽  
Transformation Method ◽  
Small Subunit ◽  
Genetically Engineered ◽  
Agrobacterium Strain ◽  
Binary Vectors ◽  
Tobacco Leaves ◽  
Cry1ac Gene ◽  
Assembly Method ◽  
Cry1ac Protein

Availability of a suitable plant transformation binary vector is necessary for the generation of transgenic crops with an adequate expression of transgenic proteins. Therefore, three binary vectors were constructed viz., pBK204, pBK205, and pBK206 harboring either a truncated or a full-length version of a Cry1Ac gene for the generation of Helicoverpa protected crops. Two different promoters viz., Arabidopsis Rubisco small subunit (AtSSU) gene promoter or CaMV35S promoters were used to regulate the various versions of Cry1Ac gene. The binary vectors were reconstructed either by the Gibson assembly method and others by ligating the restriction enzyme digested fragments. The reconstructed binary vectors were mobilized into Agrobacterium strain AGL1 and validated by Agrobacterium infiltration assays of Nicotiana benthamiana. The amount of Cry1Ac protein accumulated in the Agroinfiltrated tobacco leaves was quantified using the quantitative ELISA assay. The expression of the Cry1Ac protein in the tobacco leaves ranged from 0.25 to 0.26 µg /g fresh weight (FW) when transformed with these three constructs. Thus, the vectors constructed in this study appeared to be suitable for generation of Helicoverpa resistant transgenic crops by Agrobacterium-mediated genetic transformation method.

scholarly journals Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Mohamed Ramadan ◽  
Muna Alariqi ◽  
Yizan Ma ◽  
Yanlong Li ◽  
Zhenping Liu ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Sequence Similarity ◽  
High Specificity ◽  
Transformation Method ◽  
High Sequence Similarity ◽  
Single Experiment ◽  
Next Generation Sequencing Technology ◽  
Cotton Transformation ◽  
Assembly Method ◽  
Multiple Genes

Abstract Background Upland cotton (Gossypium hirsutum), harboring a complex allotetraploid genome, consists of A and D sub-genomes. Every gene has multiple copies with high sequence similarity that makes genetic, genomic and functional analyses extremely challenging. The recent accessibility of CRISPR/Cas9 tool provides the ability to modify targeted locus efficiently in various complicated plant genomes. However, current cotton transformation method targeting one gene requires a complicated, long and laborious regeneration process. Hence, optimizing strategy that targeting multiple genes is of great value in cotton functional genomics and genetic engineering. Results To target multiple genes in a single experiment, 112 plant development-related genes were knocked out via optimized CRISPR/Cas9 system. We optimized the key steps of pooled sgRNAs assembly method by which 116 sgRNAs pooled together into 4 groups (each group consisted of 29 sgRNAs). Each group of sgRNAs was compiled in one PCR reaction which subsequently went through one round of vector construction, transformation, sgRNAs identification and also one round of genetic transformation. Through the genetic transformation mediated Agrobacterium, we successfully generated more than 800 plants. For mutants identification, Next Generation Sequencing technology has been used and results showed that all generated plants were positive and all targeted genes were covered. Interestingly, among all the transgenic plants, 85% harbored a single sgRNA insertion, 9% two insertions, 3% three different sgRNAs insertions, 2.5% mutated sgRNAs. These plants with different targeted sgRNAs exhibited numerous combinations of phenotypes in plant flowering tissues. Conclusion All targeted genes were successfully edited with high specificity. Our pooled sgRNAs assembly offers a simple, fast and efficient method/strategy to target multiple genes in one time and surely accelerated the study of genes function in cotton.

Genetically Engineered Saccharomyces cerevisiae Strain that Can Ultilize Both Xylose and Glucose for Fermentation

2013 ◽  
Vol 448-453 ◽  
pp. 1637-1643
Author(s):  
Jing Ping Ge ◽  
Lu Yan Zhang ◽  
Wen Xiang Ping ◽  
Meng Yun Zhang ◽  
Yan Shen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Pathway ◽  
Ethanol Yield ◽  
Transformation Method ◽  
Fuel Ethanol ◽  
Genetically Engineered ◽  
Lithium Acetate ◽  
Expression Plasmid ◽  
Primary Problem

The primary problem in producing fuel ethanol through microorganism fermentation with lignocellulose is the strain. We constructed a URA3-directed low copy integration-expression plasmid pZMYBX1 and rDNA-directed high copy integration-expression plasmid pZMYX2. Using the lithium acetate transformation method, we co-transformed the linearized plasmid pZMYBX1 (StuI) and pZMYX2 (HpaI) into theS. cerevisiaecells. Ultimately, we obtain three recombinants: HDY-ZMYWBG1, HDY-ZMYWBG2 and HDY-ZMYWBG3. The ethanol yield for HDY-ZMYWBG1 and HDY-ZMYWBG3 are 0.368 g/g and 0.365 g/g, respectively, which are higher than the 0.330 g/g yield for W5. This findings show that the xylose metabolic pathway could be introduced into theS. cerevisiaeto produce an alternative strain for the production of biological ethanol from lignocellulose substrate.

