Sari ~~ Ilprovelaa ot~~~rco plant (~~~ to bcg~by_ll,8~pbd ~t~~~T~P.a.c.eo erhiqit~ee~i~i~s~ U~S~ef~llcr ~ lar~!P~ It is apprapriJte to begin by idantlfyina in uhat area ilnprovenents to the tobacco plant are sought. There are It least three broad factions for uhon the tobacco plant has inportnnce · the fsrler, the cigarette nanufacturcr and the consllner. Aithcrto the ~~in focus of tobacco dl~elop~cat has been agronalic - considerations of leaf yield and basic qualitier hl~e duninated. It uill be of increasing inportmoc in the future development of tobacco cultivJrl that the ~r~ing of tobacco is seen in the context of its ultimate use, that is for the production of cia~rcttcs, CDnscgutntly if sa iool; to the future ilprovenents in tobacco they na! be Jgrononically t,lrgetted, (oifering benefits largely to the grover) but ue can also expect to see developnents that offer ilproued processing by the cigarette nanufacturer or inproved products for the snaker, These 'dovnstrean' attributes are recogniaed as the consequence of Ir~in~, harvesting, curing practice, leaf laturatlon and blending of tohdcco~ In this respect ue oust acknowledge that any attenpts to ~chicre lodificJtlans in tobacco leaf genetics that result in dovnstrasn inprovelentJ l;ili present a big challenge. There is 1 considerable body of enpirical evidence to suggest that different tobacco cultivarltypes have substantial effects on the end product gualities. Fron this position it uould seen reasonable to assune that ~~kr chlmistrr is ~ubrtantially influenced by leaf EhORiltrY VhiCh in turn is a function of the plants genetic co~olitlon, Naturally the process is further conplicoted by the influence of cn~iro~lntal factors on genetic cxpresaion, although even the ability of a plant to respond to its environment is genetically bas~d~ The technisues of plant biotechnology provide a series of enabling technologies that generate the potential for plant inprovement at the genetic Ilrtl, and as tobacco plant genetics seens to underpin nest aspects of tobacco leaf and product ~lalitiar vC can exanine the potential to apply these technigues for specific inprovenent of tob~cco~ ~3 ClibPDF - v~~fastio.soni -1· ~: For ease of presentation and discussion, the technologies for plant improvement can be suulitcd under 5 Pri~ry headings: 1) plant breeding 2) cellular and tissue culture technologies 3) specific genetic engineering 4) molecular technologies; gene mapping 5) S!mbictic soil micro-organism intcrlction, Collectively, the first four broad areas constitute the basis of plant biotech~lo~y. Ih~v should be conlidered Ir m intelrlted uhole and not necesllrilr all discrete or isolated processes for plant inprovcllnr, For instance it is inconceivable todar that use of the sophisticated technologies of 2 · 1 could result in a commercially useful innoultion without recourse to 1, conventional plant breeding. indeed 15 a note of caution it should be stressed that in~Lst~lnt in the sophisticated modern biotechnologicai technigues is cxpenJiva and that there Ixirtr a danger that scientists sill turn to these technigues in situations vhcre further thought might hs~l rho~n that less glamorous traditional approaches are appropriate. In addition to the conventionally accepted areas of biotechnolog)' uithin the context of plants there is an area of increasing inportance, that of Isbiotic roil micro-organisms and their interaction between plant and rhilosphere for efficient uptake of trace nutrients will also he discussed. plant breeding for tobacco pngncpt: current position plant breeding is the established, con~ention~l science used to great effect throushout historr far the ilprovc~nt of agriculturally important crept. The process relies on the natural combination and segregation of genetic material and the selection of plants with desirable attributes that occurs during rslull reproduction of plants, The freguencp with which some commercially significant improvement occurs, partlcultrlv in multlgenic traits, often necessitates the use of large population rtudier, all;ars ersuminl that the plant trait can be ilproved within the scope of natural biological variability within a species, The process can he further complicated where selection criteria cannot be applied or identified at an early stage of the plants de~elopncnt as is the case with some mature plant attributes e,g, fruit colour, rite, past·haiv~st