Transfer of bacterial blight resistance from <Emphasis Type="Italic">Oryza meyeriana</Emphasis> to <Emphasis Type="Italic">O. sativa</Emphasis> L. by asymmetric somatic hybridization

Asymmetric somatic hybrid plants were produced between cultivated rice (Oryza sativa L.) and wild species [O. meyeriana (Zoll. etMor, exSteud.)] with high resistance to rice bacterial blight. X-ray-irradiated protoplasts of the wild species were used as donor and chemically fused with iodoacetamide-inactivated protoplasts of rice cv. 02428 to produce hybrids. Seventy-two plants were regenerated from 623 calli based on metabolic complementation. The morphological characters of the plants closely resembled that of the rice. Simple sequence repeats were employed to identify their hybridity. Cytological analysis of root-tips revealed that their chromosome number varied in the range of 27--38. The somatic hybrids were inoculated with strains of Xanthamonas oryzae pv. oryzae at adult growth stage and demonstrated the resistance to bacterial blight introgression from the O. meyeriana.     

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.     

Analysis of F1 hybrid and BC1 monosomic alien addition line plants from Brassica oleracea × Sinapis alba by GISH

Sterile and semi-fertile F₁ plants were obtained by intergeneric sexual hybridization between paternal Brassica oleracea var. alboglabra (genome CC, 2n=18) and maternal Sinapis alba (genome SS, 2n=24), BC₁ plants were obtained by backcrossing between paternal B. oleracea and maternal semi-fertile F₁ plants. Genomic in situ hybridization (GISH) combined with dual-colour fluorescence in situ hybridization (dcFISH) showed that sterile F₁ plants contained 21 chromosomes consisting of one B. oleracea chromosome set and one S. alba chromosome set, belonging to expected hybrids, and semi-fertile F₁ plants contained 30 chromosomes consisting of two B. oleracea chromosome sets and one S. alba chromosome set. It is obvious that the semi-fertile F₁ plants belong to unexpected hybrids. 1―3 trivalents were detected at meiotic metaphase I of semi-fertile F₁ pollen mother cells (PMCs). Different separation ratios of S chromosomes were detected at anaphase I. A monosomic alien addition line (MAAL) was identified by GISH-dcFISH from BC₁ plants; it contained 19 chromosomes consisting of 18 C chromosomes and 1 S chromosome. At meiotic metaphase I, 9 divalents from B. oleracea and one univalent from S. alba could be detected. Sometimes, one putative C-S trivalent could also be detected. The achievement of B. oleracea-S. alba monosomic alien addition lines lays a foundation for gene introgression, location and cloning.     

普通小麦与玉米的体细胞杂交再生完整植株

从小麦济南177制备得到两种原生质体，一种来源于具有一定分化能力的愈伤组织但其分裂能力较低；一种来源于生长迅速的悬浮细胞但不能分化，将二种原生质体混合，共同作为受体与经过紫外线（UV）照射的玉米（Zea mays L)原生质体在PEG诱导下融合，培养后获得再生克隆并进一步分化为植株，而它们单独与玉米原生质体融合均不能再生植株，通过染色体、同工酶及5SrDNA分析及生长习性分析证明再生植株均为体细胞杂种。     

Functional characterization and mapping of two MADS box genes from peach （Prunus persica）

Previously an AGAMOUS gene homologue PpMADS4 and a FRUITFULL gene homologue PpMADS6 were isolated from peach （Prunus persica）, and both genes were shown to express in the developing floral and fruits. To gain insight into their function, the two genes were constitutively expressed in Arabidopsis thaliana and their effects on plant growth and floral organ development were studied in this work. The transgenic plants all displayed early flowering and conversion of inflorescence to floral meristem. However, the two genes had different effects on the floral organ structures in A. thaliana. The transgenic plants overexpressing PpMADS4 displayed homeotic conversion of floral organs, and particularly the perianth abscission was inhibited. The plants overexpressing PpMADS6 showed early flowering, produced higher number of carpels, petals, and stamens than nontransgenic plants, and pod shatter was prevented; significantly, the transgenic plants yielded more than one siliques from a single flower. A SSR molecular marker was developed for PpMADS4, and it was then assigned into the G5 linkage group of Prunus sp. Both PpMADS4 and PpMADS6 genes were located at the same region in the G5 linkage group. Our results showed the potential application of these two MADS box genes for crop and fruit tree improvement.     

