Agroinfection and nucleotide sequence of cloned wheat dwarf virus DNA

Cloned DNA of the geminivirus wheat dwarf virus (WDV) was successfully used to infect seedling wheat plants. The clone was derived from circular double-stranded viral DNA isolated from naturally infected tissue. The initiation of infection was mediated by Agrobacterium tumefaciens using cloned dimeric WDV genomes in a binary Agrobacterium vector. The WDV DNA which comprised the infectious clone was sequenced and is compared with the published sequence of a Swedish isolate of the same virus. The results confirm that the single WDV genome component of 2.75 kb carries all the information necessary for production of viral symptoms, virus particles and viral double- and single-stranded DNA forms.     

A single treatment of rice seedlings with 5-azacytidine induces heritable dwarfism and undermethylation of genomic DNA

Summary A single exposure of germinated rice seeds (Oryza sativa) to either of the DNA demethylating agents 5-azacytidine (azaC) or 5-azadeoxycytidine (azadC) induced dwarf plants. At maturity, seeds treated with azaC exhibited normal morphological characteristics in comparison with untreated controls except that their height (total stem length) was reduced by about 15%. The M₁ progeny, obtained by self-fertilization of an azaC-induced dwarf plant, segregated into dwarf (35%) and apparently tall types (65%). The M₂ progenies, obtained by self-fertilization of dwarf M₁ plants, were also dwarf, while those from tall M₁ plants were only tall. Genomic DNA isolated from mature leaves of azaC-treated seeds showed about a 16% reduction in the 5-methylcytosine (m⁵C) content in comparison with DNA from untreated samples. A similar reduction in the m⁵C content was also observed in the M₁ and M₂ progenies. Thus, both undermethylation and dwarfism induced by azaC treatment were heritable. The results suggest that azaC induced demethylation of genomic DNA, which caused an altered pattern of gene expression and consequently a reduction in plant stem length.     

Transgenic wheat progeny resistant to powdery mildew generated by Agrobacterium inoculum to the basal portion of wheat seedling

To improve the transformation efficiency of wheat (Triticum aestivum L.) mediated by Agrobacterium tumefaciens, we explored the possibility of employing the basal portion of wheat seedling (shoot apical meristem) as the explants. Three genotypes of wheat were transformed by A. tumefaciens carrying β-1, 3-glucanase gene. After vernalization, the seeds to be transformed were germinated. When these seedlings grew up to 2∼5 cm, their coleoptile and half of the cotyledon were cut out, and the basal portions were infected by A. tumefaciens. A total 27 T₀ transgenic plants were obtained, and the average transformation efficiency was as high as 9.82%. Evident segregation occurred in some of the T₁ plants, as was indicated by PCR and Southern blotting analysis. Investigation of the T₂ plants revealed that some transformed plants had higher resistance to powdery mildew than the controls. Northern blotting revealed that β-1, 3-glucanase gene was normally expressed in the T₂ plants, which showed an increased resistance to powdery mildew. The results above indicate that the exogenous gene has been successfully integrated into the genome of wheat, transmitted and expressed in the transgenic progeny. From all the results above, it can be concluded that Agrobacterium inoculum to the basal portion of wheat seedling is a highly efficient and dependable transformation method. It can be developed into a practicable method for transfer of target gene into wheat.Tong-Jin Zhao and Shuang-Yi Zhao contributed equally to this paper.     

A moderately repeated DNA sequence of wheat and rye genomes

A 371 base pair segment (bordered by Hind III and Eco RI cutting sites) of wheat embryo nuclear DNA has been cloned and sequenced. It is AT-rich (68%), shares some sequence features with autonomously replicating sequence (ARS) elements, and occurs in approximately 7600 copies per haploid genome. When used as probe for blot hybridization to Hind III-digested wheat DNA, it gives an irregular series of hybridization bands. Essentially the same hybridization pattern was observed for rye DNA. It is concluded that this segment is distributed irregularly but, apparently, according to the same rule in both wheat and rye genomes.     