phrriolo~~ etc. The situation is further complicated for tobncco where some traits can only he assessed following the multistage processing of tobacco for product lanfulactutc~ Under there circunrtlncc~, the cost and tinescales for improvements in genetically based product attributes becomes prohibitively large and expensive, ClibPDF - v~~fastio.soni -------~- --- I~- in addition to the classical breedins technisucs of selection, the in-breeding far plant trait improvement and stabiliration is a proven if slow process for plant developnent ~i;hi~ the context of sexual colpatability between varieties and current availability in nature of traits that we wish to introduce in 1 ri~en variety. The lajor restriction for the use and application of plant brecdinh in all crops is the Ienlth of tile it takes to Ichi~~e the dcvelopn~nt and release of 1 new co·ercial cultivar; for tobacco this would take in the order of I - 10 ~eers~ The pri~lry application of plant bretdinr for tobacco inprovencnt has been for the developlaent of asronomic irpro~enent, due in part to the defined needs of the Brower/industry and to the ~oEt practical use of this tcchnololy. There are eninent tobacco research institutes throulhout the world that have pioneered the developroent and impro~encnt of tobacco eultivars, the loot notable beinll N C 9tate ~nivlrsitr, USI, Delhi, Canada and Kutsala, Zilbabwc~ The contribution of the 1S land rrant universities in applyinl breedinl effort for the inpro~ement of Burley and Yirlinia tobacco, has been uutstandin~. They are responsible for lost colllercial cultivars in use todav, The nnjor Coals of these '~nstitute' funded prolrallles for the reasons outlined have been to ilpro\'e vield and perfornsnce of corcrcinl cultfvars with the najor a~ph~~il being in terns of yrononic inprove~lnt particularly disease tolerance and resistance, in addition there are two issues that influence the dfvclopn~nt of new tobacco cultivars: (i) ~uction floor purchjsinl, tolether with intervention support by Covernnent tends to impose constraints on the introduction of novel tobacco cul~i~arr (particularly those servinl the interests of a specific end-usrr)~ (ii) There Is a sense of reduction in 6overnment support for tobacco research for plant inprovcment, There would appear to be a need therefore for Ireater industry initiative in the developnent of future tobacco brerdinl proarlres, if the full potential for product i~prorcmlnt is to be achieved throunh conventional and sophisticated use of te~hnolo~ics. ClibPDF - v~~fastio.soni ___ For the new technologies to impact an conventional plant breeding programmes and strategies in the development of new culti~ars they must: 1) provide the basis for reduced timescal~s for plant selection and trait incorporation 21 overcome sexual barriers and current natural limitations 3) provide a basis for selection criteria for plant, leaf and product qualities preferably at the seedling stage. I) create novel and directed plant trait combinations 5) provide a basis for highly specific notifications of plant processes to achieve defined changes in both leaf and product. Tobacco as a model~plant for biotech_nolpey No other plant has been used so extensively Is tobacco in pioneering the development of plant biotechnologies in fields of cellular, molecular and genetic engineering techniques. There is an extensive literature documenting its I~s~ as a model plant in the derclopa~ent of methodologies anti processes (1). The primary reason why tobacco has developed as a model plant is the ease with which it will grow in a variety of culture environments eg microproplgption, as single cells, as protoplasts and in anther culture (1), 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 transfonnjtion system (dgrobaetcriun~ Tulebciens) has greatly facilitated the development of cellular transformation generally and our understanding of the laoecallar basis of genetic engineering today (2), In light of the foreeoing, it may be of considerable surprise to hear that almost without encption there has been little or no attempt to ulilize these technologies by the Tobacco Industry for commercial benefit, The limited exception is the reported use of anther culture derived plants in the development of a Chinese cultivar, Tan·Yu (3), Of the two major world successes in the area of genetically engineered plants, notably herbicide resistance (b) and BT gene transfer (5) both were achieved initially using tobacco, Tn neither case did the companies concerned appear to have 1 defined commercial goal for tobacco in mind. These innovations may hue relevance colpnercially where weed control is a major problem for tobacco or indeed where crop damage from Iepidoptera insects occurs. Today it appears that these genetic transformation plncesses, altblgh stable from generation to generation, have not been introLlced into commercial tobacco cultivars by the tobacco seed suppliers, (ft nav well be the case, of calrse, that this work is being preerusse~l in the undisclosed breeding plans of these companies). a Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill 5 · ~·, Specific App~ic~io_nqfPI~LBi9~~~h?plpRF to Tob~zZ_cp~lt~ero_ye?enE 1, Plant Breeding The role of conventional plant breeding is taken as implicit in the development of new cultivars using new technplDgies~ The key and central issue is the development of the relationship and understanding of genetics and its implications for tobacco growing, cul~~ leaf, tobacco blends (filler and flsvour gracles) and product guality~ 2~ Cellular and Tissue~T~g~es a) Tissue culture pl0pal?li~ Micropropagation, the technique whereby Jpical or lateral bud or even other plant structures can be steriliscd and aseptically grovn on 1 culture medium has had considerable commercial impact in some areas of agriculture and floriculture. Its primary benefits are: (i) cional propagation of plants, particularly applicable for the maintenance and mass propagation of elite genotypcs by vegetative propagation, It is lost frequently applied where maintenance of an individual plant's specific characteristics are required (usually identified from a segregating plant populltien), To date it has been applied successfullv to a range of plants e,g, orchids, carnations, roses, ornamental plants, pineapple, ginger, pepper, rootstocks etc. (ii) The other primary application of this form of lass propagation is for the eradication of plant diseases~ Where apical and lateral bud meristems are excised land suitably treated e,g, heat) and cultured any residual or latent viruses present in the plant can be eradicated with significant ilnprovemenr. in plant vigour and performance (e~8, yield or fruit ~ualitr)~ 'Ihis approach has been applied to many plant types in the generation of nuclear stock aterial e,a, potatoes and strak~berries~ There art essentially two 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 where tissue culture propagation is practicable is Fhere this technology is used to generate parental material, subsequently bulked by traditional vegetative neans~ 1Si n Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill -6- ii· As a mass propagation technology for the production of tobacco plantlets, meristcm micropropasation could not be justified en a need or cost basis, Rowever, it has significance for the Indus~rr as a research tool that could provide the basis of an 'internal controll for establishing some key factors for cultivar use and development: a) true genetic stability and effect of local environment for regional comparisons and on the development of cultural practices for product improvement, b) season to season and agronomic factor effects on cropipreduct quality. Cloned genetic stability across a plant poF:!la:isn rould provide In excellent basis for studying and op:lir:ne harvesting and curing processing practices, c) clhte to climate factors and their effect on tobacc: qualities, d) effects of different and nominally established curing p~jciices~ In addition, paralleling the practice uith other crops, tissue cuirare techniques could be utilized to maintain elite tobacco breeding li;les in the production of hybrid seed, This technology can and has been used under t~o 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-breeding, (ii) use of CMS plants via tissue culture maintenance in the production of technologically based hybrid seed proC~~i:3~ b. Uscand.PPssible aPeli~alia~ of Lther Cul ture Anther culture tor haploid plant production) methodology relies on ereisian Inn propagation of parts of the male and female floFe: that hare undergone meiosis in the generation of haploid (n) tissue. The regenerated diploid (Zn) plants can under certain circumstances be produced from chromosome doubling using colchicine (6)~ ~hcre this can be induced, true