scholarly journals AGROBACTER1UM-MED1ATED TRANSFORMATION OF APPLE

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 661e-661
Author(s):  
Rudaina Alrefai ◽  
Schuyler S. Korban ◽  
Leslie L. Domier
Keyword(s):  
Binary Vector ◽  
Gus Activity ◽  
Hygromycin Resistance ◽  
Gus Gene ◽  
Regeneration Medium ◽  
Helper Plasmid ◽  
Integrative Transformation ◽  
Transgenic Lines ◽  
Callus Lines

Two strains of Agrobacterium tumefaciens carrying a disarmed Ti-binary vector, pZA-7, were used as vectors for transformation of apple leaf segments, EHA101:pZA-7 carries a helper plasmid derived from pTiB0542, and C58Z707:pZA-7 carries a helper plasmid derived from pTiC58. The binary vector provides two selection markers, neomycin phosphotransferase (nptII) and hygromycin resistance genes, and a screening marker, β-glucuronidase (GUS) gene. Preliminary experiments were conducted to determine the effect of different concentrations of kanamycin, carbenicillin, and cefotaxime on regeneration of apple leaf sections and inhibition of A. tumefaciens strains. In vitro-derived leaf sections of `Royal Gala' apple were grown on a regeneration medium containing thidiazuron and NAA; these were then dipped into a suspension culture of A. tumefaciens and transferred to a fresh regeneration medium. Callus lines exhibiting kanamycin and hygromycin resistance were obtained mostly with agrobacterium strain EHA101:pZA-7. Expression of GUS activity was also determined in putative transformed calli. Southern blot analysis was used for confirming integrative transformation in transgenic lines.

scholarly journals Modification of plant regeneration medium decreases the time for recovery of Solanum lycopersicum cultivar M82 stable transgenic lines

2016 ◽  
Author(s):  
Sarika Gupta ◽  
Joyce Van Eck
Keyword(s):  
Plant Regeneration ◽  
Solanum Lycopersicum ◽  
Gene Function ◽  
Marker Gene ◽  
Transformation Method ◽  
Induction Medium ◽  
Root Induction ◽  
Regeneration Medium ◽  
Transgenic Lines

AbstractTomato (Solanum lycopersicum) has rapidly become a valuable model species for a variety of studies including functional genomics. A high-throughput method to obtain transgenic lines sooner than standard methods would greatly advance gene function studies. The goal of this study was to optimize our current transformation method by investigating medium components that would result in a decreased time for recovery of transgenics. For this study, 6-day-old cotyledon explants from Solanum lycopersicum cultivar M82 in vitro-grown seedlings were infected with the Agrobacterium tumefaciens strain LBA4404 containing the binary vector pBI121. This vector contains the β-glucuronidase reporter gene and the neomycin phosphotransferase II selectable marker gene that confers resistance to kanamycin. Modification of our standard plant regeneration medium with indole-3-acetic acid (IAA) at concentrations of either 0.05 mg/l or 0.1 mg/l decreased the recovery time for transgenic lines by 6 weeks as compared to our standard medium that contains zeatin as the only plant growth regulator. We observed 50% and 54% transformation efficiency on plant regeneration medium containing 0.05 mg/l and 0.1 mg/l IAA, respectively. Moreover, addition of 1 mg/l IAA to the root induction medium resulted in earlier root development than medium that did not contain IAA. Addition of IAA to the plant regeneration and rooting media did not have any negative effects on plant development. Recovery of transgenic lines in a shorter time results in higher throughput for the introduction of gene constructs and has the potential to decrease the time and resources needed to complete investigations of gene function.

scholarly journals Dissection of a Synthesized Quantitative Trait to Characterize Transgene Interactions

Genetics ◽  
1997 ◽  
Vol 147 (1) ◽  
pp. 315-320
Author(s):  
Jan-Peter Nap ◽  
Anthony J Canner ◽  
Ludmila Mlynárová ◽  
Willem J Stiekema ◽  
Ritsert C Jansen
Keyword(s):  
Gene Action ◽  
Plant Traits ◽  
Single Copy ◽  
Gus Activity ◽  
Single Locus ◽  
Gus Gene ◽  
Wild Type ◽  
Transgenic Lines ◽  
Insight Into ◽  
Chicken Lysozyme

