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.     

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.     

Identification of SNPs and development of functional markers for LMW-GS genes at <Emphasis Type="Italic">Glu-D3</Emphasis> and <Emphasis Type="Italic">Glu-B3</Emphasis> loci in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Low-molecular-weight glutenin subunits (LMW-GS) have great effect on wheat processing quality, but were numerous and difficult to dissect by SDS-PAGE. The development of functional markers may be the most effective way for a clear discrimination of different LMW-GS genes. In the present study, three different approaches were used to identify SNPs of different genes at Glu-D3 and Glu-B3 loci in bread wheat for the development of six STS markers (3 for Glu-D3 and 3 for Glu-B3 genes) that were validated with distinguished wheat cultivars. Firstly, seven LMW-GS gene sequences ( AY585350, AY585354, AY585355, AY585356, AY585349, AY585351 and AY585353 ) from Aegilops tauschii, the diploid donor of the D-genome of bread wheat, were chosen to design seven pairs of AS-PCR primers for Glu-D3 genes. By amplifying the corresponding genes from five bread wheat cultivars with different Glu-D3 alleles (a, b, c, d and e) and Ae. tauschii, a primer set, S13F2/S13R1, specific to the gene AY585356, was found to be positive to cultivars with alleles Glu-D3c and d. Nevertheless, the other five pairs of primers designed from AY585350, AY585349, AY585353, AY585354 and AY585355, respectively, did not produce specific PCR products to the cultivars tested. Secondly, all the PCR products from the five primer sets without specific characteristics were sequenced and an SNP from the gene AY585350 was detected in the cultivar Hartog, which resulted in the second STS marker S1F1/S1R3 specific to the allelic variant of AY585350. Thirdly, three Glu-D3 sequences (AB062851, AB062865 and AB062872) and three Glu-B3 sequences (AB062852, AB062853 and AB062860) defined by Ikeda et al. (2002) were chosen to query wheat EST and NR databases, and DNA markers were developed based on the putative SNPs among the sequences. Using this approach, four STS markers were developed and validated with 16-19 bread wheat cultivars. The primer set T1F4/T1R1 was also a Glu-D3 gene-specific marker for AB062872, while T2F2/T2R2, T5F3/T5R1 and T13F4/T13R3 were all Glu-B3 gene specific markers for AB062852, BF293671 and AY831800, respectively. The chromosomal locations of the six markers were verified by amplifying the genomic DNA of Ae. tauschii (DD), T. monococcum (AA) and T. turgidum (AABB) entries, as well as Chinese Spring and its group 1 chromosome nulli-tetrasomic lines. The results are useful to discriminate the corresponding Glu-D3 and Glu-B3 genes in wheat breeding programs.     

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.     

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     

Fertilizer-dependent efficiency of Pseudomonads for improving growth,yield, and nutrient use efficiency of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Acquisition of nutrients by plants is primarily dependent on root growth and bioavailability of nutrients in the rooting medium. Most of the beneficial bacteria enhance root growth, but their effectiveness could be influenced by the nutrient status around the roots. In this study, two 1-aminocyclopropane-1-carboxylate (ACC)-deaminase containing plant-growth-promoting rhizobacteria (PGPR), Pseudomonas fluorescens and P. fluorescens biotype F were tested for their effect on growth, yield, and nutrient use efficiency of wheat under simultaneously varying levels of all the three major nutrients N, P, and K (at 0%, 25%, 50%, 75%, and 100% of recommended doses). Results of pot and field trials revealed that the efficacy of these strains for improving growth and yield of wheat reduced with the increasing rates of NPK added to the soil. In most of the cases, significant negative linear correlations were recorded between percentage increases in growth and yield parameters of wheat caused by inoculation and increasing levels of applied NPK fertilizers. It is highly likely that under low fertilizer application, the ACC-deaminase activity of PGPR might have caused reduction in the synthesis of stress (nutrient)-induced inhibitory levels of ethylene in the roots through ACC hydrolysis into NH₃ and α-ketobutyrate. The results of this study imply that these Pseudomonads could be employed in combination with appropriate doses of fertilizers for better plant growth and savings of fertilizers.     

