We have constructed a nuclear genomic library from the cruciferous plant Arabidopsis thaliana ecotype Columbia in a cosmid vector, pLZO3, and a host organism, Agrobacterium tumefaciens AGL1, which can directly DNA-transform the parent organism, Arabidopsis. The broad host range cosmid pLZO3 carries a gentamicin acetyltransferase gene as bacterial selective marker and tandem, chimeric neomycin and streptomycin phosphotransferase genes as plant selective markers. Agrobacterium AGL1 carries the hypervirulent, attenuated tumor-inducing plasmid pTiBo542 from which T-region DNA sequences have been precisely deleted, allowing optimal DNA transformation of many dicotyledonous plants. Agrobacterium AGL1 also carries an insertion mutation in its recA general recombination gene, which stabilizes the recombinant plasmids. The Arabidopsis genomic library consists of some 21,600 clones gridded onto 96-well microtiter dishes and, if random, carries at least three genomic equivalents. When probed for the presence of several Arabidopsis low copy-number genes, the genomic library seems representative. As with the unicellular organisms Escherichia coli and Saccharomyces cerevisiae, this DNA transformation competent genomic library should expedite gene isolation, by gene rescue, in multicellular organisms like Arabidopsis.

A DNA Transformation–Competent Arabidopsis Genomic Library in Agrobacterium

Plant breeders have used disease resistance genes (R genes) to control plant disease since the turn of the century. Molecular cloning of R genes that enable plants to resist a diverse range of pathogens has revealed that the proteins encoded by these genes have several features in common. These findings suggest that plants may have evolved common signal transduction mechanisms for the expression of resistance to a wide range of unrelated pathogens. Characterization of the molecular signals involved in pathogen recognition and of the molecular events that specify the expression of resistance may lead to novel strategies for plant disease control.

https://science.sciencemag.org/content/sci/268/5211/661.full.pdf

Molecular genetics of plant disease resistance

This review bridges functional and evolutionary aspects of plastid chromosome architecture in land plants and their putative ancestors. We provide an overview on the structure and composition of the plastid genome of land plants as well as the functions of its genes in an explicit phylogenetic and evolutionary context. We will discuss the architecture of land plant plastid chromosomes, including gene content and synteny across land plants. Moreover, we will explore the functions and roles of plastid encoded genes in metabolism and their evolutionary importance regarding gene retention and conservation. We suggest that the slow mode at which the plastome typically evolves is likely to be influenced by a combination of different molecular mechanisms. These include the organization of plastid genes in operons, the usually uniparental mode of plastid inheritance, the activity of highly effective repair mechanisms as well as the rarity of plastid fusion. Nevertheless, structurally rearranged plastomes can be found in several unrelated lineages (e.g. ferns, Pinaceae, multiple angiosperm families). Rearrangements and gene losses seem to correlate with an unusual mode of plastid transmission, abundance of repeats, or a heterotrophic lifestyle (parasites or myco-heterotrophs). While only a few functional gene gains and more frequent gene losses have been inferred for land plants, the plastid Ndh complex is one example of multiple independent gene losses and will be discussed in detail. Patterns of ndh-gene loss and functional analyses indicate that these losses are usually found in plant groups with a certain degree of heterotrophy, might rendering plastid encoded Ndh1 subunits dispensable.

/pdf/the-evolution-of-the-plastid-chromosome-in-land-plants-gene-4mwl68mgqz.pdf

The evolution of the plastid chromosome in land plants: gene content, gene order, gene function.

Selectable marker genes in transgenic plants: applications, alternatives and biosafety

A chimeric gene was constructed consisting of the promoter of the nopaline synthase gene of Agrobacterium and the structural gene of the aminoglycoside phosphotransferase of Tn5. This chimeric gene was recombined into the T-DNA of Agrobacterium. When this strain was used in a cocultivation experiment, transformed calli could be selected out of a pool of non-transformed calli when kanamycin was added into the growing medium

Chimeric genes as dominant selectable markers in plant cells

A bacterial streptomycin resistance gene (SPT) was engineered to make it possible to detect visually the transposition of the maize transposon Activator (Ac) in tobacco. In the presence of streptomycin, transgenic seedlings carrying the SPT gene appear green, whereas those carrying an SPT:: Ac construct display clones of green cells on a white background. Fully green seedlings arise in the progeny of SPT:: Ac transformants as a result of excision of Ac before fertilization. About half of these germinal revertants carry a transposed Ac element. Therefore, SPT:: Ac constitutes an efficient marker for selecting plants that have undergone transposition. In maize, there is a negative effect of increasing Ac dosage on the frequency and timing of Ac transposition. This negative effect is not observed in tobacco with the streptomycin variegation assay.