Characterisation of germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy

Experiments were conducted to characterise the changes, especially of water status in germinating and non-germinating wheat seeds by nuclear magnetic resonance (NMR) spectroscopy. NMR relaxation time (T₂) measurements showed tri-phasic or bi-phasic characteristics during different stages of hydration, depending on the seed's ability to germinate. Component analysis of T₂ data revealed the existence of only two components, bound and bulk water, in dry seeds. In contrast, both the germinating and non-germinating wheat seeds had a three-component water proton system (bound, bulk and free water) in phase I of hydration. During the lag phase (phase II) of hydration, bulk water component of non-germinating seeds disappeared completely, resulting in a two component water proton system. Nevertheless, the three component water proton system was observed in the germinating seeds in phase II. Following phase II, rapid hydration (phase III) was observed in germinating seeds only. Water protons were re-organised and there were increases in bulk and free water but decreases in bound water concomitantly. Comparison of the physical state of water in these seeds by NMR spectroscopy with that of tissue leachate conductivity measurement suggests that the seed membrane system was affected more evidently in non-germinating seeds, leading to the disorganised cell structure. The present study provides evidence that the reorganisation of physical state of water in germinating wheat seeds during hydration is essential for its subsequent event of germination.     

Analysis of DNA methylation during the germination of wheat seeds

DNA methylation is known to play a crucial role in regulating plant development and organ or tissue differentiation. Here, we focused on the DNA methylation dynamics during the germination of wheat seeds using the adapted AFLP technique so called methylation-sensitive amplified polymorphism (MSAP). The MSAP profiles of genomic DNA in embryo and endosperm tissues of germinating seeds, as well as dry seeds were characterized and notable changes of cytosine methylation were detected. Comparisons of MSAP profiles in different tissues tested showed that the methylation level in dry seeds is the highest. The alteration analysis of cytosine methylation displayed that the number of demethylation events were three times higher than that of de novo methylation, which indicated that the demethylation was predominant in germinating wheat seeds, though the methylation events occurred as well. Sixteen differentially displayed DNA fragments in MSAP profiles were cloned and the sequencing analysis confirmed that nine of them contained CCGG sites. The further BLAST search showed that four of the cloned sequences were located in coding regions. Interestingly, three of the sixteen candidates were homologous to retrotransposons, which indicated that switches between DNA methylation and demethylation occurred in retrotransposon elements along with the germination of wheat seeds.     

A large DNA repeat of the dispersion pattern common to wheat and rye genomes

A Hind III-generated fragment of wheat embryo nuclear DNA has been cloned and sequenced. The cloned fragment corresponds to a 1241 bp long, moderately repeated (60 000 copies/genome) segment of the genomic DNA. The repeat is AT-rich (67%), contains an open reading frame for 151 amino acids and several nucleotide blocks resembling the consensus domain of autonomously replicating sequences. Southern blot hybridization analyses indicate that the repeat is scattered through the wheat genome. A sequence homologous to this repeat occurs also in rye embryo nuclear DNA where it shows the same dispersion pattern as that observed for the wheat repeat.     

