The response of anther culture to culture temperature in Triticum aestivum

Summary The response of anther culture to culture temperature was studied in detail using many varieties, F₁ hybrids and pollen-derived lines of wheat (Triticum aestivum) as materials. The suitable culture temperature for inducing pollen callus (or embryoids) in wheat anther culture ranged from 26 °C to 30 °C, varying with genotypes. But for the great majority of wheat genotypes the suitable culture temperatures lay between 28 °C and 30°C. The most significant genotypic variation in the response to culture temperature was observed in the comparison between the culture at 33 °C for eight days followed by culture at 25 °C (or 26 °C) and the continuous culture at 25 °C (or 26 °C). This genotypic variation in the response to culture temperature is a heritable character which may be controlled by multiple genes. The effect of culture at 30 °C for eight days followed by culture at 26 °C was similar to, or in some cases, better than that of continuous culture at 28 °C, and the effect of culture at 32 °C for eight days followed by culture at 28 °C was similar to that of continuous culture at 30 °C. In the range from 26 °C to 32 °C, the overwhelming majority of pollen calli emerged before the 40th day after anther inoculation, and the higher the culture temperature, the earlier and more concentrated the emerging period of the pollen callus. The pollen callus obtained at high temperatures above 28 °C should be transferred in time onto the regeneration medium at 25°–27°C to induce shoots.     

Aluminium induces rapid changes in cytosolic pH and free calcium and potassium concentrations in root protoplasts of wheat (Triticum aestivum)

Addition of aluminium chloride (50 μM Al) caused different effects on the transmembrane electrical potential (PD) of root cells in Al-tolerant wheat (Triticum aestivum) cv. Kadett and Al-sensitive cv. WW 20299. As changes in PD of plant cells may depend on transient fluxes of protons, potassium and/or calcium through cell membranes, the effect of Al was investigated on the cytosolic concentrations of these ions in protoplasts isolated from root tips of the same cultivars. The tetra[acetoxymethyl] esters of the fluorescent dyes bis-carboxyethyl-carboxyfluorescein, BCECF, K⁺-binding benzofuran isophthalate, PBFI, and the stilbene chromophore Fura 2-AM were used to determine pH, K⁺ and Ca²⁺, respectively. Changes in fluorescence ratios, directly reflecting changes in [H⁺], [K⁺] and [Ca²⁺] in the cytosol, were determined by photometry fluorescence microscopy. Additions and removals of Al to and from both cultivars caused hyperpolarizations and depolarizations, respectively, but only in the sensitive cv. WW 20299 did the resting PD decrease gradually. Addition of Al to the protoplasts caused rapid changes in cytosolic pH, free [K⁺] and [Ca²⁺]. In both cultivars Al caused a transient oscillating increase in cytosolic [Ca²⁺] for 1 or 2 min and a rapid pH-dependent change in cytosolic [K⁺]. At pH 5 the presence of K⁺ in the medium diminished the Al-induced decrease in cytosolic [K⁺]. Aluminium (50 μM) induced a transient increase in cytosolic [H⁺] (pH decreased) in both cultivars, but the cytosolic pH returned to its initial value only in the Al-tolerant cv. Kadett. In the Alsensitive cv. WW 20299, repeated additions of Al caused a gradual decline in pH. Moreover, in the presence of 1 mM KCl, pH recovered completely in both cultivars. Since only the effect on pH differed in the two cultivars, the more toxic effect of Al on the cv. WW 20299 should be related to the change in pH.     

A 23-kDa, root exudate polypeptide co-segregates with aluminum resistance in Triticum aestivum

