How To Grow Tobacco At Home

Tobacco Growing Made Easy

Everything you need to know is explained in Tobacco Growing Made Easy. There is no time like the present to start your tobacco crop. You will however, need the information in this guide to get off to the best possible start. You could hunt the internet for months without even coming close to the amount of good information and tips in this guide. You will learn: Which seeds produce the best tobacco How to make a sand mixture to disperse tobacco seeds. How much light you should allow for optimum results. How to water your seedlings so they don't drown. The easiest way to germinate tobacco seeds Simple techniques for producing the largest tobacco plants Hands free maintenance allowing you to set it and forget it The very best time for harvesting Drying and curing for maximum flavour and quality The different types of tobacco available to you. How to choose the best seeds for the best plants. The truth about soil types and how they affect your plants. How to handle seedlings so that you do not damage them. How to avoid fungus and mould. More here...

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First Phase Use of Foreign Serine PI Genes

The first gene of plant origin to be transferred to another plant species to produce enhanced insect resistance was isolated from cowpea, and encoded a Bowman-Birk type serine PI with two inhibitory sites active against bovine trypsin (CpTI).35 We produced a fUll-length cDNA clone of CpTI, and placed its coding sequence under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter in the final construct prepared for transfer to plants. Transgenic tobacco plants were produced by a standard Agrobacterium tumefaciens-mediated transformation protocol, using a binary vector system. Transformants were screened for CpTI expression, which showed that many of the resulting plants expressed CpTI at levels greater than 0.1 of total soluble protein, a general observation for expression of proteins of this type in transgenic plants. Plants expressing CpTI at the highest levels (approximately 1 of total soluble protein) were clonally propagated, and used for insect bioassays....

Definition of Steps Involved in the Signaling Process of Cell Death Induction during Plant Pathogen Interactions

One of the earliest biochemical events subsequent to pathogen recognition is the alterations of ion fluxes characterized in particular by an uptake of H+ and Ca2+. Pharmacological studies as well as pH measurements have revealed a rapid proton uptake in cells infected with an avirulent bacterial pathogen or treated with cell death elicitors such as cryptogein or harpin (Glazener et al., 1996 Viard et al., 1994). More recent studies using transgenic tobacco plants expressing the bacterial proton pump, bacterioopsin (bO), suggested that proton fluxes might have an important role in the induction of cell death. Such plants exhibit HR-like spontaneous lesions and mutations in the bO gene that affect the proton channeling capacity abolish this phenotype. These data indicate that the ectopic expression of the bO protein is likely to induce cell death by modifying the proton fluxes rather than by an indirect effect (Mittler and Lam, 1995b Pontier et al., 2002). The important role of calcium...

Molecular Components for Cell Death Control during Plant Pathogen Interactions

Of which derives from their specificity of cleaving after an asparatic acid at the P1 position of their substrate recognition sites. When activated, these proteases begin a proteolytic cascade that irreversibly commits the cell to undergo cell death. The activity of caspases is under stringent control by paired pro-apoptotic and pro-survival signals through the positive regulator called CED4 APAF-1 and the negative regulator BCL2 and its related proteins (BLPs). CED4 APAF-1 proteins apparently activate caspases through proteinprotein interactions while BLPs can regulate caspases through multiple mechanisms (Raff, 1998). In mammals, a large family of BLPs has been shown to control PCD and is comprised of genes showing pro-apoptotic (i.e. Bax, Bak and Bid) as well as pro-survival (i.e. Bcl2 and Bcl-xL) activities. No BLPhomologue has been detected in plants yet. However, two recent studies involving the ectopic expression of BLPs in plants suggest the existence of similar regulatory...

Regulation of Chloroplast Senescence

The molecular reasons underlying premature senescence at elevated CO2 are not clear. In principle a down-regulation of photosynthesis at elevated CO2 levels may be achieved by either an end product inhibition or by a redeployment of nitrogen (Bowes, 1991). Most scientists favor the idea that at elevated CO2 levels carbohydrate accumulation exerts a negative feedback inhibition on photosynthesis, which most likely occurs at the level of Rubisco (Bowes, 1991 Webber et al, 1994 Pearson and Brooks, 1995 Miglietti et al, 1996 Sicher and Bunce, 1997). Increases in sugar and starch contents indeed correlate with a clearly reduced level of the rbcS transcripts under conditions of high CO2 (Van Oosten and Besford, 1995 Ono and Watanabe, 1997). An acceleration of Rubisco degradation at elevated CO2 is not necessarily an indication of senescence but might just reflect the lower need for Rubisco at elevated CO2 (Webber et al., 1994). On the basis of determinations of yield and nitrogen...

