Isozyme analysis on progenes of somatic hybrids of Oryza sativa L.and O.officinalis

Some wild species of Oryza possess agronomi-cally useful traits such as disease/insect resis-tance and salt tolerance.However,the use ofwild Oryza species for rice improvement hasbeen hampered by the sexual incompatibility orsterility of hybrids obtained by conventionalmethods.We have used protoplast fusion as amethod to incorporate useful trait of wild O-ryza species into cultivated rice and~ obtainedsomatic hybrid plants between O.sativa and

Abortive Process of a Novel Rapeseed Cytoplasmic Male Sterility Line Derived from Somatic Hybrids Between Brassica napus and Sinapis alba

Somatic hybridization is performed to obtain significant cytoplasmic male sterility （CMS） lines, whose CMS genes are derived either from the transfer of sterile genes from the mitochondrial genome of donor parent to the counterpart of receptor or production of new sterile genes caused by mitochondrial genome recombination of the biparent during protoplast fusion. In this study, a novel male sterile line, SaNa-IA, was obtained from the somatic hybridization between Brassica napus and Sinapis alba. The normal anther development of the maintainer line, SaNa-IB, and the abortive process of SaNa-IA were described through phenotypic observations and microtome sections. The floral organ of the sterile line SaNa-IA was sterile with a shortened filament and deflated anther. No detectable pollen grains were found on the surface of the sterile anthers. Semi-thin sections indicated that SaNa-IA aborted in the pollen mother cell （PMC） stage when vacuolization of the tapetum and PMCs began. The tapetum radically elongated and became highly vacuolated, occupying the entire locule together with the vacuolated microspores. Therefore, SaNa-IA is different from other CMS lines, such as ogu CMS, pol CMS and nap CMS as shown by the abortive process of the anther.

Analysis of drought tolerance and genetic and epigenetic variations in a somatic hybrid between Ipomoea batatas (L.) Lam. and I. triloba L.

The somatic hybrid KT1 was previously obtained from protoplast fusion between sweetpotato （Ipomoea batatas （L.） Lam.） cv. Kokei No. 14 and its wild relative I. triloba L. However, its genetic and epigenetic variations have not been investigated. This study showed that KT1 exhibited significantly higher drought tolerance compared to the cultivated parent Kokei No. 14. The content of proline and activities of superoxide dismutase （SOD） and photosynthesis were significantly increased, while malonaldehyde （MDA） content was significantly decreased compared to Kokei No. 14 under drought stress. KT1 also showed higher expression level of well-known drought stress-responsive genes compared to Kokei No. 14 under drought stress. Amplified fragment length polymorphism （AFLP） and methylation-sensitive amplified polymorphism （MSAP） analyses indicated that KT1 had AFLP and MSAP band patterns consisting of both parent specific bands and changed bands. Fur- ther analysis demonstrated that in KT1. the proportions of Kokei No. 14 specific genome components and methylation sites were much greater than those of I. triloba. KT1 had the same chloroplast and mitochondrial genomes as Kokei No. 14. These results will aid in developing the useful genes ofI. triloba and understanding the evolution and phylogeny of the cultivated sweetpotato.

Molecular characterization of LMW-GS genes from a somatic hybrid introgression line Ⅱ-12 between Triticum aestivum and Agropyron elongatum in relation to quick evolution

In order to exploit the evolution and find novel low-molecular-weight glutenin subunit （LMW-GS） for improvement of common wheat quality, thirteen variants from a somatic hybrid introgression line II-12 between Triticum aestivum cv. Jinan 177 （JN177） and Agropyron elongatum were characterized via genomic PCR. Four clones were pseudogenes because they contained an internal stop codon. The remaining nine variants contained intact open reading frames （ORFs）. Sequence alignment indicates that the proteins deduced from the nine ORFs have similar primary structure with LMW-GS cloned from its parents previously. However, they have some unique modifications in the structures. For example, EU292737 contains not only an extra Cys residue in the C-terminal domain but also a long repetitive domain. Both EU 159511 and EU292738 start their first Cys residue in the N-terminal repetitive domain, but not in the N-conserved domain traditionally. These structural alterations may have positive contributions to wheat flour quality. The results of phylogeny showed that most LMW-GS variances from 11-12 were homologous to those from parent JN177 and other wheat lines. The reason for quick evolution of LMW-GS in 11-12 was discussed.

Mapping of Microsatellite SW943 to Porcine Chromosome 12p11-(2/3p13) Using Primed in situ Synthesis and Somatic Cell Hybrid Panel

The porcine microsatellite SW943 was regionally localized on 12p11-(2/3p13) by the two methods: the Primed in situ (PRINS) labelling on the pachytene bivalents of pigs using the Dig-11-dUTP as the report molecule and pig X rodent Somatic Cell Hybrid Panel (SCHP) which contains 27 cell lines through PCR amplification. Advantages and disadvantages of the two methods for physical mapping of microsatellites were also discussed.

Molecular identification of plants regenerated from somatic hybridization between rice and apomictic Panicum

We attempted to introduce apomictic gene(s)into rice via somatic hybridization by usingapomictic Panicum maximum Jacq.as thedonor of apomictic gene(s).Protoplasts of rice derived from suspen-sion cells were inactivated with indoacetamide(IOA)and protoplasts of Panicum maximum

Progress of chromosome engineering mediated by asymmetric somatic hybridization

Plant somatic hybridization has progressed steadily over the past 35 years. Many hybrid plants have been generated from fusion combinations of different phylogenetic species, some of which have been utilized in crop breeding programs. Among them, asymmetric hybrid, which usually contains a fraction of alien genome, has received more attention because of its importance in crop improvement. However, few studies have dealt with the heredity of the genome of somatic hybrid for a long time, which has limited the progress of this approach. Over recent ten years, along with the development of an effective cytogenetical tool ＂in situ hybridization （ISH）＂, asymmetric fusion of common wheat （Triticum aestivum L.） with different grasses or cereals has been greatly developed. Genetics, genomes, functional genes and agricultt, ral traits of wheat asymmetric hybrids have been subject to systematic investigations using gene cloning, genomic in situ hybridization （GISH） and molecular makers. The future goal is to fully elucidate the functional relationships among improved agronomic traits, the genes and underlying molecular mechanisms, and the genome dynamics of somatic introgression lines. This will accelerate the development of elite germplasms via somatic hybridization and the application of these materials in the molecular improvement of crop plants.

