Indian Journal of Biotechnology Vol 3, April 2004, pp 171-184 Tobacco (Nicotiana tabacum L.)-A model system for tissue culture interventions and genetic engineering T R Ganapathi', P Suprasanna', P S Rao' and V A Bapat'? IPlant Cell Culture Technology Section, Nuclear Agriculture and Biotechnology Division Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India 2lndo-American Hybrid Seeds (India) Pvt Ltd, Bangalore 560 070, India Tobacco (Nicotiana tabacum L.) has become a model system for tissue culture and genetic engineering over the past several decades and continues to remain the 'Cinderella of Plant Biotechnology', An ill vitro culture medium (Murashige and Skoog, 1962), based on the studies with tobacco tissue cultures, has now been widely used as culture medium formulation for hundreds of plant species. Studies with tobacco tissue culture have shed light on the control of ill vitro growth and differentiation. Further, induction of haploids, microspore derived embryos and selection of mutant cell lines, have been achieved successfully. Tobacco has also been employed for the culture and fusion of plant protoplasts, providing invaluable information on way to explore the potential of somatic hybridization in other crops. Optimization of genetic transformation, using Agrobacterium tumefaciens and A. rhizogenes, has been central to the cascade of advances in the area of transgenic plants. Developments in the field of molecular farming for the expression and/or production of recombinant proteins, vaccines and antibodies are gaining significance for industrial use and human healthcare. Keywords: genetic transformation, molecular farming, plant biotechnology, plant cell and tissue culture, recombinant proteins, tobacco IPC Code: lnt. CI.7 A 01 H 4/00, 5/00, A 61 K 35174,35176,39/002,39/02,39/12; C 12 N 15/00, 15/01, 15/05, 15/08, 15/09 Introduction fusion and plant genetic engineering, which have Advances in plant biotechnology have made a shown tremendous potential for application in crop significant impact in the area of in vitro culture, improvement. genetic manipulation and newer approaches in Majority of the discoveries in the field of plant cell, experimental plant biology!". Tn the past 2-3 decades, tissue culture and molecular biology have originated plant tissue culture has undergone an exciting from the experimentation with tobacco plants 1,7. As a development, providing knowledge about totipotency, result, tobacco has become a model system as the differentiation, cell division, cell nutrition, meta- 'Cinderella of Plant Biotechnology'. This plant has bolism, radiobiology, mutations and cell preservation. been found to be an extremely versatile system for all While microbial systems have been successfully used aspects of cell and tissue culture research. Working for the production of antibiotics and other useful with tobacco tissue cultures, Murashige and Skoog" compounds, it is now becoming possible to use plant devised an in vitro culture medium that has become tissue culture system to produce a wide range of the widely used nutrient formulation for an ever- biologically active compounds, like alkaloids, growing range of plant species. In vitro studies with steroids, phenolics, vitamins and other useful tobacco tissue culture, using different physical and chemicals. Further, cell culture could be used for the chemical factors, have provided insight into the purposes of specific biotransformation of organic control of growth and differentiation. Induction of molecules. Other more sophisticated approaches of haploids and selection of mutant cell lines, owing to tissue culture include in vitro selection, protoplast the experiments with tobacco tissue cultures, have become useful tools. Isolation, culture and regeneration of plants from protoplasts as well as * Author for correspondence: somatic hybridization, all have been accomplished Tel: 91-22-25593276; Fax: 91-22-25505151 E-mail: [email protected],n using tobacco system, which has provided invaluable 172 INDI,\N J 1110TITIINOL, i\1'1{1I. 200,\ information on ways to explore the potential or 18°C was optimum. l ligl: light intensity was found to somatic hybridiz.uion in other crops. be inhibitory 1'01' shoot-bud formation in tobacco". Tobacco (Nicotiana /(/!J(fCIIIII L.; 211=4,,=:18) is a Shoot-bud regeneration was observed in the presence natural allotetraploid Iormcd through hybridiz.uion or blue ligill, whereas rooting occurred in red light. between two diploid (211=2--1) progenitors, N. Extensive studies were carried out on various sylvestris and N. torncntosijonnis npproxim.ucly (j parameters coni roll i ng flower bud format ion and million years ago'), Within J months or time, ;1 induction Irom epidermal cells or tobacco dihnploids, tobacco plant goes from seed to next generation seed which were ohtaiucd from Icrrilc flowers raised [rom and generates up to a million seed per plant. Scaling peels or mule sterile pl.uus ':'. Thus, hypohaploid', with up to hundred or thousands or acres is wry rapid. less ih.u: (dihuploidx) chromosomes or epidermal