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1

Micropropagation in in vitro culture efficiency of selected protected species Asteraceae

100%

Trejgell A. , Tretyn A.

Biotechnologia

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2010

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nr 3

202-209

EN

The aim of the present research was the assessment of efficiency of micropropagation system for selected species that belong to Asteraceae family, as well as analysis of morphological traits and plantlets ability to flower. The experimental material were shoot tips isolated from 7-day-old seedlings of Leontopodium alpinum, Senecio macrophyllus, Carlina acaulis, and Cirsium pannonicum. The shoot tips were transferred on the medium supplemented with BA (1 mg.dm-3), and NAA (0,1 mg.dm-3). The obtained shoots were transferred onto fresh medium with the same combination of growth regulator (3 subcultures). The shoots with 4 or more leaves were rooted on the half-strength MS medium without growth regulators for 4 weeks. The plantlets were acclimatized to ex vitro conditions and planted to the field. The analysis of flowering ability, leaves and flower morphology, and survival level were the objectives of the study. The plantlets were acclimatized in a greenhouse and planted to the field condition. From 10 seeds of initial material one can obtain 24 278 plants of Leontopodium alpinum, 1507 of Carlina acaulis, 1261 of Senecio macrophyllus, and 37 of Cirsium pannonicum taking into consideration the percentage of seed germination, proliferation rate in three subcultures, frequency of microshoots rooting and survival rate during acclimatization. The regenerated plants demonstrated traits of donor plants and were able to flower and produce viable seeds.

2

The effect of cytokinins on morphogenetic response of Polemonium coeruleum seedlings explants

100%

Trejgell A. , Tretyn A.

Biotechnologia

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2010

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nr 2

125-131

EN

The aim of the presented research was to examine the morphogenetic response of Polemonium coeruleum explants. The donor material were 10-day-old seedlings. Surface sterilized seeds were germinated on MS medium supplemented with GA3 (1 mg?dm-3). Seedling explants (shoot tips, fragments of cotyledons, hipocotyls and roots) were isolated and transferred onto solidified MS medium supplemented with different types of cytokinins (BA, KN, ZEA, 2iP) at concentrations 1.0, 3.0 and 5.0 mg?dm-3 in combination with NAA (0.1 mg?dm-3). All explant types were characterized by callus proliferation. It was observed that calli developed on the entire surface of hipocotyl and root fragments. On the other hand, shoot tips and cotyledonary petioles formed callus tissue at the cut ends, and petioles only at abaxial ends. The growth of calli on all explant types was strongly stimulated by ZEA. Among the explants tested, only shoot tips exhibited shoot organogenesis. The highest frequency of shoot organogenesis was observed when the explants were cultured on a medium supplemented with 5.0 mg?dm-3 BA (100%) or 5.0 mg?dm-3 ZEA (97%). The highest shoot number per explant was obtained in the presence of 5.0 mg?dm-3 ZEA (8.4 on average). The presence of BA or ZEA in the proliferation medium inhibited rhizogenesis and the elongation growth of shoots. However, root organogenesis was supported by KN added into the medium.

3

Ability to make flowers of Pharbitis nil plants regenerated from flower buds and immature zygotic embryos

100%

Trejgell A. , Tretyn A.

Biotechnologia

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2001

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nr 2

118-121

EN

Flower buds and immature embryos of P. nil Chois., which were grown in vivo , were material for the study. Flower bud (2-3 mm size) were treated with osmotic/trophic shock (12% sucrose) for 24h. After this time these explants were exposed on regeneration medium (MS supplemented with BAP in concentration 5 mg.dm-3 and NAA in concentration 0,1 mg.dm-3). After 4-6 weeks organogenesis of shoots was observed. Plantlets were isolated and transferred on MS medium with addition of GA3 [0,5 mg.dm-3] and NAA [0,1 mg.dm-3]. The plantlets developed into whole plants. Those plants were able to produce flower without photoperiodic induction, because these shoots regenerated from inducted tissue and ?remembered? this information. Immature embryos were isolated from previously sterilised fruit and afterwards transferred to MS without plant hormones. The embryos were cut across their axis. After 4-6 weeks of cultivation somatic embryos were formed in the injury place (hypocotyl region). These embryos were isolated and transferred on the same medium, which was used in shoots regenerated from flower buds. Embryos were converted into complete plants, but they weren?t able to flower without photoperiodic induction. However even the smallest embryos, which were used to our study (1 mm long) were able to flower, after a single induction cycle (16 hour of darkness), when the induction was given directly after isolation of embryos.

