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1

Formation and characterization of hairy roots

100%
Wasilewska A., Krolicka A.
|
|
issue 4
173-188
EN
Agrobacterium rhizogenes is a soil phytopathogen, which infects wounded plant tissue and generates overproliferation of neoplastic roots. Hairy-root cultures obtained by transformation of plant tissue by A. rhizogenes have evolved as an important tool for biosynthesis of plant secondary metabolites. This review discusses the methods for efficient plant transformation and hairy root formation for secondary metabolites production.
2

Cucumber transformation methods - the review

80%
Yin Z., Bartoszewski G., Szwacka M., Malepszy S.
Biotechnologia
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2005
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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.
3

Transfer and transgene expression

80%
Szopa J., Wrobel M.
Biotechnologia
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2001
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issue 1
63-72
EN
The development of transgenic plants strongly depends on the stable introduction of foreign nucleic acid into the plant genome. Recently, various transformation methods have been developed and successfully used for the development of transgenic plants of agricultural importance. Various vectors have been prepared, however the binary vector based on agrobacterial T-DNA is most commonly used. Of vectorless gene transfer systems, particle bombardment seems to be most useful. In both approaches, stable integration of DNA is based on random hybridization. The transformation efficiency is measured by the level of transgene expression and it may be potentially improved by different modifications, including insertion of multiple copies of promoter into a particular gene, insertion of procaryotic enhancer, increasing of mRNA stability, insertion of SAR/MAR sequences at the ends of gene, etc. Very recently it has been found that the increase of histone synthesis enhances transgene expression possibly as a result of increased transformation efficiency. Transgenic plants production also depends on the efficiency of the plant regeneration system which is used. There is no universally applicable method for regeneration of different tissues from various sources, thus regeneration protocol should be modified appropriately for each tissue and species.
4

Polish transgenic cereal crops

80%
Zimny J., Sowa S., Menke-Milczarek I., Czaplicki A., Sowa A.
Biotechnologia
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2000
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issue 4
80-87
EN
First transgenic cereal plants have been obtained in Poland seven years ago. Within the time other cereals like wheat, rye and barley have been also transformed. The prerequisite for that was a very efficient regeneration system by somatic embryogenesis. Generally the basic study on transgenic cereals are quite advanced but the question is how to include transgenic lines in to practical breeding process? Most of genes, promoters and transformation methods are patented and probably Polish breeders will never afford to buy the licences. Though there is a need to concentrate the future work in Polish institutes on identification and isolation of genes of interest. Than to transfer them to plants and register transgenic varieties. According to the Polish law it is allowed to carry out the field experiments, but it is not possible to register the plant variety.
5

Flow cytometry applied in plant biotechnology

80%
Sliwinska E.
Biotechnologia
|
2002
|
issue 1
122-128
EN
Flow cytometry (FCM) is a rapid and exact method for estimating the nuclear DNA content. Thus, it can be used for ploidy screening of different plant materials cultured in vitro (plantlets, callus, cell suspensions and somatic embryos) as well as haploids and somatic hybrids. In addition, it can be applied as a tool to analyse the events of genetic transformation. The application of FCM in biotechnology will be discussed.
6

Efficiency of transformation of Polish cultivars of pea (Pisum sativum L.) with various regeneration capacity by using hypervirulent Agrobacterium tumefaciens strains

80%
Pniewski T., Kapusta J.
Journal of Applied Genetics
|
2005
|
vol. 46
|
issue 2
139-147
EN
An Agrobacterium-mediated transformation method of pea has been developed for several edible and fodder cultivars of pea (Pisum sativum L.), characterized previously in their potential for regeneration via organogenesis. The most appropriate explant, which was susceptible to Agrobacterium infection and capable of regenerating transgenic plants, turned out to be a slice of an immature embryo, including the embryo axis and the basal part of a cotyledon. Three hypervirulent strains of A. tumefaciens were tested: AgL0, AgL1 and EHA105. Each carried the binary vector pP35SGIB containing the uid gene, with an intron under control of the 35S promoter, and the bar gene conferring resistance to phosphinotricin. Strain AgL0 was found to be efficient for the majority of cultivars, followed by AgL1 and EHA105. Transformation efficiency varied from 0.7 to 4.1%, depending on cultivar and Agrobacterium strain. The transformation efficiency of particular pea cultivars did not clearly correspond to their regeneration capacity, which ? although indispensable ? was not a critical parameter of successful transformation. The presence of integrated genes in pea genomic DNA was detected by the PCR. T-DNA was stably transmitted to the progeny, as it was confirmed by Southern hybridization. The activity of introduced genes was analysed by the histochemical GUS assay and by painting leaves or by spraying transgenic plants with the herbicide Basta.
7

An attempt at the transformation of embryogenic genotypes of alfalfa using gene pPR97/GUS/Intron-LLpr10.1a promoter

80%
Broda Z., Morawska J.
Biotechnologia
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2000
|
issue 4
106-111
EN
The transformation was led by pPR97/GUS/Intron LLpr10.1a promoter gene, which codes the acid protein. Two different media were used in the experiment. On Blayds medium, the highest efficiency of soamatic embryogenesis (25%) was observed in the Kometa variety. The culture media developed by Guelph University appeared to be the best for the Kama variety (18%).
8