Molecules that Activate the OD Pathway

WIPK transcript rapidly accumulated in tobacco plants after wounding,43 and co-suppression of WIPK in transgenic tobacco plants abolished wound-induced JA synthesis and PI expression, indicating that this kinase regulates the early response of plants leading to activation of the OD pathway.

Future Targets For Engineering New Lignins

Its deduced amino acid sequence is similar to that of the LIM protein family that contains a zinc finger motif. Transgenic tobacco plants with antisense Nt lim 1 showed low expression levels of phenylalanine ammonia-lyase, 4-coumarate CoA ligase, and cinnamyl alcohol dehydrogenase and a decrease in lignin content. This simultaneous down-regulation of several enzymes is particularly interesting because it can prevent the unwanted accumulation of phenolic intermediates along the phenylpropanoid and lignin pathways and facilitate the reorientation of carbon flux into primary metabolism.

Indirect Effects on Honeybees

Paul et al28 observed insects visiting a field plot of tobacco transformed with a kanamycin resistance marker. They found no difference in the range of animals or the frequency of visits between modified and non-modified tobacco plants. Insects observed included thrips, aphids, pollen beetles, hoverflies, butterflies, honeybees and several species of bumblebees. Scheffler et al29 evaluated pollen dispersal in oilseed rape engineered to contain a herbicide resistance (bar) gene. To ensure effective pollination, honeybee hives were placed near the field site for this study, which consisted of a 1 m-circle of non-transgenic plants located in the centre of a 9-m circle of transgenics, surrounded by 1.1 ha of non-transgenic plants. Sampling involved recording numbers of flowering plants, recording numbers and types of insect visitors and collecting seed at various distances, radiating out from the transgenic circle. Interactions between the pollinating insects and the transgenic plants...

Multigene Defense Mechanisms

In the first section, strategies were applied that are based on the expression of a single gene (or a few genes) and a single target mechanism. From epidemiological studies it is evident that such strategies have a high chance of being overcome by pathogens, similar to resistance against chemical pesticides. From this point of view, more complex defense strategies should be beneficial for enhanced long-term pathogen tolerance. One obvious strategy is to make use of the plant's own defense system, for instance, by lowering the threshold level above which a plant mounts an efficient set of defense reactions to combat microbial pathogens. Verbene et al. (47) described the expression of two bacterial genes in plant chloroplasts that lead to salicylic acid biosynthesis from chorismate. Transgenic tobacco plants have high levels ( 100 M) of salicylic acid glucoside (the plant's vacuolar storage form) and are resistant against the fungus Oidium lycopersicon. Similarly, lesions after...

Competence and Determination Are Two Stages in Floral Evocation

In a day-neutral tobacco, for example, plants typically flower after producing about 41 leaves or nodes. In an experiment to measure the floral determination of the axillary buds, flowering tobacco plants were decapitated just above the thirty-fourth leaf (from the bottom). Released from apical dominance, the axillary bud of the thirty-fourth leaf grew out, and after producing 7 more leaves (for a total of 41), it flowered (Figure 24.13A) (McDaniel 1996). However, if the thirty-fourth bud was excised from the plant and either rooted or grafted onto a stock without leaves near the base, it produced a complete set of leaves (41) before flowering. This result shows that the thirty-fourth bud was not yet florally determined.

The Relationship of Cytokinins and Senescence A Manipulation of Cytokinin Levels

One approach is based upon inducible promoter systems to provide for conditional IPT expression. Heatshock promoters have often been used. Smart et al. (1991) reported that transgenic tobacco plants transformed with IPT under the control of a soybean heatshock promoter showed phenotypes associated with higher cytokinin levels such as shorter stature, lateral shoot growth, and delayed leaf senescence even without heatshocks. These pheno-types were more profound after several heatshocks of whole plants or defined areas of leaves. However, Medford et al. (1989) did not observe a phenotypic difference before and after heatshocks in transgenic tobacco and Arabidopsis plants transformed with IPT under the control of a maize heatshock promoter. With or without heatshock, the transgenic plants exhibited reduced apical dominance and a delay in leaf senescence. As expected from the phenotypic change in non-heatshocked plants, cytokinin levels in transgenic plants were higher than control plants...

