A Model System for Tissue Culture Interventions and Genetic Engineering
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. shorter period as compared with the conventional breeding practice. In China, new varieties of tobacco Studies on Genetic Variation and Mutant Selection (Tangu 1, 2 and 3) have been released using anther Mutant selection through tissue culture has become culture by Shangtung Institute of Tobacco and are possible due to the occurrence of a high degree of being cultivated in about 20,000 acres. variability (sornaclonal variation) in cell cultures. The First pollen mitosis was reported to be the critical fact that millions of cells can be cultured in shake stage for Nicotiaua anther culture". The physical flasks has permitted the selection for specific factors, like light, temperature and pH, also mutations at cellular level and to regenerate plants influenced the pattern of response. For example, from selected cell lines for specific mutations at anthers of tobacco kept in the dark at the beginning of cellular level, and to regenerate field plants", Various the experiment responded better". The nitrogen types of chemical and physical mutagens have been starvation of anther donor plants increased the anther applied to callus, cell suspensions or protoplasts in response and embryo yield'". Chilling of anthers prior attempts to isolate desirable mutants. to culture or a cold treatment of flower buds also Plant cell and tissue cultures have been shown to 26 28 enhanced embryogenesis in tobacc0 . . Further, cause or allow many genetic changes to take place. 29 Deaton et 0/ studied the vigour and variation Variability in DNA content and nuclear volume was expressed by pollen plantlets of tobacco and noticed in regenerating cultures of tobacco". Increase conducted their evaluation trials. However, in ploidy, chromosomal rearrangements, and 30 Zeppernick et al studied the relationship between abnormal chromosomes and altered DNA content in ploidy level, morphology and concentration of regenerating plantletshave been effectively used in nicotine of haploid and doubled haploids raised tobacco improvement programmes. The fate and through anther culture. possible role of aberrations (dicentric chromosomes) Depending upon species and other factors, pollen were studied on the development of the different grains either developed into embryos or formed embryonic phases ", Extensive chromosomal callus, which then differentiated into embryos upon chimeras were observed in callus derived regenerants transfer to an appropriate medium. Normal embryo- of tobacco and transmission of chromosomal genic process was usually observed, showing chimeras was reported to first and second selfed 36 globular, heart and torpedo stages of embryos:". progeny plants . Haploid plants produced large number of flowers and For mutant selection, tobacco cells are a favourite these were generally smaller in size. However, in material due to the regenerative ability coupled with tobacco anther cultures of Nicotiana, the pollen grains the capability of tobacco callus to form fine cell directly produced haploid plantlets without suspensions that grow rapidly. Carlsorr" isolated intervention of a callus mass. Moreover, besides the auxotrophic mutants of tobacco using selection development of normal embryos, numerous abnormal technique developed for animal cell culture. The embryos in various stages of differentiation were nucleotide 5' brornodeoxyuridine was used to select detected". Albino plants have also been observed in six tobacco clones showing partial growth require- 32 tobacco anther cultures . ments ". Clones were selected in N. plutnbaginifolia 38 In general, success in the production of embryos requiring isoleucine, leucine and uracil . Tempera- from anther cultures depends to a large extent on ture sensitive variants of N. tabacutn were also various factors such as method of culture, nutritional isolated successfully'". Of the specific selection 174 INDIAN J BIOTECHNOL, APRIL 2004
agents, chlorate has been found to be the most foreign genetic material, such as organelle or DNA, effective with plant cells. Nitrate reductase converted into genome. For the first time, Nagata and Takebe55 chlorite that kills plant cells. The cells lacking nitrate descri bed the regeneration of whole plants from reductase survived and could be isolated. This system mesophyll protoplasts of N. tabacum. Later, others was so effective that Muller and Grafe" isolated also reported regeneration of entire plants from 56 double recessive mutants in N. tabacum. mesophyll protoplasts of tobacc0 .57. Protoplasts Like auxotrophic mutants, there are many reports derived from haploid tobacco have also shown the wherein tobacco cell lines resistant to various regeneration potential ". Leaves are considered an compounds have been isolated. Widhlom41 utilized ideal source of protoplasts for tobacco because they tryptophan analogue,S methyltryptophan to select N. can be kept in abundant supply by ill vitro shoot tip tabacum resistant cell lines. In another case, culture or from greenhouse grown plants. Thus, a suspension cultures resistant to ethionine were large quantity of mesophyll protoplasts of tobacco can isolated'", Selection for resistance to several be isolated from diploid or haploid plants. Moreover, antibiotics, initially with the streptomycin, produced high percentage of the protoplasts can reform cell 43 organelle encoded mutations in Nicotiana . wall and divide to produce a callus. Protoplasts have Tobacco cell lines tolerant to high NaCI levels also been isolated from epidermis, cell suspensions (0.88%) have been isolated":". The regenerated and stem callus, beside mesophyll cells. Further, plants from these line also retained tolerance through parameters affecting the initiation of protoplast two successive sexual generations with the enhanced division of haploid and diploid tobacco have been levels of tolerance. Dix46 obtained NaCI tolerant evaluated ". plants in N. syLvestris and observed that the character Somatic hybridization has assumed great was transmitted to the next generation. Different cell significance among many applications of protoplast lines of N. tabacum resistant to various herbicides, technology. The experiment to select somatic hybrids such as Arnitrole'", Bentazone'", Paraquat", with the aid of albino mutant was first performed on Picloram5o have also been isolated. Moreover, tobacco