萝卜与苤蓝属间杂种胚离体培养…

以萝卜雄性不育系DH303A为母本,以苤蓝为父本进行属间远缘杂交,萝卜与苤蓝杂交的不亲和性发生在胚珠发育过程中,杂交荚荚长快速生长期在授粉后的第2~5天,而横径快速生长期在授粉后第2~4天。在杂种胚发育后第6天和第7天进行胚抢救,用诱导培养基:MS+2,4-D 2.0 mg/L+KT 0.5 mg/L+蔗糖3%+琼脂0.6%;继代培养基:MS+2,4-D 2.0 mg/L+KT 0.5 mg/L+蔗糖3%+琼脂0.6%;分化培养基:MS+IAA 0.5 mg/L+KT 1.5 mg/L+蔗糖3%+琼脂0.6%;生根培养基:MS+NAA 0.2 mg/L+IAA 0.5 mg/L+蔗糖3%+琼脂0.6%,能成功获得幼苗。     

猪附红细胞体PCR-微孔板杂交-酶免疫检测方法的研究

以猪附红细胞体和多种血营养菌16S rRNA基因的保守区设计合成引物HM-f/HM-r,在反向引物的5′端用生物素标记,以猪附红细胞体广东株16S rRNA基因的可变区序列设计种特异性寡核苷酸探针,探针的5′端用地高辛标记,成功地建立了猪附红细胞体PCR-微孔板杂交-酶免疫检测方法。通过测定不同浓度PCR产物对应的微孔板杂交-酶反应显色OD值,用SPSS统计软件进行回归分析,得到回归方程为y=2.1012-0.4492×logx,其相关系数R为0.977,显示对未知样品可进行定量检测。该方法与猪的多种细菌性病原、猫血巴尔通氏体CA株和E.wenyonii无交叉反应,其敏感性比常规PCR琼脂糖电泳检测提高了近100倍。用建立的方法对38份临床样品进行检测,结果有12份检测结果为阳性。     

A new approach to evaluate relative resistance and tolerance of tomato cultivars to begomoviruses causing the tomato yellow leaf curl disease in Spain

A simple procedure to evaluate relative resistance and tolerance of tomato cultivars to the begomoviruses causing tomato yellow leaf curl (TYLC) disease in Spain was developed. To estimate the resistance and tolerance levels of a cultivar, several formulae were developed based on the ratio of infected plants, virus titre (estimated by tissue–print hybridization) and symptom intensity. The formulae were applied to five commercial tomato cultivars (Amoretto, Birloque, Royesta, Tovigreen and Ulises) naturally infected by TYLC viruses. The analyses showed that Ulises, Birloque and Tovigreen exhibited a moderate resistance, and Ulises was also highly tolerant. There was a positive correlation between symptom intensity and virus titre in infected plants, suggesting that the hybridization technique could also be used as an early estimator of tolerance. Finally, molecular hybridization and nucleotide sequence analyses of the begomovirus intergenic region showed that the local TYLC virus population consisted of a single species, Tomato yellow leaf curl virus (TYLCV, formerly TYLCV-Israel), with low genetic variation (nucleotide identity between isolates higher than 97%).     

普通西瓜3个变种45S rDNA和5S rDNA的染色体定位

【目的】为了探明普通西瓜种(Citrullus lanatus)3个变种间的亲缘关系和进化与驯化过程,【方法】利用双色荧光原位杂交技术(fluorescence in situ hybridization,FISH),将核糖体45S rDNA和5S rDNA在11份栽培变种(Citrulluslanatus subsp.vulgaris)、2份Egusi变种(C.lanatus subsp.mucosospermus)和3份Citroides变种(C.lanatus subsp.lana-tus)有丝分裂中期染色体上进行了定位研究。11份栽培变种西瓜和2份Egusi类型西瓜都含有2对45S rDNA位点和1对5S rDNA位点,3份Citroides变种都含有1对45S rDNA位点和2对5S rDNA位点。【结果】结果表明,普通西瓜种3个变种内部各基因型间具有相同的45S rDNA和5S rDNA分布模式,栽培变种和Egusi变种具有相同的分布模式,但它们均与Citroides变种存在明显差异,说明栽培变种与Egusi变种之间的亲缘关系更近一些。【结论】rDNA序列分布分析为在分子细胞学水平开展栽培西瓜的进化与驯化分析提供了证据。     

采用胚挽救获得Ogura CMS甘蓝与甘蓝型油菜的种间杂种

为将甘蓝型油菜细胞质雄性不育系(Ogura CMS)的恢复基因转至甘蓝Ogura CMS材料上,以6份甘蓝Ogura CMS材料为母本,以含恢复基因的甘蓝型油菜为父本,人工杂交结合胚挽救培养,研究取材时期、培养基成分和杂交组合对胚珠成苗的影响,同时对胚挽救培养获得的植株是否为真实F1杂种进行鉴定。结果发现,胚珠培养以剥蕾授粉后16d取材时胚珠成苗数最多,成苗率为4.56%;培养基以MS+GA 0.1 mg·L^-1+NAA 0.1 mg·L^-1+0.5%水解酪蛋白(CH)+0.5%活性炭(AC)成苗效果最好,成苗率高达6.24%;M09CMS×RFO-46组合成苗数最多,成苗率为5.96%。67株胚挽救培养得到的植株经流式细胞仪、SSR分子标记和形态学鉴定,得出65株为真杂种,真杂种率高达97%。     

