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

▪ Abstract Plastids of higher plants are semi-autonomous organelles with a small, highly polyploid genome and their own transcription-translation machinery. This review provides an overview of the technology for the genetic modification of the plastid genome including: vectors, marker genes and gene design, the use of gene knockouts and over-expression to probe plastid function and the application of site-specific recombinases for excision of target DNA. Examples for applications in basic science include the study of plastid gene transcription, mRNA editing, photosynthesis and evolution. Examples for biotechnological applications are incorporation of transgenes in the plastid genome for containment and high-level expression of recombinant proteins for pharmaceutical and industrial applications. Plastid transformation is routine only in tobacco. Progress in implementing the technology in other crops is discussed.

Plastid transformation in higher plants.

We describe here the development of a reproducible plastid transformation system for potato and regeneration of plants with uniformly transformed plastids. Two distinct tobacco-specific plastid vectors, pZS197 (Prrn/aadA/TpsbA) and pMON30125 (Prrn/GFP/Trps16:PpsbA/aadA/TpsbA), designed for integration into the large single copy and inverted repeat regions of the plastid genome, respectively, were bombarded into leaf explants of potato line FL1607. A total of three transgenic lines were selected out of 46 plates bombarded with pZS197 and three transgenic lines out of 104 plates were obtained with pMON30125. Development of a high frequency leaf-based regenera- tion system, a stringent selection scheme and optimization of biolistic transformation protocol were critical for recovery of plastid transformants. Plastid-expressed green fluorescent protein was used as a visual marker for identification of plastid transformants at the early stage of selection and shoot regeneration. The establishment of a plastid transformation system in potato, which has several advantages over routinely used nuclear transformation, offers new possibilities for genetic improvement of this crop.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.1999.00508.x

Stable chloroplast transformation in potato : use of green fluorescent protein as a plastid marker

Plastid transformation in higher plants is accomplished through a gradual process, during which all the 300-10,000 plastid genome copies are uniformly altered Antibiotic resistance genes incorporated in the plastid genome facilitate maintenance of transplastomes during this process Given the high number of plastid genome copies in a cell, transformation unavoidably yields chimeric tissues, which requires the identification of transplastomic cells in order to regenerate plants In the chimeric tissue, however, antibiotic resistance is not cell autonomous: transplastomic and wild-type sectors both have a resistant phenotype because of phenotypic masking by the transgenic cells We report a system of marker genes for plastid transformation, termed FLARE-S, which is obtained by translationally fusing aminoglycoside 3"-adenyltransferase with the Aequorea victoria green fluorescent protein 3"-adenyltransferase (FLARE-S) confers resistance to both spectinomycin and streptomycin The utility of FLARE-S is shown by tracking segregation of individual transformed and wild-type plastids in tobacco and rice plants after bombardment with FLARE-S vector DNA and selection for spectinomycin and streptomycin resistance, respectively This method facilitates the extension of plastid transformation to nongreen plastids in embryogenic cells of cereal crops

Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants.

Incorporation of a selectable marker gene during transformation is essential to obtain transformed plastids. However, once transformation is accomplished, having the marker gene becomes undesirable. Here we report on adapting the P1 bacteriophage CRE-lox site-specific recombination system for the elimination of marker genes from the plastid genome. The system was tested by the elimination of a negative selectable marker, codA, which is flanked by two directly oriented lox sites (>codA>). Highly efficient elimination of >codA> was triggered by introduction of a nuclear-encoded plastid-targeted CRE by Agrobacterium transformation or via pollen. Excision of >codA> in tissue culture cells was frequently accompanied by a large deletion of a plastid genome segment which includes the tRNA-ValUAC gene. However, the large deletions were absent when cre was introduced by pollination. Thus pollination is our preferred protocol for the introduction of cre. Removal of the >codA> coding region occurred at a dramatic speed, in striking contrast to the slow and gradual build-up of transgenic copies during plastid transformation. The nuclear cre gene could subsequently be removed by segregation in the seed progeny. The modified CRE-lox system described here will be a highly efficient tool to obtain marker-free transplastomic plants.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313x.2001.01068.x

Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system

Soluble metabolic products, such as ethanol, are produced by growing modified algal cells in a growth medium and recovering the products from the growth medium. The algal cells are modified to overproduce the metabolic product by providing for overexpression of at least one enzyme in the metabolic pathway for the product. For the production of ethanol, the alcohol dehydrogenase gene, the pyruvate decarboxylase gene, or both, are overexpressed, typically under the control of a heterologous promoter. The algal cells may be modified by transformation with a DNA construct including coding sequence(s) of the enzyme(s) under the control of a heterologous promoter.

Methods and compositions for producing metabolic products for algae

Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.

Benjamin A. Moll

Papers

Methods and compositions for producing metabolic products for algae

Streptomycin and lincomycin resistances are selective plastid markers in cultured Nicotiana cells