scholarly journals Optimization of Agrobacterium-mediated transformation conditions of prospective genotypes of winter bread wheat by in planta method

2021 ◽  
Vol 28 ◽  
pp. 66-71
Author(s):  
O. V. Dubrovna ◽  
L. V. Slivka
Keyword(s):  
Transgenic Plants ◽  
Bread Wheat ◽  
Optical Density ◽  
Dna Transfer ◽  
Plant Genome ◽  
In Planta ◽  
Agrobacterium Mediated Transformation ◽  
The Third ◽  
Winter Bread Wheat ◽  
Inoculation Medium

Aim. Optimization of conditions for genetic transformation of new promising genotypes of winter bread wheat (T. aestivum L.) by in planta method. Methods. Agrobacterium-mediated transformation by in planta method using the strain AGL0 and vector construct pBi2E. Results. The influence of air temperature, optical density of cells of agrobacterial suspension, inoculation day and composition of inoculation medium on the frequency of obtaining transgenic plants of new winter wheat genotypes was studied. The dependence of the frequency obtaining of transgenic plants from environmental conditions, in particular temperature, has been established. It was found that the temperature regime of 20-22°C provided the largest number (4.8%) of wheat transformants, and when the temperature is reduced to 16-18°C there is a decrease in the efficiency of T-DNA transfer into the plant genome and the lowest frequency of transformation (0.7%). Conclusions. The largest number of transformants was obtained using a inoculation medium without sucrose, the optical density of cells of the agrobacterial suspension of 0.6 op.od. and inoculation on the third day after castration of ears. Keywords: T. aestivum, Agrobacterium-mediated transformation in planta, optimization of conditions.

scholarly journals Genetic transformation of promising genotypes of winter bread wheat by in planta method

2021 ◽  
Vol 28 ◽  
pp. 106-111
Author(s):  
L. V. Slivka ◽  
O. V. Dubrovna
Keyword(s):  
Triticum Aestivum ◽  
Transgenic Plants ◽  
Genetic Transformation ◽  
Bread Wheat ◽  
Optical Density ◽  
Triticum Aestivum L ◽  
In Planta ◽  
Agrobacterium Mediated Transformation ◽  
Winter Bread Wheat ◽  
Inoculation Medium

Aim. Optimization of conditions and genetic transformation of new promising genotypes of winter bread wheat (Triticum aestivum L.) by in planta method. Methods. Agrobacterium-mediated transformation by the in planta method using strain AGL0 and vector construct pBi-OAT. Results. The influence of air temperature, optical density of cells of agrobacterial suspension, inoculation day and composition of inoculation medium on the frequency of obtaining transgenic plants of new promising genotypes of winter wheat was studied. The dependence of the frequency of obtaining transgenic plants on environmental conditions, in particular temperature, has been established. It was found that the temperature regime of 20-22 °C provided the largest number (4.4%) of wheat transformants, and when the temperature is reduced to 16-18 °C there is a decrease in the efficiency of T-DNA transfer into the plant genome and the lowest frequency of transformation is observed. Conclusions. The largest number of transformants was obtained using a inoculation medium without sucrose, the optical density of cells of the agrobacterial suspension of 0.4 op.od. and inoculation on the third day after castration of ears. Keywords: Triticum aestivum L., Agrobacterium-mediated transformation, ornithine-δ-aminotransferase gene.

scholarly journals Receiving of genetic-modified wheat plants with heterolous ornitin-δ-aminotransferase gene

2018 ◽  
Vol 22 ◽  
pp. 222-227
Author(s):  
O. M. Honcharuk ◽  
O. V. Dubrovna
Keyword(s):  
Triticum Aestivum ◽  
Transgenic Plants ◽  
Bread Wheat ◽  
Binary Vector ◽  
Triticum Aestivum L ◽  
Callus Cultures ◽  
Pcr Analysis ◽  
Genetic Construct ◽  
Agrobacterium Mediated Transformation

