Hybrid plant regeneration from interfamilial somatic hybridization between grapevine (vitia vinifera L.) and Red Thorowax (Bupleurum scorzonerifolium willd)

The protoplasts of Red Thorowax ( Bupleurum scorzonerifolium) irradiated by ultraviolet light (UV) at an intensity of 260μW/cm2 for 0, 1,2 and 3 min respectively were fused with that of grapevine ( Vitia vinifera). The regenerated 19 clones, every one derived from a single fused cell, were identified as hybrids by phenotype, isozyme, chromosome and 5S rDNA spacer region analysis. The results reveal that all of them are somatic hybrids. 11 hybrid calli including asymmetric and symmetric products regenerated somatic embryos and young leaves after 5 months of culture, of which 4 hybrid cell lines derived from asymmetric fusion regenerated plants with roots after 8-10 months of culture. Inspection of chromosome showed that regeneration of whole plant was related to the decrease of chromosome number. Identification of 5S rDNA spacer region of the plants confirmed that they were interfamilial hybrid plants.     

Molecular identification of asymmetric somatic hybrids between wheat and Italian ryegrass

Sexual incompatibility between common wheat and Italian ryegrass was an obstacle for transferring useful traits from italian ryegrass to wheat. In order to use those desirable genetic resources to improve wheat and to create new cytoplasmic germplasm, the protoplasts of wheat and Italian ryegrass were successfully electrofused and the somatic hybrid plants were regenerated. Examination with 6 restriction enzymes, 13 probes including 9 mtDNA probes (H454, Pst24, B30, Pro I, 490, B342, pHJ2-7-1, B376, 7), 3 cpDNA probes (pHvc p1, pHvc p5 and pHvc p8) and one nuclear DNA probe-- pTA71 (rDNA) in total 73 enzyme/ probe combinations revealed rich polymorphism between the fusion partners. RFLP analysis indicated that approximately 93.4% of the regenerated plants were true somatic hybrids. AFLP analysis implied that the somatic hybrids were highly asymmetric. The RFLP analysis using mt- and cpDNA specific probes also demonstrated the non-coexistence of mitochondria and chloroplasts from the fusion partners in the somatic hybrid cells.     

Production of bacterial blight resistant lines from somatic hybridization between Oryza sativa L. and Oryza meyeriana L

Novel bacterial blight (BB) resistance gene(s) for rice was (were) introduced into a cultivated japonica rice variety Oryza sativa (cv. 8411), via somatic hybridization using the wild rice Oryza meyeriana as the donor of the resistance gene(s). Twenty-nine progenies of somatically hybridized plants were obtained. Seven somatically hybridized plants and their parents were used for AFLP (amplified fragment length polymorphism) analysis using 8 primer pairs. Results confirmed that these plants were somatic hybrids containing the characteristic bands of both parents. The morphology of the regenerated rice showed characters of both O.sativa and O.meyeriana. Two somatic hybrids showed highest BB resistance and the other 8 plants showed moderate resistance. The new germplasms with highest resistance have been used in the rice breeding program for the improvement of bacterial blight resistance.     

Testes and chromosomes in interspecific hybrids betweenHelicoverpa armigera (Hübner) andHelicoverpa assulta (Guenée)

The interspecific hybridization betweenHelicoverpa armigera females andHelicoverpa assulta males yieldedF ₁ hybrids (RS), fertile males and sterile individuals with abnormal genitals. The reverse hybridization betweenH. assulta females andH. armigera males yielded F₁ hybrids (SR)-fertile males and fertile females. The morphology of testes and the karyotype of chromosomes of larvae in the hybrids were investigated. Among the 2d old fifth-instar SR larvae, individuals without testes were fertile females and those with testes were fertile males. The length and breadth of testes between SR and parental species were not significantly different (p>0.05). Among the 2d old fifth-instar RS larvae, the testes were observed in all the individuals, but it could be classified into two types. The length and the breadth of testes in Type 1 larvae were not significantly different from those of their parental species (p>0.05), while those in Type 2 were significantly less than those of their parental species (p<0.01). Mitotic metaphase I of brain cells showed the diploid chromosomes number of both reciprocal hybrids was 2n=62, as many as their parents. The haploid number of 31 was confirmed by counts from spermatocytes at meiotic metaphase from SR male larvae and Type 1 larvae of RS. Meiosis was not observed in spermatocytes of Type 2 larvae of RS. Considering the characteristics of adult hybrids of RS, it was concluded that Type 1 individuals in RS were fertile and those of Type 2 were sterile. The sterility of Type 2 individuals in RS is attributed to the abnormity in development of testes and the failing meiosis of spermatocytes. As a result, the normal spermatozoon could not been produced.     

