Variation between glaucous and non-glaucous near-isogenic lines of rye (Secale cereale L.) under drought stress.

Glaucous (811, L35, and RXL10) and non-glaucous (811bw, L35bw, and RXL10bw) near-isogenic lines (NILs) of rye (Secale cereale L.) forming three pairs of inbred lines were the subject of the research. The research aimed to study the relationship between wax cover attributes and the physio-biochemical drought reactions and yield of rye NILs and to uncover the differences in drought resistance levels of these lines. The greatest differences between glaucous and non-glaucous NILs were observed in the RXL10/RXL10bw pair. Of particular note were the stable grain number and the thousand grain weight of the non-glaucous line RXL10bw under drought and the accompanying reactions, such as an approximately 60% increase in MDA and a two-fold increase in wax amount, both of which were significantly higher than in the glaucous line RXL10 and in other NILs. The surprisingly high level of MDA in the RXL10bw line requires further analysis. Moreover, additional wax crystal aggregates were found under drought conditions on the abaxial leaf surface of the glaucous lines 811 and RXL10. The use of rye NILs indicated that line-specific drought resistance could be associated with wax biosynthetic pathways involved in physiological and biochemical responses important for increased drought resistance.

The effect of 2,4-dichlorophenoxyacetic acid on the production of oat (Avena sativa L.) doubled haploid lines through wide hybridization.

BACKGROUND: Development of new cultivars is one of the vital options for adapting agriculture to climate change, and the production of doubled haploid (DH) plants can make a significant contribution to accelerating the breeding process. Oat is one of the cereals with particular health benefits, but it unfortunately still remains recalcitrant to haploidization. Our previous studies have clearly demonstrated that post-pollination with hormone treatment is a key step in haploid production through wide hybridization and indicated it as the most effective method for this species. Therefore, we subsequently addressed the problem of the influence of 2,4-dichlorophenoxyacetic acid (2,4-D) concentration on consecutive stages of DH production. METHODS: Twenty-nine genotypes were tested, 9,465 florets were pollinated with maize pollen 2 days after emasculation and then treated with 2,4-D at 50 mg/L and 100 mg/L. RESULTS: The applied treatments did not reveal any differences in the number of obtained haploid embryos. However, almost twice as many haploid plants formed on MS medium after applying a higher auxin concentration and 20% more successfully acclimatized. Moreover, 100 mg/L 2,4-D treatment resulted in twice as many DH lines that produced almost three times more seeds compared to 50 mg/L treatment. Nevertheless, the results have confirmed the existence of strong genotypic variation, which may significantly limit the development of an effective and economically feasible method that could be incorporated into breeding programs.

Anatomical and hormonal factors determining the development of haploid and zygotic embryos of oat (Avena sativa L.).

A critical step in the production of doubled haploids is a conversion of the haploid embryos into plants. Our study aimed to recognize the reasons for the low germination rate of Avena sativa haploid embryos obtained by distant crossing with maize. Oat cultivars of 'Krezus' and 'Akt' were investigated regarding embryo anatomy, the endogenous phytohormone profiles, and antioxidant capacity. The zygotic embryos of oat were used as a reference. It was found that twenty-one days old haploid embryos were smaller and had a less advanced structure than zygotic ones. Morphology and anatomy modifications of haploid embryos were accompanied by extremely low levels of endogenous auxins. Higher levels of cytokinins, as well as tenfold higher cytokinin to auxin ratio in haploid than in zygotic embryos, may suggest an earlier stage of development of these former. Individual gibberellins reached higher values in 'Akt' haploid embryos than in the respective zygotic ones, while the differences in both types of 'Krezus' embryos were not noticed. Additionally to the hormonal regulation of haploid embryogenesis, the poor germination of oat haploid embryos can be a result of the overproduction of reactive oxygen species, and therefore higher levels of low molecular weight antioxidants and stress hormones.

Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei.

The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.

Impact of Ascorbate-Glutathione Cycle Components on the Effectiveness of Embryogenesis Induction in Isolated Microspore Cultures of Barley and Triticale.