We previously reported that treatment with aluminum (Al) leads to the accumulation of several polypeptides (12-, 23-, and 43.5-kDa) in root exudates of an Al-resistant cultivar of Triticum aestivum. In this report, we examine the segregation of the 23-kDa, Al-induced polypeptide and the Al-resistant phenotype in single F₂ plants arising from a cross between Al-resistant and Al-sensitive doubled-haploid (DH) lines. Single plants and plant populations were screened for sensitivity/resistance to Al using synthesis of 1,3-β-glucans (callose) as a sensitive marker for Al injury. Callose production in the Al-sensitive cv. Katepwa was approximately 3-fold higher than observed in the Al-resistant cv. Maringa, or a near-isogenic line derived from Katepwa and Maringa (Alikat), over a broad range of Al concentrations (0–100 μM). Similar results were observed with DH lines developed from cv. Katepwa, which produced two–four times more callose than DH lines developed from cv. Alikat. When single plants from F₁ and F₂ populations derived from a cross between DH Katepwa and DH Alikat were scored for Al-induced callose production after 4 days exposure to 100 μM Al, all F₁ plants were Al-resistant and F₂ plants segregated approximately 3:1 for Al-resistance/sensitivity. A backcross population derived from crossing Al-resistant F₁ with Al-sensitive Katepwa, segregated 1:1 for Al-resistance/sensitivity. Thus, the Al-resistant phenotype is inherited in a monogenic, dominant fashion in our DH lines. Enhanced accumulation of the Al-induced, 23-kDa polypeptide in root exudates was a trait which co-segregated with the Al-resistant phenotype in F₂ populations. This polypeptide was strongly labeled with S-methionine after 3 days of Al exposure and 6 h labeling. When root exudate polypeptides were separated by immobilized metal ion affinity chromatography, the 23-kDa polypeptide demonstrated significant Al-binding capacity. This polypeptide has been purified to near-homogeneity, providing an opportunity to isolate the gene(s) encoding this polypeptide.     

Root proliferation in response to neighbouring roots in wheat (Triticum aestivum)

It has been hypothesized that plants compete actively by allocating more resources to competitive organs and activities in response to neighbours, and this can reduce population performance, such as yield in crops. Root proliferation and reduced aboveground growth in response to the presence of roots of a neighbouring plant in experiments with vs. without root dividers between pairs of plants has been reported in several studies, but this result has been criticized as a possible artefact resulting from differences in soil volume available to roots in the two treatments. To address this possible confounding effect, we conducted a pot experiment with a traditional landrace and a modern cultivar of wheat (Triticum aestivum). Pairs of spring wheat plants were grown in pots with two types of root dividers (a) film, which completely divides the soil into two volumes, and (b) fine nylon net, through which roots cannot grow but chemical cues can move. We hypothesized that the root proliferation in response to root interactions would reduce aboveground growth. Wheat plants produced significantly more belowground and less aboveground biomass when interacting through the net dividers than when roots were completely separated. This effect was smaller, but still significant, in the modern cultivar. Our results confirm neighbour-induced root proliferation resulting in a so-called “tragedy of the commons” in an important crop species. The results also suggest that this response has decreased over the course of crop breeding, due to inadvertent “group selection”, and that there is further potential to increase yields by reducing or eliminating this response.     

Lignin deposition induced by aluminum in wheat (Triticum aestivum) roots

We investigated the relation between the toxic effect of aluminum (Al) on root growth and the lignin deposition in wheat ( Triticum aestivum L. cvs Atlas 66 and Scout 66). In the Al-tolerant cultivar Atlas 66, control treatment without AlCl₃ at pH 4.75, cell length increased dramatically in the portion of the root that was 0.6 to 3.2 mm from the root cap junction (approximately 1.0 to 3.6 mm from the root tip). However, treatment with 20 μ M AlCl₃ for 24 and 48 h completely inhibited root elongation and markedly decreased the length and increased the diameter of the cells in the same portion of the root. Moreover, marked deposition of lignin was observed in the cells that corresponded to the portion 1.5 to 4.5 mm from the root tip in Atlas 66 roots treated with 20 μ M AlCl₃, while no deposition of lignin was detected in control roots. Treatment with 5 μ M AlCl₃ slightly inhibited root growth and there was no deposition of lignin in the root. On the other hand, in roots of the Al-sensitive cultivar Scout 66, treatment with 5 μ M AlCl₃ completely inhibited root growth and markedly induced deposition of lignin. These results suggest that lignification in the elongating region coincided with the extent of inhibition of root growth by Al in two wheat cultivars that differed in their sensitivity to Al.     