Abstract Six transgenic tobacco lines, each homozygous for the β-glucuronidase (GUS) gene at a different locus, and wild type were selfed and intercrossed to evaluate GUS activity in all possible hemizygous, homozygous and dihybrid combinations of GUS alleles. The transgenic lines are characterized by their GUS activity (two low, three intermediate, one high), T-DNA complexity (four single-copy, two more complex single-locus) and the presence of the chicken lysozyme matrix-associated region (MAR) around the full T-DNA (two lines). Gene action and interaction was analyzed by weighted linear regression with parameters for additivity, dominance and epistasis. The analysis showed that each of the four single-copy lines acted fully additively. In contrast, the two complex single-locus lines showed classical single-locus overdominance and were epistatic dominant over all other GUS alleles. The latter is manifested in severe suppression of GUS activity in dihybrid lines, irrespective of the presence of MAR elements around the GUS gene. Such elements apparently do not protect against epistatic dominance. The quantitative data suggested that the epistatic dominance and overdominance are based on the same molecular mechanism. Our approach of a genetic analysis of quantitative variation in well-characterized transgenic lines provides a powerful tool to gain insight into complex plant traits.

AN AGROBACTERIUM-MEDIATED TRANSFORMATION SYSTEM FOR THE TOMATO CULTIVAR KECSKEMÉTI 262

2000 ◽  
Vol 48 (3) ◽  
pp. 221-226
Author(s):  
Csaba Bánfalvy ◽  
Zoltán Szabó
Keyword(s):  
Transformation Method ◽  
Transformation System ◽  
Regeneration Medium ◽  
Tomato Cultivar ◽  
Agrobacterium Strain ◽  
Cotyledon Explants ◽  
Glucuronidase Activity ◽  
Transgenic Lines ◽  
Foreign Genes

Cotyledon explants of tomato (Lycopersicum esculentum Mill.) cv. Kecskeméti 262 were excised from 9-day-old in vitro seedlings. The transformation method of Arrillaga et al. (1998) was tested and found to be effective for the introduction of foreign genes into this variety. Using an Agrobacterium strain carrying the plasmid 35S GUS INT, ten independent transgenic lines retaining all the phenotypic markers of cv. Kecskeméti 262 were isolated on regeneration medium containing kanamycin and were tested further for ß -glucuronidase activity. The results show that the method of Arrillaga et al. (1998) is suitable for the introduction of agronomically important genes into the cultivar Kecskeméti 262.

Agrobacterium-mediated transformation of Australian rice cultivars Jarrah and Amaroo using modified promoters and selectable markers

2000 ◽  
Vol 27 (3) ◽  
pp. 201 ◽  
Author(s):  
Narayana M. Upadhyaya ◽  
Brian Surin ◽  
Kerrie Ramm ◽  
Judy Gaudron ◽  
Petra H. D. Schünmann ◽  
...  
Keyword(s):  
Selectable Marker ◽  
Binary Vector ◽  
Marker Gene ◽  
Gus Activity ◽  
Hygromycin Resistance ◽  
Rice Cultivars ◽  
Selectable Marker Gene ◽  
Ubiquitin Promoter ◽  
Camv35s Promoter ◽  
Derivatives Of