Enhanced antioxidant defence plays an essential role in plant survival under stress conditions. However, excessive antioxidant activity sometimes suppresses the signal necessary for the initiation of the desired biological reactions. One such example is microspore embryogenesis (ME)-a process of embryo-like structure formation triggered by stress in immature male gametophytes. The study focused on the role of reactive oxygen species and antioxidant defence in triticale (×Triticosecale Wittm.) and barley (Hordeum vulgare L.) microspore reprogramming. ME was induced through various stress treatments of tillers and its effectiveness was analysed in terms of ascorbate and glutathione contents, total activity of low molecular weight antioxidants and activities of glutathione-ascorbate cycle enzymes. The most effective treatment for both species was a combination of low temperature and exogenous application of 0.3 M mannitol, with or without 0.3 mM reduced glutathione. The applied treatments induced genotype-specific defence responses. In triticale, both ascorbate and glutathione were associated with ME induction, though the role of glutathione did not seem to be related to its function as a reducing agent. In barley, effective ME was accompanied by an accumulation of ascorbate and high activity of enzymes regulating its redox status, without direct relation to glutathione content.

Oat (Avena sativa L.) Anther Culture.

Production of doubled haploids (DHs) by androgenesis is a promising and convenient alternative to traditionally used breeding techniques. Low response of anther culture and strong genotype dependency in the development of embryo-like structures (ELS) was reported for oat (Avena sativa L.). Total homozygosity has been reached in one generation. This chapter describes a step-by-step protocol that can be useful for androgenesis studies and oat DH line production through anther culture.

Oat Doubled Haploid Production Through Wide Hybridization with Maize.

Wide hybridization is one of the haploid-inducing techniques that can accelerate the breeding process. Obtaining new cultivars is crucial to solve the problem of the constantly growing world population and global increase in demand for food, feed and renewable energy under changing environmental conditions. Here, we present a detailed protocol for obtaining oat (Avena sativa L.) doubled haploids (DHs) by pollination with maize (Zea mays L.). After fertilization, not only oat homozygotes, but also oat × maize hybrid zygotes can be formed, and during early embryo development, maize chromosomes are preferentially eliminated, which ultimately results in haploid plant formation. This chapter describes a method to produce oat DHs by crossing oat with maize, covering all steps from crossings to haploid plant regeneration and chromosome doubling.

The Effect of Zinc, Copper, and Silver Ions on Oat (Avena sativa L.) Androgenesis.

Oat (Avena sativa L.) cultivars 'Bingo' and 'Chwat' were used to compare the embryogenesis competence of another culture. Despite the embryo-like structures obtained from both tested cultivars, only 'Chwat' produced green plantlets, which confirmed the cultivar dependency. 'Chwat' produced the highest number of embryo-like structures and green plantlets (0.7/100 anthers and 0.1/100 anthers, respectively). The embryo-like structure formation also depended on cold pretreatment combined with Cu2+, Zn2+, or Ag+ ion supplementation, which was applied during the tiller pretreatment or added to the induction media. The highest number of embryo-like structures (2.1/100 anthers) were observed on anthers derived from the tillers kept in a 50% Hoagland medium with the addition of 10 µM of CuSO4. In turn, the induction media supplemented with the ions Cu2+, Zn2+, or Ag+ increased neither the number of embryo-like structures nor the green plantlet production compared to the control conditions. However, such ion applications turned out to be most effective when the induction medium was enriched with 25 µM of AgNO3 and left to obtain the highest number of embryo-like structures and green plantlets (0.8/100 anthers and 0.2/100 anthers, respectively). Therefore, more attention should be paid to the possibilities of adjusting the media nutrient composition, as this may be the only way to significantly increase the efficiency of this method.

Functioning of the Photosynthetic Apparatus in Response to Drought Stress in Oat × Maize Addition Lines.

The oat × maize chromosome addition (OMA) lines, as hybrids between C3 and C4 plants, can potentially help us understand the process of C4 photosynthesis. However, photosynthesis is often affected by adverse environmental conditions, including drought stress. Therefore, to assess the functioning of the photosynthetic apparatus in OMA lines under drought stress, the chlorophyll content and chlorophyll a fluorescence (CF) parameters were investigated. With optimal hydration, most of the tested OMA lines, compared to oat cv. Bingo, showed higher pigment content, and some of them were characterized by increased values of selected CF parameters. Although 14 days of drought caused a decrease of chlorophylls and carotenoids, only slight changes in CF parameters were observed, which can indicate proper photosynthetic efficiency in most of examined OMA lines compared to oat cv. Bingo. The obtained data revealed that expected changes in hybrid functioning depend more on the specific maize chromosome and its interaction with the oat genome rather than the number of retained chromosomes. OMA lines not only constitute a powerful tool for maize genomics but also are a source of valuable variation in plant breeding, and can help us to understand plant susceptibility to drought. Our research confirms more efficient functioning of hybrid photosynthetic apparatus than oat cv. Bingo, therefore contributes to raising new questions in the fields of plant physiology and biochemistry. Due to the fact that the oat genome is not fully sequenced yet, the mechanism of enhanced photosynthetic efficiency in OMA lines requires further research.