homozygous plants can be produced (of:a referred to as instant 'in·brcds') that can have a variety of jppli:~ri~ns for plant breeding generallf (7), These Ire prkrilr: (i) identification and selection of haploid plants vith 'drsinble characteristics' that con be chromosome d:ubled to produce honozygous tin-bred) plants for breeding purposes or direct use, till for use in the development of breeding lines for hrb:id plant production, Plants produced in this manner ;hould in theory exhibit greater heterosis tvieour) because haploid derived in-breds are truly hoPotrsous uhereas those;roduced by conventional breeding are not, This is probably not highly relevant to tobacco, a plant that shoes limited hybrid vigour, suggested to be because Commercial tobacco cullivlrs are derived from a naturally occurring hrSrid (that gives rise to an amphidipioid plant), Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill _ ___I (iii) as a possible breeding tool for chrc~ron~l additions and substitutions. whrre ob~ious phenotypic selection criteria 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 'downstream' attributer hou~~e! becomes practically limiting when considering individual plant changes. C) C~l~l~rCChP~d~ The major research Effort in this area again using tobacco simply as a model plant has been directed towards creating genetic di~crsit~ thraurh aU~s ·dC~~· Cdt~ry q~~l~e 11 r~a fusi~ns with some research effort focused on insertion and ~nion of lanonic fragments (8,9,10), in addition, effort has been focused on utili;ing the spontaneous or induced variability that can be achieved in certain circumstances of plant cell culture, so called somatic variance. There are essentially two typer of somatic variance: (i) ~Sg~?_t~_nl~Be~~L~j4I~d somatic variation: sonaclonal variation t'nder certain conditions of plant culture, the CXCiSCd, organised tissue becomes de-differentiated as in the case of callus and simple cell culture, In this form, cells are encouraged to divide essentially as single cells, an event that leads to a degree of spontaneous genetic variation, the fre~ucpcy of which can vary with culture conditions (11,12,13). The expcri~~ntor will select culture conditions to enhance the specific Ireguc~cy of cellular variation in the pursuit of new or novel biological ~~riation. The value of the spontaneous variation or sonaclonal variation that exists in a population 15 I function of the ability to select for improvement. In the case of tobacco, its ~alue is limited primarily to selections that can take place at the in-vitro level. (ii) ~p&ail~l~Lcsl ~~biliSu In essence this is analogous to the above Lxeapt that the ~~riobility is induced or enhanced by the use of chemical lutp~ens, ThiJ approach extends pr enhances the freguencY of mutation and can be applied to plant tissue directlv increasing its potential value over the plant systems for which single cell regeneration protocols exist (1~,15,16), ClibPDF - v~~fastio.soni ·__·_ -A· The following conclusions an be made regarding the strcrurths and weaknesses of the use of cellular technologies, i) it overcomes any sexual barriers iii its major benefit is that it can potentially provide new genetic variability for tobacco since regeneration potential has been validated. iii) the major challenge is to identify selection criteria against Fhich commercially significant improvements fan be identified (cutrently this approach has relevance for tobacco improvement where selection criteria can be applied directly to the culture conditions such as climatic and nutritional stresses and disease tolerance and resistance. i spontaneous or induced variability is not always stable generation to generation Inevitably with :~a cellular approach, breeding strategies have to be identified for incorporating the new attributes of the new variety so produced into commercial cultivars. In practice, this technique although capable of creating new variations in the plant is limited currently by the selection criteriat plant improvements post growing ie leaf and final product improvement would necessitate unacceptably hish plant populations from which improvements would have to be selected, having been cured, aged and processed to cigarettes, 3, ss,erCf~c,C~ltir~9~~g In broad teres, plant genetic engineering is the process uherebv 'useful' genes can be identified in nature and functionally inserted into agriculturally