用RAPD技术分析烟草与曼陀罗体细胞杂种

利用RAPD技术对普通烟草(Nicotiana tabacum L.cv Xanthi nc)和毛曼陀罗(Datura innoxia Mill)原生质体融合获得的体细胞杂种进行了分析.使用了9个10-mer随机引物对7个体细胞杂种和双亲进行RAPD指纹图谱分析,从7个杂种的9组DNA指纹图谱上共得到570条扩增带,其中与烟草特征谱带相同的带数占了67.8%,与双亲共有的谱带相同的带数为24.6%,而与曼陀罗特征谱带相同的带数仅有2.8%.由此可见,杂种组织倾向于排除毛曼陀罗的DNA,融合体是一个高度不对称的体细胞杂种.此外,杂种中还出现了双亲没有的谱带(新带占4.8%).     

小麦与高冰草体细胞杂种F5代麦谷蛋白及SDS沉降值分析

通过对普通小麦与高冰草体细胞杂种F5代 175个株系的麦谷蛋白及SDS沉降值分析 ,发现杂种F5代有 10种不同的麦谷蛋白组成 ,并出现了 1Ax1,1Ax2 ,1Bx13,1By16 ,1By8,1Dx5等 6种不存在于亲本小麦中的高分子量麦谷蛋白亚基及 1Bx13 1By16 ,1Bx7 1By8,1Dx5 1Dy12等 3种新亚基组合 ,部分杂种中出现了高冰草的麦谷蛋白特征谱带 ;杂种株系的SDS沉降值表现出较大的差异 ,其中一些杂种的SDS沉降值明显高于亲本普通小麦 .结果表明 ,通过体细胞杂交有可能将野生禾草的优良性状引入普通小麦 ,创造出多种不同组成及组合的新种质 ,为小麦品质育种开辟新途径     

A minimum of four human class II alpha-chain genes are encoded in the HLA region of chromosome 6

The major histocompatibility complex (MHC) in man, also called the HLA region, is located on the short arm of chromosome 6 and encodes antigens involved in immunological processes. The class II HLA antigens consist of two noncovalently associated polypeptide chains, one of molecular weight 34,000 (alpha) and the other of molecular weight 29,000 (beta). The extensive polymorphism of the beta chain(s) has allowed the genetic mapping of the corresponding beta gene(s) to the HLA-DR region. cDNA clones for the HLA-DR alpha chain have been used to map the non-polymorphic DR alpha-chain gene to chromosome 6 using mouse-human somatic cell hybrids. Similarly, the DR alpha-chain gene has been mapped to the short arm of chromosome 6 centromeric to the HLA-A, -B and -C loci by in situ hybridization experiments. We isolated a cDNA clone that is related to the DR alpha chain and encodes the class II antigen DC alpha chain. We describe here how this DC alpha clone was used to find two or three additional alpha-chain genes by cross-hybridization and how HLA-antigen loss mutants of a human lymphoblastoid cell line (LCL) were used to ascertain that these additional class II antigen alpha-chain genes are also located in the HLA region.     

Cloning oflea cDNA fragment of carrot (Daucus carota L.) and analysis of its expression features

Addition of concentrated sucrose to MS culture arrests the development of carrot somatic embryo at the stage of cotyledon embryo and, with the sucrose concentration restored to normal level, the embryo thus arrested is reactivated into post-embryonic development. Using the method of RT-PCR, the cDNA fragment of a new member of the Dc3 family of lea has been obtained from carrot somatic embryo under regulated state. As revealed by Northern blotting, strong expression has been observed in carrot somatic embryo under regulated state but the expression was much reduced 12 h after deregulation, and nearly disappeared 24 h after. Based on this finding as well as results of related studies, it is surmised that changing the sucrose concentration in culture enabled carrot somatic embryo under suspension culture to undergo a specific course of development which is comparable to the dormancy-germination process of seeds.     