4

Suspension cultures of Pharbitis nil

100%

Szyp I. , Tretyn A.

Biotechnologia

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2001

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nr 1

127-130

EN

Suspension cultures are more suitable for physiological, biochemical and molecular investigations than callus cultures grown on solid media, because the former provide more homogeneous system than the latter ones. A large number of plants were found suitable for establishing cell suspension cultures. In this study we describe the obtaining of cell suspension cultures derived from callus induced from cotyledons of Pharbitis nil. Explants isolated from plants grown under inductive and non-inductive conditions were cultured on MS basal medium containing various concentrations and combinations of growth regulators. To initiate the cell suspension culture, small clumps of friable callus obtained from cotyledons were suspended in liquid callusing medium. An initial inoculum density was 2104cells/ml. Every 20 days the cultures were transferred to a fresh medium. The cell number in suspension was determined by direct microscopic counting with haemocytometr. The cell suspension cultures contained both single cells and small cell aggregates.

5

Real Time PCR. The idea of the method and strategies of reaction monitoring

80%

Studzinska A. , Tyburski J. , Daca P. , Tretyn A.

Biotechnologia

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2008

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nr 1

71-85

EN

Real-time PCR has become one of the most widely used methods of gene quantitation in molecular biology and medical diagnostics. This technique combines PCR amplification and the detection of the PCR product into a single step. In real-time PCR, the amount of product formed is measured during the course of the experiment by monitoring the fluorescence of dyes or probes introduced into the reaction. Fluorescence data are generated by the use of intercalating dyes such as SYBR Green I or molecular probes, the most important of which are: TaqMan, Molecular Beacons, Hybridization Probes, and Scorpion Probes. The real-time PCR reactions are characterized by the PCR cycle in which the target amplification is first detected. This cycle is referred to as a treshold cycle (Ct), at which fluorescence intensity becomes greater than background fluorescence. Consequently, the greater the quantity of target DNA in the starting material, the faster the significant increase in fluorescence intensity will appear, yielding lower Ct. The relation between Ct and the concentration of the target sequence allows for a precise quantitation of the genetic material in the sample.

6

Application of callus tissue in the studies on molecular mechanism of flower induction

80%

Szyp I. , Dabrowska G. , Tretyn A.

Biotechnologia

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2001

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nr 2

165-169

EN

Flowering is a crucial turning point in the life cycle of most plants. The process of flowering is controlled by external factors such as light and temperature. Floral induction is the first step in the transition from the vegetative to reproductive stage of development. In the photoperiodically sensitive plants this process is regulated by the duration of light and darkness during a 24-h cycle. The aim of our study was to determine whether the undifferentiated callus tissue obtained from cotyledons, is suitable for molecular investigations on the mechanisms of flower induction. The callus tissue was obtained from cotyledons of Pharbitis nil plants, which were cultivated in inductive or non-inductive conditions. The callus obtained after two subcultures was used for isolation of RNA. The total RNA was extracted as described by Chomczynski (1993). We have examined the changes in the pattern of RNA in these two types of callus, using the technique of differential display by the polymerase chain reaction (PCR). Differential display is a method for the identification and cloning of differentially expressed eucaryotic genes. We have found the differences between patterns of RNA derived from callus tissue cultivated under non-inductive conditions and callus tissue cultivated under inductive conditions. In conclusion we can suggest that the tested callus preserved the information on the photoperiodic induction in cells. The process of undifferentiation did not result in the loss of the properties acquired by cotyledon tissue during the photoperiodic treatment.

7

PCR in real time. The methods of data analysis

80%

Tyburski J. , Studzinska A. , Daca P. , Tretyn A.

