~LN~i~i~l~i ior Im_lrovernent of the lobacco_4lant (I~paler to be presented CI'·~.L. Heard at the Tobaece Exhibition, Beijinp 23. September 'Bi introduction It is appropriate to begin by identifvins in what area improvements to the tobacco plant ate sought. There are at least three broad factions for uham the tobacco plant tas importance · the farmer, the cigarette manufacturer and the consumer. %itherto the main focus of tobacco development has been agronomic - considerations of leaf yield and basic qualities have do~inared. It will be of increasing importance in the future development of tobacco cultira:s that the plpui~ of tobacco is Seen in the context of its ultimate use, that is for the production of ciga:ettes~ Consequentle if ve look to the future imprD\'eruents in tobacco thev mas be aeronPnicalll Larlerted, (3fiering benefits largely to the prover) but ve Can also ~xpec: to see developments that offer improved processing by the cigarette manufacturer PI improved products for the smoker, These 'dovnstream' attributes are recognised as the consequence elf groaing, harvesting, curing practice, leaf maturation and blending of tobacco. In this respect we must acknowledge that any attempts to achieve modifications in tobacco icai genetics that result in downstream improvements Fill present a big challenge. There is a considerable body of empirical evidence to suggest that different tobacco caltirarl~ypes have substantial effects on the end product ~ualilies. from ttis position it vould seen reasonable to assume that smoke chemistry is substantially influenced by leaf chemistry which in turn Is a function of the plants genetic composition, Naturally the process is further complicated by the influence of environmental factors on genetic expression, although even the ability of a plant to respond to its environment is geneticail~ based, The techniques of plant biotechnology provide a series of enabling technologies that generate the potential for plant improvement at the genetic level, and as tobacco plant genetics seens to underpin most aspects of tobacco leaf and product qualities we can examine the potential to applr these techniques for specific improvement of tobacco. U1 BATCo document for Legal Services : Health Canada 21 May 1999 ___._ - 2 · ~ For ease of presentation and discussion, the technologies for 3lant Improvement can be summarized under i primary headings: 1) plant breebi~ 21 cellular and tissue culture technologies 3) specific genetic engineering b) molecular technologies; gene Lapping j) Symbiotic soil micro-organism interaction. Collectively,:he first four broad areas constitute the basis oi plant bio:echnolog~. They should be considered as an integrated rhsie and not necessarily as discrete or isolated processes for plant impro~·enent~ For instance ii is inconceivable today that use of the sopbis~iea~r~ technologies of 2 - 4 could result in a commercially useful isnovation ~lthOUt recourse to 1, conventional plant breeding, Indeed as a note of caution it should be stressed that investment in the sophisticated modern blotechnoioeical techniques is expensive and that there exists a danger that scientists uill turn to these techniques in situations u~ere further thought might have shoan that less glamorous traditional approaches are appropriate, In addition to the conventionally accepted areas of b:otechnolog~ k'ithin the center; of plants there is an area of !ncreasii~ importance, that of symbiotic soil micro-organisms and their interaction beti;een plan: and rhi2Dsphe:e for efficient uptake of trace nu::ients vill also be discussed, Plant breeding f9rlohacf6 improvement;~_currenr PD~rion Plant breeding is the established, conventional science used to great eifect throughout hlsr.Drv for the improvelpent of ~8;iculiuraiir important: crops. :he process relies on the natural combination and segregation of genetic material and the selection of plants vit~ desirable a ibutes that occurs during sexual reproduction of plants. T~o ::e~tlcncy k.ith vhiCh S[lrm commercially significant impror~mat accurs, particularly in Puitipenie traits, often necessitates the use of large PopFiation studies, alFays arseing that the plant trait can be improvel Fithin the scope of natural Liological variability vithin a species, The process can be further complicated aherr selection criteria cannot be applied or identified at an early stage of the plants development as is the case vith some pature plant attributes e.g, irui; colour, rile, post-harvest physiology etc, The situation is further complicated for too~cco vhe:e lome traits can onl) be asJe55ed MloFing the mulrisraee processing of tobacco for product manfufacture, under these eircuotstar,ces, the cost and tipescale~ for improvements in penoticaliy based product attributes becPses prohibitively large and expensive. 3 BATCo document for legal Services : Health Canada 21 Yay 1999 -]· in additi3n to the classical breeding techniques of selection,:ne in-breeding or plant trait improvement and stab:iizarion is a proven if slou process for plant developnent Fithin the context of sexuai compatabi!i~y bervecn varieties and current Ivallani:itv in aa:P:e of traits that we Fish to introduce in a given variety. The male; restriction or the use and application of plant breeding in ai; crops ;s the length of t~me it ta~es to achieve the development and release of a neF commt:clai cuitivar: for tobacco this woull take in the c!al! Of i . 10 rears, The primary application of plant breeding for tobacco improvement has been for the de~eiPpnent of apronomic improvement, due in parr to the deiine~ needs of :he g:overiindustry and to the most practical use pi t!is technology. There are eminent tobacco research institutes throughout the ~orld that have pioneered the development and improvement of tobacco cul::rars, tie most notaoie being i; C State University, CSA, Delhi, Canada and Ltsaga, lilbabve. ~he cont:ibation of the CS land grant universities :n appli'lng Lre~dinp effort for the imptovement of Burley and Virginia tobacco, has been suts;anding. They are responsible for most commercial cui:ivarr In use ~oda!'