Transgenic Central American, West African and Asian Elite Rice Varieties Resulting from Particle Bombardment of Foreign DNA into Mature Seed-derived Explants Utilizing Three Different Bombardment Devices

We developed a novel system based on particle bombardment forthe transfer of foreign genes into eight important rice varietiesgrown in Central America, West Africa and Asia. Novel featuresof this improved protocol, which we believe will replace alternativesystems currently utilizing immature embryos, embryogenic celllines or suspension cultures include: (a) bombardment of targettissue 5 d after seed germination; (b) no requirement to selectfor embryogenic callus prior to bombardment; (c) high transformationfrequencies (up to 14%); (d) ability to recover transgenic materialat similar frequencies irrespective of bombardment devices used.Southern blot analysis and enzymatic assays in primary transformantsand progeny confirmed the stable integration and expressionof introduced transgenes in all engineered cultivars.Copyright1998 Annals of Botany Company. Oryza sativa, transgenic rice, particle bombardment, transformation.     

Parameters Influencing Stable Transformation of Rice Immature Embryos and Recovery of Transgenic Plants using Electric Discharge Particle Acceleration

Recovery of transgenic rice plants from elite varieties wasreported from the author's laboratory. By using electric dischargeparticle acceleration, recombinant technology was extended tocommercially important japonica and indica rice cultivars notamenable to conventional transformation methodologies. Criticalparameters influencing the recovery of transformed embryogeniccallus from which transgenic plants were recovered have beendefined. Such parameters included the physiological conditionof explants prior to bombardment, DNA and gold particle loadingrates, accelerating voltage, and depth of particle penetration.Selection for recovery of stable transformants was not criticalin this transformation/regeneration system; both selected andnon-selected tissues yielded transformed embryogenic callusand plants at approximately similar frequencies.Copyright 1995,1999 Academic Press Oryza sativa L., transformation parameters, transgenic indica and japonica plants, particle bombardment, ß-glucuronidase     

Protamine-mediated DNA coating remarkably improves bombardment transformation efficiency in plant cells

We have developed a method by which remarkably higher efficiencies of transient and stable transformation were achieved in bombardment transformation of plants. Over fivefold increase in transient gus gene expression was achieved when rice or maize suspension cells were bombarded with gold particles coated with plasmid DNA in the presence of protamine instead of the conventional spermidine. A 3.3-fold improvement in stable transformation efficiency was also observed using rice suspension cells with the new coating approach. The coated protamine-plasmid DNA complex resisted degradation by a DNase or by rice cell extract much longer than the spermidine-plasmid DNA complex. The results from this study suggest that protamine protects plasmid DNA longer than spermidine when being delivered inside the cells, probably by forming a nano-scale complex, and thus helps improve the efficiency of particle bombardment-mediated plant transformation.     

Direct DNA transfer using electric discharge particle acceleration (ACCELL™ technology)

Direct DNA transfer methods based on particle bombardment have revolutionized plant genetic engineering. Major agronomic crops previously considered recalcitrant to gene transfer have been engineered using variations of this technology. In many cases variety-independent and efficient transformation methods have been developed enabling application of molecular biology techniques to crop improvement. The focus of this article is the development and performance of electric discharge particle bombardment (ACCELL™) technology. Unique advantages of this methodology compared to alternative propulsion technologies are discussed in terms of the range of species and genotypes that have been engineered, and the high transformation frequencies for major agronomic crops that enabled the technology to move from the R&D phase to commercialization. Creation of transgenic soybeans, cotton, and rice will be used as examples to illustrate the development of variety-independent and efficient gene transfer methods for most of the major agronomic crops. To our knowledge, no other gene transfer method based on particle bombardment has resulted in variety-independent and practical generation of large numbers of independently-derived crop plants. ACCELL™ technology is currently being utilized for the routine transfer of valuable genes into elite germplasm of soybean, cotton, bean, rice, corn, peanut and woody species.     

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.     

Advances in cereal gene transfer

Over the past five years, transgenic strains of various major cereals have been produced, with transformation of rice and maize being most common. A majority of the cereal transformants obtained to date has been generated by the particle bombardment technique, but Agrobacterium-mediated transformation is rapidly becoming the method of choice. Rice, the plant in which transformation-related technology is most advanced, appears to be the model monocotyledon for basic and applied studies.     

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.     

农杆菌介导法与基因枪轰击法结合在植物遗传转化上的应用

根癌农杆菌介导法(Agrohacterium mediated transformation)和基因枪轰击法( particle bombardment transformation)是植物遗传转化的主要方法。两种方法各有优缺点．农杆菌介导法是一种天然的植物遗传转化系统,外源基因在转基因植物中的拷贝数低,遗传稳定性好;基因枪转化法不受材料基因型的限制。通过结合两种方法的优点,发展了3种农杆菌介导和基因枪轰击法相结合的遗传转化方法,分别为农杆枪法、基因枪轰击／农杆菌感染法、金粉或钨粉包裹菌体细胞作为微弹轰击法。对3种结合转化方法的技术途径、原理、转化受体及研究进展等方面进行了综述。     

