Marc Van Montagu

A general method for the transfer of cloned genes to plant cells

This paper describes a method for the transfer to plant cells of any cloned gene, regardless of its termini or internal restriction enzyme cleavage sites. A broad host-range intermediate vector, pGV1117, was constructed containing HindIII-23, a right-end T-region fragment of the nopaline plasmid pTiC58. Using in vivo protection by EcoRI methylase and EcoRI linker ligation, a fragment of rabbit chromosomal DNA, carrying the beta-globin gene, was inserted into plasmid pGV1117. Following transmission to Agrobacterium tumefaciens, insertion of the gene into the T-region of pTiC58 occurred via in vivo recombination. Infection of axenic tobacco seedlings resulted in the transfer to the plant genome of an intact beta-globin gene, as part of the T-DNA. Although the gene was stably maintained during tissue culture, beta-globin-specific transcripts were not detected in the transformed plant cells.

Petunia plants escape from negative selection against a transgene by silencing the foreign DNA via methylation

SummaryTransgenicPetunia hybrida clones harbouring the T-DNA gene2 ofAgrobacterium tumefaciens were used to test a strategy for the trapping of plant transposable elements. In thePetunia line used, floral variegation is due to the presence of the non-autonomous transposable elementdTph1 at theAn1 locus. The gene2 product converts the auxin precursor indole-3-acetamide and its analogue 1-naphthalene acetamide into the active auxins indole-3-acetic acid and 1-naphthalene acetic acid. Plant cells that express gene2 can use a low concentration of the precursors as auxins and become sensitive to the toxicity of high concentrations of these compounds. By selecting protoplast-derived microcalli or seedlings able to grow on medium with high precursor concentrations, variant plants were obtained in which gene2 was no longer expressed. Southern analysis, using gene2-specific probes, revealed that in one variant the T-DNA was deleted. For 30 other variants no alteration in gene2 structure was observed, indicating that transposable element insertion was not responsible for the inactivation of gene2. Analysis with restriction enzymes allowing discrimination between methylated or non-methylated DNA sequences showed that the inactivated gene2 sequences were methylated. Addition of the in vivo methylation inhibitor 5-azacytidine to the medium led to reactivation of gene2 expression in some of the variants. These observations demonstrated that reversible DNA methylation was the main cause of silencing of gene2 in this system.

Introduction and expression of the octopine T-DNA oncogenes in tobacco plants and their progeny

Abstract The tumour-inducing T-DNA genes 1, 2 and 4 of the octopine Ti-plasmid pTiAch5 were cloned and introduced into tobacco cells by cocultivation or leaf disk transformation using pTi derived vectors. When a selectable marker was needed, we used a aminoglycoside phosphotransferase II (nos-APH(3′)II) chimeric gene conferring kanamycin resistance to plant cells. The expression of gene 4 in transformed tissue cultures precluded the regeneration of normal transformed plants. Normal transformed plants were obtained with the construction carrying genes 1 or 2. We report in vivo complementation of genes 1 and 2 after crosses of transformed plants. Strategies are described for the use of genes 1 and 2 as selection or screening markers in plant cells or regenerated plants.

Selectable and screenable markers

The transfer of foreign DNA to plant cells can be achieved in a variety of ways that are described in several chapters of this manual. This chapter describes the various selectable and screenable marker genes that are presently used in plant transformation experiments. Screenable markers have been widely used in gene constructs to study the regulation of plant gene expression.

The Use of the Ti Plasmid of Agrobacterium to Study the Transfer and Expression of Foreign DNA in Plant Cells: New Vectors and Methods

Agrobacterium tumefaciens has long been proposed as a vector for the introduction of genetic material into plant cells. Recently, this natural gene transfer system has been modified to allow the transfer of any foreign gene of interest to plant cells in a simple and efficient manner. We will describe some of the new Agrobacterium vectors currently in use in our laboratory, some of the interesting genes which have been introduced into plant cells, as well as the different plant culture methods used for the DNA transfer.

Ti Plasmids as Gene Vectors for Plants

The formation of so-called “crown gall” tumors on dicotyledonous plants is the direct result of the introduction into the nuclear genome of plant cells of a set of genes that regulate cell and organ development. In other words, in nature, a mechanism exists that not only efficiently introduces foreign genes into the plant nucleus, but also contains a set of genes that regulate plant-cell development and differentiation. As a result of this gene transfer, crown gall cells, unlike untransformed plant tissues, can be cultured under axenic conditions on synthetic media in the absence of growth hormones, i.e., cytokinins and auxins.

The development of host vectors for directed gene transfers in plants

For the study of plant developmental biology nature has provided us with an unexpected system with the double advantage of being an efficient gene-vector that already contains a set of purified genes directly involved in the control of developmental processes in plants. This system is the crown gall tumor inducing Ti plasmid of Agrobacterium tumefaciens.

1-Aminocyclopropane-1-carboxylate synthase genes: Present and future

Hormonal regulation of plant growth and development has always received major attention in plant physiology research. In the past half decade this topic has become a highlight amongst plant molecular biologists. Featuring as the smallest, but by far not the least important hormone, ethylene has been studied in quite some detail from a molecular viewpoint (Van Der Straeten and Van Montagu, 1991). The importance of ethylene in plant development and in response to environmental stimuli, has focused the interest of several laboratories on the molecular regulation of ethylene biosynthesis. Genes encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase, the key regulatory enzyme in ethylene biosynthesis, have been cloned and sequenced from tomato (Van Der Straeten et al. 1990), winter squash (Nakajima et al. 1990), and zucchini (Huang et al. 1991; Sato et al. 1991). A putative gene encoding ACC oxidase or Ethylene Forming Enzyme (EFE), catalyzing the final step in ethylene formation, has also been isolated (Hamilton et al. 1990). In addition, the ethylene signal transduction pathway is being dissected by a genetic strategy i.e. by isolation of mutants with altered ethylene responses (Bleecker et al. 1988; Van Der Straeten et al. 1989; Guzman and Ecker, 1990). The corresponding genes may be isolated by the combined use of the restriction fragment length polymorphisms, overlapping cosmids, and yeast artificial chromosome libraries. Together, these molecular approaches to ethylene physiology will not only allow a more profound understanding of basic processes of hormonal gene regulation, but also offer possibilities for interesting agricultural applications.

Purification and Amino-Acid Sequence Analysis of 1-Aminocyclopropane-1-Carboxylic Acid Synthase from Tomato Pericarp

1-Aminocyclopropane-l-carboxylic acid (ACC) synthase (EC 4.4.1.14) was purified 5000-fold from LiCl-induced tomato fruit slices by conventional and high-performance liquid chromatography. In the final preparation the enzyme is estimated 50% pure. Two-dimensional gel electrophoresis indicates that ACC synthase activity is associated with a 45-kD polypeptide.