. The major goals of these Institute' funded programmes for the reasons outilned iia~e beer, to improve rield and perfor~ance of commercial eultivars ~ith the major emphasis being in terms of agronPmic improvement particuiar:~ disease tolerance and resistance, In addition there are tse issues tbar Influence the development of neu tobacco cultivars: (il ;uclion floor purchaslr$, together vith intervention Support by :oserment tends to impose constraints a the introda:ior; of Jo~lel tobacco cultivars (particularly those serving the interests oi a specific end-user), (ii) 'Ihtre is a sense of reduction in Government support fo: tobacco research for plant improvement, There Would appear to be a need therefore for greater industry initiative In the development of future tobacco breeding programmes, if the full potential for product improvement is to be achieved through concenticlnal and sophisticated use of technologies, BATCo document for Legal Services : Health Canada 21 May 1999 -I- For the nev technologies to impact on conventional plant breeding prol(rammes and strategies in the development of ne~ cultivars they must: 1) p!p~~ide the basis for reduced timescales for plant selection and trait incorporation 2) overcome sexual barriers and current natural limitations 3) provide a basis for selection criteria for plant, ieai and product qualities preferably at the seedling stage, $) create novel and directed plant trait combinations 51 provide a basis for highly specific modifications of plant processes to achieve defined changes in both leaf and product, Tobacco as i made! plant for biotechnolagy Oo other plant has Seen used so extensively as tobacco in pioneering the development of plant bioreehnologies in fields of cellular, molecular and genetic engineering technirlues. There is an extensive literature documenting its use as a model plant in the development of methodologies and processes (1). The primary reason uhy tobacco has developed as a model plan: is the ease I;ifh k'hiCh it vill grow in a variety of culture environments ee micropropagation, as single cells, as protoplasts and in anther culture (li. These properties of rapid propagation, ability to form cellular and nuclear fusion products and the formation of 'instant in breds' from anther culture have all contributed to its extensive use. The ease with which tobacco can be regenerated from a single cell to form whole plants together with the availability of a naturally occurring cell transformation Svsiea (~robacrtri~p Tumefaciens) has greatly facilitated the development bi ccllullr t:ansforn~ation generally and oII! underrtandinl Of the molecular basis of genetic engineering today (2), In light of the foregoing, it pay be of considerable surprise to hear that almost without exception there has been little or no attempt to utilite these technologies by the Tobacco Industry for commercial benefit, The limited exception is the reported use of anther culture deri\'ed plants in the development of a Chinese cultivar, Tan-Yu (3), Of the two major vorld successes in the area of genetically engineered plants, notably herbicide resistance (O~ and 93 gene transfer (j) both ~e:e achieved initially using tobacco, In neither case did the companies concerned appear to have a defined commercial goal for tobacco in mind. These innovations may have relevance corPmerciall~ where weed control is a major problem for tobacco or indeed There crop damage from lepidoptera h insects occurs. Today it appears that these genetic transformation O processes, although stable from generation to generation, have not been O introduced into commercial tobacco cuitivars by the tobacco seed 03 slppliers. lit may veil be the case, of course, that this work is being U1 progressed In the undisclosed breeding plans of these companies). tn 't` BATCo document for Legal Senriees : Health Canada 21 May 1999 ·j- i~ SPecific hppiication oi Plant~Giotechnolosr to_~l~!eaccrl~era\·ement i. Plant Breeding The role of conventional plant breeding is taiien as implicit in the development of new ccl~ivars using new technologies. The key and central issue is the development of the reiationship and understanding of genetics and its implications for tobacco growing, C~I;1 leaf, tobacco blends (iilier and flavour grades) and product ~lalitr, ?, Cellular and T~ssue Cu!:urr Technologies a) Tissue culture propagation HjClOpropagatior,, the technique whereby apical or lateral bud or ecen other plant structures can be srerilised and aseptically 8rou on a culture medium has had c3nsiderahie commercial impact in some areas of agriculture and iloricuiture, its primary benefits are: ii) clonal propaga~;on of plants, particularly applicable for the maintenance and mass propagation of elite genorypes iv vegetative propagation. I: is most frequently applied where maintenance of an individual plant's specific characteristics are required (usually identified from a segregating plant population). To dare it has been applied successfully to a range of plants e,g. orchids, carnations, roses, ornamental plants, pineapple, ginger, pepper, roocstPds etc. (ii) The other priPar~ application of this form of mass propagation is for the eradication of plant diseases. Where apical and lateral bud meristems ate excised land suitably treated e.g, heati and cultured any residcal or latent viruses present in:he plant can be eradicated ~irh significant improvement in plant vigour and performance (e.g~ Field or fruit quality). This approach has been applied to many plant types in the generation of nuclear stock material e.g. potatoes and strawberries, There are essentially tvo circumstances under which this technology has commercial application. In the first case individual finished products have high value and can bear the high cost of tissue culture propagation, The other circumstance uhere tissue culture propagation is practicable is there this technology is used to generate parental material, subsequently bulked by traditional vegetative means~ jl V1 BATCo document far Legal Services : Health Canada 21 May 1999 -6· i;: ds a mass propagation technoiogv for the production of tobacco plantlets, mer,5telc mic:opropagation could not be justified on a need or cost basis. Hovever, it has significance for the Industry as a research tool that could proride the basis of ar,'inte:nal contro:' for establishing some key factors for Cuitiral use and development: a) true genetic stability and effect of local enviro~ent for reg:onal comparisons and on the development of c31 'turai practices for prod~rr improvement. b) season to season and agronpmic facto; effects or, croplproduct quality. Cloned genetic stabllit), across i plant population uould provide an excellent basis for studying and optinislng harvesting and curing processing practices, C) climate to climate factors and their effect os tobacco qualities, dj eiiects of different and nominally established curing practices, In addition, paralleling the practice with otter crops, tissue culture techniques could be utilized to maintain elite tobacco breeding lines in the production of hybrid seed. This technology can and has been used under two scenarios: (i~ to short-circuit the selection and maintenance Of plants from a segregating population that have relevance for hybrid plant seed Production prior to extensive in-b:eedins. (ii) use of CHS plants via tissue culture maintenance in the Production of technologically based hv~rid seed production. b, ~se and Possible Agplicarinr~ of dnt~cr Culture ~nther culture (oy haploid plant production) lethodolppy relies on excision and propagation of parts of the ~aie and female flower that have undergone meiosis in the generation of haploid (nJ tissue, The regenerated diploid 12n) plants can under certain circumstances be produced from chromosome doubling using colchicine (6), Uhere this can be induced, true haozyeous plants can be produced ioften referred to IS instant 'in-breds') that can have a variety of applications for plant breeding generally ii), These are primarily: (i) identification and selection of hapioid plants with 'desirable characteristics' that can be chromosome doubled to produce holooryg0us