important plants for which a defined need has been identified. This process usually follows the outlined sequence of events: (il identification and characterisation of a significant gene or genes. (ii) isolation of the DNA responsible for the identified gene and development of a gene construct in order to have functional significance in the recipient plant~ This will include 'prep9ntion' of appropriate regulatory, initiating and termination seoucnces in order that the gene operates and is expressed efficiently, a Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill ·· ·_ ·9- (iii) the potentially functional gene construct has to be inserted into the recipient plant using appropriate vector svstems~ This can necessitate the development and use of appropriate amplification and transformation vector systems to achieve an efficient number of transformed plant cells, (ieJ regeneration of the 'transfoned' plant cell population to whole plants needs to be followed by appropriate selection critP.ria, The molecular biologist is making considerable progress in facilitating the use and operation of these processes (2,it), He can define appropriate regulatory sequences such as light sensitive triggers which lead to direct apression of the gene in the leaf, This is an example of an increasing capability of tissue or organ specific expression, flovever, this technology by definition delaands a precise understanding of plant and biological processes and identified coeercial peals, hlthough this is In cleiting technology of the future the reality today 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 uhether the inserted gene is operating effecticeiy~ The challenge for the molecular biologist today is to be able to insert or delete genes in a highly specific manner, The rellit~ 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 phrsiolo8ial, biochcaical and molecular processes that control plant regulation and development. This is particularly true of tobacco and there is an urgent need to conduct basic research to define how these basic processes influence cownercially significant plant Jttriblltes such as basic plant and leaf qualities, smoke flsvour and other organoleptic sensations and to use this knowledge to engineer desired changes in the tobacco plant (1 similar approach has been referred to at international tobacco conference (IB)), To date genetic engineering a~ethodolaeies have been used to achieve el~phosate resistance and to produce plants capable of producing DT torin, Neither of these achievements needed a fundamental understanding of plant metabolism but rather are relatively simple single gene traits identified in bacterial cells, svsterns that were Fell studied and amenable to direct investigation and analysis, The immediate prospect of further copercklly significant single gene trait manipulations in the future POUld Seen relatively limited. Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill ·10· however, the process of genetic engineering has the lonn·term potential to substantially influence manfuacturinB costs, product qualities and plant attributes that will pioneer novel product changes and indeed it is possible to set some broad goals for the industry such as: (i) Hanieulatior~ sf_the nitrate_r~~a~e reeler cii, M_od_ifiE~a~~~f~a~_fii~~p~Fne ratios bLgenptir engi?~Z~e (iii) HoPific_a_ti~e ~s~qn4~~fl a~lpur..ll'.?~.~!etjc_ pa_t_hg~r ( iv ) ,R~e~E~J.aBF~if ultur a~kne~ts If Fe had 1 better understanding of the fundamental nature of disease tolerance and insect resistance in plants and the uptake and utilization of nutrients and fertilizers we could develop strategies for their mote efficient utilization. In order to focus the R b D effort and develop the appropriate technologies it Fill be necessary for the Industry to clearl~ define its commercial goals for the future. 