Aim. Receiving of genetically modified plants of bread wheat with heterologous ornithine‑δ‑aminotransferase gene. Methods. Agrobacterium-mediated transformation of callus cultures in vitro, PCR-analysis. Results. By Agrobacterium-mediated transformation of the morphogenic calluses of bread wheat (Triticum aestivum L.) using the AGLO strain containing the binary vector pBi-OAT with the target ornithine-δ-aminotransferase (oat) and selective neomycinphosphotransferase II (nptII), transgenic plants-regenerators have been obtained. Conclusions. As a result of the genetic transformation of Zimoyarka variety, 12 wheat regenerants were obtained in the genome which revealed a complete integration of the genetic construct containing the oat and nptII transgenes. Keywords: Triticum aestivum L., Agrobacterium-mediated transformation, ornithine‑δ‑aminotransferase gene, PCR-analysis.

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.

Tolerance to, and Uptake and Degradation of 2,4,6-Trinitrotoluene (TNT) are Enhanced by the Expression of a Bacterial Nitroreductase Gene in Arabidopsis thaliana

2005 ◽  
Vol 60 (3-4) ◽  
pp. 272-278 ◽  
Author(s):  
Mami Kurumata ◽  
Misa Takahashi ◽  
Atsushi Sakamoto ◽  
Juan L. Ramos ◽  
Ales Nepovim ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Transgenic Plants ◽  
Degradation Products ◽  
Plant Genome ◽  
Pcr Analysis ◽  
Wild Type ◽  
In Planta ◽  
Gene Encoding ◽  
Plant Body ◽  
Wide Range

Abstract Arabidopsis thaliana was transformed with a gene encoding a nitroreductase (NTR, E.C. 1.6.99.7) with activity against a wide range of nitroaromatic compounds. The gene was transferred from Escherichia coli by an Agrobacterium-mediated in planta method. The ob­tained seeds were sowed to produce T1 plants, and they were assayed for the integration of the transgene in the plant genome. Transgenic plants that were positive with the PCR analysis were self-pollinated to produce T2 generation plants. Seven lines obtained were assayed for the NTR activity. While the noil-transformed wild-type plants showed no detectable NTR activity, the enzyme activity of the transgenic plant lines was approx. 20 times higher. Using the line with the highest NTR activity, the phytoremediation characteristics of plants against 2,4,6-trinitrotoluene (TNT) was investigated. While the wild-type plants did not grow in the presence of 0.1 mᴍ TNT, the transgenic plants grew almost normally in this condition. The uptake of TNT by seedlings of transgenic plants increased by 7 to 8 times when they were floated on TNT solution. HPLC analysis showed that the peak due to TNT taken up into plant body was much smaller in the transgenic plants as compared with that of the wild type, and that a number of peaks attributable to the degradation products of TNT, including 4-amino-2,6-dinitrotoluene, were detected in the extract from the transgenic plants. This indi­cates that the expression of bacterial NTR improved the capability of plants to degrade TNT.

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 Optimization of conditions of Agrobacterium-mediated transformation of bread wheat by the in planta

2019 ◽  
Vol 51 (4) ◽  
pp. 283-294
Author(s):  
O.V. Dubrovna ◽  
◽  
S.S. Kulesh ◽  
L.V. Slivka ◽  
Keyword(s):  
Bread Wheat ◽  
In Planta ◽  
Agrobacterium Mediated Transformation

scholarly journals IN-PLANTA TRANSFORMATION METHOD MEDIATED WITH Agrobacterium tumefaciens FOR T-DNA TRANSFER IN TABLE GRAPE (Vitis vinifera L.)

