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 2～5cm, their coleop tile and half of the cotyledon were cut out, and the basal portions were infected by A. tumefaciens. A total 27 To transgenic plants were obtained, and the average transfor mation efficiency was as high as 9.82%. Evident segrega tion 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 por-tion of wheat seedling is a highly efficient and dependable transformation method. It can be developed into a practi-cable method for transfer of target gene to wheat.

We report the production and characterization of somatic hybrids between Triticum aestivum L. and Agropyron elongatum (Host) Nevishi (the synonym is Thinopyrum ponticum). Asymmetric protoplast fusion was performed between Agropyron elongatum protoplasts irradiated with a low UV dose and protoplasts of wheat taken from nonregenerable suspension cultures. More than 40 green plantlets were obtained from 15 regenerated clones and one of them produced seeds. The phenotypes of the hybrid plants and seeds were intermediate between wheat and Agropyron elongatum. All of the regenerated calli and plants were verified as intergeneric hybrids on the basis of morphological observation and analysis of isozyme, cytological, 5SrDNA spacer sequences and random amplified polymorphic DNA (RAPD). RFLP analysis of the mitochondrial genome revealed evidence of random segregation and recombination of mtDNA.

Intergeneric somatic hybridization between wheat (cv Jinan 177) protoplasts that have 24 28 chromosomes and Hapnaldi villosa protoplasts con-taining 11 14 chromosomes was carried out by the polyethylene glycol (PEG) method A high frequency of hybrid calli and plants were obtained frpin the fusion products, as revealed by cytological and biochemical techniques and by PCR analysis of 5S rDNA spacer sequences GISH (genomic in situ hvbridization) anal-ysis confirmed the presence of chromosomes frpin both parents in the hybrid clones and the COlBitlon occur-rence of translocations between them The RFLP analysis of the organellar DNA using mitochondrion-and chloroplast-specific probes revealed that mito-chondria frpin both parents existed in the cells of hybrid calli and their recombination. whereas chloro-plasts segregated and recombined randomly The gross momhology of hybrid plants resembled that of wheat, but the gross morphologv of their ovaries and anthers were intermediate between those of the two parents The relationship between hybrid plant regeneration and the balance of genetic materials in hybrid clones is discussed.

Wheat productivity is severely affected by soil salinity mainly due to Na+ toxicity to plant cells. To improve the yield performance of wheat in saline soils, we have generated transgenic wheat expressing a vacuolar Na+/H+ antiporter gene AtNHX1 from Arabidopsis thaliana, to enhance the plant capacity of reducing cytosolic Na+ by sequestering Na+ in the vacuole. The AtNHX1 transgenic wheat lines exhibited improved biomass production at the vegetative growth stage and germination rates in severe saline conditions. A field trial revealed that the transgenic wheat lines produced higher grain yields and heavier and larger grains in the field of saline soils with the electrical conductivity values of soil saturation extracts (ECe) of 10.6 and 13.7dSm−1. The transgenic lines accumulated a lower level of Na+ and a higher level of K+ in the leaves than non-transgenic plants under saline conditions (100 and 150mM NaCl). These results indicate that the salt tolerance of wheat and grain yield in saline soils can be improved by enhancing the level of the vacuolar Na+/H+ antiporter.

The vast majority of angiosperms are (or were once) polyploid, and as hexaploid bread wheat has undergone two ploidy events separated by approximately 0.5 million years, it represents an elegant model to study gene silencing over time in polyploids. Using an SSCP platform, we have analysed patterns of transcriptional silencing (frequency, genome identity and organ specificity) within 236 single-copy genes, each mapping to one locus on one of the three homoeologous chromosomes within groups 1, 2, 3 and 7 of wheat. In about 27% of unigenes expressed in leaf, and about 26% of those in root, one (rarely two) members of a gene set (homoeoalleles) were not present in the cDNA template. Organ-specific regulation is commonplace, with many homoeoalleles transcribed in leaf but not root (and vice versa). There was little indication of extensive bias towards selective silencing of a particular genome copy. Expression of some of the silenced homoeoalleles was restored in certain aneuploid lines and varieties, and these displayed a significant degree of genetic variation for the silencing of a given homoeoallele. We propose that a substantial proportion of this phenomenon is effected by an epigenetic mechanism, and suggest that this form of genetic variation may be a significant player in the determination of phenotypic diversity in breeding populations.

Two types of protoplasts of wheat (Triticum aestivum L. cv. Jinan 177) were used in fusion experiments-cha9, with a high division frequency, and 176, with a high regeneration frequency. The fusion combination of either cha9 or 176 protoplasts with Russian wildrye protoplasts failed to produce regenerated calli. When a mixture of cha9 and 176 protoplasts were fused with those of Russian wildrye, 14 fusion-derived calli were produced, of which seven differentiated into green plants and two differentiated into albinos. The morphology of all hybrid plants strongly resembled that of the parental wheat type. The hybrid nature of the cell lines was confirmed by cytological, isozyme, random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analyses. GISH analysis revealed that only chromosome fragments of Russian wildrye were transferred to the wheat chromosomes of hybrid calli and plants. Simple sequence repeat (SSR) analysis of the chloroplast genome of the hybrids with seven pairs of wheat-specific chloroplast microsatellite primers indicated that all of the cell lines had band patterns identical to wheat. Our results show that highly asymmetric somatic hybrid calli and plants can be produced via symmetric fusion in a triparental fusion system. The dominant effect of two wheat cell lines on the exclusion of Russian wildrye chromosomes is discussed.

