ho s for thentifi ion of

aoco Cultivars

Dr. Peter RangeLandesanstalt fOr Pflanzenbau Forchheim

Chern. Ing. Savvas DivanidisTobacco Institute of Greece

Scientific Series of the International Bureau Volume 28

Forschungszentrum JOlich GmbHScientific Series of the International Bureau

GREEK AND GERMANTOBACCO VARIETIES

Chemical Methods for theIdentification ofTobacco Cultivars

Dr. Peter RangeLandesanstalt fOr Pflanzenbau Forchheim

Chem. Ing. Savvas DivanidisTobacco Institute of Greece

German-Greek-Cooperationin Scientific Research and Technological Development

Die Deutsche Bibliotl1ek - CIP - Einl1eitsaufnal1me

Greek and German tobacco varieties: chemical methods for the identification oftobacco cultivars : German Greek cooperation in scientific research and tecl1nologicaldevelopment / Forscl1ungszentrum Julicl1 GmbH. Peter Range; Sawas Divanidis. ­Jullch : Forscl1ungszentrum, Zentralbibliotl1ek, 1995(Scientific series of tile International Bureau / Forscl1ungszentrum JOIicl1 GmbH; Vol.28)ISBN 3-89336-145-6NE: Range, Peter:; Forschungszentrum <Julich~ Ilnternationales BOro: Scientific series of ..,

Herausgeberund Vertrieb:

Druck:

Copyright:

Forschungszentrum JOlich GmbHZENTRALBIBLIOTHEK0·52425 JOlichTelefon (02461) 61·5368· Telefax (02461) 61·61 03

Graphische Kunstanstalt Dieter Gehler, DOren-Birkesdorf

Forschungszentrum JOlich 1995

Scientific Series of the International Bureau, Volume 28

ISSN 0938·7676

ISBN 3·89336·145-6

TABLE of contents Page

Preface 7

Abstract 8

1 Introduction 9

2 Tobacco Plantation 102.1 Soil and Growing Conditions 102.1.1 Flue Cured Tobacco 112.1.2 Oriental Tobacco 122.1.3 Burley Tobacco . 122.2 Plantation Areas in Greece 122.2.1 Thessaloniki-Soxos and Karditza-Mitropoli 132.2.2 Thessaloniki-Plati 142.2.3 Komotini and Kozani-Chirolimni 142.2.4 Katerini-Vrias/N.Efessos 152.2.5 Agrinion-Sfina 16

3 Tobacco Quality 173.1 Outer and Inner Quality 173.2 Regulations of the European Union 183.3 Methods 183.3.1 Nitrate 193.3.2 Carbon Monoxide 193.3.3 Nitrogen Oxides 19

4 Greek Tobacco Breeding Lines 224.1 Breeding Line 5/R 224.2 Breeding Line 4N 254.3 Breeding Line 1IB 284.4 Breeding Line 2/K 314.5 Breeding Line 3/S 314.6 Discussing the Results 31

5. German Tobacco Cultivars 345.1 Geudertheimer 345.2 Burley 355.3 The Effects of Nitrogen-Fertilization 375.3.1 Condensate 375.3.2 Nicotine 395.3.3 Carbon Monoxide 405.3.4 Nitric Oxide 415.3.5 Nitrate 435.3.6 Nicotine and Nornicotine 435.4 Discussing the Results 436 Tobacco Breeding 446.1 Identification of Tobacco Varieties 456.2 Secondary Plant Compounds of Content 456.3 Isoenzymes and Alloenzymes 456.4 Methods 466.4.1 Protein Extraction Procedures 466.4.2 Electrophoresis Procedure 47

5

6.4.3 Isoelectric Focusing Procedure 486.4.4 Protein and Enzyme Staining Techniques 486.4.5 Documentation 496.5 Results and Discussion 506.5.1 Tobacco Seed 506.5.2 Green Leaf : 516.5.3 Dry Tobacco 52

7 Conclusion 53

EXPERIMENTAL 57

8 Nitric Oxide Analysis 578.1 BAT Nitric Oxide Analyzer in Combination with Smoking Machine Filtrona

SM 302 578.1.1 Additional Equipment required 578.1.2 Setting Up and Calibration Instructions 578.1.3 Smoking Procedure 588.2 Monitor Labs. Nitrogen Oxides Analyzer 8440E in Combination with Smoking

Machine Borgwaldt RM 20 CS and Automatically Working Nitrogen FlushingUnit 59

8.2.1 Additional Equipment required 598.2.2 Setting Up and Calibration 608.2.3 Smoking Procedure 60

9 Electrophoresis 619.1 Protein and Enzyme Extraction 619.1.1 Equipment 619.1.2 Buffers 629.1.3 Reagents 629.1.4 Staining Salts 629.1.5 Sample Preparation 629.1.6 Cleaning Procedure for the Dialysis Tube 649.1.7 Rapid Determination of the Protein Concentration 649.2 Vertical Polyacrylamide Slab Gel Electrophoresis 659.2.1 Equipment 659.2.2 Buffers 669.2.3 Reagents 669.2.4 Staining Salts 669.2.5 Stock Solutions 669.2.6 Simple Gels 689.2.7 SDS-Gradient Gels 699.2.8 Application of Samples 719.2.9 Operating the Electrophoresis 719.2.10 Removing the Gels 719.3 Isoelectric Focusing 729.3.1 Agarose Gels 729.3.2 Preparation of Solutions 729.3.3 Sample Preparation 739.3.4 Operating the Isoelectric Focusing 739.3.5 Staining of Samples 749.4 Protein and Enzyme Staining Procedures 759.4.1 Unspecific staining of proteins 759.4.2 Rapid Staining for Native Gels and IEF-Gels 76

6

9.4.3 Silver Staining 769.4.4. Destaining and Drying of Polyacrylamide Slab Gels 779.4.5 Specific Staining of Enzymes 779.5 Enzyme Staining Procedures of Agarose Gels 789.5.1 Chemicals 789.5.2 Preparation of Enzyme Buffers 789.5.3 Transfer Membrane Preparation Solutions : 799.5.4 Stain Solutions 799.5.5 Enzyme Staining 80

10 Summary 81

BIBLIOGRAPHy 83

7

8

Preface

In the framework of the German-Greek co-operation in Science and Technology, a projectwas implemented by the Landesanstalt fur Pflanzenbau Forchheim, Rheinstetten and theTobacco Institute of Greece, Drama, directed to the plantation control of tobacco varietiesin the Common Market, to the development of methods for the determination of nitric oxidein tobacco smoke and to the improvement of identification procedures for tobacco var­ieties in seed, green and dried leaves and in tobacco powder.

Joint work also comprised the plantation of tobacco varieties at five tobacco stations in thenorthern and middle part of Greece, having different soil and climate conditions.

Preliminary results have been presented at the 29th Workshop of the Technical Committeefor Tobacco Plantation of the Federal Association ofthe German Tobacco Farmers, Bruch­sal-Buchenau, Germany, 1989, at the XXXII. Tobacco-Colloquy, in Toulouse, France, 1990,at the Coresta Symposium, Kallithea (Halkidiki), Greece, 1990 and at the 1st German-GreekWorkshop on Environmental Engineering in Oberpfaffenhofen, 1991.

The authors wish to express their gratitude to Dr. P. Schweiger, Director of the Landesan­stalt fur Pflanzenbau Forchheim, to Prof. A. G. Sficas, Department of Agriculture, Universityof Thessaloniki and to Mr. Mour Paschal ides, the former Directors of the Tobacco Instituteof Greece, for their helpful discussions and their kindness, to Prof. R. Preussmann, Instituteof Toxicology and Chemotherapy, German Cancer Research Center, Heidelberg, for thekind help in measuring the nitrosamines, to Mr. H. Drescher and Mr. U. Krohn from H. Borg­waldt, Hamburg, to Mr. K. RUdy, Mr. Weindl, Mr. A. Zangenberg, Mr. H. P. Eismann and Mr.E. Aichele, from Kontron Elektronik, Eching - MOnchen, for their support and helpful dis­cussions during the development of the nitric oxide method for tobacco smoke, to Prof. K.Wegmann, Institute of Chemical Plantphysiology, University of TObingen, to Dr. H. Delinceeand Mrs. Sigrid Delincee, Institute of Biochemistry, Federal Agency of Food (BFE), Karls­ruhe, and to Mrs. Maria MOiler from Desaga, Heidelberg, for their support and helpful dis­cussions during the development of the electrophoretic methods for the tobacco proteindetermination. The authors are deeply indebted to Mrs. Maria MOiler for reading the elec­trophoresis chapters and offering valuable comments, also to Mrs. Litsa Divanidis for thetranslation of several chapters and reading the report.

Acknowledgments are gratefully extended, to Dr.Andrea Schumacher and to Dr. N. Billenk­amp, Breeders of the Landesanstalt fur Pflanzenbau Forchheim, in carrying out the plan­tation and selection of the tobacco varieties, to Mr. Sp. Halivopoulos and Mrs. Vasiliki Tsak­maki, Chemists of the Tobacco Institute of Greece, for their valuable assistance in carryingout this project, to Mrs. Elisabeth Fischer, in carrying out the electrophoretic and thin layerisoelectric focusing separation of tobacco proteins, and to Mr. P. Kappler and P. Klein, incarrying out the tobacco and tobacco smoke analysis, the nitric oxide calculations and thedata processing, Chemistry Technicians of the Landesanstalt fur Pflanzenbau Forchheim,and to Mr. B. Turkochorlis and to Mr. D. Hatzigeorgiou, Chemistry Assistants of the To­bacco Institute of Greece, in carrying out the tobacco and tobacco smoke analysis and thedata processing and last to Mr. U. Ziegler, agronomist of the Landesanstalt fOrPflanzenbauForchheim, who showed us the possibilities to work with different standard graphic pro­grams to create our graphs.

The authors feel deeply indebted for the equipment support and the grants of the FederalMinistry for Research and Technology, Bonn, and the grants of the Ministry of Industry,Energy and Technology, Athens.

9

Abstract

Seventeen German and five Greek cultivars from the harvest 1987 and 25 German Geuder­theimer cultivars, - new breeding lines and common types from Oriental-, Clqar-, Burley­and Virgintobacco -, from the harvest 1990 were analyzed according to nitrogen fertiliz­ation for the leaf constituents nitrate, alkaloids, sugar, and nitrogen, for the smoke consti­tuents condensate, nicotine, carbon monoxide and nitric oxide: The limitation to 15 mgcondensate per cigarette, asked by the European Commission for 1993, could only be ful­filled with the lower stalk positions of the cultivars. Nicotine was changing according to thetobacco types. Carbon monoxide was found in a middle range equal for all types. Nitricoxide was changing according to the nitrogen fertilization and the cultivars. A correspond­ing increase of the nitric oxide percentage in the tobacco smoke was observed in varietiesneeding more nitrate fertilizer. Nitrate, nicotine and the sum of nornicotine and myosmine inthe leaf correlated with cultivar, climate and fertilization. The nitrosamines in the tobaccoleaf and the tobacco smoke varied with the nitrate content in the cultivars, the harvestingand curing conditions.

Cigarettes, made of tobacco leaves and containing small amounts of nitrate, generatedless nitric oxide than cigarettes made of tips, which contained more nitrate.

Twenty-five breeding lines of dark air cured tobacco from the harvest 1990 were identifiedwith Polyacrylamide Gel Electrophoresis.

10

1 Introduction

Irakllon~"'·

n I k I

II Na Ii 0 n

..

..Arta

Picture 1: Tobacco Plantation Areas in Greece

In Greece tobacco is in the first rank of the national income; the main plantation areas areplotted in picture 1.

In Germany tobacco belongs to the special-purpose crops1-6); the main plantation areasare plotted in picture 2.

In Baden-Wurtternberq for example with its small family farms, 1987 2.5% of the area wascultivated with these crops, but the profit amounted to 73% of all crops/!

The cultivation of tobacco in the EU is restricted to regions with well-defined cultivars andcertain yields. According to the region, cultivar and classification, the tobacco farmers geta premium of more than50% of the tobacco pricefrom the EU. The premiumsfor the cultivars are paid ac­cording to the regional plan­tation conditions.

In the Common Market 1988the situation for the Greektobacco farmers was char­acterized by only a littlechance of selling the localdark varieties especially ofthe Agrinion area. The Ger­man tobacco farmers hadalso problems, caused byan Italian overproduction oftheir Geudertheimer var­ieties in South Italy.

In the year 1989, there was aseparation of the Geuder­theimer plantation areas inan area "GeudertheimerNorth" in Germany and anew area "GeudertheimerSouth" in Italy. Before 1989it was attractive to thefarmers in southern regionsof Italy to plant GermanGeudertheimer varieties toget the high premium. Thispremium was much higherthan the premium for theown regional tobacco cultlv­ars. In consequence toomuch Geudertheimer to­bacco of bad quality wasproduced, which couldn'tbe sold and had to bebought at the intervention price from the Common Market. Consequently, for the followingyear the EU authorities reduced the plantation area of the Geudertheimer cultivars. Thisarea restriction was made at the German farmers' expense.

11

II Dresden

Berlin

III

NCirnb'fQ

IIIStuttgart

IIIBremen

IIIacnn

On the other hand all to­bacco producing countriesof the EU were asked by theEU Commission to lower thehazardous compounds, es­pecially the tar content. Ac­cording to the directive "Eu­rope against Cancer" the taryield of all cigarettes mar­keted in the European Unionshould not be greater than15 mg beyond 1993 and notgreater than 12 mg beyond1998. Therefore, all high tarproducing tobacco cultivarswould have no chance ofselling in the Common Mar­ket after this date. ForGreece there is an exceptiontill the year 2006.

For the produce control it isnecessary to develop newbreeding lines with low tarcontent and low contents ofcarbon monoxide and ni­trogen oxides, and it isnecessary to develop newlaboratory methods, too, todifferentiate between the to­bacco cultivars.

Picture 2: Tobacco Plantation Areas in Germany There is a special interest forGreece and Germany and forthe other tobacco producing

countries, too, in developing identification methods to detect whether the offered tobaccoin the Common Market is from the declared plantation area or not. This has to be done witha "cultlvar card" characterizing the tobacco according to its outer and inner quality and ananalytical description of the tobacco cultivar too.

2 Tobacco PlantationGreek and German tobacco planters look for new fields for the plantation of flue cured to­bacco. Planting tobacco, it is necessary to understand how the manifestation of soil char­acteristics is influenced by the environment. In potential new growing areas"" this can com­plicate the problem and possibly mislead the choice of soils for early trials?'.

2.1 Soil and Growing Conditions

Reasonable moisture-holdin~ capacity and free drainage, pH-, organic matter- and salin­ity-values (see table Nr. 1)9-1 ), as the contents of nitrogen, phosphorous, potassium, cal­cium, magnesium, manganese and chlorine, are the only basic requirements needed inchoosing suitable soils (see tables Nr. 2 and 3).

12

Table 1: Recommended pH-, Electric Conductivity-, and Organic Matter-Values for SoilSamples from Fields for Tobacco Plantation

Electric OrganicpH Conductivity Matter

mS/cm %C

sand, loamy sand 5,3 - 6,0 < 0,75 1-4

sandy loam 6,1 - 6,7 < 0,75 1-4

loam 6,8- 7,2 < 0,75 1-4

Table 2: Recommended P-, K- and Mg-values for soil Samples from Fields for TobaccoPlantation

P K Mgmg/kg mg/kg mg/kg

light soils 92 -109 108 -166 42 - 60

medium soils 92 -109 174 - 249 78 -120

strong soils 92 -109 257 - 291 90 -150

Table 3: Recommended Mn- Values for Soil Samples from Fields for Tobacco Plantation

Active Mn mg/kg

pH < 6,0 ph 6,0 - 6,5 ph> 6,0

low <3 < 5 < 7

medium 3-4 5 -10 7 -14

high >4 >10 >14

According to the type of tobacco and the part of the growing season the plant uptake of nu­trition elements is different. Nitrogen uptake for virgin cultivars for instance must be low ornonexistent in the latter part of the growing season. On the opposite acceptable growth,yield and quality are not obtained without adequate nitrogen nutrition during early growth.Similar conditions are found for oriental tobaccos, while Burley cultivars need four timesmore nitrogen during the growing season.

2.1.1 Flue Cured Tobacco

Flue-cured tobacco is a low-nitrogen product. In an open soil, tobacco roots have beenfound at a depth of 120 em, so concern is not only with the topsoil. Subsoil will much in­fluence the moisture status, according to whether its permeability is excessive or very re­stricted. Not so obvious is the subsoil potentiality for releasing nitrogen towards the end ofthe season. Increasing fineness of texture and increasing redness of color are indications ofsuch a possibility. Color is a more certain guide than texture.

The basic requirements will obviously be met by medium-coarse sands or sandy loams onthe surface, underlain by fine sandy loam or sandy clay, which extends to a depth of 120 ­150 em. The pH-values will normally be in the range 5.5 - 5.6.

13

The typical flue-cured soils are light-colored sandy loams and fine sandy loams, 15 - 25 cmdeep, with yellowish or reddish sandy clay subsoil. They will have greater latitude in ab­sorbing variations of the climate and giving a reasonable return from a range of weatherconditions-which, of course, is why they are considered particularly suitable. Growing flue­cured tobacco on a light, well-drained soil provided care has been taken in making up thefertilizer dressing, the optimum cultural treatments can be used to extract the maximumpossible crop from the environment in the reasonable expectation that as yield increasesso will the degree of usability8).

2.1.2 Oriental Tobacco

The finest, most gentle, feminine, classical tobaccos are all Yakas (upland). Soils in thefoothills, in semi-arid climates with heavy winter storms, are likely to be eroded and thereforthin and low in nitrogen reserves. These conditions combined with the rest of environmentgive the products. The soil consists of stony, sandy, calcareous clays of grey to reddishcolor. Soil depth is limited, over a calcareous subsoil, and organic matter is low. Converselythe Ova (plain) are richer, deeper soils, which have received some deposit from the moreupland areas. The greater fertility, higher organic matter content and better water-holdingcapacity produce the somewhat rougher more masculine tobaccos which are the Ovas,and entirely acceptable as such. If one gets right down to the very deep alluvial floodplains, growth is stronger and questions of quality acceptability become more doubtful.However the greater productivity makes these areas more attractive to farmers8).

2.1.3 Burley Tobacco

It is the typical tobacco of the deep fertile open soils.Reddish-brown silt loams of limestone origin with subsoils of well-structured silty clayscomplete a picture of fertility, excellent drainage and good aeration. Good water holdingcapacity to maintain growth through periods of erratic rainfall. Other brown or yellow­brown soils of limestone origin are very usable but often need more fertilizer. Heavier loamshave all the necessary attributes of depth, fertility and open texture'".

2.2 Plantation Areas in Greece

Greece is a country well known for his famous oriental tobaccos and is predominantlysuited for this type of tobacco. It has mountain soils of the lithosolic group occurring as"rock soils" on the slopes or near the base of mountains. The surface layers are thin or shal­low, are very stony throughout, are underlain with rock, and are of low productivity.

There is an extensive zone of forest soils, - from which the forests have been largely re­moved -, which have been derived from the disintegration of granitic and limestone parentrocks. The A horizon is feebly developed, and the B horizon is underlain with parent ma­terial of white chalk. The soil was named "Terra Rossa" according to its red color due to thecontent of ferric hydroxide.

The soils of the Macedonian and Thracian area on which the best quality of oriental to­bacco is being grown are upland forest soils, designated brown podsols, red and yellowpodsols, and Mediterranean dry forest soils. These podsolic soils generally light texturedand of low to medium productivity. The brown soils are high in calcium carbonate, granular,dark brown to reddish brown in color, shallow and of medium productivity. The red and yel­low podsols have a grayish surface layer but the clay subsoils are red or yellowish. They areacid, low to medium in organic matter and productivity, and of good soil structure. The soilof the dry Mediterranean group range from very sandy to clay, and are alkaline to slightlyacid. Some of them are developed from limestone, and all are shallow and low in humus.

14

Others have arisen from non-limestone parent material and have a reddish brown B hor­izon11). The more fertile ova and plains soils provide suitable burley soils, given avoidanceof tight pockets with poor drainage8),

The inland areas of Macedonia, Thessaly and Thrace are transitional between Mediterra­nean and the continental climates. Spring is wetter and the dry season is largely confinedto July and August though rainfalls up to 25 mm are not uncommon eyen in those months8).For some areas irrigation is necessary.

To find out, if a field fits for the tobacco plantation or shows possible nutrition deficiencies,soil samples must be taken. One hundred and eight soil samples were taken in the autumn1990 and in the spring 1991 from five experimental tobacco fields in the northern andmiddle part of Greece. They were analyzed for pH-, salinity- (expressed as Electrical Con­ductivity in mS/cm) and organic matter-value. They were analyzed for the content of ni­trogen, of phosphorous, of potassium, of magnesium, of manganese and of chlorine, too.

2.2.1 Thessaloniki-Soxos and Karditza-Mltropoll

Fields for the Virgin tobacco plantation in the areas Thessaloniki-Soxos and Karditza-Mit­ropoli are found. The soil samples from Thessaloniki-Soxos show low to acceptable pH-,low organic matter-values, a low salinity (expressed as Electrical Conductivity in mS/cm),low phosphorous-, low to high potassium-, very high magnesium-, high manganese- andlow chlorine-contents (see table Nr. 4); the soil samples from fields of Karditza-Mitropolishowed high pH-, low organic matter-values, a low salinity (expressed as Electrical Con­ductivity in mS/cm), low phosphorous-, low potasslum-, very high magnesium- and lowchlorine-contents (see table Nr. 5).