Cloning of lea cDNA fragment of carrot (Daucus carota L.) and analysis of its expression features

Addition of concentrated sucrose to MS culture arrests the development of carrot somatic embryo at the stage of cotyledon embryo and, with the sucrose concentration restored to normal level, the embryo thus arrested is reactivated into post-embryonic development. Using the method of RT-PCR, the cDNA fragment of a new member of the Dc3 family of lea has been obtained from carrot somatic embryo under regulated state. As revealed by Northern blotting, strong expression has been observed in carrot somatic embryo under regulated state but the expression was much reduced 12 h after deregulation, and nearly disappeared 24 h after. Based on this finding as well as results of related studies, it is surmised that changing the sucrose concentration in culture enabled carrot somatic embryo under suspension culture to undergo a specific course of development which is comparable to the dormancy-germination process of seeds.     

Study on an Organic Hybrid Made from Chlorinated Polyethylene and 2,2''''-methylene-bis- (4-methyl-6-cyclohexylphenol)

The dynamic mechanical properties and miscibility of an organic hybrid made from chlorinated polyethylene (CPE) and 2, 2'-methyIene-bis-( 4-methyl-6-cyclohexylphenol) ( ZKF ) are mainly discussed in this paper. It is found that ZKF acts as an antiplasticizer in CPE matrix and has good miscibility even with large ratio in CPE /ZKF hybrids. The glass transition temperature of various CPE /ZKF hybrids determined by DSC give a good fit to Wood's equation. Bifunctional ZKF is supposed to improve the intermolecular force of CPE, and the improvement is developed when the ZKF content increases. On the other hand, the viscoelastic properties are highly improved with the addition of ZKF. TA and tanδ peak values increase when the ZKF content increase in the CPE /ZKF hybrids, the damping capacity has been improved during the glass transition of CPE /ZKF hybrids. In addition, the glass transition temperature shifts to room temperature from the low temperature with the continuous addition of ZKF to CPE.     

Fusion of isolated plant protoplasts

A first step towards somatic hybridization in plants has been achieved. Protoplasts have been isolated and seen to fuse.     

Comparative analysis and evolutionary significance of the HMG1 gene in crucian carp,blunt snout bream,and their polyploid progeny

The full-length mRNA of the high mobility group protein 1 coding gene (HMG1) was obtained by RACE-PCR from red crucian carp (Carassius auratus red var.),blunt snout bream (Megalobrama amblycephala),and their triploid and tetraploid progeny.The sequence contained an open reading frame of 579 nucleotides coding for 193 amino acids.The nucleotide identity of HMG1 was higher between the tetraploid hybrid and the maternal red crucian carp (99%) than between the tetraploid hybrid and the paternal blunt snout bream (97%).The nucleotide identity between the triploid hybrids and each parent (95%) was lower than that between the parents (98%).The protein identity between the tetraploid hybrid and each parent (100%) was higher than that between the triploid hybrid and each parent (97%).Our results suggest that interspecific hybridization generates a shock to the HMG1 gene in triploid hybrids,causing divergence of nucleotides.The HMG1 protein of the tetraploid hybrids was consistent with that of its parents,which reduced the barrier of cross incompatibility between alleles,providing the basis for the bisexual fertile tetraploid hybrids forming a new polyploid species in nature.The secondary and tertiary structures of the HMG1 protein contain eight helices,three switches,two DNA-binding domains in the N-terminus,and a long acidic tail in the C-terminus.Together,these data suggest that the HMG1 protein plays a role of protein-DNA interactions,facilitating various DNA-dependent activities in the nucleus.We also investigated the phylogeny of fish,amphibian,reptilian,bird,and mammalian HMG1 proteins.Our results suggest that HMG1 is an ancestral protein that has been highly conserved.These data provide clues as to how interspecific hybridization may form polyploid hybrids.     