To study the usefulness of low-molecularweight glutenin subunits (LMW-GS) of Agropyron elongatum (Host) Nevski to wheat (Triticum aestivum L.) quality improvement, we characterized LMW-GS genes of A. elongatum. Nine LMW-GS genes of A. elongatum, which were named AeL1 to AeL9, were cloned by genomic PCR. After sequencing, we obtained complete open reading frames from AeL2 to AeL8 and partial genes of AeL1 and AeL9. All nine sequences are homoeologous to those of wheat and related grasses. Comparison of the deduced amino acid sequences with those of published LMW-GS suggests that the basic structures of all the subunits are very similar. However, except for AeL4 and AeL5, which contain the identical N-terminal sequence with LMW-m, other LMW-GS sequences separated from A. elongatum cannot be classified according to previous criteria for the three types: LMW-m (methionine), LMW-s (serine), and LMW-i (isoleucine), and then 12 groups. In addition, there are some characters in the LMW-GS sequences of A. elongatum: AeL2, AeL3, and AeL6 involve a Cys residue in the signal peptide respectively, which is absent in most of LMW-GS; AeL3, AeL6, AeL8, and AeL9 start their first Cys residues in the N-terminal repetitive domains, respectively; both AeL2 and AeL5 have nine Cys residues, with an extra Cys residue in the N-terminal repetitive domain and the repetitive and glutamine-rich domain; AeL2, AeL3, AeL6, and AeL9 comprise long repetitive domains. Phylogenetic analysis indicates that there is a relatively weak sequence identity between the LMW-GS genes from A. elongatum cloned in this study and those reported from other plants. Three LMW-GS sequences, AeL2, AeL3, and AeL6, are clustered to Glu-A3 from wheat than to those from other plants. The possible use of these genes in relation to the high quality of hybrid wheat is discussed.

Suspension-derived protoplasts of Agropyron elongatum irradiated by ultra-violet light (UV) were fused with the suspension-derived protoplasts of Triticum astivum using PEG. Fertile intergeneric somatic hybrid plants were produced and various hybrid lines have been selected and propagated in successive generations. Their hybrid nature was confirmed by analysis of profiles of isozymes, RAPDs, and 5S rDNA spacer sequences, and via GISH analysis. By the procedure described, the phenotype and chromosome number of wheat could be maintained besides transfer of a few chromosomes and chromosomal fragments from the donor A. elongatum. The results above indicated that highly asymmetric fertile hybrid plants and hybrid progenies of wheat were produced via somatic hybridization.

In this paper, we describe how Bupleurum scorzonerifolium/Triticum aestivum asymmetric somatic hybrids can be exploited to study the wheat genome. Protoplasts of B. scorzonerifolium Willd were irradiated with ultraviolet light (UV) and fused with protoplasts of common wheat (T. aestivum L.). All cell clones were similar in appearance to those of B. scorzonerifolium, while the regenerated plantlets were either intermediate or B. scorzonerifolium-like. Genotypic screening using isozymes showed that 39.3% of cell clones formed were hybrid. Some of the hybrid cell clones grew vigorously, and differentiated green leaves, shoots or plantlets. DNA marker analysis of the hybrids demonstrated that wheat DNA was integrated into the nuclear genomes of B. scorzonerifolium and in situ karyotyping cells revealed that a few wheat chromosome fragments had been introgressed into B. scorzonerifolium. The average wheat SSR retention frequency of the RH panel was 20.50%, but was only 6.67% in fusions with a nonirradiated donor. B. scorzonerifolium chromosomes and wheat SSR fragments in most asymmetric hybrid cell lines remained stable over a period of 2.5–3.5 years. We suggest the UV-induced asymmetric somatic hybrids between B. scorzonerifolium Willd and T. aestivum L. have the potential for use in the construction of an RH map of the wheat genome.

The introgressed small-chromosome segment of Agropyron elongatum (Host.) Neviski (Thinopyrum ponticum Podp.) in F5 line II-1-3 of somatic hybrid between common wheat (Triticum aestivum L.) and A. elongatum was localized by sequential fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and karyotype data. Karyotype analysis offered basic data of arm ratios and relative lengths of 21 pairs of chromosomes in parent wheat Jinan177 and hybrid II-1–3. Using special high repetitive sequences pSc119.2 and pAs1 for FISH, the entire B- and D-genome chromosomes were detected. The FISH pattern of hybrid Ⅱ-1-3 was the same as that of parent wheat. GISH using whole genomic DNA from A. elongatum as probe determined the alien chromatin. Sequential GISH and FISH, in combination with some of the karyotype data, localized the small chromosome segments of A. elongatum on the specific sites of wheat chromosomes 2AL, 1BL, 5BS, 1DL, 2DL and 6DS. FISH with probe OPF-031296 from randomly amplified polymorphic DNA (RAPD) detected E-genome chromatin of A. elongatum, which existed in all of the small chromosome segments introgressed. Microsatellite primers characteristic for the chromosome arms above were used to check the localization and reveal the genetic identity. These methods are complementary and provide comprehensive information about the genomic constitution of the hybrid. The relationship between hybrid traits and alien chromatin was discussed.