A mannose selection system for production of fertile transgenic maize plants from protoplasts.

Maize (Zea mays L.) callus cultures cannot use mannose as a sole carbohydrate source, but can utilize fructose for that purpose. Phosphomannose isomerase (PMI) can convert mannose to fructose. Transgenic maize plants were obtained by selecting polyethylene glycol (PEG)-mediated transformed protoplasts on mannose (20 g/l) containing medium. Transgenic calluses and plants carrying the PMI structural gene, manA, were able to convert mannose to fructose. The PEG-mediated protoplast transformation frequency was 0.06%. Stable transformation was confirmed by PCR, PMI activity, germination tests, and by histochemical staining with 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (X-Gluc). Stable integration of the transgenes into the maize genome was demonstrated in T1 and T2 plants. Results indicate that the mannose selection system can be used for maize PEG-mediated protoplast transformation.

Interaction of the mutagenic metabolite of sodium azide, synthesized in vitro, with DNA of barley embryos.

The in vitro synthesized sodium azide mutagenic metabolite (azidoalanine) produced single-strand breaks and proteinase K-sensitive sites in isolated, germinating barley embryos. In contrast with sodium azide, the efficiency of DNA damage induction was lower, and both types of DNA lesions were totally or partially repaired in the course of subsequent 24 h incubation of the embryos. The mutagenic azide metabolite did not inhibit DNA replication, while azide did so even at doses which are not highly mutagenic. The metabolite labelled with 14C at the amino acid residue was taken up with a similar efficiency both into barley embryos germinating for 2 days and into cells of Salmonella typhimurium TA100. The majority of the radioactivity was incorporated into proteins, less into RNA and a negligible amount into DNA.

Comparative induction of nitrate reductase by nitrate and nitrite in barley leaves.

The comparative induction of nitrate reductase (NR) by ambient NO3- and NO2- as a function of influx, reduction (as NR was induced) and accumulation in detached leaves of 8-day-old barley (Hordeum vulgare L.) seedlings was determined. The dynamic interaction of NO3- influx, reduction and accumulation on NR induction was shown. The activity of NR, as it was induced, influenced its further induction by affecting the internal concentration of NO3-. As the ambient concentration of NO3- increased, the relative influences imposed by influx and reduction on NO3- accumulation changed with influx becoming a more predominant regulant. Significant levels of NO3- accumulated in NO2(-)-fed leaves. When the leaves were supplied cycloheximide or tungstate along with NO2-, about 60% more NO3- accumulated in the leaves than in the absence of the inhibitors. In NO3(-)-supplied leaves NR induction was observed at an ambient concentration of as low as 0.02 mM. No NR induction occurred in leaves supplied with NO2- until the ambient NO2- concentration was 0.5 mM. In fact, NR induction from NO2- solutions was not seen until NO3- was detected in the leaves. The amount of NO3- accumulating in NO2(-)-fed leaves induced similar levels of NR as did equivalent amounts of NO3- accumulating from NO3(-)-fed leaves. In all cases the internal concentration of NO3-, but not NO2-, was highly correlated with the amount of NR induced. The evidence indicated that NO3- was a more likely inducer of NR than was NO2-.

Source of endoproteolytic activity associated with purified ribulose bisphosphate carboxylase.

The association of endoproteolytic activity with purified ribulose bisphosphate carboxylase (RuBPCase) from barley (Hordeum vulgare var Numar) leaves was investigated. RuBPCase purified by chromatography on agarose gel and diethylaminoethyl-cellulose was free of associated endoproteolytic activity. The addition of leupeptin and casein to the extraction buffer completely eliminated proteolysis during initial extraction of RuBPCase. Endoproteolytic activity previously found associated with RuBPCase was identified as being due to contamination of endoproteinases from broken vacuoles.

A mutagenic metabolite synthesized by Salmonella typhimurium grown in the presence of azide is azidoalanine.

A mutagenic azide metabolite was purified from the medium in which Salmonella typhimurium cells were grown in the presence of azide. This metabolite was identified to be azidoalanine based on infrared and mass spectroscopy and elemental analysis. This compound appeared to be identical to the mutagenic compound synthesized in vitro from azide and O-acetylserine by partially purified O-acetylserine sulfhydrylase. The metabolite (azidoalanine) mutagenic efficiency and spectrum in S. typhimurium was similar to that of inorganic azide. The compounds 2-azidoethylamine, 2-bromoethylamine, 3-bromopropionic acid and N-(azidomethyl) phthalimide were also mutagenic with a similar spectrum to azide and azidoalanine, but with lower efficiency. The compounds 3-azidopropylamine, 4-azidobutylamine, 3-chloroalanine and ethylamine were only weakly or nonmutagenic. Numerous other chloro, bromo and azido phthalimide derivatives tested were nonmutagenic. It is suggested that the lack of azide mutagenicity (and perhaps carcinogenicity) in mammalian cells may be due to their inability to convert azide to azidoalanine.

In vitro production of azide mutagenic metabolite in Arabidopsis, Drosophila and Neurospora.

The ability of Arabidopsis, Drosophila and Neurospora to convert azide to its mutagenic metabolite was investigated. Cultures of these organisms all contained significant levels of O-acetylserine sulfhydrylase activity. Extracts from each organism produced a product from O-acetylserine and azide in vitro which was mutagenic in Salmonella typhimurium TA1530.

Pollen genetic markers for detection of mutagens in the environment.

To utilize and exploit pollen for in situ mutagen monitoring, screening and toxicology, the range of genetic traits in pollen must be identified and analyzed. Traits that can be considered include ornamentation, shape and form, male sterility viability, intraspecific incompatibility, proteins and starch deposition. To be useful for the development of mutagen detection systems proteins should be: (1) activity stainable or immunologically identifiable in the pollen, (2) the products of one to three loci, and (3) gametophytic and nuclear in origin. Several proteins including alcohol dehydrogenase in maize, which meet those criteria will be discussed. The waxy locus in barley and maize which controls starch deposition has been characterized genetically and methods have been developed for pollen screening and mutant detection. At Washington State University a waxy pollen system is being developed in barley for in situ mutagen monitoring. The basis is an improved method for staining and scoring waxy pollen mutants. Specific base substitution, frameshift, and deletion mutant lines are being developed to provide information about the nature of the mutations induced by environmental mutagens. Thirty waxy mutant lines, induced by sodium azide and gamma-rays have been selected and are being characterized for spontaneous and induced reversion frequencies, allelism, karyotype, amylose content, and UDP glucose glucosyltransferase (waxy gene product) activity. Twelve mutant alleles are being mapped by recombinant frequencies.

Ontogeny of the barley plant as related to mutation expression and detection of pollen mutations.

Clustering of mutant pollen grains in a population of normal pollen due to premeiotic mutational events complicates translating mutation frequencies into rates. Embryo ontogeny in barley will be described and used to illustrate the formation of such mutant clusters. The nature of the statistics for mutation frequency will be described from a study of the reversion frequencies of various waxy mutants in barley. Computer analysis by a "jackknife" method of the reversion frequencies of a waxy mutant treated with the mutagen sodium azide showed a significantly higher reversion frequency than untreated material. Problems of the computer analysis suggest a better experimental design for pollen mutation experiments. Preliminary work on computer modeling for pollen development and mutation will be described.