Factors influencing successful<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated genetic transformation of wheat

The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like bread wheat requires the identification and optimisation of the factors affecting T-DNA delivery and plant regeneration. We have used immature embryos from range of wheat varieties and the Agrobacterium strain AGL1 harbouring the pGreen-based plasmid pAL156, which contains a T-DNA incorporating the bar gene and a modified uidA (beta-glucuronidase) gene, to investigate and optimise major T-DNA delivery and tissue culture variables. Factors that produced significant differences in T-DNA delivery and regeneration included embryo size, duration of pre-culture, inoculation and co-cultivation, and the presence of acetosyringone and Silwet-L77 in the media. We fully describe a protocol that allowed efficient T-DNA delivery and gave rise to 44 morphologically normal, and fully fertile, stable transgenic plants in two wheat varieties. The transformation frequency ranged from 0.3% to 3.3%. Marker-gene expression and molecular analysis demonstrated that transgenes were integrated into the wheat genome and subsequently transmitted into progeny at Mendelian ratios.     

Efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br. using shoot apices as explant source

A critical step in the development of a reproducible Agrobacterium tumefaciens mediated transformation system for a recalcitrant species, such as pearl millet, is the establishment of optimal conditions for efficient T-DNA delivery into target tissue from which plants can be regenerated. A multiple shoot regeneration system, without any intervening callus phase, was developed and used as a tissue culture system for Agrobacterium-mediated transformation. Agrobacterium super virulent strain EHA105 harboring the binary vector pCAMBIA 1301 which contains a T-DNA incorporating the hygromycin phosphotransferase (hpt II) and β-glucuronidase (GUS) genes was used to investigate and optimize T-DNA delivery into shoot apices of pearl millet. A number of factors produced significant differences in T-DNA delivery; these included optical density, inoculation duration, co-cultivation time, acetosyringone concentration in co-cultivation medium and vacuum infiltration assisted inoculation. The highest transformation frequency of 5.79% was obtained when the shoot apex explants were infected for 30 min with Agrobacterium O.D.₆₀₀ = 1.2 under a negative pressure of 0.5 × 10⁵ Pa and co-cultivated for 3 days in medium containing 400 μM acetosyringone. Histochemical GUS assay and polymerase chain reaction (PCR) analysis confirmed the presence of the GUS gene in putative transgenic plants, while stable integration of the GUS gene into the plant genome was confirmed by Southern analysis. This is the first report showing reproducible, rapid and efficient Agrobacterium-mediated transformation of shoot apices and the subsequent regeneration of transgenic plants in pearl millet. The developed protocol will facilitate the insertion of desirable genes of useful traits into pearl millet.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

The effect of temperature on Agrobacterium tumefaciens-mediated gene transfer to plants

Agrobacterium tumefaciens is generally used to achieve genetic transformation of plants. The temperatures that have been used for infection with Agrobacterium in published transformation protocols differ widely and, to our knowledge, the effect of temperature on the efficiency of T-DNA transfer to plants has not been investigated systematically. Agrobacterium tumefaciens strains harbouring a binary vector with the β-glucuronidase ( uidA ) gene and either a nopaline-, an octopine- or an agropine/ succinamopine-type helper plasmid were tested in two transformation systems at temperatures between 15 and 29°C. One system involved cocultivation of Phaseolus acutifolius callus whereas in the other system Nicotiana tabacum leaves were vacuum-infiltrated. In both situations, irrespective of the type of helper plasmid, the levels of transient uidA expression decreased notably when the temperature was raised above 22°C. Expression was low at 27°C and undetectable at 29°C. We anticipate that the efficiency of many published transformation protocols can be improved by reconsidering the factor of temperature.     