‘On-farm’ seed priming with zinc in chickpea and wheat in Pakistan

A series of on-station trials was implemented between 2002 and 2006 to assess the response of wheat (Triticum aestivum L.) and chickpea (Cicer arietinum) to zinc (Zn) added by soaking seeds (priming) in solutions of ZnSO₄ before sowing. Wheat seed was primed for 10 h in 0.3% Zn and chickpea for 6 h in 0.05% Zn. Seed treatments increased the seed concentration in wheat from 27 to 470 mg/kg and in chickpea from 49 to 780 mg/kg. Priming wheat seeds with 0.3% Zn significantly increased the mean shoot dry mass, Zn concentration and Zn uptake of 15-day-old seedlings relative to non-primed controls and seeds primed with water alone. Using 0.4% Zn further increased shoot Zn concentration but depressed shoot dry mass to the level of the non-primed control. In seven trials, mean grain yield of wheat was significantly increased from 2.28 to 2.42 t/ha (6%) by priming with water alone and to 2.61 t/ha (14%) by priming with 0.3% Zn. Mean grain yield of chickpea in seven trials was increased significantly from 1.39 to 1.65 t/ha (19%) by priming seeds with 0.05% Zn. The effect of priming chickpea seeds with water was intermediate (1.49 t/ha) and not statistically separable from the non-primed and zinc-primed treatments. Increased grain yield due to priming in both crops was associated with increases in total biomass but there was no significant effect of priming on harvest index. In addition to increasing yield, priming seeds with Zn also significantly increased grain zinc concentration, by 12% in wheat (mean of three trials) and by 29% in chickpea (one trial) and the total amount of Zn taken up by the grain (by 27% in wheat and by 130% in chickpea). Using ZnSO₄ to prime seeds was very cost-effective, with net benefit-to-cost ratios of 75 for wheat and 780 for chickpea. An erratum to this article can be found at     

Influence of aFusarium culmorum inoculation of wheat onin sacco dry matter degradation of wheat straw and wheat chaff

The aim of this study was to investigate the effect of aFusarium culmorum inoculation of wheat on thein sacco dry matter degradation (DG) of wheat straw and wheat chaff in dairy cows. The ruminal disappearance of dry matter was measured with thein situ nylon bag technique. Samples of wheat straw and wheat chaff from non-inoculated andFusarium-inoculated wheat were used to examine the ruminal dry matter degradability. Samples were subjected to ruminal incubation in two dairy cous fitted with a permanent rumen fistula and incubated for 4, 8, 16, 24, 48, 72, 96 and 120 h. To describe the degradation kinetics, the equation by Ørskov and McDonald (1979) was used. DG rates obtained for contaminates straw and chaff were higher compared to the corresponding rates of the non-contaminated samples, which is assumed to be due to the activity of fungal enzymes. It can be concluded that an infection of wheat withF. culmorum may have an influence on the dry matter degradation of straw and chaff.     

Effect of Tilemsi phosphate rock-solubilizing microorganisms on phosphorus uptake and yield of field-grown wheat (Triticum aestivum L.) in Mali

With the broad aim of biologically improving P uptake by wheat fertilized with Tilemsi phosphate rock (TPR), we investigated the effect of inoculation with TPR-solubilizing microorganisms isolated from Malian soils and with a commercial isolate of the arbuscular mycorrhizal (AM) fungus Glomus intraradices (Gi). AM root length colonization, and growth yield and P concentration of the cultivar Tetra of wheat were measured under field conditions in Mali. Experimental plots were established in Koygour (Diré) during the 2001–2002 cropping season. Inoculation treatments included two fungal isolates, Aspergillus awamori (C1) and Penicillium chrysogenum (C13), and an isolate of Pseudomonas sp. (BR2), used alone or in fungus-bacterium combinations in the presence or absence of the AM fungus Gi. In fertilized treatments, 0 or 30 kg P ha⁻¹ was applied as TPR or diammonium phosphate (DAP). In 45-day-old wheat plants, the highest root length AM colonization (62%) was observed with TPR fertilized wheat inoculated with Gi and BR2. Our results suggest that BR2 is a mycorrhizal-helper bacteria and a good plant growth-promoting rhizobacteria. In fact, inoculation of wheat Tetra fertilized with TPR with a combination of Gi, BR2 and C1 produced the best grain yield with the highest P concentration. This work shows that by inoculating seeds with TPR-solubilizing microorganisms and AM fungi under field conditions in Mali it is possible to obtain wheat grain yields comparable to those produced by using the expensive DAP fertilizer.     

