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

We report here a 100-fold increased frequency of plastid transformation in tobacco by selection for a chimeric aadA gene encoding aminoglycoside 3"-adenylyltransferase, as compared with that obtained with mutant 16S rRNA genes. Expression of aadA confers resistance to spectinomycin and streptomycin. In transforming plasmid pZS197, a chimeric aadA is cloned between rbcL and open reading frame ORF512 plastid gene sequences. Selection was for spectinomycin resistance after biolistic delivery of pZS197 DNA into leaf cells. DNA gel-blot analysis confirmed incorporation of the chimeric aadA gene into the plastid genome by two homologous recombination events via the flanking plastid gene sequences. The chimeric gene became homoplasmic in the recipient cells and is uniformly transmitted to the maternal seed progeny. The ability to transform routinely plastids of land plants opens the way to manipulate the process of photosynthesis and to incorporate novel genes into the plastid genome of crops.

https://www.pnas.org/content/pnas/90/3/913.full.pdf

High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene.

The invention provides for identification and use of certain chloroplast transit peptides for efficient processing and localization of dicamba monooxygenase (DMO) enzyme in transgenic plants. Methods for producing dicamba tolerant plants, methods for controlling weed growth, and methods for producing food, feed, and other products are also provided, as well as seed that confers tolerance to dicamba when it is applied pre- or post-emergence.

Chloroplast transit peptides for efficient targeting of DMO and uses thereof

The invention provides methods for weed control with dicamba and related herbicides. It was found that pre-emergent applications of dicamba at or near planting could be made without significant crop damage or yield loss. The techniques can be combined with the herbicide glyphosate to improve the degree of weed control and permit control of herbicide tolerant weeds.

Methods for weed control using plants having dicamba-degrading enzymatic activity

The invention provides a modified variant of dicamba monooxygenase (DMO). The invention relates to the unexpected finding that cells expressing this DMO exhibit high levels of tolerance to the herbicide dicamba. Compositions comprising DMO-encoding nucleic acids and methods of use are provided.

Modified dmo enzyme and methods of its use

The bacterial gene aadA encodes the enzyme aminoglycoside-3"-adenyltransferase that confers resistance to spectinomycin and streptomycin in Escherichia coli. Chimeric genes have been constructed for expression in plants, and were introduced into Nicotiana tabacum by Agrobacterium binary transformation vectors. Spectinomycin or streptomycin in selective concentrations prevent greening of N. tabacum calli. Transgenic clones, however, formed green calli on selective media containing spectinomycin, streptomycin, or both drugs. Resistance was inherited as a dominant Mendelian trait in the seed progeny. Resistance conferred by the chimeric aadA gene can be used as a color marker similar to the resistance conferred by the streptomycin phosphotransferase gene to streptomycin.

Aminoglycoside-3''-adenyltransferase confers resistance to spectinomycin and streptomycin in Nicotiana tabacum.

This invention relates to improved methods for transposon tagging as a route to plant gene isolation. The specific improvements comprise efficient means for preselecting plants which have undergone transposition. Through use of this preselection, it is possible to substantially reduce the number of plants which must be inspected to locate individuals having the transposon excised and inserted into a gene of interest. The result is a substantial savings in time, costs and efficiency associated with growing and screening large numbers of plants for tagging and isolation of a desired plant gene.

Transposon tagging of genes in transformed plants

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.

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

This invention relates to the discovery that the prokaryotic enzyme, aminoglycoside 3"-adenyltransferase (AGAT), in particular as encoded by a bacterial aadA gene, is useful as a selectable marker for transformed plants. The enzyme conveys resistance to spectinomycin and streptomycin. Such markers are particularly advantageous because they are non-lethal, provide rapid visual identification of transformed cells and permit selection in media containing either spectinomycin or streptomycin. In addition, AGAT may be used as a selectable marker which differentiates by enabling survival on selective media.

Pal Maliga

Papers

Aminoglycoside-3''-adenyltransferase confers resistance to spectinomycin and streptomycin in Nicotiana tabacum.

Transposon tagging of genes in transformed plants

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

Method of introducing spectinomycin resistance into plants