Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation

When Agrobacterium was used to transform Nicotiana plumbaginifolia protoplasts and Arabidopsis thaliana roots and seedlings, a large number of plants were found in which not only the T-region defined by the border repeat sequences but the entire binary vector was integrated, as determined by both PCR and Southern analysis techniques. N. plumbaginifolia protoplast co-cultivation experiments yielded 3 out of 5 transformants with collinear sequence past the left border. In Arabidopsis root transformation experiments, 33% (6/18) of the transformants had T-DNA which exceeded the left border repeat. Vacuum infiltration of Arabidopsis seedlings produced even a greater percentage of transformants with sequences outside the left border repeat (62%, 39/63). The long transfer DNA cosegregated with the T-region encoded hygromycin resistance in the T2 progeny eliminating the possibility that long transfer DNA was of extrachromosomal or Agrobacterium origin. The high frequency of long transfer after vacuum infiltration of A. thaliana needs to be considered when analyzing T-DNA tagged mutants.     

pBINPLUS: An improved plant transformation vector based on pBIN19

We describe the construction of a new plant transformation vector, pBINPLUS, based on the popular pBIN19 vector. Improvements over pBIN19 include location of the selectable marker gene at the left T-DNA border, a higher copy number inE. coli, and two rare restriction sites around the multiple cloning site for easier cloning and analysis of T-DNA insertions in plant genomes.     

Radiation-sensitive Arabidopsis mutants are proficient for T-DNA transformation.

Stable transformation of plants by Agrobacterium T-DNAs requires that the transgene insert into the host chromosome. Although most of the Agrobacterium Ti plasmid genes required for this process have been studied in depth, few plant-encoded factors have been identified, although such factors, presumably DNA repair proteins, are widely presumed to exist. It has previously been suggested that the UVH1 gene product is required for stable T-DNA integration in Arabidopsis. Here we present evidence suggesting that uvh1 mutants are essentially wild type for T-DNA integration following inoculation via the vacuum-infiltration procedure. Received: 23 June 1998 / Accepted: 21 February 1999     

pORE: a modular binary vector series suited for both monocot and dicot plant transformation

We present a series of 14 binary vectors suitable for Agrobacterium-mediated transformation of dicotyledonous plants and adaptable for biolistic transformation of monocotyledonous plants. The vector size has been minimized by eliminating all non-essential elements from the vector backbone and T-DNA regions while maintaining the ability to replicate independently. The smallest of the vector series is 6.3 kb and possesses an extensive multiple cloning site with 21 unique restriction endonuclease sites that are compatible with common cloning, protein expression, yeast two-hybrid and other binary vectors. The T-DNA region was engineered using a synthetic designer oligonucleotide resulting in an entirely modular system whereby any vector element can be independently exchanged. The high copy number ColE1 origin of replication has been included to enhance plasmid yield in Escherichia coli. FRT recombination sites flank the selectable marker cassette regions and allow for in planta excision by FLP recombinase. The pORE series consists of three basic types; an ‘open’ set for general plant transformation, a ‘reporter’ set for promoter analysis and an ‘expression’ set for constitutive expression of transgenes. The sets comprise various combinations of promoters (P _HPL, P _ENTCUP2 and P _TAPADH), selectable markers (nptII and pat) and reporter genes (gusA and smgfp).     

A plant transformation vector with a minimal T-DNA

Plant transformation, viaAgrobacterium tumefaciens, is usually performed with binary vectors. Most of the available binary vectors contain within the T-DNA (which is transferred to the plant genome) components not required for the intended modification. These additional sequences may cause potential risks during field testing of the transgenic plants or even more in the case of commercialization. The aim of this study was to produce a plant transformation vector which only contains a selectable and screenable marker gene and a multiple cloning site for insertion of promoter::foreign gene::terminator cassettes from other plasmids.     

