Jolanta Biesaga-Kościelniak

The role of oxidative stress induced by growth regulators in the regeneration process of wheat

As part of work to optimize the regeneration processes of winter wheat callus culture the effects of two auxins (2,4-D, IAA), two cytokinins (kinetin, zeatin), and the fungal mycotoxin zearalenone, were tested individually in vitro using embryo-, and inflorescence-derived callus. To determine the role of oxidative stress in cell regeneration, changes in the basic antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and peroxidases (PODs) were investigated. In general, zearalenone (ZEN) was found to be more effective than cytokinin treatments for inducing shoot production, whereas auxins suppressed the regeneration process. Regenerating callus showed higher induction of these antioxidant enzymes in comparison with non-regenerating callus. SOD, CAT and POD activities were higher in callus derived from inflorescence than in callus derived from immature embryo. Activities of SOD, CAT and POD in culture derived from immature embryos were depending on type of growth regulator in medium. The highest enzyme activities were observed in non-regenerating tissues after auxins treatment and in regenerating tissues after cytokinins treatment. The effect of ZEN was similar to that of cytokinins. One MnSOD band and two Cu/ZnSOD bands were detected in all cultures. Changes in SOD izoform patterns occurred in callus culture on media with auxins and ZEN, but not on media with cytokinins. Our results suggest that callus regeneration is associated with reactive oxygen species production induced by specific growth regulators. Reactive oxygen species under the control of cellular antioxidant machinery can mediate signalling pathways between exogenously applied growth regulators and the induction and/or creation of the direction of morphogenesis.

The influence of animal sex hormones on the induction of flowering in Arabidopsis thaliana: comparison with the effect of 24-epibrassinolide

The influence of selected steroids on the in vitro generative development of Arabidopsis thalianawas investigated. The activity of the animal steroids androsterone, androstenedione, progesterone, estrone, estriol, and 17β-estradiol was compared to 24-epibrassinolide, a member of the regulatory family of brassinosteroids. A. thaliana plants were cultured in vitro in media containing these steroids. The stimulatory effect of the tested substances was evaluated by measurement of the percentage of generative plants versus vegetative plants in the experimental group. It was established that androstenedione, the main testosterone precursor, and androsterone, a typical male hormone, were more effective in stimulating flowering in A. thaliana than the female hormones, estrogens and progesterone. Androsterone at a concentration of 0.1 μM increased the percentage of generative plants up to 96% (control 41%). Estrogens at the same concentration decreased the number of generative plants and 24-epibrassinolide did not stimulate A. thalianagenerative development.

Cytokinins in shoot apices of Brassica napus plants during vernalization

The putative role of cytokinins in processes leading to reproductive development of plants was investigated by analysing the shoot apical parts of a winter cultivar of oilseed rape (Brassica napus L. var. oleifera, cv. Górczański). The endogenous cytokinin levels were measured by liquid chromatography-tandem mass spectrometry (LC-MS) in the shoot apices of vegetative plants (grown at 20/17°C with a 16/8h day/night regime) and vernalized plants (56 days at 5/2°C with a 16/8h photoperiod) at different times during floral transition. During vernalization, the content of all isoprenoid cytokinins increased significantly, coinciding well with the onset of the early stages of reproductive development. Cytokinin levels reached their maxima when most of the plants became irreversibly reproductive (after 42 days of cold treatment). cis-Zeatin riboside (unequivocally identified by quadrupole-time-of-flight MS) accounted for ca. 87-89% of the total isoprenoid cytokinin content in control and vernalized plants, whilst N(6)-isopentenyladenosine ( approximately 6% in control and approximately 8% in vernalized plants) and cis-zeatin (approxiamtely 2% in control and approximately 1% in vernalized plants) were the next most abundant cytokinins. In the post-vernalization period, endogenous cytokinin levels decreased, but remained significantly higher in the reproductive plants than in the vegetative controls. These results suggest that cytokinins, especially those of the cis-zeatin type, are involved in vernalization-induced reproductive development of B. napus.

