High Efficiency of Genetic Transformation of Rice Using Agrobacterium Mediated Procedure

Four japonica varieties and two indica varieties were used for the genetic transformation of rice （Oryza sativa L.） by using Agrobacterium tumefaciens (Smith et Townsend) Conn EHA101 harboring binary vector containing GUS gene and selectable marker gene of NPTⅡ and HPT. Calli derived from mature and immature embryos of rice were infected and cocultured with Agrobacterium at logarithmic phase. The highest transformation frequency was 55.1% (indica) and 85.2% (japonica) respectively according to the estimation of hygromycin resistant calli produced. The ratio of transgenic plants regenerated from the calli of indica and japonica varieties was 37.8% and 69.0% respectively. The putative transformed plants were confirmed by GUS assay, PCR analysis and Southern blotting. The segregation of foreign genes in T1 progeny corresponded to the Mendelian ratio. This transformation procedure of rice will provide an efficient model for the transformation of monocots.     

农杆菌介导的苏云金杆菌抗虫基因cryIA(b)和cryIA(c)在水稻中的遗传转化及蛋白表达

通过农杆菌介导法用含有抗潮霉素和 Ｇ Ｕ Ｓ 基因的双元载体将杀虫结晶蛋白基因ｃｒｙ Ｉ Ａ（ ｂ） 和ｃｒｙ Ｉ Ａ（ｃ） 导入到籼、粳稻幼穗愈伤组织中,然后经过在含有不同浓度潮霉素的培养基上进行数次筛选,获得一批 Ｂｔ 转基因株。经 Ｐ Ｃ Ｒ、 Ｓｏｕｔｈｅｒｎ 杂交及 Ｗｅｓｔｅｒｎ 印迹分析证实此二基因已整合进水稻中,饲虫试验结果表明,转基因株具有１００ ％ 杀虫率。     

A procedure for regenerating Japonica and Indica varieties of rice from protoplasts

Fertile rice plants have been regenerated from protoplasts of two japonica rice varieties (Radon and Baldo) using a protocol initially developed for plant regeneration from protoplasts of an indica rice. Embryogenic calli were developed from immature embryos of Radon and Baldo rice on a callus induction medium, and then used to establish cell suspensions. Protoplasts were isolated from the cell suspensions, and cultured on a Millipore filter placed on a Kao/agarose medium that contained cell clusters from suspensions of IR52 or IR45. The protoplasts grew vigorously on Kao medium and developed into embryogenic calli within two to three weeks. Somatic embryo development occurred during a subsequent transfer of the calli to an LS medium for two to three weeks. The calli were then transferred to MS or N6 plant regeneration medium, and within one to three weeks, plants regenerated from 21 to 32% of the Radon calli, and 33 to 35% of the Baldo calli. Based upon these results and the previous success in regenerating an indica variety from protoplasts, this procedure has great promise for regenerating a range of rice varieties, and probably for regeneration of other monocotyledonous plants from protoplasts     

提高农杆菌转化水稻频率的研究

以16种重要的籼稻和粳稻栽培品种为材料,研究了影响农杆菌转化水稻频率的有关因素,结果表明,CC培养基是绝大多数水稻全国组织的最适诱导与继代培养基;添加2．5－5mg/L ABA可以有效地改善水稻愈伤组织的质量,籼稻愈伤组织所需的筛选剂浓度低于粳稻愈伤组织所需的浓度,根癌农杆菌EHA105菌株对水稻的转化效果优于LBA4404和AGL1菌株的效果,头孢霉素对农杆菌的抑制效果优于羧苄青霉素的效果,共培养后进行适当的干燥处理既可增强脱菌效果,又可提高转化频率,应用我们所优化的农杆菌转化技术体系,获得了10个品种的水稻转基因植株。     

基于TAC载体的水稻转化系统的建立

将含有大约50kb水稻基因组片段的TAC17克隆(NK15)通过电击转化到农杆菌LBA4404中,经多次继代培养,该克隆在农杆菌中是稳定的。用常规的农杆菌介导方法将该克隆转化粳稻品种农垦58S成熟胚的愈伤组织,对T0代进行PCR和Southern杂交分析表明,TAC17所携带的50kb外源DNA片段已完整地整合到水稻基因组上,整合方式多数为单位点插入,整合位点是随机的。经T1代分析表明,外源基因可以稳定地遗传,而且进一步确定外源大片段的整合方式为为单位点插入。     

Agrobacterium tumefaciens－mediated transformation of rice with the spider insecticidal gene conferring resistance to leaffolder and striped stem borer

Immature embryos of rice varieties "Xiushuill" and "Chunjiang 11" precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticidal gene). The resistant calli were transferred onto the differentiation medium and plants were regenerated. The transformation frequency reached 56% approximately 72% measured as numbers of Geneticin (G418)-resistant calli produced and 36% approximately 60% measured as numbers of transgenic plants regenerated, respectively. PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome. Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder (Cnaphalocrasis medinalis) after 7d of leaf feeding reached 38% approximately 61% and the corrected mortality of the striped stem borer (Chilo suppressalis) after 7d of leaf feeding reached 16% approximately 75%. The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests.     