Detection of a non-structural protein of M r 11 000 encoded by the virion DNA of maize streak virus

A polypeptide of approximately 11 000 daltons (11 kDa protein) encoded by an open reading frame (10.9 ORF) from the virion sense of maize streak virus (MSV) DNA has been detected among the products of in vitro translation reactions programmed with RNA from infected maize plants and also in total protein extracts from infected leaves. The 11 kDa protein has not been detected in virions and is therefore proposed to have a nonstructural role.Viral DNA with an additional in-frame translation stop codon in the 10.9 ORF was not infectious when transmitted to maize plants via Agrobacterium tumefaciens agroinfection, suggesting that the 10.9 ORF may be essential for virus function. Computer comparison data show that equivalent ORFs in wheat dwarf virus (WDV) and digitaria streak virus (DSV) have some sequences in common with the 10.9 ORF of MSV. Further-more, the absence of similar sequences in geminiviruses which infect dicotyledonous plants suggests that the 11 kDa protein and its putative homologs in WDV and DSV have a function necessary only for those geminiviruses which infect the Gramineae.The significance of the 11 kDa protein in relation to expression of the virion sense DNA of MSV is discussed.     

Molecular characterization of Haynaldia villosa chromatin in wheat lines carrying resistance to wheat curl mite colonization

Wheat-Haynaldia villosa (L.) Schur, hybrid lines were tested as potential sources of resistance to colonization by the wheat curl mite, the vector of wheat streak mosaic virus. Two lines, Add 6V-1 and Sub 6V-1, were found to be mite-resistant. Fluorescence in situ hybridization using total genomic DNA, from H. villosa in the presence of unlabelled wheat DNA, confirmed that Add 6V-1 is a disomic wheat-H. villosa chromosome addition line. Sub 6V-1 turned out to be a homoeologous wheat-H. villosa chromosome translocation line rather than a substitution. The translocation in Sub 6V-1 occurred between a wheat chromosome and a chromosome from H. villosa through Robertsonian fusion of misdivided centromeres. Only the short arm of the group 6 chromosome of H. villosa was involved in the genetic control of mite resistance, a conclusion based on the genomic in situ hybridization signal and specific DNA fragments obtained by polymerase chain reaction.LRC Contribution No. 3879542     

Biological control of seedling blight of wheat caused by Fusarium graminearum with beneficial rhizosphere microorganisms

Fusarium graminearum is associated with the cereal damping-off complex which reduces germination, seedling stand and yield. Fifty-two bacterial strains and six Trichoderma spp. isolated from the wheat rhizosphere were evaluated for biocontrol of seedling blight of wheat caused by F. graminearum. Their potential as biocontrol agents was tested in vitro and in the greenhouse. Isolates varied in their ability to inhibit the mycelial growth of F. graminearum in agar plate bioassays by 0–79%. This parameter was not related with biocontrol efficacy of in vivo assays. In greenhouse trials, all isolates were initially evaluated for reducing disease on wheat cultivars Klein Centauro (moderately resistant to F. graminearum) and Pro INTA Oasis (susceptible) planted in sterilized soil artificially infested with the pathogen. Among the 25 bacteria and six fungal isolates that exhibited a pronounced suppressive effect, the most efficient 10 for both cultivars were further assayed on eight cultivars (Buck Candil, Buck Catriel, Buck Chambergo, Buck Poncho, Buck Topacio, Klein Cacique, Klein Centauro and Pro INTA Oasis) potted in cultivated–inoculated soil. Three weeks after sowing, plant stand, percentage of diseased emerging seedlings, plant height and dry weight were evaluated. Among the antagonists only Stenotrophomonas maltophilia was significantly better than the control for the average of the eight cultivars for plant stand, height and dry weight. Stenotrophomonas maltophilia also caused a non-significant decrease in the percentage of diseased plants. Three strains of Bacillus cereus and one isolate of Trichoderma harzianum gave also a good control in some cultivars. The ability of these isolates to affect the infection of wheat seedlings by F. graminearum may be of potential value in field trials.     

