Progress 10/01/10 to 09/30/15

Outputs
Target Audience:Tobacco farmers, Extension Specialist, County Agents,and Agri-businesses in Kentucky and Tennessee Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?KTTII is currently providing training to two Ph.D. and one MS graduate students. In the past two years, two visiting scientists from China have worked with KTTII for one year; one of the scientists has returned to work with KTTII for an additional two years. In addition, KTTII typically hosts one to three student interns from Brazil for four to six months each year. For professional development, KTTII researchers attend tobacco research conferences including the Tobacco Workers' Conference, the Tobacco Science Research Conference, and the CORESTA congress. How have the results been disseminated to communities of interest?Research findings are presented at the Tobacco Workers' Conference, the Tobacco Science Research Conference, and the CORESTA congress and published in appropriate refereed journals. Applied research results are presented to tobacco producers at approximately six to eight grower meetings, field days, and industry tours conducted in Kentucky and Tennessee each year; results are also published in bulletins and fact sheets for use by tobacco producers. Quarterly and/or annual reports of sponsored research results are also provided directly to sponsoring companies and agencies. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The original objectives of the Kentucky-Tennessee Tobacco Improvement Initiative (KTTII) were to enhance the development of disease resistant tobacco varieties, support tobacco genetics research, and more efficiently deliver new technologies for tobacco producers in Kentucky and Tennessee. From 2010 through 2015, KTTII developed and released seven new burley and one new dark tobacco cultivar. Burley cultivars KT 210LC, KT 212LC, and KT 215LC were released in 2010, 2012, and 2015, respectively. KT 210LC has good black shank resistance and moderate fusarium wilt resistance. It also has high resistance to black root rot, wildfire, and tobacco mosaic virus, but it is susceptible to tobacco vein mottling virus and tobacco etch virus. KT 212LC is an early maturing cultivar that has high resistance to race 0 and low-medium resistance to race 1 black shank. It also has moderate fusarium wilt resistance and high resistance to black root rot, wildfire, and tobacco mosaic virus, but it is susceptible to tobacco vein mottling virus and tobacco etch virus. KT 215LC was released because of its combined high resistance to race 1 black shank and fusarium wilt. It is a high yielding cultivar that has higher race 1 black shank and fusarium wilt resistance than existing burley cutivars. It also has high resistance to race 0 black shank, black root rot, and wildfire; however, it is susceptible to blue mold and tobacco mosaic virus, tobacco vein mottling virus, tobacco etch virus, and potato virus Y. Dark tobacco cultivar KT D14LC, which was released in 2014, has high resistance to race 0 black shank and higher resistance to race 1 black shank than any other commercial dark tobacco cultivar. KTTII cultivars are widely utilized by tobacco farmers. Approximately 80-85% of burley tobacco and 20-25% of dark tobacco crops in the United States are produced annually from varieties developed and released from KTTII. Based on seed sales, average yields, and prices for burley and dark tobacco, annual sales of US crops produced from KTTII varieties exceed $200 million. Recently the focus of the KTTII breeding program has been on harm reduction in burley tobacco. For the last six years, the primary objective has been the transfer of CYP82e4, CYP82e5v2 and CYP82e10e4 mutant alleles that reduce nicotine to nornicotine conversion (developed by researchers at North Carolina State University) into commercial burley tobacco varieties developed by KTTII. In 2015, new cultivars KT 209Z, KT 210Z, KT 212Z, and ms TN 86Z were released. These new varieties contain extremely low levels of nornicotine and nitroso-nornicotine (reduced by approximately 85%) in comparison to their previous low converting (LC) predecessors. High levels of nornicotine are undesirable in tobacco products due to its detrimental effects on smoke flavor. Even low levels of nornicotine in tobacco have recently become a concern because nornicotine is a precursor of nitroso-nornicotine (NNN). NNN, formed via the nitrosation of nornicotine, is one of the most harmful tobacco specific nitrosamines (TSNA), which are known carcinogens in laboratory animals. It is anticipated that three additional varieties, (TN 90Z, KT 204Z, and KT 206Z) will be released in 2016. KTTII has also been active in the development of molecular markers to facilitate the development of new tobacco varieties, the development of mutation breeding populations, and the optimization of doubled haploid breeding techniques for burley tobacco. New molecular markers developed by KTTII include gene-specific, co-dominant CAPS and dCAPS markers for the CYP82e4, CYP82e5v2 and CYP82e10e4 mutant alleles discussed earlier in this report. A repulsion SCAR marker for blue mold and a repulsion SSR marker for race 0 black shank have also been identified by KTTII. In 2015, new markers