The impact of zearalenone and thidiazuron on indirect plant regeneration of oilseed rape and wheat

The aim of the study was to test the synergistic activity of zearalenone and thidiazuron with commonly used regulators (6-benzylaminopurine, 2,4-dichlorophenoxyacetic acid) in callus tissue growth and the regeneration processes of winter oilseed rape and winter wheat. Oilseed rape callus was obtained from the upper fragments of seedling hypocotyls. Wheat callus tissue was obtained from immature embryos. Zearalenone, in combination with 6-benzylaminopurine, stimulated the regeneration of oilseed rape and, in combination with 2,4-dichlorophenoxyacetic acid, the regeneration of wheat. An additional application of thidiazuron to these media increased the percentage of shoot regeneration from callus in both species tested. Zearalenone is proposed as a new regulator in in vitro plant cultures for use in protocols of plant regeneration.

Transfer of the ability to flower in winter wheat via callus tissue regenerated from immature inflorescences

Using two wheat varieties (Triticum aestivum L., winter ‘Grana’ and spring ‘Jara’) the ability of callus from immature inflorescences to differentiate into new plants was studied. In the case of the winter wheat the requirement for vernalization of the newly developed plants to attain the ability for heading was investigated.Callus was developed from 1–2 mm fragments of immature inflorescences, 5–10 mm in length, on Murashige and Skoog medium containing 1 mg l-1 2,4-d, 3% sucrose and 0.6% agar, at 25° C and in continuous light of about 6.4 W m-2 PAR energy. After 6 weeks of culture green centres of differentiation were observed. During the following 4 weeks culture on MS medium free of 2,4-d, leaf-like structures as well as a small number of roots were obtained. The regenerants were rooted on a half-strength MS medium, then transferred to pots of soil and placed in a glasshouse with 16 h photoperiod at a temperature day/night 23/17° C.After about 6 weeks of culture in the glasshouse, almost all regenerants (98%) headed and were fertile, producing normal seeds, including the winter variety. The heading of winter plants under conditions excluding vernalization indicates that the callus tissue derived from a generative organ transmits the state of generative induction onto the developing new plants.

The effect of zearalenone on PSII photochemical activity and growth in wheat and soybean under salt (NaCl) stress

The effects of mycotoxin zearalenone (ZEN) on the photochemical activity of photosystem II (PSII) in wheat and soybean leaf discs incubated in ZEN solutions as well as the after-effects of pre-sowing soaking of seeds in solutions containing ZEN on the photochemical activity of PSII and on the seedlings growth under salt stress (NaCl solutions were investigated). The incubation of wheat leaf discs in ZEN solutions strongly inhibited the energy flux per cross section (CS) for absorption (ABS/CS), trapping (TRo/CS) and electron transport (ETo/CS), while the effects of ZEN action on soybean discs were opposite and the values of those parameters significantly increased with the increase in ZEN concentration. Incubation of seeds in a ZEN solution resulted in an increase in photochemical efficiency of PSII in soybean seedlings, but did not induce any response of PSII in those of wheat at medium illuminations. Only at the stronger illumination for both species did ZEN induce an increase in efficiency of excitation energy capture by open PSII reaction centers, photochemical quenching of chlorophyll a fluorescence and quantum yield of PSII electron transport. Pre-sowing soaking of seeds in a ZEN solution decreased the photoinhibitory injuries of PSII in wheat and soybean due to safe scattering of the excess excitation energy through an increase in energy-dependent quenching (qE) and state transition quenching (qT). ZEN when added to NaCl solutions during the period of germination contributed to reduction in the growth inhibition of wheat seedlings. The incubation of wheat leaf discs in ZEN solutions strongly inhibited CS, ABS/CS, TRo/CS and ETo/CS. Possible effects of ZEN on some physiological processes in plants have been discussed especially in the context with photochemical activity of PSII and a salt stress.

Direct electric current partly replaces the chilling effect in vernalisation of winter wheat

Summary Partly vernalised plantlets of Triticum aestivum var. Grana, cultivated in vitro , were subjected for varying lengths of time to electric current of different voltages with the anode (+) attached to the first leaf and the cathode (-) to the medium and vice versa . Twenty-four hours after the treatment, the plantlets were planted into the pots. The percentage of reproductive plants and their time to heading was recorded. The positive electric current greatly increased (in the range of 60 to 90 %) the percentage of generative plants as compared to the controls (5 %) almost irrespective of the voltage and duration of the treatment. The reverse polarity (-) exerted a much weaker effect. The time to heading was almost unchanged by the electric treatments. It was deduced that the effect of electric current replaced part of the chilling requirement and shifted the developmental state to reproductive phase. The experiments are in progress to elucidate the interaction between electric current and chemical signals that may be involved in flower induction.

