利用gfp报告基因优化农杆菌介导的水稻遗传转化

为提高农杆菌介导的水稻遗传转化效率,以晚粳97为转化材料,绿色荧光蛋白gfp基因为报告基因,采用正交试验L9(33)对影响农杆菌介导水稻的遗传转化因子进行优化。通过观察愈伤组织荧光表达情况,分析菌液浓度、共培养温度与共培养时间对农杆菌转化水稻的影响。结果表明,在OD660值为0.1、共培养21℃～23℃黑暗条件下,农杆菌与水稻愈伤共培养72 h,最有利于水稻的遗传转化,该条件下晚粳97愈伤组织荧光表达率达到70.9%。     

Genetic transformation of Linum by particle bombardment

Summary Linseed flax (Linum usitatissimum L.) was transformed by bombarding hypocotyl tissues with gold particles coated with plasmid DNA carrying the β-glucuronidase (GUS) (uid-A) and neomycin phosphotransferase II (npt-II) genes. Transient expression of the introduced β-glucuronidase gene was used to study factors influencing the DNA delivery, while progeny analyses confirmed stable transformation. The efficiency of DNA delivery, uptake and expression was significantly affected by the duration of hypocotyl preculture, bombardment distances, the level of chamber vacuum, the quantity of DNA, and the size of particles. Nineteen independent GUS-positive shoots were recovered and regenerated into whole plants, from which 10 plants successfully produced viable seeds. Analysis of T₁ and T₂ self pollinated progeny for histochemical and fluorometric GUS assays and polymerase chain reaction (PCR) analyses for uid-A, plus npt-II PCR and germination assays in progeny plants demonstrated that the transgenes were expressed in selected plants and transmitted to progeny, usually via a single Mendelian locus. The results show that particle bombardment can be used to produce transgenic Linum plants. The system is rapid, simple and offers an alternative to Agrobacterium methods.     

根癌农杆菌介导的水稻转化研究进展

农杆菌介导的水稻基因转化是水稻基因转化的热门。本文对由农杆菌介导转化获得的水稻品系（品种）,影响农杆菌介导转化的因素,农村菌浸染的方法,外源基因的检测和遗传等方面作综合论述,并提出了农杆菌介导转化水稻的前景。     

Transformation of nuclear and plastomic plant genomes by biolistic particle bombardment

Microprojectile bombardment is a powerful method for the transformation of various organisms and tissues. For plants, the biolistic approach is primarily used for transformation of cereals and other monocotyledons, as well as for dicotyledonous plants shown to be recalcitrant to Agrobacterium-based transformation of organellar genomes, and transformation of plant and algal chloroplasts has recently been reported. In this protocol paper we provide methods for nuclear and plastomic transformation of plants using the biolistic technique.     

Agrobacterium-mediated transformation of Javanica rice

Difficulties frequently encountered using direct DNA transfer methods for transformation of Javanica varieties of rice (Oryza sativa L.) have limited the application of biotechnology to these varieties. We now reportAgrobacterium-mediated transformation of Javanica cultivars Gulfmont and Jefferson that are, respectively, widely used or about to enter commercial cultivation in the southern USA. Vigorous, phenotypically normal, fertile plants expressing both the selectable marker and the gene of interest were obtained. Southern analysis showed that only one or two copies of the T-DNA insert were present. Sequence analysis of right border fragments of one line confirmed that insertion was into a coding region of rice nuclear DNA. This analysis also revealed the presence of relatively short regions of permuted T-DNA border sequences, similar to those found afterAgrobacterium-mediated transformation of dicots. Progeny analysis of lines bearing two copies showed co-segregation, indicating that they were located relatively closely on the same chromosome. The introduced genes were transmitted to the R₁ and R₂ generations in a Mendelian fashion, confirming the suitability of this approach for biotechnological improvement of elite rice cultivars.     