We report the first successfulAgrobacterium-mediated transformation of Australian elite rice cultivars, Jarrah and Amaroo, using binary vectors with our improved promoters and selectable markers. Calli derived from mature embryos were used as target tissues. The binary vectors contained hph(encoding hygromycin resistance) or bar (encoding herbicide resistance) as the selectable marker gene and uidA (gus) or sgfpS65T as the reporter gene driven by different promoters. Use of Agrobacterium strain AGL1 carrying derivatives of an improved binary vector pWBVec8, wherein the CaMV35S driven hph gene is interrupted by the castor bean catalase 1 intron, produced a 4- fold higher number of independent transgenic lines compared to that produced with the use of strain EHA101 car-rying the binary vector pIG121-Hm wherein the CaMV35S driven hph is intronless. The Ubiquitin promoter produced 30-fold higher ß-glucuronidase (GUS) activity (derivatives of binary vector pWBVec8) in transgenic plants than the CaMV35S promoter (pIG121-Hm). The two modified SCSV promoters produced GUS activity com-parable to that produced by the Ubiquitin promoter. Progeny analysis (R1) for hygromycin resistance and GUS activ-ity with selected lines showed both Mendelian and non-Mendelian segregation. Lines showing very high levels of GUS activity in T0 showed a reduced level of GUS activity in their T1 progeny, while lines with moderate levels of GUS activity showed increased levels in T1 progeny. Stable heritable green fluorescent protein (GFP) expression was also observed in few transgenic plants produced with the binary vector pTO134 which had the CaMV35S promoter-driven selectable marker gene bar and a modified CaMV35S promoter-driven reporter gene sgfpS65T.

scholarly journals Morpho-Physiological Testing of NaCl Sensitivity of Tobacco Plants Overexpressing Choline Oxidase Gene

Plants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1102
Author(s):  
Galina N. Raldugina ◽  
Sergey V. Evsukov ◽  
Liliya R. Bogoutdinova ◽  
Alexander A. Gulevich ◽  
Ekaterina N. Baranova
Keyword(s):  
Choline Oxidase ◽  
Culture Methods ◽  
Gene Encoding ◽  
Oxidase Gene ◽  
Bacterial Enzyme ◽  
Transgenic Lines ◽  
High Regeneration ◽  
Whole Plants ◽  
Implicit Response

In this study the transgenic lines (TLs) of tobacco (Nicotianatabacum L.), which overexpress the heterologous gene encoding the bacterial enzyme choline oxidase were evaluated. The goal of our work is to study the effect of choline oxidase gene expression on the sensitivity of plant tissues to the action of NaCl. The regenerative capacity, rhizogenesis, the amount of photosynthetic pigments and osmotically active compounds (proline and glycine betaine) were assessed by in vitro cell culture methods using biochemical and morphological parameters. Transgenic lines with confirmed expression were characterized by high regeneration capacity from callus in the presence of 200 mmol NaCl, partial retention of viability at 400 mmol NaCl. These data correlated with the implicit response of regenerants and whole plants to the harmful effects of salinity. They turned out to be less sensitive to the presence of 200 mmol NaCl in the cultivation medium, in contrast to the WT plants.

scholarly journals Efficient Cryopreservation of Populus tremula by In Vitro-Grown Axillary Buds and Genetic Stability of Recovered Plants

Plants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 77
Author(s):  
Elena O. Vidyagina ◽  
Nikolay N. Kharchenko ◽  
Konstantin A. Shestibratov
Keyword(s):  
Survival Rate ◽  
Axillary Buds ◽  
Long Term Storage ◽  
Average Survival ◽  
Transgenic Lines ◽  
Ssr Loci ◽  
One Step ◽  
The One

Axillary buds of in vitro microshoots were successfully frozen at –196 °C by the one-step freezing method using the protective vitrification solution 2 (PVS2). Microshoots were taken from 11 transgenic lines and three wild type lines. Influence of different explant pretreatments were analyzed from the point of their influence towards recovery after cryopreservation. It was found out that the use of axillary buds as explants after removal of the apical one increases recovery on average by 8%. The cultivation on growth medium of higher density insignificantly raises the regenerants survival rate. Pretreatment of the osmotic fluid (OF) shows the greatest influence on the survival rate. It leads to the increase in survival rate by 20%. The cryopreservation technology providing regenerants average survival rate of 83% was developed. It was based on the experimental results obtained with explant pretreatment. Incubation time in liquid nitrogen did not affect the explants survival rate after thawing. After six months cryostorage of samples their genetic variability was analyzed. Six variable simple sequence repeat (SSR) loci were used to analyze genotype variability after the freezing-thawing procedure. The microsatellite analysis showed the genetic status identity of plants after cryopreservation and of the original genotypes. The presence of the recombinant gene in the transgenic lines after cryostorage were confirmed so as the interclonal variation in the growth rate under greenhouse conditions. The developed technique is recommended for long-term storage of various breeding and genetically modified lines of aspen plants, as it provides a high percentage of explants survival with no changes in genotype.