Table 4: Chemical Analysis of Soil Samples from Fields for Virgin Tobacco Plantation,Thessaloniki - Soxos 1991

Organic ElectricMatter Conduct. P K Ca Mg Mn CI

pH %C mS/cm mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg

5,3 1,04 0,12 28 50 1840 270 27 4,7

5,1 1,38 0,09 18 50 1500 250 22 4,7

4,9 1,00 0,13 24 100 1350 190 27 4,7

5,6 1,35 0,10 28 230 2500 306 29 7,1

5,6 1,49 0,20 28 203 1430 145 57 7,1

Table 5: Chemical Analysis of Soil Samples from Fields for Virgin Tobacco Plantation,Karditza - Mitropoli 1991

Organic ElectricMatter Conduct. P K Ca Mg CI

pH %C mS/cm mg/kg mg/kg mg/kg mg/kg mg/kg

7,7 1,17 0,16 16 47 3480 320 2,4

7,1 1,73 0,26 36 80 3080 398 16,6

5,1 1,15 0,12 18 50 1310 545 4,7

7,5 1,37 0,15 10 50 9000 462 2,4

15

Organic ElectricMatter Conduct. P K Ca Mg CI

pH %C mS/cm mg/kg mg/kg mg/kg mg/kg mg/kg

6,4 1,26 0,15 22 47 2310 852 2,45,5 1,28 0,10 26 63 1610 697 7,17,6 1,55 0,12 32 60 9250 264 2,4

7,6 1,53 0,15 18 47 13000 150 2,46,6 1,31 0,11 16 50 2800 895 7,1

2.2.2 Thessaloniki-Plati

Fields for the Burley tobacco plantation in the area Thessaloniki-Plati are found. The soilsamples show acceptable pH-, low to medium organic matter-values, a low salinity (ex­pressed as Electrical Conductivity in mS/cm), low phosphorous-, medium to very high po­tassium-, high magnesium- and low chlorine-contents (see table Nr. 6).

Table 6: Chemical Analysis of Soil Samples from Fields for Burley Tobacco Plantation,Thessaloniki - PIaU 1991

Organic ElectricMatter Conduct. P K Ca Mg Mn

pH %C mS/cm mg/kg mg/kg mg/kg mg/kg mg/kg

8,0 1,37 0,15 7 205 12500 360 7,18,0 1,27 0,20 10 220 13000 310 14,78,4 1,05 0,24 8 145 13125 397 11,8

7,0 1,82 0,15 11 300 13000 360 7,1

7,8 1,85 0,16 10 345 13000 410 7,17,9 1,75 0,17 9 340 12250 435 4,77,8 2,30 0,16 6 540 12250 537 2,4

2.2.3 Komotini and Kozani-Chirolimni

The soil samples from fields for the Oriental tobacco plantation of Komotini and Kozani­Chirolimni show acceptable to high pH-, low to medium organic matter-values, a lowsalinity (expressed as Electrical Conductivity in mS/cm), low phosphorous-, medium tohigh potassium-, very high magnesium-, high manganese- and low chlorine-contents (seetables Nr. 7 and 8).

Table 7: Chemical Analysis from Soil Samples from Fields for Oriental Tobacco Plantation,Komotini 1991

Organic ElectricMatter Conduct. P K Ca Mg Mn Cl

pH %C mS/cm mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg

5,2 1,91 0,15 14 237 2730 612 53 14,2

5,6 2,04 0,11 22 283 2510 565 41 14,2

16

Organic ElectricMatter Conduct. P K Ca Mg Mn CI

pH %C mS/cm mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg

7,2 1,98 0,20 26 367 3075 243 59 7,1

6,9 1,90 0,20 14 307 4080 260 44 4,7

5,4 1,85 0,09 28 330 2650 424 46 9,5

5,1 1,60 0,12 22 190 1850 247 24 4,7

Table 8: Chemical Analysis of Soil Samples from Fields for Oriental Tobacco Plantation,Konzani - Chiromlimni1991

Organic ElectricMatter Conduct. P K Ca Mg CI

pH %C mS/cm mg/kg mg/kg mg/kg mg/kg mg/kg

7,9 2,15 0,17 14 320 14375 477 11,8

7,2 2,39 0,19 9 180 6250 540 7,1

8,0 1,37 0,15 7 205 12500 360 7,1

8,0 1,27 0,20 10 220 13000 310 14,2

8,4 1,05 0,24 8 145 13125 397 11,8

7,8 1,82 0,15 11 300 13000 360 7,1

7,8 1,85 0,16 10 345 13000 410 7,1

7,9 1,75 0,17 9 340 12250 435 4,2

7,8 2,30 0,16 6 540 12250 537 2,4

2.2.4 Katerini-Vrias/N.Efessos

The soil samples from fields for the Oriental and Burley tobacco plantation of Katerini­Vrias/N.Efessosshow acceptable to high pH-, medium organic matter-values, a low salinity(expressed as Electrical Conductivity in mS/cm), low phosphorous-, low to medium po­tassium-, medium to high magnesium-, high manganese- and low chlorine-contents (seetable Nr. 9)

Table 9: Chemical Analysis of Soil Samples from Fields for Oriental and Burley TobaccoPlantation, Katerini - Vrias/N. Efessos 1990

OrganicMatter P K Ca Mg Mn CI

pH %C mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg

7,2 1,93 26 163 2290 153 65 4,7

5,5 1,97 26 283 1200 149 61 7,1

4,9 1,95 22 307 870 115 88 7,1

5,7 2,20 22 197 1410 142 63 4,2

7,3 2,16 34 247 2580 146 69 9,5

7,0 2,00 28 260 2010 143 68 7,1

17

OrganicMatter P K Ca Mg Mn CI

pH %C mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg

4,7 2,27 28 160 970 120 70 7,17,3 2,26 40 190 2740 159 60 4,76,6 2,00 22 267 1770 164 59 4,76,4 1,94 22 160 1560 132 39 9,57,4 2,00 34 170 2990 173 53 7,15,5 2,06 22 233 1280 149 64 11,87,0 1,79 22 230 1770 131 53 4,76,0 1,56 22 233 3240 131 57 7,17,5 1,68 32 180 2480 120 91 4,75,1 1,79 49 263 2390 138 83 14,26,1 1,79 24 273 755 95 93 4,77,7 1,93 22 223 1270 114 59 2,47,7 1,83 32 243 3260 152 81 7,17,0 1,83 32 247 1770 126 60 2,4

2.2.5 Agrinion-Sfina

The soil samples from fields for Oriental and Burley tobacco plantation of Agrinion-Sfina,Mg-Trial, 0,20 em, show acceptable to high pl-l-, low to medium potasslurn-, medium tohigh magnesium- and high manganese-contents (see tables Nr. 10 and 11)

Table 10: Chemical Analysis of Soil Samples from Fields for Oriental and Burley TobaccoPlantation, Agrinion - Sfina, Mg-Trial, 0,20 em, 1990

K Ca Mg K Ca MgpH mg/kg mg/kg mg/kg pH mg/kg mg/kg mg/kg

5,9 147 1040 232 7,4 170 3700 202

6,0 153 1380 80 7,5 160 2960 159

5,9 160 1540 306 6,9 173 2460 237

6,4 267 1460 289 7,8 137 4300 201

7,6 193 2760 151 7,7 163 1240 907

5,7 157 1030 116 6,0 130 2360 126

6,4 193 1610 108 7,2 167 2630 138

7,6 200 2280 148 6,8 170 1890 172

5,7 150 1590 290 5,2 153 2800 349

6,4 137 2280 126 6,6 160 3120 146

18

K Ca Mg K Ca MgpH mg/kg mg/kg mg/kg pH mg/kg mg/kg mg/kg

7,0 150 2320 159 7,5 153 3100 163

7,0 153 1970 290 7,6 160 13700 208

Table 11: Chemical Analysis of Soil Samples from Fields for Oriental and Burley TobaccoPlantation, Agrinion - Sfina, 0,20 em, 1990

Ca Mn Ca MnpH mg/kg mg/kg pH mg/kg mg/kg

5,6 1390 21 7,8 5850 59

5,1 940 42 7,2 2900 51

6,1 1820 32 6,4 1950 33

7,2 2150 36 7,7 5675 56

6,6 1490 45 7,2 2460 52

7,6 2850 45 7,3 2500 38

7,0 2300 42 5,5 1210 26

6,9 2070 25 6,0 1520 22

7,0 1990 28 4,7 800 14

7,7 3780 44 7,0 1570 19

5,6 1100 25 6,5 3880 40

6,6 1750 29 6,8 1930 30

7,8 3250 39 7,6 2940 34

7,6 2780 42 7,5 3740 37

3 Tobacco QualityAll tobacco cultlvars, especially the oriental types in Greece, are well fitted to their growingsite and its environment. They are divided in aromatic, taste and neutral tobaccos. Theirquality is influenced by the climate and the soil. Also the cultural practice such as seedlingproduction, fertilization, water requirement and last harvesting, curing and the handling ofthe produce has a great influence to the quality.

3.1 Outer and Inner Quality

Characterizing the tobacco cultivars according to outer and inner quality, the outer qualityis described by the kind, form and appearance of the leaves; sensorial and physicalmethods are used; the inner quality is correlated on one hand with positive tobacco-ownsubstances of value and on the other hand with negative intrinsic and extrinsic com­pounds, hazardous to smokers and co-smokers. The inner quality itself is subdivided in apositive quality and a negative quality: the positive quality is referring to the simple lnor-

19

ganic and organic elements necessary for the tobacco nutrition, - the so-called micro- andmacroelements -, but also to the more complex organic plant-own compounds essentialfor the smell and taste of tobacco smoke, such as the amount of proteins and sugars or thekind of aromatic compounds; the negative quality implies the hazardous smoke sub­stances and the residues from soil, water and air pollution, from fertilizers and plant pro­tection agents and their metabolites. All these compounds can be found in the leaf or ontheleafsurtace12-1~.

3.2 Regulations of the European Union

The handling and use of the plant protection agents and their metabolites is regulated bynational and international guidelines and laws17), which establish amounts of the toleratedresidues of the plant protection agents and their metabolites. According to the EU directive91/414/EWG the harmonization of national regulations in plant protection matters has totake place18).

For hazardous smoke substances the EU Commission had demanded the limitation ofcondensate to 12 mg per cigarette in 1988. Most of the produced tobaccos didn't fulfill thisdemand. The lower stalk positions only of the cultivars showed a tar content below 15 mgper cigarette. This is the case for Greek and for German cultivars, too. Till 1993 the limi­tation therefore was raised to 15 mg condensate per cigarette.

By this regulation, certain tobacco cultivars are more seriously affected than others, par­ticularly the local varieties grown and smoked in Greece. The oriental varieties "Tsebelia"and "Mavra" are endangered as will be the dark air-cured Havanna, Badischer Geuder­theimer and Paraguay. Many areas in Greece, where dark air-cured tobacco and orientalvarieties are grown, are not at all suited to other cultivars and crops in terms of soil andclimate conditions.

In these regions the cultivation of adopted tobacco cultivars with an acceptable tar/nic­otine ratio and diminished carbon monoxide- and nitric oxide-content may only be suc­cessful. Especially the aromatic oriental tobacco as it is planted in Greece with its low nic­otine and medium sugar content, is valuable, to balance blend cigarettes in taste andaroma. Neutral types improve blends by reducing nicotine content and making cigarettesmilder in taste and of better combustibility.

The cultivars Virgin and Burley are of interest for the Greek and German plantation, Geu­dertheimer only for the German plantation.

3.3 Methods

Since the early twentieth the chemical examination of tobacco in leaf and smoke is in prog­ress. Many data about nitrogen, nitrate, sugars, alkaloids, condensate, nitric oxide and car­bon monoxide for different tobacco varieties have been published. They are reviewed forinstance from A. Wenusch, 1939 and 194013) and T. C. Tso, 1972 and 199016).

Under the interaction aspect is the relationship among nitrate in the tobacco leaves and ni­tric oxide, and carbon monoxide in the tobacco smoke, and nitrosamines, too, in the leavesand in the smoke is of interest.

Total nitrogen was determined according to the Kjeldahl method19), total sugar accordingto the Schorl method19) and alkaloids according to DIN 10241 or by the gaschroma­tography method according to DIN ISO 1031520). Nitrosamines were determined in the In­stitute for Toxicology of the German Cancer Research Center, Heidelberg, by gaschroma­tography-thermal energy analyzer method (GC-TEAf1-24).

20

Nicotine and condensate (free from water) were determined according to DIN ISO 3402,DIN ISO 3308, DIN ISO 4387, DIN ISO 10315, DIN ISO 10362-1 or ISOTC 126 N 47120), al­kaloids in tobacco smoke as nicotine were determined according to DIN ISO 1031520) andcarbon monoxide according to DIN 1024820).

3.3.1 Nitrate

Nitrate was determined by gaschromatography according to the "xylene" method25): afterextraction with water and filtration the nitrate reacts in presence of sulfuric acid with p-xy­lene to 2-nitro-p-xylene according to the reaction mechanisms shown below:

_ H2S04CH3 - C6H4 - CH3 + N03 > CH3 (N02) - C6H 4 - CH3-H+

3.3.2 Carbon Monoxide

The gas carbon monoxide in the tobacco smoke is measured in a flow cell with an air - tightnondispersive infrared detector (NDIR) after stimulation at the absorption wavelength in theinfrared region in comparison with a reference carbon monoxide sealed inside a separate'cell. The depletion of energy at the absorption wavelength gives rise to a change in thepressure of the detector, causing a small movement of a thin metallic diaphragm, - the oneelectrode of a variable electrical capacitor -, and a capacitance change of the capacitorsolely related to the amount of the energy absorbing sample undergoing measurement andin turn related to the amount of carbon monoxide of the sample.

3.3.3 Nitrogen Oxides

To compare different results in the determinations of the gaseous nitric oxide in tobaccosmoke, it is necessary to check the device of the used nitric oxide analyzers; also it is of in­terest to understand, how the used smoking machines in combination with the used ana­lyzers are working. Last it is necessary to consider the smoking parameters for the smokingprocedure.

The measurement of the hi~h~ reactive nitric oxide in the gasphase of tobacco smoke hasbeen proved to be difficult 6, ,28).

The cigarette manufacturers in Germany and Greece mainly use the BAT. nitric oxide ana­lyzer from the company BAT.29), R. & D. D. Group R. & D. Center, Southampton, UK, com­bined with the smoking machine SM 302 from the company Filtrona Instruments & Auto­mation Ltd.30), Harpenden, UK, working on the basis of a chemiluminescence detector,where the photomultiplier electrical output signal is buffered by an amplifier attenuated anddisplayed in combination with a chart recorder.

Nitrogen oxides analyzers based on the chemiluminescence detector use the high reactiv­ity of nitrogen oxide in that way that a definite small fraction of the nitric oxide moleculesreacts with ozone to form activated nitrogen dioxide species N02*; as the activated oremitting species (N02*) reverts to a lower energy state, it emits a broadband radiation from500 to 3000 nm with a maximum intensity at approximately 1100 nm.

NO + 03 ~ N02* + 02N02* ~ N02 + hv

Since one nitrogen oxide molecule is required to form one N02* molecule, the chemilumi­nescence emission intensity is proportional linearly to the nitrogen oxide concentration inthe sample. The photornultipller tube current is proportional directly to the chemilumines­cence emission intensity31, 32). Under reducing conditions, as they are found in tobacco

21

smoke, most of these analyzers measure less nitric oxide. Carbon monoxide, hydrogen,ammonia, sulfur dioxide, methane and other hlqher aliphatic hydrocarbons reduce nitricoxide by the reaction mechanisms shown below"33):

2CO + 2NO <II ~ N2 + 2C02CO + 2NO iii ~ N20 + CO25H2 + 2NO <II ~ 2NH3 + 2H20

2NH3 + 4NO iii .. 2N2 + N20 + 3H20

Negative nitric dioxide values are found, too, in nitrogen oxides analyzers as the MonitorLabs. Nitrogen Oxides Analyzer 8440E.,4), Atlanta, USA, working on that way that thesample stream entering the analyzer is split into a nitrogen oxides and a nitric oxide stream.The one difference between the nitrogen oxides and the nitric oxide stream in this device, ­in comparison with B. A.1. nitric oxide analyzer29) -, is that in the nitrogen oxides stream isplaced a converter with molybdenum as catalyst, converting the nitric dioxide to nitricoxide. Nitric dioxide concentration level is derived by electronically subtracting the nitricoxide signal from the nitrogen oxides signal.

Positive nitric dioxide signals in tobacco smoke were found with the Monitor Labs. Ni­trogen Oxides Analyzer 8440E34), Atlanta, USA, after the molybdenum converter was re­placed by a converter containing carbon. The positive nitric dioxide signals can be used asfinal check before starting the nitric oxide measurement and during the nitric oxidemeasurement in cigarette smoke, too35).

Calculation

The calculation of the nitric oxide yield in tobacco smoke for puff to puff smoking of ciga­rettes was derived according to the calculation of the carbon monoxide yield of DIN ISO3402 20) or CORESTA recommended method Nr. 536), assuming nitric oxide to be an idealgas37), with a defined puff volume of 35 ml per two seconds and a draw intermission of 10seconds.

Let: Cobs = observed nitric oxide concentration, vpmN = total number ofpuffs inthe measured sample

(including three clearing puffs)V = puff volume, mlq = number ofcigarettes to produce the measured samplet = ambient temperature, 'Cop = ambient pressure, kPa

Calculation of the nitric oxide yield - volume per cigarette basis - at 101,3 kPa and O°C:

Cobs x V x N x P x 273III nitric oxide per Cigarette = ---3-------­

q x 10 x 101,3 x (t + 273)

Short formula:

with:

III nitric oxide per CigaretteCobs x N x p

-::------ X F103x (t+273)

22

F = 35 x 273

103x101,3

0,012572 (one cigarette)= 0,050289 (four cigarettes)

0,100576 (eight cigarettes)

Calculation of the nitric oxide yield - mass per cigarette basis - at 101,3 kPa and O°C:

Cabs x V x N x P X 273 x 30/1g nitric oxide per Cigarette

qx103x101,3x (t+273) x 22,4

Short formula:

with:

/1g nitric oxide per CigaretteCobsxNxp-----xFqx(t+273)

F == 35 x 273 x 30

103 x 101,3 x 22,4

== 0,01684 (one cigarette)0,06736 (four cigarettes)

== 0,13472 (eight cigarettes)

Balance Method

Tocheck the method of BAT. nitric oxide analyzer29) in combination with the smoking ma­chine M 302 of the com~any Filtrona30) there was the need of a balance method. The ni­trogen oxides rnethod'' ) developed with the Monitor Labs. Nitrogen Oxides Analyzer8440E34), Atlanta, USA, in combination with the diluting stack sampler for monitoring gas­eous emissions Model 797 of the com~any Kipp Analytica39, 40), and the smoking machineRM 20 of the company H. Borqwaldt" ), was modified for the use as balance method. Thediluting stack sampler of the company Kipp Analytica, rarefying the nitrogen oxides withdried and purified air to avoid the chemical reaction with other smoke components37), wasreplaced for a nitrogen flushing unit, using two three-way valves, the one electrically oper­ated from the pump stroke unit of the smoking machine combined with a back pressurevalve, the other mechanically operated (see graph 1).

Graph 1Valve Circuit Oiagramm for Puff by PuffAnalysis of Nitric Oxide in TobaccoSmoke with Nitrogen Flashing of theSmoking Machine and the Nitric OxideAnalyzer

SMOKINGMACHINE

TO NO-ANALYZER

PISTON PUMP

23

During the draw intermission the connecting tubes and the nitrogen oxides analyzer isflushed with nitrogen gas of highest purity between every puff of the smoking machine: infresh cigarette smoke, - on a puff by puff basis -, nitrogen oxides are directly measurablewithout dilution35).

Smoking Machine Borgwaldt RM 20 CS

The Borgwaldt RM 20 CS smoking machine is a rotating machine with 20 channels. For thedetermination of nitrogen oxides with the central Cambridge filter holder every channel canbe used as single port, so that the nitrogen oxides content is measurable in the puff volumepuff by puff per single cigarette. During the draw intermission the smoking machine and ni­trogen oxides measuring apparatus is automatically flushed with nitrogen (see graph Nr. 1).Depending on the stagnant volume of the smoking machine and the nitrogen oxidesmeasuring apparatus up to five cigarettes can be smoked per smoking procedure withoutinterfering peaks on the chart recorder.

Smoking Machine Filtrona M 302

The smoking machine M 302 of the company Filtrona30) is a linear smoking machine witheight channels, ports and separate Cambridge filter holders working in a way that the nitricoxide content is determined in the puff volume puff by puff per eight cigarettes together.The really drawn puff volume per puff is therefore 35 ml times eight (the used number of thechannels for smoking eight cigarettes toqether), After the calculation formula from thehandbook for B. A. 1. nitric oxide analyzer<T2)

Nitric oxide (1l9/Cigarette) A x N ~ P x 0T0168473 +

was corrected with the factor of eight in

Nitric oxide (llg/Cigarette)

where A =average concentration (vpm)N = number of lit puffs per cigaretteP = barometric pressure (mm Hg)T =room temperature ('C).

the nitric oxide measurement took place.

