scholarly journals Glyphosate selection of maize transformants containing CP4epsps gene

2020 ◽  
Vol 26 ◽  
pp. 239-244
Author(s):  
I. O. Nitovska ◽  
B. V. Morgun ◽  
O. Ye. Abraimova ◽  
T. M. Satarova

Aim. To study the selection conditions of maize transformants containing the CP4epsps gene using glyphosate as a selective agent. Methods. Tissue culture in vitro, Agrobacterium-mediated transformation, selection of transgenic plants, isolation of total plant DNA, analysis of plant DNA by polymerase chain reaction (PCR). Results. The morphogenic maize callus of immature embryos of the hybrid (PLS61)R2×PLS61 was produced, which had a high regeneration rate (up to 95%), that persisted over long cultivation. Agrobacterium mediated transformation of the morphogenic callus and selection of the transgenic material using glyphosate yielded maize transformants containing the CP4epsps gene at a frequency of 1%. Conclusions. Maize genotype (PLS61)R2×PLS61 is promising for studies on the maize genetic transformation, in particular for the production of transgenic maize resistant to glyphosate herbicide. The use of morphogenic maize callus (PLS61)R2×PLS61 and glyphosate as a selective agent at a concentration of 0.1 mM and 0.25 mM in media for callusogenesis and 0.01 mM in the medium for regeneration was effective for the selection of transgenic plants with the gene CP4epsps. Keywords: Zea mays L., morphogenic callus, Agrobacterium-mediated transformation, PCR, genetic engineering.

Author(s):  
I. O. Nitovska ◽  
I. K. Komarnytskyi ◽  
B. V. Morgun

Aim. Glyphosate selection has a number of advantages over other commonly used selectable markers for maize. There is some natural variability within maize germplasm for degree of sensitivity to glyphosate. We investigated the selective effect of glyphosate for production transgenic maize callus after Agrobacterium-mediated transformation among geno-types of Ukrainian plant breeding. Methods. Agrobacterium-mediated transformation, glyphosate selection in vitro, and PCR analysis were used to obtain transgenic maize callus and to confirm its status. Results. An efficient selectable marker system for production transgenic maize callus lines tolerant to herbicide glyphosate was proposed. Calluses of four maze genotypes of Ukrainian plant breeding and pCB135 vector containing CP4epsps gene were used in Agrobacterium-mediated transformation experiments. Three callus maize lines of DK267×PLS61 genotype containing CP4epsps gene were obtained. Conclusions. The use of glyphosate as a selective agent after Agrobacterium-mediated transformation proved to be effective for transgenic maize callus lines production containing the gene CP4epsps. The success of Agrobacterium-mediated transformation of maize callus strongly depended on the genotype of source ma-terial. Keywords: Agrobacterium-mediated maize transformation, CP4epsps gene, glyphosate selection, PCR analysis.


2018 ◽  
Vol 22 ◽  
pp. 222-227
Author(s):  
O. M. Honcharuk ◽  
O. V. Dubrovna

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.


2000 ◽  
Vol 66 (3) ◽  
pp. 1237-1242 ◽  
Author(s):  
Kaare M. Nielsen ◽  
Jan D. van Elsas ◽  
Kornelia Smalla

ABSTRACT Here we show that horizontal transfer of DNA, extracted from transgenic sugar beets, to bacteria, based on homologous recombination, can occur in soil. Restoration of a 317-bp-deleted nptIIgene in Acinetobacter sp. strain BD413(pFG4) cells incubated in sterile soil microcosms was detected after addition of nutrients and transgenic plant DNA encoding a functionalnptII gene conferring bacterial kanamycin resistance. Selective effects of the addition of kanamycin on the population dynamics of Acinetobacter sp. cells in soil were found, and high concentrations of kanamycin reduced the CFU ofAcinetobacter sp. cells from 109 CFU/g of soil to below detection. In contrast to a chromosomalnptII-encoded kanamycin resistance, the pFG4-generated resistance was found to be unstable over a 31-day incubation period in vitro.


