Edyta Skrzypek

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.

Alleviation of Osmotic Stress Effects by Exogenous Application of Salicylic or Abscisic Acid on Wheat Seedlings

The aim of the study was to assess the role of salicylic acid (SA) and abscisic acid (ABA) in osmotic stress tolerance of wheat seedlings. This was accomplished by determining the impact of the acids applied exogenously on seedlings grown under osmotic stress in hydroponics. The investigation was unique in its comprehensiveness, examining changes under osmotic stress and other conditions, and testing a number of parameters simultaneously. In both drought susceptible (SQ1) and drought resistant (CS) wheat cultivars, significant physiological and biochemical changes were observed upon the addition of SA (0.05 mM) or ABA (0.1 μM) to solutions containing half-strength Hoagland medium and PEG 6000 (−0.75 MPa). The most noticeable result of supplementing SA or ABA to the medium (PEG + SA and PEG + ABA) was a decrease in the length of leaves and roots in both cultivars. While PEG treatment reduced gas exchange parameters, chlorophyll content in CS, and osmotic potential, and conversely, increased lipid peroxidation, soluble carbohydrates in SQ1, proline content in both cultivars and total antioxidants activity in SQ1, PEG + SA or PEG + ABA did not change the values of these parameters. Furthermore, PEG caused a two-fold increase of endogenous ABA content in SQ1 and a four-fold increase in CS. PEG + ABA increased endogenous ABA only in SQ1, whereas PEG + SA caused a greater increase of ABA content in both cultivars compared to PEG. In PEG-treated plants growing until the harvest, a greater decrease of yield components was observed in SQ1 than in CS. PEG + SA, and particularly PEG + ABA, caused a greater increase of these yield parameters in CS compared to SQ1. In conclusion, SA and ABA ameliorate, particularly in the tolerant wheat cultivar, the harmful effects and after effects of osmotic stress induced by PEG in hydroponics through better osmotic adjustment achieved by an increase in proline and carbohydrate content as well as by an increase in antioxidant activity.

Influence of auxins on somatic embryogenesis and alkaloid accumulation in Leucojum aestivum callus

In vitro cultures of Leucojum aestivum are considered as an alternative for the production of galanthamine, which is used for the symptomatic treatment of Alzheimer’s disease. We studied the effects of auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), 3,6-dichloro-o-anisic acid (dicamba) at concentrations of 25 and 50 µM on the induction of embryogenic callus and its capacity to induce somatic embryogenesis and alkaloid accumulation. The embryogenic response of the explants was from 30% for 25 µM of dicamba to 100% for picloram (for both 25 and 50 µM). 2,4-D (50 µM) stimulated greater callus proliferation and somatic embryo induction as compared to the other auxins. Polyethylene glycol (PEG) stimulated somatic embryo maturation. Callus grown on media containing 50 µM of auxins produced fewer phenolic compounds as compared with callus grown on media containing 25 µM of auxins. GC-MS analyses showed seven alkaloids in the in vivo bulbs and two to four in callus culture. Galanthamine was detected in callus cultivated with 2,4-D (25, 50 µM), picloram (25 µM), and dicamba (50 µM). Other alkaloids, trisphaeridine, tazettine, and 11-hydroxyvittatine were accumulated only in callus growing on medium with picloram (50 µM).

Production of double haploids in oat (Avena sativa L.) by pollination with maize (Zea mays L.)

The aim of the study was to optimize the method of oat haploid production by pollination with maize. Seventeen oat genotypes were used in the experiment. Various factors influencing the growth and development of ovaries and embryo production were investigated: genotype, time of pollination, growth regulators and time of their application. Emasculated before anthesis, oat florets were pollinated with maize pollen after 0, 1 or 2 days. Next, one of two auxins analogues (2,4-D or dicamba) were applied to oat pistils. These auxins had no significant influence on the number of enlarged ovaries and embryos. The time of application of these growth regulators had a significant influence on embryo production. Haploid embryos were obtained from all used genotypes, although the frequency of enlarged ovaries and obtained embryos did not differ markedly between the genotypes. On average, 85% of ovaries were enlarged and 11.7% of them produced haploid embryos. Depending on the regeneration medium, 24–41% of embryos were germinated, of which 12% had developed into green plants. A strong significant difference in the number of germinating embryos and haploid plants was observed between the kind of regenerating medium used. There were no albino plants and all the obtained plants were haploid.