tin-bred) plants for breeding purposes or direct use. (ii) for use in the Oevelopleat of breeding lines for hybrid Plant productiaa. Plants produced in this manner should in theory exhibit greater heterosis j~igdur) because haplPid derived in-breds are truly ho~ozrpous ~hereas these produced b by conventional breeding are not. This is probably nor O highly relevant to tobacco, a plant that shows limited O hybrid rigour, suggested to be because commercial tobacco 03 culti~ars are derived from a naturally occurring hybrid '' (that gives rise to an amphidiploid plant), U1 BATCo document for Legal Services : Health Canada 21 May 1999 (iii) as a possible breeding too: for chromosomal additions and substitut ions. Where obvious phenotypic selection criteri; can be applied to the experimental plants derived in this manner, or some in-vitro selection pressure can be applied such as water stress, salt or nutrient tolerance, or disease resistance this technology has practical relevance. The opportunity for identifying plants with 'do~nstrea~' at:ributes hovcve: becomes p;ac:icall~ limiting uhen considerinp individual plant changes. cl Cellular Technologies The major research effort in this area again using tobacco simply as a model plant has been directed touards creating genetic diversity through cellular manipulation particuiar:~ cftoplasmic and nuclear fusions with some research effort focused on insertion and fusion of genomic fragments (8,9, 10), In addition, effort has been focused on u'.:li:ing the spontaneous or induced ~a:iabilirv that can be achieved in certain circumstances of plant cell cclture, so called somatic variance. There are essentially two types of somatic variance: (1) Soontaneour peneracipgof somatic ra:iaticn; soplaclonal variation Ender certain conditions of plant culture, the excised, organised tissue becomes de·difierentiated as in the case of callus and simple cell culture. In this fort, cells are encouraged to divide essentially as single cells, an event that leads to a degree of spontaneous genetiC variation, the frequency of which can vary with culture conditions (11,1?,13), The experimentor rill select culture conditions to enhance the specific frequer,cy of cellular variation in the pursuit of neF or novel biological variation. The value of the spontaneous variation or somaclonal variation that exists in a population is a function of the ability to select for improvement. in the case of tobacco, its value is limited primarily to selections that can take place at the in-vitro level, (ii) t~_agenesis or induced variability In essence this is analogous to the above except that the rariability is induced or enhanced by the use of chemical mutagens, This approach extends or enhances the frequency of mutation and can be applied to plan: tissue directly increasing :ts potential value over the plant systems for which single cell regeneration protocols exist (1l,1j,16). Cn 3' ~D BATCo document f~r Legal Senrices : Health Canada 21 May 1999 ·8· I·r The joljok~ing conclusions:an be made regarding the strengths ane uealinesres of the use of Cellular technologies. i) it orercones anT sexual barriers ii) its major beneiir is that it can potenliall~ provide net genetic uariabilirv for tobacco since regeneration potential has been validated. iiil the major challerge is ~p identifl selection criteria against chlch commerciallT; significant improvements can be identified icurrently this approach ~as relevance for tobacco improvement there selection criteria can be applied directly to the culture conditions such as climatic and nutritional stresses and disease tolerance and resistance.l ic) sp6firaneous or induced variability is not aluays 5:able generation to generation Inevitabi~ titii :~· cellular approach, breeding strategies have to be identiiieb for incorporating the ne~ attributes of the lec ~aliet~ 50 produced into commercial culticars~ In practice, this technique although capable of creating neu n:i~tions in the plant is limited currently b' the selection criteria; plant improaements post S:aving re leaf and fit product ioprPrement Fwld necessitate unacceptably high plant populations from vhieh improvements Would have to be selected, having been cured. aged and processed to cigarettes. Specific Genetic Enp:neerinp In broad terms, plant genetic engineering is the process thereby 'useiul' genes can be identified in nature and functionallY inserted into agricPlturalll important plants for uhich a defined need has been identified, This process usually follows the outlined sequence of events: (i) identification and characterisation of a significant gene or genes. (ii) isolation of the D#a responsible for the identified gene and deveiopmen: o[ a gene construct in order to have functional significance in:he recipient plant, This Fill include 'preparationl of appropriate regulatory, initiating and termination sequences in order that:he gene operates and is expressed efficienilv, 1~ ill BATCo document for Legal Services : Health Canada 21 May 1999 -0- (iiil :he potentially i~n~tianal gene construct has to be inserted into:he recipient plant using appropriate rectar s;slems. This can necessitate the development ane use of appropriate ivPp:ificatjon and transformation vector systems sc achieve an efficient number of transformed plant celis~ ~iui regeneration of the 'transioraed' plant cell population to whole plants needs to be follooed by appropriate selection criteria. The molecular biologist is making considerable progress in facilitating the use and operation of these processes (2,17). lie can define approp:late regulatcrv sequences such as light sensitive triggers which lead to direct expression of the gene in the leaf. This is an example of in increasing capability cf tissue or organ specific expression. Eotecer, :nis technolo&~ aP definition demands a precise understandln( of plans and biological processes and identified commercial goals. ~thouOh this is an exciting technoiogy of the uture the reality :odaF is that only single gene trait manipulations are possible and this seriously restricts its immediate useful application. The success of these molecular techniques depends on increasing the statistical chance of inserting genes effectively and on selection criteria for identifying chether the inserted gene is operating effecri~elv, The challenge for ;he molecular biologist today is to be able to insert or delete genes in a highly specific manner. The reality of the situation is that there has been a tendency for the genetic engineering capabilites of the molecular biologist to outstrip our understanding of the physiolPgical, LiochePical and molecular processes that control plant regulation and developent. This is particularly true of tobacco and there is an urgent need to conduct basic resea!ch:o define nop these basic processes influence commercially significant plant attributes such as basic plant and leaf qualities, smoke flavour and other organoieplic sensations and to use this knowledge to engineer desired changes in the tobacco plant [a similar approach has been referred to at international tobacco conference (18)1. ID date genetic engieeering methodologies hare beer, used to achieve pl~phosate resistance and to produce plants capable of producing BT toxin, Neither of these achievements needed a fundamental understanding of plant metabolism but rather are relatively simple single gene traits identified in bacterial cells, svstems that were well studied and amenable to direct investigation and analrsis. The immediate prospect of further commercially significant single gene trait manipulations in the future would seem relatively limited. ,." Lr, BATCa document for Legal Senriees : Health Canada 21 May 1999 ·10- However, the process of genetic e~pineerinp has the lone-term potential to substantially infiuence manfuacturing costs, product qualities and plant attributes that vil; pioneer nove; product changes and indeed it is possible to set some broad goals for the industry such as: (i) nanipulat!pn ~f_lhe nitrate reduerase ccmelex (iii ~odifhtion of :ai nicotine ratios by genetic e?pjne!