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 Final Health and Environmental Risk Assessment of Genetically Modified Soybean MON 87701

2021 ◽  
pp. 132-133
Author(s):  
Åshild Kristin Andreassen ◽  
Nana Yaa Ohene Asare ◽  
Anne Marie Bakke ◽  
Knut Kelkås Dahl ◽  
Knut Thomas Dalen ◽  
...  
Keyword(s):  
Environmental Risk ◽  
Nutritional Assessment ◽  
Current Knowledge ◽  
Field Trials ◽  
Genetically Modified Soybean ◽  
Bioinformatics Analyses ◽  
Cry1ac Gene ◽  
Soybean Cultivars ◽  
Conventional Counterpart ◽  
Cry1ac Protein

Soybean MON 87701 expresses the cry1Ac gene from Bacillus thuringiensis. The encoded Cry1Ac protein confers resistance against specific lepidopteran pests. Updated bioinformatics analyses of the inserted DNA and flanking sequences in soybean MON 87701 have not indicated a potential production of harmful toxins and allergens or polypeptides caused by the genetic modification. Genomic stability of the functional insert and consistent expression of the cry1Ac gene, have been shown over several generations of soybean MON 87701. Data from several field trials performed in USA, Canada, Chile and Argentina during 2005-2006 show that soybean MON 87701 is compositionally, morphologically and agronomically equivalent to its conventional counterpart and other commercial soybean cultivars. Subchronic feeding studies with rats as well as nutritional assessment with broilers have not revealed relevant adverse effects of MON 87701. These studies indicate that MON 87701 is nutritionally equivalent to and as safe as conventional soybean cultivars. The Cry1Ac protein produced in soybean MON 87701 do not show sequence resemblance to known toxins or IgE-dependent allergens, nor has the whole GM plant been reported to cause changes in IgE-mediated allergic reactions in patients reactive to soybean or in non-ectopic control individuals. Soybean is not cultivated in Norway, and there are no cross-compatible wild or weedy relatives of soybean in Europe.  Based on current knowledge and considering the intended uses, which exclude cultivation, the VKM GMO Panel concludes that soybean MON 87701 with the Cry1Ac protein:    -   Is compositionally, morphologically and agronomically equivalent to its conventional counterpart and other commercial soybean cultivars  -   Is unlikely to introduce a toxic or allergenic potential in food or feed compared to conventional soybean cultivars  -   Is nutritionally equivalent to and as safe as its conventional counterpart and other conventional soybean cultivars  -    Does not represent an environmental risk in Norway.

Efficacy of Cry1Ac protein against gypsy moth and fall webworm in transgenic poplar (Populus davidiana × Populus bolleana) by bioassay

2018 ◽  
Vol 98 (4) ◽  
pp. 844-850 ◽  
Author(s):  
Liping Ding ◽  
Yajuan Chen ◽  
Hongzhi Wang ◽  
Jianhua Wei
Keyword(s):  
Transgenic Plants ◽  
Gypsy Moth ◽  
Enzyme Linked Immunosorbent Assay ◽  
Hyphantria Cunea ◽  
Cry1ac Gene ◽  
Populus Davidiana ◽  
Transgenic Lines ◽  
Insect Mortality ◽  
Fall Webworm ◽  
Cry1ac Protein

