The endophytic fungi from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In order to study the species composition of endophytes from wheat healthy plants in Buenos Aires Province (Argentina) and to determine their infection frequencies from leaves, stems, glumes and grains, wheat plants were collected from five cultivars at five growth stages from crop emergence to harvest. A total of 1,750 plant segments (leaves, stems, glumes and grains) were processed from the five wheat cultivars at five growth stages, and 722 isolates of endophytic fungi recovered were identified as 30 fungal genera. Alternaria alternata, Cladosporium herbarum, Epicoccum nigrum, Cryptococcus sp., Rhodotorula rubra, Penicillium sp. and Fusarium graminearum were the fungi that showed the highest colonization frequency (CF%) in all the tissues and organs analysed. The number of taxa isolated was greater in the leaves than those in the other organs analysed.     

Molecular genetic improvement of cereals: transgenic wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Only modest progress has been made in the molecular genetic improvement of wheat following the production of the first transgenic plants in 1992, made possible by the development of efficient, long-term regenerable embryogenic cultures derived from immature embryos and use of the biolistics method for the direct delivery of DNA into regenerable cells. Transgenic lines expressing genes that confer resistance to environmentally friendly non-selective herbicides, and pests and pathogens have been produced, in addition to lines with improved bread-making and nutritional qualities; some of these are ready for commercial production. Reduction of losses caused by weeds, pests and pathogens in such plants not only indirectly increases available arable land and fresh water supplies, but also conserves energy and natural resources. Nevertheless, the work carried out thus far can be considered only the beginning, as many difficult tasks lie ahead and much remains to be done. The challenge now is to produce higher-yielding varieties that are more nutritious, and are resistant or tolerant to a wide variety of biotic as well as abiotic stresses (especially drought, salinity, heavy metal toxicity) that currently cause substantial losses in productivity. How well we will meet this challenge for wheat, and indeed for other cereal and non-cereal crops, will depend largely on establishing collaborative partnerships between breeders, molecular biologists, biotechnologists and industry, and on how effectively they make use of the knowledge and insights gained from basic studies in plant biology and genetics, the sequencing of plant/cereal genomes, the discovery of synteny in cereals, and the availability of DNA-based markers and increasingly detailed chromosomal maps.     

Tolerance of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) to high soil and solution selenium levels

The fertilisation of wheat crops with Se is a cost-effective method of enhancing the concentration of organic Se in grain, in order to increase the Se intake of animals and humans. It is important to avoid phytotoxicity due to over-application of Se. Studies of phytotoxicity of Se in wheat grown in Australia, where rainfall and grain yield are usually relatively low, have not been reported previously, and overseas studies have had varied results. This study used trials conducted in the field, glasshouse and laboratory to assess Se phytotoxicity in wheat. In field trials that used rates of up to 120 g ha^–1Se as selenate, and in pilot trials that used up to 500 g ha^–1 Se soil-applied or up to 330 g ha^–1 Se foliar-applied, with soils of low S concentrations (2–5 mg kg^–1), no Se toxicity symptoms were observed. In pot trials of four weeks duration, the critical tissue level for Se toxicity was around 325 mg kg^–1 DW, a level attained by addition to the growth medium of 2.6 mg kg^–1 Se as selenate. Solution concentrations above 10 mg L^–1 Se inhibited early root growth of wheat in laboratory studies, with greater inhibition by selenite than selenate. For selenite, Se concentrations around 70 mg L^–1 were required to inhibit germination, while for selenate germination % was unaffected by a solution concentration of 150 mg L^–1 Se. Leaf S concentration and content of wheat increased three-fold with the addition of 1 mg kg^–1 Se as selenate to the growth medium. This effect is probably due to the induction of the S deficiency response of the main sulphate transporter. This study found wheat to be more Se-tolerant than did earlier studies of tobacco, soybeans and rice. We conclude that Se phytotoxicity in wheat will not be observed at the range of Se application rates that would be used to increase grain Se for human consumption (4–200 g ha^–1 Se as selenate, which would result in soil and tissue levels well below those seen in the above studies), even when – as is common in Australia – soil S concentration and grain yield are low.     

