Tobacco Explants Research Articles

The Effect of Sodium Butyrate on Adventitious Shoot Formation Varies among the Plant Species and the Explant Types.

Histone acetylation plays an important role in plant growth and development. Here, we investigated the effect of sodium butyrate (NaB), a histone deacetylase inhibitor, on adventitious shoot formation from protoplast-derived calli and cotyledon explants of tobacco (Nicotiana benthamiana) and tomato (Solanum lycopersicum). The frequency of adventitious shoot formation from protoplast-derived calli was higher in shoot induction medium (SIM) containing NaB than in the control. However, the frequency of adventitious shoot formation from cotyledon explants of tobacco under the 0.1 mM NaB treatment was similar to that in the control, but it decreased with increasing NaB concentration. Unlike in tobacco, NaB decreased adventitious shoot formation in tomato explants in a concentration-dependent manner, but it did not have any effect on adventitious shoot formation in calli. NaB inhibited or delayed the expression of D-type cyclin (CYCD3-1) and shoot-regeneration regulatory gene WUSCHEL (WUS) in cotyledon explants of tobacco and tomato. However, compared to that in control SIM, the expression of WUS was promoted more rapidly in tobacco calli cultured in NaB-containing SIM, but the expression of CYCD3-1 was inhibited. In conclusion, the effect of NaB on adventitious shoot formation and expression of CYCD3-1 and WUS genes depended on the plant species and whether the effects were tested on explants or protoplast-derived calli.

Efficacy of transgenic tobacco carrying synthetic plant-preferred codon-optimized novel VIP3AA44 Gene towards Helicoverpa armigera and Spodoptera litura

Vip3A proteins are synthesized during vegetative growth of Bacillus thuringiensis and are toxic against a wide range of lepidopteran insects. Since the mode of action of vip3A toxins is different from Cry proteins, vip3A proteins are good candidates for gene pyramiding in transgenic crops to combat development of resistance against the currently deployed genes. A synthetic plant-preferred codon-optimized novel vip3Aa44 gene (NCBI accession number HQ650163) was cloned into pBINAR plant transformation vector and tobacco explants were transformed with leaf disc co-cultivation method to evaluate toxicity of this gene against Helicoverpa armigera and Spodoptera litura. The putative transgenics were confirmed by PCR and RT-PCR analysis. The bioassays were performed on detached leaves from putative transgenics using lab-grown population of H. armigera and S. litura. Mortality after 72 hr ranged from 30–56% for H. armigera and 40–60% for S. litura, indicating potential of vip3Aa44 gene against these lepidopteran pests in transgenic development.

Agrobacterium- Mediated Transformation of Tobacco Plants using Walnut Polyphenol Oxidase Gene

Polyphenol oxidase (PPO) is a well-recognized copper-containing enzyme which can catalyze oxidation of a great variety of phenolic compounds. PPO is found in diverse microorganisms, plants, and animals. To examine the anti-pathogenic role of PPO in walnut (Juglans regia L.), Agrobacterium tumefaciens strain LBA4404 was used to transform tobacco (Nicotiana tobacum L.) explants. Recombinant binary vector pBI121, containing the walnut JrPPO gene and the nptII gene as a selectable marker, was incorporated into A. tumefaciens. MS medium supplemented with 50 mg/l of kanamycin and 200 mg/l of cefotaxime was used as a selection medium. Plantlets were regenerated from putatively transgenic calli and polymerase chain reaction and Southern blot analysis were performed to confirm the integration of JrPPO into the genome. To evaluate the function of PPO in pathogen resistance, transgenic tobacco plants were exposed to Pseudomonas syringae pv. tabaci. According to symptom progress and quantitative analyses, although there was no significant difference in transgenic tobacco, but mean comparison showed that disease severity of transgenic tobacco was less than wild plants. Finally, it may be concluded that PPO has a notable function in the resistance process in walnut, but tobacco transgenic plants might not be a suitable test plant to examine the resistance role of PPO in walnut.

Biologically active recombinant human erythropoietin expressed in hairy root cultures and regenerated plantlets of Nicotiana tabacum L.