Ear culture as a technique to overcome hybrid necrosis in wheat

Hybrid necrosis in wheat is a problem for gene transfer in wheat breeding. Hybrid necrosis occurs due to dominant complementary interaction of two genes, Ne1 and Ne2. A cross between wheat (Triticum aestivum L.) varieties C306 (drought tolerant, Ne1 carrier) and WL711(high yielding, Ne2 carrier) produced necrotic F1 hybrids, which died before ear emergence and produced no seeds. To overcome the problem of hybrid necrosis, ears enclosed in the leaf sheath were taken and cultured to maturity in liquid medium containing 5% sucrose and 0.04% glutamine. The necrotic hybrids produced only a few seeds per ear compared to parents, but individual grain weight was similar in the hybrid and the parents. The F1 ear culture study has been repeated for three years and F2 seeds obtained. In 1996–97, the cultured ears of F1 hybrids produced 62 seeds, of which only 52 showed germination and were grown under normal field conditions. Out of the 52 seeds, 50% were non-necrotic and showed segregation for various physiological traits. The results reveal that hybrids ears had the potential to form viable seeds. Culturing of wheat ears before ear emergence and production of viable F2 seeds from necrotic hybrids is a simple and efficient method for overcoming the problem of hybrid necrosis. This revised version was published online in June 2006 with corrections to the Cover Date.     

A model wheat cultivar for transformation to improve resistance to Fusarium Head Blight

Fusarium head blight (FHB), caused primarily by Fusarium graminearum, is a major disease problem in wheat (Triticum aestivum). Genetic engineering holds significant potential to enhance FHB resistance in wheat. Due to the requirement of screening for FHB resistance on flowers at anthesis, the number of screens carried out in a year is limited. Our objective was to evaluate the feasibility of using the rapid-maturing dwarf wheat cultivar Apogee as an alternative genotype for transgenic FHB resistance research. Our transformation efficiency (number of transgenic plants/number of embryos) for Apogee was 1.33%. Apogee was also found to exhibit high FHB susceptibility and reached anthesis within 4 weeks. Interestingly, microsatellite marker haplotype analysis of the chromosome 3BS FHB resistant quantitative trait locus (QTL) region indicated that this region maybe deleted in Apogee. Our results indicate that Apogee is particularly well suited for accelerating transgenic FHB resistance research and transgenic wheat research in general. C.A. Mackintosh and D.F. Garvin contributed equally to the article and should be considered co-first authors     

Field response of wheat to arbuscular mycorrhizal fungi and drought stress

Mycorrhizal plants often have greater tolerance to drought than nonmycorrhizal plants. This study was conducted to determine the effects of arbuscular mycorrhizal (AM) fungi inoculation on growth, grain yield and mineral acquisition of two winter wheat (Triticum aestivum L.) cultivars grown in the field under well-watered and water-stressed conditions. Wheat seeds were planted in furrows after treatment with or without the AM fungi Glomus mosseae or G. etunicatum. Roots were sampled at four growth stages (leaf, tillering, heading and grain-filling) to quantify AM fungi. There was negligible AM fungi colonization during winter months following seeding (leaf sampling in February), when soil temperature was low. During the spring, AM fungi colonization increased gradually. Mycorrhizal colonization was higher in well-watered plants colonized with AM fungi isolates than water-stressed plants. Plants inoculated with G. etunicatum generally had higher colonization than plants colonized with G. mosseae under both soil moisture conditions. Biomass and grain yields were higher in mycorrhizal than nonmycorrhizal plots irrespective of soil moisture, and G. etunicatum inoculated plants generally had higher biomass and grain yields than those colonized by G. mosseae under either soil moisture condition. The mycorrhizal plants had higher shoot P and Fe concentrations than nonmycorrhizal plants at all samplings regardless of soil moisture conditions. The improved growth, yield and nutrient uptake in wheat plants reported here demonstrate the potential of mycorrhizal inoculation to reduce the effects of drought stress on wheat grown under field conditions in semiarid areas of the world.     

