多个抗虫基因转化水稻两用系培矮-64S

应用基因枪法对水稻两用系培矮-64S进行了转化。质粒载体pKC-3串联了三个抗虫基因,将其转化成熟胚诱导形成的愈伤组织,共获得33株转基因植株。分别对R_0代植株不同的基因进行PCR及Southern blot分析,并对R_1代植株进行了PCR分析。结果表明,三个抗虫基因均已整合到水稻基因组并获得稳定遗传。     

水稻白叶枯病广谱抗性基因Xa21导入两用不育系培矮64S

以克隆的Xa21基因为外源基因,成熟胚愈伤组织为转化受体,应用农杆菌介导法对水稻两用型核不育系培矮64S进行转化,获46株转基因植株。PCR和Southern分析结果表明,Xa21已整合到受体基因组。用稻白叶枯病病原菌(Xanthomonasoryzaepv.oryzae)菲律宾小种6号接种鉴定,结果表明大多数转基因植株获得了抗病性。已整合的Xa21基因能够稳定地遗传,在所检测转基因株系的T1代中,Xa21基因显示3:1的分离。     

不育系培矮64S衍生的近等基因系的育性感温性及基因初定位研究

培矮64S是光敏核不育水稻农垦58S衍生出的应用面积最大、不育临界温度最低的不育系.但其不育临界温度高于温敏核不育系株1S,杂交制种也没有株1S安全.为了探究培矮64S育性感温的机制,选育不育临界温度更低、杂交制种更安全的不育系,本研究以培矮64S为母本与丰源B杂交,再以杂交后代的不育株与丰源B回交两次制备近等基因系,...     

温度对双低两用核不育水稻96-5-2S与培矮64S育性的影响

在自然变温、人工控温及冷水灌溉条件下,比较研究了温度对双低两用核不育水稻96－5－2S与两用核不育水稻培矮64S育性影响的差异。结果表明：（1）当它们在雄性育性转换温敏感期1－12d平均自然日均温23.0-23.8℃的低温时,96－5－2S表现不良,套袋自交结实率为0,而培矮46S可育,套袋自交结实率为0．1％－4．5％;（2）在它们雄性育性转换温敏感期用22℃恒温处理5d,96－5－2S败育彻底,套袋自交结实率为0,而培矮64S可育,套袋自交结实率为10．7％;用17℃恒温处理6d,96－5－2S与培矮64S均可育,但96－5－2S套袋自交结实率（6．8％）显著高于培矮64S（2．5％）;（3）在它们雄性育性转换温和不同温度的冷水串灌15d,水深维持在20cm左右,当水温为22－22．5℃时,96－5－2S不育,结实率为0,而培矮64S可育,结实率为18．5％;当水温为19．5－21．5℃时,96－5－2S与培矮64S均可育,但96－5－2S结实率（2．5％－45．1％）显著或极显著低于培矮64S（50．4％－56．9％）以上结果说明：导致双低两用核不育水稻96－5－2S雄性不育的起点温度与导致其生理不育的下限温度均低,其不育性比培矮64S更稳定,耐寒性比培矮64S更强,即可确保制种安全,又可确保自身繁殖,对加快两系法杂交水稻的发展步伐将起到重要的促进作用。     

水稻IR36及光温敏核雄性不育系培矮64S小孢子母细胞减数分裂期间微管骨架的变化

对水稻（Oryza sativa L.)IR36及光温敏核雄性不育系培矮64S小孢子母细胞减数分裂期间微管骨架的变化的研究显示,微管在小孢子母细胞正常发育的情况下,每一个发育阶段（即造孢细胞时期、细线期、偶线期、粗线期、双线期、终变期、中期Ⅰ、后期Ⅰ）都出现不同的组织结构形态和分布。在IR36中,还发现了一些新的和特殊的 管组织结构和分布。主要包括：（1）在细线期,从核膜向胞质射的微管呈螺旋状,显示核具旋转功能;（2）在偶线期,微管的分布呈极性分布现象;（3）在终变期,核被一组微管形成的宽带围绕着（perinuclear broad band）。而培矮64S小孢子母细胞分裂期间,各发育阶段的细胞内的;管管骨架结构呈现许多不正常现象。如：（1）在偶线期具极性分布的微管分布不存在;（2）在小孢子母细胞减数分裂期间的细胞内出现许多特别粗的微管束;（3）在终变期围绕核的微管宽带结构松散及内含微管密度低。由于微管骨架的分布变化可以使人们较早地在雄性不育细胞内看到败育现象,故此微管的分布和构型变化应可作为一个早期确立雄性不育现象的视觉标记(visual marker）或形态指标予以利用。     

早籼稻培矮64S愈伤组织形态及植株再生

研究了早籼稻品种培矮64S种子胚愈伤组织诱导的再生的条件。调节培养基中的2,4－D,KT及NAA等激素浓度,胚性愈伤组织诱导频率可达到48．5％,愈伤组织学再生频率接近60％。结果表明,合适的激素浓度可显著提高籼稻组织培养的效率。     