~ng liii) ~odiiiehtior; of rasle andflavour s~therie pathva~5 (ir] Reducticn in us o: al:icultuiai inputs Ii ::c had a better understanding of the fundamental na:url of disease tolerance and insect resistance in plants and the uptake and u~ilizarion or nut:ients and fertilizers we could develop strategies for their more efficient utilization, in order to focus the R i D effort and develop the apprspr:dre teeino.ogie5 it cil: be necessarr for ;he:ndusrry to clearl~ define Its colm~erc:al goals for the future. ~olecu!ar Blolo9ll.~ene~raPFinn techniques :his area has been separated from specific genetic engineering to indicate some of :he spic off benefits of fundamental molecular research that are finding commercial application, The work relates primarily to the use and aplilcatian of RFLP technology. In ~impir terms the technique utili2es naturally occurring changes in the DNd strands that occur at sexual cell division, which in combination vith highly Specific endonucleases gives rise to an array of fragments unigue to an iodisidual plant, ~ series of moiecular markers can be developed to cbancterise a particular fragment. in this manner it may be possible to obtain a molecular profile of an individual plant and m3re ilpporrantiy through the appropriate c~~ination of plant breeding and molecular biolon establish relationships between plant generic information and observable traits, Such an approach will provide the basis for: al definition of traits in ~olecular terms providing a basis for trait enhancement b) facilitate the combination of traits into specific cultii.arr c) monitoring the introduction of traits into commercial culti~ars d) to provide the basis of a selection technologg either in te:a of mltchine to original parent types or for novel genetic combinations ei begin to provide !ocsr for specific genetic engineering chere more O pragmatic approaches fail, O f) of primary importance allow a shortening of plant breedi~~ time scales Lr In I~oring agrPnomic or product based traits. LTI BATCo document for Legal Services : Health Canada 21 May 1999 _· -n- 61 it is believed that the use and application of this technolog~ ~ill provide a ~or~ iptdilre application for lloleoular based techni~ues that has relevance for irproving the tobacco plant both for agrPnolic and dovnsrre~ benefit, i. S\mbiotic Soil ~ic:o-o~znisls for4lant Benefit There has been increasing e\lidence that a range of soil aicro-br~anisl~ other than the :hizobiun type can facilitate the uptake of nutricnrs and in some cases partially protect against disease and v~tlr stress. Those of primar~ interest are of the lycorrhizal funlai type ci uhich there are essentialll. tv~ t~es, ecto- and cndoPychorriza, It appears that ect~ycorrhial are of pri~~ry benefit to iarelr trees ~nilst V~ Indom~corrhila tend to be associated ~ith a vide variety of Igricultcrllly inportant crops including sooe annually planted laterial. hlthoogh it has pro~cd possible to culture cct~~corrhi~l in-vitro using continuous culture conditions it has not been possible to bulk Droduce VI endorycotrhiz? in this v~F~ Production svs~cas for Y~ mvcorrhlla have been developed ~rilizi~ host plant syste$s in Eontrolleh cnviro~ents. The potential for use of this system for colnercial benefit depends on: i) base nterial cost ii) application rites required for specific plant types iii) cost savings on reduced fertirifer use The use of vh pvcorrhiza has not been co~erci~l!y ~llidatcd for tobacco, To dire, the full benefits of using such 6~biOtiC soil I1CrO-OIIPniSIS has vet to be fully ~llulted bur vould appear to offer sent potential under defined condi~ons Ind aisht provide a basis for ensineerina additional beneficial changes in the organisas for Dutual benefit, Coaclo~ Renar)ts The future is technically ch~lll~ing, but the acrpi~ technologies can and should be utilized to develop novel tobacco breeding stf~tc~ies, designed to net the future needs of the industry. There vill ~1 need for the Indastrv to carefully define its goals and to laintain a conelitnent to iundanental research If fort to achieve substantive ioprovenents in tobacco, .O lCC ~a i I~ I V: BATCo dcclnsnt for legal 8,nkes : He~t~ Canada 21 YIY lggg Referrncrrr i. ~~ori~n~ Procedures f3: 4p~ri~enr~ Use. USOalcch~clll~!rnn ~c, lj86, ?, 3. Schell el II, Flint CL" ~ransfor~rions and Generic insineerina in Ilanl IrPlovepenr and So~a:le Cell Ccncticr~ Ed~ F~sll ~cldllic PIess, (1982) i, k'atanlbc, Y, (19ij) Jan, J, o: Breedinl 25 (1) 79. 6~ Illli(erlee, I.F.~ Belling, 1~ (1926) J, Hered, lj, 19j. i, Chih-chi~ Chu, Hlploids :~ Plmt IDprovepent in Plant Ilprovemcot and Sonaric Cell Genltics~ ~C· \:asil acldemic Press (19821, 8. H~cbess, 6., and labib, 6. ilFil) M~ec. Gm. Lenet. ~j, ?~· 9. Uh~c, D.~L, and \:asiI, .. (197~). Thc~~ Ippl. Genct ~1 134. lO.:ocl;ins, ~C, Genrle, ~~ ?dce-Joner, n~·I and Poue:, J.L [197i) Flint Scil lett~ 10, i, 11. 5aeristln, I.~, and Helche:5, ~ (19691~ Lo~c. Ic~ Ccnrt. 10j, 31i, It Ill~iP, ~J·1 and ScDvcroi:, ~~~ [1901). Iheor. ~ppl. GlPrt, iO, 13. lurt, bG, and &tti~rr, 3.F. (1976). J. Hered, 67, 381. In ~~lnt Tissue CuIrure and itJ BioIolical ~pplications' Ed, Barl, SP:inser, Berlin. Ij, Sco~croft, U.R. (19ii), hd~. ~rono~~ ?9,39~ Ik Thomas, i, ii:ne, P·~~ and PDtrykllr, I,(\P19), i, Pfl~rc~ Zucht~, 82,1~ ii. PrPeccdin~s of 'CORESTA' c3nierenfe, Crcece (Scpt, '85), 18. Second Inter~atioP~1 Tobacco Exhfbiticn, Richond virrinia (1986), ·O ;O ~h .U BATCo document fer legal Services : Health Canada 21 May Isss btten:ion Y: h.l. Heard Deve!opent of a Group raee::h~progranrme for obacco improvement winE p!at biotcchnP!qE Backlround n resesrch prograrmne is under development between BhT and Ikyford Plant laboratories to achieve tobacco improvements through the development and we of emerging plant biotechnPlopica: methodologies that have benefit for the Group. The p:opan~e that is being evolved is fundamental in its approach but has the clear abjective of bring focused to satisfy current and future Group needs, Outline of the research strategy The initial programme of R 6~ D is predominantly molecular based and aimed at identifying and establishing the relationships between plant genetics and plant attributes