1 l Altria Client Services l Tobacco & Leaf Technologies l 11/8/2011

Reduction of nornicotine

in experimental burley

tobaccos and impact on

N-nitrosonornicotine in

lamina and smoke

CORESTA Agronomy and Phytopathology

November 7, 2011

Alec Hayes

2011

_AP

05_H

ayes

.pdf

AP

2011

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Outline

Review recent efforts to impact the levels of nornicotine in

Burley tobaccos.

Discuss the effect of experimental low nornicotine Burley

tobaccos on 100% Burley cigarette smoke NNN.

Discuss the role of nitrate level and its interaction with

nornicotine level in affecting cigarette smoke NNN in 100%

Burley cigarettes.

2 l Altria Client Services l Tobacco & Leaf Technologies l 11/8/2011

2011

_AP

05_H

ayes

.pdf

AP

2011

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

3 l Altria Client Services l Tobacco & Leaf Technologies l 11/8/2011

Nicotine Demethylase Gene Family

Conversion of nicotine to nornicotine in Nicotiana tabacum is mediated by

CYP82E4, a cytochrome P450 monooxygenase

Balazs Siminszky, Lily Gavilano, Steven W. Bowen, and Ralph E. Dewey

PNAS October 11, 2005 vol. 102 no. 41 14919-14924

Biochemical and molecular characterizations of nicotine demethylase in tobacco

Dongmei Xu, Yanxin Shen, Joe Chappell, Mingwu Cui, Mark Nielsen

Physiologia Plantarum Volume 129, Issue 2, pages 307–319, February 2007

CYP82E

N-nitrosonornicotine

NNN

Nornicotine

Nicotine

2011

_AP

05_H

ayes

.pdf

AP

2011

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

4 l Altria Client Services l Tobacco & Leaf Technologies l 11/8/2011

2006 Field Study – Experimental Transgenic Burley

P= 0.1246 P=

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

TN90 LC Exp Trx 1 Exp Trx 2

nornicotine (mg/g)

NNN lamina (ug/g)

NNN (ug/dg TPM)

2006 Field Study

5 l Altria Client Services l Tobacco & Leaf Technologies l 11/8/2011

Lexington, KY Field Study

A

B B Green = analyte level in

filler/lamina

Gray = analyte level in

smoke

* Letters indicate significant differences for smoke NNN based on Tukey-Kramer HSD (P

Triple Mutant Knockout – 2010 Report

6 l Altria Client Services l Tobacco & Leaf Technologies l 11/8/2011

Single mutant

Double mutant

Transgenic

Triple mutant

2011

_AP

05_H

ayes

.pdf

AP

2011

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

2010 Field Study

7 l Altria Client Services l Tobacco & Leaf Technologies l 11/8/2011

A A A

B

Green = analyte level in

filler/lamina

Gray = analyte level in

smoke

* Letters indicate significant differences for smoke NNN based on Tukey-Kramer HSD (P

2010 Field Study

8 l Altria Client Services l Tobacco & Leaf Technologies l 11/8/2011

A A A

B

Green = analyte level in

filler/lamina

Gray = analyte level in

smoke

* Letters indicate significant differences for smoke NNN based on Tukey-Kramer HSD (P

Transgenic RNAi – 2010 Field Evaluation

9 l Altria Client Services l Tobacco & Leaf Technologies l 11/8/2011

0,00

0,50

1,00

1,50

2,00

2,50

3,00

Nornicotine (mg/g)

NNN (ppm)

2010 Field Evaluation - 2 locations, 4 replications

No significant

differences in

nitrate, yield, or

grade index

A A

B B B

* Letters indicate significant differences for lamina nornicotine and NNN based on Tukey-Kramer HSD (P

NNN from 2010 Burleys

10 l Altria Client Services l Tobacco & Leaf Technologies l 11/8/2011

Conventional Inventory Burley

TN90 NDM KT204 NDM

Experimental Burley

Conventional and Experimental Burley

B A C

D E

Green = analyte level in

filler/lamina

Gray = analyte level in

smoke

* Letters indicate significant differences for smoke NNN based on Tukey-Kramer HSD (P

NNN from 2010 Burleys

11 l Altria Client Services l Tobacco & Leaf Technologies l 11/8/2011

Conventional Inventory Burley

TN90 NDM KT204 NDM

Experimental Burley

Conventional and Experimental Burley

B A C

D E

What is the impact of

blending on smoke

NNN?

Blend Hypothesis:

Components will be

additive in smoke.

* Letters indicate significant differences for smoke NNN based on Tukey-Kramer HSD (P

NNN from Burley blends

12 l Altria Client Services l Tobacco & Leaf Technologies l 11/8/2011

A

C B A

B A

* Letters indicate significant differences for smoke NNN based on Tukey-Kramer HSD (P

NNN from Burley Blends

13 l Altria Client Services l Tobacco & Leaf Technologies l 11/8/2011

Five blends evaluated for Smoke NNN:

• Two blends suggested additive trend

• Three blends suggested blend interaction (i.e. blend

not significantly different from 100% LC)

* Letters indicate significant differences for smoke NNN based on Tukey-Kramer HSD (P

Summary

Significant reductions of nornicotine in experimental Burleys can lead

to significant reductions in smoke NNN in 100% Burley cigarettes.

Variation in nitrate level is an important contributor to the smoke NNN

levels observed in 100% Burley cigarettes.

Data suggests blend components may interact and thus

implementing NDM across tobacco types could be important to

achieve significant NNN reductions across the blend.

Next Steps: Further investigate the interaction and relative

contribution of nornicotine level and nitrate in 100% Burley cigarettes

and in combination with other blend components ( e.g. Flue-cured

varieties) .

14 l Altria Client Services l Tobacco & Leaf Technologies l 11/8/2011

2011

_AP

05_H

ayes

.pdf

AP

2011

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A

Acknowledgements

Altria Client Services

–Mark Nielsen

–Marcos Lusso

–Andrew Adams

–Kenny Lion

–Raquel Olegario

–Mohammad Hajaligol

–Susan Plunkett

–Raheema Muhammad

Kah

North Carolina State

University

–Ramsey Lewis

University of Kentucky

–Andy Bailey

–Anne Jack

Virginia Tech

–Danny Peek

15 l Altria Client Services l Tobacco & Leaf Technologies l 11/8/2011

2011

_AP

05_H

ayes

.pdf

AP

2011

- D

ocum

ent n

ot p

eer-

revi

ewed

by

CO

RE

ST

A