Complementation in monocyte hybrids revealing genetic heterogeneity in chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare syndrome, found predominantly in male children and characterized by life-threatening, recurrent infections. The superoxide (O2-)/hydrogen peroxide (H2O2) generating system in the granulocytes and monocytes of CGD patients is completely defective. Furthermore, a novel type of cytochrome b, detected by the optical spectrum of phagocytes from healthy subjects, is lacking in those of most male CGD patients. In female CGD patients, the cytochrome b is present, but cannot, as in normal cells, be reduced on metabolic stimulation of the phagocytes in anaerobic conditions. Here, to demonstrate the importance of cytochrome b in this system and to investigate the genetic background of the various forms of CGD, we have hybridized monocytes from a cytochrome b negative, X-linked male CGD patient with monocytes from a cytochrome b positive, male CGD patient with unknown genetic background. Monocytes were used because they are the only blood phagocytes that show an active protein synthesis, whereas fibroblasts or lymphocytes do not express the O2-/H2O2 generating system. The heterologous hybrids were positive in the nitroblue tetrazolium (NBT) slide test, indicating the complementation of the O2-/H2O2 generating system, whereas the homologous hybrids remained negative, as did the non-fused cells of these patients. We thus conclude that cytochrome b is part of the O2-/H2O2 generating system and that somatic cell hybridization experiments with monocytes provide a means of studying the genetic background of CGD patients. We believe this to be the first report of genetic complementation by somatic cell hybridization experiments using monocytes instead of fibroblasts.     

Localisation of the G gamma-, A gamma-, delta- and beta-globin genes on the short arm of human chromosome 11.

Human-mouse somatic cell hybrids have proved invaluable in assigning human genes to their respective human chromosomes. To date, the success of this approach has depended on identifying human proteins which are synthesised in hybrid cells containing a small number of human chromosomes. Consequently, chromosome assignment has been limited mainly to human proteins which are expressed in man-mouse somatic cell hybrids and for which a suitable assay, usually electrophoretic or immunological, exists to distinguish between the human and murine homologous proteins. This technique is therefore unsuitable for the assignment of those human genes which are expressed only in differential cells and not in hybrid cells. Here, we describe how nucleic acid hybridisation and restriction endonuclease mapping of DNA can be combined to test for the presence of human structural gene sequences within hybrid cell DNA. This method can be used to assign any purified human DNA sequence to a human chromosome, and does not require the DNA sequence to be expressed in man-mouse hybrid cells.     

Cloning and expression profiles of 15 genes encoding WRKY transcription factor in wheat (Triticum aestivem L.)

WRKY proteins are involved in various physiological processes, including biotic and abiotic stress responses, hormone responses and development. However, no systematic identification, expression and function analysis of WRKY genes in wheat were reported. In this study, we isolated 15 wheat cDNAs with complete open reading frame (ORF) encoding putative WRKY proteins using in silico cloning. Phylogenetic analysis indicated that the 15 wheat WRKY genes belonged to three major WRKY groups. Expression analysis revealed that most genes expressed drastically in leaf, except TaWRKY10 which expressed in crown intensively. Four genes were strongly up-regulated with the senescence of leaves. Eight genes were responsive to low temperature, high temperature, NaCl or PEG treatment. Moreover, differential expression patterns were also observed between wheat hybrid and its parents, and some genes were more responsive to PEG treatment in the hybrid. These results demonstrated that wheat WRKY genes are involved in leaf senescing and abiotic stresses. And the changed expression of these WRKY genes in hybrid might contribute to the heterosis by improving the stress tolerance in hybrids.     

用流式细胞仪和RAPD快速鉴定柑橘体细胞杂种

利用流式细胞仪和随机扩增多态DNA（RAPD）方法对获得的3例柑橘体细胞杂种进行鉴定,结果表明,两者相结合可快速有效地鉴定体细胞杂种．所检测的3个组合共9株再生植株,有8株是四倍体体细胞杂种,1株为二倍体叶肉亲本型杂种．体细胞杂种一般表现为具有双亲的特征带,且综合双亲的所有带,但在部分杂种中检测到了双亲都没有的新带,也发现有丢失亲本的特征带或共有带的现象,说明融合后染色体发生了重组和交换;有的引物只检测到叶肉亲本的特征带,根据柑橘叶肉细胞无论单独培养还是共培养均不能再生的事实,可推测其为体细胞杂种或二倍体叶肉亲本型胞质杂种．对这两种方法相结合用于体细胞杂种鉴定的可行性进行了讨论．     