Author(s):  
Н.В. Пролётова ◽  
Л.П. Кудрявцева

Цель исследований – оптимизация селективных сред для проведения отбора in vitro каллусных клеток льна, устойчивых к культуральному фильтрату штаммов возбудителя антракноза и создание in vitro новых генотипов, устойчивых к болезни. В результате исследований уточнен состав культурального фильтрата штаммов антракноза. Выявлено, что токсичность культуральных фильтратов не зависела от вирулентности используемых штаммов – более токсичными оказались культуральные фильтраты штаммов 784 (сильновирулентного) и 780 (средневирулентного) (загнивание и отмирание первичных корешков на 5 сутки наблюдали у 67 – 88% проросших семян), менее токсичны – штаммы 793 (сильновирулентный) и 788 (слабовирулентный) (на 5 сутки загнивание и отмирание первичных корешков отмечено у 9 – 15% проросших семян). Установлено, что морфогенные очаги формировались активнее у генотипов, морфогенный каллус которых переносили на среду с аналогичной или более высокой концентрацией культурального фильтрата. Показано, что на 14 сутки во втором пассаже с большей частотой формировались морфогенные каллусы, почки и побеги при использовании в первом и втором пассажах селективной среды, содержащей культуральный фильтрат в концентрации 40 мл/л, или в первом пассаже – 40 мл/л, а во втором – 44 мл/л. Выделены генотипы, сохраняющие устойчивость к антракнозу в течение трёх поколений на уровне 50 – 60%: НО-78 х Ленок, HJI-103-2 х Ленок, НЛ-40-1 х Ленок, HЭ-38 х Росинка, НЭ-36 х Ленок, НЭ-17 х Ленок, HЭ-16-2 х Росинка. Research objective – optimization of selective media for in vitro selection of flax callus cells resistant to culture filtrate of anthracnose pathogen strains and in vitro creation of new disease-resistant genotypes. As a result of the research, the composition of the culture filtrate of anthracnose strains was clarified. It was revealed that the toxicity of cultural filtrates did not depend on the virulence of the strains used - cultural filtrates of strains 784 (highly virulent) and 780 (medium virulent) turned out to be more toxic (decay and death of primary roots on day 5 was observed in 67 - 88% of germinated seeds), less toxic - strains 793 (strongly virulent) and 788 (weakly virulent) (on the 5th day, decay and death of primary roots was noted in 9-15% of germinated seeds). It was found that morphogenic foci were formed more actively in genotypes, the morphogenic callus of which was transferred to a medium with a similar or higher concentration of the culture filtrate. It was shown that on the 14th day in the second passage, morphogenic callus, buds and shoots were formed with a greater frequency when using in the first and second passages a selective medium containing a culture filtrate at a concentration of 40 ml/l, or in the first passage - 40 ml/l, and in the second - 44 ml/l. Genotypes were identified that retain resistance to anthracnose for three generations at a level of 50 - 60%: NO-78 x Lenok, HJI-103-2 x Lenok, NL-40-1 x Lenok, NE-38 x Rosinka, NE-36 x Lenok, NE-17 x Lenok, NE-16-2 x Rosinka.


Author(s):  
Mohammad Naim Noori

Cell and tissue selection in vitro allows targeted selection of the desired traits under severe selective conditions at the level of individual cells and tissues. On the basis of multistage cell and tissue selection with the use of a selective agent - neutral osmotic polyethylene glycol 6000 in increasing concentrations -5%, 10%, 15%, 20% of the final volume of the nutrient medium, callus of the Zhansaya soybean variety that are stably resistant to osmotic stress have been obtained. Regeneration from callus was noted only in8.3 % of the planted callus 6 soybean regenerants plants resistant to osmotic stress were obtained from callus.


2020 ◽  
Vol 26 ◽  
pp. 270-275
Author(s):  
L. V. Slivka ◽  
O. V. Dubrovna

Aim. Optimization of conditions for genetic transformation of new perspective winter wheat genotypes and production of transgenic plants. Methods. Agrobacterium-mediated transformation in vitro culture using callus cultures. Results. The influence of the optical density of cells of agrobacterial suspension, the concentration of the antibiotic cefotaxime, the duration of coculture on the frequency of obtaining kanamycin-resistant regenerants of new winter wheat genotypes by genetic transformation of callus cultures were investigated. By Agrobacterium-mediated transformation of morphogenic calluses of new perspective winter wheat genotypes were obtained plant-regenerants in the genome of which revealed the complete incorporation of a genetic construct containing oat and nptII transgenes. Conclusions. Agrobacterium-mediated transformation of callus cultures of new perspective winter wheat genotypes was optimized, and transgenic plants with the target gene of ornithine-δ-aminotransferase were obtained. Keywords: Triticum aestivum, Agrobacterium-mediated transformation, callus cultures, ornithine-δ-aminotransferase gene.