2019 ◽  
Vol 6 (2) ◽  
pp. 95
Author(s):  
Rindang Dwiyani ◽  
Hestin Yuswanti ◽  
Yuyun Fitriani ◽  
Bambang Sugiharto
Keyword(s):  
Transformation Method ◽  
Table Grape ◽  
Dna Transfer ◽  
Plant Genome ◽  
Nptii Gene ◽  
In Planta Transformation ◽  
In Planta ◽  
Simple Method ◽  
Leafy Shoots ◽  
Sweeping Method

The aim of the research is to investigate a simple method of in planta transformation method for T-DNA transfer in table grape.  The T-DNA harbored the S0SPS1 gene under the control of promoter of the 35S CaMV from the Cauliflower Mosaic Virus and contained the NPTII gene, a kanamycin-resistant gene as a selectable marker for transformant selection.  Six-month plants originated from cuttings were used as target plants.  We explored two methods of in planta transformation, namely ”dipping” and “sweeping”.  For both methods, the leaves of the target plants were removed and those of shoots without leaves were used as the target of  transformation.  In the “dipping method”, those shoots were dipped with the agrobacterial suspension for 60 seconds.  However, for the “sweeping method”, the scars (the spots where leaves were removed) were swept with agrobacterial suspension using a cotton bud.  Those treated non-leafy-shoots (from both methods) then were grown to be leafy shoots.  Those leafy shoots then were cut and transplanted into the soil and grown to be a whole plant. The leaves of those plants then were taken as samples for DNA extraction and PCR using primers of NPTII gene (Forward: 5’-GTCATCTCACCTTCCTCCTGCC-3’; Reverse: 5’ GTCGCTTGGTCGGTCATTTCG-3’) with expected amplified band of 550 bp. We found that only the “sweeping method” plants amplified the 550 bp bands, while those of the “dipping method”  did not.  We suggest that the T-DNA was successfully integrated into the genome of plants treated with the “sweeping method” but not with the “dipping method”.  Leaf sugar content (oBrix) of PCR-positive vines was higher than those of the wild-type vines, ensuring the integration of the T-DNA into the plant genome.

scholarly journals Citrus Cell Suspension Culture Establishment, Maintenance, Efficient Transformation and Regeneration to Complete Transgenic Plant

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 664
Author(s):  
M. Moniruzzaman ◽  
Yun Zhong ◽  
Zhifeng Huang ◽  
Huaxue Yan ◽  
Lv Yuanda ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Suspension Culture ◽  
Cell Suspension ◽  
Cell Suspension Culture ◽  
Culture System ◽  
Transformation Rate ◽  
Suspension Cell Culture ◽  
Citrus Reticulata ◽  
Agrobacterium Mediated Transformation ◽  
Solid Media

Agrobacterium-mediated transformation of epicotyl segment has been used in Citrus transgenic studies. The approach suffers, however, from limitations such as occasionally seed unavailability, the low transformation efficiency of juvenile tissues and the high frequency of chimeric plants. Therefore, a suspension cell culture system was established and used to generate transgenic plants in this study to overcome the shortcomings. The embryonic calli were successfully developed from undeveloped ovules of the three cultivars used in this study, “Sweet orange”-Egyptian cultivar (Citrus sinensis), “Shatangju” (Citrus reticulata) and “W. Murcott” (Citrus reticulata), on three different solid media. Effects of media, genotypes and ages of ovules on the induction of embryonic calli were also investigated. The result showed that the ovules’ age interferes with the callus production more significantly than media and genotypes. The 8 to 10 week-old ovules were found to be the best materials. A cell suspension culture system was established in an H+H liquid medium. Transgenic plants were obtained from Agrobacterium-mediated transformation of cell suspension as long as eight weeks subculture intervals. A high transformation rate (~35%) was achieved by using our systems, confirming BASTA selection and later on by PCR confirmation. The results demonstrated that transformation of cell suspension should be more useful for the generation of non-chimeric transgenic Citrus plants. It was also shown that our cell suspension culture procedure was efficient in maintaining the vigor and regeneration potential of the cells.

Sign in / Sign up

Export Citation Format

Share Document