Maria Wędzony

Progress in Doubled Haploid Technology in Higher Plants

In the early 1990s, many basic protocols were developed for haploidy and doubled haploidy, but most were inefficient. During the last decade, progress in technology has been achieved mainly by empirical, time and cost consuming testing of protocols; as a consequence success was proportional to the number of laboratories involved. In the most frequently studied crops (barley, wheat, triticale, maize, rice and rapeseed) improved protocols are now used routinely in breeding and although several problems remain the benefits make doubled haploidy well worthwhile. Significant advances have also been achieved in vegetable, fruit, ornamental, woody and medicinal species, though responses in many remain low with legumes being particularly recalcitrant. There has been resurgence in doubled haploids over the last few years with protocols published for almost 200 plant species. The present review aims to show the recent progress in haploid and doubled haploid technology of higher plants.

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.

Stress-related variation in antioxidative enzymes activity and cell metabolism efficiency associated with embryogenesis induction in isolated microspore culture of triticale (x Triticosecale Wittm.)

Isolated microspore cultures of two spring triticale (x Triticosecale Wittm.) cultivars were used to examine the effect of various stress treatments (either high—32°C or low—5°C temperature with or without nitrogen/carbohydrate starvation) applied to excised anthers on the effectiveness of microspore embryogenesis induction. To quantify the effects of pretreatment conditions, the activity of antioxidative enzymes (catalase, peroxidase and superoxide dismutase) together with respiration rate and heat emission were measured. It was observed that heat shock treatment applied as the only one stress factor increased the activity of antioxidative enzymes which suggests intensive generation of reactive oxygen species. Such pretreatment effectively triggered microspore reprogramming but drastically decreased microspore viability. After low temperature treatment, the activity of antioxidative enzymes was similar to the control subjected only with the stress originated from the transfer to in vitro culture conditions. This pretreatment decreased the number of microspores entering embryogenesis but sustained cell viability and this effect prevailed in the final estimation of microspore embryogenesis effectiveness. For both, low- and high-temperature treatments, interaction with starvation stress was beneficial increasing microspore viability (at 5°C) or efficiency of embryogenesis induction (at 32°C). The latter treatment significantly reduced cell metabolic activity. Physiological background of these effects seems to be different and some hypothetical explanations have been discussed. Received data indicate that in triticale, anther preculture conditions could generate oxidative stress and change the cell metabolic activity which could next be reflected in the cell viability and the efficiency of microspore embryogenesis.

Stress-induced changes important for effective androgenic induction in isolated microspore culture of triticale (× Triticosecale Wittm.)

The accumulation of abscisic acid (ABA) and the activities of antioxidative enzymes along with cell metabolic activity were monitored during androgenesis induction in triticale (×Triticosecale Wittm.). Tested cultivars ‘Mieszko’ and ‘Wanad’ were selected due to their significantly different responses to androgenic induction. Significant variation was observed in respect of superoxide dismutase activity and endogenous ABA content in anthers isolated from freshly cut tillers. For both cultivars, tillers pretreatment with low temperature decreased peroxidase activity by 36%, highly accelerated respiration rate and reduced heat production. At the same time, the level of ABA in ‘Mieszko’ was increased to the level measured in ‘Wanad’. This effect was associated with higher microspore culture viability and increased stress tolerance in ‘Mieszko’. Low temperature and metabolic starvation during 4-day anther preculture did not influence activities of antioxidative enzymes, while it resulted in slight decrease in respiration rate and heat emission. The importance of these changes for effective androgenesis induction is discussed.

Microtubule configurations and nuclear DNA synthesis during initiation of suspensor-bearing embryos from Brassica napus cv. Topas microspores

In the new Brassica napus microspore culture system, wherein embryos with suspensors are formed, ab initio mimics zygotic embryogenesis. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during early stages of plant embryogenesis. Here, we studied in this new culture system both the temporal and spatial distribution of nuclear DNA synthesis places and the organization of the microtubular (MT) cytoskeleton, which were visualized with a refined whole mount immunolocalization technology and 3D confocal laser scanning microscopy. A ‘mild’ heat stress induced microspores to elongate, to rearrange their MT cytoskeleton and to re-enter the cell cycle and perform a predictable sequence of divisions. These events led to the formation of a filamentous suspensor-like structure, of which the distal tip cell gave rise to the embryo proper. Cells of the developing pro-embryo characterized endoplasmic (EMTs) and cortical microtubules (CMTs) in various configurations in the successive stages of the cell cycle. However, the most prominent changes in MT configurations and nuclear DNA replication concerned the first sporophytic division occurring within microspores and the apical cell of the pro-embryo. Microspore embryogenesis was preceded by pre-prophase band formation and DNA synthesis. The apical cell of the pro-embryo exhibited a random organization of CMTs and, in relation to this, isotropic expansion occurred, mimicking the development of the apical cell of the zygotic situation. Moreover, the apical cell entered the S phase shortly before it divided transversally at the stage that the suspensor was 3–8 celled.

Visualisation of microtubules and actin filaments in fixed BY-2 suspension cells using an optimised whole mount immunolabelling protocol

Excellent visualisation of microtubules and actin filaments was obtained in fixed tobacco BY-2 suspension cells after optimising a protocol for whole mount immunolabelling. The procedure is based on modification of fixation, cell wall digestion, dimethyl sulfoxide (DMSO) treatment, post fixation, and blocking. The most critical aspects of successful preservation and visualization of cytoskeletal elements appeared to be: a two-step fixation with paraformaldehyde and glutaraldehyde before enzymatic cell wall digestion and a post fixation with aldehydes thereafter. The method allows the improved visualization of the organisation of the microtubular and actin filament arrays during the successive stages of cell division and at interphase. Although we present the application of our protocols for cytoskeleton labelling, the excellent results show the potential of using this method for the analysis of various proteins and molecules in plant cells.