Transgenic Plants With Modified Antioxidant Enzyme Levels

The first report on transgenic plants overproducing SOD was, however, not promising. Transgenic tobacco plants with 30- to 50-fold increased SOD activity levels due to the production of a petunia chloroplastic Cu ZnSOD were not more resistant against methyl viologen (MV) (49). The tolerance toward oxidative stress is easily tested in vitro with MV (also known as paraquat), which is a light-activated herbicide. In the light, MV becomes an electron acceptor from photosystem I (PSI), subsequently reducing dioxygen to 02 In this way, the herbicide strongly enhances the formation of superoxide radicals and is a fairly good mimic of the superoxide-forming process that occurs in vivo in illuminated chloroplasts. The MV also accepts electrons from the respiratory electron transport chain in the mitochondria and forms superoxide radicals in the dark too. Transgenic tomato plants with two- to fourfold increased SOD levels by producing the same chloroplastic Cu ZnSOD were not better protected...

Genetic Alterations of Light Control of Senescence

Engineering tobacco plants to overexpress PHYA inhibits leaf senescence (Cherry et al, 1991 Jordan et al, 1995), but overexpression of PHYB in potato (Thiele et al, 1999) has similar effects. The excess PHYA also counteracts the senescence-promoting effects of continuous FR (Rousseaux et al, 1997). Although these observations do not themselves produce a general explanation of how light controls senescence, it is clear that the use of mutants and genetically-engineered plants will enable the dissection of the roles of the different photoreceptors in the light control of senescence.

Pathways of Androgenesis

The significance of microspore division symmetry for vegetative cell-specific transcription and generative cell differentiation has been addressed in microspores of transgenic tobacco plants transformed with promotor of vegetative cell-specific tomato lat52 gene fused to reporter gus gene 29 . In vitro maturation, in the presence of high concentrations of colchicine, blocks the first pollen mitosis effectively, resulting in the formation of uninucleate pollen grains expressing both the abovementioned genes which are capable of germination and a pollen tube growth, despite the absence of a generative cell. Lower amounts of colchicine induced symmetric division

Potential Side Effects And Compensation Mechanisms Associated With Lignin Modifications

Tent of plant cell walls may theoretically exert pleiotropic effects on plant functions through changes in the strength of plant organs, sap conduction through the xylem, or permeability of cell wall barriers (caspary band). Changes in plant development have been observed for strongly depressed CCR tobacco lines even though it has not been clearly demonstrated whether the resulting phenotype (reduced growth) is due to changes in cell wall properties or to changes of soluble phenolic profiles. Such a reduction in size has also been demonstrated for CCoAOMT down-regulated tobacco plants. Surprisingly, CCR CAD double transformants exhibit a strong decrease in lignin content but no changes in morphology (at least in culture room conditions). This last observation suggests that the decrease in lignin content per se is not directly responsible for the developmental changes observed but that other unknown metabolic changes are probably involved. From the functional point of view, plants with...

Transcription Factors

The AP2 EREBP-type transcription factor gene, Tsil, was identified as a salt-responsive transcript in tobacco (70). Transgenic tobacco plants over-expressing Tsi exhibited increased salt tolerance. In addition, these plants showed constitutively elevated transcript levels of defense-related genes and enhanced resistance to the virulent bacterial pathogen, Pseudomonas syringae pv. tabaci. A cold-responsive zinc finger protein from soybean, SCOF-1, appears not to act directly as a transcription factor but rather associates with a soybean G-box binding bZIP transcription factor, SGBF-1, in the nucleus and thereby dramatically enhances its binding affinity to the ABA responsive cis-acting element (ABRE) of cold-responsive genes (90). Tobacco plants con-stitutively overexpressing SCOF-1 had normal phenotypes and did not exhibit altered cold sensitivity. However, cold-stressed SCOF-1-transgenic plants recovered significantly faster than wild-type plants under normal growth conditions....

Requirements For Plant Transformation

DNA transfer to plants was first attempted in the 1960s, although the lack of selectable markers and molecular tools to confirm transgene integration and expression made the outcome of such experiments unclear (5). A breakthrough came in the late 1970s with the elucidation of the mechanism of crown gall formation by Agrobacterium tumefaciens (6). The discovery that virulent strains of A. tumefaciens carried a large plasmid that conferred the ability to induce crown galls and that part of the plasmid (the T-DNA) was transferred to the plant genome of crown gall cells provided a natural gene transfer mechanism that could be exploited for plant transformation (7). Tobacco plants carrying recombinant T-DNA sequences were first generated in 1981, although the foreign genes were driven by their own promoters and were not expressed in plant cells (8). The first transgenic tobacco plants expressing recombinant genes in integrated T-DNA sequences were reported in 1983 (9). The technique of...