AxNxPxO.13470273+ T

4 Greek Tobacco Breeding LinesThe Greek tobacco breeding lines were cultivated in 1987. Twenty-eight samples of fiveGreek breeding lines have been analyzed for compounds in leaf and smoke. Up to sevenstalk positions of the breeding lines 1/B, 2/K, 3/S, 4N and 5/R have been recorded. The to­bacco leaf components nitrate, nicotine, sugar and total nitrogen are given in% (see graphsNr. 2, 3, 4, 5 and 6).

In the tobacco smoke condensate, nicotine, carbon monoxide and nitric oxide have beenevaluated. The smoke components are specified per cigarette (see graphs Nr. 7, 8, 9, 10,and 11).

4.1 Breeding Line 5/R

The condensate amounts of the breeding line 5/R (see graph Nr. 11) fulfill the directions ofthe EU in the harvesting grades from "lugs" to "cutter". This breeding line is somewhatbetter than the German Burley cultivars, grown in Forchheim (see graphs Nr. 26a and 27a).

24

Graph 2: Compounds in Tobacco from Greek Breeding Une 11B 1987Harvesting Grades 1 - 7, N-Fertilization 30 kg Nlha

14..---------------------------,

12

10

8

6

4

o2 3 4 5 6 7

Nitrate GC % III 0,74 0,41 0,21 0,17 0,1 0,06 aTotal N % III 2,23 2,37 2,67 2,61 2,78 2,22 3,2

Nicotine GC % IS] 2,46 2,97 3,32 4,17 3,72 2,74 3,32

Total Sugar % 0 7,63 6,96 5,93 6,3 6,1 12,1 6,46..Reducing Sugar % I&l 5,43 5,1 4,26 4,93 4,43 9,36 5,1

Sucrose % I'ZJ 2,09 1,76 1,58 1,3 1,58 2,6 1,29

Graph 3: Compounds in Tobacco from Greek Breeding Une 21K 1987Harvesting Grades 1 - 4, N-Fertilization 40 kg Nlha

25.----------------------------,

20

15

10

5

2 3 4

Nitrate GC % II 0 0 0 0

Total N % tzl 1,6 1,68 1,6 2,43Nicotine GC % II 0,51 0,46 0,46 1,32Total Sugar % ~ 19,1 17,66 18,73 10,2Reducing Sugar % D 16,63 14,7 15,93 8,16..Sucrose % f2] 2,34 2,81 2,66 1,93

The nicotine rates correspond with a good climate in Greece and equal to good German to­bacco cultivars. 4 mg nicotine per cigarette have been found in the "tips"; the carbon mon­oxide content is a little bit higher than in the German cultivars.

The nitric oxide amounts have been found in all harvesting grades to be higher than 200 Ilgper cigarette: in the "lugs" the nitric oxide amount has been found to be 385 Ilg per ciga­rette, significantly higher than the amounts of German Burley cultlvars (see graphs Nr. 26aand 27a).

25

Graph 4: Compounds in Tobacco from Greek Breeding Line 3/5 1987Harvesting Grades 1 - 6, N-Fertilization 30 kg N/ha

25,-------------------------------,

Nitrate GC % III 0,2 0,1 0,1 0,1 0,1 0,1

Total N % I7J 2,24 1,91 2,12 2,24 1,95 1,7

Nicotine GC % III 1,37 2,32 2,06 1,32 1,43 1,14

Total Sugar % ~ 12,96 22,26 15,4 12,8 16,63 16,8

Reducing Sugar % 0 18,56.. 10,56 13,5 10,73 14,9 15,4

Sucrose % [2l 2,28 3,51 1,8 1,96 1,64 1,33

Graph 5: Compounds in Tobacco from Greek Breeding Line 4/V 1987Harvesting Grades 1 - 6, N-Fertilization 30 kg N/ha

30 ,--------------------------------,

25

20

15

10

5

2 3 4 5 6

Nitrate GC % II 0,1 0,1 0,1 0,1 0,1 °Total N % [Z] 1,19 1,12 1,23 1,22 1,49 1,13Nicotine GC % II 1,77 1,43 1,89 2 2,86 3,26Total Sugar % ~ 19,96 23,86 27,46 25,66 25,13 23,46Reducing Sugar % 0 17,86 20,13 24,6 22,63 23,33 22,46

Sucrose % Ell 1,99 3,54 2,71 2,87 1,71 1,33

Altogether the content of smoke compounds of the Greek tobacco breeding line is signifi­cantly higher than of the German cultivars, except for the condensate content.

Regarding to the nitrate amounts of the tobacco leaf, it has been found out, that the nitraterates of 3% (see graph Nr. 6) in tobacco leaf are high and responsible for the high nitricoxide content.

The nicotine amounts with up to 5.15% nicotine in tobacco leaf (see graph 6) are very hightoo. Such high nicotine amounts could only be found in German Burley cultivars during hotand dry tobacco years (see graphs Nr. 26a and 27a).

26

Graph 6: Compounds in Tobacco from Greek Breeding Line 51R 1987Harvesting Grades 1 - 5, N-Fertilization 200 kg Nlha

6.-----------------------~--___,

5

4

3

2

o2 3 4 5

Nitrate GC % II 3 2,4 1,2 0,6 1,3Total N % ~ 3,16 3,2 3,85 4,16 3,44Nicotine GC % 0 1,89 1,89 3,95 5,03 5,15Total Sugar % ~ 0 0 0 0 0

Graph 7: Compounds in Smoke from Greek Breeding Line 11B 1987Harvesting Grades 1 - 7, N-Fertilization 30 kg Nlha

40,----------------------,

30

20

10

2 3 4 5 6 7

Condensate mg/cig II 15,67 21,75 28,51 33,85 36,16 32,94 33,48

Nicotine mg/cig ~ 1,63 2,25 3,06 3,67 3,61 3,06 3,34

Carbon monoxide mg/cig 0 11,83 14,58 17,34 19,07 19,52 19,8 15,79:;:

Nltr. oxide mg/cigxO,1 ~ 1,6 1,45 1,31 1,24 1,2 1,1 1,16

4.2 Breeding Line 4N

The breeding line 4N has been investigated in all harvesting grades from 1 to 6. The con­densate amounts of 25 mg per cigarette in the lower harvesting grades are exceeding thedirections of the EU (see graph Nr. 10).

The nicotine amounts from 1.4 to 3.1 mg nicotine per cigarette have been found in themiddle to high range (see graph Nr. 10).

27

Graph 8: Compounds in Smoke from Greek Breeding Line 2/K 1987Harvesting Grades 1 - 4, N-Fertilization 40 kg N/ha

40,---------------------------,

25

20

15

10

5

° 2 3 4

Condensate mg/cig III 23,12 22,96 24,65 34,16Nicotine mg/cig [83 0,49 0,49 0,37 1,39Carbon monoxide mg/cig 0 13,55 13,73 14,71 16,15Nitr. oxide mg/cigxO,1 ss 0,72 0,79 0,92 0,12

65432

= r-r-

""r-r-

Iiii;y:; Ii "" xx. """ 00 e-e-e = ~ )0( bz:x

°5

25

20

30

15

10

Graph 9: Compounds in Smoke from Greek Breeding Line 3/S 1987Harvesting Grades 1 - 6, N-Fertilization 30 kg N/ha

35

Condensate mg/cig III 20,05 21,29 25,41 25,15 31,55 30,52Nicotine mg/cig I2lI 1,35 1,11 1,54 1,17 1,6 1,2Carbon monoxide mg/cig 0 11,11 11,95 12,88 13,84 14,25 14,29Nitr. oxide mg/cigxO,1 1S1 0,92 0,84 0,77 0,72 0,69 0,68

The carbon monoxide amounts from 15.8 to 19.9 mg per cigarette are much higher thanthe amounts of the German cultivars (see graph Nr. 10, 12a and 13a).

The nitric oxide contents with amounts from 76 to 115 /1g per cigarette are in the low tomiddle range (see graph Nr. 10).They are corresponding to the amounts in the German cul­tivar Virgin D.

The low nitric oxide contents in the smoke are referring to the low nitrate amounts in the to­bacco leaf. Here are the nitrate amounts lower than 0.1% or not detectable.

28

65432

."...,

"'" "'"=

~iiZR) ~ ~ ~ 50< """ 122 h ""

15

10

5

°

25

20

Graph 10: Compounds in Smoke from Greek Breeding Line 4/V 1987Harvesting Grades 1 - 6, N-Fertilization 30 kg N/ha

40

35

30

Condensate mg/cig • 25,9 29 28,47 30,13 34,14 32,09Nicotine mg/cig 123 1,5 1,42 1,73 1,67 2,66 3,11Carbon monoxide mg/cig 0 19,96 18,87 15,85 15,36 18,01 16,81Nitr. oxide mg/cigxO,1 ~ 0,76 0,77 0,8 0,85 0,95 1,15

Graph 11: Compounds in Smoke from Greek Breeding Line 5IR 1987Harvesting Grades 1 - 5, N-Fertilization 200 kg N/ha

25,-------------------------,

2 3 4 5

Condensate mg/cig • 10,17 12 14,6 18,5 20,52Nicotine mg/cig ~ 0,83 1,01 2,28 3,43 3,98Carbon monoxide mg/cig 0 9,02 9,6 11,18 13,41 13,58Nitr. oxide mg/cigxO,1 ~ 3,86 3,58 2,72 2,15 2,55

The nicotine rates have been found to be in a middle to high range from 1.4. to 3.2%.

The sugar amounts have been found in an upper range from 19.9 and 27.4%.

The total nitrogen contents are low and showing a little variation only from 1.1. to 1.4% (seegraph Nr. 5).

29

Graph 12: Nitrosoalcaloids and Nitrate Contents in Cigarettes of Greek Breeding Lines1987; Harvesting grades 1 - 6

2.000

1.500

1.000

500

J1lh .nnJ1JLrl1.n.m~ri! • .1\ ~ 1-

NNN MS ng/olg 121 40 37 35 32 18 8 4 3 2 1 0 0 0 0 0 24 232 264 328 315 458 0 0 0 0 0 0

NNN pret ng/ol9 IZJ 107 96 69 75 33 16 63 0 0 0 0 0 0 0 0 81 424 632 1.12 1.13 1.63< 0 0 0 0 0 aNNK MS ng/olg 028 15 18 15 14 5 9 7 6 8 4 0 a a 0 7 30 50 46 46 52 a a a 0 a aNNK pre! ng/olg E'I 59 41 16 a a a 15 0 a a a a a a 0 a 50 46 71 73 114 a 0 a 0 a aNAT/B MS ng/olg o 47 22 50 46 51 12 6 3 6 4 a a a 0 a 23 359 300 365 384 392 a 0 0 a a aNAT/S prel ng/olg [] 10 54 75 63 68 0 25 0 0 0 0 0 0 0 0 20 336 322 650 931 576 a 0 0 a a aN03 mn/oi II 6,1 4 2,2 1 0,5 0,1 1,6 1,1 0,6 0,4 0,2 0,1 0 0,1 0,3 1,5 22,5 19,2 10,1 5,4 9 0 0,05 0,1 0,15 0,2 0.3

65432

~ It. ~ ~ ~ ~rl :::~ ...

r; jVrm ~nW y;, :..~ ~mlo

20

80

40

60

100

1. Harvesling Grade I2. HaNesling Grade II3, Harvesling Grade III4, Harvesling Grade IV5, Harvesling Grade V6 Harvesling Grade VI

Graph 13: Nitrosoalcaloids and Nitrate Contents in Cigarettes of Greek Breeding Line 1/B1987; Harvesting grades 1 - 6, Fertilization 30 kg N/ha

120

NNN MS ng/cig E3 40 37 35 32 18 8

NNN prst, ng/cig l?L1 107 96 89 75 33 18

NNK MS ng/clg [J 26 15 18 15 14 5

NNK pret, ng/cig ~ 69 41 18 0 0 0

NAT/S MS ng/cig [] 47 22 50 48 51 12

NAT/S pret. ng/clg CD 10 54 75 53 68 0

Nitrate mg/cig II1II 6,1 4 2,2 1 0,5 0,1

4.3 Breeding Line 1/B

The breeding line 1/8 is a type of tobacco with high condensate amounts in the upper har­vesting grades. More than 33.4 mg condensate per cigarette are observed (see graphNr.7).

30

Graph 14: Nitrosoalcaloids and Nitrate Contents in Cigarettes of Greek Breeding Line 21K1987; Harvesting grades 1 - 4, N- Fertilization 30 kg Nlha

100

80

1. HaNestJng Grade I602, HaNesting Glade II

3, H8IVesting Grade III4, Harvesting Grade IV 40

20

02 3 4

NNN MS ng/clg [] 0 0 0 24"

NNN pret. ng/clg [Z] 0 0 0 81

NNK MS ng/clg IQI 0 0 0 7

NNK pret. ng/cig ~ 0 0 0 0

NAT/S MS ng/clg [] 0 0 0 23

NAT/S pret. ng/clg CD 0 0 0 20

Nitrate mg/cig II 0 0,1 0,3 1,5

Graph 15: Nitrosoalcaloids and Nitrate Contents in Cigarettes of Greek Breeding Line 31S1987; Harvesting grades 1 - 6, N- Fertilization 30 kg Nlha

60 ~---------------------------,

501, HaNesting Grade I2, HaNesling Grade II 403, Harvesting Grade III4, HaNesting Grade W 305, HaNesting Grade V6, HaNesting Grade VI '

10

o2 3 4 5 6

NNN MS ng/clg I2l 4 3 2 1 0 0

NNN pret. ng/clg IZJ 53 0 0 0 0 0

NNK MS ng/cig 0 9 7 8 6 4 0

NNK pret. ng/clg [SJ 15 0 0 0 0 0

NAT/S MS ng/clg II 6 3 6 4 0 0

NAT/S pret. ng/clg CD 25 0 0 0 0 0Nitrate mg/cig II 1,6 1,1 0,6 0,4 0,2 0,1

The nicotine content has been found in the middle to high range from 1.6 and 3.6 mg percigarette.

The carbon monoxide amount is also high. It has been found in the range from 11.8 to 19.8mg per cigarette.

The nitric oxide contents are in the middle range from 110 to 160 /1g per cigarette (seegraph Nr. 7).

31

Graph 16: Nitrosoalcaloids and Nitrate Contents in Cigarettes of Greek Breeding Line 4/V1987; Harvesting grades 1 - 6, N- Fertilization 30 kg Nlha

2,5 r------------------------------,

2

1. Harvesting GradeI2. HarveslingGrade II3. HarveslingGrade III 1,54. Harvesling Grade IV6. Harvesting Grade V6. Harvesting GradeVI

0,5

2 3II1II

411/

5I

6I

NNN MS ng/olg III 0 0 0 0 0 0

NNN pret. ng/olg I'ZI 0 0 0 0 0 0

NNK MS ng/olg 0 0 0 0 0 0 0

NNK pref. ng/o1g ~ 0 0 0 0 0 0

NAT/S MS ng/olg D 0 0 0 0 0 0

NAT/S pref. ng/olg ~ 2 0 0 0 0 0

Nitrate mg/olg III 0 0,05 0,1 0,15 0,2 0,3

Graph 17: Nitrosoalcaloids and Nitrate Contents in Cigarettes of Greek Breeding Line SIR1987; Harvesting grades 1 - 5, N- Fertilization 30 kg Nlha

NNN MS ng/olg I'ZJ 232 284 328 315 456

NNN pret. ng/olg D 424 632 1.123 1.132 1.633>:

NNK MS n9/0lg I2J 30 60 48 46 52

NNK pret. ng/ol9 0 60 48 71 73 114'"

NAT/S MS ng/olg ~ 359 300 365 384 392

NAT/S pref. ng/olg [] 336 322 660 931 676

Nitrate mg/olg III 22,5 19,2 10,1 5,4 9

The high nitrate contents in the tobacco leaf (see graph Nr.2) can only be used partly as anexplanation of the extreme nitric oxide contents. The nitrate contents are decreasing fromthe lower to the higher harvesting grades. According to the indirect correlation between ni­trate content and stalk positions in the first harvesting grade 0.7% nitrate has been foundand in the 6. harvesting grade only 0.1% nitrate.

The nicotine contents are ranging from high levels to very high ones, from 2.4 to 3.7%.

The sugar content has been found in a low to middle range from 5.9 to 12.1%.

32

The total nitrogen amount has been found in a middle range from 2.2. and 3.2% (see graphNr.2).

4.4 Breeding line 2/K

This breeding line 2/K is an oriental tobacco type. The condensate content has been foundin an extremely high range from 22.9 to 34.1 mg per cigarette.

The nicotine content is observed to be in a low to middle range from 0.3 to 1.3 mg per ciga­rette.

The carbon monoxide amounts are high and ranging from 13.5 to 16.1 mg per cigarette.

The nitric oxide amounts are ranging from low to middle rates: in the lower harvestinggrades from 72 to 92 I-lg per cigarette, in the upper harvesting grades 120 I-lg per cigarette(see graph Nr. 8).

In the tobacco of the breeding line 2/K no nitrate is detectable. The nicotine contents havebeen found in the low to middle range from 0.4 to 1.3%.

The sugar amounts are referring to a middle range from 10.2 to 19.1%, in which the upperharvesting grade is showing the lowest sugar amount.

The total nitrogen has been found to be 1.6% with exception of the upper harvesting grade,in which the content is referring to 2.4%. The total nitrogen has only a slight variation (seegraph Nr. 3).

4.5 Breeding line 3/5

The breeding line 3/8 is an oriental tobacco type with a condensate content ranging from20 to 31.5 mg per cigarette. The nicotine content is ranging from 1.1 to 1.6 mg per cigaretteand the amount is medium.

The carbon monoxide contents have been found in a middle range from 11.1 to 14.2 mgper cigarette.

The nitric oxide contents are found in a low to a middle range from 68 to 92 I-lg per cigarette(see graph Nr. 9).

The nitrate contents of the breeding line 2/K have been found in a low range of 0.1%.

The nicotine amounts are in the middle range from 1.1 to 2.3%.

The sugar contents have been also found in a middle range from 12.8 to 16.8% except thesecond harvesting grade, which has been found to be 22.2%.

The total nitrogen content is medium and is ranging from 1.7 to 2.2% (see graph Nr.4).

4.6 Discussing the Results

High rates of nitrate fertilizer given to tobacco are responsible for high contents of nitrogen,total alkaloids, nitrate and volatile bases in tobacco, for the high percentage of volatile nl­trosamines, too, as it has been proved. N-nitroso-nornicotine (NNN) is the mostly found ni­trosamine in the mainstream of the smoke. A high percentage of N-nitroso-nornicotine isobserved in cured and especially in fermented tobacco, which contain a lot of nitrate22, 23).

A survey of the rates of nitrosamines N'- nitrosonornicotine (NNN), 4-(methylnitrosamino)­1-(3-pyridil)-1-butanone (NNK) and N'-nitrosoanabasine / N'-nitrosoanatabine (NAB/NAT)with nitrate is shown from all primes of the five Greek breeding lines in graph Nr. 12.

33

Additionally from every prime of five different Greek breeding lines the contents of conden­sate, nicotine, carbon monoxide and nitric oxide on the one hand (seegraphs Nt:7 -11), ni­trate, nitrosamines in the cigarette tobacco and in the mainstream smoke on the other hand(see graphs Nr. 13 - 17) are shown as graph and diagram.

Discussing the results and the correlations, the rates of carbon monoxide increase in everyprime in all tobacco breeding lines except 4N breeding line in which an irregularity in therates is observed. Breeding lines with high nitrate percentage (5/R) have high percentage innitric oxide and nitrosamines. Corresponding decrease of nitrate and nitric oxide is foundout in every prime in the breeding lines 1/B, 3/8 and 5/R and corresponding increase inevery prime in 2/K and 4N. In 5/R breeding line the highest percentage of nitrosaminesboth in tobacco leaves and in the mainstream smoke is observed. In 4N, 2/K and 3/8 no ni­trosamines are observed, while 1/B is in a middle position.

In 5/R breeding line the rates of carbon monoxide are much lower (9 - 13 mg/cig) than thecorresponding rates of 4N breeding line (15 - 20 mglcig). 1/B, 2/K and 3/8 breeding linesare in a middle position (11 - 18 mg/cig). Generally the rates of carbon monoxide increasein every prime of all tobacco breeding lines except 4N breeding line, in which an irregularityin the rates of carbon monoxide is observed.

It has been found out that in breeding lines with high percentage of nitrate such as 5/Rthere is most of both nitric oxide and nitrosamines. Breeding lines showing low nitrate con­tents, such as 4N, 2/K and 3/8, have low contents of nitric oxide and nitrosamines, too. The1/B breeding line is in the middle position both in nitrate, nitric oxide and nitrosamines.Generally a corresponding decrease of nitrate and nitric oxide is found out in every prime inthe breeding lines 1/B, 3/8, 5/R and a corresponding increase of the same parameter inevery prime in 2/K and 4N breeding lines.

Regarding the nitrosamines in 5/R breeding line the highest percentage is observed in themain stream smoke and in the tobacco leaves, too. This result is in contrast to the breedinglines 4N, 2/K and 3/8, in which no nitrosamines are observed, while the breeding line 1/B isin a middle position.

