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

Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5

A Ti plasmid mutant was constructed in which all the on-cogenic functions of the T-DNA have been deleted and replaced by pBR322 This Ti plasmid, pGV3850, still mediates efficient transfer and stabilization of its truncated T-DNA into infected plant cells Moreover, integration and expression of this minimal T-DNA in plant cells does not interfere with normal plant cell differentiation A DNA fragment cloned in a pBR vector can be inserted in the pGV3850 T-region upon a single recombination event through the pBR322 region of pGV3850 producing a co-integrate useful for the transformation of plant cells Based upon these properties, pGV3850 is proposed as an extremely versatile vector for the introduction of any DNA of interest into plant cells

Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity.

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 F(1) generation.

/pdf/direct-gene-transfer-to-plants-1cqds2cwmi.pdf

Direct gene transfer to plants.

Foreign genes introduced into plant cells with Ti-plasmid vectors are not expressed. We have constructed an expression vector derived from the promoter sequence of nopaline synthase, and have inserted the coding sequences of the octopine synthase gene and a chloramphenicol acetyltransferase gene into this vector. These chimaeric genes are functionally expressed in plant cells after their transfer via a Ti-plasmid of Agrobacterium tumefaciens.

R. Nagaraja Rao

Papers

Plasmid pKC7: a vector containing ten restriction endonuclease sites suitable for cloning DNA segments.

Cloning of restriction and modification genes in E. coli: The HhaII system from Haemophilus haemolyticus

A thermoinducible λ phage-ColE1 plasmid chimera for the overproduction of gene products from cloned DNA segments

Characterization of plasmids from Haemophilus parainfluenzae and Haemophilus haemolyticus.