Protoplasts were isolated from cultured cells of wheat and maize and fused using PEG. Calli and green plants were regenerated following irradiation of the maize, and some tested positive for hybridity using morphological, isozymic and various DNA-based marker systems. Genomic in situ hybridization (GISH) of selected maize-carrying regenerants showed that some maize chromatin was dispersed throughout the wheat nuclear genome. Cytogenomic analysis, using RFLP and SSR, demonstrated the presence of both wheat and maize loci in the mitochondrial genome, so that, in the hybrid individuals, either distinct mitochondria from each parent coexist and/or recombinant mitochondria have been generated. With respect to the chloroplast genome, there was no evidence of the presence of any introgression from maize.

Introgression lines derived from somatic hybrids between Thinopyrum ponticum Podp. (Agropyron elongatum (Host) Nevishi) and Triticum aestivum L. cv. Jinan 177 were screened for salt-tolerance in hydroponic experiments. Their growth rates, salt-tolerance index and the content of free proline, Na+, K+ and Na+/K+ in the leaf were compared with those of wheat cv. Jinan 177. The lines were tested under natural saline conditions in two locations. One line expressed higher salt-tolerance than its parental wheat and a check salt-tolerant cultivar. Karyotype analysis showed that the two lines possessed 42 chromosomes of wheat introgressed with small chromosome segments from Th. ponticum. We suggest that these two lines show stable inheritance of salt tolerance, and that this salt-tolerance has been transferred from Th. ponticum into wheat.

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.

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.