It is characteristic that the rates of nitrosamines in 5/R breeding line are ten-fold higher thanin 1/B breeding line. These rates of nitrosamines in smoke and in leaf are in a completecorrespondence to the rates of nitrate in tobacco. Comparing the percentages of N'-nitro­sonornicotine (NNN), L-(methylnitrosamino)-1-(3-pyridil)-1-butanone (NNK) and N'-nitro­soanabasine IN' -nitrosoanatabine (NAB/NAT) it is observed, that the half to one third of theN' -nltroso-nornlcotlne (NNN) and L-(methylnitrosamino)-1-(3-pyridil)-1-butanone (NNK)rates of the mainstream smoke are found in the tobacco leaf, while the rates of N'-nitro­soanabasine I N'-nitrosoanatabine (NAB/NAT) in the mainstream smoke and in the leavesare equal.

The nitric oxide rates of in 5/R breeding line are the highest (385 - 215 flg/cig). In the 1/Bbreeding line a decrease to half the rates is observed. In 5/R lines nitric oxide rates arebetween 160 - 110 flg/cig, while in the rest of the breeding lines the rates are in lowerpercentage (68 - 120 flg/cig).

Considering the results in detail, it is found out, that the 1/B breeding line shows a decreaseof the rates of nitrate in every prime (6.1 - 0.1 mg/cig). Corresponding decrease takes placein rates of nitric oxide in the smoke (160 -110 g/cig) and N-nitroso-nornicotine in the main­stream. In contrast an increase of the rates of carbon monoxide in every prime (11.8 -19.8mg/cig) is observed.

In 318 breeding line a decrease of the rates of nitrate, - corresponding to the rates of nitricoxide and N'-nitrosonornicotine -, combined with an increase of the carbon monoxiderates is observed.

34

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5, Perega6, Perega 234

Graph 18a: Compounds in Smoke from Geudertheimer Cultivars 1987,LUGS, N-Fertilization 180 kg N/ha

20

Condensate mg/cig III 13,36 14,09 14,15 15,71 14,54 17,41Nicotine mg/cig IF§j 0,49 0,46 0,37 0,34 0,56 0,19Carbon monoxide ml/cig [J 9,1 8,5 8,8 8,4 8,4 8,6:::

Nitr. oxide mg/cigxO,1 bSl 0,84 0,76 0,67 0,62 0,73 0,78

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1, Geudertheimer III2, Bad, Geudertheimer 25

3, Bad, GeudertheimerW 20

4, Bad, Geudertheimer K5, Perega 15

6, Perega 234 10

Graph 18b: Compounds in Smoke from Geudertheimer Cultivars 1987,LUGS, N-Fertilization 180 kg N/ha

35

Condensate mglg tdm III 26,28 24,88 27,36 30,36 28,71 30,09Nicotine mg/g tdm fi2J 0,96 0,81 0,72 0,66 1,11 0,33CO ml/g tdm 0 17,8 15 17 16,2 16,6 17,5"

NO mg/g tdm x 0,1 ~ 1,66 1,34 1,29 1,2 1,45 1,35

Compared with 1/8 and 3/8 breeding lines the 2/K and 4N breeding lines - with an in­crease in the rates of nitrate and nitric oxide in every prime - show an inverse correlation tothe nitrate and nitric oxide.

In 5/R breeding line a decrease of the rates of nitrate and nitric oxide from the first to thefourth prime and then an increase is observed. In carbon monoxide an increase is observedin every prime. The decrease of nitric oxide and nitrate is much bigger in this breeding line,than in 1/8, 2/K and 3/8 breeding lines.

35

5. German Tobacco OultivarsSeventeen German cultivars of the harvest 1987 and twenty-eight new breeding lines ofdark air cured tobacco of the harvest 1990 have been analyzed in leaf and smoke for ni­trate, alkaloids, condensate, nicotine, carbon monoxide and nitric oxide. Nitric oxide hasbeen determined separately in a second smoke procedure. To have a comparison with thecommercial brands, the contents of tobacco smoke compounds are calculated in milli­grams per cigarette (see graphs Nr. 18a - 27a) and to have a comparison within the cultiv­ars the values are additionally calculated per one gram smoked tobacco, related to the drymatter (see graphs Nr. 18b - 27b).

In previous investigations the cultivar Virgin D showed a low rate of nitric oxide. Therefore,no German Virgin cultivar has been analyzed.

Six Geudertheimer cultivars, - lugs and leaf, N-fertilizer 180 kg/ha -, eleven Burley cultiv­ars, - leaf and tips, N-fertilizer 180 kg/ha -, and the cultivar Badischer Burley E, - N-ferti­lizer variation 0 kg and 250 kg/ha -, have been investigated. All tobacco samples were un­fermented.

For the first investigation one thousand six hundred and twenty cigarettes (twenty-sevensamples) and for the second investigation one thousand six hundred and eighty (twenty­eight samples) from every sample sixty tipples cigarettes, well defined in paper, fillingpower, and hardness were made by hand, under standardized conditions.

5.1 Geudertheimer

The Geudertheimer cultivars Geudertheimer III, Badischer Geudertheimer W, BadischerGeudertheimer K and Perega 234 were planted in 1987 in Forchheim (see graphs Nr. 18a­19b). The stalk positions "lugs" and "leaf" were only air dried. The graphs Nr. 18a and 19ashow the results of the analysis of condensate, nicotine, carbon monoxide and nitric oxide.

The EU limitation for 1993 to fifteen milligrams condensate per cigarette is fulfilled for thecultivars Geudertheimer III and Badischer Geudertheimer W in the stalk position "lugs". Inthe stalk position "leaf" no Geudertheimer cultivar is within this limitation: all cultivars havevalues of nineteen milligrams per cigarette and have therefore clearly four milligrams con­densate more (see graphs Nr. 18a and 19a).

The nicotine level in the stalk position "lugs" of the Geudertheimer cultlvars is very low. Thestalk position "leaf" has with 0.19 milligram nicotine per cigarette the lowest nicotine con­tent (see graphs Nr. 18a and 19a). The tobacco year 1987 showed low temperatures com­bined with wet and rainy weather. Therefore, the low nicotine content shows more theweather conditions than the Geudertheimer properties: In dry and warm years the Geuder­theimer cultlvars produce high nicotine contents.

The carbon monoxide contents from eight to ten millimeters per cigarette in the Geuder­theimer cultivars are very low. They are comparable with the contents of the commerciallow carbon monoxide brands (see graphs Nr. 18a and 19a).

The Geudertheimer cultivars have surprisingly low nitric oxide contents: They are in the re­gion from sixty-seven to eighty-eight micrograms per cigarette (see graphs Nr. 18a and19a).

The Geudertheimer cultivars planted in 1990 in Forchheim show generally higher contentsof tobacco leaf and smoke compounds (see graphs Nr. 20 - 25b). Nitric oxide contents arein the region from two hundred and forty-eight (PB SB 5) to five hundred and fifty-six (Ita­lien. Havanna) micrograms per cigarette (see graph 24a) according to the high nitrate con­tents from 3 and ca. 4% respectively (see graph Nr. 21).

36

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5

Graph 19a: Compounds in Smoke from Geudertheimer Cultivars 1987,LEAF, N-Fertilization 180 kg N/ha

25

Condensate mg/cig l1li 19,48 19,31 18,25 18,78 18,37 19,63Nicotine mg/cig ~ 0,74 0,86 0,47 1,29 1,01 0,19Carbon monoxide ml/cig [J 10,2 9,3 9 9,5 10,1 10Nitr. oxide mg/cigxO,1 ~ 0,88 0,77 0,66 0,73 0,82 0,79

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3, Bad, GeudertheimerW4, Bad, Geudertheimer K 20

5, Perega6, Perega 234 10

Graph 19b: Compounds in Smoke from Geudertheimer Cultivars 1987,LEAF, N-Fertilization 180 kg N/ha

50

Condensate mg/g tdm l1li 38,13 38,14 31,71 32,98 33,61 34,97Nicotine mg/g tdm f2I 1,45 1,7 0,82 2,27 1,85 0,34

CO ml/g tdm 0 20 18,3 15,7 16,8 18,5 17,8:::

NO mg/g tdm x 0,1 ~ 1,72 1,52 1,15 1,29 1,49 1,41

The other compounds in tobacco smoke, tar and carbon monoxide, don't differ so muchand have less variability according to parameters, as to be seen in 25 breeding lines of theGeudertheimer cultivars of the harvest 1990 (see graphs Nr. 23a - 25b).

5.2 Burley

In the year 1987 the cultivars Badischer Burley E, Bursanica 217 Pereko, B 535, Burley 21,G 37 and Burley CA were planted. The Maryland cultivar MD 609 was planted for thepurpose of comparison (see graphs Nr. 26a and 26b). All Burley cultivars have in the stalk

37

Graph 20: Compounds in Tobacco from New Breeding Lines of Dark Air-Cured TobaccoCultivars 1990; LEAF, N-Ferlilization 180 kg N/ha

Nitrate % • 3,11 3,99 3,5 3,22 2,6 2,9 2,32 1,92 2,5

Nicotine % ~ 1,04 0,05 0,11 0,09 0,03 1,79 0,23 0,91 0,13

Sum of Nom. W 0,06 1,08 1,32 1,43 0,77 0,07 0,75 0,09 0,55+Myosm.% ;:;;.

Graph 21: Compounds in Tobacco from New Breeding Lines of Dark Air-Cured TobaccoCultivars 1990; LEAF, N-Ferlilization 180 kg N/ha

5

10. Havanna lie 411. Havanna dunkelrol12. Iialien. Havanna13.PB SB 5 314. Pay S10215. Phillippiin16. Semois 217. Z14918. Z149 NA

015 16 17 1810 11 12 13 14

• 3,63 3,72 3,9 3 3,77 1,97 3,03 2,6 2,46Nitrate %

~ 0,13 0,03 0,76 1,12 1,6 0,21 1,76 0.3 0,35Nicotine %

Sum of Nom. W 0,72 0.6 0,1 0,05 0.06 0,68 a 0.54 0,37+Myosm.% .;.;.

position "leaf" condensate values between nineteen and twenty-two milligrams per ciga­rette. They are clearly outside the EU-Iimitation for 1998. Therefore, these Burley cultivarshave no chance of getting any promotion by the EU-authorities in the future, too.

The Burley cultivars as well as the Geudertheimer cultivars show very low nicotine con­tents. Bursanica 217, Pereco and B 535 have a nicotine content of 0.25 milligram per ciga­rette, the cultivar Badischer Burley E of 0.93 milligrams per cigarette, the cultivar Burley CA

38

Graph 22: Compounds in Tobacco from New Breeding Lines of Dark Air-Cured TobaccoCultivars 1990; LEAF, N-Fertilization 180 kg N/ha

4

19Z 228 320Z32821 Z328 NA22, Havanna lI{Ve'rP.90}23Z 234 224 Z89125 Z89228 Z89327 Z 228xMo 609·1.'·628Sao Gecdennemer

019 20 21 22 23 24 25 26 27 28

Nitrate % • 2,4 3,02 3,74 2,29 2,29 1,49 2,37 2,2 1,83 1,77 2,6

Nicotine % ~ 1,05 0,04 0,06 0,36 0,36 0,48 0,78 0,1 0,1 0,41 0,73

Sum 01 Norn. 0° 0,74 0,84 0,45 0,45 0,78 0,24 0,85 0,83 0,83 0,9

+Myosm.%

of 0.99 milligrams per cigarette and the cultivar Burley 21 of 1.33 milligrams per cigarette(see graphs Nr. 26a and 26b).

The Burley cultivars show very low carbon monoxide contents, too. The amounts between8.6 and 9.8 milliliters per cigarette are still somewhat lower than the amounts of the Geu­dertheimer cultivars (see graphs Nr. 26a and 26b).

The amounts of nitric oxide in the Burley cultivars are low. They have with amounts from 85up to 211 micrograms per cigarette higher contents of nitric oxide than the Geudertheimercultivars. The nitric oxide content of the cultivar Badischer Burley E with 211 microgramsper cigarette is only somewhat lower than that of the cultivar Maryland MD 609 with 227micrograms per cigarette (see graphs Nr.26a and 26b). This content equals to commercialbrands with very high content of nitric oxide.

5.3 The Effects of Nitrogen-Fertilization

For the Geudertheimer and Burley cultivars a N-fertilization of 180 kg per hectare is recom­mended. The N-fertilization of more than of 180 kg per hectare is a special experiment toshow the effect of high N-fertilization in the smoke contents. In the case of 0 kg per hectarethe tobacco plant can only use the N-amount that is created by the mineralization in the soilduring the year. The harvesting grades of "leaf" and "tips" have been investigated in the to­bacco leaf and smoke.

5.3.1 Condensate

The condensate amounts of the Burley-tobacco with 250 kg N-fertilization per hectare iscorresponding to 180 kg N per hectare. It has been found, that without N-fertilization theBurley tobacco with 15.18 mg condensate per cigarette has already too much condensateaccording to the EU demand. The Bad Geudertheimer creates less condensate with 250 kgN per hectare (only 14.93 mg per cigarette). This amount seems to be false because the180 kg N-fertilization creates already an amount of 19.31 mg per cigarette. In the Bad

39

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Graph 23a: Compounds in Smoke from New Breeding Lines of Dark Air-Cured TobaccoCultivars 1990; LEAF, 180 kg N/ha

30

Condensate mg/cig II 17,79 17,42 22,34 22,74 24,22 20,57 22,86 17,55 18,79

Nicotine mg/cig [] 2,32 0,24 0,24 0,34 0,17 3,48 0,22 2,29 0,16Carbon monoxide ml/cig 0 9,53 10,39 10,87 11,49 11,27 10,7 10,49 9,48 9,68Nitr, oxide mg/clgxO,l ~ 3,87 4,7 3,63 3,2 ° 2,89 4,73 3,95 3,94

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Graph 23b: Compounds in Smoke from New Breeding Lines of Dark Air-Cured TobaccoCultivars 1990; LEAF, 180 kg N/ha

40

Condensate mg/g tdm II 27,06 27,04 33,71 34,64 36,27 33,73 33,7 26,81 30,51

Nlootlne mg/g tdm lSJ 3,53 0,37 0,36 0,52 0,26 5,7 0,32 0,5 0,26"

CO ml/g tdm 0 14,48 16,11 16,93 17,41 16,88 17,55 15,46 14,48 15,72

NO mg/g tdm x 0,1 E3 6,15 7,27 5,76 5,48 ° 5,01 8,03 5,67 7,52

Burley E, "tips", 20.6 mg condensate per cigarette are found with the fertilization of 250 kgN per hectare. In comparison without N-fertilization the 250 kg N-fertilization makes thecondensate content high, while in comparison with 180 kg N-fertilization the condensatecontent is not so high. The cultivar Bad Burley E, "tips", and the cultivar Burley NA, "Ieaf",­having 250 kg N-fertilization per hectare - are showing the same condensate amount of 20mg per cigarette. The same has been observed in the cultivar Burlina T 89, "leaf", with acondensate content of 18.93 mg per cigarette. Comparing the Burley cultivars no differ­ences are found with equal N-fertilization and within the same harvesting grade (see graphsNr. 27a and 27b).

40

181716151413121110

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Graph 24a: Compounds in Smoke from New Breeding Lines of Dark Air-Cured TobaccoCultivars 1990; LEAF, N-Fertilization 180 kg N/ha

25

Condensate mg/cig I11III 23,04 21,51 19,56 21,14 20,88 21,69 21,49 21,1 19,86

Nicotine mg/clg GJ 0,21 0,16 1,62 0,38 2,37 3,34 3,38 0,56 0,83Carbon monoxide ml/cig 0 10,71 9,58 9,86 10,48 10,8 10,48 11,11 10,22 11,02Nltr. oxide mg/clgxO,1 ESI ° ° 5,56 2,48 5,01 4,4 5,01 4,14 3,56

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Graph 24b: Compounds in Smoke from New Breeding Lines of Dark Air-Cured TobaccoCultivars 1990; LEAF, N-Fertilization 180 kg N/ha

60

Condensate mg/g tdm I11III 37,73 31,42 32,04 34,8 33,57 32,05 34,62 34,08 35,09

Nicotine mg/g tdm lSI 0,35 0,24 2,65 0,62 3,82 4,94 54,5 0,9 1,48

CO ml/g Idm [J 17,54 13,98 16,15 17,26 17,37 15,49 17,9 16,52 19,65

NO mg/g IdmxO,l IS3 ° ° 9,36 4,4 8,83 7,72 9,27 7,7 6,1

5.3.2 Nicotine

The cultivar Bad. Burley E, "leaf", having a N-fertilization of 250 kg per hectare, is creating ahigh nicotine amount of 2.53 mg nicotine per cigarette. Without N-fertilization a low contentof 0.37 mg nicotine per cigarette has been found. In the cultlvar Bad. Burley E, "tips", thecontent of 0.49 mg nicotine per cigarette has been found without N-fertilization and with anN-fertilization of 250 kg per hectare the an amount of 2.72 mg nicotine per cigarette (seegraph Nr. 27a). In this experiment it is documented that without N-fertilization low nicotinecontents are got.

41

28272625242322212019

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23 Z 23424. Z 89125. Z 89226. Z 89327. Z 228xMd 609-1-1-628. Bad.Geuderth.

Graph 25a: Compounds in Smoke from New Breeding Lines of Dark Air-Cured TobaccoCultivars 1990; LEAF, N-Fertilization 180 kg N/ha

25

Condensat mg/clg II 21,45 19,7 16,2 22,04 18,64 20,06 21,64 20,15 20,99 20,38

Nicotin mg/cig [] 2,08 0,15 0,14 0,83 1,18 1,39 1,66 0,33 0,07 0,3

Carbon monoxide ml/cig 0 11,31 9,09 7,72 9,12 10,78 11,58 11,73 11,25 11,04 9,7

Nitr. oxide mg/cigxO,l ~ 3,46 4,15 4,61 2,74 2,9 3,5 3,63 2,82 3,4 3,05

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10

Graph 25b: Compounds in Smoke from New Breeding Lines of Dark Air-Cured TobaccoCultivars 1990; LEAF, N-Fertilization 180 kg N/ha

50

Condensate mg/g tdm I11III 39,52 29,66 24,51 39,57 33,94 36,08 35,61 36,47 38,05 4,3Nicotine mg/g tdm D 3,82 0,23 0,21 1,49 2,13 2,51 2,73 0,6 0,13 0,6CO mi/g tdm 0 20,83 13,69 11,67 16,37 19,35 20,84 19,31 20,34 20,02 19,34NO mg/g tdm x 0,1 ISJ 5,92 6,99 7,17 4,18 5,85 6,96 6,63 5,6 6,74 5,53

The special cultivar Burlina T 89 has been found with high nicotine content, the otherspecial cultivar Burlina NA has been found with low till middle nicotine content with a N ­fertilization of 250 kg per hectare. That is why Burlina T 89 is a high-Ievel-nicotine-cultivarand the Burlina NA is a low-Ievel-cultivar.

5.3.3 Carbon Monoxide

Different N-fertilization is influencing the carbon monoxide content only slightly. A compari­son between the N-fertilization of 250 kg N per hectare and no fertilization is creating the

42

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3, Pereko4, B 535 15

5, Burley 216, MD 609 10

7, G 378, Burley CA 5

Graph 26a: Compounds in Smoke from Burley Cultivars 1987;LEAF, N-Fertilization 180 kg N/ha

25

Condensate mg/cig III 19,22 20,42 19,05 21,12 21,17 21,21 22,01Nicotine mg/cig !SJ 0,93 0,25 0,25 0,25 1,33 1,11 0,99Carbon monoxide ml/cig D 9,8 9,5 8,6 9,2 9,8 8,6 9Nitro oxide mg/cigxO,1 E8J 2,11 0,95 0,94 1,03 0,94 2,27 0,85 1,16

8765432u r rr:

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40

20

10

30

1, Bad Burley E2. Bursanica 2173. Pereko4. B 5355. Burley 216. MD 6097. G 378, Burley CA

Graph 26b: Compounds in Smoke from Burley Cultivars 1987;LEAF, N-Fertilization 180 kg N/ha

50

Condensate mg/g tdm III 35,1 35,77 35,55 39,09 35,74 31,79 39,27Nicotine mg/g tdm l23 1,7 0,44 0,47 0,46 2,25 1,66 1,77CO ml/g tdm 0 18 16,6 16,1 17,1 16,5 12,9 16,1NO mg/g tdm x 0,1 ~ 3,85 1,66 1,76 1,91 1,59 1,27 2,06

same carbon monoxide content of 10.4 - 11.3 ml per cigarette. The only exception is thecultivar Burlina 183, "tips", with a N-fertilization of 250 kg per hectare and 1301 ml per ciga­rette (see graphs Nr. 27a).

