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1

Transgene expression in plant cell

100%
Niemirowicz-Szczytt K., Bartoszewski G.
Biotechnologia
|
2000
|
issue 4
11-23
EN
Transgene incorporation in plant genome does not mean that the gene will be active and will show expression at the required level. It is often the case that transgenic plants do not exhibit the activity of the gene introduced. Gene expression is known to be dependent on many factors such as: chimeric gene structure, proper and stable integration to the genome and proper transcription and translation. The knowledge of transgene regulation is essential for the understanding of the transcription and translation mechanisms. Each plazmid component is vital for later transgene expression. This refers to the size of T-DNA introduced, location of particular elements, leader sequences length, AUG sequences, introns presence, optimum coding gene synthesis, removal of RNA instability signals and many other possible modifications.
2

Agrobacterium-mediated tomato transformation

100%
Bartoszewski G., Niemirowicz-Szczytt K.
|
|
issue 1
43-63
EN
Tomato is one of the model species used for crop transformation. The first transgenic tomato variety, known as Flavr Savr?, was approved for sale in the USA in 1994. The introduced trait of this cultivar is delayed ripening. In 1996, its acreage was reported to be 10,000 acres. Another variety characterised by delayed ripening is Endless Summer?, approved in 1995. There are some other cultivars with new traits, such as thicker skin, altered pectin or resistance to viruses (TSWV, ToMV) either approved or pending approval. In addition, a wide range of basic research on tomato transformation has been carried out, including studies on resistance to herbicides, viruses, fungi, bacteria, insects as well as on altered transcription regulation, ripening, carotenoid synthesis, level of auxines and cytokinines, carbohydrates, proteins and specific vaccines. Further improvement of tomato varieties is expected with the use of Agrobacterium tumefaciens.
3

Effectiveness of Agrobacterium-mediated transformation in selected forms of tomato (Lycopersicon esculentum Mill.) and preliminary characteristics of transgenic plants.

81%
Bartoszewski G., Malepszy S., Niemirowicz-Szczytt K.
Biotechnologia
|
1997
|
issue 4
62-70
EN
Three forms of Lycopersicon esculentum Mill. variety Beta, line ls and line nor were subject to Agrobacterium-mediated transformation with two constructs: pBI121 and pRUR528. In both cases transgenic plants were obtained which formed roots in a medium with kanamycin and showed expected band in the PCR analysis. The effectiveness of the transformation, measured as the number of explants forming structures on selection medium, was different for the used genotypes when a bacterial strain carrying plasmid pRUR528 was used. No genotype-dependent difference in effectiveness of the transformation was observed in the case of pBI121. The genotype-dependent differences in the effectiveness of the transgenic shoots regeneration and rooting were observed. 76% of transgenic plants showed unchanged ploidy level.
4
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Cecha męskiej sterylności u rzodkiewki (Raphanus sativus L.) – podłoże genetyczne i molekularne

81%
Hawliczek-Strulak A., Bartoszewski G., Korzeniewska A., Niemirowicz-Szczytt K.
Postępy Nauk Rolniczych
|
2011
|
issue 4
5

Cucumber transformation methods - the review

81%
Yin Z., Bartoszewski G., Szwacka M., Malepszy S.
Biotechnologia
|
2005
|
issue 1
95-113
EN
Several aspects of cucumber transformation including the ways of transgene introduction, factors influencing the transformation efficiency and the fate of the introduced genes were reviewed. Various transgenes have been introduced into the cucumber genome mostly via the Agrobacterium-mediated transformation. The frequency of Agrobacterium-mediated transformation ranged from 0.8 to 10% and was influenced by the selection agent, the regeneration efficiency, activation of vir genes expression, the explant size, bacteria cell density, the length of exposure and the co-cultivation period. The transgenes were integrated mostly as single copy in the Agrobacterium-mediated transformation and as multiple copies in direct transformation. Variable levels of the transgene expression were observed. The transmission of the transgenes as well as the transgenic phenotype follow the Mendelian, and rarely non-Mendelian, ratio. The production of marker-free transgenic cucumber and use of an alternative transformation method are recommended.
6

