Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants.

A two-component cloning system to transfer foreign DNA into plants was derived from the octopine Ti plasmid pTiB6S3. pGV2260 is a non-oncogenic Ti plasmid from which the T-region is deleted and substituted by pBR322. pGV831 is a streptomycin-resistant pBR325 derivative that contains a kanamycin resistance marker gene for plant cells and a site for cloning foreign genes between the 25-bp border sequences of the octopine T-region. Conjugative transfer of pGV831 derivatives to Agrobacterium and cointegration by homologous recombination between the pBR322 sequences present on pGV831 and pGV2260, can be obtained in a single step. Strains carrying the resulting cointegrated plasmids transfer and integrate T-DNA into the genome of tobacco protoplasts, and transformed tobacco calli are readily selected as resistant to kanamycin. Intact plants containing the entire DNA region between the T-DNA borders have been regenerated from such clones. In view of these properties we present pGV831 and its derivatives as vectors for efficient integration of foreign genes into plants.     

Chimeric vector construction for higher-plant transformation

A chimeric vector pKR612B1 was developed containing the neomycin phosphotransferase (APH) gene from the Tn5 transposon under the control of the gene VI promoter of cauliflower mosaic virus (CaMV), and was used to transform higher plant protoplasts. Plasmid pDOB612, the parental vector of pKR612B1, has two unique restriction sites, SmaI and BamHI, positioned just downstream of the CaMV gene VI promoter sequence. These unique cloning sites can be used for any kind of gene insertion into this vector. Using the polyethylene glycol transformation procedure, a large number of turnip and tobacco protoplasts were transformed and proved to be resistant to kanamycin (Km). From tobacco protoplasts whole Km-resistant plants were regenerated and shown to contain the integrated foreign gene. APH activity was detected in both transformed calli and in regenerated plants. DNA from transformed clones was analysed by Southern blot hybridization, showing the presence of the Tn5-derived gene.     

Genetic transformation in higher plants

Successful transformation of plant cells has been obtained utilizing vectors and DNA delivery methods derived from the plant pathogen, Agrobacterium tumefaciens. This soil bacterium is capable of transferring a DNA segment (T‐DNA), located between specific nucleotide border sequences, from its large tumor inducing (Ti) plasmid into the nuclear DNA of infected plant cells. The exploitation of the Agrobacterium/Ti plasmid system for plant cell transformation has been facilitated by (1) the construction of modified Agrobacterium strains in which the genes responsible for pathogenicity have been deleted; (2) the design of intermediate vectors containing selectable drug markers for introducing foreign genes into the Ti plasmid and subsequently into plant cells; and (3) the development of efficient in vitro methods for transforming plant cells and tissues with engineered Agrobacterium strains. These modifications have led to the development of a simple, efficient, and reproducible transformation system from which morphologically normal transformed plants can be readily regenerated. The foreign genes are stably maintained and expressed in the resulting plants and are inherited by progeny as typical Mendelian traits. The availability of transformation systems has already facilitated numerous studies on gene expression and regulation in plants and should eventually allow for the modification of various crop species in an agronomically significant manner. The needs and possibilities for the development of alternate vectors and transformation procedures will be discussed.     

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.     

Gene targeting in plants using theAgrobacterium vector system

In the past decade several methods have been developed for the introduction of foreign DNA into plant cells to obtain transgenic plants. In some of these methods, purified DNA is directly introduced into protoplasts that for some species can be regenerated into mature plants. The more commonly used protocols, however, employ the natural capacity ofAgrobacterium tumefaciens to transfer a defined peice of DNa, called T-DNA, to the nucleus of plant cells that are more easy to regenerate than protoplasts. In plant cells, like in animal cells, foreign DNA (including T-DNA) is readily inserted into the genome via illegitimates recombination. In contrast, targeted integration via homologous recombination, referred to as ‘gene targeting’, can only be obtained at relatively low frequencies. Nevertheless, gene targeting has become a standard strategy for reverse genetics studies in animals. In plants, the occurrence of gene targeting was only reported recently. This review focuses on the use of theAgrobacterium vector system to achieve gene targeting in plants. Recent experimental data concerning gene targeting in plants are presented and the overall suitability ofAgrobacterium T-DNA transfer for this purpose is assessed in light of contemporary views on the mechanism of T-DNA transfer.     