Hairy root culture is a potential alternative to conventional mammalian cell culture to produce recombinant proteins due to its ease in protein recovery, low costs and absence of potentially human pathogenic contaminants. The current study focussed to develop a new platform of a hairy root culture system from Nicotiana tabacum for the production of recombinant human EPO (rhEPO), which is regularly produced in mammalian cells. The human EPO construct was amplified with C-terminal hexahistidine tag from a cDNA of Caco-2 cells. Two versions of rhEPO clones, with or without the N-terminal calreticulin (cal) fusion sequence, were produced by cloning the amplified construct into gateway binary vector pK7WG2D. Following Agrobacterium rhizogenes mediated transformation of tobacco explants; integration and expression of constructs in hairy roots were confirmed by several tests at DNA, RNA and protein levels. The amount of intracellular rhEPO from hairy root cultures with cal signal peptide was measured up to 66.75 ng g-1 of total soluble protein. The presence of the ER signal peptide (cal) was essential for the secretion of rhEPO into the spent medium; no protein was detected from hairy root cultures without ER signal peptide. The addition of polyvinylpyrrolidone enhanced the stabilization of secreted rhEPO leading to a 5.6 fold increase to a maximum concentration of 185.48 pg rhEPOHR g-1 FW hairy root cultures. The rhizo-secreted rhEPO was separated by HPLC and its biological activity was confirmed by testing distinct parameters for proliferation and survival in retinal pigment epithelial cells (ARPE). In addition, the rhEPO was detected to an amount 14.8 ng g-1 of total soluble leaf protein in transgenic T0 generation plantlets regenerated from hairy root cultures with cal signal peptide.

Modulation of viral infection in plants by exogenous guanosine

To evaluate the role of purines in antiviral treatments in plants, ribavirin (RB) and tiazofurin (TZ) were applied in combination with guanosine (GS) or adenosine (AS) in in vitro grapevine or tobacco explants infected by Grapevine leafroll associated virus 3 (GLRaV-3) and Cucumber mosaic virus (CMV), respectively. Using a microelectrochemical (trans-plasma membrane electron transport, t-PMET) technique, in vivo assay of free reduced nicotinamide adenine dinucleotide (NADH) was also carried out to estimate the inosine monophosphate dehydrogenase inhibition caused by drugs. Antiviral effectiveness of TZ, evaluated as virus-free explants or virus copies, was significantly hindered by GS in both species, while AS did not interfere with the drugs. GS, but not AS, slightly hindered the antiviral effectiveness of RB. With regard to NADH tests, t-PMET inhibition caused by RB and TZ was dose dependent and the interference of drugs with the NAD+/NADH conversion was confirmed by NADH content. Findings indicate that exogenous GS up to 0.50 mM replenished the GS pool depleted by drugs, contrasting antiviral action. At higher doses of GS, the TZ antiviral action was completely inhibited and exogenous GS caused a feedback that reduced t-PMET activity. The reversal was partially against RB, suggesting that the reduction of the GS pool contributed to the antiviral activity of RB, but it was not the only cause of antiviral effectiveness.

Complex, non-monotonic dose-response curves with multiple maxima: Do we (ever) sample densely enough?

We usually expect the dose-response curves of biological responses to quantifiable stimuli to be simple, either monotonic or exhibiting a single maximum or minimum. Deviations are often viewed as experimental noise. However, detailed measurements in plant primary tissue cultures (stem pith explants of kale and tobacco) exposed to varying doses of sucrose, cytokinins (BA or kinetin) or auxins (IAA or NAA) revealed that growth and several biochemical parameters exhibit multiple reproducible, statistically significant maxima over a wide range of exogenous substance concentrations. This results in complex, non-monotonic dose-response curves, reminiscent of previous reports of analogous observations in both metazoan and plant systems responding to diverse pharmacological treatments. These findings suggest the existence of a hitherto neglected class of biological phenomena resulting in dose-response curves exhibiting periodic patterns of maxima and minima, whose causes remain so far uncharacterized, partly due to insufficient sampling frequency used in many studies.

The MYB Transcription Factor Family Genes in Sugarcane (Saccharum sp.)

Transcription factors are a family of proteins that control the physiological and biochemical process of a plant. The MYB family of proteins is the largest family of transcription factors (TF) that plays an important role in controlling growth and development and stress responses. These genes have been extensively studied in many dicot and monocot plants. Among the grasses, sugarcane has a complex polyploidy genome, and only little information is available about the MYB TF family. With increasing importance of this biofuel crop, 57 R2R3-MYB, 20 MYB-like, and 1 3R1-MYB genes were computationally predicted and 51 ScMYBs were isolated by RT-PCR. Subgroup-specific conserved motifs outside the MYB domain demonstrated their phylogeny and functional conservation in sugarcane. Based on their biological process, the isolated ScMYB genes were functionally categorized into cell growth and maintenance (39.74 %), stress response (33.33 %), metabolism (14.10 %), and developmental regulation (12.82 %). Expression analysis by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) revealed a significantly higher expression trend of ScMYB26/AS1 > ScMYB54 > ScMYB66 > ScMYB75 transcripts in response to water deficit, whereas in salt stress, ScMYB1 and ScMYB58 showed higher expression during 12 days of stress (DOS) in sugarcane leaves. Further, the stress-responsive ScMYB26/AS1 was functionally characterized by agroinfiltration-mediated transient expression in tobacco. Semi-qRT-PCR demonstrated the increased expression of introduced ScMYB26/AS1 transcript in transformed tobacco explants during stress induction. Taken together, our comparative genomics analysis and isolation of ScMYB genes from sugarcane illustrated the viable, future solid foundation for improvement of agricultural crops to stress tolerance by a transgenic approach.