land will draw m RPlp a gene mapping techniques). These molecular approaches have an increasing role to play in the improvement of agriculturally important crops through establishing relationships between plant genetics and quantitative or readily definable phenotypic traits. To date these approaches have not been applied to tobacco and in addition some of the IndustrylGroup Feeds nill necessitate establishing relationships between plant genetics and subjectively based product attributes. These challenging demands will require specific method development and validation w a means of achieving significant plant/product ~mprovaent. In summary the primary objective will be to establish the contribution of plant genetics to tobacco attributes and to achieve identified imprwaeptr at the agronomic, processor and consumer level, The major long-term cmbphasis will initicllr he aimed at improving taste, flavour and other product quality st:ributel, but the progren~e will be developed in a manner to ma~ilise the potential for spin-off benefits that nigh: not be justifiable in isolation, BATCo document for legal Services : Health Canada 21 May 1999 The research direction will require orientation b!~ the Group and integration into the Group's conventional research act!citics at the following levels: (i) Plant Sreedind To maximioe the chance of success of the mPlcesiar programme it is of paramount importance to establish a strong working relationship between lab:esearch and plant breeding activiites, This is for several reasons, Initially the contact is necessary to provide a knowledge base of put and present plant breeding progranrmeo and available germplasm sources the: could have relevance for plant improvement in satisfying Group needs. The other major area of interaction is at the level Of experimental breeding prog~z~i~es, plant selection and tria:!ing in supper: of identified progrann~es, h meeting is proposed between TPI and the Group's plant breeders to initiate working relationships that contribute to and progress the molecular based R & 9 p:ogra~es. The significance of the interaction of molecular scientists and plant breeders cannot be over emphaised in te~s of achieving quantita:ive plant improvement ~ (ii) leaf chemistry and tobacco flevour research In establishing a molecular basis for plant and tobacco attributes progress can be enhanced where information exists or can be obtained that identifies or characterises the association between leaf chemistry and plant/product attributes generally, h the research programme is developed there will be a growing potential requirement for significant interaction and specific research effort aimed at advancing our understanding the effect of leaf chemistry on plant attributes, There is a reciprocal benefit from the use of molecular approaches in providing a possible basis for focussing-in on significant pathways that contribute to plantlproduct quality attributcs~ It is significant that there may be a need for research into elucidating plant metabolic pathways generally using conventional bioche~cical means particularly where specific component modification is identified as a commercial goal (e,g, modification of nitrosamlne levels). BATCo document for Legal Services : Health Canada 21 May 1999 (iii' ?ian: IProduct evaluation Exironmental and cultural practices together with conventional processing and blending methodblogics substantlallv influence leaf Iproduct, qualities, In the dereioppent of an improvement programme there will be a need for specific experimental strategies that will facilitate evaluation of plants at the individual level isee experimental design section; tissue culture cl3nal propagation), Knee establishing links between plant genetics and subjective plant characteristics there will be a need for evaluation of ex3trlmenta! products using highly discriminet~r)' consumer paael regimes. proposed eDtrimer;tal approaches ~wo initial experimental approaches are being utilised to develop methodologies and strategies that will begin to identify the potential for plant improvement through the use of molecular technology and used to establish a pragmatic basis for plantlproduct development, evaluation and improvement , The definition of future goals will in part be a function of the success of this approach under development but equally upon identifieatidn of long-term Crwp needs, i, RFLP method development: si~hifiea~ce:cr eharac;eristinp retorted tobacco flavour attributes There are numerous reports in the literature that indicated that specific tobacco cultivars or leaf chemistries can be attributed to particular tobacco or product Tlavourlquality attributes, it is proposed that the following cultivars are zed to characterise at the molecular level, genetic differences that can be associated with reported product/leaf qualities, RPe Cultivar!breedipg line Burleu 8Y21, LABY;I1, KY14, Fi15, I(Y1? Flue cured NC2326, Ti1113, pp964, PD1SB Cigar filler Ti165, Ti1341 Other Mihara, Ti1408, Ti1127135 In addition, where these associations can be identified, breeding strategies will be developed in parallel to test the strength of these relationships and to define their characterisation. This approach will preside a fi;m Coundation fcr establishing the validity of the Pethodolog~ for characterising tobacco plant attributes generally which can then be applied to attributes of particular commercia! si~nificance. BA~Co document for Legal Senrices : Health Canada 21 May 1999 -4- ?. Development and Characterisat;on of l'irginia tobaccos_k·ith associated oriental smoking proPer:ies. It is proposed that two c~tivsrs are selected that give rise to and typify virginia and oriental tobacalslppking qualities, These two eultivars will be crossed to I'ield hybrid tobacco seed, The derived h~rid plants will be se!l-pollinatcd to generate seed yielding a segregating plant population. d seleeticm of plants will be taken from this population based on a number of parameters, aponomif, phenotypic and where appropriate a statistical sample where no direct selection criteria can be used to establishlipdicate plant attributes, The individual plants identified from this population will be clonallv propagated to generate sufficient genetically identical pleats to prepare pure blends from defined curing regimes. The products generated on this basis will be subject to panel test evaluation and molecular characterisstipn. This methodological approach will provide a basis for validating or otherwise the specific relationships between accepted plant attributes and their genetics, BATCo document for Legal Services : Health Canada 21 May 1999 ,· ? i CI-·L· r Section j ~:i7e of a series of Strategies for a h~D Ibjarcs Im~ovanwt P~o~am~ P!