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.

Gametophytic development of Brassica napus pollen in vitroenables examination of cytoskeleton and nuclear movements

Isolated microspores and pollen suspension of Brassica napus “Topas” cultured in NLN-13 medium at 18°C follow gametophytic pathway and develop into pollen grains closely resembling pollen formed in planta. This culture system complemented with whole-mount immunocytochemical technology and novel confocal laser scanning optical technique enables detailed studies of male gametophyte including asymmetric division, cytoskeleton, and nuclear movements. Microtubular cytoskeleton configurationally changed in successive stages of pollen development. The most prominent role of microtubules (MTs) was observed just before and during nuclear migration at the early and mid-bi-cellular stage. At the early bi-cellular stage, parallel arrangement of cortical and endoplasmic MTs to the long axis of the generative cell (GC) as well as MTs within GC under the plasmalemma bordering vegetative cell (VC) were responsible for GC lens shape. At the beginning of the GC migration, endoplasmic microtubules (EMTs) of the VC radiated from the nuclear envelope. Most cortical and EMTs of the VC were found near the sporoderm. At the same time, pattern of MTs observed in GC was considerably different. Multiple EMTs of the GC, previously parallel aligned, reorganized, and start to surround GC, forming a basket-like structure. These results suggest that EMTs of GC provoke changes in GC shape, its detachment from the sporoderm, and play an important role in GC migration to the vegetative nucleus (VN). During the process of migration of the GC to the VC, multiple and thick bundles of MTs, radiating from the cytoplasm near GC plasma membrane, arranged perpendicular to the narrow end of the GC and organized into a “comet-tail” form. These GC “tail” MTs became shortened and the generative nucleus (GN) took a ball shape. The dynamic changes of MTs accompanied polarized distribution pattern of mitochondria and endoplasmic reticulum. In order to confirm the role of MTs in pollen development, a “whole-mount” immunodetection technique and confocal laser-scanning microscopy was essential.

Changes of electric potential in pistils of Petunia hybrida Hort. and Brassica napus L. during pollination

The pattern of electric signals accompanying compatible and incompatible pollination were studied in pistils of petunia (Petunia hybrida L.) and rape (Brassica napus L). Electric potential was recorded for 4–7 hours with non-polarizable Ag/AgCl electrodes implanted into the ovary and beneath the sigma. At the end of measurements, pistils were fixed and the growth of pollen tubes was analyzed under a fluorescent microscope.Action potentials appeared in both species. In rape the potential dropped by 10 mV for few minutes after pollination regardless of the compatibility of the cross. In this species, during compatible pollination action potentials with amplitudes of 15–20 mV were recorded up to one hour after pollination. They were followed by a long lasting decrease of the potential by 10 to 50 mV. Contrary, after the self-incompatible pollination, action potentials were rare and of lower amplitudes and the potential gradually raised in comparison to the initial level. During the first hour after the compatible pollination of Petunia hybrida series of action potentials with amplitudes reaching 10–20 mV were recorded. At the time corresponding to the pollen tubes entrance to the transmitting tissue of the style, action potentials reaching up to 40 mV were followed by a steady decrease of the potential. The electric signals traveled along the style with velocity of 25 mm/s. Incompatible pollination in petunia resulted only in minor oscillation and gradual increase of the potential up to 100 mV in comparison to the initial level.The present investigation demonstrated that each phase of pollen-stigma recognition events, germination and growth of pollen tubes within the style have its characteristic pattern of electric changes which was species specific and depended on compatibility of the cross.

Genetic Map of Triticale Integrating Microsatellite, DArT and SNP Markers

Triticale (×Triticosecale Wittm) is an economically important crop for fodder and biomass production. To facilitate the identification of markers for agronomically important traits and for genetic and genomic characteristics of this species, a new high-density genetic linkage map of triticale was constructed using doubled haploid (DH) population derived from a cross between cultivars ‘Hewo’ and ‘Magnat’. The map consists of 1615 bin markers, that represent 50 simple sequence repeat (SSR), 842 diversity array technology (DArT), and 16888 DArTseq markers mapped onto 20 linkage groups assigned to the A, B, and R genomes of triticale. No markers specific to chromosome 7R were found, instead mosaic linkage group composed of 1880 highly distorted markers (116 bins) from 10 wheat chromosomes was identified. The genetic map covers 4907 cM with a mean distance between two bins of 3.0 cM. Comparative analysis in respect to published maps of wheat, rye and triticale revealed possible deletions in chromosomes 4B, 5A, and 6A, as well as inversion in chromosome 7B. The number of bin markers in each chromosome varied from 24 in chromosome 3R to 147 in chromosome 6R. The length of individual chromosomes ranged between 50.7 cM for chromosome 2R and 386.2 cM for chromosome 7B. A total of 512 (31.7%) bin markers showed significant (P < 0.05) segregation distortion across all chromosomes. The number of 8 the segregation distorted regions (SDRs) were identified on 1A, 7A, 1B, 2B, 7B (2 SDRs), 5R and 6R chromosomes. The high-density genetic map of triticale will facilitate fine mapping of quantitative trait loci, the identification of candidate genes and map-based cloning.