Regeneration of transgenic shape Vitis vinifera L. Sultana plants: genotypic and phenotypic analysis

Different approaches to producing transgenic grapevines based on regeneration via embryogenesis were investigated. Embryogenic callus was initiated from anther tissue of Vitis vinifera cv. Sultana and three embryogenic culture types (embryogenic callus, tissue type I; proliferating embryos, tissue type II; and a suspension) were established. The three culture types were incolucaled with Agrobacterium tumefaciens harbouring a binary vector which contained a uidA reporter gene and either a hpt or nptII selectable marker gene or the cultures were bombarded with microprojectiles carrying a uidA/nptII binary vector. Transgenic plants were produced only from Agrobacterium transformation experiments. Transformed embryos were selected with kanamycin or hygromycin antibiotics and recovered with the highest efficiency from inoculated type I cultures. Southern analysis of genomic DNA extracted from ten transgenic plants showed that the number of T-DNA insertions in the genome ranged from 1 to at least 4. Evidence for methylation of the T-DNA at cytosine and adenine residues in transgenic plants was found by Southern analysis of DNA digested with two isoschizomer pairs of restriction endonucleases. No evidence for genotype alterations or somatic meiosis was found when DNA from 80 somatic embryos and seven plants regenerated from embryogenic culture were analysed at six sequence-tagged sites which are heterozygous in cv. Sultana. Expression of the uidA gene in in vitro grown leaves of transgenic plants was most often high and uniform but GUS staining was occasionally observed to be low and/or patchy. Transgenic plants and all plants regenerated from embryogenic culture produced red veined, lobed leaves which are uncharacteristic of the accepted ampelographic phenotype of Sultana. It is suggested that this phenotype may represent a juvenile growth stage.     

Acetosyringone,pH and temperature effects on transient genetic transformation of immature embryos of Brazilian wheat genotypes by Agrobacterium tumefaciens

Low transformation efficiency is one of the main limiting factors in the establishment of genetic transformation of wheat via Agrobacterium tumefaciens. To determine more favorable conditions for T-DNA delivery and explant regeneration after infection, this study investigated combinations of acetosyringone concentration and pH variation in the inoculation and co-cultivation media and co-culture temperatures using immature embryos from two Brazilian genotypes (BR 18 Terena and PF 020037). Based on transient expression of uidA, the most favorable conditions for T-DNA delivery were culture media with pH 5.0 and 5.4 combined with co-culture temperatures of 22 °C and 25 °C, and a 400 μM acetosyringone supplement. These conditions resulted in blue foci in 81% of the embryos. Media with more acidic pH also presented reduced A. tumefaciens overgrowth during co-culture, and improved regeneration frequency of the inoculated explants. BR 18 Terena was more susceptible to infection by A. tumefaciens than PF 020037. We found that it is possible to improve T-DNA delivery and explant regeneration by adjusting factors involved in the early stages of A. tumefaciens infection. This can contribute to establishing a stable transformation procedure in the future.     

Light strongly promotes gene transfer from <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> to plant cells

Light conditions during Agrobacterium-based plant transformation, the most routinely used method in plant genetic engineering, differ widely and, to our knowledge, have not been studied systematically in relation to transformation efficiency. Here, light effects were examined in two already optimized transformation procedures: coculture of Agrobacterium tumefaciens with callus from two genotypes of the crop plant Phaseolus acutifolius (tepary bean) and coculture of root segments from two ecotypes of Arabidopsis thaliana. Except for the light conditions during coculture, all steps followed established procedures. Coculture was done either under continuous darkness, under a commonly used photoperiod of 16 h light/8 h darkness or under continuous light. beta-glucuronidase (GUS) production due to the transient expression of an intron-containing uidA gene in the binary vector was used to evaluate T-DNA transfer. In all situations, uidA expression correlated highly and positively with the light period used during coculture; it was inhibited severely by darkness and enhanced more under continuous light than under a 16 h light/8 h dark photoperiod. The promotive effect of light was observed with Agrobacterium strains harboring either a nopaline-, an octopine- or an agropine/succinamopine-type non-oncogenic helper Ti plasmid. The observed positive effect of light has obvious implications for developing and improving transient and stable transformation protocols, specifically those involving dark coculture conditions.     

Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana

The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.     

Agrobacterium T-DNA integration: molecules and models

Genetic transformation mediated by Agrobacterium involves the transfer of a DNA molecule (T-DNA) from the bacterium to the eukaryotic host cell, and its integration into the host genome. Whereas extensive work has revealed the biological mechanisms governing the production, Agrobacterium-to-plant cell transport and nuclear import of the Agrobacterium T-DNA, the integration step remains largely unexplored, although several different T-DNA integration mechanisms have been suggested. Recent genetic and functional studies have revealed the importance of host proteins involved in DNA repair and maintenance for T-DNA integration. In this article, we review our understanding of the specific function of these proteins and propose a detailed model for integration.     