Biological Activity of thetzsGene of NopalineAgrobacterium tumefaciensGV3101 in Plant Regeneration and Genetic Transformation

Agrobacterium tumefaciens has been widely used in plant genetic transformation. Hormone-encoding genes residing in the T-DNA region have been removed, resulting in disarmed Agrobacterium strains that are used in various transformation experiments. Nopaline Agrobacterium strains, however, carry another hormone gene, trans-zeatin synthesizing (tzs), that codes for trans-zeatin in the virulence region of the tumor-inducing plasmids. We investigated the activity and function of the tzs gene of a nopaline Agrobacterium sp. strain GV3101 in plant in vitro regeneration. Leaf explants of tobacco and Nicotiana benthamiana co-cultured with strain GV3101 exhibited active shoot regeneration in media without added plant growth regulators. On medium without plant growth regulators, transgenic shoots were also induced from explants co-cultured with GV3101 containing a binary vector. Enzyme-linked immunosorbent assay showed that cell-free extracts of Agrobacterium sp. strain GV3101 culture contained the trans-zeatin at 860 ng/liter. Polymerase chain reaction using tzs-specific primers showed that the tzs gene was present in strain GV3101 but not in other Agrobacterium strains. The study showed that the tzs gene in GV3101 was actively expressed, and that trans-zeatin produced in the Agrobacterium strain can promote plant shoot regeneration.

Combination of ammonium nitrate, cerium chloride and potassium chloride salts improves Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum

The frequency of plant transformation can be improved by addition of various chemical into transformation media. In the past, we showed that exposure of tobacco, wheat and triticale explants to ammonium nitrate, cerium and lantanium chloride and potassium chloride resulted in an increase in the frequency of transformation. Here, we tested whether a combination of increased concentrations of the aforementioned salts yielded a higher transformation frequency. We found that exposure to 61.8 mM ammonium nitrate caused a 5.0-fold increase in transformation frequency, whereas exposure to 1.0 μM cerium chloride or 47.0 mM potassium chloride resulted in 1.2- and 2-fold increases, respectively. Exposure to 61.8 mM ammonium nitrate and 1.0 μM cerium chloride led to a 4.8-fold increase in transformation frequency, whereas exposure to 61.8 mM ammonium nitrate and 47.0 mM potassium chloride let to a 5.2-fold increase. Finally, exposure to 61.8 mM ammonium nitrate, 1.0 μM cerium chloride and 47.0 mM potassium chloride produced a 5.1-fold increase. The analysis of the intactness of T-DNA borders showed that plants exposed to ammonium nitrate and a combination of ammonium nitrate with other salts had the more intact right borders and the less intact left borders. The best results were observed when all three salts (ammonium nitrate, potassium chloride and cerium chloride) were used. Thus, we concluded that the addition of cerium chloride and potassium chloride does not substantially improve the transformation rate beyond the improvement observed upon treatment with 61.8 mM ammonium nitrate, but may slightly improve the intactness of T-DNA borders.

Changes in cytokinin levels and metabolism in tobacco (Nicotiana tabacum L.) explants during in vitro shoot organogenesis induced by trans-zeatin and dihydrozeatin

The uptake and metabolism of trans-zeatin and/or dihydrozeatin, in correlation with cytokinin oxidase/dehydrogenase (CKX) and β-glucosidase activity, were studied in leaf segments derived from wild-type (WT) and transgenic (T) tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) during in vitro induction of shoot organogenesis. T explants harbored the maize gene Zm-p60.1β-glucosidase. Higher levels of shoot regeneration were observed on T explants in the early stages of cultivation. In WT explants, the content of cytokinin (CK)-O- and N-glucosides increased. In T explants, a higher content of Z-9-riboside and Z-9-riboside-5′-monophosphate and higher CKX activity during the early stage of cultures were found. A positive correlation was obtained for bioactive CK content and the organogenic response in T explants. Our results indicate a connection between the organogenic capacity of tobacco explants, metabolism of endogenous CKs and uptake of exogenous CKs from the cultivation medium.