Introduction of antisensewaxy gene into main parent lines ofindica hybrid rice

Amylose content in rice endosperm is one of the key determinants of rice eating and cooking quality, and the poor quality ofindica hybrid rice is closely related to the high amylose level in rice grains. In order to improve the grain quality of theindica hybrid rice by genetic engineering, an antisense fragment of ricewaxy gene, driven by the 5′-franking sequences of the ricewaxy gene, was successfully introduced into three major parent lines ofindica hybrid rice, all contain a high amylose level in the grains, viaAgrobacterium, and more than 100 hygromycinresistant plants were regenerated. The analysis of PCR amplification and Southern blots indicated that the T-DNA containing the antisensewaxy gene had been integrated into the genome of transgenic rice plants. Most of the primary transgenic rice plants grew normally, and the mature seeds from these transgenic plants were performed for analysis of the amylose content. The results showed that the amylose content in the endosperm of some grains was reduced and the lowest reached 7.02% in one homozygous transgenic line, 72.4% lower than that of the wild type. The influence of the altered amylose content on the gelatinization temperature and gel consistency was also observed in several homozygous transgenic rice plants. The two authors contributed equally to this work.     

Vegetation evolution on the central Chinese Loess Plateau since late Quaternary evidenced by elemental carbon isotopic composition

There are many controversial issues in loess studies such as natural vegetation types on the Chinese Loess Plateau during the historical periods and the spatial and temporal evolution of C3/C4 plants. Elemental carbon isotopic composition （δ^13Cec） in the loess section may offer new evidence for these problems. Elemental carbon （EC） is produced by incomplete combustion of vegetation, and its carbon isotopic composition has a very small difference from that of the formal vegetation, then δ^13Cec can be used as a record to recover the changes of vegetation. Elemental carbon was extracted by applying the oxidation method from the Ioess-paleosol sequence in the central Chinese Loess Plateau, and its car- bon isotope composition was analyzed by the isotope mass spectrometer. The results showed that the vegetation in this region was a mixed type of C3 and C4 plants, dominated with C3 plants in most of the time. Since late Quaternary, C3/C4 plants may not follow a simple glacial-interglacial cycle mode on the Chinese Loess Plateau, but showing fluctuations. C3 plants increased gradually in L4 period, and more C3 plants occurred during $3 period, and C4 plants increased again during L3-- L2 periods, after that, Cs plants dominated again during S1 --S0 periods. During periods of paleosol development, C3 plants were abundant in S3 and S1, and there were more Ca plants in S2 and SO. During periods of loess sedimen- tation, there were more C3 plants in L4 and L1, and there were more C4 plants in L3 and L2. On the orbital timescale, the vegetation variations revealed by δ^13Cec record are consistent with the results of pollen data and also similar to the results obtained by organic carbon isotopic composition since the last glacial period.     

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．     

单倍体小麦与簇毛麦的不对称体细胞杂交

以小麦杂２４２（Ｔｒｉｔｉｃｕｍａｅｓｔｉｖｕｍ,ｃｖ．２４２）花药来源的愈伤组织分离原生质体作受体;簇毛麦（Ｈａｙｎａｌｄｉａｖｉｌｏｓａ）幼胚来源的愈伤组织原生质体经２２０μＷ／ｃｍ２紫外线照射３０ｓ作供体,用ＰＥＧ法诱导融合,最后获得再生愈伤组织．对融合再生的４个最先长大的细胞系进行染色体,同工酶和ＲＡＰＤ分析．结果表明,这些细胞系均为体细胞杂种,ＲＡＰＤ技术可作为小麦体细胞杂种早期鉴定的有效方法     

Identification of a novel tillering dwarf mutant and fine mapping of the TDDL（T） gene in rice （Oryza sativa L.）

