Accelerated production of transgenic wheat (Triticum aestivum L.) plants

We have developed a method for the accelerated production of fertile transgenic wheat (Triticum aestivum L.) that yields rooted plants ready for transfer to soil in 8–9 weeks (56–66 days) after the initiation of cultures. This was made possible by improvements in the procedures used for culture, bombardment, and selection. Cultured immature embryos were given a 4–6 h pre-and 16 h post-bombardment osmotic treatment. The most consistent and satisfactory results were obtained with 30 g of gold particles/bombardment. No clear correlation was found between the frequencies of transient expression and stable transformation. The highest rates of regeneration and transformation were obtained when callus formation after bombardment was limited to two weeks in the dark, with or without selection, followed by selection during regeneration under light. Selection with bialaphos, and not phosphinothricin, yielded more vigorously growing transformed plantlets. The elongation of dark green plantlets in the presence of 4–5 mg/l bialaphos was found to be reliable for identifying transformed plants. Eighty independent transgenic wheat lines were produced in this study. Under optimum conditions, 32 transformed wheat plants were obtained from 2100 immature embryos in 56–66 days, making it possible to obtain R3 homozygous plants in less than a year.     

Isolation and analysis of endophytic microorganisms in wheat (Triticum aestivum L.) leaves

The present investigation was undertaken in order to select the surface-sterilization technique most efficient for eliminating epiphytes, to document the spectrum of endophytes of healthy leaves from three wheat cultivars in Buenos Aires Province (Argentina) and to determine their infection frequencies at three growth stages. Surface-sterilization with undiluted commercial solution of sodium hypochlorite was reaffirmed as adequate for removing epiphytes on wheat leaves. From the 450 wheat leaf segments incubated, three bacterial isolates and 130 fungal isolates were obtained. From all the isolates, 19 fungal species were identified. Bacterial isolates were characterized as Bacillus sp. There were significant differences between microorganisms, stages of growth, and stages × microorganisms interaction. Differences between cultivars, stages × cultivars, microorganisms × cultivars and for the triple interaction were not significant. Frequency of microorganisms isolated increased with crop age, but it was statistically similar for the three wheat cultivars tested (Klein Centauro, Klein Dragón and Buck Ombú). Rhodotorula rubra, Alternaria alternata, Cladosporium herbarum and Epicoccum nigrum were isolated in the highest frequency. The other microorganisms were present at intermediate or low values. The species isolated may be assigned to three groups: (a) well-known and economically important pathogens of wheat, (b) commonly abundant phylloplane fungi considered to be primary saprobic and minor pathogens and (c) species occasionally present in wheat.     

Distribution of magnesium in wheat (Triticum aestivum L.) in relation to supply

The aims of this study were to describe the distribution of magnesium (Mg) and its retranslocation within wheat, in order to develop diagnostic procedures for Mg deficiency. Plants were grown in solution culture with both constant supply (0, 5, 10, 20, 40, 80, 160 MMg) and discontinued supply (40 M and 160 M decreased to nil).Magnesium was depleted from old leaves when Mg supply to the roots was halted. However, initial deficiency symptoms occurred on young leaves under constant but inadequate supply, contrasting with previous reports. Magnesium concentrations were also lower in young leaves compared to old leaves. Symptoms of yellowing and necrosis occurred if the leaf tissue contained <1194 gg^–1, irrespective of leaf age. The minimum Mg concentration in whole shoots associated with maximum shoot weight was 932 gg^–1; for the youngest emerged blade (YEB) it was 861 gg^–1. Symptoms were apparent on the young leaf before a reduction in shoot weight was measurable. The concentration of Mg in the YEB and whole shoot were better related to solution Mg concentration than was the Mg concentration in the old leaf.     

Identification of three Wx proteins in wheat (Triticum aestivum L.)

Nullisomic analysis of waxy (Wx) protein of hexaploid wheat (Triticum aestivum L.) cv. “Chinese Spring” using two-dimensional polyacrylamide gel electrophoresis revealed that threeWx loci,Wx-A1, Wx-B1, andWx-D1, located on chromosome arms 7AS, 4AL, and 7DS, produce three distinct Wx subunit groups, subunit group-A (SGA), SGB, and SGD, respectively. SGA has a higher molecular weight and a more basic isoelectric point (pI) than the other two. SGB and SGD have the same molecular weight but a slightly different pI range. Owing to the detection of these three subunit groups, we were able to identify the expression of three waxy genes in wheat endosperm and to find two types of mutants among Japanese wheat cultivars, one lacking SGA and the others SGB. These results suggest the possibility of breeding a waxy wheat.     