Comparative effectiveness of <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Serratia</Emphasis> sp. containing ACC-deaminase for improving growth and yield of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) under salt-stressed conditions

Ethylene synthesis is accelerated in response to various environmental stresses like salinity. Ten rhizobacterial strains isolated from wheat rhizosphere taken from different salt affected areas were screened for growth promotion of wheat under axenic conditions at 1, 5, 10 and 15 dS m⁻¹. Three strains, i.e., Pseudomonas putida (N21), Pseudomonas aeruginosa (N39) and Serratia proteamaculans (M35) showing promising performance under axenic conditions were selected for a pot trial at 1.63 (original), 5, 10 and 15 dS m⁻¹. Results showed that inoculation was effective even in the presence of higher salinity levels. P. putida was the most efficient strain compared to the other strains and significantly increased the plant height, root length, grain yield, 100-grain weight and straw yield up to 52, 60, 76, 19 and 67%, respectively, over uninoculated control at 15 dS m⁻¹. Similarly, chlorophyll content and K⁺/Na⁺ of leaves also increased by P. putida over control. It is highly likely that under salinity stress, 1-aminocyclopropane-1-carboxylic acid-deaminase activity of these microbial strains might have caused reduction in the synthesis of stress (salt)-induced inhibitory levels of ethylene. The results suggested that these strains could be employed for salinity tolerance in wheat; however, P. putida may have better prospects in stress alleviation/reduction.     

An integrative genetic linkage map of winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

We constructed a genetic linkage map based on a cross between two Swiss winter wheat (Triticum aestivum L.) varieties, Arina and Forno. Two-hundred and forty F₅ single-seed descent (SSD)-derived lines were analysed with 112 restriction fragment length polymorphism (RFLP) anonymous probes, 18 wheat cDNA clones coding for putative stress or defence-related proteins and 179 simple-sequence repeat (SSR) primer-pairs. The 309 markers revealed 396 segregating loci. Linkage analysis defined 27 linkage groups that could all be assigned to chromosomes or chromosome arms. The resulting genetic map comprises 380 loci and spans 3,086 cM with 1,131 cM for the A genome, 920 cM for the B genome and 1,036 cM for the D genome. Seventeen percent of the loci showed a significant (P < 0.05) deviation from a 1:1 ratio, most of them in favour of the Arina alleles. This map enabled the mapping of QTLs for resistance against several fungal diseases such as Stagonospora glume blotch, leaf rust and Fusarium head blight. It will also be very useful for wheat genetic mapping, as it combines RFLP and SSR markers that were previously located on separate maps. S. Paillard and T. Schnurbusch contributed equally to the work     

Quantitative structure analysis of genetic diversity among spring bread wheats (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) from different geographical regions

Genetic diversity in spring bread wheat (T.␣aestivum L.) was studied in a total of 69 accessions. For this purpose, 52 microsatellite (SSR) markers were used and a total of 406 alleles were detected, of which 182 (44.8%) occurred at a frequency of <5% (rare alleles). The number of alleles per locus ranged from 2 to 14 with an average of 7.81. The largest number of alleles per locus occurred in the B genome (8.65) as␣compared to the A (8.43) and D (5.93) genomes, respectively. The polymorphism index content (PIC) value varied from 0.24 to 0.89 with an average of 0.68. The highest PIC for all accessions was found in the B␣genome (0.71) as compared to the A (0.68) and D␣genomes (0.63). Genetic distance-based method (standard UPGMA clustering) and a model-based method (structure analysis) were used for cluster analysis. The two methods led to analogical results. Analysis of molecular variance (AMOVA) showed that 80.6% of the total variation could be explained by the variance within the geographical groups. In comparison to the diversity detected for all accessions (H _e = 0.68), genetic diversity among European spring bread wheats was H _e = 0.65. A comparatively higher diversity was observed between wheat varieties from Southern European countries (Austria/Switzerland, Portugal/Spain) corresponding to those from other regions.     

Genotyping of somatic hybrids between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.     