Rice plant architecture is an important agronomic trait that affects the grain yield. To understand the molecular mechanism that controls plant architecture, a tillering dwarf mutant with darker-green leaves derived from an indica cultivar IR64 treated with EMS is characterized. The mutant, designated as tddl（t）, is nonallelic to the known tiilering dwarf mutants. It is controlled by one recessive nuclear gene, TDDL（T）, and grouped into the dn-type dwarfism according to Takeda＇s definition. The dwarfism of the mutant is independent of gibberellic acid based on the analyses of two GA-mediated processes. The independence of brassinosteroid （BR） and naphthal-3-acetic acid （NAA） of the tddl（t） mutant, together with the decreased size of parenchyma cells in the vascular bundle, indicates that the TDDL（7） gene might participate in another hormone pathway. TDDL（T） is fine mapped within an 85.51 kb region on the long arm of rice chromosome 4, where 20 ORFs are predicted by RiceGAAS （http：//ricegaas.dna.affrc. go.jp/rgadb/）. Further cloning of TDDL（T） will benefit both marker assisted selection （MAS） of plant architecture and dissection of the molecular mechanism underlying tillering dwarf in rice.     

Development of Triticum aestivum-Leymus racemosus ditelosomic substitution line 7Lr#1S（7A） with resistance to wheat scab and its meiotic behavior analysis

Leymus racemosus is highly resistant to wheat scab （Fusarum head bright）. The transfer of scab resistant gene from L. racemosus to Triticum aestivum is of great significance for broadening the base of wheat resistance. In the present study, the pollen of T. aestivum-L, racemosus monosomic addition line with scab resistance was treated by irradiation with 1200 R ^60Co-γ-rays prior to pollinating to emasculated wheat cv. Mianyang 85-45. Nine plants with a telocentric chromosome 7Lr#1S were observed in M1, and one ditelosomic substitution line 7Lr#1S was selected from selfcrossing progenies and confirmed by chromosome C-banding and GISH. Furthermore, a co-dominant EST-SSR marker CINAU 31 was employed to identify this substitution line. A pair of chromosome 7A of common wheat were found to be replaced by a pair of telocentric chromosome 7Lr#1S, and further investigation showed that chromosome configuration of the substitution line at MI of PMCs after GISH was 17.50Ⅱ^w ＋ 2.19Ⅱ^w ＋ 0.42Ⅱ^7Lr#1S ＋ 1.08 Ⅰ^7Lr#1S ＋ 0.69 Ⅰ^w. Two telocentric chromosomes paired as a bivalent in 59.7% of PMCs. Abnormal chromosome behaviors of telocentric chromosomes were observed in part of PMCs at anaphase I and telophase I, including the moving of two telocentric chromosomes to the same pole, lagging and earlier separation of their sister chromatid. All these abnormal behaviors can be grouped into three distinct types of tetrads according to different numbers of 7Lr#1S in their daughter cells and various micronucleus in some tetrads. However, due to the high transmission frequency of the female and male gametes with a 7Lr#1S, 84% of the selfcrossing progeny plants had ditelosomic substitution. The substitution line showed high resistance to wheat scab in a successive two-year test both in the greenhouse and field; hence, the line will be particularly valuable for alien gene mapping, small fragment translocation induction and telosomic cytological behavior analysis.     

Inheritance of resistance toHelicoverpa armigera of 3 kinds of transgenicBt strains available in upland cotton in China

There are 3 kinds of transgenicBt strains, Shanxi 94-24, Zhongxin 94, and R19, in upland cotton in China. Their transgenicBt insect-resistance cultivars or hybrids have been developed and grown by farmers. Genetic studies indicate that the resistance of the 3 transgenicBt cotton strains toHelicoverpa armigera is controlled by one pair of non-allelic dominant genes. Linkage relationship between the resistant genes of R19 and Shanxi 94-24 transgenicBt strains shows that they may be inserted in the same chromosome. F₁ hybrids crossed among the 3 strains show that high levels of protection from feeding damage are the same as that of their parents. Therefore, there is no co-suppression phenomenon in many transgenic plants. The results presented here afford a fundamental reliance in developing transgenicBt insect-resistant cultivars and exploiting the heterosis of hybrids in upland cotton.     

