Michael W. Saul

Direct gene transfer to cells of a graminaceous monocot

SummaryDefinitive evidence is presented for the first time for stable gene transfer to cultured cells in a plant of the family Gramineae, Lolium multiflorum (Italian Ryegrass), using DNA transformation of protoplasts from a non-morphogenic cell culture. A construction consisting of expression signals from gene VI of Cauliflower Mosaic virus joined to the aminoglycoside (neomycin) phosphotransferase gene (APH(3′)II) from transposon Tn5 conferred resistance to the antibiotic G-418 to cell colonies arising from transformed protoplasts. By demonstrating a tight correlation between the resistant phenotype, the physical presence of the foreign gene and the presence of the active gene product we have shown that these colonies are true transformants and that a gene which is expressed well in dicotyle-denous plants is also expressed in cells of graminaceous monocots.

Molecular and general genetics of a hybrid foreign gene introduced into tobacco by direct gene transfer

SummaryTwo clones of N. tabacum, transformed to kanamycin resistance by direct transfer to protoplasts of a hybrid gene, consisting of the protein coding region from the bacterial gene for aminoglycoside phosphotransferase under the control of 5′/3′ expression signals from cauliflower mosaic virus gene VI, in the bacterial plasmid pUC8, have been subjected to a detailed genetic crossing analysis accompanied by Southern blot analysis and enzyme activity assays of representative offspring. The genetic data obtained from large populations of R1/F1 and R2/F2 offspring as well as from more than 20 subclones of each of the original transformants confirm that (a) one functional copy of the hybrid gene was stably integrated into chromosomal DNA of the original transformants, (b) that the gene normally was stably maintained during clonal proliferation, (c) that normally it is transmitted in a regular fashion (with exceptions) to sexual offspring, and (d) that it is inherited as a single dominant trait. Data from DNA hybridisation and enzyme assays confirm this interpretation. The functional gene is integrated together with several non-functional copies and bacterial plasmid sequences, which are inherited as one block together with the functional gene.

A functional map of plasmid ColE1

SummaryExamination of the properties of ColE1 derivatives containing either deletions or insertions of transposable genetic elements, has enabled a functional map of plasmid ColE1 to be constructed.

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.

Combination of kanamycin resistance and nitrate reductase deficiency as selectable markers in one nuclear genome provides a universal somatic hybridizer in plants

SummaryThe combination in the nuclear genome of a dominant resistance marker (to select against unfused wild-type cells) and a recessive deficiency marker (to select against unfused mutant cells) in a cell line should provide a system for selecting fusion hybrids between the mutant line and any wild-type line. To test this idea, we fused protoplasts from a non-morphogenic cell line of Nicotiana tabacum which was kanamycin resistant (by transformation) and deficient in nitrate reductase (NR-K+) with protoplasts from N. tabacum cv. Petit Havana clone SR1, which provided resistance against streptomycin as an additional selectable marker (NR+K-SR+). Putative hybrids were selected using a culture medium containing no available reduced nitrogen source and 50 mg/l kanamycin sulphate. After regeneration into plants, the hybrid character was demonstrated from: (i) the morphological variation of the regenerants; (ii) the chromosome number; (iii) the ability to grow on medium without a reduced nitrogen source and containing kanamycin sulphate at 50 mg/l; (iv) the presence of nitrate reductase activity; (v) the presence of the gene coding for neomycin phosphotransferase, which provides resistance to kanamycin sulphate; (vi) callus formation from leaves on medium containing 1 g/l streptomycin or 50 mg/l kanamycin sulphate; (vii) F1 plants containing nitrate reductase and the gene for neomycin phosphotransferase. Fusions between the mutant cell line (NR-K+) and three wild-type tobacco species and subsequent cultivation on medium containing no available nitrogen source but 50 mg/l kanamycin sulphate resulted in callus formation with all combinations, while hybrid plants were only regenerated when N. sylvestris was the fusion partner.