Comparative study of symmetric and asymmetric somatic hybridization between common wheat and Haynaldia villosa

Symmetric and asymmetric protoplast fusion between long term cell suspension-derived protoplasts of Triticum aestivum (cv. Jinan 177) and protoplasts of Haynaldia villosa prepared from one-year-old embryogeneric calli was performed by PEG method. In asymmetric fusion, donor calli were treated with gamma ray at a dose of 40, 60, 80 Gy (1.3 Gy/min) respectively and then used to isolate protoplasts. Results of morphological, cytological, biochemical (isozyme) and 5S rDNA spacer sequence analysis revealed that we obtained somatic hybrid lines at high frequency from both symmetric and asymmetric fusion. Hybrid plants were recovered from symmetric and low dose g-fusion combinations. GISH (genomic in situ hybridization) analysis proved exactly the existence of both parental chromosomes and the common occurrence of several kinds of translocation between them in the hybrid clones regenerated from symmetric and asymmetric fusion. And the elimination of donor DNA in hybrid clones regenerated from asymmetric fusion combinations was found to increase with the increasing gamma doses. It is concluded that transference and recombination of nuclear DNA can be achieved effectively by symmetric and asymmetric fusion, hybrids with small fragment translocation which are valuable in plant breeding can be obtained directly by asymmetric fusion.

Mitochondrial Genome of Callus Protoplast Has a Role in Mesophyll Protoplast Regeneration in Citrus: Evidence From Transgenic GFP Somatic Homo-Fusion

Protoplast fusion has great potential in citrus improvement. Although citrus mesophyll protoplasts usually cannot divide and regenerate,symmetric protoplast fusion of embryogenic callus protoplast + mesophyll protoplast sometimes results in the regeneration of mesophyllparent-type cybrids. It suggested that mitochondrial DNA(mt DNA) from protoplasts of embryogenic callus parent plays an important role in stimulating division and regeneration of mesophyll protoplasts. Herein, somatic fusion was conducted via electrofusion between callus protoplasts isolated from Valencia orange [Citrus sinensis(L.) Osbeck] cell suspension cultures and transgenic GFP-tagged mesophyll protoplasts from the same genotype, i.e. transgenic Valencia orange plants containing the green fluorescent protein(GFP) gene, in an effort to elucidate whether mt DNA of callus line could stimulate the division and regeneration of mesophyll protoplasts from the same genotype. Two embryoids and one plantlet with GFP expression were successfully obtained and subsequent ploidy analysis by flow cytometry indicated that they were all diploids. The regenerated diploid embryoids and plantlet with GFP expression could be considered as ‘cybrids' with mt DNA from the callus protoplasts of Valencia orange. The result indicated that citrus mesophyll-parent-type cybrid regeneration needed the stimulation of mt DNA from protoplasts of embryogenic callus parent regardless of their origin either from another genotype or the same genotype as the mesophyll parent.

RAPD-based genetic analysis of offsprings from the sexual cross using allotetraploid citrus somatic hybrid as pollen parent

Thirty-one polymorphic decamer primers were selected to genotype 92 progenies from the cross be- tween Yiben No.4, a monoembryonic diploid F1 hybrid of Citrus reticulata Blanco cv Huanongbendizao tangerine and C. ichangensis Swingle, and [Hamlin sweet orange + Rough lemon], an allotetraploid somatic hybrid of Citrus sinensis Osbeck cv. Hamlin and C. jambhiri Lush cv. Rough Lemon. χ2 (Chi-square) analysis of RAPD markers in the progenies indicated they were randomly transmitted from the four donor parents, without significant difference between the diploids and triploids. However, these progenies were clustered into three major groups using dendrogram constructed by UPGMA, skewed to three parents in certain degrees, 15 (13 triploids and 2 diploids) to Hamlin, 16 (9 and 7) to Yiben No. 4, and 61 (57 and 4) to [Hamlin sweet orange + Rough Lemon] from which genomic contribu- tion was predominant in progenies, respectively.

Cloning of the Full-length cDNA of the Wheat Involved in Salt Stress：Root Hair Defective 3 Gene （RHD3）

The full-length cDNA of the wheat (Triticum aestivum L.) root hair defective 3 gene (RHD3) has been cloned from the salt-tolerant hybrid wheat variety Shanrong No. 3 (Za3) using the mRNA differential display and 5’rapid amplification of cDNA ends (RACE) methods. Analysis of the amino acid sequence deduced from the wheat RHD3, gene shows that two conservative GTP-binding motifs, namely GXXXXGKS and DXXG, in eukaryotes also exist at the N-terminal of wheat RHD3. In addition, an 18 amino acid residue transmembrane domain, namely FYLAVMFVVFLVGKAIWV, exists at positions 701—718 of the C-terminal of the deduced protein of wheat RHD3 obtained, but this domain is absent in another three proteins aligned, including rice RHD3, Arabidopsis RHD3, and yeast homologue SEY1. Northern blot revealed that transcription of the wheat RHD3, gene is down-regulated in both the salt-tolerant line and in JN177 under saline stress. A possible stress-responsive mechanism for this gene is discussed.