haploids'". Similar results were obtained with glycerol and lactose were successfully utilized for the interspecific combinations of non-allelic light detecting mutant cells of tobacco". sensitive albino mutants of N. sylvestris": Non-allelic Other application of mutant selection includes albino mutants were also used for N. tabacum and N. selection for disease resistance. Plant resistance to knightiana, which was unable to produce shoots virus and its transmission to the progeny of tobacco forming potential of tobacco and chlorophyll has been reported.". Single clones of tobacco tissue synthesis of N. knghtiana. The first somatic hybrids varied in their susceptibility to virus and fungal plant was reported after a fusion of N. glauca x N. infections and their multiplications. Population of langsdorffii protoplasts with the help of sodium rnutagenized haploid cells of tobacco was plated in a nitrate'". Protoplasts from cell line of N. sylvestris medium containing inhibitory concentrations of resistant to kanamycin that have no ability to form methionine since Pseudomonas tabacii, causal shoots were fused with the protoplasts from N. bacteria of wild fire disease, produces methionine. In knightiana that do not form shoots in vitro. Somatic vitro survived cells, produced on inhibitory levels of hybrids were obtained on the basis of kanamycin methionine, were further used for generating disease resi.stance an d on thee capaoaciti y to regenerate sooh ts 63-. resistant varieties ". In callus derived plants of an Vigorous growth patterns of hybrid colonies were also interspecific hybrid of tobacco, variability for plant used to isolate hybrids from N. glauca and N. height, number of days to flower, number of leaves langsdorfii". Several fusion experiments on tobacco and leaf area were also reported.". was conducted using plastorne mutants in relation to Studies on Isolation, Culture and Fusion of chlorophyll synthesis and cytoplasmic male sterility Protoplasts (CMS) and segregation of mixed cytoplasm into 65 Isolation, culture, fusion of plant protoplasts and mutant and wild type plastorne were observed ,66. regeneration has generated great hopes for plant Gamma irradiated protoplasts, which were carrying a improvement. Absence of cell wall allows the fusion functional nptII gene, were fused with un-irradiated between protoplasts, derived from two diverse plants kanamycin-sensitive recipient protoplasts to yield that are sexually incompatible, as well as uptake of a asymmetric hybrids 67. GANAPATHI et al: TOBACCO FOR TISSUE CULTURE INTERVENTIONS 175
The somatic hybrid plants generally have chromo- compounds and referred to as biotransformation, for some number more than 4n due to the fusion of more example stereospecific reduction of codeine." and than one protoplast. The hybrid nature of most of the conversion of N-diphenylurea into Dvglucose". somatic fusion products could be demonstrated by Microsomes from tobacco tissue cultures were also their chromosome analysis, isoenzymes, morpho- found to convert squalene 2, 3 epoxide into cyclo- logical comparisons or growth characteristics. Prat68 artenol'". These examples opened up the possibilities examined mutations arised following protoplasts of identifying a high yielding nicotine cell line. The culture of highly inbred line of N. sylvestris and also a work on biotransformation in Nicotiana pertaining to line derived from it after five consecutive cycles of several compounds has shown the possibilities of androgenesis and chromosome doubling. A system utilizing plant tissue and cell cultures for the isolation was devised in N. tabacuni" and N. sylvestri/o where of several useful compounds. somatic embryos were directly produced from cultured protoplasts without callus formation. The Studies on Transgenic Tobacco and Applications feasibility of this technique at the application level Studies undertaken by Uchimiya and Murashige will further determine the practicability of somatic with tobacco DNA and tobacco protoplasts showed hybrids as to be complementary to classical plant 81 uptake of homologous DNA . Suzuki and Takebe breeding methods. Besides protoplast fusion, demonstrated the insertion of viral DNA into extensive experiments have been conducted on mesophyll protoplasts of tobacco'", Agrobacterium tobacco protoplasts for various other aspects of tumefaciens, a soil bacterium, has been known to genetic manipulation. induce crown gall disease in many plants. During infection process, the bacterial plasmid integrates into Studies on Secondary Metabolites and Biotrans- plant genome and influences the plant tissue to form formation galls. Using A. tumefaciens, tobacco cell suspensions Plant cell cultures synthesize secondary were transforrnedv''" and showed the presence of metabolites (biochemical totipotency) and this has nopaline, the plasmid DNA encoding amino acid. The significance not only for basic research but also for initial experiments with tobacco generated interest in industrial processes. Ohta and Yatazawa" reviewed the use of A. tumefaciens system for transformation the work on nicotine production in tobacco tissue and, in recent years, its plasmid has become an cultures. Nicotine in the range of 0.1-1 mg/mg dry wt important vector for gene transfer. Venkateswarlu and 85 was detected in cell cultures of tobacco; whereas, 29 Nazar presented evidence by using tobacco mg/mg of dry wt was found in the roots of intact chloroplasts that Agrobacterium-mediated transfor- plants 72. It was observed that nicotine synthesis in mation could be used to introduce foreign genes into tobacco could be regulated by exogenous supply higher plant chloroplasts by site-specific homologous without recourse to organogenesis". A close relation- recombination. ship has also been demonstrated between cell organi- An unusual approach to transfer genes was zation and nicotine production in tissue cultures of demonstrated in Nicotiana using irradiated pollen'". tobacco". By obtaining single cell clones, high Seeds were produced in N. forgetiana by pollinating it yielding strains of nicotine were isolated. The with irradiated pollen of N. alta. Although, the technique of single cell plating was used and a irradiated pollen produced pollen tube but failed to number of cell colonies were isolated from cell fertilize the ovule. However, most of the plants cultures of N. cellrustica, which showed wide produced showed flower colour and other characters variations in their growth characteristics and ability to of N. alta. In another approach, it has been shown that synthesize nicotine". Further, the nicotine content in swelling of germinating pollen grains could take up tobacco showed relationship with the ploidy level of DNA or bacteriophage", The progenies of N. glauca, the planr'". Besides nicotine, a number of other derived from N. glauca pollen treated with N. secondary products, such scopoletin, esculetin, langsdorffi DNA, produced tumours. The tobacco bergapten, cycloartenol, citrostradienol, citroastradiol, protoplasts could also be transformed with disarmed cycloeulenol, obtusifoliol " and aliphatic alkanes 77, Ti plasmid vector pG 3850. Further, PN CAT have been detected in tobacco cell cultures. Nicotiana containing a chimeric PNOS CAT gene construct cell cultures also have the ability to transform organic gave rise to chloroamphenicol resistant calli. 176 INDIAN J BIOTECHNOL, APRIL 2004