Mapping of esterase loci in <Emphasis Type="Italic">Aegilops uniaristata</Emphasis> and homoeologous group 3 chromosomes of wheat

This study was planned to identify the chromosomal location of esterase loci in wheat (Triticum aestivum), in comparison to Aegilops uniaristata, using wheat Ae. uniaristata disomic addition and translocation lines. Two loci (Est-N1 and Est-N8) were identified on 3N chromosome of Ae. uniaristata and their probable homoeoloci were, for the first time, mapped close to three RFLP probes (Xpsr56, Xpsr394, and Xpsr1196) on homoeologous group 3 wheat chromosomes.     

Efficient and rapid <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of durum wheat (<Emphasis Type="Italic">Triticum turgidum L. var. durum)</Emphasis> using additional virulence genes

Genetic transformation of wheat, using biolistics or Agrobacterium, underpins a range of specific research methods for identifying genes and studying their function in planta. Transgenic approaches to study and modify traits in durum wheat have lagged behind those for bread wheat. Here we report the use of Agrobacterium strain AGL1, with additional vir genes housed in a helper plasmid, to transform and regenerate the durum wheat variety Ofanto. The use of the basic pSoup helper plasmid with no additional vir genes failed to generate transformants, whereas the presence of either virG⁵⁴² or the 15 kb Komari fragment containing virB, virC and virG⁵⁴² produced transformation efficiencies of between 0.6 and 9.7%. Of the 42 transgenic plants made, all but one (which set very few seeds) appeared morphologically normal and produced between 100 and 300 viable seeds. The transgene copy number and the segregation ratios were found to be very similar to those previously reported for bread wheat. We believe that this is the first report describing successful genetic transformation of tetraploid durum wheat (Triticum turgidum L. var. durum) mediated by Agrobacterium tumefaciens using immature embryos as the explant.     

A new PCR-based marker on chromosome 4AL for resistance to pre-harvest sprouting in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Pre-harvest sprouting (PHS) is a complex trait controlled by multiple genes with strong interaction between environment and genotype that makes it difficult to select breeding materials by phenotypic assessment. One of the most important genes for pre-harvest sprouting resistance is consistently identified on the long arm of chromosome 4A. The 4AL PHS tolerance gene has therefore been targeted by Australian white-grained wheat breeders. A new robust PCR marker for the PHS QTL on wheat chromosome 4AL based on candidate genes search was developed in this study. The new marker was mapped on 4AL deletion bin 13-0.59-0.66 using 4AL deletion lines derived from Chinese Spring. This marker is located on 4AL between molecular markers Xbarc170 and Xwg622 in the doubled-haploid wheat population Cranbrook × Halberd. It was mapped between molecular markers Xbarc170 and Xgwm269 that have been previously shown to be closely linked to grain dormancy in the doubled haploid wheat population SW95-50213 × Cunningham and was co-located with Xgwm269 in population Janz × AUS1408. This marker offers an additional efficient tool for marker-assisted selection of dormancy for white-grained wheat breeding. Comparative analysis indicated that the wheat chromosome 4AL QTL for seed dormancy and PHS resistance is homologous with the barley QTL on chromosome 5HL controlling seed dormancy and PHS resistance. This marker will facilitate identification of the gene associated with the 4A QTL that controls a major component of grain dormancy and PHS resistance.     

Asymmetric somatic hybridization between wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) and <Emphasis Type="Italic">Avena sativa</Emphasis> L.

Protoplasts from cell suspensions of young-embryo-derived calli, whichwere non- regenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% of the same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity of less than 10%) irradiated with UV at an intensity of300 μW/cm2 for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts of the recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.     