The Influence of Growth Regulators on Membrane Permeability in Cultures of Winter Wheat Cells

Abstract The effect of plant growth substances (IAA, 2,4-D, zeatin, kinetin, zearalenone) were studied on membrane properties of the cells of embryogenic (E) and non-embryogenic (NE) calli derived from immature inflorescences (inf) or embryos (emb) of winter wheat. Calli initiated from inflorescences show higher permeability. The ion leakage from cells of E calli was higher than from cells of NE calli. Growth regulators were used in concentrations of 2-30 mg/l (about 10-140 μm). All tested growth substances increased ion leakage from NE emb cells, IAA, zeatin and kinetin being most effective. In NE inf cells the effect of growth substances was similar as in NE emb, but much weaker. In E cells of both types (inf and emb) growth substances decreased ion leakage. Changes in the leakage of potassium and calcium ions were similar to those in total ion leakage. The uptake of labelled auxins (IAA and 2,4-D) was higher in NE cells (especially in NE inf) than in E cells. The endogenous level of IAA was higher in E cells than in NE cells and in inf cells than in emb cells. The importance of auxin in determining permeability of cell membranes is discussed.

Contents of polyamines during vernalization in wheat and the effect of zearalenone

The contents of endogenous free and conjugated polyamines, putrescine (Put) and spermidine (Spd), were determined during 9 week of vernalization (at 5 °C) in winter wheat seedlings cultivated on Murashige and Skoog media without (MS0) and with 2 mg dm−3 zearalenone (MSZEN). At the 4th week of chilling treatment, which is sufficient to induce generative development in 30 % of plants, the marked increase in free and conjugated forms of Put and free Spd were observed. The presence of ZEN in medium significantly accelerated the vernalization. About 20 % of plants treated with ZEN flowered already after 2 weeks and 40 % after 3 weeks of chilling. Significantly higher content of free Put was determined in roots grown on MSZEN compared with MS0 during the first 5 weeks of vernalization with maximum at the 4th week. After germination, a marked decrease in free Spd content was observed both in plants grown on MS0 and MSZEN. Application of ZEN significantly slowed down the Spd decline in leaves and roots during the first and second week of vernalization. The content of Spd and its conjugates decreased in vernalized plants after 1 week of cultivation at 20 °C.

Can the Giberella zeae toxin zearalenone affect the photosynthetic productivity and increase yield formation in spring wheat and soybean plants

The seeds of soybean cv. Aldana and spring wheat cv. Torka were soaked for 24 h in solution of zearalenone [ZEN, 2,4-dihydroxy-6-(10-hydroxy-6-oxo-trans-1-undecenyl)-benzonic acid lactone, 4 mg dm−3] and then they were sown in the pot experiment in an open vegetation hall. The after-effects of ZEN on growth of plants, net photosynthetic (PN) and transpiration (E) rates, stomatal conductance (gs), photochemical efficiency of photosystem II (PSII) and on final seeds yield, were determined. A significant increase of seeds yield was revealed in plants of both cultivars i.e. by 22% and 19% of seed (grain) number and by 28 and 24% of seed (grain) mass, in soybean and in wheat, respectively. The photosynthetic rate (PN) was stimulated during the juvenile and final phase by about 13.6% (average) in soybean plants. During other developmental stages, assimilation of CO2 was retarded. The response of CO2 assimilation in wheat plants was less pronounced as compared to that in soybean, but an increase of PN by over 24% near the final stage of development was observed. The quantum yield of PSII electron transport (ΦPSII) in soybean plants was changed after the treatment of seeds by ZEN similarly as for the rate of CO2, whereas in wheat it continued to gradually increase i.e. during the whole growth period. Changes of ΦPSII both in soybean and in wheat plants, as the response to ZEN treatment, were accompanied with an increase in the efficiency of changes occurring within the antenna (Fv′/Fm′) as well as within centres of photochemical reactions (qp). The conclusion is that ZEN can affect plant growth and development in many ways, as well as in the status and functioning of the photosynthetical apparatus. Some of the effects can be very longlasting, as e.g. stimulation of production of seed yield in response to treatment of seeds with this substance.