Ramsey Lewis

Collection and preservation of genetic variability in Nicotiana by the United States Nicotiana Germplasm collection is important for maintaining the economic efficiency and stability of tobacco production as well as for maintaining flexibility for manufacture of future tobacco products. Although the necessity for studying and utilizing the Nicotiana Germplasm Collection may be greater than ever before, financial support is needed to maintain this resource into the future. The USDA withdrew financial support for all aspects of applied tobacco research in 1994, including support for germplasm maintenance. Most recently, N.C. State University ceased funding for the salary of the curator position due to internal budgetary problems that have occurred in the last few years. This proposal concerns a possible three-year funding arrangement to support continued maintenance of the collection and to provide PMI access to seeds of desired accessions.

The formation of undesirable tobacco-specific nitrosamines (TSNAs) occurs during the curing, storage and processing of the tobacco leaf and requires two distinct components, an alkaloid substrate and a nitrosating agent. The prevailing consensus for the air-cured burley tobaccos is that the reduction of free nitrate pools in the leaf by surface microbes accounts for the formation of the highly reactive nitrite molecules that are believed to interact with plant alkaloids to form TSNAs. Our recent results demonstrated that it is possible to alter the nitrogen assimilation pathway in a manner that greatly decreases both nitrate levels and TSNAs. The experiments outlined in this proposal build on these results and will further define the optimal means by which the nitrate and TSNA contents of the cured leaf can be reduced.

This proposal concerns six tobacco breeding and genetic research projects with the objectives of germplasm enhancement, enhancement of cured leaf quality, and modification of tobacco cured leaf chemistry.

Tobacco breeding and genetics research will be performed.

The formation of undesirable tobacco-specific nitrosamines (TSNAs) occurs during the curing, storage and processing of the tobacco leaf and requires two distinct components, an alkaloid substrate and a nitrosating agent. The prevailing consensus for the air-cured burley tobaccos is that the reduction of free nitrate pools in the leaf by surface microbes accounts for the formation of the highly reactive nitrite molecules that are believed to interact with plant alkaloids to form TSNAs. Our recent results demonstrated that it is possible to alter the nitrogen assimilation pathway in a manner that greatly decreases both nitrate levels and TSNAs. The experiments outlined in this proposal build on these results and will further define the optimal means by which the nitrate and TSNA contents of the cured leaf can be reduced.

Main objective: Identify biochemical reactions in plants that can be blocked or reduced to cause plant growth reduction or death. Specific objectives: 1) Screen Arabidopsis thaliana mutants for existing knock-down and/or knock-out mutations that generate visible phenotypes of negative impacts on plant metabolism and growth. 2) Use select target genes to reproduce phenotypes in tobacco using gene editing approaches.

AVOCA has a dominant position in the business of extracting the diterpene, sclareol, from clary sage (Salvia sclarea) and marketing this compound for use in the fragrance industry. The worldwide demand for sclareol and its chemical derivatives has been growing, and is expected to continue to grow, because of increased use in the manufacture of home goods such as laundry and detergent products. AVOCA currently has the infrastructure to produce 800 tons of sclareolide (a sclareol derivative) using a plant-based extraction process. Increasing clary sage acreage is one way to meet future market demands for sclareol. Eighty-thousand acres of clary sage would be required to meet this demand, however. More cost effective approaches might involve use of genetics-based research to increase the amount of sclareol that can be produced per acre, and/or affecting factors contributing to improved stability of sclareol yields. Consideration of these possibilities may be of increased importance as competitors appear to be attempting to enter the marketplace through development of microbe-based production platforms. This research proposal outlines three areas of proposed research related to genetics-based efforts to improve plant yields of sclareol: I. Interference with metabolic pathways that may compete with the sclareol biosynthetic pathway II. Overexpression of genes directly involved in the sclareol biosynthetic pathway III. Altered expression of a transcription factor that may affect global expression of genes in the sclareol biosynthetic pathway

Energy canes broadly refer to species and hybrids within the genus Saccharum that are specifically developed for bioenergy applications. These perennial grasses have considerable potential for biomass production. Our recent evaluations of cold hardy energy cane hybrids have shown dry biomass yields can exceed 25 T/acre. However, many energy canes are highly fertile and could become weedy if cultivated. Novel gene editing approaches have the potential to develop seedless forms of high yielding cultivars. This project will build on prior work that focused on breeding high biomass, cold hardy hybrids and the development of embryogenic regeneration systems. Efforts will now focus on refining regeneration systems and the development of transformation and gene editing technologies with initial application for the development of seedless cultivars. These technologies will not only address immediate needs and lead to valuable, improved bioenergy crops, but will provide a platform for bioengineering and enhancement of bioprocessing and value added traits far into the future.

Genetic variability for reducing nicotine content in cured tobacco has been of interest to tobacco breeders for decades. Interest has become heightened within the last two years, however, due to recommendations by the World Health Organization (WHO) to lower cigarette nicotine levels to below concentrations designated as ���������������sub-threshold levels of addiction.������������������ This proposal concerns the investigation of alternative genetic variability affecting lower nicotine content in tobacco.

This proposal involves two main tobacco breeding projects. One relates to understanding and using genetic variability to influence the stem to lamina ratio. The second project relates to the development of a strategy to convert female parental lines of new F1 hybrid tobacco cultivars to cytoplasmic male sterility.