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.     

Mineralization of chlorpyrifos by co-culture of <Emphasis Type="Italic">Serratia</Emphasis> and <Emphasis Type="Italic">Trichosporon</Emphasis> spp.

A bacterial strain (Serratia sp.) that could transform chlorpyrifos to 3,5,6-trichloro-2-pyridinol (TCP) and a TCP-mineralizing fungal strain (Trichosporon sp.) were isolated from activated sludge by enrichment culture technique. The fungus could also degrade 50 mg chlorpyrifos l(-1) within 7 days. Co-cultures completely mineralized 50 mg chlorpyrifos l(-1) within 18 h at 30 degrees C and pH 8 using a total inocula of 0.15 g biomass l(-1).     

Quantitative studies on phosphorus transference occuring between <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and its attached bacterium (<Emphasis Type="Italic">Pseudomonas</Emphasis> sp.)

Phosphorus release from Microcystis aeruginosa and attached bacterium (Pseudomonas sp.) isolated from Lake Taihu was examined using a phosphorus isotope tracer in order to investigate the phosphorus transference between the two species. Our results reveal that the amount of phosphorus released form ³²P-saturated M. aeruginosa is determined by its growth phase and most of phosphorus is assimilated by Pseudomonas finally while the amount of phosphorus released from ³²P-saturated Pseudomonas is also determined by the growth phase of M. aeruginosa and most of them are assimilated by M. aeruginosa. The results suggest that phosphorus transference occurs between M. aeruginosa and its attached Pseudomonas . This process makes microenvironment of mucilage of M. aeruginosa attached bacteria maintain relative high amounts of phosphorus. Attached bacteria may be a temporary phosphorus bank to the growth of M. aeruginosa, and assimilation of phosphorus by M. aeruginosa becomes easy when M. aeruginosa is in lag growth phase. Thus, the phosphorus exchange between M. aeruginosa and attached Pseudomonas in microenvironment may be important to microfood web and cyanobacteria bloom.     

Synthesis of nanophase hydroxyapatite by a <Emphasis Type="Italic">Serratia</Emphasis> sp. from waste-water containing inorganic phosphate

Synthesis of nanophase hydroxyapatite (HA) on a bacterial surface was achieved at the expense of CaCl₂ and inorganic phosphate (Pi). After initial nucleation, calcium was precipitated on and around the cells as calcium phosphate at the expense of inorganic phosphate in the challenge solution, with no precipitation in cell-free controls. HA was also biomanufactured using inorganic phosphate ions scavenged from a phosphate-containing waste-water. With additional Ca²⁺, the concentration of phosphate was decreased from 0.27 (25ppm) to 0.02m (2ppm) in the waste-water. Crystals of calcium phosphate manufactured by the cells were located by scanning electron microscopy (SEM) and identified as HA by X-ray powder diffraction, with an average crystal size calculated as 25nm. Possible application of bioHA as a biomaterial and implications for one-step `waste-into product' are discussed.     

Degradation of ethylbenzene by free and immobilized <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">fluorescens-</Emphasis>CS2

Pseudomonas fluorescens-CS2 metabolized ethylbenzene as the sole source of carbon and energy. The involvement of catechol as the hydroxylated intermediate during the biodegradation of ethylbenzene was established by TLC, HPLC and enzyme analysis. The specific activity of Catechol 2,3-dioxygenase in the cell free extracts of P. fluorescens-CS2 was determined to be 0.428 μmoles min⁻¹ mg⁻¹ protein. An aqueous-organic, Two-Phase Batch Culture System (TPBCS) was developed to overcome inhibition due to higher substrate concentrations. In TPBCS, P. fluorescens-CS2 demonstrated ethylbenzene utilization up to 50 mM without substrate inhibition on inclusion of n-decanol as the second phase. The rate of ethylbenzene metabolism in TPBCS was found enhance by fivefold in comparison with single phase system. Alternatively the alginate, agar and polyacrylamide matrix immobilized P. fluorescens-CS2 cells efficiently degraded ethylebenzene with enhanced efficiency compared to free cell cultures in single and two-phase systems. The cells entrapped in ployacrylamide and alginate were found to be stable and degradation efficient for a period of 42 days where as agar-entrapped P. fluorescens was stable and efficient a period of 36 days. This demonstrates that alginate and polyacrylamide matrices are more promising as compared to agar for cell immobilization.     