5.3.4 Nitric Oxide

The nitric oxide amounts are directly correlated with the N-fertilizationo The variant withoutN-fertilization has been found in the cultivar Bad. Burley E, "leaf", with the lowest amount of

43

Graph 27a: Compounds in Smoke from Burley Cultivars 1987 Changing with NitrogenFertilization; LEAF and TIPS, N-Fertilization 0 - 250 kg N/ha

25

I~EAF

1, Bad Burley E(0 kg N/ha)2, Bad Burley E(250 kg N/ha) 20

3, Burlina T89 (250 kg N/ha)4, Burlina NA (250 kg N/ha) 15

5, Bad Geuderth, (250 kg N/ha)6, Bad Burley E(0 kg N/ha) 10

7, Bad Burley E(250 kg N/ha)5

°5Zb-~[~~ Rl ~

2 3 4 5

TIPS

6 7

Condensate mg/clg • 15,18 19,74 18,93 20,9 14,93 20,23 20,6

Nicotine mg/clg IT8l 0,37 2,53 2,22 0,5 0,62 0,49 2,72

Carbon monoxide ml/cig CJ 10,7 10,6 10,8 11,3 10,4 10,8 13,1

Nltr. oxide mg/cigxO,1 rs 0,61 2,32 2,06 1,41 1,7 0,93 2,77

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"'" !soL ~ d ~ ss Iv; ~ it m~°

1. Bad Burley E(0 kg N/ha)2, Bad Burley E(250 kg N/ha) 30

3, Burlina T89 (250 kg N/ha)4, Burlina NA (250 kg N/ha)5, Bad Geuderth, (250 kg N/ha) 20

6, Bad Burley E(0 kg N/(ha)7, Bad Burley E(250 kg N/ha)

10

Graph 27b: Compounds in Smoke from Burley Cultivars 1987 Changing with NitrogenFertilization; LEAF and TIPS, N-Fertilization 0 - 250 kg N/ha

40

Condensate mg/g tdm • 25,58 31,84 31,82 34,93 24,71 32,76 35,62

Nicotine mg/g tdm ~ 0,62 4,08 3,73 0,84 1,03 0,79 4,7

CO ml/g tdm 0 18 17,1 18,1 18,9 17,2 17,5 22,6NO mg/g tdm x 0,1 ~ 1,04 3,74 3,47 2,36 2,82 2,1 4,79

61 /1g nitric oxide per cigarette, the variant with 250 kg N per hectare has been found with232 /1g nitric oxide per cigarette (see graph Nr. 27a). The variant with 180 kg N per hectarehas been found with 211 g per cigarette (see graph Nr. 26a). The cultivar Burlina T 89,"leaf", has been found with the very high content of 206 /1g nitric oxide per cigarette with aN-fertilization of 250 kg per hectare. 180 kg N per hectare create a nitric oxide content of 95/1g per cigarette in the cultivar Bursanica, "leaf" (see graph Nr. 26a). The cultivar Burlina NAhas been found with a slightly increased nitric oxide content of 141 /1g per cigarette with aN-fertilization of 250 kg per hectare. The cultivar Bad. Geudertheimer is producing an

44

amount of 170 g nitric oxide per cigarette, with a N-fertilization of 250 kg per hectare, 180kg N are producing only 67 fig nitric oxide per cigarette. All amounts are related to the "leaf"(see graphs Nr. 18a and 27a). A surmounted N-fertilization has been found in the cultivarBad Geudertheimer to create the double nitric oxide content. Very high nitric oxideamounts have been found in the cultivar Bad Burley E, "leaf" and "tips", in the variant with250 kg N per hectare: 232 and 277 fig nitric oxide per cigarette (see graph Nr. 27a).

5.3.5 Nitrate

The components of the tobacco smoke are influenced by the amounts and composition ofthe tobacco leaf substances. In the cultivar Bad Burley E with a N-fertilization of 250 kg perhectare a nitrate amount of 1.2% has been found, creating a nitric oxide amount of 232 figper cigarette. Therefore it is now clearly seen, that the cultivar Bad Geudertheimer createsa high nitric oxide content of 170 fig per cigarette with a N-fertilization of 250 kg per hectareand a nitrate content of 1.1% in the leaf: it is the same nitrate content found in the leaf of theBad Burley Ecultlvar that is responsible for a high nitric oxide content. The cultivars BurlinaT 89 and Burlina NA have been found with a N-fertilization of 250 kg per hectare to createnitrate amounts of 1.1% or 0.9%, which correlate with the high nitric oxide contents of 206fig per cigarette and 140 fig per cigarette (see graphs Nr. 27a and 28).

5.3.6 Nicotine and Nornicotine

Independently of the cultivars low nicotine contents in the tobacco leaves have beenfound. Only the cultivar Burlina T 89 has been found with a slightly higher content of 1.58%nicotine from the cultivar Bad Burley E with a content of 1.3% nicotine and aN-fertilizationof 250 kg per hectare. The cultivar Burlina NA is creating only 0.2% nicotine together with ahigh nornicotine content of 1.92%. The low amounts of the tobacco leaf components aredirectly correlated with the low amounts of the tobacco smoke contents. These lowamounts are due to the tobacco year 1987, a cold and rainy year.

Graph 28: Compounds in Smoke from Burley Cultivars 1987 Changing with NitrogenFertilization; LEAF and TIPS, N-Fertilization 0 - 250 kg Nlha

2,5

1, Bad Burley E(0 kg N/ha) LEAF TIPS

2, Bad Burley E(250 kg N/ha)2

3, Burlina T89 (250 kg N/ha)4, Burlina NA (250 kg N/ha) 1,5

5, Bad Geuderth, (250 kg N/ha)6, Bad Burley E(0 kg N/ha)

0,5

° 2 3 4 5 6

Nitrate % • 0,1 1,2 1,1 0,9 0,1 1,1

Nicotine % [ZJ 0,28 1,3 1,58 0,2 0,42 0,33

Sum of Nornic.+Myosm.% • 0,05 0,03 0,01 1,92 0,01 °45

5.4 Discussing the Results

A graphic plot between N-fertilization and nitrate contents in tobacco leaves and nitricoxide in tobacco smoke is showing the casual relationship between high nitrogen fertiliz­ation and the extreme high nitric oxide amounts in the tobacco smoke (graph Nr. 29). AllBurley cultivars have been evaluated with the fertilizer concentration 0 kg N per hectare,180 kg N per hectare and 250 kg N per hectare. The graph is showing clearly increasingrates of nitric oxide in tobacco smoke with increasing nitrogen fertilization. Without N-fer­tilization the tobacco plant could only use the soil nitrogen created during the vegetationperiod by mineralization. The nitric oxide content starts at different levels according to theharvesting grades. The curve "Ieaf" goes from 60 Ilg nitric oxide per cigarette without N fer­tilization to 230 Ilg nitric oxide with a N-fertilization of 250 kg per hectare (see graph Nr.29,curve "Ieaf"). In the harvesting grade "tips" 100 Ilg nitric oxide per cigarette without N-fer­tilization are observed and 280 Ilg nitric oxide per cigarette with 250 kg N-fertilization perhectare (see graph Nr. 29, curve "tips"). Considering the results the rates of N-fertilizationare in good correspondence with the rates of nitric oxide. Appropriate to the harvestinggrade there is a linear proportion to be seen.

A summary of the nitric oxide rates shows that they usually range in accordance with N-fer­tilization of the cultivars. It has been found out that in the harvesting year 1987 the Geuder­theimer cultivar with a N-fertilization of 180 kg per hectare has a very low nitric oxide range.This nitric oxide range corresponds with the low ranges of the Greek breeding lines 4N and3/8 with a N-fertilization of 30 kg per hectare, by which the breeding lines 2/K and 1/B areobserved to have significantly higher nitric oxide ranges with the common fertilization of30 - 40 kg per hectare (see graph Nr. 30).

From this it is clearly to be seen, that an excessive N-fertilization in tobacco cultivationused for an increase in the yield is creating high levels of nitric oxide in tobacco smoke. Thisis shown by the German cultivar Bad Burley E in the harvesting grade "Ieaf" and "tips". Al­together the Burley cultivars are very high in nitric oxide content. An unexpectedly low con­tent was found in the German Geudertheimer cultivars and in the Greek breeding line 3/8from the year 1987. Independently of fertilization and harvesting grades it has been foundthat the nitric oxide content is significantly correlated to differences in the tobacco cultlv­ars. Therefore, the nitric oxide content has to be considered as a quality and breeding at­tribute.

NO pg/cig300,---------------------------,

250

200

150

+

-1-

TIPS

LEAF

Graph 29: Nitric Oxidein Smoke from BurleyCultivars 1987;LEAF and TIPS, N­Fertilization 0 - 250 kgNlha

100

50

200 250

N kg/ha

15010050O'--------------------------!

o

46

NO j.lg/ha400

Graph 30: Nitric Oxide

Din Tobacco Cu/tivars

3001987; Overall Range GREECE GERMANYfrom all HarvestingGrades; N-Fertilization 200 D D0- 250 kg N/ha

D100 c::::J D c:J

01/B 2/K 3/S 4/V 5/R Burley Geudert30 40 30 30 180 180 180

N kg/ha

6 Tobacco BreedingThe main goals in tobacco breeding43, 44, 45) are high yields, differentiation from existingvarieties, rigidity of the plant and resistance to tobacco diseases. Characterization canadditionally be done according to the chemical point of view in analyzing the chemicalcompounds.

After a three years lasting plantation trial the breeder will get from the German FederalAgency of Varieties a cultivar protection for ten years, if there is a significant improvementin breeding about the outer and inner quality of the new tobacco cultivar compared to oldervarieties. Due to the close relation between the modern tobacco cultivars, this is withoutanalytical methods rarely to achieve.

6.1 Identification of Tobacco Varieties

In Germany in three cases the chemical compounds of tobacco varieties are used for theidentification of new breeding lines: Varieties that are low in nicotine content and varietiesthat produce nornicotine from nicotine during the air drying process, they are described ac­cording to the alkaloid contents; Virgin varieties are described according to the duvatrien­diol amount.

6.2 Secondary Plant Compounds of Content

Because the metabolism of the tobacco plant, - responsible for the existing amounts -,chemical compounds as alkaloids and duvatrienediols, which pertaine to the quality of thetobacco sample, are secondary plant compounds of content and vary according to the cli­mate, the environment, the plantation and harvesting procedures; normally they can't beused for an identification of the tobacco varieties. This is only achieved by the analysis ofthe genetic code, - the DNA in chromosomes -, or by the analysis of a special group ofplant cell proteins, which are directly generated by the DNA.

6.3 Isoenzymes and Alloenzymes

The differences found in these specialized protein types as peroxidase and polyphenol ox­idase directing the metabolism in the plant cell are called "isoenzymes" or "alloenzymes".

47

They are genetically determined and the result of the expression of the allelic genes46). Thewhole genotypical expression of the isoenzymes is best visible in the ripe tobacco plant.Electrophoretically separated tobacco peroxidase and polyphenoloxidase show onlyquantitative and no qualitative expression differences in different stages of the plant devel­opment as S. J. Sheen has pointed out47).

The isoenzymes are determined in the green tobacco leaf shortly before their maturity. Forthe identification of the cultivars, the quantity of the isoenzymes in the patterns is not so im­portant as the quality differences in the patterns produced by the single cultivar or appear­ing as additional patterns in the electrophoregram of the F7-generation in comparison tothe parents. According to the complexity of these protein compounds they are named ac­cording to their function; the therm "isozyme system" is often used for them.

6.4 Methods

6.4.1 Protein Extractipn Procedures

Seed Proteins

According to H. Stegemann et a1.48) the seed proteins were ground in a mortar, washedwith cold acetone (- 30°C, dried over Na2S04 free from water) and extracted withtris-borate buffer pH 8.9. Until to the further preparation the residue was stored in a freezer(- 20°C).

Green Tobacco Leaves

The protein extraction of green tobacco leaves was done according to M. Abet et aI.49), thedialysis and concentration procedure according to T. Cooper50) and the measurement ofthe protein concentration according to N. E. Bradford 51).

Picture 3: Working Place for Electrophoresis of Proteins Consisting of Desaphor VA 150Chamber, Desatronic 3000 / 200 Power Supplyand Frigostat

48

Using a cold mortar (- 30°C) six frozen green tobacco leaves without midrib, cut in smallpieces, were ground to a fine powder with liquid nitrogen, stored over night in a freezer,filtrated and washed with a cold acetone-water mixture (4:1, - 30°C) and cold acetone(- 30°C). The tobacco powder was homogenized in Mcilivaine Buffer 0,1 M, pH 2.8, con­taining dithiothreitol; the mixture then centrifuged at 15000 rpm, the supernatant filtrated ina dialysis tube, dialyzed against tris 5 mM-glycine 38,5 mM buffer pH 2,8 to pH 4, concen­trated against polyethylene glycol 20000 to a content of 2 ml, centrifuged at 1500 rpm, theprotein content measured to 0,1 %, and stored in a freezer at - 30°C.

Air-Dried and Milled Tobacco Samples

The procedure used for the extraction of the proteins from air-dried and milled tobaccosamples equals the Rrocedure for proteins of green tobacco leaves. "Acetone dry powder"according to A. Gorg52) had to be prepared additionally: Using a cold mortar (- 30°C) the to­bacco meal was ground with cold acetone (- 30°C, dried over Na2S04 free from water), thetobacco slurry filtrated, washed twice with a cold acetone-water mixture (4:1 vt»,- 30°C), three times with cold acetone (- 30°C), dried by aspiration, freed from smell in avacuum-desiccator and stored in a freezer (- 20°C).

6.4.2 Electrophoresis Procedure

After previous work done with dry tobacco showing the degradation of tobacco proteins indark air cured tobacco during the natural fermentation process, for which an isoelectric fo­cusing method with Sephadex G 75, pH gradient 2 - 11, was developed53), and after firstattempts to separate proteins of tobacco seeds using the isoelectric focusing method withpolyacrylamide gel slabs, pH gradient 6 - 954), the electrophoresis method was used forthe separation of proteins and enzymes.

The electrophoresis with polyacrylamide gel slabs, with its fewer number of bands in theprotein and enzyme profiles than in that ones of the isoelectric focusing method, is easierfor the characterization of tobacco cultivars. For tobacco cultivar identification seven pub­lications in the literature were found using the polyacrylamide gel electrophoresismethod55). The most publications describe the isoelectric focusing method as the methodof choice for other cultivars56).

Picture 4: Desatronic IT Integrator for the Quantitative Estimation of Protein Profiles

49

Since 1992 - considering the toxicity of polyacrylamide gel slabs and the waste problem ­for tobacco cultivar identification the isoelectric focusing method with commercial avail­able agarose gel slabs is used, too.

Electrophoresis was performed using a Desaphor VA 150 chamber, a polymerization stand,a peristaltic pump Desaga STA, a magnetic stirrer, a Desatronic 3000/200 power supplyand a Frigostat (see picture Nr.3) in combination with a Desatronic IT integrator (see pictureNr.4).

The electrophoresis chamber was designed to hold two vertical polyacrylamide gel slabs.Each slab could hold up to twenty four samples.

The procedure for casting the gels was adapted from the operating instructions for theelectrophoresis equipment of the company Desaga57).

The used solutions were modified according to H. Stegemann et a1.48)and K. Altland 58) tofit the procedure.

The concentration of the acrylamide and bisacrylamide (T %) and the amount percentageof the N, N'-Methylene-bis-acrylamide (bisacrylamide (C %)) was calculated as follows:

T = (A + B) x 100 1V C =B x 100 1(A + B)A =g Acrylamide B =g Bisacrylamide V =Volume of the solution

For proteins from tobacco seed a SDS gradient gel, T =8 % till 25 %, C =5 %, electro­phoresis system was used.

For proteins from green tobacco leaves, air dried and milled tobacco samples a discon­tinuous gel electrophoresis system was used consisting of a separation gel (TG : T =10 %,C =5 %, pH =9.1) and a concentration gel (KG: T =2.5 %, C =7.5 %, pH =6,7).

A tris-HCI buffer (pH 9,1) was used in the separating gel and a tris-HsP04 buffer (pH 6,7) inthe concentration gel. All gels were ammonium persulfate catalyzed. Sample density wasincreased with 40 % sucrose helping to prevent samples from migration between the wells.

The lower electrode buffer was tris-HCI (pH 8.1) and the upper electrode buffer tris-glycine(pH 8,9).

Gels were run from cathode to anode at a voltage of 500 volts and a current of 100 mA.Electrophoresis was ended when the marker dye (0,01 % bromophenol blue) had migratedto the bottom of the gel.

The gels were removed from the cells and appropriately stained.

6.4.3 Isoelectric Focusing Procedure

Isoelectric focusing was performed using a horizontal electrophoresis unit Desaphor HF, a3000-Volt power supply Desatronic, a circulating water bath Frigostat, a third electrode forDesaphor HF (see picture Nr. 5).

The procedure for isoelectric focusing was adapted from the operating instructions for theelectrophoresis equipment of the company Desaga57) and with commercial available aga­rose gel slabs, pH gradient 3 - 10 plus spread at pH gradient 3 - 7 (HyPure Gel Desaga Nr.150009) according to the procedures for the identification of corn varieties from the com­pany Isolab inc.59Jand the identification of potato varieties from "HyPure Procedures" sub­mitted from the company Desaga60).

6.4.4 Protein and Enzyme Staining Techniques

For total protein staining Coomassie Brilliant Blue was used normally, but for small proteinamounts the very sensitive silver staining according to E. Hempelmann, M. Zwanzig and

50

Picture 5: Desaphor HF for Isoelectric Focusing of Proteins

M. MUller61) was used. Silver staining can be used for SDS gels, IEF gels and PAGE gels(native).

For malate dehydroqenase, esterase and peroxidase different staining techniques accord­ing to G. J. Brewer6'2) were used.

Malate DehydrogenaseThe staining solution for malate dehydrogenase contained 0.05 M tris pH 7.0, p-nitrobluetetrazolium, phenazine methosulfate, malic acid, and diphosphopyridine nucleotide.

EsteraseFor esterase 1% 1,2-naphthyl acetate solution was used as the substrate that a goodisoenzyme profile resulted. Since 1,2-naphthyl acetate is not very soluble, it was made upin 70% acetone-aqua dest. with the 1,2-naphthyl acetate being dissolved in the acetonefirst.

PeroxidaseThe substrate used for the detection of peroxidase isoenzymes was benzidine. Benzidineproved to be good in both the number and clarity of bands produced.

6.4.5 Documentation

After extraction, clean-up and concentration procedures, the isoenzymes and alloenzymeswere electrophoretically separated and densitometrically scanned (see picture Nr. 6).

Photographs were taken and diagrams were drawn of all the gels. The estimation wasmade from the electrophoregrams, the relative mobility (Rt) for each band calculated and

51

protein profiles drawn with the graphic program Chart from the company Microsoft (seefigures Nr. 1 and 2). Rf can be defined as:

distance the protein migratedRf = distance the dye migrated

1980; -+9

2. 00 (l'-r-----'--Itt-'--'-----'-----'-__+_ 2. (l 0 0

1982

1 • 6 (1 '~'--.J 1.600

1.2C'u) 1. ,0- _ 1.200

,,

L~. 80.3---1

I

O.40aJ

(t.oe0

75.0

: j

; :

:100.0

1~ ii.: '. ! ~9: ~

O.8(1(l

0.400

0.000

~3. 200

144.9

o,,~ i \ ~ , .• "

,.,,~ u&.~.I.~.J.Lffi!>_ e , '"

Picture 6: Densitometric Estimation of Electrophoregrams of Tobacco Seed Proteins Be­discher Geudertheimer of two Different Years

6.5 Results and Discussion

6.5.1 Tobacco Seed

To their genuineness different dark or bright tobaccos were examined first in protein bandsof the tobacco seeds of two crops, whose harvest had a difference of two years. Seeds offormer crops were examined because during the second year after the harvest there is anoptimum of germination. The above examined seeds were proved to be genuine becauseof the identical protein bands with an exception of Geudertheimer III and Badischer BurleyE (see figures Nr. 1 and 2).

52

1 Bad. Geuderth.2 Geuderth. III3 Pereg 2344 Perega5 Bad. Burl. E6 B 57 Bursanica 2178 Burlina 1839 Virgin Aurea10 Bel 10

1098765432

°

0,4

0,2

0,3

0,1

0,7

0,6

0,9

0,8

Rf 0,5

Figure 1: Seed proteine profiles from different Cigar tobaccos,Burley and Virgin cultivars; harvested 1980

Seed protein profilesfrom the harvestingyears 1980 (see figureNr. 1) and 1982 (seefigure Nr. 2) show thesame profiles with theonly exception of thecultivar Virgin Aurea(profile Nr. 9), due tothe impurity of theseed.

From the protein pro­files, no significantidentification of singlecultivars is possible,only the tobacco typesare distinguishableand their correlation tothe blue mold resis­tance (Rf-region 0.4).Varieties resistant against the blue mold disease from varieties not resistant could be ident­ified. So the resistant varieties Bel 10, Burlina 183, Bursanica 217, Perega and Pereg 234appear to have one protein band in Rf-rate 0,6 (see figures Nr. 1 and 2).

6.5.2 Green Leaf

1 Bad. Geuderth.2 Geuderth. III3 Pereg 2344 Perega5 Bad. Burl. E6 B 57 Bursanica 2178 Burlina 1839 Virgin Aurea10 Bel 10

°

0,9

0,8

0,7

0,2

0,1

0,4

0,6

0,3

Rf 0,5

1 2 3 4 5 6 7 8 9 10

Figure 2: Seed proteine profiles from different Cigar tobaccos,Burley and Virgin cultivars; harvested 1982

The Burley tobaccowas harvested fromthe experimental fieldof Forchheim, Baden­WUrttemberg, in 1989,as mature green to­bacco of the stalkposition "lugs".

Individual varieties orrelative breeding linesin dark and light to­baccos could be ident-ified because of protein and isoenzyme bands in the green leaf. A relation to the proteinbands was observed just before the ripening of the green tobacco leaves in the prime "tip".The varieties Badischer Burley E, Burlina '83 and Bursanica 217 could be identified only bytheir protein bands while the breeding lines B 520, B 21, Pereco, B 535 and B 535 Bursa

Green tobacco protein and isoenzyme profiles of a cultivar vary with the plantation area,the harvesting year, the stalk position and the maturity of the leaf. Leaf protein profiles fromdifferent Burley cultiv-ars are shown in fig­ures Nr. 3 and 5. Addi­tionally, leaf isoenzymeprofiles of peroxidaseare shown in figuresNr. 4 and 6.