2019 ◽  
Vol 20 (2) ◽  
pp. 279 ◽  
Author(s):  
Dengxiang Du ◽  
Ruchang Jin ◽  
Jinjie Guo ◽  
Fangdong Zhang

Several approaches have recently been adopted to improve Agrobacterium-mediated transformation of maize; however, about eight months of in vitro culture are still required to isolate transgenic plants. Furthermore, genetic transformation of maize depends on immature embryos, which greatly increases costs. Here, we report a method that ensures the competency of an embryogenic callus secondary culture under laboratory conditions for Agrobacterium-mediated transformation. Moreover, pretreatment of the cell wall with a mixed lytic enzyme solution prior to Agrobacterium infection, significantly improved transformation efficiency and stability. Average stable transformation efficiency was approximately 30.39%, with peaks of 94.46%. Expression and phenotypic analysis of the Rsc reporter gene were tested in the T0 generation of transgenic plants. Using this system, we successfully regenerated transgenic maize plantlets within three months of the emergence of the embryogenic callus. Additionally, we reduced somaclonal variation accompanying prolonged culture of maize cells in the dedifferentiated state, thus facilitating the molecular breeding of maize.


Agronomy ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1279
Author(s):  
Wojciech Marecki ◽  
Jadwiga Żebrowska

The soil pathogenic fungus Phytophthora cactorum causes the most dangerous diseases occurring in strawberry plantations—strawberry crown rot and leather rot. Modern biotechnology methods, e.g., in vitro culture selection and molecular diagnostics can be utilized in the selection of cultivars that are less susceptible or resistant to Phytophthora diseases. In this study, in vitro selection of four strawberry microclones: ‘Elsanta’, ‘Feltar’, ‘Teresa’ and ‘Plena SVdT’ against Phytophthora cactorum (Lebert and Cohn) J. Schröt was carried out. Molecular analysis with inter simple sequence repeat (ISSR) markers was also used to evaluate genetic similarity of the selected resistant plants. None of the analyzed microclones showed complete resistance to the selection factor, but there were plants in all tested microclones that survived the pressure of the pathogen. Results showed that susceptibility to this pathogenic fungus was significantly differentiated and depended on the microclone. The ‘Feltar’ microclone had the significantly lowest susceptibility to Phytophthora disease, followed by the microclones ‘Elsanta’ and ‘Teresa’ with significantly higher susceptibility. The ‘Plena SVdT’ microclone showed the highest susceptibility to Phytophthora disease. This differentiation was linked to the genetic similarity observed at deoxyribonucleic acid (DNA) level between the resistant plants selected from microclones. Cluster analysis revealed that microclones with similar susceptibility to phytophthorosis, i.e., ‘Elsanta’, ‘Feltar’ and ‘Teresa’, appeared to be genetically similar. The microclone ‘Plena SVdT’ revealed a different course of phytophthorosis from the aforementioned microclones, being the least genetically similar to them.


2020 ◽  
Vol 15 (3) ◽  
pp. 10-15
Author(s):  
Elena Vinogradova

The studies were carried out with the aim of studying the effect of various concentrations of sucrose and mannitol on seeds, immature embryos, and callus cultures of flax to develop a method for obtaining genotypes resistant to osmotic stress. The work was carried out in the Tver region in the laboratory of breeding technologies in 2017–2019. Flax varieties Barbara, Belinka, LM-98, Aurore, Tverskoy, Svetoch, Diplomat, Symfonia were used as objects of research. The seeds were obtained from the National Flax Collection of the Federal Scientific Center for Bast Crops. The effect of sucrose solution on the length of the primary root was detected at concentrations - 0; 8.7; 14.9%. To assess the germination energy of seeds under osmotic stress, the concentration of sucrose was reduced and the range 0 (control) ... 9% was considered. Immature embryos removed from the capsules on the 10th day after pollination were cultivated on MS medium with sucrose, as a selective agent, at a concentration of 5.0 ... 7.0%. Callus tissues were cultured using mannitol as an osmotic at concentrations of 0; 30.0; 36.4; 37.0; 37.4; 38.0 mg/l. Concentrations of 5.0, 6.0 and 7.0% of sucrose can be used as an osmotic differentiator for seeds (10 ... 80% of seeds germinated in the Belinka variety, 80 ... 100% in the Varbara variety, 80 ... 90% in the variety LM-98). Sucrose, as a selective agent, in a culture of immature flax embryos in vitro at a concentration of 5.0 ... 7.0% can be selective only for certain genotypes, for example, the Aurore variety. The selection of resistant callus cells, followed by the formation of adventive buds and shoots in the meristematic foci, can be carried out on media containing 30.0 or 36.4 mg / L of osmosis, which allows obtaining morphogenic callus, buds, shoots in all studied genotypes, as well as in the Aurore variety 1.1 ... 1.2 byp./callus, in the Tverskoy variety - 0.6 ... 0.8, in the Barbara variety - 1.0 ...1.1


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