Carbohydrate metabolism and phosphorus/salinity interactions in wheat (Triticum aestivum L.)

Foliar inorganic ion and carbohydrate concentrations were determined in wheat plants treated with factorial combinations of phosphorus fertilizer and NaCl in a glasshouse experiment. Growth reductions and visual symptoms of salt toxicity were minimized when phosphorus nutrition was adequate, and were intensified by phosphorus deficiency. Foliar sodium and chloride accumulated up to 4.0–5.5% d.w. with salinity treatment. However, ionic concentrations within corresponding leaves or distributions between leaves of plants with different phosphorus treatments were not influenced by phosphorus treatment and had no relationship to the severity of salt toxicity symptoms. This suggests that phosphorus deficiency reduced the cellular tolerance for ion accumulation. A combination of phosphorus deficiency and salinity induced an accumulation of foliar starch and sucrose despite substantial reductions in net CO₂ assimilation rates. This accumulation did not occur if phosphorus nutrition was adequate, which is consistent with the roles of phosphorus in carbohydrate metabolism. It is proposed that adequate phosphorus nutrition is essential for effective ion compartmentation by contributing to efficient carbohydrate utilization in salt-stressed wheat.     

Silicon absorption by wheat (Triticum aestivum L.)

Although silicon (Si) is a quantitatively major inorganic constituent of higher plants the element is not considered generally essential for them. Therefore it is not included in the formulation of any of the solution cultures widely used in plant physiological research. One consequence of this state of affairs is that the absorption and transport of Si have not been investigated nearly as much as those of the elements accorded 'essential' status. In this paper we report experiments showing that Si is rapidly absorbed by wheat (Triticum aestivum L.) plants from solution cultures initially containing Si at 0.5 mM, a concentration realistic in terms of the concentrations of the element in soil solutions. Nearly mature plants (headed out) 'preloaded' with Si absorbed it at virtually the same rate as did plants grown previously in solutions to which Si had not been added. The rate of Si absorption increased by more than an order of magnitude between the 2-leaf and the 7-8 leaf stage, with little change thereafter. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Golgi apparatus in developing endosperm of wheat (Triticum aestivum L.)

The ultrastructure and distribution of the Golgi apparatus in developing wheat endosperm was investigated using a zinc iodide-osmium tetroxide staining complex in conjunction with low and high voltage electron microscopy. Dictyosomes were numerous in starchy endosperm and aleurone at 15 days after anthesis, and during the period of rapid storage protein deposition 25 d after anthesis. Fewer dictyosomes were seen in maturing endosperm. Two types of vesicles were associated with the dictyosomes; small, heavily-stained vesicles were sited at the ends of fine tubules which extend from the cisternae, and larger less-stained vesicles were associated with the periphery of the cisternae. Stereo-pairs of micrographs up to 1 m thick were taken to demonstrate the interconnections between cisternal and tubular endoplasmic reticulum. Elements of tubular ER were closely associated with dictyosomes, but connections were not observed. These results are discussed in relation to the transport of endosperm storage proteins from their site of synthesis on the cisternal ER to their site of storage, the protein bodies.     

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.     

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.)

The activity of a range of endo- and exopeptidase enzymes have been measured in the glumes, flag leaf and stem during the period of grain development in wheat. The enzymes show a sequential pattern of appearance with activity peaks occurring at a number of intervals from anthesis until just prior to the cessation of grain growth. Of the enzymes studied only the haemoglobin- and casein-degrading activity and alanylglycine-dipeptidase activity increased during the period of rapid protein loss, while aminopeptidase, carboxypeptidase and leucyltyrosine dipeptidase reached maximum activity prior to this period.     

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.)