A simple and general method for transformation, using tobacco leaf disks, was developed", which has Tobacco leatpieces were infectedwi1h become the standard method for producing transgenic Agrobacterium (5 X 10' cells/mlJ and co- cultivated on MS + SAP (1 mgAt) + NAA tobacco. Leaf disks (1 cnr') were excised from in vitro (0.1 mg/lt). Alter 3 days the same were' shoot cultures for preculture on MS medium with BA transterred to sam e media containing CEtotaxime (400 mglltJ and NAA. After 2 days, the leaf disks were co- cultured with overnight grown A. tumefaciens culture. After 30 min of co-infection, the leaf disks were Transterto MS + SAP (1 mglltJ + NAA blotted to remove excess bacteria and transferred to (0.1 mgAt) + Cet
Table 2-Production of biopharmaceuticals for human health in transgenic tobacco plants
Protein Application Expression level Reference
lll Human Protein C Anticoagulant 0.01% SLP Crammer et al l14 Human granulocyte macrophage calony Neutropenia Giddings et al stimulating factor Human somatotropin Growth hormone 7% SLP (chloroplasts) Staub et alliS l16 Human erythropoietin Anemia 0.01%SLP Kusnadi et al ill Human epidermal growth factor Wound repair and cell proliferation 0.01%SLP Crammer et al Human interferon beta Hepatitis Band C 0.01% FW Kusnadi et all16 Human haemoglobin alpha, beta Blood substitute 0.05% SP Crammer &
Weissenborn I 17 Human homotrimeric collagen Collagen 0.01%FW Ruggiero et alliS l14 Angiotensin converting enzyme Hypertension Giddings et al Alpha Trichosanthin from TMY-U1 HIY therapy 2% SLP Giddings et all14 subgenomic coat protein 11l GIucocerebrosi dase Gauchers disease 1-10% SLP Crammer et al
FW: fresh weight, SLP: soluble leaf protein, SP: seed protein, -: not reported plant synthesized product is a tobacco derived incidence of infectious diseases and the expense of antibody targeting gum disease. Most of the tobacco immunization programme limit the use of available types secrete sticky compounds on the outer leaf vaccines for large segments of population. Vaccine/ surface as a first line of defence and some varieties antigen coding genes for traveller's diarrhoea, produces 16% of the leaf dry weight as gum. hepatitis B, gastroenteritis, foot and mouth disease, Scientists of the University of Kentucky isolated a mink enteritis, swine fever, hog cholera, rabies, swine promoter to control the type of compounds that are transmissible gastroenteritis, dental caries, auto- secreted by tobacco leaf hair. Targeting the immune diabetes and cholera have been expressed in recombinant proteins in tobacco gum offered several different plant systems including tobacco (Table 3)124. advantages and purification would be easier and much 137. The first study on the production of plant-based cheaper!". vaccine was done in tobacco with the hope of Various types of antibodies are used in human developing a less expensive product 130. It was also medicine for diagnostic as well as for therapeutic shown that rHBsAg self-assembled into sub viral purposes 122. Recombinant antibodies have been particles, identical to the plasma and yeast derived 122 produced in transgenic tobacco plants. The expression HBsAg specific antibodies in mice . The expression and assembly of immunoglobulin (lgG heavy and of cholera toxin B was demonstrated in transgenic light chains) led to the production of other antibodies, tobacco chloroplast, resulting in the accumulation of like IgG-IgA antibody against a surface antigen of 4.1 % of total soluble leaf protein as functional CTB Sterptococcus mutans designed to prevent tooth oligomers'i". Further, binding assays confirmed the decay. Estimates of production costs for an antibody correct folding and disulfide bond formation of the in plants indicate as much as 10 to 20-fold lower costs plant derived CTB pentamers. The expression of C- per gram compared to produced by cell culture. Since terminal region of merozite surface protein plant based expression levels are low, chloroplast (PfMSP119), a potential malaria vaccine candidate, transformation may offer as an alternative. As there was reported in tobacco 128. Immunoblot assay are 10,000 copies of chloroplast genomes per cell, this indicated that transformed plant expressed MSP119 can facilitate the introduction of 10,000 copies of displayed structural and immunological charac- foreign genes per transformed cell and subsequently teristics identical to the E. coli expressed protein. This can boost several hundred-fold increase in gene presents a significant step towards the development of expression compared to nuclear transformation 123. low-cost subunit vaccine against malaria. . Vaccines are of prime significance for the human The tobacco cell line (NT -1) was transformed with health. However, in many developing countries, the hepatitis B virus's' gene, coding for surface GANAPATHI et 01: TOBACCO FOR TISSUE CULTURE INTERVENTIONS 179
Table 3-Vaccines produced in transgenic tobacco plants
Disease/Antigen Origin Expression level Reference
Traveller's Diarrhoea Enterotoxigenic E. coli 0.001% SLP Haq et 01124 (Heat labile enterotoxin B) 125 Hepatitis B (HBsAg) Hepatitis B virus 0.0066% SLP Mason et 01 Gastroenteritis Norwalk virus 0.23% SLP Mason et 01125 (Norwalk virus capsid protein) Immunocontraception (ZP 3) Murine Fitchen et 01126 Malaria (epitopes derived Plasmodium sporozoites 0.0035% SLP Turpen et aim from sporozoites) Plasmodi um falciparum Ghosh et ai128
Swine fever (Hog cholera, EO, El & E2) Swine fever virus Kapusta et ctf129.