Development of transgenic rice pure lines by particle bombardment‐mediated transformation and genetic analysis‐based selection

Mature seed‐derived callus from an elite Chinese japonica rice cv. Eyl 105 was transformed with a plasmid containing the selectable marker hygromycin phosphotransferase (hpt) and the reporter β‐glucuronidase (gusA) genes via particle bombardment. After two rounds of selection on hygromycin (30 mg/l)‐containing medium, resistant callus was transferred to hygromycin (30 mg/l)‐containing regeneration medium for plant regeneration. Twenty‐three independent transgenic rice plants were regenerated from 127 bombarded callus with a transformation frequency of 18.1%. All the transgenic plants contained both gusA and hpt genes, revealed by PCR/Southern blot analysis. GUS assay revealed 18 out of 23 plants (78.3%) proliferated on hygromycin‐containing medium had GUS expression at various levels. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parent plants showing 3:1 Mendelian segregation, we identified three independent homozygous transgenic rice lines. The homozygous lines were phenotypically normal and fertile compared to the control plants. We demonstrate that homozygous transgenic rice lines can be obtained via particle bombardment‐mediated transformation and through genetic analysis‐based selection.     

基因枪介导小麦成熟胚遗传转化的影响因素

小麦成熟胚作为转化受体可克服小麦幼胚存在的受季节和幼胚发育阶段限制的缺点。以湖北省小麦品种‘鄂麦12’和模式品种‘Bobwhite’为材料,成熟胚为转化受体,优化基因枪转化法的轰击压力、轰击距离、选择剂等因素,建立以小麦成熟胚为转化受体的高效转化系统。结果表明:小麦成熟胚作为转化受体时,适宜轰击压力和轰击距离组合是900 psi、6 cm;成熟胚对选择剂G418的敏感性强于幼胚,轰击后需要延长恢复时间,选择剂G418的适合浓度为20～40 mg/L。在以上优化条件下小麦成熟胚转化频率达0.3%～0.9%,已初步建立基因枪介导的小麦成熟胚遗传转化系统。     

Molecular Characteristics of Transgenic Wheat and the Effect on Transgene Expression

A population of R0 transgenic wheat plants, generated by particle bombardment, was analyzed to define molecular, genetic and phenotypic properties resulting from transformation with a cointegrate vector, or cotransformation with two separate plasmids. By evaluating the progeny of 70 independently-derived transgenic plants, we also identified rare events such as chimerism and transgene elimination, which provide valuable information concerning the development of transgenic cereal plants following bombardment experiments. The frequency of chimerism in our transgenic wheat plants was very low. Furthermore, while transgene elimination did occur, this was also a very rare event. We determined the copy numbers of integrated transgenes and the levels of transgene expression. Comparisons to transgenic rice plants generated in the same manner demonstrated some similarities, but also important differences in transgene behavior. Whereas in rice there is no evidence for any direct relationship between transgene copy number and transgene expression or stability, multicopy populations in wheat demonstrated a bias towards higher levels of expression for the two genes and the maize ubiquitin promoter evaluated in the present study.     

The green fluorescent protein (GFP) as a vital screenable marker in rice transformation

 An engineered green fluorescent protein (GFP) from the jellyfish Aequora victoria was used to develop a facile and rapid rice transformation system using particle bombardment of immature embryos. The mgfp4 gene under the control of the 35s Cauliflower Mosaic Virus promoter produced bright-green fluorescence easily detectable and screenable in rice tissue 12–22 days after bombardment. Visual screening of transformed rice tissue, associated with a low level of antibiotic selection, drastically reduced the quantity of tissue to be handled and the time required for the recovery of transformed plants. GFP expression was observed in primary transformed rice plants (T₀) and their progeny (T₁). We describe various techniques to observe GFP in vitro and in vivo. The advantages of this new screenable marker in rice genetic engineering programmes are discussed. Received: 6 October 1997 / Accepted: 9 October 1997     

Particle bombardment and the genetic enhancement of crops: myths and realities

DNA transfer by particle bombardment makes use of physical processes to achieve the transformation of crop plants. There is no dependence on bacteria, so the limitations inherent in organisms such as Agrobacterium tumefaciens do not apply. The absence of biological constraints, at least until DNA has entered the plant cell, means that particle bombardment is a versatile and effective transformation method, not limited by cell type, species or genotype. There are no intrinsic vector requirements so transgenes of any size and arrangement can be introduced, and multiple gene cotransformation is straightforward. The perceived disadvantages of particle bombardment compared to Agrobacterium-mediated transformation, i.e. the tendency to generate large transgene arrays containing rearranged and broken transgene copies, are not borne out by the recent detailed structural analysis of transgene loci produced by each of the methods. There is also little evidence for major differences in the levels of transgene instability and silencing when these transformation methods are compared in agriculturally important cereals and legumes, and other non-model systems. Indeed, a major advantage of particle bombardment is that the delivered DNA can be manipulated to influence the quality and structure of the resultant transgene loci. This has been demonstrated in recently reported strategies that favor the recovery of transgenic plants containing intact, single-copy integration events, and demonstrating high-level transgene expression. At the current time, particle bombardment is the most efficient way to achieve plastid transformation in plants and is the only method so far used to achieve mitochondrial transformation. In this review, we discuss recent data highlighting the positive impact of particle bombardment on the genetic transformation of plants, focusing on the fate of exogenous DNA, its organization and its expression in the plant cell. We also discuss some of the most important applications of this technology including the deployment of transgenic plants under field conditions.     