The flag leaf of wheat was examined for changes in quantity and activity of ribulose-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39), in the proteolytic degradation of RuBPCase and other native proteins, and in the ultrastructure of the leaf cells during grain development. Proteolytic degradation of RuBPCase at pH 4.8 increased until 8–10 d after anthesis, then declined, and increased again 16–18 d after anthesis. The second peak coincided with the onset of a preferential loss of immunologically recognizable RuBPCase. The specific activity and number of active sites per molecule of RuBPCase did not change during senescence. Examination of ultrastructure with the electron microscope showed little change in the appearance of the mitochondria as the flag leaf aged. Prominent cristae were still evident 35 d after anthesis. In contrast, the chloroplasts showed a progressive disruption of the thylakoid structure and an increasing number of osmiophilic glubules. The double membrane envelope surrounding the chloroplast appeared intact until at least 20 d after anthesis. The tonoplast also appeared intact up to 20 d. At later stages of senescence of the leaf the outer membrane of the chloroplast adjacent to the tonoplast appeared to break but the inner membrane of the envelope appeared intact until at least 35 d after anthesis.Abbreviation RuBPCase ribulose-1,5-bisphosphate carboxylase (EC. 4.1.1.39) I=Waters et al. 1980     

Cell shaping and microtubules in developing mesophyll of wheat (Triticum aestivum L.)

Summary Differentiated mesophyll cells ofTriticum aestivum (cv. Star) exhibit a lobed outline resembling tube-shaped balloons with almost regularly spaced constrictions. It was shown that these constrictions are probably the result of hoops of wall reinforcements laid down during early stages of cell expansion. It appears that these hoops prevent expansion in the corresponding regions and thus give rise to the peculiar cell shape. The comparatively thin cell walls of the bulges are uniformly reinforced after the lobed shape is established.By using immunofluorescence techniques a change in the pattern of cortical microtubule arrangement was observed which corresponded to the pattern of cell wall deposition. Discrete bands of microtubules were found beneath the sites of hoop reinforcement. These bands disintegrated during late stages of cell expansion with microtubules fanning out into the almost empty regions of the bulges.Abbreviations DMSO dimethyl sulfoxid - EGTA ethylene glycol bis-(-aminoethyl ether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanat - MSB microtubule stabilizing buffer - PBS phosphate buffered saline - PIPES 1,4-piperazine diethanesulfonic acid - PMSF phenylmethyl sulfonylfluoride     

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.     

Enzyme activities associated with developing wheat(Triticum aestivum L.) grain amyloplasts

Intact amyloplasts from endosperm of developing wheat grains have been isolated by first preparing the protoplasts and then fractionating the lysate of the protoplasts on percoll and ficoll gradients, respectively. Amyloplasts isolated as above were functional and not contaminated by cytosol or by organelles likely to be involved in carbohydrate metabolism. The enzyme distribution studies indicated that ADP-glucose pyrophosphorylase and starch synthase were confined to amyloplasts, whereas invertase, sucrose synthase, UDP-glucose pyrophosphorylase, hexokinase, phosphofructokinase-2 and fructose-2,6-P₂ase were absent fro the amyloplast and mainly confined to the cytosol. Triose-P isomerase, glyceraldehyde-3-P dehydrogenase, phosphohexose isomerase, phosphoglucomutase, phosphofructokinase, aldolase, PPi-fructose-6-P-1 phosphotransferase, and fructose-l,6-P₂ase, though predominantly cytosolic, were also present in the amyloplast. Based on distribution of enzymes, a probable pathway for starch biosynthesis in amyloplasts of developing wheat grains has been proposed.     

The control of respiration in wheat (Triticum aestivum L.) leaves

The aim of this work was to discover whether the respiration of wheat (Triticum aestivum L. cv. Huntsman) leaves, transferred to darkness after 7 h photosynthesis, showed an initial period of wasteful respiration. For young and old leaves, CO₂ production and O₂ uptake after 7 h photosynthesis were up to 56% higher than at the end of an 8-h night. The maximum catalytic activities of citrate synthase (EC 4.1.3.7), aconitase (EC 4.2.1.3), fumarase (EC 4.2.1.2) and cytochrome-c oxidase (EC 1.9.3.1) at the end of the day did not differ from those at the end of the night. Changes in the contents of glucose 6-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, and -ketoglutarate did not as a group parallel the changes in the rate of respiration. The detailed distribution of label from [U-¹⁴C] sucrose supplied to leaves in the dark was similar at the end of the day and the end of the night. No correlation was observed between the rates of leaf respiration and extension growth. It is argued that the higher rate of respiration at the beginning of the night cannot be attributed to wasteful respiration.Abbreviation RQ respiratory quotient We thank Dr H. Thomas and Professor C.J. Pollock, Institute for Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, UK for their generous help in measuring leaf extension. R.H.A. thanks the Science and Engineering Research Council for a studentship.     