水稻两用核不育系培矮64S花药培养条件的研究

研究了水稻培矮６４Ｓ花药培养的几个条件,认为低温预处理天数以７～８ｄ为宜,诱导愈伤组织的培养基宜采用Ｎ６或ＳＫ３．     

花药培养对籼稻两用核不育系培矮64S的提纯与改良

以籼稻两用雄性核不育系（简称两用系）培矮６４Ｓ为供体,通过花药培养,获得了２６个愈伤系共１９６９株花粉植株。根据花粉一代（Ｈ１）的倍性和育性,可把它们分为三种类型,即ａ）单倍体：有１个愈伤系４１株全为单倍体植株;ｂ）不分离型：有１９个愈伤系的群体育性不分离,全部植株均在不育期表现败育,其它性状有差异但不显著;ｃ）分离型：其余６个愈伤系表现分离,其中３个各有２－７株单倍体植株出现,并且这些愈伤系中均     

培矮64S中不育临界温度低的新株系筛选

在自然与控温条件下,研究了培矮６４Ｓ育性对温度反应的个体差异,从中筛选出１个不育临界温度低的新株系,即９６－５－２Ｓ．在此株系育性转换敏感期内,用２２℃恒温处理,结实率为０．０％;而用１８．５℃的冷水串灌处理,结实率为５７．６％     

修饰的cry1Ac基因导入籼稻明恢81获得抗虫纯合系

采用细胞内定位技术对cryl Ac基因进行修饰,其表达产物定位于内质网及衍生自内质网的蛋白体。通过基因枪法成功地通过修饰的cryl Ac基因导入到优良杂交籼稻有恢81中。对潮霉素抗性植株的PCR和Southern检测及ELISA分析证实,修饰的cryl Ac基因已整合到受体水稻品种中并得以表达,自交加代结合潮霉素筛选于T2代获得转基因纯合系。抗虫性测试表明,部分转基因纯合系高抗二化螟(Chilo suppressalis)。     

DNA methylation changes in photoperiod-thermo-sensitive male sterile rice PA64S under two different conditions

Epigenetic modification can occur at a high frequency in crop plants and might generate phenotypic variation without changes in DNA sequences. DNA methylation is an important epigenetic modification that may contribute to environmentally-induced phenotypic variations by regulating gene expression. Rice Photoperiod-Thermo-Sensitive Genic Male Sterile (PTGMS) lines can transform from sterility to fertility under lower temperatures and short-day (SD) conditions during anther development. So far, little is known about the DNA methylation variation of PTGMS throughout the genome in rice. In this study, we investigated DNA cytosine methylation alterations in the young panicles of PTGMS line PA64S under two different conditions using methylation sensitive amplified polymorphism (MSAP) method. Compared with the DNA methylation level of PA64S under lower temperatures and SD conditions (fertility), higher methylation was observed in PA64S (sterility). The sequences of 25 differentially amplified fragments were successfully obtained and annotated. Three methylated fragments, which are homologous to D2, NAD7 and psaA, were confirmed by bisulfite sequencing and their expression levels were also evaluated by qPCR. Real time quantitative PCR analysis revealed that five of the six selected methylated genes were downregulated in PA64S (sterility). These results suggested that DNA methylation may be involved in the sterility–fertility transition of PA64S under two different environmental conditions.     

影响籼稻基因枪法遗传转化效果的几个因素的研究（简报）

Four factors influence on transformation of indica rice, which were high osmotic treatment; different explant as the target tissue; pressure of rupture disk and quantity of plasmid DNA, were investigated in this experiment. High osmotic treatment of target tissue prior to and after bombardment increased 3.2-fold for Gus transient expression than control. The best treatment of high osmotic was that the target tissues were kept in the target-bed medium which contained 0.4-0.6 mol/L sorbitol and manitol each for 4 h prior to bombardment and for 16 h after bombardment. Four explants: scutellum from mature seed, young panicle, embryogenic callus and suspension cells of indica rice were tested as target explant by particle bombardment. The results of Gus transient showed that the highest expression was scutellum and for other three explants, the order from high to low was young panicle, embryogenic callus and suspension cell. Transgenic plants were obtained from all of the explants except young panicle. For the pressure of rupture disk on transformation, 1100 psi or 1300 psi of the pressure of rupture disk were best one for the transformation and higher than 1300 psi could damage the target tissue which become black and died in the following culture duration. For the quantity of plasmid DNA, the results showed that 0.83 microgram of plasmid DNA per bombardment was preferred for the transformation of indica rice.     