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens

A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. The explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the [beta]-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants in soil was 2.5 to 3 months. So far, more than 100 transgenic events have been produced. Almost all transformants were morphologically normal. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to five copies of the transgene were integrated into the wheat genome without rearrangement. Approximately 35% of the transgenic plants received a single copy of the transgenes based on Southern analysis of 26 events. Transgenes in T1 progeny segregated in a Mendelian fashion in most of the transgenic plants.     

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。     

Agrobacterium-mediated transformation of élite indica and japonica rice cultivars

A rapid, efficient, routine system has been established forAgrobacterium tumefaciens-mediated production of hundreds of fertile transgenic plants from commercially important rice cultivars, including an indica cultivar, Pusa Basmati 1. Calli induced from embryos of mature rice seeds were cocultivated withA. tumefaciens strain LBA4404 carrying the plasmid pTOK233, then exposed to hygromycin selection followed by an efficient regeneration system. Based on the total number of calli co-cultivated, the transformation frequencies of independent transgenic rice plants including cultivars Pusa Basmati 1, E-yi 105, E-wan 5 and Zhong-shu-wan-geng, were 13.5, 13.0, 9.1, and 9.3%, respectively. T1 seeds were harvested within 7–8 mo of initiation of mature embryo cultures. Data from Southern hybridization analysis proved that foreign genes on T-DNA were stably integrated into the rice genome at low copy/site numbers. Mendelian inheritance of the transgenes was confirmed in T1 progeny.     

农杆菌介导将白叶枯病抗性基因Xa21导入水稻获得转基因植株

利用农杆菌介导的高效遗传转化系统,将白叶枯病抗性基因Xa21转入黄淮稻区主栽品种豫粳6号的胚性愈伤组织,获得转基因植株,GUS染色和PCR分析证明Xa21基因已整合到水稻基因组中,其自交T1代植株经GUS染色和白叶枯病接种鉴定呈现3：1分离,研究为培育抗白叶枯病水稻品种奠定了基础。     

Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis

Brachypodium distachyon is a promising model system for the structural and functional genomics of temperate grasses because of its physical, genetic and genome attributes. The sequencing of the inbred line Bd21 ( http://www.brachypodium.org ) started in 2007. However, a transformation method remains to be developed for the community standard line Bd21. In this article, a facile, efficient and rapid transformation system for Bd21 is described using Agrobacterium -mediated transformation of compact embryogenic calli (CEC) derived from immature embryos. Key features of this system include: (i) the use of the green fluorescent protein (GFP) associated with hygromycin selection for rapid identification of transgenic calli and plants; (ii) the desiccation of CEC after inoculation with Agrobacterium ; (iii) the utilization of Bd21 plants regenerated from tissue culture as a source of immature embryos; (iv) the control of the duration of the selection process; and (v) the supplementation of culture media with CuSO₄ prior to and during the regeneration of transgenic plants. Approximately 17% of CEC produced transgenic plants, enabling the generation of hundreds of T-DNA insertion lines per experiment. GFP expression was observed in primary transformed Bd21 plants (T₀) and their progeny (T₁). The Mendelian inheritance of the transgenes was confirmed. An adaptor-anchor strategy was developed for efficient retrieval of flanking sequence tags (FSTs) of T-DNA inserts, and the resulting sequences are available in public databases. The production of T-DNA insertion lines and the retrieval of associated FSTs reported here for the reference inbred line Bd21 will facilitate large-scale functional genomics research in this model system.     

Transformation of rice mediated by Agrobacterium tumefaciens

Agrobacterium tumefaciens has been routinely utilized in gene transfer to dicotyledonous plants, but monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they were outside the host range of crown gall. Various challenges to infect monocotyledons including rice with Agrobacterium had been made in many laboratories, but the results were not conclusive until recently. Efficient transformation protocols mediated by Agrobacterium were reported for rice in 1994 and 1996. A key point in the protocols was the fact that tissues consisting of actively dividing, embryonic cells, such as immature embryos and calli induced from scutella, were co-cultivated with Agrobacterium in the presence of acetosyringonc, which is a potent inducer of the virulence genes. It is now clear that Agrobacterium is capable of transferring DNA to monocotyledons if tissues containing competent cells are infected. The studies of transformation of rice suggested that numerous factors including genotype of plants, types and ages of tissues inoculated, kind of vectors, strains of Agrobacterium, selection marker genes and selective agents, and various conditions of tissue culture, are of critical importance. Advantages of the Agrobacterium-mediated transformation in rice, like on dicotyledons, include the transfer of pieces of DNA with defined ends with minimal rearrangements, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes, and high quality and fertility of transgenic plants. Delivery of foreign DNA to rice plants via A. tumefaciens is a routine technique in a growing number of laboratories. This technique will allow the genetic improvement of diverse varieties of rice, as well as studies of many aspects of the molecular biology of rice.     