were also developed for the Nic 1 and Nic 2 genes that control alkaloid levels in tobacco. By using these new markers, utilized in conjunction with pre-existing markers, differentiation between heterozygous versus homozygous plants for various desirable traits can now be made in segregating breeding populations. KTTII began a project in 2012 to develop mutation populations for use in future variety development. Both fast neutron irradiation (FN) and ethyl methanesulfonate (EMS) are being utilized to achieve mutagenesis. During 2013, a mutation population was developed by exposing approximately 150,000 seeds of breeding line TKF 2002 to 20Gy of FN irradiation. At the 20Gy dose, a large percentage of plants died shortly after germination or failed to grow to transplant size. Many of the surviving plants displayed gross morphological mutations, while other plants appeared to have a morphologically normal phenotype. Most plants displaying gross morphological abnormalities either failed to flower or did not set seed, although a few abnormal plants did produce seed. Among the normal appearing plants, there was a wide range in flowering time and seed set, indicating that many, if not all, of the plants contained mutant alleles that did not affect plant morphology. Selections were made from 4876 plants to establish the initial FN mutation population. A project to optimize the production of doubled haploid breeding lines, initiated by KTTII in 2012, was completed in 2015. In tobacco, haploid plants can be derived by two techniques. Androgenic (paternal) haploids (ADH) can be derived from in-vitro anther culture, while gynogenic (maternal) derived haploids (MDH) can be derived via an interspecific cross between Nicotiana tabacum L. and Nicotiana africana Merxm. The main advantage of these methods for a breeding program is the time saved in obtaining pure lines compared to traditional selection. A disadvantage of ADH shown in flue-cured tobacco was inferior agronomic performance compared to the original cultivars and MDH; in comparison to ADH, MDH plants more closely resembled the original cultivar. Results from the KTTII optimization research project have demonstrated that although many ADH lines are in fact inferior, individual ADH lines can be identified that perform as well as or better than the source cultivar. More importantly, the research demonstrated that both ADH and MDH lines performed as well or better that their source cultivar when used as parental lines for the development of new hybrid cultivars. The research project also determined that in cases where the identification of DH lines containing quantitatively inherited traits for soil-borne diseases is the primary objective, it is more effective to delay the DH process until the F2 generation. Segregating F2 plants can be grown in field nurseries having high levels of the soil-borne disease of interest, with only those plants displaying high disease resistance being selected for use in the DH process. In essence, the plants from segregating F2 line are "prescreened" for quantitatively inherited disease resistance. Research results demonstrated that this approach resulted in a much higher percentage of DH lines that had high resistance to the targeted soil-borne pathogen.

Publications

• Type: Journal Articles Status: Published Year Published: 2012 Citation: Ritchey, E.L., R.D. Miller, and R.L. Ellis. 2014. The influence of nitrogen rate and foliar fertilization on yield, quality, and leaf chemistry in burley tobacco. Tobacco Science. 51:8-12. Li, Dandan, Ramsey S. Lewis, Anne M. Jack, Ralph E. Dewey, Steve W. Bowen and Robert D. Miller. 2012. Development of CAPS and dCAPS markers for CYP82E4, CYP82E5v2 and CYP82e10 gene mutants reducing nicotine to nornicotine conversion in tobacco. Molecular Breeding. Online, March, 2012. (doi:10.1007/s11032-011-9575-9)
• Type: Conference Papers and Presentations Status: Published Year Published: 2010 Citation: Yang, S., X.Wu, X. Sui, Y. Bao, N. Martinez, D. LI, and R. Miller. 2014. Genetic dissection of blue mold resistance in burley tobacco. Paper presented at the 46th Tobacco Workers Conference, Pinehurst, NC. Proceedings. Yang, S., Y. Bao, X. Sui, X. Wu, N. Martinez, D. LI, and R. Miller. 2014. Genetic mapping of black shank resistance in burley tobacco. Paper presented at the 46th Tobacco Workers Conference, Pinehurst, NC. Proceedings. De Oliveira, E. and R.D. Miller. 2014. Evaluation of doubled haploid production in burley tobacco. Paper presented at the 46th Tobacco Workers Conference, Pinehurst, NC. Proceedings. Shelton, C.G. and R.D. Miller. 2014. Field evaluation of burley lines containing alleles minimizing the conversion of nicotine to nornicotine. Paper presented at the 46th Tobacco Workers Conference, Pinehurst, NC. Proceedings. Miller, R.D., L.P. Bush, and A.M. Jack. 2012. Effects of variety and harvest management on cured leaf quality and TSNA content of burley tobacco. Paper presented at the 66th Tobacco Science Research Conference, Concord, NC. Proceedings. Miller, R.D., L.P. Bush, and A.M. Jack. 2012. Effects of variety and harvest management on cured leaf quality and TSNA content of burley tobacco. Paper presented at the 2012 CORESTA Conference, Sapporo, Japan. Proceedings. Miller, R.D.; B.R. Neal, B.R., G.A. Weinberger, and R.A. Hensley. 