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1

Suspension cultures of Pharbitis nil

100%
Szyp I., Tretyn A.
Biotechnologia
|
2001
|
issue 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.
2

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

100%
Trejgell A., Tretyn A.
Biotechnologia
|
2001
|
issue 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.
3

Screening of the EST database for identification of the complete cDNA sequence of the gene encoding aquaporin of Pharbitis nil Choisy (PnPIP1)

100%
Dabrowska G., Mordaka P. M.
Biotechnologia
|
2008
|
issue 2
190-198
EN
Aquaporins are membrane proteins that facilitate water transport across the membranes in various microorganisms, plants and animals. Plant aquaporins are divided into four groups based on the amino acid sequence similarities and intracellular localization: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-like intrinsic proteins (NIPs) and small basic intrinsic proteins (SIPs). We found 35 EST sequences homologous to 3' and 5' termini of the partial cDNA of P. nil in the GenBank NCBI database. cDNA encoding full length aquaporin of P. nil was cloned with the use of the reverse transcription-polymerase chain reaction (RT-PCR). The 1189 bp full length cDNA sequence of aquaporin P. nil (PnPIP1) was obtained. Analysis of protein hydropathy indicated that cloned part of PnPIP1 contained the NPA motif (Asn-Pro-Ala) that is present in all known aquaporins. The amino acid sequence of the PnPIP1 protein exhibits 90, 89 and 88% sequence similarity to Petunia x hybrida, Nicotiana excelior and Fraxinus excelsior aquaporins respectively. We showed that the EST database is a useful tool for identification of the complete cDNA of known genes.
4

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

100%
Szyp I., Dabrowska G., Tretyn A.
Biotechnologia
|
2001
|
issue 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.
5

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

88%
Trejgell A., Wisniewska J., Tretyn A.
Biotechnologia
|
2004
|
issue 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.
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