The Wounding Response in Plant Defense

These wound-induced inhibitors have been well characterized. Both PI-I and PI-II are potent inhibitors of chymotrypsin and subtilisin, and both inhibit trypsin, but less strongly. More recently, a wound-induced trypsin inhibitor has been isolated and characterized from alfalfa leaves. This particular inhibitor was identified as a member of the Bowman-Birk inhibitor family.23 Following these initial reports, genes encoding the wound-inducible inhibitors from both potato24 and tomato25 have been isolated and characterized. Transformation of tobacco plants with a gene encoding potato PI-II resulted in a systemic induction of the transgene expression after wounding,26 showing that the signal inducing PI gene expression was similar in the two species.

Insect Responses to Dietary Serine PIs

Serine Proteinase

Despite successes in enhancing the resistance of transgenic plants towards insects by the expression of foreign PIs, other examples of this technology have given disappointing results, with little or no protection observed. For example Jongsma et al50 produced transgenic tobacco plants expressing the chymotrypsin trypsin-specific potato inhibitor II (PI-II) constitutively. In contrast to results reported above in Section 2.4, the growth of Spodoptera exigua larvae fed with detached leaves of plants expressing PI-II was not affected. These results, with other observations, enabled the authors to show that the insect could respond to the

Preventing Establishment By Transgenic Mitigation

Dwarfing would be disadvantageous to the rare weeds introgressing the TM construct, as they could no longer compete, but is desirable in many crops, preventing lodging and producing less stem with more leaves. The dwarf and herbicide resistant TM transgenic hybrid tobacco plants (simulating a TM introgressed hybrid) were more reproductive than the wild type when cultivated alone (without herbicide). They formed many more flowers than the wild type when cultivated by themselves, which is indicative of a higher harvest index. Conversely, the TM transgenics were weak competitors and highly unfit when co-cultivated with the wild type in ecological simulation of competition. The inability to achieve flowering on the TM plants in the competitive situation resulted in zero reproductive fitness of the TM plants grown in an equal mixture with the wild type at typical field spacing of plants resulting from seed rain of volunteer weeds 23 . 33 Tian, JL Shen, RJ and He, YK (2002) Sequence...

Chimeras Of P450 Enzymes

These fusion proteins offer the interesting possibility of engineering animal P450s into plants. This idea has been used for purposes of herbicide resistance by engineering Rat CYP 1Al Yeast CPR fusion protein into tobacco plants 92 . The fusion protein was shown to provide the plants with resistance to the herbicide chlortoluron. Another group of investigators 93 have produced a protein consisting of Rat CYP 1A1 fused to The active form of Rat CYP 1A1 has already been expressed in the chloroplasts of tobacco plants 93 . The chloroplasts exhibit a P450 dependent mono-oxygenation activity when exposed to light. This suggests CYP 1A1 is coupled to some of the photosynthetic proteins. This maybe possible because Fd and FNR have potentials to form an ET chain similar to that found in the mitochondrial mono-oxygenase system which consists of Fd and NADPH-ferredoxin-oxidoreductase.

Aspartate

Other approaches can also be proposed to increase the methionine content of legume seeds, some of which have been tested in seeds of model species or other crop species. Saalbach et al. (53,57,58) showed that a me-thionine-enriched 7S globulin from Vicia was stably accumulated at levels of 1.5 to 2.2 of the total protein in seeds of transgenic tobacco plants, whereas methionine-enriched 11S globulin from the same species did not accumulate and was subsequently shown to be degraded (59). Similarly, Utsumi and co-workers (60,61) have shown that wild-type and methionine-enriched 11S globulins of soybean accumulate stably in seeds of transgenic rice. De Clercq et al. (62) constructed modified 2S albumins from Arabidopsis and chimeras comprising parts of the Brazil nut and Arabidopsis albumins and expressed them in seeds of tobacco, Arabidopsis, and oilseed rape. Down-regulation of the sulfur-poor 11S globulins (cruciferins) in oilseed rape has also been shown to result in compensatory...

