Stephen G. Rogers

Engineering Herbicide Tolerance in Transgenic Plants

The herbicide glyphosate is a potent inhibitor of the enzyme 5-enolpyruvylshikimate- 3-phosphate (EPSP) synthase in higher plants. A complementary DNA (cDNA) clone encoding EPSP synthase was isolated from a complementary DNA library of a glyphosate-tolerant Petunia hybrida cell line (MP4-G) that overproduces the enzyme. This cell line was shown to overproduce EPSP synthase messenger RNA as a result of a 20-fold amplification of the gene. A chimeric EPSP synthase gene was constructed with the use of the cauliflower mosaic virus 35S promoter to attain high level expression of EPSP synthase and introduced into petunia cells. Transformed petunia cells as well as regenerated transgenic plants were tolerant to glyphosate.

Inheritance of functional foreign genes in plants.

Morphologically normal plants were regenerated from Nicotiana plumbaginifolia cells transformed with an Agrobacterium tumefaciens strain containing a tumor-inducing plasmid with a chimeric gene for kanamycin resistance. The presence of the chimeric gene in regenerated plants was demonstrated by Southern hybridization analysis, and its expression in plant tissues was confirmed by the ability of leaf segments to form callus on media containing kanamycin at concentrations that were normally inhibitory. Progeny derived from several transformed plants inherited the foreign gene in a Mendelian manner.

Gene transfer in plants: Production of transformed plants using Ti plasmid vectors

Publisher Summary This chapter discusses the production of transformed plants using Ti plasmid vectors. Transformed plants are produced by introducing foreign genes into either single-cells (protoplasts) or intact tissues using genetically modified strains of A. tumefaciens , where the tumor-causing genes encoding phytohormone biosynthetic enzymes are specifically deleted and replaced with an appropriate selectable marker gene. The disarmed A. tumefaciens strains retain a full complement of vir -region genes required for transfer DNA (T-DNA) transfer into plant cells. The transformed plant cells are then selected and regenerated into intact plants using special tissue culture methods. The production of transgenic plants can be divided into four main steps: introduction of foreign genes into modified A. tumefaciens strains, cocultivation of A. tumefaciens strains with plant cells or tissues, selection and regeneration of transformants, and analysis and verification of gene expression in transformed plants.

Improved vectors for plant transformation: expression cassette vectors and new selectable markers

Publisher Summary The chapter reviews the vectors and methods based on the Agrobacterium tumefaciens Ti plasmid. The chapter also describes the construction and use of several new cassette plasmids for the expression of gene-coding sequences in plants. Improvements in the basic vectors and selectable markers for the identification of transformants are appearing at an increasing pace. These are leading to increased efficiency and ease of producing transgenic plants as well as for an extension of the range of plant species, which may be transformed. The Agrobacterium tumefaciens Ti plasmid-derived vectors are the easiest and most utilized of the various schemes for the introduction of DNAs into plants. In nature, A. tumefaciens infects most dicotyledonous and some monocotyledonous plants by entry through wound sites. Methotrexate (mtx) is an antimetabolite that inhibits eukaryotic dihydrofolate reductase (dhfr), ultimately preventing the biosynthesis of glycine, thymine, and purines. The expression cassette vectors described in the chapter are used to express coding sequences for a wide range of bacterial, mammalian, and plant genes.

Leaf disc transformation

Leaf disc transformation of tobacco is the paradigm for Agrobacterium-mediated transformation of plant tissues and subsequent selection and regeneration of transgenic plants. This system permits efficient gene transfer, selection and regeneration to be coupled together in a simple process. Tobacco is an excellent host for A. tumefaciens, and also responds exceedingly well in culture. While the technique is most easily practiced with tobacco, it has been applied to a number of other species (Table 1). This example will be described for tobacco, using a vector that confers kanamycin resistance, pMON200 [5].

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 whi...

Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens.

