Summary
A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens The efficiency of transformation was similar to that obtained by the methods used routinely for transformation of dicotyledons with the bacterium Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R0, R1 and R2 generations Sequence analysis revealed that the boundaries of the T-DNA in transgenic rice plants were essentially identical to those in transgenic dicotyledons Calli induced from scutella were very good starting materials A strain of A tumefaciens that carried a so-called ‘super-binary’ vector gave especially high frequencies of transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.1994.6020271.x

Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.

We attempted to overexpress chalcone synthase (CHS) in pigmented petunia petals by introducing a chimeric petunia CHS gene. Unexpectedly, the introduced gene created a block in anthocyanin biosynthesis. Forty-two percent of plants with the introduced CHS gene produced totally white flowers and/or patterned flowers with white or pale nonclonal sectors on a wild-type pigmented background; none of hundreds of transgenic control plants exhibited such phenotypes. Progeny testing of one plant demonstrated that the novel color phenotype co-segregated with the introduced CHS gene; progeny without this gene were phenotypically wild type. The somatic and germinal stability of the novel color patterns was variable. RNase protection analysis of petal RNAs isolated from white flowers showed that, although the developmental timing of mRNA expression of the endogenous CHS gene was not altered, the level of the mRNA produced by this gene was reduced 50-fold from wild-type levels. Somatic reversion of plants with white flowers to phenotypically parental violet flowers was associated with a coordinate rise in the steady-state levels of the mRNAs produced by both the endogenous and the introduced CHS genes. Thus, in the altered white flowers, the expression of both genes was coordinately suppressed, indicating that expression of the introduced CHS gene was not alone sufficient for suppression of endogenous CHS transcript levels. The mechanism responsible for the reversible co-suppression of homologous genes in trans is unclear, but the erratic and reversible nature of this phenomenon suggests the possible involvement of methylation.

Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans.

Rice (Oryza sativa), a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage, especially in tropical areas. The remaining edible part of rice grains, the endosperm, lacks several essential nutrients, such as provitamin A. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in at least 26 countries, including highly populated areas of Asia, Africa, and Latin America. Recombinant DNA technology was used to improve its nutritional value in this respect. A combination of transgenes enabled biosynthesis of provitamin A in the endosperm.

https://science.sciencemag.org/content/sci/287/5451/303.full.pdf

Engineering the Provitamin A (β-Carotene) Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm

A “plant gene vector cassette” to be used in combination with various Escherichia coli gene-cloning vectors was constructed. This cassette contains a replication and mobilization unit which allows it to be maintained and to be transferred back and forth between E. coli and Agrobacterium tumefaciens hosts provided these hosts contain plasmid RK2 replication and mobilization helper functions. The cassette also harbors a transferable DNA unit with plant selectable marker genes and cloning sites which can be combined with different bacterial replicons, thus facilitating the reisolation of transferred DNA from transformed plants in E. coli. The vector cassette contains two different promoters derived from the T-DNA-encoded genes 5 and nopaline synthase (NOS). By comparing the levels of expression of the marker enzymes linked to each of these promoter sequences, it was found that the gene 5 promoter is active in a tissue-specific fashion whereas this is not the case for the NOS promoter. This observation provides the first documented instance of a gene derived from a procaryotic host the expression of which is apparently regulated by plant growth factors.

https://link.springer.com/content/pdf/10.1007/BF00331014.pdf

The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector

The soil bacterium Agrobacterium tumefaciens is a plant pathogen that causes crown-gall tumours after infection of wounded dicotyledonous plants. Large plasmids (Ti-plasmids) are responsible for the oncogenicity of the bacterium1–3. Crown-gall tumours contain a DNA segment, called the T-DNA, which is homologous with a defined part of the Ti-plasmid present in the tumour-inducing bacterium, and is stably integrated into the plant genome4–7. Apart from the T-DNA another region of the Ti-plasmid-called the vir-region, is essential for tumour induction8–11. We report here the interaction of two compatible plasmids, one containing the vir-region, the other carrying the T-DNA on a wide host-range replicon. An A. tumefaciens strain harbouring both plasmids has a normal tumour-inducing capacity, although neither plasmid is functional alone. With this approach, the T-DNA on one plasmid can, because of its size, be easily genetically manipulated using Escherichia coli as a host. Transfer of this plasmid into an A. tumefaciens strain harbouring the plasmid with the vir-region allows introduction of the manipulated T-DNA into plant cells. In this way, sophisticated binary vector systems for plant genetic engineering can be developed.