Dental caries (SpA antigen) Streptococcus inutans 0.02% SLP Mason & Arntzen 130 Cholera(CT-B) Vibrio cholerae Arakawa et ai131 Colon cancer Verch et ai132 Influenza Influenza virus Beachy et ail33 134 Lymphoma (Tumour derived ScFv epitopes) Mc Cormick et ctf , Post--surgical/burn infections Pseudomonas aeruginosa Stackzek et ai135 Epitope of outer membrane protein F Human cytomegalo virus Cytomegalo virus 0.02% SLP Tackaberry et 01136 Swine Transmissible gastroenteric virus Corona virus 0.20% SLP Tuboly et ai137
SLP: soluble leaf protein, -: not reported anti•gen':".138 T_IwoC pant transrorrnanon. vectors Conclusions pHERlOO and pHBSlOO with and without endo- Tobacco has become the plant system of choice for plasmic reticulum retention signal, respectively were almost all the aspects of cell and tissue culture used for transformation. The integration of the research. Majority of the experimental discoveries in transgene was analysed by PCR and southern blot the field of plant cell, tissue culture and plant hybridization, and expression level was determined by molecular biology owe their inception to the studies ELISA. The maximum expression of 2 ug/g fresh with tobacco. Trends in plant biotechnology research weight and 10 ng/ml of spent medium was reported in show a substantial increase in research publications pHERI00 transformed cells. Western blot analysis on tobacco from 123 to 1396 from 1980-1990 to confirmed the presence of 24 kDa band specific to 1990-2000. Next to tobacco, arabidopsis has been HBsAg in the transformed cells. The buoyant density now the model plant for molecular research. in CsCI of HBsAg deri ved from pHBS 100 However, researchers all over the world still continue transformed tobacco cells was determined and found to use tobacco for a wide variety of plant research to be l.095 gm/mJ. The secretion of HBsAg particles programmes. Tobacco as a model system has played a by plant cells into the cell culture medium was major role in the advancement of plant science and reported for the first time. has been used as a tool in gaining fundamental knowledge in diverse areas of plant biology. Recent The availability of genetic transformation methods advances in the field of molecular farming have used of. plants has broadened the type of experimental tobacco as a 'plant factory' for the purposes of problems, the diversity of cell types that can be developing production system for recombinant approached and the transfer of useful genes. These proteins, pharmaceuticals, vaccines, industrial investigations promise to make the plant based enzymes and antibodies. Several companies are systems extremely attractive and powerful for crop already using this technology for commercial improvement, besides studying gene regulation and production. Vector Tobacco Inc., Durham, USA has developmental biology in higher plants. developed a variety of genetically engineered tobacco 180 INDIAN J BIOTECHNOL, APRIL 2004
plants. One of them is nearly nicotine-free. Based on 18 Prabhudesai V R & Narayanaswamy S, Organogenesis in the present developments, it appears that, in future, tissue cultures of certain asclepiads, Z Pflarizenphysiol, 91 (1974) 181-185. tobacco being a non-food crop and having apathy 19 Lloyd R, Tissue culture as a means of circumventing from non-cigarette lovers, will assume the role of a lethality in an interspecific Nicotiana hybrid, Tob Sci, 19 most useful crop for molecular farming, leading to (1975) 4-6. better industrial and human healthcare options. 20 Arai M, Saito T, Kaneko Y & Matsuhima H, Cellular and ultra-structural changes of regenerating shoots from tobacco References iNicotiana tabacum) internodes cultured in vitro, Physiol Plant, 99 (J 997) 523-528. I Brar D S & Khush G S, Cell and tissue culture for plant improvement, in Mechanisms for plant growth and 21 Maheshwari S C, Rashid A & Tyagi A K, Haploids from improved productivity-Modern approaches, edited by A S pollen grains-Retrospect and prospect, Alii J Bot, 69 Basra (Mercel Dekker Inc, New York, USA) 1994, 229- (1982) 865. 278. 22 Nitsch J P, Experimental androgenesis In Nicotiana, 2 Chopra V L et al, Applied plant biotechnology (Oxford & Phytomorphol, 19 (1969) 390-404. IBH Publishers, New Delhi) 1999. 23 Nakamura A, Yamada T, Kadotani N & Itagaki R, 3 Ghosh S P, Biotechnology and its application ill Improvement of flue-cured tobaco variety MC 1610 by horticulture (Narosa Publishing House, New Delhi) 1999. means of haploid breeding methods and some problems of 4 Rao P S, Suprasanna P & Ganapathi T R, Plant this method, in Haploids ill higher plants-Advances and biotechnology and agriculture prospects for improving and potential, edited by KJ Kasha (University Guelph Press, increasing plant productivity, Sci Cult, 62 (1996) 185-191. Guelph, Ontario) 197:1,277-278. 5 Rao P S & Suprasanna P, Augmenting plant productivity 24 Wark D C, Doubled haploids in Australian tobacco through plant tissue culture and genetic engineering, J Plant breeding, SABRO}, 2 (1977) 19-23. Bioi, 26 (1999) 119-127. 25 Sunderland N & Wicks F M, Embryoid formation in pollen 6 Soh W & Bhojwani S S, Morphogenesis in plant tissue grains of Nicotiana tabacum, J Exp Bot, 22 (70) (1971) 213- cultures (Kluwer Academic Publishers, Dordrecht) 1999. 226. 