Visual detection of transposition of the maize element activator (ac) in tobacco seedlings.

Brassica cybrids were obtained after fusing protoplasts of fertile and cytoplasmic male sterile (CMS) B. napus lines carrying the original b. napus, and the Ogura Raphanus sativus cytoplasms, respectively. Iodoacetate treatment of the fertile line and X-irradiation of the CMS line prevented colony formation from the parental protoplasts. Colony formation, however, was obtained after protoplast fusion. Hybrid cytoplasm formation was studied in 0.5 g to 5.0 g calli grown from a fused protoplast after an estimated 19 to 22 cell divisions. Chloroplasts and mitochondria were identified in the calli by hybridizing appropriate DNA probes to total cellular DNA. Out of the 42 clones studied 37 were confirmed as cybrids. Chloroplast segregation was complete at the time of the study. Chloroplasts in all of the cybrid clones were found to derive from the fertile parent. Mitochondrial DNA (mtDNA) segregation was complete in some but not all of the clones. In the cybrids, mtDNA was different from the parental plants. Physical mapping revealed recombination in a region which is not normally involved in the formation of subgenomic mtDNA circles. The role of treatments used to facilitate the recovery of cybrids, and of organelle compatibility in hybrid cytoplasm formation is discussed.

Rapid chloroplast segregation and recombination of mitochondrial DNA in Brassica cybrids

X-irradiated protoplasts of a Brassica napus line carrying the Ogura Raphanus sativus male sterile cytoplasm were fused to protoplasts of male fertile B. napus cv. Olga. Plants were regenerated from six out of 34 randomly selected clones. In one clone, Bn(RS)26, a plant with male sterile flowers was obtained. Mitochondria of this plant are non-parental as revealed by DNA-DNA hybridization using a species specific probe. Its chloroplasts, however, derive from the fertile parent which results in loss of the sensitivity to low temperatures associated with R. sativus plastids in the male sterile parent. The novel cytoplasm of the Bn(RS)26 cybrid was transmitted through seed.

Fusion-mediated combination of Ogura-type cytoplasmic male sterility with Brassica napus plastids using X-irradiated CMS protoplasts.

Plant cells in photoheterotrophic culture respond to streptomycin by bleaching and retarded growth but no cell death. A new genetic marker for plant cell transformation has been developed that is based on the expression of the enzyme streptomycin phosphotransferase (SPT), and confers the ability to form green colonies on a selective medium. Coding sequences of SPT from the bacterial transposon Tn5 were placed under the control of gene expression signals derived from the Agrobacterium Ti plasmid Ach5. The 5′ end of the SPT gene has been replaced with the promoter region of the gene coding for the first enzyme of agropine biosynthesis, the 3′ end with that of the enzyme octopine synthase. The chimeric SPT gene has been linked to a selectable kanamycin resistance gene, and introduced into Nicotiana tabacum and Nicotiana plumbaginifolia by selection for the linked kanamycin resistance marker. Streptomycin resistance was expressed in some but not all of the kanamycin-resistant lines and was transmitted to the seed progeny as a dominant nuclear trait.

A dominant nuclear streptomycin resistance marker for plant cell transformation

Previous studies have shown that a chimeric streptomycin phosphotransferase (SPT) gene can function as a dominant marker for plant cell transformation. The SPT marker previously described by Jones and co-workers has a limited value since it conferred a useful level of resistance only to a fraction (10%) of Nicotiana plumbaginifolia transgenic lines. Expression of resistance was species specific: no such resistant transformants were found in N. tabacum. In this paper we describe an improved SPT construct that utilizes a mutant Tn5 SPT gene. The mutant gene, SPT*, encodes a protein with a two amino acid deletion close to its COOH-terminus. In N. tabacum cell culture the efficiency of transformation with the improved streptomycin resistance marker was comparable to kanamycin resistance. When the chimeric SPT* gene was introduced linked to a kanamycin resistance gene, streptomycin resistance was expressed in most of the transgenic N. tabacum lines.

Pal Maliga

Papers

Visual detection of transposition of the maize element activator (ac) in tobacco seedlings.

Rapid chloroplast segregation and recombination of mitochondrial DNA in Brassica cybrids

Fusion-mediated combination of Ogura-type cytoplasmic male sterility with Brassica napus plastids using X-irradiated CMS protoplasts.

A dominant nuclear streptomycin resistance marker for plant cell transformation

Improved expression of streptomycin resistance in plants due to a deletion in the streptomycin phosphotransferase coding sequence.