Direct gene transfer to plants

Evidence for direct, gene-mediated stable genetic transformation of plant cells of Nicotiana tabacum is presented. A selectable hybrid gene comprising the protein coding region of the Tn5 aminoglycoside phosphotransferase type II gene under control of cauliflower mosaic virus gene VI expression signals was introduced into plant protoplasts as part of an Escherichia coli plasmid. The gene was stably integrated into plant genomic DNA and constitutively expressed in selected, drug resistant, protoplast-derived cell clones. The mode of integration of the foreign gene into the plant genome resembled that observed for DNA transfection of mammalian cells. Plants regenerated from transformed cell lines were phenotypically normal and fertile, and they maintained and expressed the foreign gene throughout the development of vegetative and generative organs. Microspores, grown in anther culture, developed into resistant and sensitive haploid plantlets. Genetic crossing analysis of one of the transformed plants revealed the presence of one dominant trait for kanamycin resistance segregating in a Mendelian fashion in the F₁ generation.     

Direct Gene Transfer in Psophocarpus tetragonolobus Resistance to Kanamycin

Epicotyl-derived protoplasts of Psophocarpus tetragonolobuswere isolated and regenerated to plants. These protoplasts weretransformed to kanamycin resistance following uptake of plasmid(pABDl or pHP23) DNA in combination with PEG treatment. Protoplast-derivedtransformed colonies were selected on kanamycin (75 mg l^–1).The transformed calli expressed NPT II activity and also exhibitedthe presence of the plasmid gene integrated into the plant genome.However, none of the transformed clones showed regenerationof shoot buds. Psophocarpus tetragonolobus, winged bean, naked DNA transformation, protoplast culture, regenerated plants     

Transformation and regeneration of Brassica oleracea mediated by an oncogenic Agrobacterium tumefaciens

A chimaeric neomycin phosphotransferase II (NPT II) gene was introduced in Brassica oleracea using an oncogenic strain of Agrobacterium tumefaciens harbouring Ti plasmid which contains Nos/NPTII in its T-DNA. The transformation of B. oleracea with the oncogenic Ti plasmid, resulted in regeneration of shoots and roots without any exogenous requirement of phytohormones. The presence of NPT II gene was determined by hybridization of Tn5 encoded NPT II gene with DNA of kanamycin resistant regenerated plants. The expression of NPT II was demonstrated by kanamycin phosphorylation assay. Several regenerated plants were obtained, a few of them were found to be morphological variants and a chlorophyll deficient mutant plant was also obtained.     

A set of novel Ti plasmid-derived vectors for the production of transgenic plants

Summary We describe in this paper the construction and use of a set of novel Ti plasmid-derived vectors that can be used to produce transgenic plants. These vectors are based on one of two strategies: 1) double recombination into the wild-type Ti plasmid of genetic information flanked by two T-DNA fragments on a wide-host range plasmid; 2) the binary vector strategy. The vector based on the double recombination principle contains a kanamycin resistance gene for use as a plant selectable marker, a polylinker for the insertion of foreign genes, and a nopaline synthase gene. The vector was constructed such that a disarmed T-DNA results from the double recombination event. The binary vector combines several advantageous features including an origin of replication that is stable in Agrobacterium in the absence of selection, six unique sites for insertion of foreign genes, an intact nopaline synthase gene, and a kanamycin resistance marker for selection of transformed plant cells. All of these vectors have been used to produce tobacco plants transformed with a variety of foreign genes.     

Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts

Summary Genetic transformation of Nicotiana tabacum protoplasts was achieved by incubation of protoplasts with a plasmid DNA-calcium phosphate coprecipitate, followed by fusion of the protoplasts in the presence of polyvinyl alcohol and subsequent exposure to high pH. A derivative of the plasmid pBR322 containing a chimaeric gene, consisting of the nopaline synthase promoter, the coding region of the aminoglycoside phosphotransferase gene of Tn5 and the polyadenylation signal region of the octopine synthase gene, was used for these transformation experiments. This chimaeric gene confers resistance of transformed plant cells to kanamycin. This novel transformation procedure reproducibly yielded transformants at frequencies of approximately 0.01%. Aminoglycoside phosphotransferase II activity was detected in both transformed calli and in regenerated plants. DNA from some of the transformed clones was analyzed by Southern blot hybridization. The input DNA appears to be integrated into high molecular weight cellular DNA. Genetic analysis of one of the kanamycin resistant plants shows that the chimaeric gene is transmitted to the progeny as a single dominant trait in a Mendelian fashion. As a comparison the input DNA was also introduced into tobacco protoplasts using Agrobacterium tumefaciens and Ti-plasmid derived gene vectors.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Superfluous Transgene Integration in Plants