Conversion of a rice CMS maintainer into a photo- or thermo-sensitive genetic male sterile line

A maintainer line of 3-line hybrid rice commonly presents a certain genetic distance to a 2-line restorer line, but in many cases, 2-line restorer lines present defects upon recovery of the object cytoplasmic male sterile (CMS) line of the maintainer line, which impedes the utilization of their heterosis. Here, we report a strategy and an example of converting a maintainer into a photoperiod/temperature-sensitive genic male sterile (P/TGMS) line with an almost identical genetic background, thus maximizing the heterosis. Firstly, through treatment of maintainer line T98B with ⁶⁰C_O-γ irradiation, we identified the TGMS line T98S, which is sterile at higher temperatures and fertile at lower temperatures. Secondly, the T98S line was proven to be identical to T98B with regard to genetic background via an examination of 48 parental polymorphous SSR markers and exhibited excellent blossom traits similar to those of T98B, with an extensive forenoon flowering rate of 75.92% and a high exertion rate of 64.59%. Thirdly, in a combination test, three out of six hybrids from T98S crossed with 2-line restorer lines showed a yield increase of 6.70–15.69% for 2 consecutive years. These results demonstrated that the strategy can generate a new P/TGMS line with strong general combining ability (converted from a maintainer line), thus helping to increase the genetic diversity of male sterile heterotic groups.     

Transgenic roots produced by introducing Ri-rol genes into cucumber cotyledons by particle bombardment

Transformed roots ofCucumis sativus were obtained from cotyledon tissues that had been bombarded with gold particles coated with plasmid pE7.4 using a pneumatic particle gun. This plasmid containsrolA, rolB, rolC genes and ORF 13 of the 7.4 kbEco RI fragment of T-DNA of pRi 1724 isolated fromAgrobacterium rhizogenes MAF 03-01724. The nature of the tissue and the composition of the culture media used greatly influenced the recovery of transformed roots. The transgenic nature of the derived roots was confirmed by the vigorous. highly-branched growth seen on a phytohormone-free medium. The stable integration ofrol genes into the cucumber genome was confirmed by Southern blot analysis.     

培矮64S/爪哇稻F1农艺性状表现和对照优势分析

本文报道了利用原始爪哇稻资源与光温敏雄性不育系培矮64S配制的27个籼爪交组合在长沙的农艺性状和杂种优势表现.从总体上来看,籼爪交组合与对照相比在每穗实粒数和理论产量上无显著差异,在其它性状上均有极显著的差异;籼爪交组合在秆高、穗长、每穗总粒数、每穗实粒数和千粒重方面有正向对照优势,在播始历期、有效分蘖数、结实率、理论产量和实际产量上存在负向对照优势.从个体上来看,籼爪交组合理论产量对照优势＞40%的比例为11.1%.实际产量对照优势＞40%的机率为3.7%,说明爪哇稻资源在籼爪交杂种优势利用中具有利用价值.本文还对爪哇稻资源在籼爪杂种优势利用中的一些问题进行了讨论.     

Optimization of particle bombardment parameters for DNA delivery into the male flowers of banana

The possibility of increasing the efficiency of banana transformation was investigated by particle bombardment of the male flowers of banana plants for constitutive expression of gfp gene. The effects of particle bombardment parameters, such as acceleration pressure, bombardment distance, chamber vacuum pressure, gold microcarrier size, gold quantity, DNA quantity, number of bombardments and pre-culture were examined. Single cauliflower-like bodies (CLBs) clusters, induced from meristemic parts of Musa sapientum cv. Nangka (AAB) male flowers, were bombarded by pCambia1304 plasmid carrying gfp gene driven by the CaMV 35S promoter. Optimal transient expression of green-fluorescent protein (GFP) was obtained when the three-day old cultured tissues were bombarded two times at 1100 psi helium pressure. However, the highest GFP expression was observed when 9 cm was applied as bombardment distance with 28 mmHg chamber vacuum pressure. Gold particle with 1 μm diameter at 60 μg/μL concentrations coated with 1.5 μg/μL of DNA have been used as the optimum bombardment parameter since GFP expression was significantly different compared to other conditions. Application of optimized condition proved effective for the generation of stable transgenic banana plants. PCR and southern blot analyses confirmed the presence and integration of gfp gene in genomic DNA of transformed plants. Transformation frequency achieved with the optimized protocol was 7.5% which was significantly higher than the conventional protocol.     

Transient expression of foreign genes in rice,wheat and soybean cells following particle bombardment

The development of an efficient transformation system is a prerequisite for the molecular analysis of gene expression in plants. In crop plants, this development has been hindered by difficulties encountered both in whole plant regeneration from protoplasts and in the general insusceptibility of monocots to Agrobacterium-mediated transformation. We have circumvented these difficulties by transferring foreign genes directly into the intact cells (with cell walls) of three important crop plants including rice, wheat and soybean by a particle bombardment device. Oryza sativa and Triticum monococcum cells were bombarded with accelerated tungsten particles coated with plasmids containing a -glucuronidase gene as the reporter. Blue transformed cells were detected in an in situ enzyme assay. The number of blue cells was next used as a convenient criterion to study several factors affecting gene transfer efficiency. After optimal conditions were defined, gene transfer into intact cells of O. sativa, T. monococcum and Glycine max was successfully carried out with chloramphenicol acetyltransferase (CAT) gene as the reporter.     

Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.