Non-oncogenic plant vectors for use in the agrobacterium binary system

Summary Agrobacterium strains harbouring the T-region and the virulence-region of the Ti plasmid on separate replicons still display efficient T-DNA transfer to plants. Based on this binary vector strategy we have constructed T-region derived gene vectors for the introduction of foreign DNA into plants. The vectors constructed can replicate in E. coli, thus the genetic manipulations with them can be performed with E. coli as a host. They can be transferred to Agrobacterium as a cointegrate with the wide host range plasmid R772. Their T-regions are transferred to plant cells from Agrobacterium strains conferring virulence functions.The plasmid pRAL 3940 reported here is 11.5 kb large, contains a marker to identify transformed plant cells and unique restriction sites for direct cloning of passenger DNA, flanked by the left- and right-hand border fragments of the T-region (including the 25 bp border repeats). The plasmid is free of onc-genes. Therefore, is does not confer tumorigenic traits on the transformed plant cells and mature, fertile plants can thus be regenerated from them.     

Construction of small binary vectors forAgrobacterium-Mediated transformation in plants

Despite the popular use of theAgrobacterium-mediated method for transforming plants, only a very limited number of binary vectors are available. In addition, those binary vectors are rather inconvenient for plasmid manipulation because of their large size. Here, we report the construction of small binary vectors, pBmin1 and pBminGFP1, forAgrobacterium-mediated transformation. The sizes of pBmin1 and pBminGFP1 are approximately 5.0 kb and 6.5 kb, respectively. These vectors contain one kanamycin resistance gene, with a double promoter for expression in both bacteria and plant cells. Vector pBminGFP1 has the 35S-GFP chimeric gene as a reporter. These small binary vectors would greatly facilitate plasmid manipulation and plant transformation.     

A plant transformation vector with a minimal T-DNA II. Irregular integration patterns of the T-DNA in the plant genome

A minimal T-DNA binary vector was used for Agrobacterium-mediated transfer of a chimeric T4 lysozyme gene located next to the left border, and transgenic potato plants which expressed T4 lysozyme protein were identified and further analysed. Frequent rearrangements of T4 lysozyme transgenes were detected. A vector derivative containing two matrix associated regions (MARs) flanking its multiple cloning site was constructed. In transgenic potato plants, reduced variability in gene expression due to position effects was detected. When either the donor vector contained MAR sequences, or when vector pPCV701 which contains a pBR322 fragment next to the left border were used, only relatively few rearrangements were observed. However, when the T4 lysozyme gene was driven by a CaMV 35S promoter modified by multiplied enhancer region carrying either 2 or 4 elements, frequent rearrangements were again obtained.     

pBIN20: An Improved Binary Vector for shape Agrobacterium-mediated Transformation

We report the construction of a binary vector for Agrobacterium tumefaciens-mediated transformation, pBIN20, which contains a superlinker region located between the left and right Ti border sequences. This vector, derived from pBI121, simplifies the cloning of plant expression cassettes and has been used in our laboratory to create lines of transgenic BY-2 tobacco cells. This new vector contains more than 20 unique restriction sites as well as the nptII selectable marker gene within the Ti-DNA borders.     

An efficient Agrobacterium-mediated transient transformation of Arabidopsis

Agrobacterium tumefaciens-mediated transient transformation has been a useful procedure for characterization of proteins and their functions in plants, including analysis of protein-protein interactions. Agrobacterium-mediated transient transformation of Nicotiana benthamiana by leaf infiltration has been widely used due to its ease and high efficiency. However, in Arabidopsis this procedure has been challenging. Previous studies suggested that this difficulty was caused by plant immune responses triggered by perception of Agrobacterium. Here, we report a simple and robust method for Agrobacterium-mediated transient transformation in Arabidopsis. AvrPto is an effector protein from the bacterial plant pathogen Pseudomonas syringae that suppresses plant immunity by interfering with plant immune receptors. We used transgenic Arabidopsis plants that conditionally express AvrPto under the control of a dexamethasone (DEX)-inducible promoter. When the transgenic plants were pretreated with DEX prior to infection with Agrobacterium carrying a β-glucuronidase (GUS, uidA) gene with an artificial intron and driven by the CaMV 35S promoter, transient GUS expression was dramatically enhanced compared to that in mock-pretreated plants. This transient expression system was successfully applied to analysis of the subcellular localization of a cyan fluorescent protein (CFP) fusion and a protein-protein interaction in Arabidopsis. Our findings enable efficient use of Agrobacterium-mediated transient transformation in Arabidopsis thaliana.     