Molecular verification of the integration of Tripsacum dactyloides DNA into wheat genome through wide hybridization

RAPD and RFLP analyses of double haploid lines which derived from hybridization between hexaploid wheat (Triticum aestivum L.2n=42) and eastern gamagrass (Tripsacum dactyloides L.2n=4x=72) are reported.Two of the 340 Operon primers have been screened,which stably amplified Tripsacum dactyloides (male parent) specific bands in the double haploid lines.These results confirm the fact that Tripsacum dactyloides DNA has been integrated into wheat genome by sexual hybridization at molecular level.This idea has been further testified by RFLP analysis.Application and potentials of transferring Tripsacum dactyloides DNA into wheat genome by sexual hybridization in wheat breeding are discussed.     

杨盘单格孢菌〔Marssonina populi (Lib.)Magn.〕的两种专化型

<正>华东地区杨树黑斑病是由杨盘单格孢菌(Marssonina populi)引起的,病害主要为害白杨派树种和黑杨派树种以及它们的杂交种。杨盘单格孢菌根据孢子萌发的形态、生理和寄主范围可以分为两个专化型。单芽管专化型(M.populi f.sp.monogermtubi)的分生孢子萌发时产生 1个芽管;在蒸馏水中基本上不萌发,在自来水中萌发良好,2小时开始萌发,9小时即达高峰;孢子萌发温度为12-28℃;孢子表面无萌发抑制物质;寄主以白杨派树种及派内杂交种为主。多芽管专化型(M.populi f.sp.multigermtubi)的分生孢子萌发时产生1-5个芽管,多为2-3个;蒸馏水中能萌发但不及自来水中萌发率高;在自来水中4小时开始萌发,13小时以后才达高峰;萌发温度为16-32℃;孢子表面有水溶性萌发抑制物质,须用清水洗涤后才能萌发;寄主以黑杨派树种及派内杂交种和黑杨派与青杨派杂交种为主。两种专化型用孢子萌发试验即可鉴别。     

Relationships between D1 protein, xanthophyll cycle and photodamage-resistant capacity in rice (Orysa sativa L.)

Relationships between D1 protein, xanthophyll cycle and subspecific difference of photodamage-resistant capacity have been studied in O. japonica rice varieties 02428 and 029 (photoinhibition-tolerance) and O. indica rice varieties 3037 and Palghar (photoinhibition-sensitivity) and their reciprocal cross F1 hybrids after photoinhibitory treatment. It was shown that PSⅡ photochemical efficiency (Fv /Fm) decreased, and xanthophyll cycle from violaxanthin (V), via anaxanthin (A), to zeaxanthin (Z) was enhanced and non-photochemical quenching (qN) increased accordingly in SM-pretreated leaves of rice when the synthesis of D1 protein was inhibited, and that there was a decrease in qN and, as a result, more loss of D1 protein and a big decrease in Fv /Fm in DTT-pretreated leaves when xanthophyll cycle was inhibited. O. japonica subspecies had a higher maintaining capacity of D1 protein and a decrease of Fv /Fm in a more narrow range, and exhibited more resistance against photodamage, as compared with O. indica subspecies. The above physiological indexes in reciprocal cross F1 hybrids, though between the values of their parents, were closer to maternal lines than to paternal lines. Experimental results support the concept that the turnover capacity for D1 protein is an important physiological basis of photoinhibition-tolerance, and will provide the physiological basis for selection of the photoinhibition-tolerant parents and develop a new approach to breed hybrids with high photosynthetic efficiency.     

Molecular verification of the integration ofTripsacum dactyloides DNA into wheat genome through wide hybridization

RAPD and RFLP analyses of double haploid lines which derived from hybridization between hexaploid wheat (Triticum aestivum L. 2n=42) and eastern gamagrass (Tripsacum dactyloides L. 2n=4x=72) are reported. Two of the 340 Operon primers have been screened, which stably amplified 处留情 (male parent) specific bands in the double haploid lines. These results confirm the fact that Tripsacum dactyloides DNA has been integrated into wheat genome by sexual hybridization at molecular level. This idea has been further testified by RFLP analysis. Application and potentials of transferring Tripsacum dactyloides DNA into wheat genome by sexual hybridization in wheat breeding are discussed.