Referee: Dr. Paul Hooykass, Institut of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333, Al Leiden, Netherlands Recent reports suggest the transfer of superfluous DNA sequences to plant genomes during transformation processes. This review investigates the evidence from the published literature for the prevalence of this phenomenon and highlights methods to limit or prevent DNA transfer and subsequent potentially detrimental evolutionary consequences. Evidence for superfluous foreign DNA transfer using both Agrobacterium-mediated transformation and direct DNA transfer methods such as microprojectile bombardment and PEG-mediated transformation of protoplasts is reported. In the case of Agrobacterium-mediated transformation, the lack of information on the integration of sequences from outside of the T-DNA borders has been due to the general belief by researchers that T-DNA processing is precise. This assumption was based on analysis of T-DNA in tumors and as a result the majority of T-DNA integration events have been identified exclusively using DNA probes, which are homologous only to DNA from within the T-DNA borders. Where direct gene transfer protocols are employed, any part of the transforming plasmid and indeed accompanying carrier DNA may become integrated into the plant genome. The main body of evidence proving that superfluous vector DNA sequences are present in plant genomes transformed using direct transfer methods is confined to the identification of plasmid concatamers integrated into plant genomes. The limited amount of recorded evidence pertaining to superfluous vector DNA integration in transgenic plants and transformed tissues makes it impossible to draw definitive conclusions as to the factors involved in promoting this phenomenon. However, there are methods available for removing superfluous sequences from transgenic plants. These have been developed for the removal of selectable marker genes, whose presence in transgenic plants has been a source of much controversy, but can equally be applied to other DNA sequences. Suggestions have been made in the review that might limit or prevent the integration of superfluous vector sequences during transformation procedures; however, these are not proven and further research is required.     

Genetic analysis of T-DNA transcripts in nopaline crown galls

Plant crown gall tumor cells result from the insertion and expression of a defined DNA sequence, called T-DNA, which is derived from the Ti plasmid, harbored by Agrobacterium tumefaciens strains. To study the function of the genes of the T-DNA of the nopaline Ti plasmid, pTiC58, a collection of mutants was isolated so that T-DNA genes are inactivated either separately or in various combinations. It was found that no single T-DNA gene or T-region border is absolutely essential for stable tumor formation. We have identified the gene responsible for synthesis in transformed cells of the phosphorylated sugar, agrocinopine, and at least three additional genes controlling the morphology of plant tumors. Two of these latter genes work together to inhibit shoot formation and ensure efficient tumorous growth. Inactivation of these genes can be suppressed by the addition of auxins. The third gene inhibits root formation and appears to play a role in the cytokinin-independent growth of transformed cells. Mutants missing all three genes do not induce tumors, nor shoot or root formation, although the mutant T-DNA sequence is transferred to plant cells.     

Expression of foreign genes in regenerated plants and in their progeny

Chimeric genes comprised of the nopaline synthase promoter and bacterial coding sequences specifying resistance to kanamycin, chloramphenicol or methotrexate, were inserted into the non-oncogenic Ti plasmid vector pGV3850 by recombination (through homologous pBR322 sequences present in the chimeric gene constructs and pGV3850). These co-integrates in Agrobacterium were used to infect single plant protoplasts of Nicotiana by co-cultivation. The resistance traits allowed the selection of transformed calli in tissue culture in the presence of the appropriate antibiotic. Furthermore, as a non-oncogenic Ti plasmid was used for the protoplast transformation, phenotypically normal and fertile plants could be regenerated from the resistant calli. We have shown that these fully differentiated plant tissues exhibit functional expression of resistance traits (Km^R and Cm^R). All plants carrying the chimeric genes developed normally, flowered, and set seeds. The inheritance of several of these resistance traits was analyzed and shown to be Mendelian. These results are model experiments to demonstrate that genes of interest can be systematically transferred to the genome of plants using non-oncogenic Ti plasmid derivatives; and that transformed plants are capable of normal growth and differentiation, thus providing a natural environment for the study of gene expression and development of plant cells.     