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。     

Enhanced resistance to two stem borers in an aromatic rice containing a synthetic cryIA(b) gene

Rice cultivars of isozyme group V include high-quality, aromatic rices that are difficult to improve by traditional methods because of the loss of quality characters upon sexual hybridization. Their low-tillering plant type predisposes them to economic loss from attack by stem borers, a group of insects to which they are susceptible. We report here the enhancement of stem borer resistance in cv. Tarom Molaii through transformation by microprojectile bombardment. Embryogenic calli derived from mature seeds were bombarded with gold particles coated with plasmid pCIB4421, carrying a synthetic truncated toxin gene based on the cryIA(b) gene from Bacillus thuringiensis, and plasmid pHygII, carrying the hygromycin phosphotransferase (hpt) selectable marker gene. Inclusion of 50 mg/l hygromycin B in culture media from bombardment through to rooting of plantlets eliminated escapes. The procedure generated three independent hpt transformants of which two also contained the cryIA(b) gene. One such line (No. 827) produced truncated (67 kDa) CryIA(b) protein equivalent to about 0.1% of total soluble protein. The cryIA(b) gene was controlled by the promoter of the maize C₄ PEP carboxylase gene and was expressed in leaf blades but was not expressed to a detectable level in dehulled mature grain. Line 827 contained about 3 copies of the cryIA(b) gene which segregated as a single dominant Mendelian locus in the second (T₁) and third (T₂) generations and co-segregated with enhanced resistance to first-instar larvae of striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas). T₂ line 827-6 homozygous for the cryIA(b) gene showed no dead hearts or whiteheads after infestation with stem borers, whereas T₂ line 827-25 lacking the gene averaged 7 dead hearts per plant and 2.25 whiteheads per plant. These results establish that transformation of high-quality rices of group V is a feasible alternative to sexual hybridization.     

Genetic transformation of <Emphasis Type="Italic">Coffea canephora</Emphasis> by particle bombardment

Stable transformation of Coffea canephora P. was obtained by particle bombardment of embryogenic tissue. Leaf explants were cultured on medium supplemented with 5 µM isopentenyl-adenosine to induce direct embryogenesis. Explants with somatic embryos were transferred to half strength MS medium with 9 µM 2,4 dichlorophenoxyacetic acid. After 2 weeks, the explants with somatic embryos and embryogenic tissue were bombarded with tungsten particles (M-25) carrying the plasmid pCambia3301 (containing the bar and uidA genes) using a high pressure helium microprojectile device. The bombarded explants were submitted to selection on medium containing 5 µM ammonium glufosinate herbicide as selective agent. After 6 months, putative transgenic embryos were transferred to a growth regulator-free medium for germination. The regenerated plantlets were β-glucuronidase (GUS) positive whereas no GUS activity was observed in non-transgenic controls. Incorporation of the bar gene into the genome was confirmed by PCR and Southern blot analysis of the regenerated transformed plants. Greenhouse grown transgenic coffee plants were found to withstand the recommended level of the herbicide Finale™ for weed control.This research was supported by the Consorcio Brasileiro de Pesquisa e Desenvolvimento do Cafe (CBP&D-Cafe).     

Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed

Here, we provide comprehensive, highly efficient protocols for Agrobacterium tumefaciens-mediated transformation of a wide range of rice genotypes. Methods that use either immature embryos (japonica and indica rice) or calli (japonica cultivars and the indica cultivar, Kasalath) as a starting material for inoculation with Agrobacterium are described. Immature embryos are pretreated with heat and centrifugal force, which significantly enhances the efficiency of gene transfer, and then infected with Agrobacterium. Callus is induced from mature seeds and infected. Transformed cells proliferated from these tissues are selected on the basis of hygromycin resistance, and transgenic plants are eventually regenerated. A single immature japonica or Kasalath embryo will produce between 10 and 18 independent transgenic plants; for other non-Kasalath indica varieties, the number of transgenic plants expected will be between 5 and 13. For japonica and Kasalath, transformants should be obtained from between 50 and 90% of calli. From inoculation with Agrobacterium to transplanting to soil will take 55 d for japonica and Kasalath, and 74 d for indica other than Kasalath using the immature embryo method, and 50 d for japonica and Kasalath using the callus method.     

Rice transformation: bombardment

Bombardment-based methodology is responsible for the effective genetic manipulation of major cereals including rice. Many groups reported significant advances on various aspects of rice molecular biology and genetic engineering using procedures based on bombardment technology. Molecular and genetic characterization of large numbers of these plants (more than 500 independent transgenic plants) provided information on structure, expression and stability of integrated DNA through multiple generations. Such evaluations were carried out in the greenhouse and in the field. Stability of expression was found to be dependent on the nature of the promoter and the transgene, and in specific cases on gene copy number. Direct DNA transfer utilizing particle bombardment for the delivery of foreign DNA into rice tissue results in the recovery of large numbers of independently derived transgenic plants in a variety-independent fashion. Gene copy number, level and stability of expression of transgenes can be compared to other DNA delivery methods, direct or indirect, including Agrobacterium-mediated gene transfer. In this paper, the technology is summarized and discussed in terms of present and future applications, including field trials and potential commercialization of transgenic rice expressing a number of genes of agronomic interest such as pest and herbicide resistance.     

