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Professor Tomasz Twardowski – 22 years devoted to Biotechnologia

100%
Malepszy S.
BioTechnologia
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2011
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issue 1
2

An amazing progress in plant transformation methods

100%
Malepszy S.
Biotechnologia
|
1997
|
issue 4
15-19
EN
A great progress in the higher plants transformation methodology has been observed. It includes the following events: increasing efficiency, development of new markers which are ecologically acceptable, transformation of new monocotyledonous species using A. tumefaciens 'supervectors', control of recombination site.
3

The transgenes are expressed with different level in plants

64%
Yin Z., Malepszy S.
Biotechnologia
|
2003
|
issue 2
236-260
EN
Variable expression for the same transgene construct has been documented in various plant species, regardless of the type of transgene. This phenomenon and the factors influencing it are reviewed. A variability in the transgene expression level was found to exist between and/or within independently derived lines, different lines of clonal replicates, siblings of the same line, a single plant, a single leaf as well as the same cell. Both the transgene dependent and the recipient dependent factors have been proposed to account for this phenomenon, the transgene dependent factors of which include transgene construct fidelity, T-DNA integration pattern, T-DNA copy number, promoter activity and the effect of nuclear matrix attachment region. In addition, different forms of epigenetic, homology-dependent gene silencing also contribute to the unstable expression of the identical transgene. The recipient dependent factors include position effect, ploidy level, genetic background, homozygosity, and developmental stage. Furthermore, environmental factors such as light intensity, temperature, field growth conditions and the season have been shown to modulate the levels of transgene expression. The approaches to stabilize the transgene expression were also discussed.
4

Plant sweetness-inducing proteins and taste-modyfying proteins

64%
Szwacka M., Malepszy S.
|
|
issue 1
64-82
EN
This work reviews the plant proteins which stimulate the sweet receptor in humans and primates. Identification of the sweet site of thaumatin is a promising target. Thaumatin as a sweetener and flavor enhancer has been used in a wide range of foods. Some of recombinant sweet proteins have been expressed in microbes below economically viable levels. Significance of high-quality production of sweet proteins in higher plant is discussed. Thaumatin and thaumatin-related family have been characterized.
5

Unintended consequences of plant transformation: a molecular insight

64%
Filipecki M., Malepszy S.
Journal of Applied Genetics
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2006
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vol. 47
|
issue 4
277-286
EN
Plant genomes are dynamic structures having both the system to maintain and accurately reproduce the information encoded therein and the ability to accept more or less random changes, which is one of the foundations of evolution. Crop improvement and various uncontrolled stress factors can induce unintended genetic and epigenetic variations. In this review it is attempted to summarize factors causing such changes and the molecular nature of these variations in transgenic plants. Unintended effects in transgenic plants can be divided into three main groups: first, pleiotropic effects of integrated DNA on the host plant genome; second, the influence of the integration site and transgene architecture on transgene expression level and stability; and third, the effect of various stresses related to tissue handling, regeneration and clonal propagation. Many of these factors are recently being redefined due to new researches, which apply modern highly sensitive analytical techniques and sequenced model organisms. The ability to inspect large portions of genomes clearly shows that tissue culture contributes to a vast majority of observed genetic and epigenetic changes. Nevertheless, monitoring of thousands transcripts, proteins and metabolites reveals that unintended variation most often falls in the range of natural differences between landraces or varieties. We expect that an increasing amount of evidence on many important crop species will support these observations in the nearest future.
6

In vitro culture and biotechnology ? fulfilled expectations?