~ biaff:1·3lcgy is a mllecticn of ~ughly sprhid:s.e~ enabling ted~cgies, ~c~i~i~ increasing c~i~~ty for tc1~h~ ard hence ckarette i~v~ne~t. Ilhe ~Tessfal a~licatia~ of ti·se techrol~jie· *ill p jepe~r: co ~he Pualirv of inptt and bel of ~a~ve ~irJ~h3 ~r c~rrerrt Cros R & O Cemres, l~f a~ process dep;-t~e~s 5 the TJlrsuir of CL'o~pwid~e mrmercial goals a~ objectives, The full rarge of tec~a~ical caPa~ilities required for su5r~ned c~bacc: ~wa~ LZ~ t~9~ietal deselc~rreri, can be ~ivi~al into ? ftnnia~ally 8~~eet areas, c3nv~ic~ oint hee~in3, tissue cult~e facilities ~ access to advanced plant cellular ad rrolecular ~e~i;i3lo~ies. I"ne camp,-cia! ai~:icatim of ;dvanced cellular and nolecular tedrolccries involves the use of tissue cul:~e ard D~UII bree?kg skills anl is im~li~ L1 sujsequw: ?rc]ee: xopsals. ~ach hncticaal are2 is ca~a~Sle of o~o~e~rin3 a Pi)ri:al idatilr, bul will rwui:e cerJal c~ordi~atim, The teci~l~0~n! required to drive t~e ~barco ir~w~ne~iI~ zc~wme can be S~Xiv~ed into 3 levels of rrateSic in~o;twce refl~tiq cirrre~a d ft~e research p3tential ard will be discussa3 as broed project areas, k'i~in BaCk, s,rate3ic area sPecific Projects will be iderr.ifit~ ard disi~ssed ir,sms of their orrwcial relevance. Identifi:a~or, of Strategic Project Areas ard Proa3sQs ~r S~cific Preects S~-ate~ I; 1.2 Year Progranmes C~iertive: the deve!opnerr: d adaptation of cucently available lec~~ fcI rhe~o;rmercia! mon~enrane~t of tDjacm. The first p~i3rity for t3bacco lant imF~ova~t re~ires Ye dweloFmen: of a oerrp~nehensive genelijrary ad gene IMF~r9 facility to ~ntide abroad-based genetic tec~a~lo~y package, Ihe approach used for the preparation of a gene library tan be described In Se~eral t~t~s u: (i) Identification ard sele~c~on of plants with desirable tobacco attributes assessed in ei~er qualitative (p· quarrtitative terms, jii) Using srec~ial exTrmes, ialatd IEm ca~ he "nrt" Lr.3 oieCes e~iv~ent tO Ilgene" lengths (apFrox 10j base pairsi, the minims size of ~I capable of ceding far a snail protein, (iii) Usinl establishaj. bacterial cc viral t~Jls~eaicn sys:ers, ~1P, is transferred to a ~st bacterial cell and incorp3rated irr,o its nuclear atericl. L ~h~ Of ~I~al edluta: pccesses this f~eiSri CNA 4 h 'c3pied' to produce a tanplate (mFWA) Ilse~ in the ~is of prs·ir,, the ultirrate gene FP~OiU6, BTiSig fT~n a CasC~de of cellular even~s, C BA'TCo document for Legal Services : Health Canada 21 May 1999 (iv) 01~ d~iol~~~ :e~1=i~es i: is ;r3ssible to isolate ~$i~~ ~da ·d ~a~ni~ ~Je ~ ~a~nents, If the omdr is cr~ on a r~~i~ ~~ it is pssSle:o ~~ca sdi~~ ~'s or prd3es a ~cy are tod. In ~s ~L: I ~e saccs~x, of ~me r~o re ~B~d md 3~ to M~dnd cw~i~:n h i r~serrwh bd~ of gc~ Ip, The me d amlic~m of t;ne gac :~j~y to esta5lish ~e basis of a oe?e ~ii~rr~ing t~o~ (CZrS) cm be ~L~~ dCIF~: (y S~icic ~s re ms3~ = ~~II ~a~se a a:~: plant's gaux~ either in ~~l~e ~n if s~idc= nro5e5 are available or, ~ relative a~ if sufficient ~~~a ~~ ~ separate one ~~ frcm ~~~, ~ gene 1~~ is ~~~v vp~atd d Ic~d ~~ cn i:r sc (iii ~ ~ are ~tr~~~ dr d~ of ~ca S ~~s :f ',4eir ~Qric lMte-ial (nJ $1l~ b ~!P daa~~ with erdxlucleases, hess a~ rise ~ lruque pieces of rolaf~~:oe ~ ~~ art rcTth 5V gb pS~ aerJOFi~LOTb~. iiii) Osin3 ~B dPF~WQI ~ ~aarren~6 me arrargd in order of d~easing size. In this ~n ~~ a'h r~atedll tained ~~ the ~lcanve F~c~es ~ b~d a ~d~s~ sc of map c~c~~·. $ ~1Fa~in rm de ~*ia, $e as5criaticn Of ~~ Qara~~i~ics d~ ~~ic gr cl~:~a cm be ~d~d d their inheritance rattens ~ sexral ~ta detenni?e3. A s~e ~de ~ h pr raia~ ard aloal~iy traits plsa Z ~ ~$ L1 gas in the off-sprin3 in ~dm to disease resistance ~1 ~~e ~~~~ia of gene-clmte=s rffiatd with that ~d~, in ~s as the ~~-~a~fe of ~ trait to w sli5sequent varie'.y oonld be ~edict~ fran 4 ~pis of any dl pm; of ~t ~I~ plat. hs is a itera:ive p.ccess imro!vi~ ~~ carpatia with ~~rle pat:err, remTnitior,, al~~ Itfinanent ard ~~emert. of do ~ac lib~h~ ~6 b~~ ~ ~is ci ~elinr4 pr y~t ~ d ga~etic base, 9uilding of ~ basic pnt i~~ ard s ~~ technnlogy will ~ "14rox~~ Ip, ddt~u3h re~~ ~ full practical ~l~da to achiwe cmw:!~ go31s dll age dr$ the t~ year for pt ~ a ·ru~e of ~oj~ with defined mjbiPw, Projeo', i, Title: ~d to ~~io~ Rb 8~~ Objective: m establish ~e Rar)rtrs ard ~o~la for c~n~ci~ll z~T3r~an: tra3 in order O ~~~~c~ a~lz~ h~q ~~ ~ inQwd ~~~a. ms gene ~$ tec~sal~ oan be ~ ~ characterise a oormaroihl ~~r or ~~ 1Le in te~ of its ga~k ~d~a ii rdtb tc ~e O 3eriornanoe, siramwrtd ~ac~srl d tit ~~ P~lbl~PI d C h~dcP capabni~as, It is pss~le thereafter to d~~ a ~4 for BATCo document for legal Services : Health Canada 21 Mg 1999 defir~i~ and Fbe~icring ola~ ~:S3ance potential in te~s 3i ~sea~e resistance traits, caxbd~se storaPe, alkaloiC wtterns, anpiricallv detecnine~l? leaf ard sroke qualities, other historical plant Sree~~9 data of #mmercial relevance, k~ SQMC1O;IBI Sele~im Te~hnolaly Projea 2. Title: Tissue O~'-ure ObjRtive: to dPel3~ scr~e~ne wocd;P-es to ide?:ify i~vid3al plan's wi~h ~y ir~rtant tTbl~S ~Cm 333~atjrx~ wi-~ ~ia:ural an? iniuce2 plant variability. g ~wious!Y discussedl tissue cult~e'ec~au~cg~ can be used to deveioP gene~ic variability throujn all fusion ~FhrmloSies ard the 3eneratien of sanaclones, QC will provide ~e basis for rapi~ se!BXlcn of plants 6'31 ~p~tiLI~ traits at the see~irr: stave, even these nxma?ly only expressed in 4e Rnture pl~, ~ne olrbind use of ·Jlese technologies will be use~ to "define" ard select for Fa~t~cular genetic c~psiticns of aslnereial reievar~e~ Proiea 3~ Title: C~I~ as a Research Tool ~bjective: ~j use Ye Cf~ as a m3at~ of ~widi~3 as aaaol~iate, di*ec~icn a~d~~fcw for cellular ard ~ol~~lar research in the identification of nnthetic mthrys of o~slmer~al significa~ce· ~ I~nen a olant's t~for;Mnce a~ behaviour has been c~aracterised albeit in empirica: terms, ~ offers t~e a:errtial for e~taiininj the genetic os~sitiin of the plant a~ ro ii~entify miow eene p~osi:iess that associate with the trait, 'R~S )mcwlejae ptwides an o~ort~mity for iaestisatir4 at fhe;rolrula: iwe!, events that at Kpra correlate kith plant traits a~ ca~ b~ usal in *see~Lg Frcgsa~n~Ps or p3tertially paa'ide the direct route for d~-,enninirg the precise seg~enc~ of events Irrx~ivd in the e~essicn of plant traits, Zis informaticn i~ovidw a route of entry for .dant trait e~z~ca~r, a~ ~i:icat~icn through the use of 2dvanced ~olecular biology, Stra:e3y II; 2-5 Year Prcgra~me objktive: t3 1RI~OP the tec~id capability ~ e~acterise ard select ~k~s~ii tern6 of tOt~ g~netk mnplsi'i~i a~ exaession· The research and dwe~o~soent dt4in this project a~ea will ~z cpncerne~ with producin; cultivnrs with rro~Ye! mokirg c~aracteristics to incllde, enhanced taste and f,avap. po~,-ties, nuel t9baEeo aroke c~a~ter, s~en~th rrplificatiwl t~L~OUj~l taT:~.CbtlM! ratio ;nanipulation· ~·s ~k a~ea will build cn the W use~ , e~nt~i~ation with anwrrticnal plant S~ee~in? ad tissue culture tedul3lcqy to describe ani exterd a plants genetic cc~sition, Ihe rJjo: research effort will k cc~emed with the dP~elo~nt of wlecular t~c~igues to mritcrr ad ehamcterise the chton;lcgical secluence of ex~a~esn3n~i3n thf3~DUt~the ol~C~S~f~ evcle, he to ~nar~cter:se cene~i~ c~os~i~n~ gen~ic exp~'es513" will ~iderably enhance the L7~ersta~inl of 16 dwel~ne~h, leaf wality ~ srol~ir~ quality End facilitate ·~,, dwei3p;e:: pf cultlvars wth:~e Oh potential to r~stnrture cigyp;te blendirg strategies to achieve tps: savirgs~ ;i series 3f Iritla! rro]ects req~urin; ;ech?olcgies for gene expression can be idwtifie~. U~ BATCo document for Legal Services : Health Canada 21 May 1999 ~:~r I. Title: ~t~~ ~i~9nte as~~es ~16ive: ~ lr,iIize tec~i~ss for aae elf~srin m:~~ r jce~r ~f~s of ~ffr~~ ~~&~~ an? ~3~31~iC faotors on c~:!:r le~ ~ bbe~i?ac~im of ~e respnse of ~~p~s a: ~e ~lsdr !!vd ~ d~f~~ hmr;rorm~8 oonditiols ~~ it's erowlm ~r:~ d~ ~~ id~ficdm of be ~rcK~l~ie~ a~~e of;v~s~~~ ~lu~e 1~ a~ty d :ac~i:a~B be da!oprt~r. of e~:~r ~~s~ for ~ffre: gro~-·q :q~~ Ccrrc~a~~ia ~~ ~doa d oualitv asrPx e!