Mark T. Nielsen

Tissue partitioning of cadmium in transgenic tobacco seedlings and field grown plants expressing the mouse metallothionein I gene

Since agricultural crops contribute >70% of human cadmium (Cd) intake, modification of crops to reduce accumulation of this pollutant metal during plant growth is desirable. Here we describe Cd accumulation characteristics of seedlings and field grown tobacco plants expressing the Cd-chelating protein, mouse metallothionein I. The objective of the transformation is to entrap Cd in roots as Cd-metallothionein and thereby reduce its accumulation in the shoot. Transformed and control seedlings were exposed for 15 days in liquid culture at a field soil-solution-like Cd concentration of 0.02 μm. Transformed seedlings ofNicotiana tabacum cultivar KY 14 contained about 24% lower Cd concentration in shoots and about 5% higher Cd concentration in roots than control seedlings. Dry weights of transformed and control tissues did not differ significantly. In the field in 1990, mature transformedN. tabacum cv. KY 14 plants exposed only to endogenous soil Cd contained about 14% lower leaf lamina Cd concentration than did controls. Differences were significant at thep≤0.1 level in 13 of 16 leaf positions. Leaf dry weight did not differ significantly but transformed field plants had 12% fewer leaves and were 9% shorter than the controls. Copper (Cu) concentration was significantly higher (ca10%) in the bottom nine leaf positions of transformed plants suggesting that reduced leaf number and plant height may be due to Cu deficiency or toxicity. Alternatively, somaclonal variation or gene position effects may be involved. No differences were found in zinc levels. WithN. tabacum cv. Petit Havana, transformed seedlings contained no less Cd in shoots but 48% higher Cd concentration in roots. However, dry weights of shoots and roots of transformed seedlings were 25% and 26%, respectively, greater than in controls. In the field, transformed and control plants of this cultivar showed little significant differences in leaf Cd content, plant height or leaf number. Although comparison of additional metallothionein-expressing tobaccos and other plants is needed, results obtained with cultivar KY 14 support the hypothesis that sequestration of Cd in roots as Cd-metallothionein may have potential for reducing Cd content of above root tissues of certain plants.

A solution culture study of manganese‐tolerant and ‐sensitive tobacco genotypes

Abstract Genotypic differences in tolerating toxic levels of heavy metals have been observed in various plant species. This research was conducted to study the effect of manganese (Mn), iron (Fe), calcium (Ca), and magnesium (Mg) accumulation on two tobacco genotypes, Tobacco Introduction (T.I.) 1112 and KY 14, that have exhibited different sensitivity to toxic levels of Mn. The investigation was carried out employing a solution culture technique in which combinations of three levels of Mn exposure at varying strengths and pH of an Hoagland solution were made. Increasing the strength of the Hoagland solution suppressed Mn uptake and the occurrence of Mn toxicity symptoms in both genotypes, suggesting the potential role of other ions on reducing Mn toxicity. A change in solution pH from 4.5 to 6.5 had no significant effect on the accumulation of Mn, Fe, Ca, and Mg in tobacco leaves as well as the occurrence of Mn toxicity symptoms in the tobacco plant. Accumulations of Mn, Fe, Ca and Mg were significantly ...

Combining a host gene and a tobacco vein mottling virus coat protein gene for broad and effective resistance to potyviruses in tobacco (Nicotiana tabacum L.)

Over 100 transgenic tobacco lines in five genetic backgrounds were transformed with the tobacco vein mottling virus (TVMV) coat protein (CP) gene. Transgenic lines were initially tested for their reaction to inoculation with a TVMV systemic strain (TVMV-S) and a potato virus Y common strain (PVY-O). Of the 104 TVMV CP lines 60% were classified as resistant to PVY-O, whereas only 30% of these same lines were resistant to TVMV-S. A subset of six PVY-O-resistant transgenic lines and four control lines were tested for their reaction to a local isolate of TVMV, tobacco etch virus (TEV) and five isolates of PVY. The same ten lines were also tested for responses to a serial dilution of inoculum for two PVY isolates, PVY-KY1 and PVY-NN. Transgenic lines carrying an endogenous resistance gene known as Virgin A mutant (VAM) had greater resistance and a broader spectrum of resistance than did transgenic lines without the VAM gene. This additive effect of the endogenous resistance gene and coat protein-mediated resistance (CPMR) was not overcome by the highest inoculum concentration. The results indicate that the additive effect of the VAM gene and CPMR could extend the effectiveness of CPMR in controlling potiviruses. These findings could have important implications for plant improvement programs using CPMR against potyvirus diseases.

Field resistance of transgenic burley tobacco lines and hybrids expressing the tobacco vein mottling virus coat protein gene

Fifty transgenic lines expressing the tobacco vein mottling virus (TVMV) coat protein (CP) gene in five genetic backgrounds were evaluated under field conditions for response to mechanic inoculation with TVMV, tobacco etch virus (TEV) and potato virus Y (PVY). TVMV CP transgenic lines conferred resistance to TVMV, TEV and PVY under field conditions. Combining two strategies, coat protein-mediated resistance (CPMR) coupled with an endogenous resistance gene (Virgin A Mutant, VAM) significantly extended the range and magnitude of virus resistance and provided a potential valuable new source of protection against potyviruses. CP transgenic lines lacking the VAM gene had high resistance to TEV, medium resistance to PVY, and a recovery phenotype to TVMV. A series of hybrids involving transgenic lines were generated and tested under field conditions for response to virus inoculation. One copy of TVMV-CP gene presented in lines homozygous for the VAM gene provided effective resistance to all three potyviruses. These studies also suggested that selection of a suitable recipient genotype was critical and that field evaluation was necessary in order to select elite resistant transgenic lines. Engineering viral CP genes into genotypes possessing some level of virus resistance could be critical to achieve an effective level of resistance.