64%
Malepszy S., Burza W.
Biotechnologia
|
2010
|
issue 2
10-17
EN
The methods for in vitro culture of plant cells, tissues and organs had focused much attention in the beginning of the last century, which resulted in setting up the first commercial laboratories over 60 years ago. These laboratories concentrated their activity on clonal propagation and their economical importance has been permanently growing. However, some of the applications of in vitro methods are still not realistic, whereas introduction of other is not satisfactory. Plant breeding is an example where application of tissue culture techniques is below expectations. There are several reasons for such situation. Two of them are of biological nature (genotypic effect, somaclonal variation), and the third reason results from the development of other molecular methods providing alternative solutions. We suggest that the main limitations in more effective usage of in vitro methods should be minimized by the development of efficient plant stem cells' culture procedures.
7

The transpositional elements as a tool for plant genome mapping

64%
Wochniak P., Malepszy S.
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|
issue 3
15-29
EN
This work aims at reviewing current progress in the field of plant transposble elements, especially those described as Ac/Ds and En/dSpm systems, first discovered in maize.We gave the molecular characteristics of plant transposones and the rules of their behaviour within a genome.The procedure for a particular gene mapping and for mapping of many genes responsible for biochemical pathway were cited.In comparison with other genome mapping methods the advantages and drawbacks of "gene tagging" were envisaged.The enclosed tables provide many documental examples of plant genes identification via "gene tagging" method.
8

Differences in the inheritance stability of kanamycin resistance between transgenic cucumbers (Cucumis sativus L.) containing two constructs

52%
Yin Z., Szwacka M., Malinowski R., Malepszy S.
|
|
issue 3
307-314
EN
A phenotypic segregation of kanamycin resistance conferred by nosnptII was investigated in two groups of transgenic cucumber lines one containing the PR-2duidA-nosnptII construct and the other with the thaumatinII-nosnptII construct to the third and fifth generation, respectively. Expression of the nptII gene was more stable and predictable in the progeny of the PR-2d lines than that of thaumatin lines. In T2 and T3 generations representing seven PR-2d primary transformants, 78% of the hemizygous progenies exhibited a segregation ratio consistent with Mendelian inheritance; non-Mendelian ratio occurred in the remaining 22%. Mendelian segregation was observed in 46% of the hemizygous progenies derived from 11 thaumatin primary transformants. The segregation ratio for two and three independent loci appeared in single PR-2d and thaumatin lines, respectively.
9

Characterization of a cucumber (Cucumis sativus L.) somaclonal variant with paternal inheritance

52%
Malepszy S., Burza W., Smiech M.
Journal of Applied Genetics
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1996
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vol. 37
|
issue 1
65-78
EN
Regeneration of cucumber from leaf explants resulted in a new species phenotype designated mosaic (msc).It is characterizes by two types of spots on teh leaves (zucchini-like and chlorophyllous) and has many altered morphological and physiological properties including slower growth, smaller organs, poorly germinating or empty seeds and smaller number of flowers per node.In msc plants the shape of the first leaf is always altered, and in about 76% of the flowers the crown is reduced and distorted to a varying degree.Chloroplasts of the zucchini-like sectors are filled with large starch grains, and some of the embryos die at various stages of development.The msc phenotype is ransmitted uniparentally only by the male plants and no segregation is observed in the F2 and subsequent generations.Possible mechanisms responsible for msc phenotype and its transmission are discussed.
10

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

52%
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.
11

Generation of selectable marker-free transgenic plants

52%
Zuzga S., Burza W., Malepszy S.
Biotechnologia
|
2003
|
issue 4
32-46
EN
Since the first reports of successful plant transformation appeared, there have been steady improvements of the transformation methods. Nowadays, transgenic plants without the incorporation of selection genes for antibiotic or herbicide resistance would be the only ones acceptable to the public, so elimination of these genes from transgenic crops prior to their field release and commercialization seems inevitable. Several strategies have been developed to generate marker-free transgenic plants, including: positive selection, alternative marker genes, co-transformation, site-specific recombination, transpozon-mediated approaches and intrachromosomal homologous recombination. The efficiency of these methods is various as comparing to the traditional marker assisted selection - higher in case of alternative procedures and lower in others.
12

Transgene inheritance in plants

52%
Yin Z., Plader W., Malepszy S.
Journal of Applied Genetics
|
2004
|
vol. 45
|
issue 2
127-144
EN
The patterns of transgene inheritance in plants and the possible explanations for non-Mendelian transmission are reviewed. The non-Mendelian inheritance of a transgene has been recorded with a frequency between 10% and 50% in transgenic plants produced either by Agrobacterium-mediated transformation or through particle bombardment. Different effects such as deletion, duplication, rearrangement, repeated sequence recombination as well as gene interaction have been observed for transgenic loci. The nature of the recipient genome, nature of the transgene and the interactions between them seem to contribute to the non-Mendelian segregation of transgenes.
13

Cucumber transformation methods - the review

52%
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.
14

Sequential fluorescent staining with CMA and DAPI for somatic chromosome identification in cucumber (Cucumis sativus L.)