Sequence analysis of the gene correlated with cytoplasmic male sterility (CMS) in rape-seed (Brassica napus) Polima and Shaan 2A

orf224 is a CMS-related mitochondrial gene discovered in Polima cytoplasm. Shaan 2A CMS line is the parent of the first rapeseed hybrid cultivar Qinyou No. 2 that has been grown in many regions of China. In this work, genomic DNA of Polima CMS line and Shaan 2A CMS line were used as templates, two primers of specific oligonucleotides at 5′ and 3′ ends were used, PCR was performed, the amplification fragments were cloned into pGEM-T Easy vectors and DNA sequences were determined. The CMS-associated gene, orf224-1 present in Shaan 2A CMS line, has a sequence highly homologous to the orf224 of the Polima CMS line, except for one nucleotide at position +398. There were only one base (AAC→AGC) and one amino acid (Asn →Ser) differences between the two. The homologies of the two sequences in nucleotide and amino acid were 99.9% and 99.6%, respectively. It is concluded that orf224 in Polima CMS line and orf224-1 of Shaan 2A CMS line are the allele at the same locus in mitochondria.     

Fine mapping of the red plant gene R1 in upland cotton(Gossypium hirsutum)

Sub 16 is a substitution line with G. hirsutum cv. TM-1 genetic background except that the 16th chromosome (Chr. 16) is replaced by the corresponding homozygous chromosome of G. barbadense cv. 3-79, and T586 is a G. hirsutum multiple gene marker line with 8 dominant mutation genes. The R ₁ gene for anthocyanin pigmentation was tagged in Chr. 16 in T586. The objective of this research was to screen SSR markers tightly linked with R ₁ by using the F₂ segregating population containing 1259 plants derived from the cross of Sub 16 and T586 and the backbone genetic linkage map from G. hirsutum×G. barbadense BC₁ newly updated by our laboratory. Genetic analysis suggested that the segregation ratio of red plants in the F₂ population fit Mendelian 1:2:1 inheritance, confirming that the red plant trait was controlled by an incomplete dominance gene. Preliminary mapping of R ₁ was conducted using 237 randomLy selected F₂ individuals and JoinMap v3.0 software. Then, a fine map of R1 was constructed using the F₂ segregating population containing 1259 plants, and R ₁ was located between NAU4956 and NAU6752, with only 0.49 cM to the nearest maker loci (NAU6752). These results provided a foundation for map-based cloning of R ₁ and further development of cotton cultivars with red fibers by transgenic technology. Supported by National Natural Science Foundation of China (Grant No. 30730067) and Programme of Introducing Talents of Discipline to Universities (Grant No. B08025)     

Identification of a novel DNA molecule associated with Tobacco leaf curl virus

A circular DNA molecule, designated as DNAβ, was identified in tobacco plants infected with Tobacco leaf curl virus (TLCV) isolates Y5 and Y8 by PCR using primers based on the conserved region of the two reported DNAβ sequences of whitefly-transmitted geminiviruses (WTGs). The complete nucleotide sequences of DNAβ of Y5 and Y8 (TLCV DNAβ) were determined. Y5 DNAβ comprises 1333 nucleotides encoding 8 predicted ORFs with 4 ORFs in virion-sense DNA and 4 ORFs in complementary-sense DNA; Y8 DNAβ consists of 1338 nucleotides encoding 7 predicted ORFs with 4 ORFs in virion-sense DNA and 3 ORFs in complementary-sense DNA. TLCV DNAβ has little sequence homology to DNA-A of TLCV., except that it shares conserved TAATATTAC loop sequence with TLCV DNA-A. Sequence comparison showed that Y5 DNAβ shared 85% sequence homology with Y8 DNAβ, and both Y5 DNAβ and Y8 DNAβ had relatively low sequence identity (51%–65%) with the reported DNAβ molecules associated with Ageratum yellow vein virus and Cotton leaf curl virus. The immunotrapping PCR and whitefly transmission tests showed that DNAβ molecule could be encapsidated in virus particle and transmitted by Bemisia tabaci. This is the first report of DNAβ associated with WTGs in China.     