Binary vectors for efficient transformation of rice

We constructed binary vectors that were designed for transfer and expression of a gene into rice chromosomes. The binary vectors contained the hygromycin-resistance gene for selection of transformants and multiple-cloning sites within the transfer DNA. In addition, vectors were designed to express foreign genes using four kinds of promoters. We also report a procedure for efficient transformation of rice plants using scutellum-derived calli and theAgrobacterium strain LBA4404.     

A focusing device for tissue transformation with the DuPont/BioRad PDS1000 helium microprojectile system

The DuPont/BioRad PDS1000/He has become an important tool in genetic transformation. With the present design, however, it is difficult to accurately predict the impact area. This report discusses targeting devices that were designed to accurately focus biolistic particles in a given area. Tobacco leaves and soybean tissue were used to evaluate the efficiency of the device. With the focusing device, GUS positive foci were confined in a defined ring on soybean cotyledonary nodes. Approximately 65 percent of the GUS positive foci on tobacco leaves were concentrated in a ring 8 to 16 mm from the center of the short, compared to 27 percent without the device. The results demonstrate that PDS-1000 biolistic particles can both be accurately and predictably delivered at target tissue.     

Biolistic transformation of animal tissue

Summary The biolistic technique transforms cells by bombardment with DNA-coated microprojectiles. It has been used to transform plants, microbes, and organelles. We adapted a standard Biolistic PDS-1000 device for use with animals and have successfully transformed tissues in live mice. The firefly luciferase gene was introduced into mouse skin and ear tissue. One day after transformation 344±74 and 1648±254 pg of luciferase were detected in skin and ear samples, respectively. Expression of the gene product was transient but detectable up to 7 days after bombardment. A further modification of the device allowed transient transformation of liver tissue in vivo. Liver contained 293±122 pg of luciferase 1 day postransformation. Expression of the gene in liver tissue was unchanged at Day 3 but declined to low levels by Day 5. This new device allowed a fourfold increase in gene expression in ear tissue extending a minimum of 14 days. This technology is applicable to a broad range of tissues and organs in situ and makes it possible to test numerous reporters and the tissue specificity of promoters. It may also be useful in protocols for somatic cell therapy. Presented in the Session-in-Depth Genetic Transformation and Genetic Analysis Using Microprojectile Bombardment at the 41st Annual Meeting of the Tissue Culture Association, Houston, Texas, June 10–13, 1990.     

基因枪转化小麦主要影响因素细述

基因枪转化是目前小麦遗传转化的主要技术之一,高效的基因枪转化系统对于转基因小麦新品种培育、候选基因功能鉴定和功能基因组学研究具有重要意义。本文综述了影响基因枪转化小麦效率的主要因素,包括基因型、外植体、植物生长调节剂、轰击参数、筛选体系等,以期为进一步改进小麦基因枪转化技术,提高基因枪转化小麦的效率提供参考。     

Distribution of zinc fractions and their transformation in submerged rice soils

Distribution of different forms of Zn in 16 acid alluvial rice growing soils of West Bengal (India) and their transformation on submergence were studied. The results showed that more than 84% of total Zn occurred in the relatively inactive clay lattice-bound form while a smaller fractionviz. 1.1, 1.6, 11.1 and 2.0 per cent of the total occurred as water-soluble plus exchangeable, organic complexed, amorphous sesquioxide-bound and crystalline sesquioxide bound forms, respectively. All these four Zn forms showed significant negative correlations with soil pH (r=−0.48^**, −0.39^*, −0.61^** and −0.67^**, respectively), while the latter two Zn forms showed significant positive correlations with Fe₂O₃ (0.68^** and 0.88^***) and Al₂O₃ (0.89^*** and 0.75^***) content of the soils. The different Zn forms were found to have positive and significant correlations amongst each other, suggesting the existence of a dynamic equilibrium of these forms in soil. Submergence caused an increase in the amorphous sesquioxide-bound form of Zn and a decrease in each of the other three forms. The magnitude of such decreases in water-soluble plus exchangeable and crystalline sesquioxide-bound forms was found to be correlated negatively with initial pH values of the soils and positively with the increase in the amorphous sesquioxide-bound form, indicating their adsorption on the surface of the freshly formed hydrated oxides of Fe, which view was supported by the existence of significant positive correlation between the increase in the amorphous sesquioxide-bound form of Zn and that in AlCl₃-extractable iron. The existence of a positive correlation between the decrease in crystalline sesquioxide-bound Zn and that in Fe₂O₃ content in soil suggested that on waterlogging the soil Zn occluded in the cry talline sesquioxide was released as a result of reduction of Fe₂O₃.     