53

Figure 3: Leafprotein profiles from different Burley cultivars; fieldexperiment 39/89, lugs, green tobacco

86: B 520-NA

665: B 5

687: G 37

694: Md B72

67B: PerekD-NA

B4: R-220lBursal

693: Md 341

646: Bad.Burl.E

665 669 678 684 686 687 693 694 646 650 660: Burley 21/.0

0.8

0.2

0.4

0.0 could be identified inrelation to proteinbands and peroxidaseband (see figures Nr. 5and 6).

All new breeding linesshow protein profiles(figure Nr. 3) differentfrom the reference cul­tivars B 5 and Bad Bur­ley E. Additionally, it isobserved that the pro­tein profiles of thebreeding lines are dif­ferent from each otherexcept Nr. 693 and 694(see figure Nr. 3).

To differentiate between these breeding lines the peroxidase profiles have to be considered(see figure Nr. 4). All Burley breeding lines show a typical band at the Rf-region of 0.70 ­0.74 (see figures Nr. 3 and 5) to the reference cultivars B5 and Bad Burley E.

Figure 4: Leaf peroxidase profiles from different Burley cultivars;field experiment 39/89, lugs, green tobacco

665: B 5

678: ParekD-NA

84: R-220 IBursal

86: B 520-NA

6B7: G 37

693: Md 34\

694: Md 872

646: Bad.Burl.E

660: Burley 21

665 669 678 684 686 687 693 694 646 650/.0

0.8

0.2

0.4

0.0

6.5.3 Dry Tobacco

After harvesting of tobacco leaves during the drying process to a water content below10%, during their curing or fermentation and storage process an enzymatic and oxidantdecomposition of proteins is occurring, described in a previous publication of H. Delinceeand P. Range53). Therefore, differing protein and isoenzyme profiles between green anddried tobacco are observed: whereas normally the profiles of dry tobacco show fewer

bands then the greenones (see figures Nr. 7and 6) and the proteinprofiles more bandsthan the isoenzymeprofiles (see figures Nr.7 and 8).

The identification ofdifferent tobacco var­ieties based on proteinand isoenzym bandswas done in dry andground tobaccosamples of the prime"leaf" coming from ex­perimental plantationsof the LAP (see figuresNr. 3 -16).

All the varieties were identified according to their protein bands (see figures Nr. 11, 12, 13and 14).

Also the question about the genuineness of "F-taler" cultivar from 1986 compared with theone of 1978, both planted in an experimental cultivation in 1988, was examined in dry to­bacco comparing the different protein bands (see figure Nr. 11, profile Nr. 775 profile Nr.

54

56: Md 609

561: B 5201B.21

565: B.211B 535

591: PerekDlB 535

696: B 535lBuree.

46: Bed.Burl.E

652: Burllne

654: Bureen Ice

46: Bed.Burl.E

50: BurlBY 21

52: Burllne

54: Bureenlce

65B: Md 609

5BI: B 5201B.21

565: B.211B 535

596591585581658654652646

0.4

1.0

0.8

0.2

0.0

0.0

0.4

0.2

Figure 5: Leafprotein profiles from different Burley cultivars; fieldexperiments 35 and 38/89, lugs, green tobacco

776): It was found outthat they are geneti-cally different to-baccos. Theisoenzymes and pro­tein bands in the greenleaf can be identifiedfrom the ones of thedry material when itcomes to their numberand their position. Inthe few bands that canfound in theisoenzymes profilesthe loss of peroxidasewas characteristic (seefigure Nr. 12).

Although protein and isoenzyme profiles of dry tobacco fewer bands than profiles of greentobacco leaves are showing, the tobacco varieties can be identified in the dry material. Forexample the breeding lines with profile Nr. 782 and Nr. 783 show no differences in the pro­tein and isoenzyme profiles: this leads to the conclusion that these breeding lines are fromthe same origin (see figures Nr. 11 and 12); the breeding lines with profile Nr. 775 and Nr.776 have been expected to be the same breeding line, but it is obvious from the proteinprofile that they are different (see figure Nr. 12).

According to this anidentification of thebreeding lines can bedone in dry tobacco asin green tobaccoleaves by the proteinprofiles in combinationwith the isoenzymeprofiles.

7 Conclusion

0.8

1.0646 650 652 654 658 581 585 591 596

591: PerekDlB 536

596: B 5351Buree.

Figure 6: Leaf peroxidase profiles from different Burley cultivars;field experiments 35 and 38/89, lugs, green tobacco

As it is shown in thisstudy, an identificationof tobacco breedinglines of the different to­bacco types is poss-ible by their protein profiles in combination with the isoenzyme profiles of peroxidase ingreen tobacco leaves and in dry tobacco, belonging to tobacco from the same area andenvironment, harvesting year and stalk position.

Based on these findings for old and new breeding lines of Cigar, Burley and Virgin tobaccopresented in figures Nr. 1 to 16, it is feasible that the electrophoretic procedures will be­come a practical meaning as a method of choice for the cultivar identification in N. taba­cum: Together with the quality analysis in tobacco and tobacco smoke an identity-card forevery culture can be made.

55

0.0

0.2

0.4

Rf0.6

0.8

1.0590 591 592 593 62

690: Bursan.217

691: Burllna 183

692: B 620

693: Bad. Burl. E

62:Md 609

Figure 7: Leaf protein profilesfrom different Burley cultivars;field experiment 37/88, leaf,dry tobacco

690: Bursan.2170.0

0.2

0.4

Rf0.6

0.8

1.0590 591 592 593 62

Figure 8: Leaf peroxidase pro­691: Burllna 183 files from different Burley cui-692: B 620 tivars; field experiment 37/88,693:Bad. Burl. E leaf, dry tobacco

62:Md 609

0.0

0.2

0.4

Rf

0.6

0.8

615: Burlay 49

619: While Burley

626: Md B72

627: Maryland

62B: Sumat.Schl.

Figure 9: Leaf protein profilesfrom different Burley cultivars;field experiment 39/88, leaf,dry tobacco

/.0

0.0

0.2

0.4

Rf0.6

0.8

1.0

56

6/5

6/5

619

6/9

626

626

6Z1

6Z1

628

628

616: Burley 49

619: While Burley

626: Md 872

627: Msryland

628: Sumat.Schl.

Figure 10: Leaf peroxidaseprofiles from different Burleycultivars; field experiment 39/88, leaf, dry tobacco

0.0

O.C

OA

0.8

1.0775 776 782 78J 786 787

775:F·lolor \978

776:F·lolor \966

782: Cobol

783: Dobltcener

786: Trumpl

787:Skronlowskl

Figure 11: Leaf profiles fromdifferent Cigar tobacco cuitiv­ars; field experiment 49/88,leaf dry tobacco

0.0

0.2

0.4

0.8

1.0

0.0

0.2

0.4

0.8

1.0

0.0

0.2

0.4

0.8

1.0

775

754

J95

776 782

759

J46 J97

78J

618

786

402

787

62J

405

776:F·leter \978

776:F·lotor \986

782: Cobol

783: Debltconor

786: Trumpf

787:SkronlOWBkl

754: Bod,Geuder,

759: Z 149

618: 8urlw 21

623: KY 6\

395; Gold A

396: Pelt'll

397: Robuolo

402: V 3

405: V It

Figure 12: Leaf peroxidaseprofiles from different Cigartobacco cu/tivars; field experi­ment 49/88, leaf, dry tobacco

Figure 13: Leaf protein pro­files from different Cigar to­baccos and Burley cu/tivars;field experiment 39 and 47/88,leaf, dry tobacco

Figure 14: Leaf protein pro­files from different Virgin cut­tivars; field experiment 29/88,leaf, dry tobacco

57

0.0

0.2

0.4

Rf

0.6

0.8

1.0

0.0

0.2

0.4

0.8

1.0

618

618

623

623

634

750

750

754

754 757

757

759

759

618: Burley 21Figure 15: Leaf protein pro-

623: KY 61 files from different Cigar to-634: K35A baccos and Burley cultivars;750; Geuderlh.11I field experiments 39 and 471754: Bad.Geudor, 88, leaf, dry tobacco757: Perege

759: Z 149

61B: Burley 2\623' KY 61 Figure 16: Leaf peroxidase

. profiles from differet Cigar to-760: Geuderlh.1I1 baccos and Burley cultivars;764: Bed.Geuder. field experiments 39 ad 47188,767: Pereg. leaf, dry tobacco769: Z 149

Figure 16: Leel peroxldeee prollle. lrom dlllerent Clger lobaccoe end Burleycuilivars; lIeld experiments 39 and 47{BB.leaf.

58

EXPERIMENTAL

8 Nitric Oxide Analysis

8.1 BAT Nitric Oxide Analyzer in Combination with Smoking Machine FiI­trona SM 302(see operating instructions30))

8.1.1 Additional Equipment required

- 1 mV or 10 mV recorder - with speed of response at least 0.5 second full scale deflec­tion. A suitable recorder can be supplied if required.

Calibration gases - suggested as follows:1 cylinder 100 vpm nitric oxide in nitrogen.1 cylinder 400 vpm nitric oxide in nitrogen.1 cylinder 700 vpm nitric oxide in nitrogen.1 cylinder 1000 vpm nitric oxide in nitrogen.Certificate of analysis to be supplied by the manufacturer,

4 stainless steel, corrosion resistant regulators suitable for use with the above mixtures.

Cylinder of oxygen.

Cylinder of nitrogen.

Regulators for oxygen and nitrogen cylinders.

Optional quick connect unions for calibration gases.4-SS-QM1-81-200 Swagelock female.1-SS-QM2-S-200 Swagelock male.With stainless steel or P.T.F.E. tubing for connection to regulators.

8.1.2 Setting Up and Calibration Instructions

- Make all connections described in the installation procedure. (1)

Switch on the nitrogen supply. (2)

Using the instrument controls, set the nitrogen pressure to 60 p.s.i.g. Measure the ni­trogen flow rate from the reaction cell, exhaust vent at the rear of the analyzer. The flowrate should be 7.2 x 10-3 m3 h(1 (120 cm3 mln"). If not correct, unlock by lifting smalllever on nitrogen flow controller dial and adjust flow rate as necessary. (3)

Switch on the oxygen supply. (4)

Using the instrument control, set the oxygen pressure to 10 p.s.i.g. Measure the com­bined nitrogen and oxygen flow rate from the reaction cell exhaust vent.The total flow rate should be 9.0 x 10-3 m3 h(1 (150 cm3 rnln"), If not correct, unlock bylifting small lever on oxygen flow controller dial and adjust flow rate as necessary. (Thisflow rate is also preset and only minor adjustment should be necessary.) Lock the flowcontroller. (5)

Switch on the MAINS and FAN/OZONE switches and leave for 20 minutes warm uptime. (6)

Switch on and zero the recorder, following the instructions given in the recorder manual.Return the recorder to the measuring mode. (7)

59

- Set the GAIN control to a suitable value. (8)

- Zero the recorder using the analyzer ZERO control. (9)

- Connect one of the standard gas mixtures to the stainless steel 3-way tap in the smok-ing machine analyzer link line, using a length of RT.F.E. tubing and stainless steel fittings.Because of the nature of RT.F.E., a short length of stainless steel tubing should be in­serted into the end of the RT.F.E. tubing before making any connections. (10)

- Check that the SLIDER SAMPLE VALVE is in the withdrawn position and the TESTswitch is in the SAMPLE position. (11)

- Turn the 3-way tap on the smoking engine to allow a standard gas to flow through thesample line, open the needle valve (if fitted) on the pressure regulator of the standard gasmixture, and set the pressure to approximately 5 p.s.i.g. (This should result in a flow gasthrough the pressure valve.) (12)

- Allow sufficient time for the lines to be thoroughly purged with gas, turn the 3-way tap tothe "Off" position, wait 3 - 5 seconds to allow the pressure in the line to come to atmos­pheric pressure, press the GAS SAMPLE VALVE firmly in. (13)

- After the peak is recorded, withdraw the gas SAMPLE VALVE, switch the 3-way valve topermit a further sample of standard gas to flow through the system. (14)

- Repeat Instruction (13). If necessary, adjust the attenuation to achieve an acceptablepeak height. (15)

- Continue to repeat Instructions 13 and 14 until three successive peaks of similar heighthave been obtained. Read off the mean peak height. (16)

- Repeat instructions 10 - 16 for the remaining three standard gas mixtures. (17)

- Switch off all the cylinders of standard gas mixtures. (18)

- Plot the calibration graph of nitric oxide concentration (vpm) certified by the manufac-turer against mean peak height (adjusted for attenuation). This graph should belinear. (19)

8.1.3 Smoking Procedure

The following instructions apply to the procedure used with a Cigarette ComponentsModel 302 smoking engine.

WARNING Ensure that the 3-way tap is turned to allow vapor phase to pass from thesmoking engine to the nitric oxide analyzer before switching on the smokingengine.

- Set up the smoking engine in the usual way. Allow at least a 15 minute warm up time be­fore checking puff volume. Zero the puff counter.

- Attach a prepared Cambridge filter holder to each of the 8 ports. Insert a cigarettemarked with the appropriate butt mark into each holder.

- Set the attenuation to the appropriate value. This value will be known beforehand afterexperience has been gained with the technique and its application to various cigarettebrands and types.

- Set the TEST switch to the RUN position. This will result in the solenoid valve operatingonce. Wait until this cycle has been completed before proceeding to the next instruction(approximately 15 seconds).

- Follow the smoking engine operating instructions to light cigarettes.

60

- The nitric oxide analyzer will sample after each puff automatically. Ensure that the peakis clearly recorded "on scale" and if necessary adjust the attenuation.

- Allow smoking to continue until the butt mark is reached. All cigarettes must be allowedto take the same number of puffs and thus the cotton puff-termination device cannot beused. Smoking is terminated when the average butt mark is reached. Typically some willbe just over and others will be just under the butt mark.

- With all the cigarettes extinguished, allow the smoking engine to take three clearingpuffs, recording the nitric oxide concentration as above.

- Calculate an average peak height (allowing for attenuation) by adding together all of theindividual peak heights, including the clearing puffs, and dividing by the number of litpuffs.

- Using the standard calibration graph, read off the average concentration of nitric oxide inthe vapor phase.

- Record barometric pressure and room temperature adjacent to the instrument.

- Calculate the delivery of nitric oxide per cigarette from the formula - mass per cigarettebasis -.

Nitric oxide (l1g/Cigarette) =A x N ~~3x+0~13470

where A = average concentration (vpm)N = number of lit puffs per cigarette (including 3 clearing puffs)P = barometric pressure (kPa)T = room temperature (C).

8.2 Monitor Labs. Nitrogen Oxides Analyzer 8440E in Combination withSmoking Machine Borgwaldt RM 20 CS and Automatically WorkingNitrogen Flushing Unit(see operating tnstructlons-'': 41))

The automatically working nitrogen flushing unit consists of two three-way valves and oneback pressure valve from stainless steel. The one in combination with the back pressurevalve is electrically operated from the pump stroke unit of the smoking machine, the othermechanically operated with the pressure of the mainstream smoke (see graph I). TheP.T.F.E. tubings for connection from the smoking machine to the nitrogens oxide analyzerhas a stagnant volume of 5 ml. During the draw intermission of the smoking machine theconnecting tubes and the nitrogen oxides analyzer are flushed with nitrogen gas of highestpurity between every puff of the smoking machine.

8.2.1 Additional Equipment required

- Electrically operated three-way valve from stainless steel.

Mechanically operated three-way valve from stainless steel.

Back pressure valve from stainless steel.Optional quick connect unions for calibration gases.With P.T.F.E. tublnq 0,4 cm in diameter for connections.

1 mV or 10 mV recorder - with speed of response at least 0.5 second full scale deflec­tion. A suitable recorder can be supplied if required.

Calibration gases - suggested as follows:1 cylinder 100 vpm nitric dioxide in nitrogen.

61

1 cylinder 100 vpm nitric oxide in nitrogen.1 cylinder 500 vpm nitric oxide in nitrogen.1 cylinder 1000 vpm nitric oxide in nitrogen.Certificate of analysis to be supplied by the manufacturer.

- 4 stainless steel corrosion resistant regulators suitable for use with the above mixtures.

- Cylinder of synthetic air.

- Cylinder of highly purified nitrogen.

- Regulators for synthetic air and nitrogen cylinders. Optional quick connect unions forcalibration gases.4-SS-QM1-B1-200 Swagelock female.1-SS-QM2-S-200 Swagelock male.With stainless steel or PT.F.E. tubing for connection to regulators.

8.2.2 Setting Up and Calibration

- Make all connections described in the installation procedure (see operating instruc­tions 34,41)).

- Calibrate from the smoking port described in the calibration procedure (see operating in­structions34,41)).

8.2.3 Smoking Procedure

The following instructions apply to the procedure used with the RM 20 CS cigarette smok­ing machine.

- Set up the smoking engine in the usual way. Allow at least a 15 minute warm up time be­fore checking puff volume. Zero the puff counter.

- Attach a prepared Cambridge filter holder to the port. Insert 5 cigarettes marked with theappropriate butt mark into each holder.

- Set the attenuation to the appropriate value. This value will be known beforehand afterexperience has been gained with the technique and its application to various cigarettebrands and types.

- Follow the smoking engine operating instructions to light cigarettes.

- The nitrogen oxides analyzer will sample after each puff automatically. Ensure that thepeak is clearly recorded and if necessary adjust the attenuation.

- Allow smoking to continue until the butt mark is reached. Smoking is terminated whenbutt mark is reached.

- With all the cigarettes extlnqulshed, allow smoking engine to take three clearing puffs,recording the nitric oxide concentration as above.

- Calculate an average peak height (allowing for attenuation) by adding together all of theindividual peak heights, including three clearing puffs, and dividing by the number of litpuffs.

- Using the standard calibration graph, read off the average concentration of nitric oxide inthe vapor phase.

- Record barometric pressure and room temperature adjacent to the instrument.

62

- Calculate the delivery of nitric oxide per cigarette from the formula. Calculation of nitricoxide yield - volume per cigarette basis - at 101,3 kPa and O°C:

Cobs x V x N x P x 273III nitric oxide per Cigarette = ---=-3------~

qx10 x101,3x (t+273)

Short formula:

CobsxNxpIII nitric oxide per Cigarette = t 273 x F

q x (+ )

with:

F = 35 x 273

103 x 101,3

0,012572 (1 cigarette)= 0,050289 (4 cigarettes)= 0,100576 (8 cigarettes)

Calculation of nitric oxide yield - mass per cigarette basis - at 101,3 kPa and O°C:

Cobs x V x N x P x 273 x 30Ilg nitric oxide per Cigarette

q x 103 x 101,3 x (t + 273) x 22,4

Short formula:

with:

Ilg nitric oxide per CigaretteCobs x N x P----~xFqx(t+273)

F = 35 x 273 x 30

103 x 101,3 x 22,4

0,01684 (1 cigarette)= 0,06736 (4 cigarettes)

0,13472 (8 cigarettes)

9 Electrophoresis

9.1 Protein and Enzyme Extraction

9.1.1 Equipment

CentrifugeMicrocentrifugeMortarPhotometerWater Yet Pump

63

FreezerSteam BathPetri DishesVacuum DesiccatorPaper Filter1,5 ml Microcentrifuge TubesPlatinum ConeDialysis Tube, 2,5 em in Diameter Serva Nr. 44144BeakersVolumetric Flasks

9.1.2 Buffers

Tris-Borate Buffer pH 8.9Mcilivaine Buffer, pH 2.8 (Merck)Glycine Buffer pH 2,8 (Merck)SDS (Sodium Dodecylsulfate)

9.1.3 Reagents

Albumin~-Mercaptoethanol

Polyethylene glycol 20000DithiothreitolEDTA (Ethylendiamine tetraacetic Acid, disodium salt)SucroseAcetone95% Ethanolcone. Phosphoric AcidSodium Sulfate free from waterSodium Hydroxide

9.1.4 Staining Salts

Bromophenol BlueCoomassie Brilliant Blue G 250 (C. I. 42655)

9.1.5 Sample Preparation

Stock Solution

0,1 M Mcilivaine Buffer, pH 2.8, 10 mM Dithiothreitol:

- make a mixture84,15 ml 0,1 M (21,0 g/I) Citric Acid * H2015,85 ml 0,2 M (35,6 g/I) Na2HP04 * 2 H200,15 g Dithiothreitol

Tris-Glycine Buffer, pH 8,3:

5 mM Tris (0,61 g/I)38,5 mM Glycine (2,89 g/I)10 mM Dithiothreitol (1,54 g/I)

64

Tobacco Seeds

- Weigh out 500 mg of tobacco seeds.

Grind in an on dry ice pre-chilled mortar.

Transfer with cold acetone (- 30°C, dried over Na2S04 water-free) to a 5 ml syringe tubeprepared at the bottom with a fast running paper filter.

Wash with cold acetone (- 30°C) until the filtrate is clear.

Dry the residue by aspiration with the aid of a water yet pump and store in a freezer(- 20°C) until use.