7 Flick C E & Evans D A, Tobacco, in Hand book of plant 26 Sunderland N & Roberts M, New approach to pollen cell culture, vol 2, edited by W R Sharp, D A Evans, P V culture, Nature (Lolld), 270 (1977) 236-238 Ammirato & Y Yamada (Collier Macmillan Publishers, 27 Sunderland N, Plant cell and tissue cultures-Principles London) 1984, 606-630. and applications (Ohio State University Press, Ohio, USA) 8 Murashige T & Skoog F, A revised medium for rapid 1978. growth and bioassays with tobacco tissue cultures, Physiol 28 Reinert J & Bajaj Y P S, Anther culture haploid production Plant, 15 (1962) 473-497. and its signi ficance, in Applied and fundamental aspects of 9 Okamura J K & Goldberg R G, Tobacco single copy DNA plant cell tissue and organ culture, edited by J Reinert & Y is highly homologous to sequences present in genomes of its P S Bajaj (Springer-Verlag, Berlin) 1977,251-267. diploid progenitors, Mol Gen Genet, 198 (1985) 290-298. 29 Deaton W R, Collins G B & Nielsen M T, Vigour and 10 Skoog F & Tsui C, Chemical control of growth and bud variation expressed by anther derived doubled haploids of formation in tobacco stem segments and callus cultured in burley tobacco tNicotiana tabacum L.) II Evaluation of first vitro, Am} Bot, 35 (1948) 782-787. and second cycle doubled haploids, Euphytica, 35 (1986) 11 Skoog F, Growth and organ formation in tobacco tissue 35-41. cultures, Amer J Bot, 31 (1944) 19-24. 30 Zeppemick B, Schaefer F, Paasch K A, Schmitt B & 12 Skoog F & Miller C 0, Chemical regulation of growth and Neumann K H, Studies on the relationship between ploidy organ formation in plant tissue cultures in vitro, Symp Sac level morphology the concentration of some phytohorrnones Exp Bioi, 11 (1957) 118-131. and the nicotine concentration of haploid and doubled 13 Skoog F, Growth and organ formation in tobacco tissue haploid tobacco tNicotiana tabacum) and NICA plants, cultures, Amer J Bot, 31 (1944)19-24. Plant Cell Tissue Organ Cult, 38 (1994) 135- 141. 14 Thorpe T A & Murashige T, Some histochemical changes 31 Ferrie A M R, Palmer C E & Keller W A, Haploid underlying shoot initiation in tobacco callus cultures, Amer embryogenesis, in In vitro embryogenesis in plants, edited ) Bot (Abst), 55 (1968) 703. by T A Thorpe (Kluwer Academic Publication, Dodrecht) 15 Tran Thanh V, Richard L & Riker A J, An experimental 1995, 309-344. model for analysis of plant/cell differentiation: Thin cell 32 Devreux M, New possibilities for the in vitro cultivation of layer, concept, strategy, methods, records and potential, in plant cells, Eur Spectra-Sci Tech Rev Eur Communities, 9 Androgenic haploids, edited by D Durzan & I K Vasil, lnt (J 970) 105-110. Rev Cytol Suppl, II A (1990) 195-223. 33 Suprasanna P & Rao P S, Selection of mutants using plant 16 Hicks G S & Sussex I M, Development in vitro of excised cell and tissue culture techniques, in Advances in plant flower primordia of Nicotiana tabacum L., Can} Bot, 48 physiology, vol 1, edited by A Hemantaranjan (Scientific (1970) 133-138. Publishers, India) 1997, 103-122. 17 Gupta G R, Guha S & Maheshwari S C, Differentiation of 34 Beriyn M B, Patterns of variability in DNA content and buds from leaves of Nicotiana tabacum L. in sterile nuclear volume in regenerating cultures of Nicotiana cultures, Phytomorphology, 16 (1966) 175-182. tabaccum, Can} Genet Cytol, 25 (1983) 354-360. GANAPATHI et al : TOBACCO FOR TISSUE CULTURE INTERVENTIONS 181
35 Toncelli F, Martini G, Giovinazzo G & Ronchi V N, Role important plants, edited by G M Reddy (Hyderabad, India) of permanent dicentric systems in carrot somatic 1987,271-276. embryogenesis, Theor Appl Genet, 70 (1975) 345-348. 55 Nagata T & Takebe I, Plating of isolated tobacco mesophyll 36 Ogura H, The cytological chimeras in original regenerates protoplasts on agar medium, Planta, 99 (1971) 12-20. from tobacco tissue cultures and their off springs, Jpn J 56 Vasil V & Vasil I K, Regeneration of tobacco and petunia Genet, 51 (1976) 161-174. plants from protoplasts and culture of corn protoplasts, In 37 Carlson P S, Methionine sulfoximide resistant mutant of Vitro Cell Dev Biol Plant, 10 (1974) 83-96. tobacco, Science, 180 (1973) 1366-1368. 57 Chupeau Y, Missonier C, Hommel M C & Goujaud J, 38 Sidorov V A, Menczel L & Maliga P, Isoleucine-requiring Somatic hybrids of plants by fusions of protoplasts- Nicotiana plant deficient in threonine deaminase, Nature Observations on the model system Nicotiana glauca-N. (Land), 294 (1981) 87-88. langsdorffi, Mol Gen Genet, 165 (1978) 239-245. 39 Malmberg R L, Biochemical cellular and developmental 58 Nitsch J P & Ohyma K, Obtention de plantes a partir de characterization of a temperature-sensitive mutant of protoplasts haploids cultivees ill vitro, CR Acad Sci Paris, Nicotiana tabacum and its second site revertants, Cell, 22 273 (1971) 801-804. (1979) 603-609. 59 Slogteren Van G M S, Planque K & Lekkerkerk J, 40 Muller A J & Grafe R, Isolation and characterization of cell Evaluation of parameters affecting the initiation of division lines of N. tabacum lacking nitrate reductase, Mol Gen of protoplasts of haploid and diploid Nicotiana. sylvestris Genet, 161 (1978) 67-76. and N. tabacum, Plant Sci Leu, 20 (1980) 35-45. 41 Widhom J M, Cultured N tabacum cells with an altered 60 Melchers G & Labib G, Somatic hybridization of plants of anthranilate synthetase which is less sensitive to feed back protoplasts I. Selection of light resistant hybrids of haploid inhibition, Biochem Biophys Acta, 261 (1972) 52-58 light sensitive varieties of tobacco, Mol Gen Genet, 135 42 Gonzales R A & Widhlom J M, Selection of plant cells for (1974) 277-294. desirable characteristics: Inhibitor resistance, in Plant cell 61 Melchers G, Microbial techniques in somatic hybridization culture-A practical approach, edited by R A Dixon (IRL by fusion of protoplasts, in International cell biology (1976- Press, Oxford) 1985, 67-78. 