Barriers to hybridization of Solanum bulbocastanum Dun. and S. Verrucosum Schlechtd. and structural hybridity in their F1 plants

Summary Results of reciprocal crosses between Solanum verrucosum (2n=2x=24) and S. bulbocastanum (2n=2x=24) are described in terms of pollen tube behaviour in styles, of berry and seed set, of fertility and of meiotic behaviour of the F₁ hybrids. Pollen tube growth of S. verrucosum is strongly inhibited in styles of S. bulbocastanum, whereas no inhibition is observed in the reciprocal cross. Therefore S. bulbocastanum x S. verrucosum fails to set berries or seeds, whereas the reciprocal cross produces both berries (54.4% berry set) and seeds (0.3 per berry). Only 14.6% of the seeds germinate. Both the diploid and corresponding tetraploid hybrid plants are vigorous, flower abundantly, have a rather regular meiosis (mainly rod bivalents), but show a high degree of cytoplasmic-genic male sterility. Crossability of the diploid hybrid plants is nil when used as pollen parents and near to zero when used as pistillate parents. The barriers to hybridization of the parent species (unilateral inhibition of pollen tube growth, somatoplastic sterility, cytoplasmic-genic male sterility and structural differences of the parental chromosomes) are discussed and methods are suggested to overcome these barriers.     

Association of a Geminivirus with a Leaf Curl Disease of Sunn Hemp (Crotalaria juncea) in India

Natural occurrence of a geminivirus causing severe leaf curl disease on sunn hemp (Crotalaria juncea) was recorded in India. The association of a geminivirus with the disease was demonstrated by whitefly transmission tests and polymerase chain reaction (PCR) amplification of DNA fragments of expected sizes with three pairs of degenerate geminivirus primers. The PCR-amplified viral DNA fragments were further characterized by Southern hybridization with a geminivirus probe consisting of the cloned coat protein (CP) gene of Indian tomato leaf curl virus (ITLCV). Restriction fragment length polymorphism analysis of a PCR-amplified CP fragment revealed that the geminivirus from sunn hemp was different than ITLCV.     

Characterization of Elicitor-inducible Tobacco Genes Isolated by Differential Hybridization

Inducible responses in plants against pathogen attack play a major role in resistance to disease. The defense responses are mostly associated with the expression of various kinds of inducible genes. We employed differential hybridization to isolate elicitor-inducible genes (EIGs) of tobacco (Nicotiana tabacum cv. Samsun NN) using the tobacco-fungal elicitor system. A cDNA library was constructed from tobacco leaves treated for 12 hr with hyphal wall components (HWC) prepared from Phytophthora infestans, and six EIGs were identified. Expression of all EIGs was induced after inoculation with the soybean pathogen Pseudomonas syringae pv. glycinea (nonpathogenic on tobacco) or treatment with salicylic acid, and a variety of expression patterns of EIG mRNAs was observed. Sequence analysis of EIG cDNAs revealed similarities to genes for SAR8.2 (EIG-B39 and EIG-D14), glycine-rich protein (EIG-G7), extensin (EIG-I30), acyltransferase (EIG-I24) and unknown protein (EIG-J7). Possible roles of EIG products in disease resistance are discussed. Received 30 August 2000/ Accepted in revised form 30 November 2000     

Induction of interspecific allo-tetraploids of Megalobrama amblycephala♀×Megalobrama terminalis♂ by heat shock

Interspecific allo-tetraploidy was induced by a combination of hybridization of Megalobrama amblycephala ♀× Megalobrama terminalis ♂ and thermal shock. A thermal shock at 40 °C for 2 min applied to eggs 33 or 26 min (τ₀: 1.40) after fertilization resulted in a significantly ( P <0.01) higher yield of allo-tetraploids than hybrid crosses without heat shock. The yield of allo-tetraploids in embryos from mid-gastrula to hatching stages was 15.7–24.4% in different thermal shock treatment groups. The allo-tetraploidy rate in 330-day-old juveniles was 9.3% and 6.7% in different year treatment groups. After 2 years of rearing, allo-tetraploidy males attained sexual maturity with milk-like milt at the age of 2+, while allo-tetraploidy females at the age of 2+, and even at 3+, did not show any signs of sexual maturation exhibiting obviously delayed maturity. Only a few allo-tetraploidy females (three individuals) showed maturation characters at the age of 4+ and these females were induced to spawn.     

Identification of differentially expressed genes associated with bud dormancy release in tree peony （Paeonia suffruticosa） by suppression subtractive hybridization

A subtractive cDNA library was developed to study genes associated with bud dormancy release in tree peonies.In order to identify genes that are highly expressed in buds released from dormancy,588 clones were examined by differential screening.Of these,185 clones were selected to be sequenced.A total of 37 unique sequences were obtained of which only 31 sequences have matches in the NCBI database or the Arabidopsis thaliana protein database.Semi-quantitative RT-PCR was used to confirm further the expression...