The effect of endogenous hydrogen peroxide induced by cold treatment in the improvement of tissue regeneration efficiency

We propose that oxidative stress resulting from an imbalance between generation and scavenging hydrogen peroxide contributes to tissue regeneration efficiency during somatic embryogenesis of hexaploid winter wheat (Triticum aestivum cv. Kamila) and organogenesis of faba bean (Vicia faba ssp. minor cv. Nadwislanski). Endogenous hydrogen peroxide content and antioxidant capacity of cells were determined in initial explants and callus cultures derived from these explants. Regeneration-competent explants (immature embryos) contained more endogenous H2O2 than explants initiated from regeneration-recalcitrant tissue (mature wheat embryos and faba bean epicotyls). Higher H2O2 levels were observed despite the higher activity of antioxidative enzymes (superoxide dismutase and catalase) and the induction of their gene expression. Calli originating from immature embryos retained the capacity of the initial explants: high H2O2 production was observed during the whole culture period. Low temperature treatment (4°C) was found to be an effective factor, which improved both regeneration ability and H2O2 production. Exogenous application to the medium of H2O2 and catalase blocker (3-aminotriazole), but not FeEDTA and superoxide dismutase blocker (diethyldithiocarbamate), also resulted in the enhancement of regeneration efficiency. These results clearly indicate that plant regeneration is specifically regulated by endogenous H2O2 and by factors, which improve its accumulation. Moreover, a study of the activity of various SOD isoforms suggests that not only the absolute concentration of H2O2, but also its localisation might be responsible for controlling regeneration processes.

QTL mapping for germination of seeds obtained from previous wheat generation under drought

The QTLs controlling germination and early seedling growth were mapped using seeds acquired from mapping population and parental lines of Chinese Spring and SQ1 grown under water-limited conditions, severe drought (SDr) and well-watered plants (C). Germination ability was determined by performing a standard germination test based on the quantification of the germination percentage (GP24) of seeds incubated for 24 h at 25°C in the dark. Early seedling growth was evaluated on the basis of the length of the root and leaf at the 6th day of the experiment. QTLs were identified by composite interval mapping method using Windows QTLCartographer 2.5 software. For the traits studied, a total of thirty eight additive QTLs were identified. Seventeen QTLs were mapped in C on chromosomes: 1A, 2A, 7A, 1B, 2B, 3B, 4B, 5B, 6B, 7B, 2D, 3D, 4D and 6D, while twenty one QTLs were identified in SDr on chromosomes: 1A, 2A, 5A, 2B, 3B, 4B, 5B, 6B, 7B, 3D, 5D and 6D. Most of the QTLs for GP and early leaf growth parameters were clustered on chromosome 4B (associated with the Rht-B1 marker) both in C and SDr plants. The results indicate the complex and polygenic nature of germination.

Application of chosen factors in the wide crossingmethod for the production of oat doubled haploids

Abstract Oat (Avena sativa L.) has recently gained importance due to the discovery of a variety of health benefits and new opportunities of use. There is no efficient protocol for the production of oat doubled haploid (DH) lines. The aim of this study was to increase the efficiency of obtaining DHs of oat by the wide crossing method. The study was performed on five oat genotypes. We have compared the induction of embryos after pollination with maize, sorghum and millet pollen as well as the development of haploid embryos isolated 2, 3 and 4 weeks after pollination and cultivated on media with different sugar content. Haploid plants were treated with colchicine after or before acclimation to natural conditions. Of the three types of pollen used, the largest number of haploid embryos was obtained using maize pollen. Three weeks after pollination was the most suitable time for the isolation and cultivation of the embryos. The most efficient medium enabling the development of embryos and conversion to plants was 190-2 containing 9% of maltose. Colchicine treatment of acclimated plants provided high survival rate.