Metabolism of 2,4-dichlorophenoxyacetic acid in cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.)

Cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.) incorporated 2,4-dichlorophenoxyacetic acid (2,4-D) into a metabolite fraction which was insoluble in ethanol, water, and hot sodium dodecylsulphate. Further treatment with hot dimethylformamide solubilized a material which by the following criteria appeared to consist of 2,4-D derivatives covalently bound to lignin: i) co-chromatography of radioactivity and of UV-absorbing material upon gel permeation chromatography; ii) spectral similarity with authentic lignins (IR- and UV-spectra, phloroglucinol reaction), 2,4-D appeared to be incorporated as the intact molecule, as shown by comparison of ring- and sidechain-labeled 2,4-D and by detection of monohydroxylated and intact 2,4-D as the major radioactive products of acid hydrolysis. The same compounds were released from the metabolite material which could not be solubilized in dimethylformamide. The incorporation of xenobiotics or their metabolites into lignin, followed by deposition in the cell wall, is suggested as a general pathway for local excretion and detoxification by plant cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4-OH-2,5-D 4-hydroxy-2,5-dichlorophenoxyacetic acid - SDS sodium dodecylsulphate - DMF dimethylformamide     

Structural homology of endosperm high molecular weight glutenin subunits of common wheat (Triticum aestivum L.)

Summary Several high molecular weight endosperm glutenin subunits, coded by genes located on chromosomes 1A, 1B and 1D of common wheat, Triticum aestivum L. em. Thell., were isolated from excised gel segments and subjected to amino acid analysis and peptide mapping; the latter was carried out following a limited digestion with trypsin, chymotrypsin or Staphylococcus aureus — V8 protease. Generally, all high molecular weight glutenins had a similar amino acid composition but several significant differences were observed in some of them. Both analyses revealed that the structural similarity among the various subunits was related to the homology of the genes coding them: subunits coded by homoalleles, i.e., different alleles of the same gene, were most similar; those coded by homoeoalleles, i.e., alleles of homoeologous genes, were less similar; whereas subunits coded either by alleles of different genes of the same gene cluster, or by nonhomoeoalleles of homoeologous clusters, were the least similar. Several small peptides derived from protease digestion of various subunits had a higher than expected staining intensity indicating that small peptide repeats may be interspersed within the glutenin subunits. The evolutionary course of the high molecular weight glutenins is discussed.     

Yield responses of Nepalese spring wheat (Triticum aestivum L.) cultivars to inoculation with Azospirillum spp. of Nepalese origin

Azosprilla were collected in wheat fields from subtropical and temperate soils of central Nepal at various elevations. Different wheat cultivars responded positively and significantly in grain yield, grain N-yield, and total N-yield in plant shoots to the inoculation with Nepalese isolate Azospirillum 10SW. Nepalese wheat cv. Seto responded significantly better with Azospirillum 10SW than with the Brasilian isolate A. lipoferum Sp 108 st, a strain which was found highly efficient in earlier experiments with German wheat cultivars, especially cv. Turbo. Yield of Turbo was increased by inoculations of both Azospirillum strains too, but it showed no significant differences depending from the inoculum used. The higher efficacy of combining Azospirillum 10SW and Seto, both collected from the same locality, indicates the possibility of improved associations using traditional cultivars and local bacteria. ei]{gnR O D}{fnDixon}     

Molecular characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.)

Molecular analysis of the transgenes bar and gus was carried out over successive generations in six independent transgenic lines of wheat, until the plants attained homozygosity. Data on expression and integration of the transgenes is presented. Five of the lines were found to be stably transformed, duly transferring the transgenes to the next generation. The copy number of the transgenes varied from one to five in the different lines. One line was unstable, first losing expression of and then eliminating both the transgenes in R3 plants. Although the gus gene was detected in all the lines, GUS expression had been lost in R2 plants of all but one line. Rearrangement of transgene sequences was observed, but it had no effect on gene expression. All the stable lines were found to segregate for transgene activity in a Mendelian fashion.     

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