The isolation of viable egg cells of wheat (Triticum aestivum L.)

The isolation of viable egg cells of wheat has been achieved without enzymatic maceration of the ovules. 2,4-D applied to the stigmas resulted 3 to 7 days later in soft ovule tissues which disintegrated upon mechanical manipulation. The isolated egg cells were viable even 2 h after isolation. Their morphology corresponded to that of the in situ egg cells. The mean isolation frequency was 20% (two egg cells per ten ovules).     

Optimisation of introducing foreign genes into egg cells and zygotes of wheat (Triticum aestivum L.) via microinjection

Summary An expeditious and highly efficient technique of microinjection has been developed with the aim of introducing exogenous DNA into egg cells and zygotes of wheat. Using a mechanical-dissection method and a novel immobilisation approach enabled us to microinject around 15 egg cells of wheat per hour. Exposing the protoplasts to a high-frequency alternating-current field for immobilisation, a significantly higher transient expression rate of the injected genes (46% and 52% for egg cells and zygotes, respectively) could be achieved than reported thus far for plant protoplasts. Whether this high transformation efficiency is due to the highfrequency electrical field applied for immobilising the protoplasts is not known. The transformation rate appeared to be a factor depending upon the time of egg cell isolation. According to the ultrastructural observations this seems to reflect a variation in competence of the egg cells during in situ development. In order to conduct studies directed towards establishing the optimal timewindow for DNA delivery into the fertilised egg cell, the time course of DNA dynamics during zygotic development has been quantified via quantitative microspectrofluorometry.Abbreviations AC alternating current - DAE days after emasculation - FDA fluorescein diacetate - HAP hours after pollination     

Cryopreservation of articular cartilage. Part 1: conventional cryopreservation methods

There is increasing interest in the possibility of treating diseased or damaged areas of synovial joint surfaces by grafts of healthy allogeneic cartilage. Such grafts could be obtained from cadaver tissue donors or in the future they might be manufactured by 'tissue engineering' methods. Cartilage is an avascular tissue and hence is immunologically privileged but to take advantage of this is the graft must contain living cells. Preservation methods that achieve this are required to build up operational stocks of grafts, to provide a buffer between procurement and use, and to enable living grafts of a practical size to be provided at the right time for patient and surgeon. Review of the literature shows that it has been relatively straightforward to cryopreserve living isolated chondrocytes, but at the present time there is no satisfactory method to preserve cartilage between the time of procurement or manufacture and surgical use. In this paper, we review the relevant literature and we confirm that isolated ovine chondrocytes in suspension can be effectively cryopreserved by standard methods yet the survival of chondrocytes in situ in cartilage tissue is inadequate and extremely variable.     

Morphological characterisation of wheat (T. aestivum L.) egg cell protoplasts isolated from immature and overaged caryopses

 The morphological features and fine structure of wheat egg cell protoplasts isolated from premature (3 days prior to anthesis) and overaged (12 days after anthesis) caryopses were compared. Except for shape, the young egg cell protoplast showed the same morphological characteristics as the egg cell in planta at the time of anthesis. Young and adult egg cell protoplasts were spherical in shape. Polarity could not be identified exactly with the methods used. During aging, the egg cell increased considerably in volume. The adult egg showed the typical features (membrane blebbing, autophagous vacuoles) of programmed cell death. It appears that after a long life-span (about18 days) cells of the female gametophyte undergo apoptosis. Received: 19 August 1998 / Revision accepted 2 November 1998     

Transformation of protoplasts and intact cells from slowly growing embryogenic callus of wheat (Triticum aestivum L.)

A procedure for culturing protoplasts from slowly growing embryogenic calli of wheat was developed. The procedure was dependent on the ability to isolate large numbers of culturable protoplasts from slowly growing embryogenic callus. Approximately 68% of the isolated protoplasts divided, and 22% formed colonies; of the latter, 67% continued to proliferate. Plating efficiency was reduced when protoplasts were transformed by polythylene glycol, electroporation, and/or Agrobacterium. Intact cells were also directly transformed by electroporation. Direct electroporation of the Agrobacterium binary vector into intact cells resulted in a significant increase of GUS activity over the control.     