2012. Effects of variety and harvest management on burley tobacco quality. Paper presented at the 45th Tobacco Workers Conference, Williamsburg, VA. Proceedings. Miller, R.D. 2011. Evaluation of burley cultivars for leaf quality. Paper presented at the 65th Tobacco Science Research Conference, Lexington, KY. Proceedings. 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. Proceedings. 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. Proceedings. Miller, R.D. and R.A. Hensley. 2010. Chemical induction of premature flowering in tobacco. Paper presented at the 2010 CORESTA Conference, Edinburgh, Scotland. Proceedings. Li, D., P. De Sa, and R.D. Miller. 2010. Development of SCAR marker linked to black shank resistance gene in tobacco. Paper presented at the 44th Tobacco Workers Conference, Lexington, KY. Tobacco Abstracts. Li, D., R. Lewis, A. Jack, R. Dewey, S. Bowen, and R. Miller. 2010. Development of dCAPS markers for CYP82E4 and CYP82E5 gene mutations in tobacco. Paper presented at the 44th Tobacco Workers Conference, Lexington, KY. Tobacco Abstracts.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Burley and dark tobacco producers in Kentucky and Tennessee; tobacco companies and commodity groups Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? KTTII is currently providing training to one Ph.D. and two MS graduate students. Two visiting scientists from China and one from Brazil also worked with KTTII for 4-12 months during 2014. In addition, KTTII typically hosts one to three student interns from Brazil for four to six months each year. For professional development, KTTII researchers attend tobacco research conferences including the Tobacco Workers’ Conference, the Tobacco Science Research Conference, and the CORESTA congress. How have the results been disseminated to communities of interest? Research findings are presented at the Tobacco Workers’ Conference, the Tobacco Science Research Conference, and the CORESTA congress and published in appropriated refereed journals. Applied research results are presented to tobacco producers at approximately six to eight grower meetings, field days, and industry tours conducted in Kentucky and Tennessee each year; results are also published in bulletins and fact sheets for use by tobacco producers. Quarterly and/or annual reports of sponsored research results are also provided directly to sponsoring companies and agencies. What do you plan to do during the next reporting period to accomplish the goals? The introgression of the CYP82e4, CYP82e5v2 and CYP82e10e4 mutant alleles that reduce nicotine to nornicotine conversion three alleles into parental lines of existing KTTII varieties is nearing completion. TN 86e3 passed the Regional Quality Trial (RQT) minimum standards in 2013, and TN 90e3, KT 209e3, KT 210e3 and KT 212e3 are being evaluated in the 2014 RQT trials. Testing of KT 206e3 in the RQT is scheduled for 2015. It is anticipated that e3 versions of all these KTTII varieties will be ready for commercial production by 2017 or 2018. Attention has now turned to incorporating these alleles into dark tobacco varieties. Dark varieties generally have relatively low nornicotine content and curing conditions play a more important level in the ultimate level of nitroso-nornicotine, particularly for dark fire-cured tobacco where barn temperatures during firing and total number of times the curing tobacco is fired significantly affect final nitroso-nornicotine content. However, it is highly likely that incorporation of the three nicotine demethylase alleles into dark tobacco varieties will also decrease nornicotine and nitroso-nornicotine levels in dark tobaccos, particularly for dark air-cured tobacco. Another current research project is using marker assisted selection techniques and an early flowering trait to add blue mold resistance to TN 90, KT 204, KT 206, KT 209, KT 210, and KT 212; all of these varieties are well adapted, black shank resistant varieties that are already being utilized by burley growers. The successful completion of this project would greatly reduce the risk of catastrophic losses due to a blue mold epidemic, and eliminate the stress and economic burden of applying fungicides when a potential blue mold outbreak is forecast. Efforts to identify additional molecular markers that will facilitate the development of improved tobacco varieties will continue in 2015. Areas of particular interest include identification of co-dominant markers for Nic-1 and Nic-2 alleles that regulate total alkaloid levels in tobacco, and the development of a gene-specific marker that can be used for selection of resistance to potato virus Y. Research is also being conducted to develop a method that will allow screening for fusarium wilt resistance in a greenhouse. A two year study is also being conducted to determine the effects of plant population on the yield and agronomic characteristics of TN 90LC and KT 206LC. TN 90 is a relatively small variety that is particularly easy to handle but had a seven year average yield of only 2,897 Lbs/A in commercial variety trials; KT 206LC is a larger variety that can be hard to handle under good growing conditions but had a seven year average yield of 3,171 Lbs/A.