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

80%
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.
9

Transgenic strawberry carrying maize IAA-glucose synthetase gene (iaglu)

80%
Wawrzynczak D., Sowik I., Michalczuk L.
Biotechnologia
|
2000
|
issue 4
103-105
EN
Strawberry leaves (Fragaria x ananassa Duch.) cv. Kaster were transformed with the aid of Agrobacterium tumefaciens, strain 4404, carrying pBIN19 plasmid with nptII gene and iaglu gene under the control of constitutive CaMV-35S promotor. The incorporation of the transgene was confirmed in PCR with iaglu-specific primers and its expression by Northern blot. The level of esterified IAA in the leaves of transgenic plants was higher and free IAA lower than in the leaves of control plants. Of 15 transgenic clones analysed, 10 have smaller leaves and 13 shorter petioles than control plants. It was also found that 8 transgenic strawberry clones started flowering 1 ? 2 weeks earlier than control plants and 5 clones did not form any runners.
10

In vitro regeneration and transformation of rye (Secale cereale L.)

70%
Sowa S., Sowa A., Zimny J.
Biotechnologia
|
2000
|
issue 4
88-92
EN
In vitro cultures are an integral part of plant transformation. Genetic manipulation can be performed only on a single cell level. Therefore in vitro culture and regeneration of plants from a single cell are very important for successful transformation In vitro culture of rye is more difficult to conduct than of others cereals. Difficulties with in vitro regeneration of rye seems to be the main factor limiting the development of rye transformation systems. Genetic transformation process includes three main steps, single cell transformation, selection of transgenic cells and regeneration of plants from single cells. Efficiency of each of these steps can influence the result of the transformation process. Therefore optimisation of those steps is very important.Transformation has been performed using the microprojecticle method and scutellum of rye embryos as a target. Two constructs have been used in cotransformation, pDB1 containing a marker bar gene (phosphinotricine acetylotransferase) for Basta herbicide resistance and a repoter uidA gen (beta-glucuronidase) and pAwact-Sec containing 196 bp fragment of a sec-1 gene in anti-sense orientation. Using pAWact-Sec construct we tried to block the expression of the endogenous gene sec-1 coding omega-secalin which is storage protein of rye grain. We were able to regenerate transgenic rye plants containing all introduced genes. The efficiency of sec-1 expression blocking was analysed by SDS-PAGE method. Among 50 analysed kernels of T1 generation we found 5 with lower omega-secalin level. Complete blocking of omega-secalin was not observed.
11

Interference in ethylene biosynthetic pathway using transformation

70%
Kepczynski J., Kepczynska E.
Biotechnologia
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2000
|
issue 2
72-82
EN
The biosynthesis of ethylene in plants and its regulation by manipulating the expression of ACC synthase or ACC oxidase genes are discussed. Ethylene synthesis can be reduced by the introduction of antisense ACC synthase or antisense ACC oxidase genes. Expression genes of SAM hydrolase from bacteriofage T3, which catalyze the conversion of SAM to methylothioadenosine, also diminished ACC availability. Another possibility of ethylene biosynthesis control is the expression of gene encoding ACC deaminase from Pseudomonas.
12

Present status of biotechnology of rye (Secale cereale L.)

70%
Bolesta E., Rybczynski J. J.
|
EN
This review paper covers the results of tissue culture and biotechnology of rye (Secale cereale L.) published between 1990 and 2000. The following subjects were raised: somatic embryogenesis, haploid production, intergenetic hybridisation and transformation. Upon conclusion of all the results published to date, we may say that the progress of biotechnology in the case of rye is very limited in comparison to other cereals. However, a lot of work and efforts were involved to obtain the aforementioned result. Taking into account the progress in plant genetics and molecular biology, the authors deeply believe that the New Millennium will bring a brake-through in rye biotechnology.
13

Preparation of pBIN19-PGIP-based genetic construct for modification of strawberry genome to obtain genotypes resistant to grey mould

70%
Korbin M., Majka J., Gruchala A., Zurawicz E.
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EN
Polygalacturonase inhibiting protein (PGIP) cDNA was isolated using RT-PCR from fruit of apple 'Golden Delicious' which was previously wounded and inoculated with Botrytis cinerea. The sequence of the cDNA showed high level of homology with the published sequence of PGIP-cDNA from apple and pear (99,7 and 97,3%) and low similarity with the sequence of cDNA from tomato (51,5%). The obtained cDNA was cloned and used for the binary construct creation. The introduction of the pBIN19-PGIP construct into strawberry plant genome seems to be a promising strategy to obtain a genotype resistant to B. cinerea, causal agent of grey mould ? the most economically important fungal disease of this species.
14

Roses breeding: in vitro culture and genetic engineering

70%
Orlikowska T., Wach I., Kucharska D.
Biotechnologia
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2000
|
issue 4
40-47
EN
Prequisites for successful transformation are developed protocols on efficient regeneration of adventitious shoots or somatic embryos. The most important works concerning regeneration, the objectives and achievements in rose transformation are discussed.
15