Autotoxicity of wheat (Triticum aestivum L.) as determined by laboratory bioassays

Wheat varietal autotoxicity and varietal allelopathy were assessed based on plant extract and root exudate bioassays under laboratory conditions. Aqueous extract of wheat differed in varietal autotoxicity and varietal allelopathy, inhibiting wheat germination by 2–21%, radicle growth by 15–30%, and coleoptile growth by 5–20%, depending on the combination of the receiver and donor. Extracts of cv Triller or cv Currawong were more allelopathic to other wheat varieties than cv Batavia and cv Federation. Triller extract was more autotoxic than Federation. Assessment of root exudates by the equal-compartment-agar-method further identified the significant differences in varietal autotoxicity and varietal allelopathy of root exudates between wheat varieties, with root exudates of Triller or Batavia showing stronger autotoxic or allelopathic effects than Currawong or Federation. The varietal autotoxicity and allelopathy of root exudates also showed a characteristic radial inhibitory pattern in the agar growth medium. These results suggest that careful selection of suitable wheat varieties is necessary in a continuous cropping system in order to minimize the negative impacts of varietal allelopathy and varietal autotoxicity. Factors affecting autotoxicity in the field and strategies in autotoxicity management are discussed. Resposible Editor: Philippe Hinsinger     

Diallelic analysis of quantitative traits in hexaploid wheat (Triticum aestivum L.)

Abstract

A complete diallel study of crosses between eight wheat varieties was carried out to determine the relative magnitude of components of genetic variation and heritability for important grain yield, quality and drought‐related traits. The data appeared adequate for the additive‐dominance model. The additive effects predominated for most traits, and consequently the narrow‐sense heritability was high to moderately high for flag leaf area, weight and venation, stomatal frequency and size, epidermal cell size, biomass, protein content, number of tillers, spike length, spike density, 1000‐grain weight and grain yield. These results appear promising for selecting better plants in the segregating populations with some degree of improvement for yield, quality and physiological efficiency.     

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.     

Arabinogalactans and arabinogalactan-proteins induce embryogenesis in wheat (Triticum aestivum L.) microspore culture

The objective of this study was to improve induction of embryogenesis in wheat microspore culture in order to obtain a high number of regenerable embryos. The arabinogalactan (AG) Larcoll and the arabinogalactan-protein (AGP) from gum arabic were tested on two spring genotypes to see if they could increase microspore viability and induce embryogenesis in the microspore culture. Adding Larcoll significantly decreased microspore mortality in both genotypes regardless of the presence or absence of ovaries in the culture. Similarly, gum arabic had a strong effect on the number of embryos produced and regenerated green plants. In fact, by using only gum arabic we were able to obtain green plants from wheat microspore cultures without the presence of ovaries. In addition to preventing a high mortality rate of the cells, our results show that the induction of embryogenesis in wheat microspore cultures is strongly affected by the use of both AG or AGP.An erratum to this article can be found at     

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 development of waterlogging damage in wheat seedlings (Triticum aestivum L.)

Summary The effects of waterlogging on concentrations of gases and various solutes dissolved in the soil water were investigated in the laboratory, to determine whether the early disruption to the growth of wheat was most closely associated with depletion of dissolved oxygen, accumulation of toxins, or changes in concentrations of nutrient ions in the soil water. Waterlogging slowed shoot fresh weight accumulation, leaf extension and nodal root growth; it also caused death of the seminal root system and early senescence of the lower leaves. However, the shoot dry weight initially increased above that of the non-waterlogged controls, and thus was not a reliable indicator of the early restriction to plant growth and development. The symptoms of damage to shoots and roots were attributed to the fall in soil oxygen concentrations, rather than to any decrease in concentration of inorganic nutrients in the soil water, or to the accumulation of any other measured solutes to toxic concentrations.     

Nuclear genes affecting albinism in wheat (Triticum aestivum L.) anther culture

Summary Inheritance of the ability to respond in wheat anther culture was studied from 6×2 reciprocal crosses between six varieties with high and two varieties with low capacity for green plant formation and their parents, replicated in two environments. Effects of genotypes dominated embryo formation and percentages of green plants, accounting for 78.4% and 85.4% of total variation, respectively, while smaller genetic effects were indicated for regeneration. Nuclear genes could explain almost all the genotype effects in this material. Embryo formation showed heterosis over high parent for 5 of the 12 hybrids, while percentages of green plants from the hybrids were intermediate to the parents. General Combining Ability (GCA) could explain 78.8% of the variation for embryo formation among the hybrids, whereas differences in percentage of green plants were dominated by Specific Combining Ability (SCA), accounting for 67.9% of hybrid variation. A positive correlation (r=0.81^**) was observed between the genetic capacity for regeneration and green plant formation. Analysis of covariance indicated that effects causing GCA for green plant formation were mainly responsible for this correlation. A regression model with two parallel lines divided the six parent lines with high green plant formation into three groups with respect to their reactions with the two testers. The results are discussed with regard to possible involvement of two sets of nuclear genes affecting the percentage of green plants obtained in wheat anther culture: one set consisting of mainly additive effects affecting green plant percentage through an initial effect on regeneration ability, and another set of two or a few more major genes with dominance or epistatic effects uncorrelated with regeneration.     