Impacts
What was accomplished under these goals? The primary objective of the Kentucky-Tennessee Tobacco Improvement Initiative (KTTII) breeding program is the development of burley and dark tobacco cultivars having improved disease resistance. KT D14LC, a new dark tobacco variety with high resistance to back shank, was released in 2014. Black shank, a soil-borne fungal disease that is widespread throughout the tobacco producing region of the United States, is responsible for millions of dollars in lost profits for tobacco farmers each year. Growers who have black shank typically grow KT D4LC, KT D6LC, KT D8LC, or DT 538LC. KT D4LC and KT D8LC have moderate black shank resistance but they are susceptible to tobacco mosaic virus, wildfire and black root rot; they also typically produce low quality cured leaf. KT D6LC is resistant to tobacco mosaic virus, wildfire and black root rot, and it produces higher quality leaf in comparison to KT D4LC and KT D8LC. However, KT D6LC has somewhat lower black shank resistance than KT D4LC and KT D8LC. DT 538LC has black shank resistance similar to KT D4LC and KT D8LC and produces leaf quality similar to KT D6LC, but it’s resistance to other tobacco diseases is unknown. KT D14LC has superior black shank resistance than current dark fire-cured tobacco cultivars; high resistance to tobacco mosaic virus, wildfire and black root rot; and cured leaf quality similar to KT D6LC and DT 538LC. KT D14LC has plant height, leaf number, internode length, and stalk size similar to other dark fire-cured tobacco cultivars. In comparison to the check varieties, KT D14LC produced higher yields than NL Madole and KY 171 and was comparable to DT 538LC, but it did not yield as well as KT D8LC or KT D6LC. Among the 12 varieties that were included in dark fire-cured tests conducted in 2012 and 2013, KT D14LC ranked third for yield, trailing only the high yielding varieties KT D8LC and KT D6LC. In 2012-2013 air-cured trials, KT D14LC again ranked third for yield among the 12 varieties tested. In black shank trials that were conducted in both Kentucky and Tennessee over three years, KT D14LC was by far the best performing variety. Based on the performance in these trials, a resistance rating of 10 to race 0 and 5 to race 1 black shank is proposed for KT D14LC. The release of this new variety offers dark tobacco producers a much better option for controlling black shank than was previously available. A recent major objective of KTTII has been harm reduction in burley tobacco. A major objective of the KTTII breeding program is the transfer of CYP82e4, CYP82e5v2 and CYP82e10e4 mutant alleles that reduce nicotine to nornicotine conversion (developed by researchers at North Carolina State University) into commercial burley tobacco varieties developed by KTTII. The introgression of these three alleles into parental lines of existing KTTII varieties is nearing completion. TN 86e3 passed the Regional Quality Trial (RQT) minimum standards in 2013, and TN 90e3, KT 209e3, KT 210e3 and KT 212e3 are being evaluated in the 2014 RQT trials. Testing of KT 206e3 in the RQT is scheduled for 2015. It is anticipated that e3 versions of all these KTTII varieties will be ready for commercial production by 2017 or 2018. Attention has now turned to incorporating these alleles into dark tobacco varieties. Dark varieties generally have relatively low nornicotine content and curing conditions play a more important level in the ultimate level of nitroso-nornicotine, particularly for dark fire-cured tobacco where barn temperatures during firing and total number of times the curing tobacco is fired significantly affect final nitroso-nornicotine content. However, it is highly likely that incorporation of the three nicotine demethylase alleles into dark tobacco varieties will also decrease nornicotine and nitroso-nornicotine levels in dark tobaccos, particularly for dark air-cured tobacco. Another current research project is using marker assisted selection techniques and an early flowering trait to add blue mold resistance to TN 90, KT 204, KT 206, KT 209, KT 210, and KT 212; all of these varieties are well adapted, black shank resistant varieties that are already being utilized by burley growers. The successful completion of this project would greatly reduce the risk of catastrophic losses due to a blue mold epidemic, and eliminate the stress and economic burden of applying fungicides when a potential blue mold outbreak is forecast.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: Ritchey, E.L., R.D. Miller, and R.L. Ellis. 2014. The influence of nitrogen rate and foliar fertilization on yield, quality, and leaf chemistry in burley tobacco. Tobacco Science. 51:8-12. Yang, S., X.Wu, X. Sui, Y. Bao, N. Martinez, D. LI, and R. Miller. 2014. Genetic dissection of blue mold resistance in burley tobacco. Paper presented at the 46th Tobacco Workers Conference, Pinehurst, NC. Proceedings. Yang, S., Y. Bao, X. Sui, X. Wu, N. Martinez, D. LI, and R. Miller. 