1977), edited by B R Brinkley & K R Porter (The 43 Maliga P, Breznovits A S & Marton L, Streptomycin- Rockefeller University Press, Boston, MA) 1977, 207-215. resistant plants from callus culture of haploid tobacco, 62 Carlson P S, Smith H H & Dearing R D, Parasexual Nature (Land), 244 (1973) 29-30. interspecific plant hybridization, Proc Natl Acad Sci USA, 44 Nabors M W, Daniels A, Nadolny L & Brown C, Sodium 69 (1972) 2292-2294. chloride tolerant lines of tobacco cells, Plant Sci Left, 4 63 Maliga P, Lazar G, Joo F, Nagy A H & Menczel L, (1975) 155-159. Restoration of morphogeneticpotential of Nicotiana by 45 Nabors M W, Gibbs S E, Bernstein C S & Meis M E, NaCI somatic hybridization, Mol Gen Genet, 157 (1977) 291-296. tolerant tobacco plants from cultured cells, Z 64 Smith H H, Kao K N & Combatti N C, Interspecific Pjlanzenphysiol, 97 (1980) 13-17. hybridization by protoplast fusion In Nicotiana, J 46 Dix P J, Environmental stress resistance selection in plant Hereditary, 67 (1976) 123-128. cell cultures, In Plant cell cultures, edited by F Sala 65 Gleba Y Y, Butenko R G & Sytnic K M, Fusion of (Elsevier, Amsterdam) 1980, 183-186. protoplasts and parasexual hybridization in' Nicotiana 47 Barg R & Umiel N, Development of tobacco seedlings and tabacum L, Dokl Akad Nauk USSR, 221 (1975) 1196 1198. callus cultures in the presence of amitrole, Z 66 Gleba Y Y & Hoffmann F, 'Arabidobrassica' plant genome Pflanzenphysiol, 83 (1977) 437-447. engineering by protoplast fusion, Naturwissenschaften, 66 48 Radin D N & Carlson P S, Herbicide tolerant tobacco (1979) 547-554. mutants selected in situ and recovered via regeneration from 67 Bates G W, Asymmetric hybridization between Nicotiana •• cell culture, Genet Res, 32 (1978) 85-89. tobaccum and N. repanda by donor-recipient protoplast fusion: Transfer to TMV resistance, Theor Appl Genet, 80 49 Miller 0 K & Hughes K W, Selection of paraquat-resistant (1990) 481-487. variants of tobacco from cell cultures, In Vitro Cell Dev Bioi .. Plant, 16 (1980) 1085-1091. 68 Prat D, Genetic variability induced in Nicotiana sylvestris by protoplasts culture, Theor Appl Genet, 64 (1983) 223- 50 Chalef R S & Parsons M F, Direct selection in vitro for 230. herbicide-resistant mutants of Nicotiana tabacum, Proc Natl 69 Lorz H, Paszkowski J, Dierks-Venting C & Potrykus I, Acad Sci USA, 75 (1978) 5104-5107. Isolation and characterization of cytoplasts and 51 Kapitska 0 S, Yulinich A V & Vinetski Y P, Dokl Akad miniprotoplasts derived from protoplasts of cultured cells, Nauk USSR, 235 (1977) 1426-1429. Physiol na«. 53 (1981) 385-391. 52 Toyoda H, Chatani K, Kita N, Matsuda Y & Ouchi S, 70 Faccioti D & Pilet P E, Plants and embryoids from haploid Multiplication of TMV in tobacco callus tissue and in vitro Nicotiana sylvestris protoplasts, Plant Sci. LeU, 15 (1979) selection for viral disease resistance, Plant Cell Rep, 8 1-6. (1989) 433-436. 71 Ohta S & Yatazawa M, Nicotiana tabacum L (tobacco)-In 53 Carlson P S, Methionine sulfoximide resistant mutant of vitro production of nicotine, in Biotechnology in agriculture tobacco, Science, 180 (1973) 1366-1368) andjorestry, vol 7, Medicinal and aromatic plants II, edited 54 Venkateswarlu T, Subhashini U & Reddy V R, Variability by Y P S Bajaj (Springer-Verlag, Berlin) 1989,367-380. In callus derived plants of an interspecific hybrid In 72 Krikorian A D & Steward F C, Biochemical differentiation Nicotiana, in Plant cell and tissue culture of economically in the biosynthetic potentialities of growing and qui scent 182 INDIAN J BIOTECHNOL, APRIL 2004
tissue, in Analysis of growth, the responses of cells and 91 Horsch R B, Fry] E, Hoffman N L, Eichholtz D, Rogers S tissues in culture, Plant Physiol, vol V, edited by F C G et al, A simple and general method for transferring genes Steward (Academic Press, New York) 1969,278. into plants, Science, 227 (1985) 1229-1231. 73 Furuya T, Kojima H & Syno K, Regulation of nicotine 92 Shillito R D, Saul M, Paszkowski M, Muller M & Potrykus biosynthesis by auxins in tobacco callus tissue, I, High efficiency direct gene transfer to plants, Phytochemistry, 10 (1972) 1529-1532. BiolTechnology,3 (1985) 1099-1103. 74 Tabata M, Yamamoto H, Hiraoka N, Marurnoto Y & 93 Deshayes A L, Herrel-Estrella L & Caboche M, Liposome Konoshima M, Regulation of nicotine production in tobacco mediated transformation of tobacco mesophyll protoplasts tissue culture by plant growth regulators, Phytochemistry, by an Escherichia coli plasmid, EMBO J, (1985) 42731- 10 (1971) 723-729. 2737. 