Influence of Rhizobial inoculation on yield of wheat (Triticum aestivum L.)

Summary Soil + charcoal (1∶3) carrier based and liquid cultures of Rhizobia were used to inoculate wheat seed cv. HD2329. The plants received 100 kg N in two equal splits and 60 kg P₂O₅ and 40 kg K₂₀ ha⁻¹. Inoculation with rhizobia had little effect on grain yield of wheat. Significant increase in straw yield and N-uptake occurred due to inoculation. A comparison of results of a similar experiment conducted during 1983–84, showed that inoculation with the same strains of rhizobia and application 50 kg N ha⁻¹ as basal dressing, was more effective in increasing yield and N-uptake in wheat cv. HD2329. It appears reasonable to assume occurrence of nitrogen fixation by root nodule bacteria in rhizosphere of wheat.     

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.     

Mapping loci associated with flour colour in wheat (Triticum aestivum L.)

 An RFLP map constructed using 150 single seed descent (SSD) lines from a cross between two hexaploid wheat varieties (‘Schomburgk’בYarralinka’) was used to identify loci controlling flour colour. Flour colour data were obtained from field trials conducted over two seasons at different sites. The estimated heritability of this trait was calculated as 0.67. Two regions identified in the preliminary analysis on chromosomes 3A and 7A, accounted for 13% and 60% of the genetic variation respectively. A detailed analysis of the major locus on 7A was conducted through fine mapping of AFLP markers identified using bulked segregant analysis (BSA). Seven additional markers were identified by the BSA and mapped to the region of the 7A locus. The applicability of these markers to identify wheat lines with enhanced flour colour is discussed. Received: 30 September 1997 / Accepted: 4 February 1998     

Analysis of gene-derived SNP marker polymorphism in US wheat (Triticum aestivum L.) cultivars

In this study, we developed 359 detection primers for single nucleotide polymorphisms (SNPs) previously discovered within intron sequences of wheat genes and used them to evaluate SNP polymorphism in common wheat (Triticum aestivum L.). These SNPs showed an average polymorphism information content (PIC) of 0.18 among 20 US elite wheat cultivars, representing seven market classes. This value increased to 0.23 when SNPs were pre-selected for polymorphisms among a diverse set of 13 hexaploid wheat accessions (excluding synthetic wheats) used in the wheat SNP discovery project (). PIC values for SNP markers in the D genome were approximately half of those for the A and B genomes. D genome SNPs also showed a larger PIC reduction relative to the other genomes (P < 0.05) when US cultivars were compared with the more diverse set of 13 wheat accessions. Within those accessions, D genome SNPs show a higher proportion of alleles with low minor allele frequencies (<0.125) than found in the other two genomes. These data suggest that the reduction of PIC values in the D genome was caused by differential loss of low frequency alleles during the population size bottleneck that accompanied the development of modern commercial cultivars. Additional SNP discovery efforts targeted to the D genome in elite wheat germplasm will likely be required to offset the lower diversity of this genome. With increasing SNP discovery projects and the development of high-throughput SNP assay technologies, it is anticipated that SNP markers will play an increasingly important role in wheat genetics and breeding applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Physiological traits associated with heat tolerance in bread wheat (Triticum aestivum L.)

Field experiments for evaluating heat tolerance-related physiological traits were conducted for two consecutive years using a mapping population of recombinant inbred lines (RILs) from the cross RAJ4014/WH730. Chlorophyll content (Chl) and chlorophyll fluorescence (CFL) were recorded under timely sown (TS) and late sown (LS) conditions. Late sowing exposes the terminal stage of plants to high temperature stress. Pooled analysis showed that CFL and Chl differed significantly under TS and LS conditions. The mean value of CFL (Fv/Fm) and Chl under both timely and late sown conditions were used as physiological traits for association with markers. Regression analysis revealed significant association of microsatellite markers viz., Xpsp3094 and Xgwm131 with coefficients of determination (R²) values for CFL (Fv/Fm) and Chl as 12 and 8 %, respectively. The correlation between thousand grain weight (TGW) with Chl and CFL were 14 and 7 % and correlation between grain wt./spike with Chl and CFL were 15 and 8 %, respectively. The genotypes showing tolerance to terminal heat stress as manifested by low heat susceptibility index (HSI = 0.43) for thousand grain weight, were also found having very low Chl, HSI (−0.52). These results suggest that these physiological traits may be used as a secondary character for screening heat-tolerant genotypes.