Research Accomplishment Reports 2011

Tobacco Breeding and Genetics

R. Miller
Department of Plant and Soil Sciences

 

Non-Technical Summary

Burley and dark tobacco production is an important component of the farm economy of Kentucky and Tennessee. Development of high yielding, disease resistant cultivars has played a major role in tripling the average yield of burley tobacco from 1920 to 2007.

However, in order for United States tobacco growers to remain profitable in the world market, they must have access to new disease resistant tobacco varieties. Considerable progress has been made in the development of disease resistant tobacco varieties since the University of Kentucky and the University of Tennessee tobacco breeding programs merged to form the Kentucky-Tennessee Tobacco Improvement Initiative (KTTII) in 1999. New burley varieties KT 200LC, KT 204LC, and KT 206LC and dark varieties KT D4LC, KT D6LC, and KT D8LC have been released since the year 2000. In addition, older varieties TN 86, TN 90, TN 97, KY14, KY17, KY 907, ms KY 14 X L8, KY 171, NL Madole, and TN D950 have been re-released as new low nicotine converting (designated as LC) varieties due to new, more stringent chemical standards throughout the tobacco industry.

The new varieties released by KTTII have been quickly accepted by tobacco growers and companies. KT 204LC became the number one burley variety in the US within three years of its release, comprising approximately 45% of the 2007 domestic crop. Overall, the top five burley varieties grown during 2007 were released by KTTII, comprising approximately 80% of the US crop. Although KT 206LC was only released in 2007 and is being produced by growers for the first time in 2008, seed sales indicate that it has been extremely well received by burley growers and will rank first in domestic production during 2008.

Although excellent progress has been maintained in providing Kentucky and Tennessee tobacco growers with improved cultivars, new diseases such as tomato spotted wilt virus, blue mold, and new strains of the causal organisms of wildfire, black shank, PVY, and TEV illustrate the continued need for effective tobacco breeding programs. The tobacco industry has also become increasingly concerned about concentrations of tobacco specific nitrosamines (TSNA), which are known carcinogens, in tobacco leaf and smoke in recent years. Because acceptable levels of these compounds have been significantly reduced, many of the older burley cultivars are not acceptable in today's marketplace.

Because the development of burley cultivars via traditional breeding methodologies is a 10-12 year process, new molecular techniques need to be developed and implemented in order to quickly develop new cultivars that have multiple disease resistance and improved chemical composition; the development of these cultivars is going to be vital in keeping Kentucky and Tennessee burley tobacco in high demand in the world market.

2011 Project Description

The objective of the Kentucky-Tennessee Tobacco Improvement Initiative (KTTII) breeding program is the development of burley and dark tobacco cultivars having improved disease resistance and acceptable chemical characteristics, with special emphasis on reduced nornicotine and tobacco specific nitrosamine (TSNA) content.

Nornicotine is a precursor of an important tobacco-specific nitrosamine, N'-nitrosonornicotine(NNN), which is reported to be a carcinogenic component of tobacco products. Nornicotine is derived predominantly from nicotine by nicotine demethylase enzymes. Three genes (CYP82E4, CYP82E5v2, and CYP82E10) with different sites and levels of activity that encode for these enzymes have been identified by researchers at North Carolina State University; ethane methyl sulfonate has since been used to introduce mutations into each of these genes to prevent production of functional gene products. An immediate objective of KTTII is the introgression of e4/e5/e10 mutant alleles into existing KTTII burley cultivars to minimize the conversion of nicotine to nornicotine.

Our research program has developed efficient DNA markers that can be used to differentiate between wild type and mutant alleles. Four dCAPS (derived cleaved amplified polymorphic sequence) markers were designed for a truncation mutation in CYP82E4, and one for a similar mutation in CYP82E5v2. Two CAPS (cleaved amplified polymorphic sequence) markers were designed for a deleterious mutation in CYP82E10. Because of the co-dominant nature of the CAPS and dCAPS markers, heterozygous and homozygous plants can be differentiated. Genotypes determined by the CAPS and dCAPS marker methods were validated by DNA sequencing and phenotyping plants carrying various mutant combinations.

These markers can be used in marker-assisted selection programs to quickly introgress the desired mutations into commercial varieties to impact nornicotine and NNN levels in tobacco leaves and derived products. At present we, have BC3 or BC4 versions of several commercial varieties that hare homozygous for the desired e4/e5 alleles; research is currently underway to also introduce the e10 alleles into these lines.