插入玉米Ds转座因子的水稻转化群体及其分子分析

转座子标签法是一种利用转座因子插入高等植物基因组中造成基因突变,然后通过分离转座因子插入的旁邻顺序,进而克隆出突变基因的策略。这种策略在高等植物功能基因组学的研究中是十分有用的。为此目的,将玉米的Ds因子及bar基因连接至载体pCAMBIA1300的T-DNA区域中,构建成重组Ti质粒pDsBar1300。pDsBar1300中T-DNA区域中的潮霉素抗性基因可在转化过程中用作水稻转化植株的选择标记。插入在Ds因子中的bar基因可追踪转化后代的Ds因子。pDsBar1300通过根瘤农杆茵介导引入水稻品种中花11号的幼胚组织。从各转化愈伤组织中获得了1400株独立的Ds水稻转化植株。通过PPT抗性检测和PCR分析证明了水稻转化植株中Ds因子的整合。Southernblot分析了转化植株基因组中Ds因子的插入拷贝数,其中单拷贝插入比率约占70％。这些插有Ds因子的水稻转化植株,当引入自主型的Ac因子反式活化Ds因子后,可使Ds因子跳跃到不同位点上,就可得到更多的突变植株。     

农杆菌介导将Bt杀虫蛋白基因导入优良玉米自交系的研究

以杂交育种中广泛使用的优良玉米自交系340、4112为材料,用带有质粒pGBIL04（Pactin-Bt-Tnos）的根癌农杆菌LBA4404转化其幼胚及其初始愈伤组织,共培养3天后,在含PPT的培养基上连续筛选培养3代,然后分化获得再生植株。PCR检测证明目的基因已整合到再生植株的基因组中。实验结果表明幼胚预培养后形成的新鲜的初始愈伤组织是比较适宜的转化受体,结果还发现将共培养温度降到22℃可以提高农杆菌介导的玉米遗传转化的筛选频率。 Abstract:Excellent inbred-lines of maize,340 and 4112,which were used largely in hybridized combination were transformed with Agrobacterium tumefaciens.The immature embryos and their original calli were infected by A.tumefaciens LBA4404 containing plasmid pGBIL04.After 3 days of co-cultivation,the immature embryos and calli were continuously selected on the medium containing phosphinothricin (PPT) for 3 generations,then plants were regenerated.It was proved by PCR analysis that the target Bt gene had been integrated into the genome of regenerated plants.The results showed that fresh original calli from the immature embryos after pre-culture were suitable acceptors.The results also showed that it could increase the frequency of selection by properly lowering the co-culture temperature to 22℃.     

Inheritance of Oryza sativa endornavirus in F1 and F2 hybrids between japonica and indica rice

We have found a 14 kbp double-stranded RNA (dsRNA) in many cultivars of japonica rice (Oryza sativa L.) but not in any cultivars of indica rice. This dsRNA is an RNA replicon with plasmid-like properties and is proposed to be a novel dsRNA virus, Oryza sativa endornavirus (OSV). Reciprocal crosses between the OSV-carrier japonica variety (Nipponbare) and the OSV-free indica variety (IR 26 or Kasalath) were performed to investigate whether OSV can be transmitted to F1 hybrids. When IR 26 and Nipponbare were used, efficient transmission of OSV from ova (93%) and pollen (89%) was observed. When Kasalath and Nipponbare were used, the OSV transmission efficiency to F1 progeny was 68% from ova and 20% from pollen. The transmission of OSV to F2 progeny plants was also complicated, showing non-Mendelian inheritance. These results suggest that the dsRNA replicon (OSV) is unstable in indica rice plants.     