Yeast-based recombineering of DNA fragments into plant transformation vectors by one-step transformation

T-DNA binary vectors are often used in plant transformation experiments. Because they are usually very large and have few restriction sites suitable for DNA ligation reactions, cloning DNA fragments into these vectors is difficult. We provide herein an alternative to cloning DNA fragments into very large vectors. Our yeast-based recombineering method enables DNA fragments to be cloned into certain types of T-DNA binary vectors by one-step transformation without the requirement of specific recombination sites or precisely positioned restriction ends, thus making the cloning process more flexible. Moreover, this method is inexpensive and is applicable to multifragment cloning.     

Identification of T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium

We have identified T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium tumefaciens (rat mutants). These mutants are highly recalcitrant to the induction of both crown gall tumors and phosphinothricin-resistant calli. The results of transient GUS (β-glucuronidase) assays suggest that some of these mutants are blocked at an early step in the Agrobacterium-mediated transformation process, whereas others are blocked at a step subsequent to translocation of T-DNA into the nucleus. Attachment of Agrobacterium to roots of the mutants rat1 and rat3 was decreased under various incubation conditions. In most mutants, the transformation-deficient phenotype co-segregated with the kanamycin resistance encoded by the mutagenizing T-DNA. In crosses with susceptible wild-type plants, the resistance phenotype of many of these mutants segregated either as a semi-dominant or dominant trait. Received: 26 October 1998 / Accepted: 8 January 1999     

Development of a binary vector system for plant transformation based on the supervirulent Agrobacterium tumefaciens strain Chry5

This report describes the disarming of Agrobacterium tumefaciens Chry5, a strain highly tumorigenic on soybean. Disarming was achieved by removing an approximately 16.5-kb segment of the 285-kb Ti plasmid pTiChry5, including approximately 4 kb of the oncogenic T-DNA and an extended region right of the T-DNA, and replacing it with a gene for carbenicillin resistance, through homologous recombination. The deletion was confirmed with Southern analysis, and the loss of tumorigenicity was verified in tobacco and tomato plant stem inoculation assays. The deletion mutant, named KYRT1, successfully transferred the β-glucuronidase (GUS) gene into tobacco leaf tissue, producing GUS-expressing callus which could be regenerated into viable plants. In a comparative study, the transformation efficiency of A. tumefaciens KYRT1, GV3850, and EHA105 was assayed by inoculating cotyledonary node explants. The results of this study revealed that, in a binary vector system, KYRT1 is equally or more effective than EHA105 or GV3850 at delivering DNA into soybean. Received: 30 January 1997 / Revision received: 10 June 1997 / Accepted: 5 July 1997     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation system for large-scale producion of transgenic chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp.<Emphasis Type="Italic">pekinensis</Emphasis>) plants for insertional mutagenesis

In order to better utilize insertional mutagenesis and functional genomics in Chinese cabbage, we have developed an improved transformation system that more efficiently produces a large number of transgenic plants. Hypocotyl explants were inoculated withAgrobacterium tumefaciens LBA4404. This strain harbors tagging vector pRCV2, which contains a hygromycin-resistance gene, an ampicillin resistance gene, and a bacterial replication origin within the T-DNA. Transformation efficiency was highest when the explants were first co-cultivated for 3 d in a medium supplemented with 5 mg L^-1 acetosyringone, then transferred to a 0.8% agar selection medium containing 10 mg L^-1 hygro-mycin. In addition, maintaining a low pH in the co-cultivation medium was critical to enhancing transformation frequency. A total of 3369 transgenic plants were obtained, with efficiencies ranging from 2.89% to 5.00%. Southern blot analysis and T, progeny tests from 120 transgenic plants confirmed that the transgenes were stably inherited to the next generation. We also conducted plasmid rescue and inverse PCR with some transformants, based on their phenotype, to demonstrate the applicability of T-DNA tagging in Chinese cabbage. The tagged sequences were then analyzed.     