~vc: to COnpfe ~ ~~ one arasi~ in ~f~d ~~VZg ~ B :e:r~ 3 tobaC;3 d moke L~~!;ac~, 4~iS k:ada evcs m~~d with these ~~~r, These ~~· dll ~~de a data base, ~~ of an ~~k~ ~~t in $e cd~ ass, tha! cc 5e ·d tO ~~!$ ~ ~l*ds his fc: i~ ~ ~Sm a;t:i5lrtes ~e will ~a~e ~us m: 1~4q~ ident~i:~~ of rtls~ pat~ap ~ld in leaf d sabe ~$~ Rwe~ Ill, ~~e: "~lr~r' b8~i~ of ~r~v ~na~ ~~I ~!e~ve: ~ ~~ig~e g~ axpessio3 in ~jacca ~~s )noh~ O k ~rd~ ~~ lwels of taste ard nwa ~p~ia~ Tbe :eseKoh ~1 ~ concerad with id~i~i~ the p~p of flavour B~!dS d the t~ ~ are ope~t~o dr~ p~ deelopn~~ mt fi~nga dll he od ro facilitate selec~i~ of c~d~rs r~ mance~ fla~~ cha;acteristios, Ms appoa8 ~1 ~ od ~ a gacr8 sense ~ id~~~ and ~abe:ise g~~ic ~cssi~ ~ pathk~ay oFeratie~ BATCo document lor Legal Services: Health Canada 21 May 1999 S'Jatec~ IJI 5 Year P!~s Prora~ Cbjective: to e~ineer tobacco pints to achieve clearly Bei-ined carmercial end-Fo~ts. Tnis research will t~ in part an ~xte~sion of d interface with sx~ aspects of F~~er~ arl~ne~ in the strategy ~ pnajer. area. Ihe research az-livi=v nee~d to a~eve these goals will rec~ire access to a full recarbinur. LN4'tea~7lwitS sclm: additiQlal specialist nseardn slxr;c~. 'ihe pr~Iy ase~s of res~3i?~v fccthe research ~c~ vill i?cluje: (ii the ca~ji;i:y to ide~ti$ ~ isolate Lrlividual g~es of cenrrercial relevan~e (U) the ability ta secr~e~ce a~ i: necessary ~fhe~ise ~NR's develop ~ve! gene c~crstrur,s CJ3 a~eve particular hrlctiens. Iiii) as part of a nwmal reo~bi~ant I~ re~ire~rrr,, develop gene transfer ad clca~irg systehs as re~red. (iv) the research and develoa~ pro~~mme Kill rwdre facilities for plant regenera:iw~ ardthe avaiia~hili:y of seldrtionlevalvatim technolc~ies . [vi tt~ ~a~ccess of amnscialisa:icn for pare plant i~hwaneclts will require cent~olled er~ir~a~ne?t selectiol with a general fiel~ tda! evallstim for a!l ~erel~pn~j~ In a~itiol to the researm ele~ts Ixltlined here, xr~ projects will reouire a~iiticnal iundrwf~a! research particularly on ~8le twulati~ ud as~edcs elf pliar.:M~i~ (bicchanical a~ p~siological )~inalv~ in plant re~iati~ and teda3lo3ies devel~nerrt. R rarge of irdividual F~oject~ i~3lvirq sirr3le gene tra~ufers a~ the develo~Rart of oenetic mfiipl:dot:e~cgies w;th imaa~ihte cam~ercial releva;lce ean;w identified. Sa~ exarrcdes a~ight be: Project 1 Title: Insect Ilesistant 'IbSaccos Dbjective: a insert a~ achieve finctirnal e~essiw of the BT g~e o: a derivative in tobacco cul:ivars, Ili~s project will pawide the o~ort~n~ity to I~edlre insect rPsir~art, pl~ts that will aeverrt leaf d~d3e, dp~ioratiofi anl qwlity loss due to inset', attack. I; is ~iigh?y likely that '~ese ergineered tobaccos ~uld Hovi~le F~otectlcn for surd leaf against insect infestatim~ The insect renistarr, tobacco plzats will lawide a ~eak in the life cycle of farrt viral icfe=ticns arising ~an insect maliatEd tran~ission~ Projectz Title: C~tiva~s Rsisccant to ~t Mediated Infes~on anj Infestation d O OSje~tive: to develoJ stra:egies to achieve Faotection of the ~bac~o rco: O structliie ~ micr63ial an3. inset: attack, 03 BATCo document for Legal Services : Health Canada 21 May 1999 ~n re a rarec of diseases ~ ~$;~ rioiq ~ dqa of the tcbaeco ~a that rb~fiaiy dtoa ~L!d d C1%I of ~~~ ~~~ have a~ard a son ~Ci3 ~t is toXio to soil ~~S ~gYl~ting be 33tential for 8m o e~· In terms d tobacco, an w~~ive g~~ ~~d b ~ d~~ a inseot toxic ~cashitd soil funlils cdpable of ~C ~~~~ ~ ~e t~cco root stn~t~rt~ me use of on ~~qr~ to ~iwe pd *0trcticn b drqe $ bt thev ate b7eMjle O a r2nrje of ~~ ~~k d~~lm rot cucrealy asmL d~ ~os In a~li~~ Ms ~~ ~~ida the p~l~ b: ~~~e~Rlen: ~aaaent in agron3mic p:~~~~ ~ projKt dl_ ialve ~e 3* d reo;cbioant g tedri~, Title: ~lo~r~n: of Tools for Gcne~ic ~~~n Qiective: to develop a series of ~anic tcc~nol~ia to ~~e ~~~ic~~ ~ ~sn'solld r~~ia ~ qatiQ of ~+ genes of oamercial nlevar~, m~t is a d ~ dsdq ~e basic nl~6 .hiolo3ical ~115 ~ identify, la~ ard isolate speoifio ga ~~ied as mmrrd~ ir~t~ ard ro c~a;ad~e~:ise gade daas oapahle of ~:3Vi~h3 q· Spe~:~~ :~~~ ~ ~cssim~ ~ dwelo;ment of ~e rcci~olb3iCkl cao~i~s is essential for Plant g~ic r~nip~aticn, Roi~ 4 Title: PlL7t ~~ ~f~dh for ccmrercial ~d~a~j~ mjective: a ~~i~ a series of g~ik ~t~r*ays that are inpatd dda~~ of led ~ snoke qaa~y and usin3 the teckco~ pad(age dsd~ in Frcjea 3 achieve ~~ ~hti~ fcr a~m~reW advan:age. b ~mch ~d i & strategic ~j~ a IL II is oonoemed ~~lr ~ c~ac~eris$ and irrFew~ bl~~se ~net the elements of gmde oncrs;=lcn ard ~~ ~nrl~ that are r~td d~ ccm~dPg J;gnificarh evarts. ~ ngaQ is ecocerned exclusively with ~~ cpecise r~~m, ~~1 ~ ~ifie~~ O ~a 9~"tU~ ~~e' in leaf and tobdooo BoFer·i·· ~s ·Ja~Hays of cvrxent mm~c~ i7terest ~ be surn~ild: ~cotine d alkaloid sjnthes~ (b) nitrate r~~e oanlexl ~~ig nitrate ~~~lh arO dtmsamine duXion (d bl~ carto~dr~e S~a3e for Ro~ifyin3 Id c~ resFccl5e (d) mntrol of Icd ~st-har;e~ ~al~ je) specifie p~kays of fIwna ~e515 C ~~ determine ~ B~~d be ~~ Y oa7 ~ aaniru! bl ~~ O fa:iiWa rdlCle to the B4T 6~1 "e ~~ orgahise ~e ~l~atim of the ~oj~~ BATCo document for legal Services : Heath Canada 21 May 1998 ldentificaticcl o:Te~!cgicd. ~ciranects crd ~la~ve ~o~ S:nrrs~s ResblPCes are ne~e~ ~ 3lant bre~di~lfield'Y*ials, tissue cul~re a & D and access t3 advanc~ c&!~a~ ard m3le~ar tec~r.oio~ a sa~isfy the identifis~ project xxramne~ i, ~odoilities ard Field Trial F;lcilities Ea~rt Bee~ Ihe B~T Csc~ B a go~ wsition w3tl~vide in these areas, with S3th ilities and established !i3ks to aca3efie research ih~use ca3a3 centres anlinr~~tes for toSaaa ~aeeiir3~ 'Ihe ~dentiiied~~t irt~ov~ilmt rrxrante will h substantial~ aided by access to tci3accll breeding li~ ~aterial and historical data for use with C~if~, Tne inte;rac~cn of -~k~se a6,ivities will ~c~ile oo~c~tvn~ities fa rapid p~opess 'Y~ be ~je in alarrt se!ecdul ard ~pe~ihive tobacco brdi~ p~cgrirmnes~ ~~ss m -mrve~ric~al pld Sree~ ia~ can h ac~iwed throujh at least tc~o routes: (a) i: is lively that sdficient tobacco t~eedi~3 data, aasi~ered ~D~ conventional ~n~F~s to be rp~conwud, muld b o~ined ,Lcan acadahic departrrwts and institutes, (b) joint var.;ttd or license agrea~s could be strlrtured beti~n~en BalI~ ard'5 seed o~arp~any prcd~ers to have access to "n~Knm~cial" taeedirg line data, Within the 9n'P?, so~ O~ ~S 3n 'in de~J~i' plant t~eedin3 anl field trial capa5ility t'iro~h i'i R & Dl;eaf Cepa~neri,~ me s~aa ~-i~ leaf o~sra~m and their current R a; a pcc~ranrre of;c~acco ~,t t"SOwi C3nventimal ir~3se breedi~ skills ~uld Fn~w~e a su3table scie?tific 52se for the Cttoup to irrrswe and dwelop tobacn? va~ieties~ hbam vzriety dwelgrent an] FwaPtarj seed pnd~on requires access to tissue ollrure faciliues. Such facilities ate available ir,Brazil t~L'DIXlh Bisdar~a, ad will re inaeasi~!y amilable as plant