2014. Genetic mapping of black shank resistance in burley tobacco. Paper presented at the 46th Tobacco Workers Conference, Pinehurst, NC. Proceedings. De Oliveira, E. and R.D. Miller. 2014. Evaluation of doubled haploid production in burley tobacco. Paper presented at the 46th Tobacco Workers Conference, Pinehurst, NC. Proceedings. Shelton, C.G. and R.D. Miller. 2014. Field evaluation of burley lines containing alleles minimizing the conversion of nicotine to nornicotine. Paper presented at the 46th Tobacco Workers Conference, Pinehurst, NC. Proceedings.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Burley and dark tobacco producers in Kentucky and Tennessee; tobacco companies and commodity groups. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? KTTII is currently providing training to two Ph.D. and one MS graduate students. Two visiting scientists from China are also currently working with KTTII for one year. In addition, KTTII typically hosts one to three student interns from Brazil for four to six months each year. For professional development, KTTII researchers attend tobacco research conferences including the Tobacco Workers’ Conference, the Tobacco Science Research Conference, and the CORESTA congress. How have the results been disseminated to communities of interest? Research findings are presented at the Tobacco Workers’ Conference, the Tobacco Science Research Conference, and the CORESTA congress and published in appropriated refereed journals. Applied research results are presented to tobacco producers at approximately six to eight grower meetings, field days, and industry tours conducted in Kentucky and Tennessee each year; results are also published in bulletins and fact sheets for use by tobacco producers. Quarterly and/or annual reports of sponsored research results are also provided directly to sponsoring companies and agencies. What do you plan to do during the next reporting period to accomplish the goals? The KTTII project to transfer the CYP82e4, CYP82e5v2 and CYP82e10e4 mutant alleles into commercial burley tobacco varieties developed by KTTII will be continued in 2014. For several of the parental line families, it was possible to make selections of individual plants that were comparable to their original LC counterparts in disease nurseries. These selections will be thoroughly evaluated in 2014 field trials; if they are determined to be comparable to the original LC lines, they will be used to release new low nornicotine converting hybrid varieties. For other parental line families evaluated in 2013, it was not possible to identify plants possessing acceptable agronomic and disease resistance traits. For these lines, additional backcrosses were made to the recurrent LC parent. These backcross lines will need to be self-pollinated and then screened via molecular markers to identify plants that are homozygous for all three mutant alleles. This will cause an approximate two year delay in the development of new low nornicotine converting hybrid varieties utilizing these parental lines. Efforts to identify additional molecular markers that will facilitate the development of improved tobacco varieties will continue in 2014. Areas of particular interest include identification of co-dominant markers for Nic-1 and Nic-2 alleles that regulate total alkaloid levels in tobacco, and the development of a gene-specific marker that can be used for selection of resistance to potato virus Y. For the mutation breeding project, an EMS procedure will be utilized to obtain a second mutagenized population that can be utilized along with the FN population developed in 2013 to search for mutant traits of interest that can eventually be bred into new tobacco cultivars. For the doubled haploid (DH) optimization project, a study will be conducted in 2014 to determine the optimal breeding generation for the initiation of the DH process in cases where obtaining high levels of quantitatively inherited disease resistance is the primary objective. DH lines are usually generated from greenhouse grown plants in the F1 generation in order to minimize the time required to reach homozygosity. The haploid or doubled haploid plants are then screened for resistance; although the best DH lines can subsequently be identified in disease nurseries, true selection is not possible because disease resistance is fixed due to the homozygosity of the DH lines. Where quantitatively inherited traits for soil-borne diseases are the primary objective, it may be better to delay the DH process until the F2 generation. Segregating F2 plants can be grown in field nurseries having high levels of the soil-borne disease of interest, with only those plants displaying high disease resistance being selected for use in the DH process. In essence, the plants from segregating F2 line are “prescreened” for quantitatively inherited disease resistance. To test this hypothesis, four experimental hybrid lines have been produced from specific combinations of parental lines having differing levels of resistance to the soil-borne diseases fusarium wilt, bacterial wilt, and race I black shank. The target of each of the crosses is the