Generation of enhancer trap lines in Arabidopsis and characterization of expression patterns in the inflorescence

Eleven thousand, three hundred and seventy enhancer/promoter trap lines in Arabidopsis were generated via T-DNA transformation utilizing the binary vector pD991 that contains a minimal promoter fused to the uidA reporter gene. Overall 31% of the lines generated exhibit a staining pattern in the inflorescence. Flanking DNA has been cloned from 15 lines exhibiting inflorescence staining patterns by either thermal asymmetric interlaced PCR (TAIL-PCR), inverse PCR (IPCR), or partial library construction. Seeds from these lines are available from the ABRC and NASC Arabidopsis stock centers and DNA pools are available from the ABRC.     

Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant

VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5--by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell--enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell.     

Optimization of biological and physical parameters for biolistic genetic transformation of common wheat (Triticum aestivum L.) using a particle inflow gun

The parameters for delivery of expression cassettes to cells of wheat morphogenic callus induced from immature embryos were optimized. Three systems (gradation, delayed, and regeneration) for in vitro selection of transgenic wheat tissue using the bar gene, providing resistance to the herbicide phosphinothricin (PPT), were compared. The efficiency of gene delivery to the cells competent for plant regeneration was assessed by comparing the number of spots transiently expressing uidA gene (encoding beta-glucuronidase) per unit surface of the morphogenic calluses treated under various conditions. The selection systems in question were evaluated by comparing the transformation efficiency frequencies. The optimal parameters for wheat biolistic transformation using a particle inflow gun were determined, namely, the distance between the particle source and the target tissue (12 cm) and helium pressure during the shot (6 atm). The optimal time of callus tissue development on the medium inducing callus formation was determined (10-14 days). Comparison of the three selection variants demonstrated that the regeneration system was the most efficient for producing true transgenic plants of common wheat.     

Arabidopsis ecotypes and mutants that are recalcitrant to Agrobacterium root transformation are susceptible to germ-line transformation

Germ-line transformation (vacuum infiltration) is frequently used to transform Arabidopsis thaliana using Agrobacterium tumefaciens. We have recently identified several Arabidopsis ecotypes and T-DNA-tagged mutants that are recalcitrant to Agrobacterium-mediated transformation of cut root segments. Some of these ecotypes and mutants are deficient in their ability to bind bacteria. Some are deficient in T-DNA integration. We report here that using a germ-line transformation protocol we transformed these ecotypes and mutants, including attachment- and integration-defective Arabidopsis plants, with a frequency similar to that of highly susceptible wild-type plants. However, we could not transform otherwise highly susceptible Arabidopsis plants by germ-line or root transformation using several vir and attachment-deficient Agrobacterium mutants. These results indicate that certain plant factors important for transformation may exist in germ-line tissue but may be lacking in some somatic cells.     

Agrobacterium proteins VirD2 and VirE2 mediate precise integration of synthetic T-DNA complexes in mammalian cells

Agrobacterium tumefaciens-mediated plant transformation, a unique example of interkingdom gene transfer, has been widely adopted for the generation of transgenic plants. In vitro synthesized transferred DNA (T-DNA) complexes comprising single-stranded DNA and Agrobacterium virulence proteins VirD2 and VirE2, essential for plant transformation, were used to stably transfect HeLa cells. Both proteins positively influenced efficiency and precision of transgene integration by increasing overall transformation rates and by promoting full-length single-copy integration events. These findings demonstrate that the virulence proteins are sufficient for the integration of a T-DNA into a eukaryotic genome in the absence of other bacterial or plant factors. Synthetic T-DNA complexes are therefore unique protein:DNA delivery vectors with potential applications in the field of mammalian transgenesis.     