The effect of light intensity on the production of oat (Avena sativa L.) doubled haploids through oat × maize crosses

Oat haploid embryos were obtained by wide crossing with maize. The effect of light intensity during the growing period of donor plants (450 and 800 μmol m−2 s−1) and in vitro cultures (20, 40, 70 and 110 μmol m−2 s−1) was examined for the induction and development of oat DH lines. Oat florets (26008) from 32 genotypes were pollinated with maize and treated with 2,4-dichlorophenoxyacetic acid. All the tested genotypes formed more haploid embryos when donor plants were grown in a greenhouse (9.4%) compared to a growth chamber (6.1%). The light intensity of 110 μmol m−2 s−1 during in vitro culture resulted in the highest percentage of embryo germination (38.9%), conversion into plants (36.4%) and DH line production (9.2%) when compared with lower light intensities (20, 40 and 70 μmol m−2 s−1). The results show that the growth conditions of the donor plant and light intensity during in vitro culture can affect the development of haploid embryos. This fact may have an impact on oat breeding programs using oat × maize crosses.

Genetic analysis of water loss of excised leaves associated with drought tolerance in wheat

Background Wheat is widely affected by drought. Low excised-leaf water loss (ELWL) has frequently been associated with improved grain yield under drought. This study dissected the genetic control of ELWL in wheat, associated physiological, morphological and anatomical leaf traits, and compared these with yield QTLs. Methods Ninety-four hexaploid wheat (Triticum aestivum L.) doubled haploids, mapped with over 700 markers, were tested for three years for ELWL from detached leaf 4 of glasshouse-grown plants. In one experiment, stomata per unit area and leaf thickness parameters from leaf cross-sections were measured. QTLs were identified using QTLCartographer. Results ELWL was significantly negatively correlated with leaf length, width, area and thickness. Major QTLs for ELWL during 0–3 h and 3–6 h were coincident across trials on 3A, 3B, 4B, 5B, 5D, 6B, 7A, 7B, 7D and frequently coincident (inversely) with leaf size QTLs. Yield in other trials was sometimes associated with ELWL and leaf size phenotypically and genotypically, but more frequently under non-droughted than droughted conditions. QTL coincidence showed only ELWL to be associated with drought/control yield ratio. Discussion Our results demonstrated that measures of ELWL and leaf size were equally effective predictors of yield, and both were more useful for selecting under favourable than stressed conditions.

Complex characterization of oat (Avena sativa L.) lines obtained by wide crossing with maize (Zea mays L.)

Background The oat × maize addition (OMA) lines are used for mapping of the maize genome, the studies of centromere-specific histone (CENH3), gene expression, meiotic chromosome behavior and also for introducing maize C4 photosynthetic system to oat. The aim of our study was the identification and molecular-cytogenetic characterization of oat × maize hybrids. Methods Oat DH lines and oat × maize hybrids were obtained using the wide crossing of Avena sativa L. with Zea mays L. The plants identified as having a Grande-1 retrotransposon fragment, which produced seeds, were used for genomic in situ hybridization (GISH). Results A total of 138 oat lines obtained by crossing of 2,314 oat plants from 80 genotypes with maize cv. Waza were tested for the presence of maize chromosomes. The presence of maize chromatin was indicated in 66 lines by amplification of the PCR product (500 bp) generated using primers specific for the maize retrotransposon Grande-1. Genomic in situ hybridization (GISH) detected whole maize chromosomes in eight lines (40%). All of the analyzed plants possessed full complement of oat chromosomes. The number of maize chromosomes differed between the OMA lines. Four OMA lines possessed two maize chromosomes similar in size, three OMA—one maize chromosome, and one OMA—four maize chromosomes. In most of the lines, the detected chromosomes were labeled uniformly. The presence of six 45S rDNA loci was detected in oat chromosomes, but none of the added maize chromosomes in any of the lines carried 45S rDNA locus. Twenty of the analyzed lines did not possess whole maize chromosomes, but the introgression of maize chromatin in the oat chromosomes. Five of 66 hybrids were shorter in height, grassy type without panicles. Twenty-seven OMA lines were fertile and produced seeds ranging in number from 1–102 (in total 613). Sixty-three fertile DH lines, out of 72 which did not have an addition of maize chromosomes or chromatin, produced seeds in the range of 1–343 (in total 3,758). Obtained DH and OMA lines were fertile and produced seeds. Discussion In wide hybridization of oat with maize, the complete or incomplete chromosomes elimination of maize occur. Hybrids of oat and maize had a complete set of oat chromosomes without maize chromosomes, and a complete set of oat chromosomes with one to four retained maize chromosomes.