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.

Following differing periods of long-term (13 years and 6 years, respectively) subculture and selection, two types of calli of the same wheat (Triticum aestivum L.) cv. Jinan 177 were obtained. The one (named cha9) grew fast and easily formed cell suspensions that were non-regenerable, but the protoplasts possessed a high division capacity. The other (named 176) was regenerable, but the derived protoplasts grew slowly. These two types of protoplasts were mixed and fused with protoplasts of Italian ryegrass (Lolium multiflorum Lam.). The protoplast of Italian ryegrass was UV irradiated. Many vigorous green plants having a wheat-like morphology were obtained. Regenerated calli and plants were recognised as somatic hybrids by a combined analysis of cytological, isozyme and random amplified polymorphic DNA (RAPD) markers. All the hybrids analysed are highly asymmetric, carrying only a few gnetic markers from Italian ryegrass. RFLP and SSR genotyping, using mitochondria- and chloroplast-specific markers, revealed a random segregation and rearrangement of the mtDNA, with a pronounced bias towards wheat, and a similarly biased segregation of cpDNA. Possible reasons for donor genome exclusion and the biased organelle segregation are discussed.

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.

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 coleop81 tile 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 transfor 84 mation efficiency was as high as 9.82%. Evident segrega85tion 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-95 tion of wheat seedling is a highly efficient and dependable 96 transformation method. It can be developed into a practi-97 cable method for transfer of target gene to wheat.

To study the effect of γ-ray treatment on donorand derived somatic hybrids, we carried out γ-ray donor treatment experiments with a wide range of γ-ray dosages and asymmetric somatic hybridization between protoplasts of wheat (Triticum aestivum L. Jinan 177) and protoplasts of Haynaldia villosa Schur. treated with different dosages of γ-rays (40, 60 and 80 Gy, respectively). We first screened the putative hybrids by isozyme analysis, followed by characterization of nuclear and organellar genome composition of the hybrids. Genomic in situ hybridization on mitotic metaphases demonstrated that the donor chromosome elimination in the hybrids increased with increased γ-ray dosage. Intergenomic chromosome recombination/translocations were observed in the hybrids from different dosages of γ-rays. PCR amplification of 5S rDNA spacer sequences showed that only some of the regenerated hybrid clones inherited donor 5S rDNA sequences, suggesting that the donor DNA was also eliminated randomly. Restriction fragment length polymorphism analysis using mitochondrion (mt) and chloroplast (cp) genespecific probes showed that the hybrid calli contained mt genomes of both parents and the cp genome of only one of the parents. Recombinations between parental mt as well as cp genes were found in the hybrid clones. Furthermore, development of the hybrid clones was dependent on the γ-ray dosage used for the donor treatment. Regenerated plants were only obtained from fusion combinations of low (40 Gy) and intermediate (60 Gy) dose irradiation. The possible role and significance of γ-rays on the introgression of small segments of donor chromosomes to the receptor is discussed.