Biotechnologia

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2008

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nr 1

86-96

EN

The accuracy of real-time PCR (RT-PCR) experiment is strongly dependent on the mathematical models of data analysis, on which the quantitative methods are based. In this review, we discuss the key steps of analysing data from real-time PCR experiments. These are the treshold cycle determination, estimation of real-time PCR amplification efficiency and amplicon quantification. The fit point method and the second derivative method are commonly used to determine a treshold cycle value, which is a cycle number in the early exponential phase of PCR that is used to calculate the initial amount of template DNA. The amplification efficiency calculation is usually based on the data collected from a standard curve. However, in the alternative methods, the amplification efficiency of an individual reaction is calculated from the kinetics of the reaction. Quantification of amplicon levels can be either absolute or relative. In absolute quantification method, the initial concentration of target template in unknowns, based on their cycle treshold values, and the construction of an absolute standard curve for each individual amplicon is required. Relative quantification can be done by the use of the relative standard curve method or the comparative method. In the first one, the initial amount of unknown samples is calculated from the standard curve of specific gene and normalized to the input amount of a reference gene which is also calculated from the standard curve. The comparative method is a mathematical model that is based on the differences in the normalized amplicon levels between the unknowns and control sample. There are also models that combine gene quantification and normalization into a single calculation.

8

Metagenomic libraries as sources of genes useful for biotechnology

80%

Deja-Sikora E. , Sikora M. , Golebiewski M. , Tretyn A.

Biotechnologia

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2007

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nr 4

125-139

EN

The vast majority of microorganisms cannot be cultured under laboratory conditions. It was estimated that over 99% of microbial genetic information are inaccessible due to the inability to isolate and culture bacteria. It is also widely known that among those uncultured organisms there are such that bear the genes which are interesting from the biotechnological point of view, i.e. coding for novel enzymes (lipases, amylases, cellulases, polymerases etc.), responsible for resistance to various chemical substances (heavy metals, aromatic compounds, pesticides, antibiotics), or the genes encoding the elements of biosynthetic pathways (for instance producing novel antibiotics). Recently, the methods have been developed that allow (i) isolation and purification of environmental DNA, (ii) construction of random fragment libraries of such DNA, and (iii) effective screening of those libraries in search for interesting genes. These methods are collectively known as ?metagenomics' or ?environmental genomics'. We aim to review the metagenomic methods, which will be done in Part I of our paper, and to present the up ? to ? date achievements and future perspectives for obtaining biotechnologically important genes from environmental samples in Part II. The main attention will be paid to soil metagenomics, as this kind of environment seems to be the most promising in terms of microbial biodiversity and the spectrum of biochemical reactions performed by inhabiting bacteria. We will treat the perspectives for isolation of novel, useful genes such as those coding for biosynthesis of antibiotics, organic compounds degrading pathways, and heavy metal resistance and prospects for their biotechnological application. Assessing the microbial biodiversity through metagenomic methods will also be covered.

9

Cell suspension obtained from callus or directly from cotyledons of Pharbitis nil Chois

80%

Trejgell A. , Wisniewska J. , Tretyn A.

Biotechnologia

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2004

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nr 2

219-224

EN

An attempt has been made to obtain cell suspension from callus as well as directly from cotyledons P. nil. Cotyledons of 7-days old sterile seedlings and flower buds excised from 3-week old plants were the material for the induction of callus. The explants were laid out on MS medium supplemented with various combination of plant hormones: BAP (5 mg/l) and NAA (1 mg/l) and supplemented with 3% sucrose or 2% glucose and 1% sucrose, BAP (5 mg/l) and IAA (0,5 mg/l), BAP (0,5 mg/l) and Picloram (1 mg/l), BAP (5 mg/l) and Picloram (0,5 mg/l), 2,4-D (0,125 mg/l).The cultures were grown in continuous white light or in darkness. The callus obtained from cotyledons cultivated in darkness and callus from flower buds cultivated in light on MS medium with BAP (5 mg/l), NAA (1 mg/l), 2% glucose and 1% sucrose were proved useful for obtaining cell suspension. Moreover, an attempt was made to obtain cell suspension directly from cotyledons. Cotyledons were cut into small fragments or were subjected to enzymatic digestion. The cell suspensions were cultivated on MS medium with the addition of BAP (5 mg/l) and IAA (0,5 mg/l) on a shaker at 140 rpm. The increase of cell number was determined by counting the cells every 5 days. In the subsequent subcultures, a decrease of the number of cell divisions was observed.