Raymond D. Shillito

Direct Gene Transfer to Plants

Evidence for direct, gene-mediated stable genetic transformation of plant cells of Nicotiana tabacum is presented. A selectable hybrid gene comprising the protein coding region of the Tn5 aminoglycoside phosphotransferase type II gene under control of cauliflower mosaic virus gene VI expression signals was introduced into plant protoplasts as part of an Escherichia coli plasmid. The gene was stably integrated into plant genomic DNA and constitutively expressed in selected, drug resistant, protoplast-derived cell clones. The mode of integration of the foreign gene into the plant genome resembled that observed for DNA transfection of mammalian cells. Plants regenerated from transformed cell lines were phenotypically normal and fertile, and they maintained and expressed the foreign gene throughout the development of vegetative and generative organs. Microspores, grown in anther culture, developed into resistant and sensitive haploid plantlets. Genetic crossing analysis of one of the transformed plants revealed the presence of one dominant trait for kanamycin resistance segregating in a Mendelian fashion in the F(1) generation.

Direct gene transfer to plants

Evidence for direct, gene‐mediated stable genetic transformation of plant cells of Nicotiana tabacum is presented. A selectable hybrid gene comprising the protein coding region of the Tn5 aminoglycoside phosphotransferase type II gene under control of cauliflower mosaic virus gene VI expression signals was introduced into plant protoplasts as part of an Escherichia coli plasmid. The gene was stably integrated into plant genomic DNA and constitutively expressed in selected, drug resistant, protoplast‐derived cell clones. The mode of integration of the foreign gene into the plant genome resembled that observed for DNA transfection of mammalian cells. Plants regenerated from transformed cell lines were phenotypically normal and fertile, and they maintained and expressed the foreign gene throughout the development of vegetative and generative organs. Microspores, grown in anther culture, developed into resistant and sensitive haploid plantlets. Genetic crossing analysis of one of the transformed plants revealed the presence of one dominant trait for kanamycin resistance segregating in a Mendelian fashion in the F1 generation.

Selection of transformed protoplast-derived Zea mays colonies with phosphinothricin and a novel assay using the pH indicator chlorophenol red

Phosphinothricin (PPT) inhibits glutamine synthetase in plant cells, resulting in an accumulation of ammonia that can be toxic. Bar, from Streptomyces hygroscopicus is a gene that codes for phosphinothricin acetyl-transferase, an enzyme that detoxifies PPT by acetylation. We used polyethylene-glycol-mediated transformation to introduce bar into protoplasts of maize (Zea mays L.). With a novel assay utilizing the pH indicator chlorophenol red we identified transformants after only two to four weeks of selection on PPT.

Direct gene transferState of the Art and Future Potential

Incubation of freshly isolated protoplasts with a gene under the control of plant expression signals leads to high levels of stable integrative transformation. The gene is transmitted to sexual offspring and inherited in a Mendelian pattern. The foreign gene is in most cases stably maintained through several sexual cycles. However, cases of somatic and meiotic instability are also found. A partially optimized protocol routinely yields transformation frequencies in the percentage range. Host range limitations were not expected and also not found in transformation experiments with protoplasts from a series of herbaceous dicots and a graminaceous monocot. Co-transformation experiments with a selectable and a non-selectable gene on separate plasmids yielded high levels of cotransfer of the non-selectable gene.

Direct DNA transfer to protoplasts with and without electroporation

The introduction of ‘naked’ DNA into plant protoplasts, in comparison to transformation methods used for bacterial, yeast and animal cells, has been a relatively recent development. The first conclusive demonstrations of uptake and integration of DNA into plant protoplasts were those of Davey et al. [1], Draper et al. [2] and Krens et al. [3] in which isolated Ti plasmid from Agrobacterium tumefaciens was applied to plant protoplasts in the presence of poly-1-ornithine or polyethylene glycol/Ca2 +. The presence of the Ti DNA in the plant genome was demonstrated both by the phenotype of hormone auxotrophic growth, production of the expected opine and by Southern blot analysis of DNA from the transformants. The DNA integrated into the genome appeared to be a random assortment of DNA derived from the Ti plasmid. The subsequent development of markers allowing positive selection in plant cells (i.e. antibiotic resistance markers) led to the development of a much simplified protoplast transformation system. Pazskowski et al. [4] constructed a simple plasmid based on pUC8 containing a selectable marker, the kanamycin resistance gene from the trans-poson Tn5 with expression signals from gene VI of the dsDNA virus cauliflower mosaic virus (CaMV). Using this plasmid (pABDl) and an uptake method derived from that of Krens et al [3], they were able to demonstrate the uptake, integration and expression of the resistance marker in protoplasts of Nicotiana tabacum.

Cryopreservation technology for plant cell cultures

Described in detail is a technique for the cryopreservation, long-term storage, thawing, recovery, and regrowth of embryogenic suspension cultures of maize (Zea mays L.) With this system we have successfully preserved many different culture lines, and recovered living material after more than 2 yr storage in liquid nitrogen. The recovered tissue is not apparently different from the starting material. With minor variations, the technique has also been used forDactylis glomerata and other suspension culture cells, as well as callus cultures. Approaches for the cryopreservation of other plant parts, such as seeds, embryos and meristems are discussed.

Direct gene transfer via polyethylene glycol

Polyethylene glycol can be used to induce DNA uptake into plant protoplasts. Procedures for isolation, culture and transformation ofN. tabacum protoplasts are described and can be adapted for other dicot and monocot species. Criteria for proof of transformation are discussed.

Crown Gall Transformation of Regenerating Protoplasts of Haploid and Diploid Petunia hybrida var. Mitchell by Agrobacterium tumefaciens

Summary Cultures of haploid and diploid leaf protoplasts of Petunia hybrida var. Mitchell were treated with 5 virulent strains of Agrobacterium tumefaciens . Transformed clones, characterised by their hormone independent growth and nopaline production, were isolated following treatment with strain T37.