75 Tabata M & Hiraoka N, Variation of alkaloid production in 94 Crossway A, Oakes J Y, Irvine J M, Ward B, Knauf Y C et Nicotiana rustica callus cultures, Physiol Plant, 38 (1976) al, Integration of foreign DNA following microinjection of 19-23. tobacco mesophyll protoplasts, Mol Gen Genet, 202 (1986) 76 Benveniste P, Hirth L & Ourisson G, La biosyenthese des 179-185. sterol dans les tissus de tabac cultive in vitro IT. 95 Klein T M, Harper E, Svab Z, Sanford J C, Fromm MEet Particularities de la biosynthese des phytosterols des tissus al, Stable genetic transformation of intact Nicotiaua cells by de tabac cultives in vitro, Phytochemistry,S (1966) 45-58. the particle bombardment process, Proc Natl Acad Sci USA, 77 Weete J D, Yenkateswaran S & Laseter ] L, Two 85 (1988) 8502-8505. populations of aliphatic hydrocarbons of teratoma and 96 Brandle J E, Labbe H, Zilkey B F & Miki B L, Resistance habituated tissue cultures of tobacco, Phytochemistry, 10 to sulfonylurea herbicides chlorosulfuron, amidisulpfuron (1971) 939-945. and DPX-R 9674 in transgenic flue-cured tobacco, Crop 78 Furuya T, Biotransformation by plant cell cultures, in Sci, 32 (1992) 1443-1445. Frontiers of plant tissue culture, edited by T A Thorpe 97 Anzai H, Yoneyama K & Yamaguchi I, Transgenic tobacco (University Calgary Press, Canada) 1978, 191-200. resistant to a bacterial disease by the detoxification of a 79 Burrows W J & Leeworthy D P, Metabolism of NN'- pathogenic toxin, Mol Gen Genet, 219 (1989) 492-494. diphenyl urea by cytokinin dependant tobacco callus, 98 Golemboski D, Lomonossoff G & Zaitlin M, Plants Biochem Biophys Res Cotnmun, 70 (1976) 1109-1114. transformed with a tobacco mosaic virus non-structural gene 80 Heintz R & Benneniste P, Cyclization of 'squalene 2, 3 sequence are resistant to the virus, Proc Nelli Acd Sci USA, epioxide by microsomes extracted from tobacco tissues 87 (1990) 6311-6315. grown in vitro, Phytochemistry, 9 (1970) 1499-1503. 99 Sardana R, Dukiandjieve S, Giband M, Cheng S, Cowan K 81 Nitsch J P & Ohyrna K, Obtention de plantes a partir de et al, Construction and rapid testing of synthetic and protoplastes haploides cultivees in vitro, CR Acad Sci Paris, modified toxin gene sequences Cryla (b & c) by expression 273 (1971) 801-804. in maize endosperm culture, Plant Cell Rep, 15 (1996) 677- 82 Uchimiya H & Murashige T, Evaluation of parameters in 681. the isolation of viable protoplasts from cultured tobacco 100 Kishor P B K, Hong Z, Miao G H, Hu C A A & Verma D P cells, Plaut Physiol, 54 (1974) 936-944. S, Overexpression of 0 pyrroline 5 carboxylic acid 83 Suzuki M & Takebe I, Uptake of single-stranded synthetase increases proline production and confers bacteriophage DNA by isolated tobacco protoplasts, Z. osmotolerance in transgenic plants, Plant Physiol, 108 Pflanrenphysiol, 78 (1976) 421-433. (1995) 1387-1394. 84 Sigee D C, Smith Y A & Hindley J, Passage of bacterial 101 Ghosh S B, Nagi L H S, Ganapathi T R, Paul Khurana S M DNA into host cells during in vitro transformation of & Bapat Y A, Cloning and sequencing of potato virus Y Nicotiana tabocum L. by Agrobacterium tumefaciens, coat protein gene from an Indian isolate and development of Microbios, 34 (136) (1982) 113-132. transgenic tobacco for PYY resistance, Curr Sci, 82 (2002) 85 Marton L, Wullems G J, Molendijk L & Schilperoort R A, 855-859. In vitro transformation of cultured cells from Nicotiana 102 Chakrabarti A, Ganapathi T R, Mukherjee P K & Bapat Y tabaccum by Agrobacterium tumefaciens, Nature (Loud), A, MSI-99 a magainin analogue imparts enhanced disease 277 (1979) 129-131. resistance in transgenic tobacco and banana, Planta, 216 86 Yenkateswarlu K & Nazar R N, Evidence for T-DNA (2003) 587-596. mediated gene targeting to tobacco chloroplasts, 103 Scorza R. Zimmerman T W, Cordts J M, Footn K ] & Biotechnology, 9 (1991) 1103-11 OS, Ravelonandro M, Horticultural characteristics of transgenic 87 Pandey K K, Sexual transfer of specific genes without tobacco expressing the rol C gene from Agrobacterium gametic fusion, Nature (Lon d), 256 (1975) 310-313. rhizogenesis, J Amer Soc Hortic Sci, 119 (1994) 1091-1998. 88 Hess D, Pollen based techniques in genetic manipulation, 104 Palazon M T, Cusido R M, Roig C & Pinol M T, Effect of Int Rev Cytol, 107 (1987) 169-190. rol genes from Agrobacterium rhizogenes TL-DNA on 89 Horsch R B, Fry J E, Hoffman N L, Eichholtz D, Rogers S nicotine production in tobacco root cultures, Plant Physiol G et al, A simple and general method for transferring genes Biochem, 35 (1997) 155-162. into plants, Science, 227 (1985) 1229-1231. 105 Song H S, Brotherton J E, Gonzales R A & Widholm J M, 90 Paszkowski J, Shillio R D, Saul M, Mandak,Y, Hohn T et Tissue culture specific expression of a naturally occurring al, Direct gene transfer to plants, EMBO J, (1984) 32717- tobacco feedback insensitive anthranilate synthase, Plant 2722. Physiol, 117 (1998) 533-543. GANAPATHI et al: TOBACCO FOR TISSUE CULTURE INTERVENTIONS 183
106 Gray M M, Yoo B Y & Charest P J, Chloroplast dependent 123 Daniell H, Guda C, McPherson D T, Zjang X, Xu, J et al, and light independent expression of the tobacco rbcs Hyper expression of a synthetic protein-based polymer promoter GUS chimeric gene in black spruce, Can J For gene, Methods Mol Bioi, 63 (1996) 359-371. Res, 26 (1996) 909-917. 124 Daniell H, Streatfield S J & Wycoff K, Medical molecular 107 Reichel C, Feltkamp D, Walden R, Steinbiss H H, Schell J farming: Production of antibodies, biopharmaceuticals and et al, Inefficient expression of the DNA binding of GAL 4 edible vaccines in plants. TIPS, 6 (2001) 219-226. in transgenic plants, Plant Cell Rep, 14 (1995) 773-776. 