Efficient transformation of wheat by using a mutated rice acetolactate synthase gene as a selectable marker

Acetolactate synthase (ALS) is a target enzyme for many herbicides, including sulfonylurea and imidazolinone. We investigated the usefulness of a mutated ALS gene of rice, which had double point mutations and encoded an herbicide-resistant form of the enzyme, as a selectable marker for wheat transformation. After the genomic DNA fragment from rice containing the mutated ALS gene was introduced into immature embryos by means of particle bombardment, transgenic plants were efficiently selected with the herbicide bispyribac sodium (BS). Southern blot analysis confirmed that transgenic plants had one to more than ten copies of the transgene in their chromosomes. Adjustment of the BS concentration combined with repeated selection effectively prevented nontransgenic plants from escaping herbicide selection. Measurement of ALS activity indicated that transgenic plants produced an herbicide-resistant form of ALS and therefore had acquired the resistance to BS. This report is the first to describe a selection system for wheat transformation that uses a selectable marker gene of plant origin.     

Tn5 transposase-assisted transformation of indica rice

Here, we describe experiments on Tn5 transposase-assisted transformation of indica rice. Transposomes were formed in vitro as a result of hyperactive Tn5 transposase complexing with a transposon that contained a 19-bp tetracycline operator (tetO) sequence. To form modified projectiles for transformation, the Tn10-derived prokaryotic tetracycline repressor (TetR) proteins, which can bind transposomes via the high affinity of TetR for tetO, were immobilized onto the surface of bare gold microscopic particles. These projectiles were introduced into cells of the indica rice cultivar Zhuxian B by particle bombardment. Once projectiles were inside the cell, tetracycline induced an allosteric conformational change in TetR that resulted in the dissociation of TetR from tetO, and thus generated free transposomes. Molecular evidence of transposition was obtained by the cloning of insertion sites from many transgenic plants. We also demonstrated that the introduced foreign DNA was inherited stably over several generations. This technique is a promising transformation method for other plant species as it is species independent.     

Transient expression of the β-glucuronidase gene in tissues ofArabidopsis thaliana by bombardment-mediated transformation

Particle bombardment has proved to be useful for the transformation of plants. We have previously reported successful transient expression of the beta-glucuronidase (GUS) gene in cultured plant cells and tissues and the stable transformation of various plants using a pneumatic particle gun. In this chapter, we describe transient expression of the GUS gene in Arabidopsis thaliana leaves and roots using the pneumatic particle gun.     

Efficient selection and regeneration of creeping bentgrass transformants following particle bombardment

We have established an efficient genetic transformation system for creeping bentgrass (Agrostis palustris Huds.) using particle bombardment. The transformation was performed using the plasmid pZO1052 which contains the reporter β-glucuronidase (uidA) gene and the selectable marker hygromycin phosphotransferase (hph) gene. Transformed calli and plants were obtained via particle bombardment followed by selection of transformants on medium containing 200 mg/l of hygromycin. An average of 4.6 resistant colonies per bombardment were obtained. Southern analysis confirmed the integration of foreign genes in 19 of 21 putative transformants, indicating that selection by hygromycin was highly effective. Received: 6 February 1997 / Revision received: 16 April 1997 / Accepted: 9 May 1997     

花生遗传转化的主要方法和研究进展

花生遗传转化研究对于花生品种改良、新品种繁育具有重要意义。农杆菌介导的遗传转化法和基因枪法是花生遗传转化的主要方法。随着对花生研究的深入开展,花生的遗传转化技术也越来越成熟。介绍了农杆菌介导的遗传转化法和基因枪法在花生遗传转化研究中的应用现状及存在的问题。     

Co-transformation of gene expression cassettes via particle bombardment to generate safe transgenic plant without any unwanted DNA

The presence of resistant selectable marker genes and other added DNAs such as the vector backbone sequence in transgenic plant might be an unpredictable hazard to the ecosystem as well as to human health, which have affected the safe assessment of transgenic plants seriously. Using minimal gene expression cassette (containing the promoter, coding region, and terminator) without vector backbone sequence for particle bombardment is the new trend of plant genetic transformation. In the present paper, we co-transformed the selectable marker bar gene cassette and non-selected cecropinB gene cassette into rice (Oryza sativa L.) by particle bombardment, then eliminated the selectable marker bar gene in R₁ generation applying the hereditary segregation strategy and attained two safe transgenic plants only harboring cecropinB gene cassettes without any superfluous DNA. This is the fist report indicating that the combination of minimal gene cassettes transformation with the co-transformation and segregation strategy can generate selectable marker-free transgenic plants, which will promote the advancement in plant genetic engineering greatly.