Transgenic tomato (Lycopersicon esculentum L.) transformed with a binary vector in Agrobacterium rhizogenes: Non-chimeric origin of callus clone and low copy numbers of integrated vector T-DNA

Summary We transformed tomato (Lycopersicon esculentum L.) by using Agrobacterium rhizogenes containing two independent plasmids: the wild-type Ri-plasmid, and the vector plasmid, pARC8. The T-DNA of the vector plasmid contained a marker gene (Nos/Kan) encoding neomycin phosphotransferase which conferred resistance to kanamycin in transformed plant cells. Transgenic plants (R ₀) with normal phenotype were regenerated from transformed organogenic calli by the punctured cotyledon transformation method. Southern blot analysis of the DNA from these transgenic plants showed that one or two copies of the vector plasmid T-DNA, but none of the Ri-plamid T-DNA, were integrated into the plant genome. Different transgenic plants derived from the same callus clone showed an identical DNA banding pattern, indicating the non-chimeric origin of these plants. We also transformed tomato by using A. tumefaciens strain LBA4404 containing a disarmed Ti-plasmid (pAL4404), and a vector plasmid (pARC8). Transgenic plants derived via A. tumefaciens transformation, like those via A. rhizogenes, contained one to two copies of the integrated vector T-DNA. The kanamycin resistance trait in the progeny (R ₁) of most transgenic plants segregated at a ratio of 3:1, suggesting that the vector T-DNAs were integrated at a single site on a tomato chromosome. In some cases, the expression of the marker gene (Nos/Kan) seemed to be suppressed or lost in the progeny.     

Mechanisms of crown gall formation: T-DNA transfer fromAgrobacterium tumefaciens to plant cells

Agrobacterium tumefaciens harbouring the Ti plasmid incites crown gall tumor on dicotyledonous species. Upon infection of these plants, T-DNA in the Ti plasmid is transferred by unknown mechanisms to plant cells to be integrated into nuclear DNA. WhenAgrobacterium is incubated with protoplasts or seedlings of dicotyledonous plants, circulation of T-DNA and expression ofvir (virulence) genes on the Ti plasmid are induced. The circularization event is efficiently induced by mesophyll protoplasts of tobacco which are highly competent for transformation by the T-DNA, and is also induced by diffusible phenolic compounds excreted from the protoplasts. The circularization and formation of crown gall both require the expression of thevirD locus, one of the induciblevir genes. These results suggest that the circularization of T-DNA reflects one of steps of the T-DNA transfer during formation of crown gall. In contrast to dicotyledonous plants, monocotyledonous plants are thought to be unresponsive to infection byAgrobacterium. We showed that monocotyledonous plants do not excrete diffusible inducers for the expression ofvir genes, while they contain a novel type of a signal substance(s). This inducer is not detected in the exudates of seedlings of monocotyledonous plants, but is found in the extracts from the seedlings, and also those from the seeds, bran and germ of wheat and oats. This finding suggests that T-DNA processing, and possibly its transfer, should take place whenAgrobacterium invades seedlings and seeds of monocotyledonous plants. Recipient of the Botanical Society Award for Young Scientists, 1987.     

Genetic analysis of integration mediated by single T-DNA borders.

Transformation of plant cells by the T-DNA of the Ti plasmid of Agrobacterium tumefaciens depends in part upon a sequence adjacent to the right T-DNA end. When this sequence is absent, the T-DNA is almost avirulent; when it is present, DNA between it and the left T-DNA border region becomes integrated in plants. To investigate further this process of DNA transfer and integration, we introduced the right border region and the nopaline synthase (nos) gene of plasmid pTiC58 into a variety of new positions around Ti plasmids. The border region functioned when separated from the remainder of the T-DNA by almost 50 kilobases. It also worked when placed outside of the T-DNA region where there were no known left-border sequences with which to interact. Indeed, the nos gene could be transferred to plants even when no other Ti plasmid sequences were present on the same plasmid. These results may indicate that the sequence requirements for the left borders are not as stringent as those for the right borders. In addition, mutants with an extra copy of the right border region within their T-DNA were found to transfer or integrate only parts of the bacterial T-DNA region. It is possible that abnormally placed T-DNA borders interfere with the normal process of DNA transfer, integration, or both.     

Opine synthesis in wild-type plant tissue

Opine production is associated with crown gall tissue, a neoplastic growth caused by infection of dicotyledonous plants with Agrobacterium tumefaciens. Recent publications have claimed that tissues of certain monocotyledonous plants can also be infected by Agrobacterium. Following infection, a part of the Agrobacterium Ti plasmid, T-DNA, is integrated into the chromosome of the infected plant. T-DNA, which codes for opine-synthesizing enzymes, is now used to add foreign genes to plants. A number of laboratories have used opine production in plant tissue, often after arginine feeding or preincubation as evidence for plant transformation by T-DNA vectors. In this report we provide microbiological, chromatographic, spectroscopic and chemical evidence indicating that opines can be formed in normal callus and plant tissue as a result of arginine metabolism. Therefore, researchers studying T-DNA should be aware of the capability of plant tissue to metabolize arginine to opines. Opine production following infection with T-DNA may not always be sufficient evidence to indicate transformation by the Agrobacterium Ti plasmid.