biotech~logy vdtutes are established thta~ut the Cfolp. Specificdweloarwts of tcbacros for parti:cla: relibfs that reppire envlracrntai i~i~aenat, can be field wal~te! in apF~opiate lo~atiens or within envircannslt~ si~latim grc~hh roms, with such facilities available in Twyfc~d Internatio~l Iaboratcsies, a b Whave recerr,ly s~engther~a~ Yleir in-fouse plant hee~i~3 skill throu3h part,i, arplo~ra?t of P, Chaolin, a world apart c~ ~nventicnal tobacco plan: ge3etks a~ ~eedin3, Fonredy of N. Caroli~3 state ~jversity, Dr, ~plin is able ..wide both expert advice far conventional oarrl deve;oprP~t and ~L;e~ access to 16 academic centrw and plant resources, Plant Sree~inl e(pertise a~i~ access to bree~ire data ~xists in other areas of the ~roup, ~iiceably Canda ard ZirrS3abke, BATC6 document for Legal Services : Health Canada 21 May 1999 2. Tissue Clr~e Q~ili:ies Formerly B~I~ d Qe & CC had these ~amjilities, zl~oug~ aAT no Icr~jP,- has irri~wse tissue culture facilities wi~ the ~ac~c, group, General 5sue c?lture excerrise is readily mailable ta B1T Cbolo thr~ i~ biotec~3lc~y pai~ners NPI a?3 ryf~d ~o~ of ~Y~i~i can I~wiBe appropriate eK31dse to stpport be detelounen: of ~jvanced rrPlecular tec~iY~lqies ~ ~iL7t bree~irr needs. It will increasi~ly ~r~ avi~~Sle c~hroush the j~iv~ual Joint vkr,iL·es in range of loca~ons e;Brasil, ~!e, Kalaysia, Sri lariKa etc. Olrrently rranv 3lant bibeci~olp~y olm3kzies particularly in the 16A ca? offer tissue culture capabilities, there is as yet no exclusive Fcsic~icr. jt a9T with ~e exception of simrxllcral ard cell ~sion t~ry~lo3y mailable thro~ IP3~P ~en:ly urder ccncJac: to 9 h W. The use of -~is forrn of tissue cult~e technology, parcic~i~sly wtMdnel vmiation as a nrans Of developing plant jlf~bVBWltS is L~eT walua~n in a range of rese~-~ ca~t~es, ard subject to validaiion will be available through B;iT's te~olo~y phrtners a~ biotechnolon ventures. The use of t~us technology will benefit ~o and becac increasingly dependant cr; ~E~~d plant screening technologies such as gene ~Fap~Lg. 3, Source of'I~ec~a~3legy's F~cili~te ~irec~ Generic Plant Et~difi:e;i~ ard Seler~on The o~tlmity to research ard dwelop both Irplecular tec~n~alagies d ~e necessary basis '3io~henistry or plant ~Sial~3y exiscs in a nm~er of cannereial and academic cer~es accessible to the BRT GIO~. Specifically: (i) NPI Irs,, B~'s US based tec~Lwlo3y partner, has a full range of dec~s ard cellular basal biological sills that 03u1d provide ~e basis of fiad~n~ntal ard noleeular tc~lo~ical sup~rt for a t7jaco irt~o~ne~t pro3ramie~ flo~ver ~ere are a n~n5er of argunerrts against using NPI in this capacity. Tneir current resolzoes are fccused predb~ar~ly cn specifically funded nseatch p~amres in su~cr: of their oJmrercial goals, with research fipding ha~jlg a high cost basis, 'Ihis arratr3ement would benefit BP~I~ in as mrh t~t NI~I could dwelcp inprwed tobacco cultivars, h~ver Nm would cxlly be usirq their existing technological skills with no substantial dweiopne~ of alditic~iai techrology for use through our tec~nolo~y agree~nts. Rwn a o3~eti~ive stard point without access to B9T's R & D resource base, Nm would be unable to develop cam~cial I~ao~lr,s fmo enabli~ techrullwies alcne, (ii) A ranFle of mrmerciat ard acadanic glO~K ~imldride have the skill base to develop the range of c~c~a~ologies nede~ to ir~n~ove tobac~ providing the goals were specified by B~. Ihe ~ast of research would be higfi, wi~t~ a strong direct control eer the research ~cgrPme~ the usua: osntrac~ual ar~an3anarts povMe access to F~nd~cts b~ ~o: :edu~lcS4'~ tkis beirg 3wnel bj respective c~a~tdc: organisdi3n, p~e is a furt~e: pstential disalvan~age, wi~ BPIT's needs will cha~we Chrer O :;ne, Yere will h a re~uranzlt for isca arj~redirecc.ihloi O BATCo document for Legal Senrices : Health Canada 21 May 1999 t~searci in 'he suc-ssU anlication of plarrt te~hnolcc:es~ Brkin3 with exter~! ccn~4al ard a·~a~ic groups I*wll rw~i:e m-IO~' p~o!e~ resu~~Lln3 tath~ t~n a Mtusal int~a~ol ci ilteres:5 arC Fooject orienta::or. in an in3ouse facility~ ~o~date there is Li-~le or no evidence of a cannerciL a cid~t~e jn~ aFplylq plur::pJno!PS1' to the dwe:o;mart d I~-J~B~t of tobacco far wm9rcizl ~3la~3ses, toSacm oily ~in3 ~sei for de; studies . (~i) 'hF~fard lntaMticne:., BRT's technology par~ for se b Erst has UK research capabilities ~ stre~ths in the area of tissue edture ard eel:alu biolo;y sac~er than at the nolecular Iwel. Ii~F~ver, B~ imto!vgpd with ny~-J~ds has be~n en the agred ~Fderst~rg that thev de~elo3 their Ir~lec~ar biolo~ical eawbilities~ Cz~e~hly ~e~ds u;l &ZT are jointly it~ilvd in b;ildim an Pdi~~e! Te~a~ology C~I'Je ~ ~E~i~e ~ta~idi~ a strong nlgulr hiol~ capability~ P~ox. 13% of the Fecgrmn~ wi:l b d~td 3 to5acco ipove~nts~ Opti~ (iii) is the a~laoa-~ ~ar stmn31y remnnerds itself f31 a~T C~~ ~a~o]ects in the nr,lecular h*iolo~y area, BA~Co document for Legal Services : Health Canada 21 May 1999 ·C;s~e ~ e3sd)4 1~ L~I ~: 1~ ~~ ~~ e~ ~ ~cc~ta~ o~atid ~~~ ~ p identifie3 ~ tea of $;7 te~i~3lqicd $d, GnlD s~e~s d tS~ Emijera~icFs :: ~BL tec~nol~ dedopnnt d 2e ~~ ~ ~bLP ddr:~;rdd· b ~39d I ml~: ~ ~~sd~ ~cu*e ) of h te4ro~i~ Cn~e~, ~-~ d ~~t C~mteTC~ divro i :,Dg obree~n! ~· j Iln"d~Fe~! I Lea-~h · I~ I i Prqpnrrr 1 Il I-- "- C -' ~-c~L- [ I ydr ~L 1 ~I~ ~m ~ITe~~ ~'~c~·-iarbl L~tro~~m ~'$ PC~~ 1 i ) I ~i~ i o: a ~i~~ Si~t~· 'e~L~a -I II ~P ~~I I BSP ~~ (i) ly~ e·id !Y P~~ T~.I c~s I i Gene~ie an~e~n~ 1 ,,j I t/dnip~~~ ,,I ~e~-in? I,I ~do~ ~B ~IB L h' I~ kcdq 'L~aa~h~s; I OPC~S'~~ ~'~I ~c,~,, cc ~ ra dst~ i ~d ~idl C~J3h bl ~ ,~~y a ~~ita .r r "I- C I LcXrm·JCMI ~~iC~I'~ tO~KXI I~rlsd The ~cc~sed ~~6~ s'uru;t~nb ~ be EePabie Cr iM~JhiSiny" J) O~XIT.lti61' sl ~C~ dbd0TCh ~ bov~nt ~o~ bC ~cdo of t'e;iPlol~ ~ tenrs oi y~id cmn~ia mjd'b· b~ is rr. Bolu :r~ ~ ~ iaepation d ,~~d~m of plan: te~nol~ 1 d~eWIR~ d ~k~l aCO~C1~ =c~c~x,8 i D d Eely rs~id onr~ci$ o5jeotives i C I f BATCo document for legal Services : Health Canada 21 May 1998 I"L~ " Cj%. ~68 CCPY #r. G,b. R~~~ ,,,~ h61~ ei 1 iba~ ~~l~ic/75 2P MU !Fei ~oblcco Plant ~ct#n~~ngy i have ~vst reed d repo~ from 6ratil on light and temperature efftctl on eurleY tobacco (SE ~~916:), me orfpins of the l~vcl~nl~on were various studies Fade by tirllil for tnE Leaf SteerinP Cormlttee on nitrogenous c~ounds In Burlg, Ut~r~ not always co;dltat, there win so~ su~~~~p ~n6ngs, in8u~~g a tendenoy fct t~~s It thapcco ta yield wre isvcura~e TY~~Oj rlfior than ied fto elsewhere in Iraril at comoerable fertilir~r levels, Iha current report doer Ivgges; that tfierc ~gh: be a ~~lne e~l~na~PL sl~olSh St:~Ph!iO~lTd CX~~tlMOb will bt as ~f~C~t as ever. PY cortrast, It dcc~ adn point to the sugges:inn of ~os for ~nrovant in real selection of varieties for spcdfic clinatic ~c~Gitionr~ The ~utfect Gf yhotoresplratio~ control is of rider interest as a route ~ oenera) crno ielrove~nt uit genetic en~lncctinp. ~ne:hcr that would he the route to ~o or ~nFly t~ to ~~tet ~net~ IWc~l for attenuated ~e~~:i~~'t~ high t~eratcrls,: am lets certain. Petnms both would be wortt trTino7 ce: Mr, A,1. Helrd J Or, I, Binnr/Dr, R,a, B~~er B~Co document for legal Services : Health Canada 21 May 1999