Characterization of cytosolic phosphoglucoisomerase from immature wheat (Triticum aestivum L.) endosperm

Phosphoglucoisomerase from cytosol of immature wheat endosperm was purified 650-fold by ammonium sulphate fractionation, isopropyl alcohol precipitation, DEAE-cellulose chromatography and gel filtration through Sepharose CL-6B. The enzyme, with a molecular weight of about 130,000, exhibited maximum activity at pH 8.1. It showed typical hyperbolic kinetics with both fructose 6-P and glucose 6-P withK _m of 0.18 mM and 0.44mM respectively. On either side of the optimum pH, the enzyme had lower affinity for the substrates. Using glucose 6-P as the substrate, the equilibrium was reached at 27% fructose 6-P and 73% glucose 6-P with an equilibrium constant of 2.7. The ΔF calculated from the apparent equilibrium constant was +597 cal mol^-1. The activation energy calculated from the Arrhenius plot was 5500 cal mol^-1. The enzyme was completely inhibited by ribose 5-P, ribulose 5-P and 6-phosphogluconate, withK _i values of 0.17, 0.25 and 0.14 mM respectively. The probable role of the enzyme in starch biosynthesis is discussed.     

Phytochrome modulation of calcium fluxes in wheat (Triticum aestivum L.) protoplasts

Employing the metallochromic dye murexide and by monitoring the uptake of radiolabelled calcium, photoreversible calcium fluxes were measured in wheat leaf protoplast suspensions. Results obtained by both methods were identical — red light promoted and subsequent far-red irradiation reversed an influx of Ca⁺⁺ ions into the protoplasts. These findings imply phytochrome regulation of Ca⁺⁺ fluxes across the plasma membrane. The influx of Ca⁺⁺ stimulated by 2 min red irradiation could be maintained in total darkness for the initial 16–18 min after illumination, after which a 6–8 min efflux process was triggered and the basal Ca⁺⁺ level restored. Verapamil, a calcium channel blocker, inhibited the red-promoted influx, whereas the far-red mediated efflux could be checked by the use of the ATPase inhibitor vanadate, and also by the calmodulin antagonist chlorpromazine, thus suggesting a role of ion channels and pumps in phytochrome-controlled Ca⁺⁺ fluxes. The possible involvement of phosphoinositides in phytochrome-modulated calcium fluxes was also investigated.Abbreviations A difference in absorbance - CPZ chlorpromazine - FR far-red (light) - MX murexide - PI phosphatidylinositol - PIP₂ phosphatidylinositol 4, 5-bisphosphate - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - POPOP 1, 4-bis [2-(5-phenyl-1, 3-oxazolyl)]-benzene - PPO 2, 5-diphenyl-1, 3-oxazole - R red (light) - SOV sodium orthovanadate     

水稻和小麦根尖细胞壁多糖的铝积累能力比较

采用水培法比较4种禾本科植物水稻（Oryza sativa L.）、玉米（Zea mays L.）、高粱（Sorghum bicolor（L.）Moench）和小麦（Triticum aestivum L.）8个基因型的抗铝（Al）能力,并对他们在Al积累后细胞壁的多糖组分进行分析。结果显示,在5~200 μmol/L Al处理下,水稻抗Al能力较强,而小麦抗Al能力较弱。在50 μmol/L Al处理下,小麦根尖的果胶和半纤维素1含量的增幅明显高于水稻。水稻基因型‘日本晴’与‘浙辐802’的细胞壁Al含量分别占根尖总Al含量的78.7%和91.6%;小麦基因型‘扬麦18’与‘扬麦16’Al含量分别占根尖总Al含量的64.9%和72.1%。Al吸附-解吸实验结果显示,小麦根尖细胞壁上Al的吸附量高于水稻。研究结果表明,细胞壁是Al积累的主要部位,对Al敏感的水稻和小麦基因型细胞壁中的Al主要分布在果胶中;而对Al耐性较强的水稻和小麦基因型细胞壁中的Al主要分布在半纤维素1中。     

Aluminum stress and protein synthesis in near isogenic lines of Triticum aestivum differing in aluminum tolerance