cell From the early experiments with tile uptake or n.ikcd culture were obtained, which were Ircc Irom DN/\ to the recent vector mediated gene trnuxfcr, endogenous hormonnl inn ucncc 1-', mak i ng it easy to tobacco has remained ;IS the most sought ;II'ICI' study tile chnngcs or single cell leading to research system. The first Iransgl'nic pl.uus arc dilfcrcnti.u iou. l licks and Sussexl(, cultured flower produced in tobacco. /\Iso, the experiments rcl.ucd to primordia or tobacco (Wisconsin J8). Then, buds with plunt trausform.uion, gl'ne expression and gene only sepal primordia were cultured, leading to the stability have all been worked out using to\):IC((1, /\11 I1L'Ld, xt.uucn and carpel primordia in acropetal the rirsl achievements or pLUlt genetic l'nginl:erillg .uc sequence on tile ;qK'X, Org;ln primordia or tile flower mostly bused on tile \\'01'1-: witl: tobacco. CIIITL'lllly, buds or tobacco were also subjected to surgical this plant is being employed in studies on the man i IHII:I tionx. production or useful recombinant proteins, aniibodicx There have been m;lI1Y reports on organogenesis in and special chemicals Ior USl~ ill medicine and tobacco tissues and calli. Rcgcncr.uion 01' planners industry, In the ;1I\:a or plun: tissue culture and gl'nL'lic W;IS reported lrom the cultured leal' cxpl.mts of m.tnipul.uion, tile work on tobacco is so en0l'l110US tobacco". Bud Iorm.uion was induced in tobacco and vast that it cannot be summed up in a single petiole cultures". Plants were regenerated Irom callus compil.uion. In this article, therefore, only those obtained j'Ol'll1 cotyledons or a hybrid (N. S{(OJlC/OIlS x aspects or plant tissue culture rcxcurch have been N. /({/)({C{(III) ;1I1d brown spot resistance W;lS trans- described in which tobacco has been a role model and tcrrcd Irom N. S{((I\'e/OIlS to N. /({b(fC{(IIII~. Cellular paved tile W;IY lor improvement ill other crop systems. and ultra structural changes in regenerating shoots in robacco h.ivc also been studicd". Similarly, experimental eLlla on the effect or growth regulators, Studies Oil ill vitro Cultures temperature and light on tobacco tissue cultures have In a series or publications, Skoog aIIII co-wer- contributed to the basic knowledge about the role or 1,..;us 10·10- reporter I on tI'ic III vitr.o sIloot lHieI' meIucuon. several Iuciors on differentiation that have been in Nicotiana with a systematic nppronch. They extended to other crops. demonstrated tilat dif'Icrcuti.uion into shoot and root could be induced by tile manipulation or the balance or an auxin (1/\/\) and a cytokinin (Kn). Tile presence Studies on Anther and Pollen Culture or adenine or Kn in the medium resulted in the /\ surveyor literature pertaining to anther culture promotion or shoot bud dilfcrcnti.uion, while tile represents that this technique has been successfully effect or cytokinin was modified by other components utilized Ior tile production or haploids and evoked in the medium particularly by 1/\/\ or N/\/\. /\ higher considerable interest among plant breeders and level or auxin induced rooting lron: tobacco callus gcncuc,.rsts I'0-.I '1'1ic maj'Ior a<.vantage was tIic recovery cultures. The other physical fuciors, such as or homozygous diploids 1'01'breeding and 1'01'cstab- temperatures, /)11, photoperiod and sucrose lishing haploid cell cultures 1'01'mut.uiounl studies ;1I1c1 concentration, influenced tile diffcrcnti.uion 1'1'0111 gellctic manipul.u ion c xpcrimcnts. Extcuxi vc studies callus cultures. For instance, Skoog':' studied tile conducted on/witl: anther culture or tobacco Il:1S led to effect or a range 01"temperatures (.'i-JJoC) 011 tobacco the idcnufir.uion or criiicn] ractors I'm succcssfu] callus growth and differentiation. I Ic observed til:lt development 01' haploids. Nilsch"2 reported haploid growth or tile callus increased with the rise in plnnttcts, 1'01'the Iirs: time, 1'1'0111N. /({baCIIIII. This was temperatures up to JJoC but 1'01'shoot-bud lonnation, followed by investigations 011 the regeneration 01' GANAPATHI et al: TOI3ACCO FOR TISSUE CULTURE INTERVENTIONS 173 haploid plantlets from anther culture of several and hormonal composition of the medium, species of Nicotiana. Three promising lines of developmental stage of microspore and ontogeny of tobacco were raised through anther culture of hybrids pollen embryos as well as the growth condition of the (line MC-161O x Coker 139)23. The new lines exhi- donor plants. The usefulness of haploids is based on bited higher resistance to bacterial wilt and black the assumption that homozygous inbred lines can be shank without loosing the agronomic and chemical readily and rapidly achieved. Haploids could offer as features of MC-161O. Similarly, double haploids of supplementary breeding lines for breeding tobacco were raised using anther culture", which programmes and the knowledge gained about culture showed high yield, good quality of curved leaves and method, treatment and growth regulators can be disease resistance characters, achieved in a much applicable for anther culture in other crops.