125 Haq T A, Mason H S, Clements J D & Arntzen C J, Oral 108 Saito K, Yamazaki M et al, Tissue specific and stress immunization with a recombinant bacterial antigen enhancing expression of TR promoter for mannopine produced in transgenic plants, Science, 268 (1995) 714-716. synthase in transgenic medicinal plants, Planta, 184 (1991) 126 Mason H S, Ball J M, Shi J J, Jiang X, Estes M K et al, 40-46. Expression of Norwalk virus capsid protein in transgenic 109 Gisel A, Iglesias V A & Sautter C, Ballistic microtargetting tobacco and potato and its oral immunogenicity in mice, of DNA and biologically active substances to plant tissue, Proc Nat Acad Sci USA, 93 (1996) 5335-5340. Plant Tissue Cult Biotechnol, 2 (1996) 33-41. 127 Fitchen J, Beachy R N & Hein M B, Plant virus expressing 110 Rasmussen J L, Kikkert J R, Roy M K & Sanford J C, hybrid coat protein with added murine epitope elicits Biolistic transformation of tobacco and maize suspension autoantibody response, Vaccine, 13 (1995) 1051-1057. cells using bacterial cells as microprojectiles, Plant Cell 128 Turpen T H, Reml S , Charoenvit Y, Hoffman S L, Fallanne Rep, 13 (1994) 212-217. V et ai, Malarial epitopes expressed on the surface of the I I I Hannik N, et al, Phytodctoxification of TNT by transgenic recombinant tobacco mosaic virus, Biotechnology, 13 plants expressing a bacterial nitroreductase, Nature (1995) 53-57. Biotechnol, 19 (200 I) I 168- I 172. 129 Ghosh S, Malhotra P, Lalirha P V, Guha-Mukherjee S & 112 Miki B L A, Mcttugh S G, Labbe H, Ouellet T, Tolman J H Chauhan V S, Expression of Plasmodium falciparum C-
et al, Transgenic tobacco gene expression and application, terminal region of merozoite surface protein (PfMSP 119), a in Biotechnology in agriculture and forestry, vol 45, edited potential malaria vaccine candidate in tobacco, Plant Sci, by Y P S Bajaj (Springer-Verlag, Berlin) 1999,336-354. 162 (2002) 335-343. 113 Dale P J, R&D regulations and field trialing of transgenic 130 Kapusta J, Modelska A, Figlerowicz M, Podkowinski J, crops, TlBTECH (1995), 13398-13403. Pniewski T et al, Biotechnological approaches for making 114 Crammer C L, Boothe J G & Oishi K K, Transgenic plants vaccine in plants, Plant Biotechnology and lnvitrobiology in for therapeutic proteins: Linking upstream and down stream the 2 I" century, edited by A Altman et al (Kluwer strategies, in Current topics ill microbiology and Academic Publishers, Dodrecht) 1999, 571-574. immunology, vol 240, Plant biotechnology: New products 131 Mason H S & Arntzen C J, Transgenic plants as vaccine and applications, edited by J Hammond, P McGarbvey & V production systems, Trends Biotechnol, 13 (1995) 388-392. Yusibov (Springer-Verlag. Berlin) 1999,95-118134. 132 Arakawa T, Yu J, Chong D K X, Hough J, Engen PC et ai, 115 Giddings G, Allison G, Brooks D & Carter A, Transgenic A plant-based cholera toxin B subunit-insulin fusion protein plants as factories for biopharmaceuticals, Nature protects against the development of autoimmune diabetes, Biotechuol, 18 (2000) 1151-1155. Nature Biotechnol, 16 (1998) 934-938. 116 Staub J M, Garcia B, Graves J, Hajdukiewicz P T J, Hunter 133 Verch T, Yusibov V & Koprowski H, Expression and P et al, A high yield production of a human therapeutic assembly of a full-length monoclonal antibody in plants protein in tobacco chloroplasts, Nature Biotechnol, 18 using a plant virus vector, J Immunol Methods, 220 (1998) (2000) 333-338. 69-75. 117 Kusnadi A R, Niklov Z L & Howard J A, Production of 134 Beachy R N, Fitchen J H & Hein M B, Use of plant viruses recombinant proteins in transgenic plants: Practical for delivery of vaccine epitopes, Ann N Y Acad Sci, 792 considerations, Bioteclinol Bioeng, 56 (1997) 473-484. (1996) 43-49. 118 Cramer C L & Weissenborn D L, HMG2 promoter 135 Mc Cormick A A, Kumagai M H, Hanley K, Turpen T H, expression system and post-harvest production of gene Hakim I et ai, Rapid production of specific vaccines for products in plants and plant cell cultures, US Patent, 5 lymphoma by expression of the tumour-derived single-chain (1997) 670, 349. Fv epitopes in tobacco plants, Proc Nat Acad Sci USA, 96 119 Ruggeiro F, Exposito J Y, Bournat P, Gruber V, Perret S et (1999) 703-708. ai, Triple helix assembly and processing of human collagen 136 Stackzek J, Bendahmane M, Gilleland L B, Beachy R N & produced in transgenic tobacco plants, FEBS Lett, 469 Gilleland Jr, H E, Immunization with a chimeric tobacco (2000) 132-136. mosaic virus containing an epitope of outer membrane 120 Hansen E, Production of recombinant antigens in plants for protein F of Pseudomonas aeruginosa provides protection animal and human immunization- a review, Brazilian J against challenge with P. aeruginosa, vaccine, 18 (2000) Genet, 20 (1997) 703-711. 2266-2274. 121 Rao P S, Suprasanna P & Ganapathi T R, Production of 137 Tackaberry E S, Dudani A K, Prior F, Tocchi M, Sardana R recombinant proteins in transgenic plants, in Role of et ai, Development of biopharmaceuticals in plant biotechnology in medicinal and aromatic plants, edited by I expression systems: Cloning, expression and immunological A Khan (UKAAZ Publisher, Hyderabad) 2000, reactivity of human Cytomegalovirus glycoprotein B 122 Anonymous, Molecular farming using tobacco GUM, (UL55) in seeds of transgenic tobacco, Vaccine, 17 (1999) Tobacco Tech, 3 (2000) 1-2. 3020-3029. 184 INDIAN J BIOTECHNOL, APRIL 2004
138 Tuboly T, Yu W, Bailey A, Degrandis S, Du S et al, 139 Sunil Kumar G B, Ganapathi T R, Revathi C J, Prasad K S Immunogenicity of porcine transmissible gastroenteritis N & Bapat V A, Expression of hepatitis B surface antigen in virus spike protein expressed in plants, Vaccine, 18 (2000) tobacco cell suspension cultures, Protein Exp Purif, 32 2023-2028. (2003) 10-17.