Aluminum (Al) stress was examined in three lines of wheat ( Triticum aestivum L.) by measuring root lengths, protein synthesis and protein accumulation in seedling root tips grown in a hydroponic system. An Al-sensitive, recurrent wheat parent (cv. Katepwa) showed very little root growth in low Al concentrations. In contrast, an Al-tolerant near isogenic line (Alikat) and Al-tolerant donor (cv. Maringa) had much greater root growth. Segregation data from an F₂ population (Katepwa × Alikat) showed that one major gene controlled Al tolerance based on root growth ( X ²= 0.651). All three lines showed an approximately 2-fold increase in [³⁵S]-Met incorporation in root tips after 3 days in Al and a comparable increase in root-tip dry weight. Maringa and Alikat root tips showed an increased total protein content while Katepwa root tips showed no increase in total protein content during the Al stress. Based on higher specific activities, insoluble proteins were preferentially translated in all three lines during Al stress. Proteinase activity in Katepwa root tips was 1.7-fold higher during Al stress, with Maringa and Alikat showing no change in proteinase activity. The Al-induced, increased proteinase activity in Katepwa appeared to inhibit soluble protein accumulation.     

Control of tissue culture response in wheat (Triticum aestivum L.)

Summary The ability of immature embryos of wheat (Triticum aestivum L.) to respond to tissue culture has been shown to involve the group 2 chromosomes. The available group 2 ditelosomic and nullisomic-tetrasomic lines of Chinese Spring wheat were used to determine the chromosome arm location and chromosome dosage effect associated with the expression of tissue culture response (TCR). Significant differences were found between the aneuploid lines and the euploid control for the expression of both regenerable callus formation and callus growth rate. A model is proposed suggesting that a major TCR gene is located on 2DL and that 2AL and 2BS possess minor TCR genes. Furthermore, a major regulatory gene controlling the expression of TCR genes may be located on chromosome 2BL.     

Antifungal activity of methanol extracts from spikes of Triticum spelta and Triticum aestivum genotypes differing in their response to Fusarium culmorum inoculation

The total concentrations of free phenolic compounds and peroxidase were determined in spikes (collected at the flowering stage) of some spelt and common wheat cultivars differing in their response to F. culmorum infection. The antifungal activity of methanol extracts obtained from spikes was also evaluated. The tested genotypes differed significantly in their response to inoculation. The most resistant were Torka and Zebra among common wheat cultivars, and Weisser Grannenspelz among spelt cultivars. The average content of free phenolic compounds in spikes of spelt and common wheat was 1246.56 μg g⁻¹ and 1236.58 μg g⁻¹, respectively. The cultivars whose spikes contained the largest amounts of phenols showed the weakest response to F. culmorum infection. No significant differences were observed with regard to peroxidase content, which was 5.22 U g⁻¹ in common wheat spikes and 5.14 U g⁻¹ in spelt spikes. Methanol extracts from spikes of all wheat cultivars contained antifungal substances. The extracts from spelt spikes inhibited the growth of F. culmorum on PDA to a lesser degree than the extracts from common wheat spikes. This corresponds to the results of field trials, in which T. spelta generally exhibited a stronger response to F. culmorum infection than common wheat. The high correlation (r = 0.816) between mycelium growth inhibition on the medium and F. culmorum infection indicates that an evaluation of the antifungal activity of extracts from spikes may be used for the selection of breeding materials directed towards increased resistance to Fusarium head blight.     

Five genes induced by aluminum in wheat (Triticum aestivum L.) roots.

Five different cDNAs (termed wali1 to wali5 for Wheat Aluminum Induced) whose expression was induced by Al stress have been isolated from the root tips of Al-treated wheat (Triticum aestivum L.) plants. Four of these genes were induced 24 to 96 h after Al treatment, and their expression is reduced when the Al is removed. Each of these four genes was induced by inhibitory levels of Al in two wheat cultivars--Warigal, an Al-sensitive cultivar, and Waalt, an Al-tolerant cultivar. The fifth gene (wali2) showed a complex bimodal pattern of induction and was induced by Al only in the sensitive cultivar. Comparison of the nucleotide sequences of these clones to those in the sequence data bases showed that wali4 is homologous to phenylalanine ammonia-lyase and wali1 is homologous to a group of plant proteins that are cysteine-rich and have homology to metallothioneins. wali2 encodes a novel protein with a repeating motif of cysteine amino acids. The remaining two wali clones (wali3 and wali5) encode related, cysteine-rich proteins that show no significant homology to any known sequences.     