A modified Bacillus thuringiensis (Bt) cry1Ac gene was introduced into poplar ‘Shanxin’ (Populus davidiana × Populus bolleana) by Agrobacterium-mediated transformation. Seventeen cry1Ac transgenic lines were regenerated. The integration and expression of the cry1Ac gene in these transgenic lines were analyzed by polymerase chain reaction (PCR) and reverse-transcription quantitative PCR, respectively. Variable amounts of cry1Ac mRNA accumulated in different transgenic plants. The expression of Cry1Ac toxin protein in transgenic plants was analyzed by enzyme-linked immunosorbent assay, the concentration ranged from 0.12 to 21.00 μg g−1. The concentration of Cry1Ac protein was highly consistent with mRNA amount in each transgenic line. Leaf section bioassays were conducted using the larvae of both gypsy moth (Lymantria dispar) and fall webworm (Hyphantria cunea). Those plants, except lines 10 and 11, caused rapid mortality of all gypsy moth and fall webworm larvae with almost no defoliation, while lines 10 and 11, with Cry1Ac protein concentrations of 0.12 and 0.76 μg g−1, respectively, provided relatively weak insect protection. Results of insect bioassays indicate that Cry1Ac protein expression over 1.3 μg g−1 consistently produced high insect mortality for both species of moths. The correlation between concentration of Cry1Ac protein and insect mortality is important to understand for managing insect pests with transgenic plants. Transgenic poplars with high insect resistance will be useful tools for managing Lepidoptera pests of these trees.

Obtaining the rapA1 genome-transformed tomato plants

Biomics ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 153-158
Author(s):  
Л.Р. Хакимова ◽  
О.В. Чубукова ◽  
З.Р. Вершинина
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transgenic Crops ◽  
Transformation Method ◽  
Root Surface ◽  
Tomato Plants ◽  
Lycopersicon Lycopersicum

Bacterial agglutinin RapA1 is involved in the attachment of rhizobia to the roots of macrosymbiont plants. Obtaining transgenic crops that produce this protein directly on the root surface is important for studying the symbiosis of these plants with rhizobia. Tomatoes (Lycopersicon lycopersicum L.) cultivar Gruntovy Gribovskiy 1180 were transformed with the gene rapA1 using the Agrobacterium tumefaciens AGL0 strain carrying the vector pCambia1301LPSLRapA1. The efficiency of the developed transformation method was about 5%.

scholarly journals Agrobacterium-Mediated Capsicum annuum Gene Editing in Two Cultivars, Hot Pepper CM334 and Bell Pepper Dempsey

2021 ◽  
Vol 22 (8) ◽  
pp. 3921
Author(s):  
Sung-il Park ◽  
Hyun-Bin Kim ◽  
Hyun-Ji Jeon ◽  
Hyeran Kim
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Capsicum Annuum ◽  
Gene Editing ◽  
Binary Vector ◽  
Transformation Method ◽  
Bell Pepper ◽  
Hot Pepper ◽  
Genome Sequences ◽  
Agrobacterium Strain ◽  
Cultivated Species

Peppers (Capsicum annuum L.) are the most widespread and cultivated species of Solanaceae in subtropical and temperate countries. These vegetables are economically attractive worldwide. Although whole-genome sequences of peppers and genome-editing tools are currently available, the precision editing of peppers is still in its infancy because of the lack of a stable pepper transformation method. Here, we employed three Agrobacterium tumefaciens strains—AGL1, EHA101, and GV3101—to investigate which Agrobacterium strain could be used for pepper transformation. Hot pepper CM334 and bell pepper Dempsey were chosen in this study. Agrobacterium tumefaciens GV3101 induced the highest number of calli in cv. Dempsey. All three strains generated similar numbers of calli for cv. CM334. We optimized a suitable concentration of phosphinothricin (PPT) to select a CRISPR/Cas9 binary vector (pBAtC) for both pepper types. Finally, we screened transformed calli for PPT resistance (1 and 5 mg/L PPT for cv. CM334 and Dempsey, respectively). These selected calli showed different indel frequencies from the non-transformed calli. However, the primary indel pattern was consistent with a 1-bp deletion at the target locus of the C. annuumMLO gene (CaMLO2). These results demonstrate the different sensitivity between cv. CM334 and Dempsey to A. tumefaciens-mediated callus induction, and a differential selection pressure of PPT via pBAtC binary vector.

Can Risk Analysis “Colorize” the Black and White of Transgenic Crops?

2001 ◽  
Vol 2 (1) ◽  
pp. 11 ◽  
Author(s):  
Michelle Marvier
Keyword(s):  
Risk Analysis ◽  
Agricultural Biotechnology ◽  
New Technology ◽  
Transgenic Crops ◽  
Genetically Engineered ◽  
The State ◽  
Crop Plants ◽  
Ecological Risks ◽  
Black And White

Scientifically-based risk analysis of transgenic crops might help this new technology to develop along paths most beneficial to society. After briefly reviewing the state of agricultural biotechnology, I focus on the ecological risks of genetically engineered crop plants. I suggest how better risk analysis could promote a more rational discussion regarding biotechnology. Accepted for publication 27 August 2001. Published 31 August 2001.

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