秀丽线虫对根际益生菌P13和S3-1传播作用及对植物生长的影响

通过缓慢杀线实验、偏好性实验和共培养毒性实验分析了秀丽线虫(Caenorhabditis elegans)对荧光假单胞菌P13(Pseudomonas fluorescens P13)和解淀粉芽孢杆菌S3-1(Bacillus amyloliquefaciens S3-1)传播的可能性.通过显微观察与平板稀释涂布对秀丽线虫细菌携带作用进行了定性、定量分析,并对细菌—线虫—植物三者交互作用进行初步探究.结果发现,P13和S3-1对秀丽线虫的慢性致死率分别为12.12%和3.00%,每10 s身体弯曲次数分别为4.68和4.33.相对于尿嘧啶缺陷型大肠杆菌(OP50),线虫对P13和S3-1选择系数分别为0.13和0.52,P13、S3-1携带菌量分别为(4.02×103±47)和(9.67×102±22)CFU/条.携细菌线虫将细菌定向传播至植物根际,细菌定殖菌量为105CFU,有效促进植物生长发育.     

Cloned goats (Gapra hircus) from foetal fibroblast cell lines

Mammalian cloning has been one of the most active research topics in the world. Cloning within vitro culured foetal fibroblast cells, in comparison with embryonic cells, can be used not only to theoretically study the embryonic or cellular development and differentiation in mammals, but also to utilize the unlimited fibroblast cells to produce large numbers of clonings. The preliminary results are as follows: (i) The division and development of the cloned embryos with embryonic donor cells and goat foetal fibroblast donor cells were 55%, 77% and 35%, 31%, respectively. There is no significant statistical difference between them, (ii) These studies result in the birth of two cloned goats derived from two 30-day foetal fibroblast cell lines, which are the first cloned mammals from somatic cells in China. This project has established a technological data base for the furture research on adult mammalian somatic cloning and nucleocytoplasmic interactions in animal development, and a novel technique for the cloning of animals with a high-level expression of transgene(s).     

Genetic analysis and gene mapping of a narrow leaf mutant in rice ( Oryza sativa L.)

A narrow leaf mutant was obtained after T-DNA transformation conducted on a rice variety Zhonghua 11. Several abnormal morphological characteristics, including semi-dwarf, delayed flowering time, narrow and inward rolling leaves, and lower seed-setting, were observed. The rate of net photosynthesis (under saturate light) of flag leaves in the mutant was significantly lower than that of the wild type. Moreover, the leaf transpiration rate and stomatal conductance in the mutant flag leaf were lower than those of the wild type at the grain filling stage. It was found that the mutant phenotype was not caused by the T-DNA insertion. Genetic analysis showed that the mutant was controlled by a single recessive gene, designated as nal3(t). A genetic linkage map was constructed using a large F₂ mapping population derived from a cross between nal3(t) and an indica variety Longtefu B with 6 polymorphic markers on chromosome 12 identified from 366 SSR markers by the BAS method. Gene nal3(t) was mapped between the markers RM7018 and RM3331. Fine mapping of nal3(t) locus was conducted with 22 newly developed STS markers based on the sequence diversity around the region harboring nal3(t) between Nipponbare and 93–11, and nal3(t) was finally mapped to a 136-kb region between the STS markers NS10 and RH12-8. Supported by National High Technology Research and Development Program of China (863 Program) (Grant No. 2006AA10A102), National Natural Science Foundation of China (Grant No. 30600349) and Natural Science Foundation of Zhejiang Province (Grant No. Y306149)     

臭氧水对草莓及拟盘多毛孢影响的研究

通过组织分离纯化和分子生物学鉴定,明确了引起草莓叶部新病的致病菌为拟盘多毛孢。以喷洒清水为空白对照,利用不同质量浓度的臭氧水直接喷洒致病菌和生长期的草莓植株,研究臭氧水对致病菌和草莓植株的浓度效应,结果显示：低浓度臭氧水（0.5~0.8 mg·L^-1）对草莓植株的生理生态变化和致病菌的生长影响较小;中浓度臭氧水（2.2~2.5 mg·L^-1）可以显著抑制致病菌的生长,并促进草莓植株的生长;高浓度臭氧水（4.0~4.3 mg·L^-1）可以很好地抑制致病菌的生长,但对草莓叶片有较严重的腐蚀作用。因此,中浓度（2.2~2.5 mg·L^-1）是喷洒草莓的最适臭氧水浓度。