52%
Plader W., Hoshi Y., Malepszy S.
Journal of Applied Genetics
|
1998
|
vol. 39
|
issue 3
249-258
EN
Each of the seven chromosomes in cucumber (Cucumis sativus L.) was identified using sequential staining with Chromomycin A3 (CMA) and 4-6-diamidino-2-phenylindole (DAPI) as DNA base-specific fluorescent dyes. The present method using enzymatic digestion produced a high level of well-spread early-metaphase chromosome complements. After CMA and DAPI staining, reproducible fluorescence bands were obtained in mitotic prometaphase chromosomes. The CMA staining method made it possible to characterize whole chromosomes from prometaphase to mid-metaphase. Chromosome 1 had the largest and widest CMA-positive (CMA+) band from the proximal region to the interstitial region on the long arm in prometaphase. A large gap separating of the short arm from the long arm was always observed in chromosome 2 during prometaphase. The banding pattern of the short arm was similar to that of the long arm in chromosome 2. Chromosomes 1 and 2 in early metaphase had sharp and large CMA-positive and DAPI-negative (CMA+DAPI-) bands at the pericentromeric regions. In early metaphase, chromosome 3 was characterized by having a narrow CMA+DAPI- band on the pericentromeric region of the short arm. Chromosomes 4 and 5 showed similar chromosome length and had a large CMA+ band at the distal region of the long arm. Chromosome 4 did not show any clear band in the short arm, while chromosome 5 showed a telomeric CMA+ band at the short arm and a clear CMA+DAPI- band at the pericentromeric region. Chromosome 6 had a CMA+ band at the distal region and a weak CMA+ band at the proximal region in each of the arms. Chromosome 7 had an evident CMA+ band in the long arm and a CMA+DAPI- band in the pericentromeric region.
15

Transient expression assay for the optimization of direct gene transfer into cucumber meristem protoplasts by electroporation

52%
Burza W., Wochniak P., Wroblewski T., Malepszy S.
Journal of Applied Genetics
|
1995
|
vol. 36
|
issue 1
1-10
EN
The paper obtained a new way of viable and very homogenous cucumber protoplasts.Protoplasts from cells formed in the shoot tip meristem culture were isolated from suspension.Plasmid pBI121 was introduced using imulse electric field.Effectiveness of transformation process was determinated by transient expression of ?-glucoronidase (GUS) gene, controlled by promotor 35S.The ativity of ?-glucoronidase enzyme as a product of GUS reporter gene was estimated by fluorometric method (Jefferson 1987).Parameters of electroporatian process were optimized.The transient expression of GUS gene was measured 24 h after electroporation.The highest effectiveness of transformation process was achieved using three electric impulses at the initial voltage of 250-350V at 10-sec.intervals as a results of discharging a 140 ?F capacitor and 50-70 ?g x cm/1000 plasmid DNA in the presence of 50 ?g x cm/1000 carrier DNA.The system presented is an effective method of exogenic DNA transfer, which is indicated by a high transient expression of the reporter gene.In comparison to Agrobacterium tumefaciens and A.rhizogenes, this alternative method of gene transfer can be used for obtaining transgenic cucumber plants.
16

Transformation of cucumber Cucumis sativus L.

52%
Szwacka M., Burza W., Palucha A., Malepszy S.
|
EN
Different transformation methods were investigated. However a lot of fertile plants were obtained only in the case of A. tumefaciens and thaumatin with kanamycin as a selective marker. Small differences in the plant morphology and development were observed in T0 generation. Most of the plants showed the presence of the transgene but the level of mRNA was different.
17

Cucumber: A model angiosperm for mitochondrial transformation?

45%
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.
18

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

45%
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.
19

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

45%
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.
20

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

45%
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.
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