development of DH lines having high levels of quantitative resistance to either fusarium wilt, race 1 black shank, or bacterial wilt, depending on the specific cross. From each of the four hybrid lines, DH lines were generated from both the F1 and F2 generations. For the F1 generation, DH lines were developed from greenhouse plants without pre-selection for disease resistance. For development of DH lines from F2 generations, segregating F2 plants from each cross were grown in field nurseries containing high infestations of either fusarium wilt, race 1 black shank, or bacterial wilt, depending on the disease targeted by a particular cross. During the 2014 growing season, DH lines developed from the F1 and F2 generations will be evaluated in the same disease nurseries used for the development of F2 DH lines. Disease survival ratings and yield data will be collected to determine if the pre-selection for quantitative resistance afforded in the F2 lines results in improved quantitative resistance.

Impacts
What was accomplished under these goals? The original objectives of the Kentucky-Tennessee Tobacco Improvement Initiative (KTTII) were to enhance the development of disease resistant tobacco varieties, support tobacco genetics research, and more efficiently deliver new technologies for tobacco producers in Kentucky and Tennessee. Since its inception in 1999, KTTII has developed and released six new burley tobacco cultivars (KT 200LC, KT 204LC, KT 206LC, KT 209LC, KT 210LC, and KT 212LC) and three new dark tobacco cultivars (KTD4LC, KT D6LC, and KTD8LC). Approximately 80-85% of burley tobacco and 20-25% dark tobacco crops in the United States are produced annually from varieties developed and released from KTTII. Based on seed sales, average yields, and prices for burley and dark tobacco, annual sales of US crops produced from KTTII varieties exceed $200 million. However, more recently the focus has been on harm reduction in burley tobacco. Currently, the primary objective of the KTTII breeding program is the transfer of CYP82e4, CYP82e5v2 and CYP82e10e4 mutant alleles that reduce nicotine to nornicotine conversion (developed by researchers at North Carolina State University) into commercial burley tobacco varieties developed by KTTII. The introgression of these three alleles into parental lines of existing KTTII varieties is nearing completion. Parental lines and hybrid varieties containing the three mutant alleles were compared with the original LC varieties in replicated field trials at three locations during 2013 to evaluate their agronomic characteristics, including yield and quality. Similar comparisons were made in two race 1 black shank nurseries to evaluate levels of black shank resistance. Within each hybrid and parental line group, there was considerable variation among the individual e3 lines for agronomic type at both Lexington and Versailles and for resistance in the black shank nurseries. In general, all of the e3 parental lines and hybrids were easily detectable, and in most cases were inferior in comparison to the original LC materials. In general, all of the e3 parental lines were inferior to the original LC parental lines. Although some of the differences were primarily cosmetic in the replicated field trials, differences were much more obvious in the black shank nurseries. Morphological problems that seemed to be more or less consistent across all e3 materials included wide leaf internode (particularly in the top third of a plant) and a tendency for upper leaves to be “rolled” or “cupped”; the severity of these symptoms varied among lines and individual plants for a given variety family, which suggests that careful plant selection or/and additional backcross followed by self-pollination and selection, may eliminate these problems. Other areas of research conducted by KTTII in 2013 include the identification of molecular markers to facilitate the development of new tobacco varieties, development of mutation breeding populations, and optimization of doubled haploid breeding techniques for burley tobacco. New molecular markers developed by KTTII include gene-specific, co-dominant CAPS and dCAPS markers for the CYP82e4, CYP82e5v2 and CYP82e10e4 mutant alleles discussed earlier in this report. A repulsion SCAR marker for blue mold and a repulsion SSR marker for race 0 black shank were also identified by KTTII during 2013; utilized in conjunction with existing dominant SCAR markers for both diseases, differentiation between heterozygous versus homozygous plants for both diseases can be made in segregating breeding populations. KTTII began a project in 2012 to develop mutation populations for use in future variety development. Both fast neutron irradiation (FN) and ethyl methanesulfonate (EMS) are being utilized to achieve mutagenesis. During 2013, a mutation population was developed by exposing approximately 150,000 seeds of breeding line TKF 2002 to 20Gy of FN irradiation. At the 20Gy dose, a large percentage of plants died shortly after germination or failed to grow to transplant size. Many of the surviving