Water-soluble phenolic compounds in the coat control germination and peroxidase reactivation in <Emphasis Type="Italic">Triticum aestivum</Emphasis> seeds

In this work, we investigated the inhibitory effects of water-soluble phenolic compounds (WSPCs) in the coat of after-ripening wheat (Triticum aestivum L.) seeds on the processes of germination and peroxidase reactivation. Wheat bran has a WSPC content of 862.5 μg gallic acid equivalent g⁻¹ dry weight. When seeds were incubated in the water extract of bran, germination, peroxidase reactivation, and coleoptile and radicle growth were suppressed in a WSPC concentration-dependent manner. The inhibitory effects were significantly ameliorated by removing WSPCs from bran extract by treating with 1% insoluble polyvinylpolypyrrolidone. Pretreatment of seeds with 0.1% H₂O₂ reduced the WSPC content in the coat, which was confirmed using Fourier transform infrared microspectroscopy. With H₂O₂ pretreatment, seed germination, peroxidase reactivation, and post-germination seedling growth were significantly stimulated. Application of the known phenolics caffeic acid, feruic acid, or vanillin to the germination medium blocked seed germination and suppressed peroxidase reactivation. The results described here indicate that WSPCs act as endogenous inhibitors in the coat to control germination of Triticum aestivum seeds, and that inhibition of germination is at least partially caused by suppressing peroxidase reactivation.     

Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) somatic embryogenesis from isolated scutellum: Days post anthesis,days of spike storage,and sucrose concentration affect efficiency

Summary To improve the efficiency of somatic embryogenesis of isolated scutella from commercial wheat (Triticum aestivum L.) cultivars, two factorial experiments were conducted to examine effects of days post anthesis (DPA), days of spike storage (DSS) at 4°C, and sucrose concentrations (SC) on the percentage of scutella producing mature embryos and the number of mature embryos produced per responsive scutellum. In the first experiment, scutella isolated from spikes collected at 10, 11, 12, 13, 14, 15, and 16 DPA and stored at 4°C for 7, 10, 13, and 16d were placed on embryo induction medium [Murashige and Skoog plus 9.96 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 110 mg l⁻¹ casamino acids], incubated in darkness for 12–14 d and then under light for 2 wk. The interaction of DPA × DSS significantly affected the percentage of scutella producing mature embryos, while only DPA affected the number of mature embryos per responsive scutellum. In the second experiment, scutella isolated from spikes collected at 12 DPA and stored for 15, 16, 17, 18, and 19d were placed on embryo induction medium containing 2, 3, 4, and 5% sucrose. The interaction of DSS × SC significantly affected both the percentage of scutella producing mature embryos and the number of mature embryos per responsive scutellum. In general, DPA/DSS/SC combinations, 12/17/3, 12/18/3, and 12/19/2, yielded the numerically highest embryogenesis efficiencies.     

<Emphasis Type="Italic">AOM3</Emphasis> and<Emphasis Type="Italic"> AOM4</Emphasis>: two MADS box genes expressed in reproductive structures of<Emphasis Type="Italic"> Asparagus officinalis</Emphasis>

The MADS box genes participate in different steps of vegetative and reproductive plant development, including the most important phases of the reproductive process. Here we describe the isolation and characterisation of two Asparagus officinalis MADS box genes, AOM3 and AOM4. The deduced AOM3 protein shows the highest degree of similarity with ZAG3 and ZAG5 of maize, OsMADS6 of rice and AGL6 of Arabidopsis thaliana. The deduced AOM4 protein shows the highest degree of similarity with AOM1 of asparagus, the SEP proteins of Arabidopsis and the rice proteins OsMADS8, OsMADS45 and OsMADS7. The high level of identity between AOM1 and AOM4 made impossible the preparation of probes specific for one single gene, so the hybridisation signal previously described for AOM1 is probably due to the expression of both genes. The expression profile of AOM3 and AOM1/AOM4 during flower development is identical, and similar to that of the SEP genes. Asparagus genes, however, are expressed not only in flower organs, but also in the different meristem present on the apical region of the shoot during the flowering season: the apical meristem and the three lateral meristems emerging from the leaf axillary region that will give rise to flowers and lateral inflorescences during flowering season, and to phylloclades and branches during the subsequent vegetative phase. The expression of AOM3 and AOM1/AOM4 in these meristems appears to be correlated with the reproductive function of the apex as the hybridisation signal disappears when the apex switches to vegetative function.