Make the solution:1 ml tris-borate buffer, pH 8,9, (see 9.2.5)2 g SDS2 g ~-mercaptoethanol

some crystals bromophenol blueaqua dest. to make to 100 ml

Add 1 ml of the solution to a 30 mg seed extract in a 1,5 ml microcentrifuge tubes.

Heat 15 minutes on a steam bath, centrifuge.

Add to an aliquot sucrose to a final concentration of 10% before applying 10 ilion theconcentration gel for electrophoresis (see 9.2.8).

Green Tobacco Leaves

- Cut in small pieces six frozen green tobacco leaves (harvested from the same parcel andstalk position) without midrib.

Grind with liquid nitrogen in a mortar until the leaf tissue becomes a fine powder.

Store over night in a freezer at - 20°C.

Pre-chill the mortar and pestle with dry ice.

Filtrate and wash with suction the tobacco powder through a filter paper (200 mm in di­ameter, Schleicher and SchOll 1460; with the aid of a platinum cone), wash twice with acold acetone-water mixture (- 30°C, 4:1 vlv) and three times with cold acetone (- 30°C).

Dry the residue by aspiration with the aid of a water yet pump, transfer to Petri dishes,dry in a vacuumdesiccator free from smell and store the sample in a freezer at - 20°C.

Homogenize in a cold mortar 50 g of the tobacco powder in 90 ml Mcilivaine Buffer0,1 M, pH 2.8, containing dithiothreitol 10 mM (1 mllg fresh tissue).

Centrifuge the mixture at 16.000 rpm, 4°C, for twenty minutes.

Filtrate the supernatant with a folded filter in a dialysis tube (50 cm in length, 0,21 cm indiameter, SERVA Nr. 44144, (cleaning procedure, see 9.1.6).

Dialyze for 4 to 5 hours against tris 5 mM-glycine 38,5 mM buffer pH 8,3, dithiothreitol1 0mM, to pH 4,3 - 4,5.

Let drip off the dialysis tube and cover several times with polyethylene glycol 20000,SERVA Nr. 33138, till a content of 2 ml.

Centrifuge at 1500 rpm.

Measure the protein content in the supernatant, - the 0,2 ml aliquot must have at least150 Ilg protein content -, (procedure, see 9.1.7) and store in a freezer at - 20°C.

Add to an aliquot sucrose to a final concentration of 10% and apply 10 fll on the con­centration gel.

65

Air-Dried and Milled Samples

- Pre-chill the mortar and pestle with dry ice.

- Weigh out 50 g of grounded leaf tissue.

- Grind with 100 ml cold acetone (- 30°C dried over Na2S04 free from water) until the to-bacco meal becomes a slurry. The mortar should be on dry ice.

- Transfer the tobacco slurry quantitatively in a beaker. Keep over night in a freezer(- 20°C).

- Transfer and filtrate with suction the tobacco slurry through a filter paper (200 mm indiameter, Schleicher and SchOll 1460; with the aid of a platinum cone), wash two timeswith a cold acetone-water mixture (- 30°C, 4:1 v/v) and three times with cold acetone(- 30°C).

- Dry the residue by aspiration with the aid of a water yet pump, transfer to Petri dishes,dry in a vacuumdesiccator free from smell and store the sample in a freezer at - 20°C.

- Homogenize in a cold mortar 10 g - 15 g of the tobacco powder in 50 - 70 ml McilivaineBuffer 0,1 M, pH 2.8, containing dithiothreitol 10 mM (1 ml/g fresh tissue).

- Centrifuge the mixture at 16.000 rpm, 4°C, for twenty minutes.

- Filtrate the supernatant with a folded filter in a dialysis tube (50 em in length, 0,21 em indiameter, Serva Nr. 44144) (cleaning procedure, see 9.1.6).

- Dialyze for 4 to 5 hours against tris 5 mM glycine 38,5 mM buffer pH 8,3, dithiothreitol1 0mM, to pH 4,3 - 4,5.

- Let drip off the dialysis tube and cover several times with polyethylene glycol 20000,SERVA Nr. 33138, till a content of 2 ml.

- Centrifuge at 1500 rpm.

- Measure the protein content in the supernatant, - the 0,2 ml aliquot must have at least150 /1g protein content - (procedure, see 9.1.7) and store in a freezer at - 20°C.

- Add to an aliquot sucrose to a final concentration of 10% and apply 10 ilion the con­centration gel.

9.1.6 Cleaning Procedure for the Dialysis Tube (make in advance)

Heat with stirring the dialysis tube for thirty minutes in EDTA 0,5 M, pH 7 - 9 (bring to sol­ution with cone. sodium hydroxide; the solution should be clear), decant, and wash eighttimes with aqua dest., using gloves against protein contamination.

9.1.7 Rapid Determination of the Protein Concentration51)

Blank Stock Solution (make four weeks before use):

100 mg Coomassie Brilliant Blue G 250 (C. I. 42655)50 ml 95% ethanol100 ml cone. phosphoric acidaqua dest. to make a final volume of 1000 mlfiltrate before use

66

Calibration

stock solution:50 mg albumin100 ml aqua dest.

aqua dest. to make a final volume of 1 ml:

1. volumetric flask: 0,2 ml containing 100 ~g albumin2. volumetric flask: 0,4 ml containing 200 ~g albumin3. volumetric flask: 0,6 ml containing 300 ~g albumin4. volumetric flask: 0,8 ml containing 400 ~g albumin5. volumetric flask: 1,0 ml containing 500 ~g albumin

Dilute 0,1 ml of every volumetric flask with 5 ml blank stock solution, measure after 2 min­utes the photometer extinction at a wavelength of 595 nm and make a standard curve:

1. volumetric flask: 0,1 ml containing 10 ~g albumin2. volumetric flask: 0,1 ml containing 20 ~g albumin3. volumetric flask: 0,1 ml containing 30 ~g albumin4. volumetric flask: 0,1 ml containing 40 ~g albumin5. volumetric flask: 0.1 ml containing 50 ~g albumin

Sample

Dilute 0,1 ml centrifuged supernatant of the protein dialyzed solution with 5 ml blank sol­ution, measure after 2 minutes the photometer extinction at a wavelength of 595 nm andestimate the protein content from the standard curve.

9.2 Vertical Polyacrylamide Slab Gel Electrophoresis

9.2.1 Equipment

Desaga Desaphor VA3000 Volt Power Supply DesatronicChamber and polymerization standCirculating Water Bath FrigostatDiffusions Destaining ApparatusStaining TraysBlotting PaperThermoplate SIlluminating BoxUltrasonic bathDesaga multiple syringe 1a ~I with 12 channelsMicrosyringes or MicropipetorsMicrotiter plateBeakersStir Bars and Stir Plate1.5 ml Microcentrifuge TubesMicrocentrifugeVortexSpatulasTimer

67

9.2.2 Buffers

Trls-Borate Buffer pH 7,1Tris-Borate Buffer pH 8,9Tris-HCI Buffer pH 6,7Tris-HCI Buffer pH 8,08Tris-HCI Buffer pH 8,9Tris-Glycine Buffer pH 9

9.2.3 Reagents

Tris (Tris(hydroxymethyl)-aminomethane)AcrylamideBisacrylamideSodium Phosphate (dibasic)Phosphoric AcidHydrochloric AcidSDS (Sodium Dodecylsulfate)TEMED (N, N, N', N'-Tetramethyl-ethylenediamine)Ammonium peroxidisulfate:SucroseGlacial Acetic AcidSodium HydroxideGlycineTrichloroacetic Acid2-Propanol

9.2.4 Staining Salts

RiboflavinCoomassie Brilliant Blue RBromophenol Blue

9.2.5 Stock Solutions

Simple Gels for Green, Dried and Milled Tobacco

Mix each of these solutions58) in a beaker using a magnetic stir bar.

Separation Gel:

Stock Solution TG 1 (toxic, avoid skin contact)38 9 acrylamide2 9 bisacrylamideaqua dest. to make a final volume of 100 ml

Stock Solution TG 218,15 9 trisaqua dest. to make a final volume of 90 mlcone HCI to titrate till pH 9,1 (about 2 ml)0,2 ml TEMEDaqua dest. to make a final volume of 100 ml

Stock Solution TG 3 (must be made daily)200 mg ammonium persulfateaqua dest. to make a final volume of 100 ml

68

Concentration Gel:

Stock Solution KG 1 (toxic, avoid skin contact)9,25 9 acrylamide0,75 9 bisacrylamideaqua dest. to make a final volume of 100 ml

Stock Solution KG 22,23 9 triscone H3P0 4 to titrate till pH 6,7 (about 1,1 ml)0,1 ml TEMEDaqua dest. to make a final volume of 100 ml

Stock Solution KG 32 mg riboflavin40 9 sucroseaqua dest. to make a final volume of 100 ml

Stock Solution KG 4 (must be made daily)100 mg ammonium persulfateaqua dest. to make a final volume of 100 ml

Stock Solution Upper Electrode Buffer (Cathode):tris-glycine pH 8.925,8 9 tris17,4 9 glycineaqua dest. to make a final volume of 5000 ml

Stock Solution Lower Electrode Buffer (Anode):tris-HCI pH 8,0872,5 9 trisaqua dest. to make a final volume of 4000 mlcone HCI to titrate till pH 8,1aqua dest. to make a final volume of 5000 ml

Stock solutions TG1, TG2, KG1, KG2 and KG3 may be made in advance and kept in brownbottles in the refrigerator when not in use.

Stock solutions Upper and Lower Electrode Buffer may be made in advance and brought to4°C.

Stock solutions TG 3 and KG 4 must be made daily.

Gradient Gels for the Separation of Tobacco Seed Proteins

Mix each of these solutions48) in a beaker using a magnetic stir bar.

Stock Solution ST1 (toxic, avoid skin contact)30 9 acrylamide0,8 9 bisacrylamideaqua dest. to make a final volume of 100 ml

Stock Solution St2: Separation gel tris - HCI Buffer pH 8.936,6 9 tris48 ml 1 N HCI to titrate till pH 8.9aqua dest. to make a final volume of 100 ml

Stock Solution St3: Concentration gel (and sample) tris-HCI- Buffer pH 6,73,54 9 NaH2P0 45,7 9 tris

69

25 ml 1 N HCI to titrate till pH 6,7aqua dest. to make a final volume of 100 ml

Stock Solution St410% SDS

Stock Solution St510% TEMED (wlv) in aqua dest.

Stock Solution St6 (must be made daily)10 % ammonium peroxidisulfate (wlv) in aqua dest.:0,5 9 ammonium persulfateaqua dest. to make a final volume of 5 ml

Stock Solution St7a: Electrode Buffer - tris (10 mM) - boric acid (200 mM) pH 7,16,055 9 tris4 I aqua dest.ca. 61,83 9 boric acid to titrate till pH 7,1aqua dest. to make a final volume of 5 Idilute 1 I with 2 I aqua dest. just before useadd 3 9 SDS

Stock Solution 7b: Electrode Buffer - tris (125 mM) - borate (19 mM) pH 8,975,69 9 trls4 I aqua dest.ca. 5,87 9 boric acid to titrate till pH 8,9aqua dest. to make a final volume of 5000 mldilute 1 I with 2 I just before use3g SDS

9.2.6 Simple Gels

Handling of the Desaphor VA (see Operating Instructions of the Co. Desaga57)

Chamber and Polymerization Stand (see operating instructions)

- Fill the lower buffer through with lower electrode buffer (5 I).

- Adjust on time the thermostat to the temperature of 5°C (native proteins).

Separation Cell (see operating instructions)

Gel Casting (see operating instructions)

Separation Gel (T=10%, C =5%; TG1 : TG2 : TG3 =1 : 1 : 2)Mix 12,5 ml TG1

12,5 ml TG2

- Degas for 2 min using an ultrasonic bath.

- Add the catalyst.

- Add 25,0 ml TG3.

- Mix and pour the gel liquid into the polymerization cuvette.

- Overlayer with 2 times 0,5 ml aqua dest. from each side and allow to polymerize for1 hour.

- Remove water layer after polymerization.

70

Concentration Gel (T= 2,5%, C = 7,5%; KG1 : KG2 : KG3 : KG4 = 1 : 1 : 1 : 1)Mix 2,5 ml KG1

2,5 ml KG22,5 ml KG32,5 ml KG4

- Degas for 2 min using an ultrasonic bath.

Pour the gel liquid on top of the polymerized separation gel and insert well former.

Allow to polymerize with UV-light.

Remove well former and rinse the wells with the upper electrode buffer.

9.2.7 SDS-Gradient Gels (T = 6 till 25%, C = 5%)

Handling of the Desaphor VA (see Operating Instructions of the Co. Desaga57)

Chamber and Polymerization Stand (see operating instructions)

- Fill the lower buffer through with electrode buffer St7a (pH 7.1, 5 I).

- Adjust on time the thermostat to the temperature of 17"C.

Separation Cell (see operating instructions)

Gel Casting (see operating instructions)

Solution A1, T = 6%Mix 5,1 ml St1

16,5 ml aqua dest.3,2 ml St20,25 ml St40,3 ml St51°III St6

Solution A2, T =7,5%Mix 6,33 ml St1

15,2 ml aqua dest.3,2 ml St20,3 ml St40,3 ml St510 III St6

Solution A3, T = 8%Mix 6,75 ml St1

14,8 ml aqua dest.3,2 ml St20,3 ml St40,3 ml St510 III St6

Solution A4, T = 10%Mix 8,5 ml St1

13,1 ml aqua dest.3,2 ml St20,3 ml St40,3 ml St51°III St6

71

Solution 81, T =20%Mix 16,9m1St1

4,7 ml aqua dest.3,2 ml St20,5 g sucrose0,3 ml St40,3 ml St51° 111 St6

Solution 82, T =25%Mix 21,2 ml St1

0,4 ml aqua dest.3,2 ml St20,5 g sucrose0,3 ml St40,3 ml St51° 111 St6

Concentration GelMix 3,22 ml St1

14,1 ml aqua dest.2,5 ml St30,2 ml St40,35 ml St53°111 St6

- Degas all gel solutions just before use.

Gel Solution Combinations used are:(for one 150 mm separation cell from every solution 30 ml)

A1 with 81A3 with 81A4 with 81A3 with 82

Concentration GelMix 3,22 ml St1

14,1 ml aqua dest.2,5 ml St30,2 ml St40,35 ml St530111 St6

- Place the gaskets onto the polymerization stand with the holes fitting over the tube con­nections for filling the cell with the gel solution.

Pump the solution of high concentration into the vessel with the low concentrated sol­ution, mix this continuously with a magnetic stirrer and pump simultaneously with twoother pump tubings the mixed solution into the cell.

Water layer and allow to polymerize for 1 hour.

Remove water layer.

Pour concentration gel and insert well former.

Allow to polymerize for 15 minutes.

Remove wellformers and drain by shaking or blotting wells with absorbent paper.

Replace the second cell by a blind cell in the case you are working with one gel only.

72

9.2.8 Application of Samples

- Place the gaskets with slids on the upper buffer trough.

Mount the separation cells - wells to the bottom - onto the upper buffer trough.

Fill the lower buffer trough with the anode buffer.

Place the upper buffer trough in oblique position into the chamber.

Avoid trapping air bubbles at the free ends of the gel.

Using discontinuous buffer system, close stopcock A.

Fill the trough with upper electrode buffer (ca. 1,0 I).

Remove any air bubbles from the sample wells with a buffer - filled small syringe.

Make a preliminary run of the gels without samples.

Switch on the buffer circulation for the lower buffer and stabilize the current on 50 mAper slab gel, limit the voltage on 500 V, run for ca. 0,5 h.

Give the concentrated fraction of the samples into the wells of a microtiter plate.

Add a bromophenol blue crystal and sucrose (10 %).

Use a well cleaned Desaga multiple syringe 10 III with 12 channels.

Apply 1aIII of each concentrated fraction in a well of the concentration gel.

Close the chamber with the lid and start the electrophoresis.

9.2.9 Operating the Electrophoresis

cathode = upper electrode

anode = lower electrode

- Adjust the power supply to the proper voltage and current.

Simple Gels

- Concentration gel: 500 V limit, 50 mA per gel slab.; ca 0,5 h until bromophenol blue hasreached the separation gel, than switch to 500 V, 100 mA for one separation gel.

Gradient-Gels

- Concentration gel: 500 V, 100 mA; 0,5 h until bromophenol blue has reached the separ­ation gel, than switch to 500 V, 150 mA (ca 2,5 h) for the separation gel.

- Stop the electrophoresis when the front of the tracking dye reaches the lower end of thecell (visible by the added dye bromophenol to the samples).

- Switch off the power supply.

- Take off the lid.

- Switch off the buffer circulation pump.

9.2.10 Removing the Gels (see operating instructions)

- Stain appropriately (see 9.4 - 9.8).

73

9.3 Isoelectric Focusing

9.3.1 Agarose Gels, pH 3 - 10 plus Spread at pH 3 - 7

Equipment

Desaga Desaphor HF Horizontal Electrophoresis Unit3000 Volt Power Supply DesatronicCirculating Water Bath FrigostatThird Electrode for Desaphor HFDiffusions Destaining ApparatusStaining TraysBlotting PaperPreCut IEF Electrode WicksThermoplate SIlluminating BoxSample Templates-Polyester 48 well (Desaga Nr. 150202)HyPure Gel (Desaga Nr. 150009)Microsyringes or MicropipetorsBeakersStir Bars and Stir Plate1.5 ml Microcentrifuge TubesMicrocentrifugeVortexSpatulasTimer

Chemicals

Glacial Acetic AcidSodium HydroxideGlycineTrichloroacetic Acid2-PropanolCoomassie Brilliant Blue R

9.3.2 Preparation of Solutions

Mix each of these solutions in a beaker using a magnetic stir bar.

Anolyte Solution

0.5 M Acetic Acid- Add 15 ml Glacial Acetic to 485 ml of distilled or deionized water.

Catholyte Solution

0.5 M Sodium Hydroxide- Add 10 9 Sodium Hydroxide to a beaker.- Bring volume up to 500 ml with distilled or deionized water.- Stir until Sodium Hydroxide is dissolved.

74

Fixative Solution

- Add 40 g Trichloroacetic Acid to a beaker.- Bring volume up to 200 ml with aqua dest.- Stir until Trichloroacetic Acid has dissolved.

Wash Solution

- Add 5.0 g Trichloroacetic Acid to a beaker.- Bring volume up to 1 I with aqua dest.- Stir until Trichloroacetic Acid has dissolved.

Brilliant Blue R-250 Stain

- Add 350 ml 2-Propanol and 80 ml Glacial Acetic Acid to 570 ml distilled or deionizedwater.

- To this solution add 1.0 g Brilliant Blue R-250.- Stir the solution at least 30 minutes to dissolve.

Glycine Extraction Solution

2% Glycine- Add 2 g Glycine to 100 ml distilled or deionized water.- Gently stir to mix.- Adjust the pH to 8.6 with 6 M Sodium Hydroxide.

9.3.3 Sample Preparation (see 9.1)

9.3.4 Operating the Isoelectric Focusing

- Turn on circulating water bath and set temperatures to 15°C, 15 minutes before the run.

Clean the cooling plate with water and towel dry.

Remove a HyPure Gel from its package. Carefully remove the protective cover sheetfrom the gel surface. Do not touch the gel or use it if it is torn or dried. Cut the bottomright-hand corner of the gel; this corner will be used as a reference point.

Pipette 3 ml of water onto the center of the Desaphor HF cooling plate.

Hold the gel by its diagonal corners to form a parabola.

Place the bottom of the parabola on the bead of water.

Distribute slowly the bead of water by using the backing of the gel (be careful not to trapair bubbles between the gel and cooling plate).

Center the gel on the Desaphor HF cooling plate.

Thoroughly blot any excess water from the periphery of the gel using a paper towel.

Place a piece of Blotting Paper evenly on the gel. Smooth the paper lightiy with finger­tips.

After five seconds, gently remove the blotting paper.

Prepare three PreCut IEF Wicks. (Twoof the wicks will be used as anode wicks, one willbe used as the cathode wick).

Place the two "anode" wicks, rough-side down on several white paper towels. (NOTE:Brown or colored paper towels may exude a dye into the electrode wick. Use whitepaper towels only)

75

- Evenly saturate each wick with 4 - 5 ml of Anolyte Solution. Gently blot the wicks to ridexcess fluid. The wicks should just begin to appear dry before being placed on the gel.

- Place each anode wick onto the edges of the gel. Run a finger along the entire length ofthe wick to ensure even contact between the wick and gel. Refer to the Alignment Dia­gram for proper placement of the wicks.

- Wash hands of residual Anolyte Solution.

- Place the remaining wick, rough-side-down, on several white paper towels.

- Evenly saturate the wick with 4 - 5 ml of Catholyte Solution.

- Gently blot the wick with another paper towel to rid any excess fluid (wick should justbegin to appear dry).

- Place the wick along the center of the gel between the two anode wicks.

- Make sure all the wicks are parallel to each other.

- Run a finger along the entire length of the wick to ensure even contact between the wickand gel.

- Position the templates 1 cm from the anodes.

- Apply 10 III of the sample to each sample well.

- Place the metal spring from the electrodes from top onto the electrode ledge and adjustthe electrodes onto the middle of the corresponding electrode strips.

- Center the electrodes evenly over the wicks.

- Load the three electrodes with the two 4 mm thick glass plates delivered.

- Slide the lid onto the chamber and lock it by turning the locking knob 90 degrees clock-wise.