plants displayed gross morphological mutations, while other plants appeared to have a morphologically normal phenotype. Most plants displaying gross morphological abnormalities either failed to flower or did not set seed, although a few abnormal plants did produce seed. Among the normal appearing plants, there was a wide range in flowering time and seed set, indicating that many, if not all, of the plants contained mutant alleles that did not affect plant morphology. Selections were made from 4876 plants to establish the initial FN mutation population. A new project to optimize the production of doubled haploid breeding lines was initiated by KTTII in 2012. In tobacco, haploid plants can be derived by two techniques. Androgenic (paternal) haploids (ADH) can be derived from in-vitro anther culture, while gynogenic (maternal) derived haploids (MDH) can be derived via an interspecific cross between Nicotiana tabacum L. and Nicotiana africana Merxm. The main advantage of these methods for a breeding program is the time saved in obtaining pure lines compared to traditional selection. A disadvantage of ADH shown in flue-cured tobacco was inferior agronomic performance compared to the original cultivars and MDH; in comparison to ADH, MDH plants more closely resembled the original cultivar. The objective of the current project is to determine the most efficient and effective method to develop doubled haploid plants of burley tobacco. ADH and MDH methodologies are being compared for relative efficiency, time required, cost for obtaining DH lines, and agronomic performance. During 2013, ten ADH and ten MDH lines derived from TN 90LC were randomly selected and compared to the original TN90LC in field trials conducted at three locations to determine the relative vigor of ADH and MDH lines versus the original cultivar. Although this study is still on-going, certain conclusions can already be drawn. It has been much easier to produce ADH plants than MDH plants. Based on field observations, in general ADH lines appeared to be inferior to MDH lines and the original TN 90LC. However, there was considerable variation among the 10 ADH lines and among the 10 MDH lines. Because TN 90LC is an F14 homozygous variety, the variability among the doubled haploid lines is likely the result of soma-clonal variation that occurred during the tissue culture process. However, based on agronomic characteristics and yield results obtained from one field trial to date, several individual ADH and MDH lines are comparable to TN 90LC. This suggests that with vigorous selection, either method can be used to produce usable doubled haploid burley tobacco breeding lines.

Publications

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: The primary objective of the Kentucky-Tennessee Tobacco Improvement Initiative (KTTII) breeding program is the development of burley and dark tobacco cultivars having improved disease resistance. KT 212LC was released in March of 2012 to provide burley producers with an early maturing variety that also possesses moderate resistance to race 1 black shank. The new variety has a yield rating of eight and disease resistance ratings of: 10 for race 0 black shank; 4 for race 1 black shank; 5 for fusarium wilt; and high for black root rot, wildfire, and tobacco mosaic virus. It is susceptible to TVMV, TEV, PVY, and blue mold. The maturity of KT 212LC is very similar to ms KY 14 X L8LC, but much earlier than previous KT varieties. The growth habit of KT 212LC is not as erect as previous KT varieties, but it is not as droopy as ms KY 14 X L8LC. KT 212LC was compared with six standard burley varieties in twelve advanced breeding line trials from 2009 through 2011. In the absence of black shank, the yield of KT 212LC was higher than the yield for TN 90LC, comparable with ms KY 14 X L8, but somewhat lower than for KT 204LC, KT 206LC, KT 209LC, and KT 210LC. KT 212LC was comparable with the check varieties for the quality index, which is indicative of cured leaf quality as judged by official USDA grades, and gross income per acre. When evaluated for percent survival in black shank nurseries, the performance of KT 212LC was similar to TN 90LC, much better than ms KY 14 X L8LC, but substantially worse than the other KT varieties. It is not recommended for use in fields having a history of black shank. KT 212LC will be available to growers for the 2013 production season. Two other KTTII breeding lines are nearing release as commercial cultivars. DFH 1101 is a dark tobacco hybrid breeding line that has excellent yield potential and significantly higher resistance to race 1 black shank in comparison to existing dark cultivars. It has received a good quality rating in performance trials and is currently being evaluated for industry usability and chemical characteristics. If it is found to meet industry standards, it will be released in early 2014. KTH 2802 is a high yielding burley hybrid breeding line that has superior combined resistance to race 1 black shank and fusarium wilt than existing burley cultivars. In performance trials conducted