A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid

Genetic engineering requires a procedure for introducing DNA into host cells, followed by integration into the host genome and gene expression. Although several procedures for DNA-medi-ated gene transfer in mammalian cells, yeast and bacteria have been reported, no such methods are yet available for plant cells. The major obstacle to DNA uptake in plant cells is the cell wall, but this can be circumvented by using plant protoplasts, cells freed of their cell walls by enzymatic digestion. However, none of the reports on the uptake of DNA into plant protoplasts1,2 has produced conclusive evidence for the integration of DNA into the host genome, that is, that stable transformation occurs. The tumour-inducing bacterium Agrobacterium tumefaciens, which causes crown gall disease, is a natural system for the introduction of foreign DNA into plants. This bacterium introduces part of its tumour-inducing (Ti) plasmid, called T-DNA, into plant cells, where it becomes integrated into the nuclear DNA of the host3,4 and is transcribed into mRNA5,6. T-DNA encodes tumour-specific enzymes responsible for the formation of amino acid derivatives such as octopine or nopaline7, which the bacterium can use as a sole source of carbon and nitrogen. The transformed cells have also acquired the ability to grow in the absence of phytohormones (autotrophy). An in vitro system for infection of Nicotiana tabacum protoplasts by A. tumefaciens has already been reported8. Transformants are selected by their ability to divide and grow in tissue culture without the addition of plant phytohormones to the synthetic culture medium. Here, we report a reproducible method for the stable transformation of tobacco protoplasts with Ti-plasmid DNA, using a similar selection procedure.

In vitro transformation of plant protoplasts with Ti-plasmid DNA

SUMMARY: A mutant of A. tumefaciens strain B6S3, carrying the R factor RP4, was able to transfer its TI plasmid to various avirulent Agrobacterium strains and to a strain of Rhizobium. Strains carrying the TI(B6S3) plasmid were selected by their ability to utilize octopine. The isolates were able to induce tumours and exclude phage AP1. The tumours induced on Kalanchoe daigremontiana were rough and contained octopine. It was concluded that octopine utilization, phage exclusion, induction of rough tumours and synthesis of octopine in the tumours are determined by the TI(B6S3) plasmid. Tumorigenicity was determined this plasmid both in Agro-bacteria and in Rhizobium, suggesting that all the genes necessary for tumour induction are plasmid-borne.

/pdf/transfer-of-the-agrobacterium-tumefaciens-ti-plasmid-to-2c7uyoij2q.pdf

Transfer of the Agrobacterium tumefaciens TI Plasmid to Avirulent Agrobacteria and to Rhizobium ex planta

Bacteria of the genus Rhizobium fix nitrogen in symbiotic association with legumes such as clover, pea and bean. If the efficiency of this plant–bacterium relationship is to be improved or the host range of the rhizobia extended, the genes involved in the symbiosis must first be identified and localized. Accumulating genetic and biochemical data support the proposal that genes on a large plasmid1 are involved in root nodulation, in analogy with the Ti genes of Agrobacterium tumefaciens, which is closely related to the fast-growing species of Rhizobium2,4 (for reviews, see refs 5, 6). Although most of the data concern Rhizobium leguminosarum7–11, some deal with Rhizobium trifolii12–14, which associates with clover. We report here that one particular plasmid in R. trifolii—which we call Sym(biosis) plasmid—not only determines host specificity, but also controls several, if not most, other steps leading to root nodule formation and nitrogen fixation on clover roots.

Sym plasmid of Rhizobium trifolii expressed in different rhizobial species and Agrobacterium tumefaciens

The invention relates to a process for the incorporation of foreign DNA into chromosomes of dicotyledonous plants by infecting the plants or incubating plant protoplasts with Agrobacterium bacteria, which contain one or more plasmids, wherein bacteria are used which contain at least one plasmid having the vir-region of a Ti (tumour inducing) plasmid but no T-region, and at least one other plasmid having a T-region with incorporated therein foreign DNA but no vir-region, as well as to a Agrobacterium bacteria wherein at least one plasmid which has the vir-region of a Ti (tumour inducing) plasmid but no T-region and at least one other plasmid which has a wild type T-region with incorporated in it foreign DNA but no vir-region.

R. A. Schilperoort

Papers

A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid

In vitro transformation of plant protoplasts with Ti-plasmid DNA

Transfer of the Agrobacterium tumefaciens TI Plasmid to Avirulent Agrobacteria and to Rhizobium ex planta

Sym plasmid of Rhizobium trifolii expressed in different rhizobial species and Agrobacterium tumefaciens

Process for the incorporation of foreign dna into the genome of dicotyledonous plants