- Connect the plugs with the outlet sockets of the power supply (the red plug with the redoutlet socket, the black plug with the black one).

- Switch on the main switch - power knob -.

- Press the key N-SET for stabilizing the power and turn the knob above until the displayshows 40 Watts.

- The voltage is limited at 1500 V, the current is set at a value of about 60 mA.

- Push the key ON/OFF for starting the separation.

- Run the gel for 60 minutes at 40 Watts.

- Remove the application strips and dry the surface of the agarose gel from exudation orcondense water with a thin sheet of filter paper.

- Focus the gel for further 60 minutes at 40 Watts constant.

- When the electrophoresis run is complete, turn the power supply off.

- Remove the Desaphor HF safety cover, the glass plates and electrodes.

- Remove the wicks from the gel's surface.

9.3.5 Staining of Samples (see also 9.5)

- Place the gel into a staining tray and pour on 200 ml of Fixative Solution.

- Rock or agitate the gel for 20 minutes.

- After the bands have been permanently fixed, discard the Fixative Solution.

76

- Wash the gel for one hour in 500 ml of Wash Solution.

- Discard the Wash Solution and repeat this wash step with 500 ml of fresh Wash Solution.

- Allow gel to dry completely by placing the gel on a flat surface overnight at room tem-perature (15°C - 30°C) or by placing it onto a gel dryer (Thermoplate S) at 60°C for twohours. The gel is dry when an even sheen appears across the gel's surface.

- When the gel is dry, place the gel in a staining tray, agarose side up.

- Cover the gel with 200 ml of Brilliant Blue R-250 Stain.

- Rock the gel gently for 30 - 60 minutes or until bands reach desired intensities. Discardstain. (Destaining is usually not necessary when using this stain.)

- Rinse the gel 10 minutes in distilled or deionized water.

- Air dry the gel.

- The gel may now be analyzed.

9.4 Protein and Enzyme Staining Procedures

9.4.1 Unspecific staining of proteins

Fixation

Solution:Mix 54 g trichhloroacetic acid

136 ml methanol340 ml aqua dest.16 g sulfosallcyllc acid (if necessary)

- Shake the gel for 0,5 h.

- Wash with 10% acetic acid for 10 min.

Standard Staining

Staining Solution:Mix 100 rnl Fixative Solution

2.5 ml 1% Coomassie Brilliant Blue R 250(Manifold use is possible.)

- Shake the gel for 3 h (or over night).

Destaining Solution

Stock Solution:Mix 63 ml 96 % acetic acid

375 ml methanol562 ml aqua dest.(For repeated use purify with charcoal, 1 g / 100 ml.)

- Shake for 3 h (or over night).

- Changing the bath several times.

Stock Solution (Modification)Mix 200 ml methanol

100 ml acetic acidaqua dest. to make to 1000 ml

77

9.4.2 Rapid Staining for Native Gels and IEF-Gels60)

Staining Solution

Mix 40 ml 0,3 % Coomassie Brilliant Blue G 250 in aqua dest. (wlv)15 ml cone. perchloric acid

- Let it stand for 10 min.

- Add 250 ml aqua dest.(Manifold use is possible.)

Destaining and Preserving Solution

Mix 100 ml cone. acetic acid20 ml glycerinaqua dest. to make to 1000 ml

9.4.3 Silver Staining61)

Fixative Solution

10% trichhloroacetic acid

- Shake for 0.5 h.

Dithiothreitol Treatment

Mix 4 mg dithiothreitol40 ml ethanol10 ml acetic acid150 ml aqua dest.

- Shake for 0.5 h.

Background Brightening Up

- Oxidize with 0,5 % K2Cr207 or 0.1 % H202 for 5 min.

- Wash with aqua dest. for 5 min.

Silver nitrate Impregnation

- Stain with 200 rnl O, 1% silver nitrate for 10 min.

- Wash with aqua dest. for 1 min.

Paraformaldehyde Treatment

Mix 6 g sodium carbonate40 mg p-formaldehydeaqua dest. to make a final volume of 200 ml

- Shake for 5 till 7 min (controlling the development on an illuminating box).

- Stop the development with 1% acetic acid.

Impregnation Solution

Mix 50 ml methanol10 ml glycerin40 ml aqua dest.

- Shake for 1 hour.

78

9.4.4. Destaining and Drying of Polyacrylamide Slab Gels

- Shake the gel in a solution of 10% acetic acid and 5% glycerin for 1 hour.

Cut 2 sheets of cellophane film (a little bit bigger than the gel and the supporting glassplate).

Swell them in 5% glycerin-aqua dest. mixture for 30 min.

Span the one sheet over a 4 mm thick glass plate.

Put the gel on it.

Span the other sheet on the gel and the glass plate.

Remove the air bubbles.

Turn the edges of the sheets around the glass plate.

Dryat room temperature on a place without draft.

9.4.5 Specific Staining of Enzymes62)

Peroxidase - PER

Staining Solution:Mix 90 mg benzidine

100 ml ethanol66 ml 3% hydrogen peroxide10 ml glacial acetic acidaqua dest. to make to 200 ml

- Shake until the bands appear (45 till 60 sec).

- Transfer gel to 7% acetic acid for 5 min.

- Rinse and hold in aqua dest.

Esterase - EST

Staining Solution:Mix 0.1 g Fast Blue RR

10 ml 0,2 M NaH2P04 * H20 (pH 4,6)50 ml 0,2 M Na2HP04 * H20 (pH 8,8)Prepare and add shortly before use the stock solution:3 ml1 % a, ~-naphthylacetate in 70% acetone-aqua dest. solution(Make first a solution in acetone and then aqua dest. to make to 100 mI.)

- Shake at room temperature until the bands appear (1.0 h).

Malate Dehydrogenase - MDH

Staining Solution:Mix 0,61 g tris (0,05 M)

2,1 g malic acid (0,2 M)

- Add 70 ml aqua dest.

- Adjust pH to 7 with 10% sodium hydroxide (ca 9 ml)

- Add 66 mg diphosphopyridine nucleotide (0,001 M)5 mg phenazine methosulfate (0,000136 M)35 mg p-nitroblue tetrazolium (0,00043 M)aqua dest. to make to 100 ml

79

- Strain through glass wool before to use.

- Shake gel in the dark at 3rC until bands develop.

- Wash and fix in 2% acetic acid.

9.5 Enzyme Staining Procedures of Agarose Gels59)

9.5.1 Chemicals

Buffers

Tris (Tris (hydroxymethyl)-aminomethane)Sodium Phosphate (monobasic)Sodium Phosphate (dibasic)Sodium CitrateSodium Acetate

Staining Salts, Co-Factors

Fast Blue RR SaltOrtho-DianisidineMMT, (3-[4, 5-Dimethyl-2-thiazolyl]-2, 5 - diphenyl-2 H-tetrazolium bromide)Magnesium ChlorideAND (P-Nicotinamide adenine dinucleotide, reduced form)NBT (Nitro Blue Tetrazolium)PMS (Phenazine Methosulfate)

Subtrates

a-Naphthyl AcetateDL-Malic AcidDL-Isocitric Acid

Reagents

MethanolEthanolHydrogen Peroxide 30%Glacial Acetic AcidHydrochloric AcidSodium ChlorideSodium HydroxideAcetone

9.5.2 Preparation of Enzyme Buffers

Mix each of these enzyme buffers in a beaker using a magnetic stir bar.

Stop Solution

Add 80 ml glacial acetic acid and 250 ml ethanol to 670 ml distilled or deionized water.

0.1 M Tris-HCI (pH 8.5), Buffer Solution

- Add 12.1 g of Tris to 900 ml of distilled or deionized water.

- Titrate with concentrated HCI to adjust solution to pH 8.5.

80

- Dilute with distilled or deionized water to 1000 ml.

0.6 M Sodium Phosphate (pH 6. 1), Buffer Solution

- Add 71.9 g sodium phosphate (monobasic) to 900 ml of distilled or deionized water.

- Titrate with 6.0 M NaOH to adjust solution to pH 6.1.

- Dilute to 1000 ml with aqua dest.

0.5 M Citrate Buffer (pH 4.4) - used in Peroxidase Stain

- Add 14.7 g sodium citrate to 90 ml of aqua dest.

- Titrate with concentrated HCI to adjust solution to pH 4.4.

- Dilute to 100 ml aqua dest.

Ortho-Dianisidine - used in Peroxidase Stain

- Add 2.75 g of o-dianisidine to 500 ml of methanol that has a concentration greater than90%.Caution: Avoid skin contact, may be carcinogenic.

This solution will keep for 9 months at room temperature.

6 M Sodium Hydroxide - used to Titrate

- Add 24 g sodium hydroxide to 90 ml of aqua dest., stir until dissolved.

- Dilute to 100 ml with aqua dest.

9.5.3 Transfer Membrane Preparation Solutions

Impregnating Solution

200 ml Methanol > 90%

Equilibration Solution

2,4 g Tris

29,0 g Sodium chloride

- To make with aqua dest. to 900 ml.

- To titrate with cone. hydrochloric acid to pH 7,5.

- To make with aqua dest. to 1000 ml.

This solution will keep for 1 month at room temperature.

9.5.4 Stain Solutions

Peroxidase - PER

To prepare stain:20 ml 0 - dianisidine buffer10 ml citrate buffer pH 4.470 ml aqua dest.

Just before staining add:5 ml 3% hydrogen peroxide

Staining is complete in 30 - 40 minutes or until bands reach strong intensities.

81

Esterase - EST

To prepare stain:

Solution I80 ml 0.6 M phosphate buffer pH 6.180 mg Fast Blue RR Salt

Just before staining add solution II:

Solution IIThis can be made up 15 minutes before use.10 ml 50% Acetone80 mg a-naphthyl acetate

Staining is complete in 60 - 90 minutes or until bands reach strong intensities.

Malate Dehydrogenase - MDH

To prepare stain:

- In a 100 ml beaker with a stir bar add:50 ml 0.1 M tris HCL pH 8.5150 mg DL-malic acid

- Stir until the DL-malic acid is dissolved.

- Re-adjust the pH to 9.2 using 6 M NaOH added dropwise.

Just before staining add:

20 mgAND10 mg MIT5 mg PMS

- Allow the staining to continue for 45 - 60 minutes or until the bands reach strong inten­sities.

9.5.5 Enzyme Staining

The Chemicals and steps listed in the first area of the section (To prepare stain) can becompleted during the electrophoretic run. The chemicals and steps listed in the secondarea (Just before staining) should be added only 3 - 4 minutes before the stains use. Therecommended time interval for proper staining is provided. The following points are to aidin achieving optimal results.

- The formulas are measured for use on transfer membranes. To stain a gel, double thevalues are given.

- Stir until all chemicals are dissolved.

- It is helpful to rock or agitate the gel while staining.

- Remove any air bubbles by gently lifting the gel corners with a spatula.

- To stop the enzymatic reaction rinse the gel with distilled or deionized water. Place in75 - 100 ml stop solution for 3 minutes. Do Not use the stop solution on Transfer mem­branes. To stop staining of transfer membranes simply rinse with distilled or deionizedwater.

- Transfer membranes fade upon drying. To avoid this, keep them in water until scoring iscompleted.

82

- Score the bands immediately. If the gel cannot be scored immediately, a photograph ofthe banding patterns is recommended.

- Diagnostic banding patterns can be observed anywhere in the 8 em inter-electrodespace.

- After the gel has been scored place the gel in 500 ml distilled or deionized water for onehour. The gel can then be air dried for permanent storage.

10 SummarySeventeen German and five Greek cultivars from the harvest 1987 and twenty-five GermanGeudertheimer cultivars from the harvest 1990 were analyzed according to nitrogen ferti­lization. New breeding lines as well as common types from oriental, cigar, Burley and Virgintypes investigated for smoke and leaf constituents: condensate, nicotine, nitric oxide andcarbon monoxide in smoke; nitrate, alkaloids, sugar, and nitrogen in leaf.

In the autumn 1990 and spring 1991 one hundred and eight soil samples were taken fromfive tobacco experimental fields in the northern and the middle part of Greece, in order tofind out their fitting for tobacco plantation or possible nutrition deficiencies.

The limitation to 12 mg condensate per cigarette, asked by the European Commission for1998, can only be fulfilled with the lower stalk positions of the cultivars. Nicotine is chang­ing according to the tobacco types. Carbon monoxide was found in a middle range equalfor all types. Nitric oxide changes according to the nitrogen fertilization and the cultivars.The leaf constituents nitrate, nicotine and the sum of nornicotine and myosmine correlatewith cultivar, climate and fertilization. The nitrosamines in the tobacco and tobacco smokevary with the nitrate content in the cultivars, the harvesting and curing conditions.

The correlation among nitrate in the tobacco leaves, nitric oxide and carbon monoxide inthe mainstream smoke, nitrosamines in the leaves and in the mainstream smoke wereexamined in every prime in the Greek breeding lines 5/R, 1/B, 2/K, 3/S and 4N.

The rates of carbon monoxide increase in every prime in all tobacco breeding lines exceptfor 4N breeding line, in which an irregularity in the rates is observed. Breeding lines withhigh nitrate percentage (5/R), there are high percentage in nitric oxide and nitrosamines. Acorresponding decrease of nitrate and nitric oxide is found out in every prime in the breed­ing lines 1/B, 3/S and 5/R and a corresponding increase in every prime in 2/K and 4N. In 51R breeding line is observed the highest percentage of nitrosamines both in tobacco leavesand in the mainstream smoke. In 4N, 2/K and 3/S no nitrosamines are observed, while 1/Bin a middle position is.

Twenty-five breeding lines of dark air cured tobacco from the harvest 1990 were identifiedwith an electrophoretic method according to their protein and isoenzyme patterns.

The genuineness of different dark or light tobaccos was examined in protein bands of thetobacco seeds of two crops whose harvest had a difference of two years. They wereproved to be genuine because of the identical protein bands with an exception of Geuder­theimer III and Badischer Burley E.

Varieties resistant to diseases from varieties not resistant could be identified. So the resis­tant varieties Bel 10, Burlina 183, Bursanica 217, Perega and Pereg 234 appear to have oneprotein band in Rf-rate 0,6.

Individual varieties or relative breeding lines in dark and light tobaccos could be identifiedbecause of protein and isoenzym bands in the green leaf in the prime "tip". The varietiesBadischer Burley E, Burlina '83 and Bursanica 217 could be identified only by their protein

83

bands. The breeding lines B 520, B 21, Pereco, B 535 and B 535 Bursa could be identifiedin relation to protein bands and peroxidase bands.

The identification of different tobacco varieties based on protein and isoenzym bands wasexamined in dry and ground tobacco samples.

All the examined varieties were taken from the prime "leaf". They were coming from experi­mental fields of the Landesanstalt fur Pflanzenbau Forchheim. They could be identifiedfrom their protein bands.

The question about the genuineness of "f-taler" varieties from 1986 compared with theones of 1978, which comes from the experimental cultivation could be examined in dry to­bacco. It was found out that they are genetically different tobaccos.

Despite of enzymatic and oxidant decomposition process, - during the drying of tobaccoand its storage -, the isoenzymes and protein bands could be identified in tobacco var­ieties in the dry material.

The isoenzymes and protein bands in the green leaf could be identified from the ones of thedry material when it comes to their number and their position. The few bands in the dry ma­terial were characterised by a loss of peroxidase bands in the isozyme patterns.

Based on our findings, it is feasible that electrophoretic procedures will become a practicalmeaning for cultivar identification in nicotiana tabacum. With the quality analysis in tobaccoand tobacco smoke an identity-card for old and new breeding lines of cigar, Burley and Vir­gin tobacco can be made.

84

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52) G6rg, Angelika (1973): "The Use of Disc-Electrophoresis and Isoelectric Focusing ofPolyacrylamide Gels for Characterizing of Plant Proteins and Enzymes and Influence ofGenetic and Ecologic Factors", Thesis, Technical University of MOnchen, Faculty ofBrewery and Food Technology, 0-85354 Freising-Weihenstephan, Germany, p. 116 ­117 (G)*

87

53) Dellncee, H. and Range, P.(1979): "Development of an Analytical Method for the Deter­mination of Protein Degradation in Tobacco": Der Deutsche Tabakbau 59 (24), p. 320­321 (G)*

54) Range, P., Fisher, Elisabeth and Muller, Maria, unpublished

55) Conklin, M. E. and Smith, H. H.(1969): "Effects of Fast Neutron Versus X-Irradiation onDevelopment, Differentiation and Peroxidase Isozymes in a genetically Tumorous Ni­cotiana Amphiploid and its Parents Int. J. Radiat. BioI. 16 (4), p. 311 - 321; Rucker, Wal­traud and Radola, B. J. (1971): "Isoelectric Patterns of Peroxidase Isoenzymes from To­bacco Tissue Cultures, Planta (Berl.) 99, p. 192 - 198; Sheen, S. J. (1972): "IsozymicEvidence Bearing on the Origin of Nicotiana Tabacum", Evolution 26, p. 143 - 154;Ham, Changyawl, Cheong YeoI Sohn, and Yung Jin Kim, (1975): "Studies on the Elec­trophoretic Variation in TetrazoliumOxidase Isozyme of Nicotiana Species": KoreanJour. Botany 18 (4), p. 150 -154; Coussirat, J. A (1983/84): "Induction of Soluble (b)Proteins by Peronospora tabacinia A. in Tobaccos Treated with Polyacrylic Acid andFungicides and in Untreated Tobaccos", A du Tabac-Sect. 2 18, p. 107 - 122; Abet, M.,Piro, F. and Tonini, A(1985): Ann. Inst. Exp. Tob. Scafati X, p. 55 - 60; Wilkinson, C. A,Mulchi, C. L. and Aycock, M. K., Jr.(1985): "Polyacrylamide Gel Electrophoresis for cui­tivar Identification in Tobacco", Crop Science 25, p. 971 - 974

56) Dellncee, H. (1986): "Advances in Isoelectric Focusing of Proteins", Izotoptechnika 29(1 - 2), p. 1 - 26; Westermeier, R. (1990): "Electrophoresis Practical Course", VCH Ver­lagsgesellschaft mbH, Weinheim (G)*; McDonald, M. B., Jr. (1991): "Blotting of SeedProteins from Isoelectrically Focused Gels for Cultivar Identification", Seed Sci. &Technol., 19, p. 33 - 40

57) Desaga GmbH, P.O.B. 101969, 0-690099 Heidelberg

58) Altland, K., Hackler, R. and Knoche, W. (1980): "Double One-Dimensional Electro­phoresis of Human Serum Transferrin: A New High-Resolution Screening Method forGenetically Determined Variation", Hum. Gen. 54, p. 221 - 231; Altland, K., Rauh, S.and Hackler, R. (1981): "Demonstration of Human Prealbumin by Double One-Dimen­sional Slab Gel Electrophoresis", Electrophoresis 2, p. 148 - 155

59) Black, J., During, Linda (1989): "Procedure for the Identification of Corn Varieties usingIsoelectric Focusing and Native Blotting", in: "Isolab inc., Innovative BiochemicalMethodology", Drawer 4350, Akron, Ohio USA 44321

60) Desaga Lab-Report Nr. 2511 (08. 05. 1981), Desaga GmbH, P.O.B. 101969, 0-690099Heidelberg)

61) Hempelmann, E., Zwanzig, M. and MUlIer, Maria (1987): "Additionally Investigations tothe Mechanism of the Polychromatic Silver Staining", Elektrophorese Forum '87", edi­tor B. J. Radola, Institute of Brewery and Analytical Chemistry, Technical University ofMunchen, 0-85354 Freising-Weihenstephan, p. 385 - 388 (G)*

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* only in German language available

88

previously published in this series

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Osnovi nauke 0 materijalima(neue Werkstoffe)by G. Ondracek and I. StarnenkovlcGERMAN-YUGOSLAV COOPERATIONISBN 3-89336-036-0

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12 Reference Materials and Methods in Environmental and Biological ResearchM. Rossbach (1992)GERMAN-SLOVENIAN COOPERATION

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15 Analysis Methods and Techniques for Hard Thin Layer Coatings Characterization ­in particular on TItanium Nitride -B. Bliznakovska, M. Milosevskiin cooperation with S. Krawczynski, Ch. Meixner, H.-A. Kotter (1993)GERMAN COOPERATION with the University "Kiril i Metodij", Skopje

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17 CVD - Main Concepts, Applications and RestrictionsB. Bliznakovska, M. Milosevsklin cooperation with S. Krawczynski, Ch. Meixner, H.-A. Kotter (1993)GERMAN COOPERATION with the University "Sv. Kiril i Metodij", Skopje

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20 A Transect from a Tectonic Melange to an Island-Arc in thePan-African of SE Egypt (Wadi Ghadir Area)Hani EI-Akhal (1993)GERMAN-EGYPTIAN COOPERATION

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22 Experimental Design and AnalysisA. Kiprijanova, L. Markovska in cooperation with S. Krawczynski (1993)GERMAN COOPERATION with the University "Sv. Kiril i Metodij", Skopje

23 Progress in Diamond and Diamondlike Coatings ProcessingB. Bliznakovska, Ch. Meixner in cooperation with S. Krawczynski (1994)GERMAN COOPERATION with the University "Sv. Kiril i Metodij", Skopje

Scientific Series of the International Bureau

24 Reference Materials on the Project Improvement of Transmission Capabilityedited by J. Krawczynski, D. Povh (1994)GERMAN-SLOVENIAN-COOPERATION

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ISBN 3-89336-145-6