in disease nurseries, KTH 2802 has displayed higher resistance to fusarium wilt than the resistant standard KT 210LC, and higher resistance to race 1 black shank than KT 209LC, which is currently the most resistant commercial cultivar available to growers. KTH 2802 was entered in the 2012 Regional Quality Test; if it meets the minimum standards established for that program it will be released as KT 213LC in February, 2013. Another major 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, a crucial step in the formation of TSNA. This project is nearing completion and it is anticipated that several breeding lines containing the mutant alleles will be entered into the 2013 RQT program. PARTICIPANTS: The Kentucky-Tennessee Tobacco Improvement initiative is a collaborative research program between the University of Kentucky and the University of Tennessee. The Principal Investigator of KTTII is supported 60% by UK and 40% by UT. TARGET AUDIENCES: The primary target audience of KTTII are tobacco producers in Kentucky and Tennessee. Research findings are presented at various grower meetings and field days in KY, TN, Ohio, Virginia, and North Carolina. Research results are also published in the Kentucky-Tennessee Tobacco Production Guide. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The objectives of the Kentucky-Tennessee Tobacco Improvement Initiative (KTTII) are to develop improved tobacco varieties, support tobacco genetics research, and more efficiently deliver new technologies for the maximum benefit of burley and dark tobacco farmers in Kentucky and Tennessee. Considerable progress has been made in the area of variety development. Six new burley varieties (KT 200, KT 204LC, KT 206LC, KT 209LC, KT 210LC and KT 212LC) have been released since the year 2002. In addition, older varieties TN 86, TN 90, TN 97, KY14, KY17, KY 907, and ms KY 14 X L8 have been re-released as new low nicotine converting (designated as LC) varieties due to new, more stringent chemical standards throughout the tobacco industry. Dark tobacco varieties developed by KTTII include KT D4LC, KT D6LC, and KT D8LC. In keeping with the mission of Land Grant Universities, varieties developed by KTTII are made available to all burley and dark tobacco growers through non-exclusive releases to participating tobacco seed companies. These new varieties have been readily accepted by tobacco growers. The top five burley varieties grown each year from 2008 through 2012 were released by KTTII, comprising approximately 75-80% of the US crop. The release of the new varieties has increased profitability of burley tobacco growers. Tobacco extension specialists in Kentucky and Tennessee estimate that the average yield increase due to these KTTII varieties is approximately 200-300 lbs per acre; for growers with black shank, yield increases would be much higher. USDA estimates indicate that 96,800 acres were planted in burley tobacco in the US during the 2012 crop year; based on seed sales records, at least 75% or 72,600 acres were planted in KTTII varieties. At an average price of $1.75 per pound, an increased yield of 200 lbs/acre due to the use of KTTII would result in over $25,000,000 in increased income for US burley tobacco growers in 2012 alone. 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, one of the primary emphases 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 development of these reduced harm varieties, coupled with other on-going KTTII research, is imperative if burley and dark tobacco grown in the US is to remain competitive on the world export market.

Publications

• Miller, R.D., L.P. Bush, and A.M. Jack. 2012. Effects of variety and harvest management on cured leaf quality and TSNA content of burley tobacco. Paper presented at the 2012 CORESTA Conference, Sapporo, Japan. Proceedings. In Press.
• Miller, R.D., B.R. Neal, B.R., G.A. Weinberger, and R.A. Hensley. 2012. Effects of variety and harvest management on burley tobacco quality. Paper presented at the 45th Tobacco Workers Conference, Williamsburg, VA. Proceedings.
• Li, D., A. Jack, and R. Miller. 2012. Selection for the low alkaloid trait: Part 2, marker assisted selection. Paper presented at the 45th Tobacco Workers Conference, Williamsburg, VA. Proceedings.
• Miller, R.D., L.P. Bush, and A.M. Jack. 2012. Effects of variety and harvest management on cured leaf quality and TSNA content of burley tobacco. Paper presented at the 66th Tobacco Science Research Conference, Concord, NC. Proceedings. In Press.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: 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. PARTICIPANTS: Dr. Robert D. Miller, Principal Investigator of the Kentucky-Tennessee Tobacco Improvement Initiative, is a Professor of Plant and Soil Science at the University of Kentucky and a Research Professor of Plant Sciences at the University of Tennessee. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
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 grower better manage harvest of their crops.

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.