Consumption of wheat seed reserves during germination and early growth as affected by soil water potential

Seed size and weight are important criteria for determining seedling vigour and stand establishment. Evolution of seed dry weight of wheat (Triticum aestivum L.) during germination and early growth was examined because poor stands are often associated with the depletion and exhaustion of seed reserves. Two laboratory experiments were conducted on filter paper and in soil at three water potentials using wheat seeds. Seed, root, and shoot dry weights were recorded at approximately one-day intervals. Coleoptile and first leaf lengths were also measured at all sampling periods. Wheat seedlings grown on filter paper in the dark grew to a length of 90 to 100 mm with 50% of the initial seed weight remaining after eight days when the experiment was terminated. In soil, wheat seedlings grew 15 mm with 25% of the initial seed weight remaining. Seed reserves were depleted more quickly when the soil was wet because seedlings grew more quickly. There were significant and similar negative relationships between seed weight and coleoptile length of wheat seedlings grown on filter paper and in soil. There was no effect of soil water potential on the relationship between seed weight and shoot length. Therefore, it was concluded that poor wheat stands are not likely to occur due to depletion of seed reserves under field conditions without mechanical obstacles.     

DNA isolation from dry and fresh samples of polysaccharide-rich plants

DNA extraction is difficult in a variety of plants because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. The chemotypic heterogeneity among species may not permit optimal DNA yield with a single protocol; thus, even closely related species may require different isolating protocols. Here we describe a modified procedure based on the hexadecyltrimethylammonium bromide (CTAB) method to isolate DNA from tissues containing high levels of polysaccharides. The procedure is applicable to both dry and fresh tissues and was tested on chickpea seeds, soybean, and wheat leaves. This method solved the problems of DNA degradation, contamination, and low yield due to binding and/or coprecipitation with starches and polysaccharides. The isolated DNA proved amenable to PCR amplification and restriction digestion.     

The 5S DNA units of bread wheat (Triticum aestivum)

A collection of 44 cloned 5S DNA units fromTriticum aestivum cv. Chinese Spring were grouped into 12 sequence-types based on sequence similarity and the respective consensus sequences were then produced. The relationship between these 12 consensus sequences (T. aestivum S 1-S 8 andT. aestivum L 1-L 4), together with two clones sequenced byGerlach andDyer, and the 5S DNA consensus sequences from diploidTriticum spp. were then determined by numerical methods. Both phenetic and cladistic analyses were carried out. The following wheat 5S DNA sequences were found to group with respective sequences from diploidTriticum spp.:T. aestivum S 4, S 6 withT. tauschii S;T. aestivum S 3 withT. monococcum S andT. monococcum S-Rus 7;T. aestivum L 1 andT. aestivum L-G&D withT. speltoides L;T. aestivum L 2, L 3 withT. tauschii L;T. aestivum L 4 withT. monococcum L andT. monococcum L-Rus 12. The analyses suggested that 5 out of the 65S Dna loci present in wheat were identified at the sequence level. The locus that could not be identified in this analysis was the5S Dna-B 1 locus. A group ofT. aestivum sequences (T. aestivum S 1, S 7, S 8, S-G&D) were found to be distinct from the other 5S DNA sequences in the data base. The existence of the distinct group of 5S DNA sequences suggests that there is a gap in our current understanding of wheat evolution with respect to the5S Dna loci.     

The contribution of different regions of the seminal roots of wheat to uptake of nitrate from soil

A technique was developed to determine the physiological activity of defined sections of seminal roots of wheat grown in sand. Wheat plants were grown for 2 weeks in narrow columns of N-deficient sand to which all other nutrients had been added. The columns were split longitudinally and ¹⁵N-labelled nitrate, in an agar medium, supplied to 2 cm sections of root. Shoots and roots were analysed after 24 h to determine the uptake of ¹⁵N. Three sections were examined on either the secondary or tertiary seminal root: 1 cm from the seed (basal segment), 35 cm from the seed (middle segment) and 4 cm from the root apex (apical segment). Total uptake was greatest from the basal and middle segments, declining by 50% from the apical segment. However, uptake per unit root length, including exposed sections of lateral roots, was not significantly different along the root.