A novel system for Agrobacterium-mediated transformation of wheat（ Triticum aestivum L.） cells

A new approach for transforming the cultured cells of wheat (Triticum aestivum L.cv.Ganmai 8)was developed vsing Agrobacterium tumefaciens. The features of the optimum procedure were:(a)both combined synthetic signal molecules and multiple natural extracts from susceptible plants were used to pretreat the primary vigorous Agrobacterium(PVA)cells for approximately 16h:(b)the gyratory magnetic field condition was used during cocultivation;(c)the cocultivating period and selecting condition were modified;(d)the recipient cells were at exuberant metabolism and active division while infected with Agrobacterium.Both neomycin phosphotransferase and nopaline synthase assays demonstrated the expression of NPT Ⅱ and NOS genes.located on the T-DNA segment of chimaeric plasmid pGV3850::1103neo.in transformed wheat cell colonies by adopting the techniques of dot blot ndPAGE or high voltage paper electrophoresis,Integration of the foreign genes into wheat genome was confirmed by Southerm blot hybridization.Moreover.a relatively rational method was described for the estimation of transformation frequencies from cultured cell levels.     

T-DNA整合的研究进展

根癌农杆菌介导的基因转化过程中,T-DNA的整合是关系到外源基因能否稳定遗传的关键步骤。影响T-DNA整合的因素很多,包括毒性蛋白、寄主因子等等,本文对此加以综述,同时阐述了T-DNA在植物基因组中的整合及染色体水平上的分布及其整合模型。     

The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation

To genetically transform plants, Agrobacterium exports its transferred DNA (T-DNA) and several virulence (Vir) proteins into the host cell. Among these proteins, VirE3 is the only one whose biological function is completely unknown. Here, we demonstrate that VirE3 is transferred from Agrobacterium to the plant cell and then imported into its nucleus via the karyopherin alpha-dependent pathway. In addition to binding plant karyopherin alpha, VirE3 interacts with VirE2, a major bacterial protein that directly associates with the T-DNA and facilitates its nuclear import. The VirE2 nuclear import in turn is mediated by a plant protein, VIP1. Our data indicate that VirE3 can mimic this VIP1 function, acting as an 'adapter' molecule between VirE2 and karyopherin alpha and 'piggy-backing' VirE2 into the host cell nucleus. As VIP1 is not an abundant protein, representing one of the limiting factors for transformation, Agrobacterium may have evolved to produce and export to the host cells its own virulence protein that at least partially complements the cellular VIP1 function necessary for the T-DNA nuclear import and subsequent expression within the infected cell.     

Floral spray transformation can efficiently generate Arabidopsis transgenic plants

In this study, floral spray and floral dip were used to replace the vacuum step in the Agrobacterium-mediated transformation of a superoxide dismutase (SOD) gene into Arabidopsis. The transgene was constructed by using a CaMV 35S promoter to drive a rice cytosolic CuZnSOD coding sequence in Arabidopsis. The transgene construct was developed in binary vectors and mobilized into Agrobacterium. When Arabidopsis plants started to initiate flower buds, the primary inflorescence shoots were removed and then transformed by floral spray or floral dip. More than 300 transgenic plants were generated to assess the feasibility of floral spray used in the in planta transformation. The result indicates that the floral spray method of Agrobacterium can achieve rates of in planta transformation comparable to the vacuum-infiltration and floral dip methods. The floral spray method opens up the possibility of in planta transformation of plant species which are too large for dipping or vacuum infiltration.     

单、双子叶植物的代谢物调节农杆菌Vir区基因表达的研究

本文研究了六种植物（三种单子叶植物,三种双子叶植物）愈伤组织的 渗出物和抽提物对农杆菌Vir基因表达的调节作用,其调节水平植物的不同而明显不同,但单,双子叶植物的代谢物对Vir基因表达的调节作用并非截然分开,即使在双子叶植物（如大豆）的抽提物与渗出物中也存在着抑制Vir基因表达的因子,而在单子叶植物（如玉米等）的抽提物与渗出物中也存在着促进Vir基因表达的调节因子,Vir位点的调节反应随渗出物与抽提物的种类不同而明显不同,不同Vir位点对同类渗出物或抽提物的反应也不同,渗出物对Vir基因表达的正调节效应优于抽提物,植物渗出物与AS对Vir区基因表达的调节并不表现简单的累加效应或协同作用,相反,在渗出物中还存在着不同程度阻抑AS对Vir基因表达正调节的因子。