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.     

Biodegradation of methyl parathion and <Emphasis Type="Italic">p</Emphasis>-nitrophenol: evidence for the presence of a <Emphasis Type="Italic">p</Emphasis>-nitrophenol 2-hydroxylase in a Gram-negative <Emphasis Type="Italic">Serratia</Emphasis> sp. strain DS001

A soil bacterium capable of utilizing methyl parathion as sole carbon and energy source was isolated by selective enrichment on minimal medium containing methyl parathion. The strain was identified as belonging to the genus Serratia based on a phylogram constructed using the complete sequence of the 16S rRNA. Serratia sp. strain DS001 utilized methyl parathion, p-nitrophenol, 4-nitrocatechol, and 1,2,4-benzenetriol as sole carbon and energy sources but could not grow using hydroquinone as a source of carbon. p-Nitrophenol and dimethylthiophosphoric acid were found to be the major degradation products of methyl parathion. Growth on p-nitrophenol led to release of stoichiometric amounts of nitrite and to the formation of 4-nitrocatechol and benzenetriol. When these catabolic intermediates of p-nitrophenol were added to resting cells of Serratia sp. strain DS001 oxygen consumption was detected whereas no oxygen consumption was apparent when hydroquinone was added to the resting cells suggesting that it is not part of the p-nitrophenol degradation pathway. Key enzymes involved in degradation of methyl parathion and in conversion of p-nitrophenol to 4-nitrocatechol, namely parathion hydrolase and p-nitrophenol hydroxylase component “A” were detected in the proteomes of the methyl parathion and p-nitrophenol grown cultures, respectively. These studies report for the first time the existence of a p-nitrophenol hydroxylase component “A”, typically found in Gram-positive bacteria, in a Gram-negative strain of the genus Serratia. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Phenanthrene degradation by bacteria of the genera <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Burkholderia</Emphasis> in model soil systems

Degradation of phenanthrene by strains Pseudomona, Moscow, KMK, 2004simova, I.A. and Chernov, I.s putida BS3701 (pBS1141, pBS1142), Pseudomonas putida BS3745 (pBS216), and Burkholderia sp. BS3702 (pBS1143) were studied in model soil systems. The differences in accumulation and uptake rate of phenanthrene intermediates between the strains under study have been shown. Accumulation of 1-hydroxy-2-naphthoic acid in soil in the course of phenanthrene degradation by strain BS3702 (pBS1143) in a model system has been revealed. The efficiency of phenanthrene biodegradation was assessed using the mathematical model proposed previously for assessment of naphthalene degradation efficiency. The efficiency of degradation of both phenanthrene and the intermediate products of its degradation in phenanthrene-contaminated soil is expected to increase with the joint use of strains P. Putida BS3701 (pBS1141, pBS1142) and Burkholderia sp. BS3702 (pBS1143).     

Characterization and role of a metalloprotease induced by chitin in <Emphasis Type="Italic">Serratia</Emphasis> sp. KCK

A metalloprotease induced by chitin in a new chitinolytic bacterium Serratia sp. Strain KCK was purified and characterized. Compared with other Serratia enzymes, it exhibited a rather broad pH activity range (pH 5.0–8.0), and thermostability. The cognate ORF, mpr, was cloned and expressed. Its deduced amino acid sequence showed high similarity to those of bacterial zinc-binding metalloproteases and a well-conserved serralysin family motif. Pretreatment of chitin with the Mpr protein promoted chitin degradation by chitinase A, which suggests that Mpr participates in, and facilitates, chitin degradation by this microorganism.     

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.     

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.