Transformation of selected genotypes of tomato with the help of A. tumefaciens

71%
Bartoszewski G., Fedorowicz O., Malinowski R., Niedziela A., Niemirowicz-Szczytt K.
Biotechnologia
|
2000
|
issue 4
99-102
EN
Tomato belongs to important crops widely cultivated all over the world. It is also one of the five most popular vegetables grown in Poland. At the same time, tomato is known to be a model species in modern biology and biotechnology. Since 1985 a lot of reports on tomato transformation with the use of Agrobacterium have been published. Recently, first transgenic varieties of this species have also been developed. Flavr Savr? obtained by Calgene, USA was the first cultivar obtained as a result of genetic engineering, officially registered in the United States. In our Department the methods of tomato transformation with Agrobacterium tumefaciens have been adapted and optimised. Numerous transgenic plants have been obtained, such as commercial varieties (Beta, Potentat), inbred lines as well as tomato mutants (non-ripening, lateral suppressor). The following genes were introduced to the above forms: beta-glucuronidase reporter gene (gusA), thaumatin gene (sweet protein), isopentenyl transferase gene (ipt, coding the key enzyme in cytokinin metabolic pathway) and thus homozygous lines were developed (T2 generation). Most recently, attempts have been made to incorporate mgfp5-ER gene coding green fluorescence protein, nucleoprotein (N) gene from tomato spotted wilt virus (TSWV) and cDNA of putative P450 cytochrome (CYP72) in order to test gene expression and interaction.
7

Cucumber: A model angiosperm for mitochondrial transformation?

71%
Havey M. J., Lilly J. W., Bohanec B., Bartoszewski G., Malepszy S.
Journal of Applied Genetics
|
2002
|
vol. 43
|
issue 1
1-17
EN
Plants possess three major genomes, carried in the chloroplast, mitochondrion, and nucleus. The chloroplast genomes of higher plants tend to be of similar sizes and structure. In contrast both the nuclear and mitochondrial genomes show great size differences, even among closely related species. The largest plant mitochondrial genomes exist in the genus Cucumis at 1500 to 2300 kilobases, over 100 times the sizes of the yeast or human mitochondrial genomes. Biochemical and molecular analyses have established that the huge Cucumis mitochondrial genomes are due to extensive duplication of short repetitive DNA motifs. The organellar genomes of almost all organisms are maternally transmitted and few methods exist to manipulate these important genomes. Although chloroplast transformation has been achieved, no routine method exists to transform the mitochondrial genome of higher plants. A mitochondrial-transformation system for a higher plant would allow geneticists to use reverse genetics to study mitochondrial gene expression and to establish the efficacy of engineered mitochondrial genes for the genetic improvement of the mitochondrial genome. Cucumber possesses three unique attributes that make it a potential model system for mitochondrial transformation of a higher plant. Firstly, its mitochondria show paternal transmission. Secondly, microspores possess relatively few, huge mitochondria. Finally, there exists in cucumber unique mitochondrial mutations conditioning strongly mosaic (msc) phenotypes. The msc phenotypes appear after regeneration of plants from cell culture and sort with specific rearranged and deleted regions in the mitochondrial genome. These mitochondrial deletions may be a useful genetic tool to develop selectable markers for mitochondrial transformation of higher plants.
8

