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Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of mannose 6-phosphate and fructose 6-phosphate. Plant cells lacking this enzyme are incapable of surviving on synthetic medium containing mannose as a carbon source. Maize, wheat and barley plants, genetically modified to express the Escherichia coli manA gene (pmi) under the control of a plant promoter, were able to survive selection on mannose-containing medium. Transformation frequencies averaged 45% for maize transformation via Biolisticse, 35% for maize Agrobacterium-mediated transformation, 20% for wheat, 3% for barley, and 2% for watermelon transformation. Moreover, the frequencies exceeded those obtained for maize and wheat using the pat or bar gene with Basta w selection. A preliminary safety assessment has been conducted for PMI. Purified PMI protein demonstrates no adverse effects in an acute mouse toxicity test. Purified PMI protein was readily digested in simulated mammalian gastric and intestinal fluids. Plants derived from sugar beet and corn cells that had been genetically modified to express the E. coli manA gene were evaluated for biochemical changes in mannose-associated pathways. No detectable changes in glycoprotein profiles were detected in PMI-transformed plants as compared to nontransgenic controls. The yield and nutritional composition of grain from PMI-transformed corn plants compared to their non-transformed isogenic counterparts were also determined and no statistically significant differences were found. The inherent safety of a system based on simple sugar metabolism coupled with high transformation frequencies for monocots make pmi an ideal selectable marker for plant transformation.

Agrobacterium rhizogenes strain K599 (pRi2659), a causative agent of hairy root disease, effectively induces hairy root formation in a variety of plant species, including numerous soybean (Glycine max) cultivars. Because Agrobacterium-mediated transformation of soybean remains challenging and labor intensive, K599 appeared a suitable progenitor for new agrobacteria strains for plant transformation. In this paper, we report the disarming and sequencing of pRi2659 and the usefulness of the resulting disarmed strain in plant transformation studies of Arabidopsis thaliana, maize (Zea mays), tomato (Lycopersicon esculentum), and soybean (G. max).

ABSTRACIEndogenous indoleacetic acid (IAA) and cytokinin concentrations were measured by high performance liquid chromatography in leaf sections of an orchardgrass (Dactylis glomerata L.) genotype which exhibited a high capacity for somatic embryogenesis in vitro and in two genotypes that did not exhibit this capacity. The nonembryogenic genotypes contained 3-to 4-fold higher concentrations of zeatin, zeatin riboside, dihydrozeatin, dihydrozeatin riboside, and total cytokinins than the embryogenic genotype. There were no significant differences in IAA concentrations between genotypes. Cytokinin concentrations between basal and distal sections of embryogenic genotype were not different, but the IAA concentration was significantly greater in basal sections. Somatic embryogenesis was inhibited in the embryogenic genotype by 0.001 micromolar exogenously added zeatin.Despite recent advances in regeneration of cereals and grasses from protoplasts (1,5,15,22), the development ofhighly efficient and repeatable regeneration systems remains a major hurdle in the advancement of biotechnological applications, including gene transfer, in these species. Somatic embryogenesis systems developed for Dactylis galomerata L. (orchardgrass or cocksfoot) are among the most advanced currently existing for the Poaceae. These include the formation of embryos directly from mesophyll cells in cultured leaf segments (4, 8) and the full development of somatic embryos in a single liquid medium (6,7,19).Somatic embryogenic capacity in orchardgrass is highly dependent on genotype (9, 13). Genotypic differences for in vitro response, including somatic embryogenesis, have also been reported for other cereal and grass species (2,11,18), but these differences have not been related to either endogenous or exogenous PGRs.2 The requirement for an exogenously added auxin to gramineous cell and tissue cultures has been long established (21); however, the influence of cytokinins is less clear. The objectives of this study were to measure and relate endogenous levels of IAA and cytokinins (Z, DHZ, ZR, DHZR) to the genotypic response for somatic embryogenesis in orchardgrass and to investigate the effect of exogenously added Z to cultured leaf segments of the embyogenic genotype. MATERIALS AND METHODSGenotypes ofDactylis glomerata L. used in this study included two clones (designated Ela and Elb) of one that exhibited a very high capacity for somatic embryogenesis (4, 8) and two (designated NEi and NE2) which exhibited no capacity to produce somatic embryos. Tillers were collected from mature plants grown in an environmentally controlled chamber with a 12 h day/night cycle at 220/1 5C. Individual leaves were separated and basal sections (0-3 cm) of the two innermost (youngest) were lyophilized. The next distal (3-6 cm) sections (nonembryogenic portion) of Ela and Elb were also collected. Plant growth regulators were extracted and purified according to a modification of existing procedures (10, 12) that is outlined in Figure 1.Cytokinins were analyzed ...

Recently, five novel fluorescent proteins have been isolated from non-bioluminescent species of reef-coral organisms and have been made available through ClonTech. They are AmCyan, AsRed, DsRed, ZsGreen and ZsYellow. These proteins are valuable as reporters for transformation because they do not require a substrate or external co-factor to emit fluorescence and can be tested in vivo without destruction of the tissue under study. We have evaluated them in a large range of plants, both monocots and dicots, and our results indicate that they are valuable reporting tools for transformation in a wide variety of crops. We report here their successful expression in wheat, maize, barley, rice, banana, onion, soybean, cotton, tobacco, potato and tomato. Transient expression could be observed as early as 24 h after DNA delivery in some cases, allowing for very clear visualization of individually transformed cells. Stable transgenic events were generated, using mannose, kanamycin or hygromycin selection. Transgenic plants were phenotypically normal, showing a wide range of fluorescence levels, and were fertile. Expression of AmCyan, ZsGreen and AsRed was visible in maize T1 seeds, allowing visual segregation to more than 99% accuracy. The excitation and emission wavelengths of some of these proteins are significantly different; the difference is enough for the simultaneous visualization of cells transformed with more than one of the fluorescent proteins. These proteins will become useful tools for transformation optimization and other studies. The wide variety of plants successfully tested demonstrates that these proteins will potentially find broad use in plant biology.