One of the primary needs of burley tobacco producers in Kentucky and Tennessee is an early maturing variety that has resistance to race 1 black shank. KTH 2901, an early maturing experimental burley hybrid that has resistance to black root rot, tobacco mosaic virus, wildfire, and race 0 and race 1 black shank was evaluated in the Minimum Standards Regional Quality Trial in 2011. If KTH 2901 meets the minimum standards of this program, it will be released as commercial variety KT 212 in March, 2012.

A study was conducted in 2011 to determine the relative impact of variety and management practices on quality of burley tobacco. Six varieties were transplanted in mid-May and again in late June at two locations in Kentucky and two locations in Tennessee; the early trials were harvested in early August and the late trials were harvested in late September. Harvest management regimens included hanging tobacco in a curing barn zero, three, six, or ten days after cutting.

2011 Impact

The emphasis of the KTTII breeding program includes cultivar and germplasm development, applied research to help growers select and integrate appropriate crop cultivars into specific farm production practices, and providing unbiased information on performance and quality of crop cultivars to producers and associated industries.

Prior to 2008, the focus of KTTII was the development of new disease resistant tobacco varieties. However, the primary emphasis of KTTII now is breeding for improved chemical characteristics that will lead to less harmful tobacco products. With the passage of The Family Smoking Prevention and Tobacco Control Act in 2009, the U.S. Food and Drug Administration (FDA) now has regulatory oversight of tobacco products in the United States. This development, coupled with the World Health Organization (WHO) Framework Convention on Tobacco Control, makes it highly likely that the importance of breeding for altered chemical composition will increase in the future.

The incorporation of the e4/e5/e10 alleles into commercial burley varieties will greatly reduce levels of NNN, which is a known carcinogen. Although this project is on-going, it is anticipated that new varieties with much reduced TSNA content will be available for commercial production within the next three to four years. The development of these reduced harm varieties, coupled with other on-going KTTII research, is imperative if burley tobacco grown in the US is to remain competitive on the world export market.

Although much of the research focus of KTTII has shifted to reduced harm varieties, the development of varieties with improved disease resistance continues to be a major objective. Black shank, a soil-borne fungal disease, is particularly devastating, leading to the loss of millions of dollars to growers each year. While approximately 75% of US burley production is comprised of black shank resistant varieties developed by KTTII, there are currently no early maturing black shank resistant varieties. The release of KT 212 will give growers who have black shank infested soils the ability to spread their crops over a longer growing season to better manage labor, equipment and barns. With the passage of the Fair and Equitable Tobacco Reform Act of 2004, there has been a shift in the number and size of burley tobacco farms in Kentucky and Tennessee.

Fewer growers with much larger acreages are no longer able to harvest tobacco only under optimal conditions; many begin harvest in late July and continue until the first frost, regardless of impending rainfall. The 2011 study to determine the relative impact of variety and management practices on quality of burley tobacco demonstrated that although there was no significant difference among varieties for cured leaf quality, rainfall occurring while tobacco remained in the field dramatically reduced leaf quality. Tobacco harvested in late September was more likely to cure with a variegated color than tobacco harvested in mid-August. This information will be presented to growers at Extension meetings and University field days to help growers better manage harvest of their crops.

2011 Publications

Li, Dandan, Ramsey S. Lewis, Anne M. Jack, Ralph E. Dewey, Steve W. Bowen and Robert D. Miller. 2011. Development of CAPS and dCAPS markers for CYP82E4, CYP82E5v2 and CYP82e10 gene mutants reducing nicotine to nornicotine conversion in tobacco. Molecular Breeding. Online, April 11, 2011.

Miller, R.D. 2011. Evaluation of burley cultivars for leaf quality. Paper presented at the 65th Tobacco Science Research Conference, Lexington, KY. Tobacco Abstracts, In Press.

Martinez, N., R.D. Miller, and G. Weinberger. 2011. Selection of resistance to multiple pathogens in tobacco assisted by markers and greenhouse screenings. Paper presented at the 65th Tobacco Science Research Conference, Lexington, KY. Tobacco Abstracts, In Press.

Li, D. A. Jack, and R.D. Miller. 2011. Marker assisted selection (MAS) for tobacco alkaloids. Paper presented at the 65th Tobacco Science Research Conference, Lexington, KY. Tobacco Abstracts, In Press.