Neuromuscular target recognition by a homophilic interaction of connectin cell adhesion molecules in Drosophila

Development ◽  
1997 ◽  
Vol 124 (8) ◽  
pp. 1433-1441 ◽  
Author(s):  
A. Nose ◽  
T. Umeda ◽  
M. Takeichi
Keyword(s):  
Cell Adhesion ◽  
Synapse Formation ◽  
Target Recognition ◽  
Surface Protein ◽  
Transgenic Lines ◽  
A Cell ◽  
Neuromuscular Connectivity ◽  
Recognition Molecule

Drosophila Connectin (CON) is a cell surface protein of the leucine-rich repeat family. During the formation of neuromuscular connectivity, CON is expressed on the surface of a subset of embryonic muscles and on the growth cones and axons of the motoneurons that innervate these muscles, including primarily SNa motoneurons and their synaptic targets (lateral muscles). In vitro, CON can mediate homophilic cell adhesion. In this study, we generated transgenic lines that ectopically expressed CON on all muscles. In the transformant embryos and larvae, SNa motoneurons often inappropriately innervated a neighboring non-target muscle (muscle 12) that ectopically expressed CON. Furthermore, the ectopic synapse formation was dependent on the endogenous CON expression on the SNa motoneurons. These results show that CON can function as an attractive and homophilic target recognition molecule in vivo.

scholarly journals In vitro transformation of pearl millet (Pennisetum glaucum (L). R. BR.): Selection of chlorsulfuron-resistant plants and long term expression of the gus gene under the control of the emu promoter

2015 ◽  
Vol 14 (46) ◽  
pp. 3112-3123 ◽  
Author(s):  
Ti eacute coura Kouakou ◽  
Bakari Kouassi Abou ◽  
Oulo N rsquo nan Alla ◽  
Gonedel eacute Bi S eacute ry ◽  
Dinant Monique ◽  
...  
Keyword(s):  
Pearl Millet ◽  
Pennisetum Glaucum ◽  
Gus Gene ◽  
In Vitro Transformation ◽  
Selection Of

scholarly journals Bixafen, a succinate dehydrogenase inhibitor fungicide, causes microcephaly and motor neuron axon defects during development

2020 ◽  
Author(s):  
Alexandre Brenet ◽  
Rahma Hassan-Abdi ◽  
Nadia Soussi-Yanicostas
Keyword(s):  
Motor Neuron ◽  
Succinate Dehydrogenase ◽  
Toxicity Testing ◽  
Mitochondrial Respiratory Chain ◽  
Modern Agriculture ◽  
Transgenic Lines ◽  
Vertebrate Model ◽  
The Central Nervous System

AbstractSuccinate dehydrogenase inhibitors (SDHIs), the most widely used fungicides in agriculture today, act by blocking succinate dehydrogenase (SDH), an essential and evolutionarily conserved component of mitochondrial respiratory chain. Recent results showed that several SDHIs used as fungicides not only inhibit the SDH activity of target fungi but also block this activity in human cells in in vitro models, revealing a lack of specificity and thus a possible health risk for exposed organisms, including humans. Despite the frequent detection of SDHIs in the environment and on harvested products and their increasing use in modern agriculture, their potential toxic effects in vivo, especially on neurodevelopment, are still under-evaluated. Here we assessed the neurotoxicity of bixafen, one of the latest-generation SDHIs, which had never been tested during neurodevelopment. For this purpose, we used a well-known vertebrate model for toxicity testing, namely zebrafish transparent embryos, and live imaging using transgenic lines labelling the brain and spinal cord. Here we show that bixafen causes microcephaly and defects on motor neuron axon outgrowth and their branching during development. Our findings show that the central nervous system is highly sensitive to bixafen, thus demonstrating in vivo that bixafen is neurotoxic in vertebrates and causes neurodevelopmental defects. This work adds to our knowledge of the toxic effect of SDHIs on neurodevelopment and may help us take appropriate precautions to ensure protection against the neurotoxicity of these substances.

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.

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