Transformed Arabidopsis thaliana plants have been produced by a modified leaf disk transformation-regeneration method. Leaf pieces from sterilely grown plants were precultured for 2 days and inoculated with an Agrobacterium tumefaciens strain containing an avirulent Ti (tumor-inducing) plasmid with a chimeric gene encoding hygromycin resistance. After cocultivation for 2 days, the leaf pieces were placed on a medium that selects for hygromycin resistance. Shoots regenerated within 3 months and were excised, rooted, and transferred to soil. Transformation was confirmed by opine production, hygromycin resistance, and DNA blot hybridization of both primary transformants and progeny. This process for producing transgenic Arabidopsis plants should enhance the usefulness of the species for experimental biology.

Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants.

Phenotypically normal petunia plants carrying chromosomal inserts of either the tomato golden mosaic virus (TGMV) A or the B component DNA, as single or tandem inserts, were obtained using an Agrobacterium tumefaciens Ti plasmid-based transformation system. Southern hybridization analysis revealed that the tandem, direct-repeat A plants contained free single and double stranded A component DNAs. No free B component DNA was detected in plants carrying tandem repeats of the B component. Progeny of self-fertilized plants appeared normal. In contrast, one-quarter of the progeny from tandem A by tandem B plant crosses showed chlorotic lesions on their leaves similar to virus symptoms. The significance of these results and the use of this method for the study of virus functions involved in TGMV replication and symptom production are discussed.

Genetic analysis of tomato golden mosaic virus: the coat protein is not required for systemic spread or symptom development.

The geminiviruses are a unique group of higher plant viruses that are composed of twin isometric particles which contain circular, single‐stranded DNA. Tomato golden mosaic virus (TGMV), a whitefly‐transmitted agent, belongs to the subgroup of geminiviruses whose members possess a bipartite genome. The TGMV A genome component has the capacity to encode at least four proteins. One of these is the viral coat protein, as inferred by homology with coat‐protein, genes of other geminiviruses and by the observation of typical geminate particles in transgenic plants that contain inserts of TGMV A DNA. We have investigated the role of the coat protein in TGMV replication and report here that its coding sequence may be interrupted or substantially deleted without loss of infectivity. However, certain coat‐protein mutants showed reproducible delays in time of symptom appearance as well as reduced symptom development, when inoculated onto transgenic Nicotiana benthamiana plants containing the TGMV B component. The most attenuated symptoms were seen with a mutant in which the coat‐protein coding sequence was almost entirely deleted. The significance of these findings for the development of plant vectors from TGMV DNA is discussed.

Maize streak virus genes essential for systemic spread and symptom development

The entire genome of single component geminiviruses such as maize streak virus (MSV) consists of a single‐stranded circular DNA of ~2.7 kb. Although this size is sufficient to encode only three average sized proteins, the virus is capable of causing severe disease of many monocots with symptoms of chlorosis and stunting. We have identified viral gene functions essential for systemic spread and symptom development during MSV infection. Deletions and gene replacement mutants were created by site‐directed mutagenesis and insertion between flanking MSV or reporter gene sequences contained in Agrobacterium T‐DNA derived vectors. Following Agrobacterium‐mediated inoculation of maize seedlings, the mutated MSV DNAs were excised from these binary vectors by homologous recombination within the flanking sequences. Our analyses show that the capsid gene of MSV, while not required for replication, is essential for systemic spread and subsequent disease development. The ‘+’ strand open reading frame (ORF) located immediately upstream from the capsid ORF and predicted to encode a 10.9 kd protein was also found to be dispensable for replication but essential for systemic spread. By this analysis, MSV sequences that support autonomous replication were localized to a 1.7 kb segment containing the two viral intergenic regions and two overlapping complementary ‘‐’ strand ORFs. Despite the inability of the gene replacement mutants to spread systemically, both inoculated and newly developed leaves displayed chlorotic patterns similar to the phenotype observed in certain developmental mutants of maize. The similarity of the MSV mutant phenotype to these developmental mutants is discussed.