4. ~lecdar BiOlDRr: p~_ne~ep:l_np_t_ec_h?IqU~s This area has been separated from specific genetic engineering to indicate some of the spin off benefits of fundamental molecular research that are finding commercial application, The work relates pliaarily to the use and application of RFIP technology, In simple terms the technique utilizes naturally occurring chafiges in the DNA strands that occur at sexual cell division, which in combination with highly specific endonucleases gives rise to an arrav of fragments unique to an individual plant, ~ series of molecular ~arke;s can be developed to characterise a particular fragment, In this manner it may be possible to obtain a molecular profile of an individual pilot and more importantly through the appropriate combination of plant breeding and molecular biology establish relationships between plant genetic information and observable traits, Such an approach will provide the basis for: 3) definition of traits in molecular terms providing a basis for trait enhancement b) facilitate the combination of traits into specific cultirars c) monitoring the introduction of traits into commercial cultirars d) to provide the basis of a selection technology either in terms of matching to original parent types or for novel genetic coabinations e) begin to provide focus for specific genetic engineering where more pragmatic approaches fail. f) of primary importance allow a shortening of plant breeding time scales in i~g~ing I(romlic II! I~Pdu~L i.~d LlliU. Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill -II- It is believed that the use and application of this technology vill provide a lore immediate application for molecular based techniques that has relevance for improving the tobacco plant both for agronomic and downstream benefit, 5, S~m_bi~lSjSSg i~~!i~P~IP~rX~?i~l?slo_LP ~ntknefit There has been increasing evidence that a range 01 bail micro-organisms other than the rhizobium type can facilitate the uptake of nutrients and in some cases partially protect against disease and water stress, Those of primary interest are of the m).corrhizal fungal type of Fhich there are essentially tvo types, ecto- and endo~nychorrizl, It appears that ectomycorrhila are of primary benefit to forest trees whilst YA endomycorrhiza tend to be associated vith a wide variety of agriculturally important crops including some annually planted material. dlthollah it has proved possible to culture ectornycbtrhiza in-vitro using continuous culture conditions it has not been possible to bulk produce T~ endomycorrhiza in this way, Production systems for YA mycorrhiza have been developed utilizing host plant spste~ns in controlled enviro~n~ents. The potential for use of this system for commercial benefit depends on: i) base material east ii) application rates required for specific plant types iii) cost savings on reduced :ertiliset use The use of YA ~ycorrhiza has not been commercially validated for tobacco, To date, the full benefits of using such s)abiotic soil micro-organisms has yet to be fully evaluated but Fould appear to offer some potential under defined conditions and night provide a basis for engineering additional beneficial changes in the organisms for mutual benefit, Co~cludiRe~ L~FtS The future is technically challenging, but the emerging technologies can and should be utilized to develop novel tobacco breeding strategies, designed to meet the future needs of the ~ndustry~ There will be a need for the Industry to carefully define its goals and to maintain a coRn~itment to fundamental research effort to achieve substantive improvements in tobacco, p Clit; PDF -!::!!::!!::!.f3 StlC.i: Dill ~ftt~r~ 1, Hicotiena, Droecdurcs for Lpcrinental Urc. USOb TEchnic~l Bulletin No. 1586~ 2, J. Schrll It II, Flint Cell TrPn~Iorn~tlon~ end Genetic Engineering in Plant ImprPveaent lad Sonltie Cell GeneticJ. Ed, Ylsil Icldclic Prers~ (1912) 3, Wttlnlbc, Y, (1975) Jlp, J, of Breeding 25 (1) ~9, 6. Blakeflee, 1,F~, Belling, J, (1921) J, Bared, 15, 195, i. Chih-thinr Chu, HlploidJ in Plant I~provacnt in Bleat Inproveocnt end Sonetic Cell GenctieJ~ Ed. Yltil Acaduie PresJ (1982)~ 8~ MelcheJI, C., end I~bib, C. (1971) Elolec, Ccn. Genlt, 133, 277. 9, White, O,U,R,, end Vesll, I, (1979), Thc~t~ Ippl, Gcnet. 54, 239. 10. Qckinl, E.C., george, O,, Price-Joncl, H,f,, end Povcr, J,B, (1977) Flint Scil Ictt~ 11, Slcrirtan, H,D,, end Helchers, C. (1969), Holec, Im, Ccnct, 105, 31i, 12, Lrkin, P,J,, end Scoveroft, W,R, (1981), Theor~ Ippl~ Flnet, 60, 197. 13, Burt, I,G,, end Natringer, D,F~ (1976), J, Hcrcd, 67, 381. 14, Widhol~, J,B. (1977). In 'Fi;lnt Tissul Culture end its Biologieal Applic~tiaos' Ed~ B~rr, Springar, Berlin, 15, Sc~c:aft, U,R. (1977), Adv, ~rono~ 29,39, 16, Thoo~l, E~, King, P.J., end eot~kus, I.(19i9), 2, Pfllnttn, Zuchtg, 82,1, ii. Proceedings of 'COBEBTI\' conference, Greece (Scpt '8j). 18, Slcond Internaticnal Tobpceo Exhibition, Fio)uond Yirninil (1986), ClibPDF - v~~fastio.soni