Transformed root cultures of Arnica montana L. ? of secondary metabolites isolation

70%
Weremczuk-Jezyna I., Wysokinska H.
Biotechnologia
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2004
|
issue 2
54-61
EN
The hairy roots of A. montana were achieved by the infection of sterile leaves with Agrobacterium rhizogenes (strain LBA 9402). The transformation was confirmed by opin and PCR analysis. In the investigation, the optimum contitions of Hairy roots growth were characterized. The best results were achieved in liquid Gamborg medium which included half-strenght macro- and microelements (1/2B5) and 50 g/l sucrose. Phytochemical analysis showed that hairy roots of A. montana produced arnifolin and chlorogenic acid.
16

Agrobacterium rhizogenes-mediated transformation of a high oil-producing filamentous fungus Umbelopsis isabellina

70%
Wei D.-S., Zhang Y.-H., Xing L.-J., Li M.-C.
Journal of Applied Genetics
|
2010
|
vol. 51
|
issue 2
225-232
EN
The Agrobacterium rhizogenes-mediated transformation procedure was developed by using the hygromycin B phosphotransferase gene (hph) as a selective marker for the oil-producing fungus Umbelopsis isabellina. Different conditions were combined to increase the transformation efficiency. The highest efficiency was obtained by using A. rhizogenes strain R105 and a vector with zygomycete promoter. Southern blot analysis demonstrated that 71% of transformants contained random integrations of T-DNA sequences under optimal conditions. We randomly selected 115 positive transformants resistant to hygromycin to analyze the amount of total fatty acid and gamma-linolenic acid (GLA). Six transformants produced a higher amount of total fatty acids than the wild strain, and one transformant also produced a higher level of GLA than the wild strain in gas chromatography analysis. This is the first report about using A. rhizogenes strain R105 and germinated conidia to transform successfully the recalcitrant zygomycetes and to obtain transformants with a stable phenotype.
17

Alfalfa (Medicago sativa) as an object for transformation by Agrobacterium tumefaciens and in vitro regeneration

61%
Lisova O., Legocki A. B., Legocki A. B.
Biotechnologia
|
2002
|
issue 3
74-90
EN
From the economical point of view alfalfa is a very valuable plant. It has very high yield potential, it is rich in protein, vitamins and minerals, so it is prized as very important feed for horses, beef cattle, sheep, goats and other domestic animals. That is why the improving of alfalfa quality by the methods of molecular biotechnology is a very interesting issue. In this review information about the crucial elements of alfalfa transformation and regeneration are gathered together. Among other following elements, genetic background of plant, A. tumefaciens strains, explant types, in vitro culture medias, plant development stages are discussed. It seems that optimalisation of the transformation and regeneration procedure are individual for every plant genotype and A. tumefaciens strain.
18

Instability of ploidy level during regeneration of transformed and non-transformed tobacco (Nicotiana tabacum L.)

61%
Sliwinska E., Zimny J., Drozdowska J.
|
2003
|
issue 3
260-266
EN
In plant tissue cultures, somaclonal variation is often observed. It can be an effect of the changes in the individual chromosome number or in the ploidy level. Flow cytometry, a fast and accurate method for the estimation of the nuclear DNA content, can be applied to study these changes. The DNA content in differentiated tissues of Nicotiana tabacum cultured in vitro was estimated using Partec CCA flow cytometer, starting from explant, through callus, up to regenerated shoots. The explant constituted stem segments of N. tabacum plants, non-transformed and transformed with gfp gene. Flow cytometric analysis showed differences in the proportion of 2C, 4C, 8C and 16C cells in plant tissue in different culture stages. Among the regenerated plantlets originated from non-transformed and transformed plants, diploid, tetraploid and mixoploid forms were observed. The transformation did not influence the share of cells representing different ploidy levels in the investigated plant material.
19

Transformation of microspore-derived embryos of winter oilseed rape (Brassica napus L.) by using Agrobacterium tumefaciens

61%
Cegielska-Taras T., Pniewski T., Szala L.
Journal of Applied Genetics
|
2008
|
vol. 49
|
issue 4
343-347
EN
Haploid microspore-derived embryos (MDEs) constitute a unique material for the introduction of new traits into winter oilseed rape (Brassica napus). MDEs have been transformed by using Agrobacterium tumefaciens strains EHA105 and LBA4404, both carrying the binary vector pKGIB containing the uidA gene encoding -glucuronidase (GUS) and the bar gene as a marker of resistance to phosphinotricin. Transformed embryos expressed GUS and regenerated plants that were resistant to herbicide Basta, as confirmed by a leaf-painting test. Progeny plants of the transformant T-39 were all transgenic, as they inherited T-DNA from their doubled haploid parental plant. Southern-blot analysis confirmed the integration and transmission of T-DNA into T1 plants. Transformation of MDEs facilitates the obtaining of winter oilseed rape homozygous for the introduced genes.
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