根癌农杆菌介导Bt基因转化水稻的研究

为了培育出无筛选标记基因的转基因水稻,试验将loxp-hpt-loxp基因与成基因连锁在-起转化水稻方法,得到loxp-hpt—loxp—Bt转基因水稻植株,再与同质的带有ere基因的水稻杂交,以定向删除潮霉素抗性筛选标记。试验表明以水稻品种“皖粳97”为供试材料,将成熟胚来源的愈伤组织用根癌农杆菌EHA105／pCAMBIA1305．1感染后,筛选出抗性愈伤组织并获得再生植株。经PCR验证,得到20棵转基因水稻植株。     

Genetic manipulation and quantitative-trait loci mapping for nitrogen recycling in rice

Immunocytological studies in this laboratory have suggested that NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in developing organs of rice (Oryza sativa L. cv. Sasanishiki) is involved in the utilization of glutamine remobilized from senescing organs through the phloem. Because most of the indica cultivars contained less NADH-GOGAT in their sink organs than japonica cultivars, over-expression of NADH-GOGAT gene from japonica rice was investigated using Kasalath, an indica cultivar. Several T0 transgenic Kasalath lines over-producing NADH-GOGAT under the control of a NADH-GOGAT promoter of Sasanishiki, a japonica rice, showed an increase in grain weight (80% as a maximum), indicating that NADH-GOGAT is indeed a key step for nitrogen utilization and grain filling in rice. A genetic approach using 98 backcross-inbred lines (BC(1)F(6)) developed between Nipponbare (a japonica rice) and Kasalath were employed to detect putative quantitative trait loci (QTLs) associated with the contents of cytosolic glutamine synthetase (GS1; EC 6.3.1.2), which is probably involved in the export of nitrogen from senescing organs and those of NADH-GOGAT. Immunoblotting analyses showed transgressive segregations toward lower or greater contents of these enzyme proteins in these BC(1)F(6). Seven chromosomal QTL regions were detected for GS1 protein content and six for NADH-GOGAT. Some of these QTLs were located in QTL regions for various biochemical and agronomic traits affected by nitrogen recycling. The relationships between the genetic variability of complex agronomic traits and traits for these two enzymes are discussed.     

农杆菌介导的小麦遗传转化几个影响因素的研究

采用携带gus和（或）bar基因双元表达载体（p3301,pBTAaB)的3个根癌农杆菌（Agrobacterium tumefaciens)菌株（AGL-1,EHA105和LBA4404）对普通小麦（Triticum aestivumL.)冬性栽培品种农大170和农大146的幼胚及幼胚愈伤组织进行了遗传转化,结果表明,菌液浓度OD6001.0和侵染时间1h对外植体的生存和转化最为有利;侵染前对外植体进行高渗处理较明显地提高了抗性愈伤获得率;乙酰丁香酮（AS）对小麦转化的作用随菌株和外植体的不同而异;菌株/质粒组合,受体基因型及外植体的类型,年龄和生理状态对转化效率有很大的影响,条件优化后,得到大量具有PPT抗性的愈伤和一些抗性植株,抗性愈伤的GUS染色阳性率在50%-60%之间,所检测的抗性苗呈GUS阳性,对6株抗性苗的PCR和Southern检测初步证明,外源基因已经整合到其中3株的基因组中。     

转基因水稻胚乳中表达铁结合蛋白提高稻米铁含量

为提高我国稻米的铁含量,通过农杆菌介导将自行克隆的菜豆(Phaseolus limensis)铁结合蛋白(Ferritin)基因导入了一个高产粳稻(Oryaz sativa L.ssp．japonuica)品种中,获得17个独立的转基因水稻株系。分子检测证明,外源基因在多数转基因水稻植株基因组中有1～3个整合位点,并可稳定遗传。在水稻种子贮存蛋白谷蛋白基因GluB-1启动子的控制下,铁结合蛋白基因可在转基因水稻的种子中高效特异地表达,不同转化子中的表达量有明显不同。在转基因水稻种子中表达铁结合蛋白后对提高精米中的铁含量有明显的效果,相对于未转化对照最多可提高64％,而锌的含量并无明显变化。     

Partial desiccation of mature embryo-derived calli,a simple treatment that dramatically enhances the regeneration ability of indica rice

Regeneration of indica rice varieties remains a limiting factor for researchers undertaking rice Iransformation experiments. As reported for japonica rice and other crops, partial desiccation of indica rice calli dramatically promotes organogenesis and leads to high regeneration ability. We are now able to obtain 66.5%, 61.1% and 73.7% of calli that regenerate plants for the indica varieties TN1, IR72 and IR64 whereas in non desiccated controls only 30.0%, 15.5% and 18.7% of calli regenerated, respectively. Plants obtained were phenotypically normal and 50% were highly fertile. Partial desiccation is a reliable and simple method for improving indica rice regeneration. It also shortens the time of in vitro culture.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-Benzyl amino purine - DTT Dithiothreitol - EDTA Ethylene diamine tetra-acetic acid - MS Murashige and Skoog - NAA Naphtalene acetic acid - PAGE Polyacrylamide gel electrophoresis - PAR Photosynthetic active radiation - SDS Sodium dodecil sulfate