Genetic control of in vitro response in wheat (Triticum aestivum L.)

Summary The present state of genetic control of in vitro responses of wheat and of the prospeets of its improvement and practical use are presented. The genetic factors affecting different stages of callus induction and of organogenesis in immature embryos are discussed. The dominant genotypie role in the in vitro processes and the cytoplasmic influence on the latter are shown. Genotype × environment and nucleus × cytoplasm interactions were observed. Some unsolved problems needing further investigations of cereals at the tissue culture level are pointed out.     

Comparative characteristic of Triticum aestivum/Triticum durum and Triticum aestivum/Triticum dicoccum hybrid lines by genomic composition and resistance to fungal diseases under different environmental conditions

The genetic diversity of common wheat hybrid lines Triticum aestivum/Triticum durum and Triticum aestivum/Triticum dicoccum (2n = 42, F_6–7) using chromosome-specific microsatellite (SSR) markers and C-banding of chromosomes was studied. Cluster analysis of data obtained by 42 SSR markers indicated that the hybrid lines can be broken into three groups according to their origin. There were two cases of complete genetic similarity between lines 183₂-2/184₁-6 and 208-3/213-1, which were obtained using common wheat as the parental plants. In cross combinations, when the stabilization of the nuclear genome of hexaploid lines occurred against a background of the cytoplasmic genome of tetraploid wheats, there was a high level of divergence between sister lines, in some cases exceeding 50%. The evaluation of the degree of susceptibility of the lines to powdery mildew, leaf and stem rust, and septoria leaf blotch was performed under different environmental conditions. It was shown that resistance to powdery mildew and leaf rust significantly depended on the region where assays were conducted. An evaluation of the field data showed that the lines 195-3, 196-1, and 221-1 with T. durum genetic material displayed complex resistance to fungal pathogens in Western Siberia and the Republic of Belarus. For lines 195-3 and 196-1, one shows a possible contribution of chromosomes 4B and 5B in the formation of complex resistance to diseases. Hybrid lines with complex resistance can be used to expand the genetic diversity of modern common wheat cultivars for genes of immunity.     

Low-temperature tolerance and genetic potential in wheat (Triticum aestivum L.): response to photoperiod, vernalization, and plant development

It is frequently observed that winter habit types are more low-temperature (LT) tolerant than spring habit types. This raises the question of whether this is due to pleiotropic effects of the vernalization loci or to the linkage of LT-tolerance genes to these vernalization loci. Reciprocal near-isogenic lines (NILs) for alleles at the Vrn-A1 locus, Vrn-A1 and vrn-A1, determining spring and winter habit respectively, in two diverse genetic backgrounds of wheat (Triticum aestivum L.) were used to separate the effects of vernalization, photoperiod, and development on identical, or near identical, genetic backgrounds. The vrn-A1 allele in the winter lines allowed full expression of genotype dependent LT tolerance potential. The winter allele (vrn-A1) in a very cold tolerant genetic background resulted in 11°C, or a 2.4-fold, greater LT tolerance compared to the spring allele. Similarly, the delay in development caused by short-day (SD) versus long-day (LD) photoperiod in the identical spring habit NIL resulted in an 8.5°C or 2.1-fold, increase in LT tolerance. The duration of time in early developmental stages was shown to underlie full expression of genetic LT-tolerance potential. Therefore, pleiotropic effects of the vernalization loci can explain the association of LT tolerance and winter habit irrespective of either the proposed closely linked Fr-A1 or the more distant Fr-A2 LT-tolerance QTLs. Plant development progressively reduced LT-acclimation ability, particularly after the main shoot meristem had advanced to the double ridge reproductive growth stage. The Vrn-1 genes, or other members of the flowering induction pathway, are discussed as possible candidates for involvement in LT-tolerance repression.     