An improvedAgrobacterium-mediated transformation protocol for recalcitrant elite indica rice cultivars

We report here a high-efficiency transformation protocol for recalcitrant indica rice cultivars IR64 and IR72 with the selectable marker genehph and thegusA reporter gene. Factors that favor high-efficiency transformation were found to be use of 2-month-old mature seed-derived embryogenic calli, maltose as a source of carbon, a higher concentration of 2,4-dichlorophenoxyacetic acid, and both phytagel and agar as gelling agents. The putative transgenic (T₀) plants were analyzed for integration of the transgene through polymerase chain reaction and Southern blotting analyses. Various factors thought to be responsible for increased transformation efficiency are discussed.     

Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice

Several approaches have recently been adopted to improve Agrobacterium-mediated transformation of rice, both to generate the large number of T-DNA insertion plants needed for functional analysis of the rice genome, and for production of rice with additional agronomical value. However, about 3 months of in vitro culture is still required for isolation of transgenic rice plants. Here, we report the competency of scutellum tissue from 1-day pre-cultured seeds for Agrobacterium-mediated transformation. Furthermore, early infection of rice seeds with Agrobacterium enhanced efficient selection of transformed calli. Using our system, we successfully regenerated transgenic rice plantlets within a month of the start of the aseptic culture of mature seeds. Our new system should reduce the somaclonal variation accompanying prolonged culture of rice cells in the dedifferentiated state and facilitate the molecular breeding of rice.     

微量元素对籼稻愈伤组织分化成苗的影响

通过研究MS培养基中碘、硼、锰、锌、钼、铜、钴以及铁8种微量无素对愈伤组织分化成苗的影响,结果表明:缺少碘、硼、锰、锌、钼、钴的培养基对籼稻愈伤组织分化成苗影响不大,而缺少铜和铁的培养基对籼稻愈伤组织分化成苗影响很大,添加不同浓度铁和铜元素的试验表明,在铁盐浓度为0.10mmo1.L-1,铜浓度为1.5×10-4mmo1.L-1时,分化成苗效果较好,而铁盐浓度分别在0.05mmol.L-1、0.15mmol.L-1、0.20mmol.L-1、0.40mmol.L-1,铜浓度分别在0.5×10-4mmol.L-1、1.0×10-4mmol.L-1、2.0×10-4mmol.L-1、4.0×10-4mmol.L-1时,对籼稻分化成苗和生长均不利。     

Rapid and efficientAgrobacterium-mediated transformation in rice

An improved protocol forAgrobacterium-mediated transformation of rice is described. The procedure permitted the regeneration of transgenic plants from callus in only two months.     

Genetic transformation of Alstroemeria using particle bombardment

Transgenic plants were obtained after particle bombardment of embryogenic callus derived from stem segments of two tetraploid Alstroemeria genotypes with plasmids containing different selection/reporter genes. Firstly, a plasmid containing a firefly luciferase reporter gene driven by the maize ubiquitin promoter (Ubi1), was bombarded into both friable embryogenic callus and proembryos. Transient and stable expression of luciferase was visually detected by a luminometer. This selection method is non-destructive and can be applied over the whole developmental process from callus to embryo and plantlet. Molecular proof of transformation was obtained both by PCR analysis and Southern hybridization. Secondly, a plasmid containing the bar gene together with an uidA gene coding for -glucuronidase both driven by the Ubi1 promoter was bombarded into proembryos. The transgenic callus was effectively selected from the callus clumps four months after bombardment on a medium containing 5 mg/l phosphinotricin (PPT). Selection by PPT was efficient and labour-saving. Stable expression of GUS was confirmed by the histochemical staining assay and molecular proof was obtained by PCR analysis.