High-frequency linkage of co-expressing T-DNA in transgenic Arabidopsis thaliana transformed by vacuum-infiltration of Agrobacterium tumefaciens

The efficiency of co-expression and linkage of distinct T-DNAs present in separate Agrobacterium tumefaciens was analysed in Arabidopsis thaliana transformed by the vacuum infiltration method. Co-expression was monitored by the synthesis of three bacterial proteins involved in the production of polyhydroxybutyrate (PHB) in the plastids. Out of 80 kanamycin- resistant transgenic plants analysed, 13 plants were co-transformed with the two distinct T-DNAs and produced PHB. Of those, 7 lines had a kanamycin-resistance segregation ratio consistent with the presence of a single functional insert. Genetic linkage between the distinct T-DNAs was demonstrated for all 13 PHB-producing lines, while physical linkage between the distinct T-DNAs was shown for 12 out of 13 lines. T-DNAs were frequently linked in an inverted orientation about the left borders. Transformation of A. thaliana by the co-infiltration of two A. tumefaciens containing distinct T-DNAs is, thus, an efficient approach for the integration and expression of several transgenes at a single locus. This approach will facilitate the creation and study of novel metabolic pathways requiring the expression of numerous transgenes. Received: 7 May 1999 / Accepted: 28 July 1999     

A risk assessment study of plant genetic transformation usingAgrobacterium and implications for analysis of transgenic plants

Agrobacterium transformation systems forBrassica, Solanum andRubus, using carbenicillin, cefotaxime and ticaracillin respectively to eliminate contamination, were examined for the presence of residualAgrobacterium. The results indicated that none of the antibiotics in question, succeeded in eliminatingAgrobacterium and the contamination levels increased in explants from 12 to 16 weeks to such an extent thatSolanum cultures senesced and died. This may be due to the fact that four times the Minimum bactericidal concentration values (concentration to be used for elimination of contaminants in culture), for the three antibiotics, were higher than the concentrations employed in the culture medium. Contamination in shoot material decreased over 16 to 24 weeks possibly due to bacteriostatis and the use only of the apical node for further culture. The presence of the binary vector was also noted under non-selective conditions, even up to 6 months after transformation, where approx. 50% of contaminated material still harboured bacterial cells with the binary vector at levels of approx. 10₇ Colony forming units per gram.     

New plant binary vectors with selectable markers located proximal to the left T-DNA border

Five new binary vectors have been constructed which have the following features: (1) different plant selectable markers including neomycin phosphotransferase (nptII), hygromycin phosphotransferase (hpt), dihydrofolate reductase (dhfr), phosphinothricin acetyl transferase (bar), and bleomycin resistance (ble); (2) selectable markers are located near the T-DNA left border and; (3) selectable marker and -glucuronidase (uidA) reporter genes are divergently organized for efficient expression, and can easily be removed or replaced as needed.     

根癌农杆菌介导库拉索芦荟遗传转化因素优化

以美国库拉索芦荟的横切薄片(transversethincelllayer,tTCL)作为转化外植体,初步研究了以根癌 农杆菌介导的多种因子对芦荟遗传转化的影响。结果表明:菌株EHA105比LBA4404及AGL1转化率高; 除了乙酰丁香酮(acetosyringone)外,菌液的预处理和重悬液的pH值也是影响转化的主要因子;菌液的预处 理和适合的蔗糖浓度对转化也有促进作用;感染时间为12～18min,共培养的温度和时间分别以25℃及5d 为佳。     

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.