Infoplant Biotech

Transgenic tobacco plants over-expressing human samdc gene driven by CaMV35S promoter were generated by Noh and Minocha 80 . These plants were found to have 2-6 fold higher SPD than the untransformed controls, there was an increase in SPM too though PUT decreased. Since high amounts of SPD is cytotoxic, the regenerants obtained might have been moderate accumulators of SPD as the increase in SPD and SPM was not comparable to the dramatic decrease in PUT. The cytotoxicity of the high amounts of SPD did not allow the regeneration of any plants over-expressing potato samdc in potato, hence a tet-inducible promoter was used to drive samdc. The sense samdc plants showed 7-fold increase in SPD, 3-fold in SPM and a decrease in PUT on tet-induction. Similarly potato plants expressing antisense samdc gene driven by both 35S promoter and tet-inducible promoter were raised 81 . The antisense samdc plants were stunted, branched, necrotic with few small tubers this was attributed to increase in...

Sequestration

A cyclic nucleotide-gated channel from tobacco (NtCBP4) has been described as a first example of a plant transporter mediating Pb2+ uptake (18). Originally identified as a calmodulin-binding protein, this channel was found to localize to the plasma membrane. NtCBP4-overexpressing tobacco plants exhibit increased sensitivity toward Pb2+, which correlates with enhanced Pb2+ accumulation. Interestingly, NtCBP4 overexpressors at the same time are more Ni2+ tolerant. Possible explanations for this effect are, as suggested by the authors, interaction of NtCBP4 with Ni2+, which attenuates uptake, or the suppression of other, more Ni2+ selective channels, by NtCBP4 overexpression.

Gametophytes

During megagametogenesis four haploid nuclei are produced. Commonly, three of the four cells resulting from meiosis die, specifically those at the micropylar end of the ovule (Barlow, 1982). Megaspore abortion is proposed to be a consequence of selective apoptosis (Bell, 1996). Gametospore degeneration in the fern Marsilea vestita exhibits characteristics of apoptosis (Bell, 1996). Mutations interpreted to cause an over expression of PCD have been identified in arabidopsis four mutants show aborted female gametospores at the one nucleate stage while gametospores of five others are affected later in development (Christensen et al., 1998). Tobacco plants transgenic for an antisense copy of a pistil-specific aminocyclopropane carboxylate oxidase failed to complete megasporoge-nesis, which was restored by the ethylene reagent ethephon (De Martinis and Mariani, 1999). During diploid parthenogenesis and early somatic embryogenesis in Norway spruce, specific nuclei die (Havel and Durzan,...

Conclusion

A recent article was published demonstrating the actual usefullness of recombinant PIs expressed intransgenic plants as a way to effectively control plant viruses (see Gutierrez-Campos R, Torress-Acosta JA, Saucedo-Arias L et al. The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants. Nat Biotechnol 1999 17 1223-1226). Transgenic tobacco lines expressing the rice cystatin OC-I were shown to be resistant to two important potyviruses, tobacco etch virus and potato virus Y, which rely on cysteine proteinase activity for the processing of their polyprotein.

Future Perspectives

Ectopic expression of pesticidal proteins presumably will be controlled by inducible promoters, such as those of PI-II85 and TobRB7, that are activated at the site of invasion by pests and nematodes, respectively.86 An ideal promoter should be highly responsive to invasion of the host plant by a pest, or regulated by inducers just prior to pest attack. The promoter should be sufficiently active to mediate a substantial defense, specially localized to the site of pest invasion. Suitable promoters such as those regulated in response to pest invasion can be identified using promoter trapping techniques.87 Recent demonstrations that tetracycline- and gluco-corticoid-inducible systems can be modulated in plant cells88,89 are indicative of how transgenes may be activated ectopically by chemical treatment to facilitate pest control. Insect resistance of transgenic tobacco plants was modulated by conditional expression of the Bt toxin gene.90 In this instance, the Bt gene was fused to the...

Lignins

Reduction of CCoAOMT alone in transgenic tobacco plants resulted in a decreased lignin content but a simultaneous reduction in CCoAOMT and COMT activities induced a further reduction in lignin content, confirming that both enzymes are indeed involved in methylation reactions in lignin biosynthesis (7). Surprisingly, although the transgenic plants showed a 40 to 60 reduction in lignin content, they appeared to grow normally under greenhouse conditions even though they exhibited a deformation of vessel elements. These results have been partly confirmed by M. Legrand's group in Strasbourg (France) (personal communication) on the same material. However, in addition to a reduction in lignin content, CCoAOMT down-regulation induced a decreased growth rate and a dramatic disorganization of vascular tissues (reduction of xylem thickness, reduction of vessel diameter). These discrepancies concerning the growth of the plants have not yet been explained. W. Boerjan et al. (personal...

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