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.

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.

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.

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.

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.