Exogenous Polyamines Only Indirectly Induce Stress Tolerance in Wheat Growing in Hydroponic Culture under Polyethylene Glycol-Induced Osmotic Stress.

The aim of the present study was to evaluate the effect of osmotic stress caused by polyethylene glycol (PEG) 6000 in hydroponic culture on wheat seedlings of drought-resistant Chinese Spring (CS) and drought-susceptible SQ1 cultivar, and to examine the alleviative role of exogenous polyamines (PAs) applied to the medium. The assessment was based on physiological (chlorophyll a fluorescence kinetics, chlorophyll and water content) as well as biochemical (content of carbohydrates, phenols, proline, salicylic and abscisic acid, activity of low molecular weight antioxidants) parameters, measured after supplementation with PAs (putrescine, spermidine and spermine) on the 3rd, 5th and 7th day of the treatment. The results indicate that PAs ameliorate the effects of stress, indirectly and conditionally inducing stress tolerance of wheat seedlings. In contrast to the susceptible SQ1, the resistant CS cultivar activated its protective mechanisms, adjusting the degree of their activation to the level of the stress, depending on the genetic resources of the plant. Increased accumulation of antioxidants in the resistant CS in response to stress after the application of PAs confirms the hypothesis that PAs are involved in the signaling pathway determining the antioxidative response and the tolerance of wheat plants to drought stress.

Chemically-induced DNA de-methylation alters the effectiveness of microspore embryogenesis in triticale.

Microspores exposed to some stress factors may display cell totipotency and could be reprogrammed towards embryogenic development. Plant breeding and genetic engineering widely use haploids/doubled haploids (DHs) derived from in vitro-cultured microspores, but the mechanism of this process remains poorly understood. Recently published data suggest that microspore embryogenesis (ME) is accompanied by changes in DNA methylation and chromatin reorganization. Here, we used two triticale DH lines (DH19 and DH28), significantly different with respect to embryogenic potential. To change DNA methylation levels, we applied two cytosine-analogs: 5-azacytidine (AC) and 2'-deoxy-5-azacytidine (DAC) treatments. We found that chemically-induced DNA demethylation caused chromatin relaxation and dysregulation of marker genes (TaTPD1-like, GSTF2, GSTA2, CHI3, Tad1, TaNF-YA7, SERK2, TaME1) related to ME. Both drugs showed significant cytotoxicity in a dose-dependent manner. We noticed that lines varied in terms of overall DNA methylation levels and responded in a different way to hypomethylation caused by the drugs. DH19 (low embryogenic) after inhibitors treatment, showed higher microspore viability, but its recalcitrancy was not overcome. For highly embryogenic DH28, we noted significantly higher effectiveness of embryo-like structure production and plant regeneration. In summary, our study provides new insight into the role of DNA methylation in ME initiation. They suggest potential benefits resulting from the utilization of epigenetic inhibitors to improve the process of DHs production.

Conversion of oat (Avena sativa L.) haploid embryos into plants in relation to embryo developmental stage and regeneration media.

Obtaining oat DH lines is only effective via wide crossing with maize. Seven hundred haploid embryos from 21 single F1 progeny obtained from wide crosses with maize were isolated, divided into four groups according to their size (<0.5 mm, 0.5-0.9 mm, 1.0-1.4 mm, and ≥1.5 mm), and transferred into 190-2 regeneration medium with different growth regulators: 0.5 mg L-1 kinetin (KIN) and 0.5 mg L-1 1-naphthaleneacetic acid (NAA); 1 mg L-1 zeatin (ZEA) and 0.5 mg L-1 NAA; or 1 mg L-1 dicamba (DIC), 1 mg L-1 picloram (PIC), and 0.5 mg L-1 kinetin (KIN). Among all isolated embryos, approximately 46.1% were between 1.0-1.4 mm, while the smallest group of embryos (7.1%) were those <0.5 mm. The ability of haploid embryos to germinate varied depending on oat genotypes and the size of embryos. Haploid embryos <0.5 mm were globular and did not germinate, whereas embryos ≥1.5 mm had clearly visible coleoptiles, radicles, and scutella, and were able to germinate. Germination of oat haploid embryos varied depending on growth regulators in the regeneration medium. Most haploid embryos germinated on medium with 0.5 mg L-1 NAA and 0.5 mg L-1 KIN, while the fewest germinated on medium with 1 mg L-1 DIC, 1 mg L-1 PIC, and 0.5 mg L-1 KIN. One hundred thirty germinated haploid embryos converted into haploid plants. Fifty oat DH lines were obtained after colchicine treatment.

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.