Selected aspects of genetic transformation in cucumber, tomato, and sweet pepper

71%
Bartoszewski G., Szwacka M., Czarny M., Niemirowicz-Szczytt K., Malepszy S.
Biotechnologia
|
2010
|
issue 4
146-154
EN
Plant transformation is a technology widely used in gene functional analysis and crop improvement. In this article we have attempted to sum up the studies on plant transformation carried out by the Department of Plant Genetics, Breeding and Biotechnology, Warsaw University of Life Sciences, pointing out to recent developments in this field. Efficient Agrobacterium-based transformation protocols for cucumber and tomato were established and applied. Several traits, including fruit taste (thaumatin gene), chilling tolerance (pGT::DHN24), parthenocarpy (DefH9::iaaM), and virus resistance (TSWV nucleoprotein gene), were modified. Transgenic cucumber lines expressing mitochondrially targeted GFP protein were developed. Sensory evaluation of fruit traits and of unintended effects of cucumber expressing thaumatin gene was made. Cucumber and tomato transformation was also applied with the aim to carry out gene functional analysis. Having introduced overexpression, silencing, and promoter gene constructs, we were able to obtain several transgenic tomato lines. Attempts have been made to set up an efficient method of sweet pepper transformation.
9

The selection of mosaic (MSC) phenotype after passage of cucumber (Cucumis sativus L.) through cell culture ? a method to obtain plant mitochondrial mutants

71%
Bartoszewski G., Havey M., Ziolkowska A., Dlugosz M., Malepszy S.
Journal of Applied Genetics
|
2007
|
vol. 48
|
issue 3
1-9
EN
Mosaic (MSC) mutants of cucumber (Cucumis sativus L.) appear after passage through cell cultures. The MSC phenotype shows paternal transmission and is associated with mitochondrial DNA rearrangements. This review describes the origins and phenotypes of independently produced MSC mutants of cucumber, including current knowledge on their mitochondrial DNA rearrangements, and similarities of MSC with other plant mitochondrial mutants. Finally we propose that passage of cucumber through cell culture can be used as a unique and efficient method to generate mitochondrial mutants of a higher plant in a highly homozygous nuclear background.
10

The selection of mosaic (MSC) phenotype after passage of cucumber (Cucumis sativus L.) through cell culture ? a method to obtain plant mitochondrial mutants

71%
Bartoszewski G., Havey M. J., Ziolkowska A., Dlugosz M., Malepszy S.
Journal of Applied Genetics
|
2007
|
vol. 48
|
issue 1
1-9
EN
Mosaic (MSC) mutants of cucumber (Cucumis sativus L.) appear after passage through cell cultures. The MSC phenotype shows paternal transmission and is associated with mitochondrial DNA rearrangements. This review describes the origins and phenotypes of independently produced MSC mutants of cucumber, including current knowledge on their mitochondrial DNA rearrangements, and similarities of MSC with other plant mitochondrial mutants. Finally we propose that passage of cucumber through cell culture can be used as a unique and efficient method to generate mitochondrial mutants of a higher plant in a highly homozygous nuclear background.
11

Chromosome number variation in somatic hybrids between transgenic tomato (Lycopersicon esculentum) and Solanum lycopersicoides

62%
Kulawiec M., Tagashira N., Plader W., Bartoszewski G., Kuc D., Sniezko R., Malepszy S.
Journal of Applied Genetics
|
2003
|
vol. 44
|
issue 4
431-447
EN
Leaf mesophyll protoplasts of Lycopersicon esculentum were fused with suspension-culture-derived protoplasts of Solanum lycopersicoides by a PEG treatment. Both species have the same chromosome number (2n = 2x = 24). The hybrid calli were selected using the full selection method ? kanamycin resistance and culture conditions critical for L. esculentum protoplast divisions. The genomic in situ hybridization analyses indicated a hypo- and hypertetraploid character of the hybrid plant with a majority of S. lycopersicoides chromosomes and a variation in chromosome number from 46 to 53. The hybrids contained a transgene derived from L. esculentum, as shown by Southern blot hybridization and PCR analyses. Their mitochondria were derived from the wild species, S. lycopersicoides. More than 60 regenerated plants were transferred into the greenhouse. They grew very slowly and were not able to flower for almost one year. The main morphological characters of the hybrids included a single shoot and small, dark-green leaves with strongly wrinkled blades. The reasons for nuclear genome asymmetry between hybrids and the possibilities of using them in a genetic and breeding programme are discussed in this paper.
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