Enhancement of phototropic response to a range of light doses in Triticum aestivum coleoptiles in clinostat-simulated microgravity

The phototropic dose-response relationship has been determined for Triticum aestivum cv. Broom coleoptiles growing on a purpose-built clinostat apparatus providing gravity compensation by rotation about a horizontal axis at 2 rev·min^-1. These data are compared with data sets obtained with the clinostat axis vertical and stationary, as a 1·g control, and rotating vertically to examine clinostat effects other than gravity compensation. Triticum at 1·g follows the wellestablished pattern of other cereal coleoptiles with a first positive curvature at low doses, followed by an indifferent response region, and a second positive response at progressively increasing doses. However, these response regions lie at higher dose levels than reported for Avena. There is no significant difference between the responses observed with the clinostat axis vertical in the rotating and stationary modes, but gravity compensation by horizontal rotation increases the magnitude of first and second positive curvatures some threefold at 100 min after stimulation. The indifferent response is replaced by a significant curvature towards the light source, but remains apparent as a reduced curvature response at these dose levels.     

Monosomic analysis of tissue culture response in wheat (Triticum aestivum L.)

Summary The ability of immature embryos of wheat (Triticum aestivum L.) to respond in cell culture was examined in crosses between the Wichita monosomic series and a highly regenerable line, ND7532. Segregation in disomic controls and 13 monosomic families showed a good fit to a monogenic ratio indicating a qualitative mode of inheritance. Segregation in the cross involving monosomic 2D showed a high frequency of regeneration (93.6%) and high callus growth rate (1.87 g/90 days) indicating that 2D is a critical chromosome. Modifying genes may be located on other chromosomes. Substitution of chromosomes from a low regenerable cultivar Vona further indicated that the group 2 chromosomes, in particular chromosome 2D, possess genetic factors promoting callus growth and regeneration.     

Molecular and biochemical characterization of cytosolic phosphoglucomutase in wheat endosperm (Triticum aestivum L. cv. Axona)

Evidence from a number of plant tissues suggests that phosphoglucomutase (PGM) is present in both the cytosol and the plastid. The cytosolic and plastidic isoforms of PGM have been partially purified from wheat endosperm (Triticum aestivum L. cv. Axona). Both isoforms required glucose 1,6-bisphosphate for their activity with K(a) values of 4.5 micro M and 3.8 micro M for cytosolic and plastidic isoforms, respectively, and followed normal Michaelis-Menten kinetics with glucose 1-phosphate as the substrate with K(m) values of 0.1 mM and 0.12 mM for the cytosolic and plastidic isoforms, respectively. A cDNA clone was isolated from wheat endosperm that encodes the cytosolic isoform of PGM. The deduced amino acid sequence shows significant homology to PGMs from eukaryotic and prokaryotic sources. PGM activity was measured in whole cell extracts and in amyloplasts isolated during the development of wheat endosperm. Results indicate an approximate 80% reduction in measurable activity of plastidial and cytosolic PGM between 8 d and 30 d post-anthesis. Northern analysis showed a reduction in cytosolic PGM mRNA accumulation during the same period of development. The implications of the changes in PGM activity during the synthesis of starch in developing endosperm are discussed.     

The phototropic response of Triticum aestivum coleoptiles under conditions of low gravity

Wheat (Triticum aestivum L. cv Broom) coleoptiles were stimulated by unilateral blue light pulses, the duration of which varied between 3 s and 30 min, under microgravity conditions on the IML-1 Spacelab mission and in ground controls. The stimuli covered first positive, indifferent and second positive response regions. When phototropic responses were observed under low-gravity conditions, slightly fewer seedlings responded compared with 1 g conditions. The latent period was similar in flight and 1g treatments (10–20 min), except for the response to 300,μmol m^?2 in the indifferent response region, where a positive response in flight plants followed a 130 min latent period, while no response was observed in 1 g plants. First positive responses at Og were slightly enhanced, both in magnitude and in duration, compared to the 1 g controls, but not to the extent predicted by clinostat studies. The response kinetics in the second positive region at 1g showed a plateau at 120 min, in contrast to the single maximum at Og. The fluence-response relationship was similar in both flight and ground controls. Only the responses to 4 and 6μmolm-2 showed significantly greater curvatures at 9g. This contrasts with previous clinostat studies, which reported substantial response enhancement at all fluence levels.