Thiopurine Prodrugs for Plant Chemotherapy Purposes

Thiopurine prodrugs are antiviral chemicals used in medical therapy whose mechanisms of action are associated with inhibition of purine biosynthesis. In terms of plant chemotherapy, previous research of 6-mercaptopurine (MP) administration in tobacco tissue culture infected by Tobacco mosaic virus (TMV) showed no inhibition of virus activity. Currently, not enough data exist to confirm thiopurine drug ineffectiveness against viruses in the plant kingdom. This paper presents a screening of MP, 6-methylmercaptopurine riboside (MMPR), 6-thioguanine (6-TG) and 1-amino-6-mercaptopurine (1A-MP) against TMV and Cucumber mosaic virus (CMV) in in vitro tobacco explants and against Grapevine leafroll-associated virus 3 (GLRaV 3) in in vitro grapevine explants. ELISA and RT-PCR were used to evaluate antiviral activity. Higher toxicity levels of MP derivatives, compared to MP, were noted in tobacco and grapevine explants. 1A-MP or 6-TG treatment resulted CMV and GLRaV 3 virus-eradicated explants as obtained with Inosine 5′-monophosphate dehydrogenase inhibitors, whereas TMV was not eradicated by any of the studied drugs.

Accumulation of xylem transported protein at pit membranes and associated reductions in hydraulic conductance

Proteins and traces of polysaccharide are the only polymeric colloids consistently transported in the xylem sap of plants. The hypothesis that such proteins could have physical inhibitory effects on xylem water transport was investigated. Ovalbumin, with a molecular weight of 45 kDa and a molecular diameter of 5.4 nm, is an inert, water-soluble protein that is midway along the size range of endogenous xylem sap proteins. Solutions of ovalbumin conjugated to a fluorescent marker and supplied to transpiring shoot explants of tobacco (Nicotiana tabacum L.) and olive (Olea europaea L.) were shown by confocal laser scanning microscopy to accumulate specifically at wall-based pit membranes that connect neighbouring xylem conduits. In addition, pressure-induced perfusion of micro-filtered ovalbumin solutions, at concentrations similar to those of endogenous xylem sap proteins, through the xylem of tobacco stem or olive twig segments resulted in the retention of c. 40% of the ovalbumin and reductions in the axial hydraulic conductance of the xylem. Smaller molecules such as Texas Red 3000 (MW 3 kDa) and Alexafluor 488–cadaverin conjugates (MW 0.64 kDa) did not show similar characteristics. The partial reduction in xylem hydraulic conductance appeared to be related to the accumulation of ovalbumin at xylem pit membranes and the consequent fouling of trans-membrane water-conducting pores with smaller diameters than those of the ovalbumin molecules. Potential implications of these novel findings for whole-plant water relations are considered.

Oligogalacturonides enhance cytokinin-induced vegetative shoot formation in tobacco explants, inhibit polyamine biosynthetic gene expression, and promote long-term remobilisation of cell calcium

Long-sized oligogalacturonides (OGs) are cell wall fragments that induce defence and developmental responses. The Ca(2+)-dependent "egg-box" conformation is required for their activity, and polyamines may prevent them from adopting this conformation. Although OGs are known to inhibit auxin-induced growth processes, their effect on cytokinin-induced ones requires investigation. In the present work OGs were shown to promote cytokinin (benzyladenine, BA)-induced vegetative shoot formation from tobacco leaf explants, independent of the presence of CaCl(2) in the medium and of auxin (indoleacetic acid, IAA) supply. The effect of polyamines, putrescine (PU) and spermidine (SD) supplied with/without their biosynthetic inhibitors (DFMO, CHA) was also investigated, and showed that spermidine enhanced adventitious vegetative shoot formation, but only on medium containing Ca(2+) and IAA. Treatments with inhibitors blocked this promotive effect. OGs did not alter free polyamine concentrations, but caused a moderate increase of conjugated ones, and exhibited an early inhibitory effect on polyamine biosynthetic gene expression. OGs, but not SD, caused long-term changes in calcium-associated epifluorescent signals in the cell walls, and, later, inside the cells of specific tissues. Electron microscopy analysis (ESI system) demonstrated that calcium accumulated in the cell walls and vacuoles of OG-cultured explants. The relationship between OGs, cytokinin, calcium, and polyamines in adventitious vegetative shoot formation is discussed.

Differential Role of Tomato Gemini Virus and Cucumovirus in Plant Organogenesis

Phenotypic symptom development in plants due to viral infection is believed to be a result of developmental defects caused by regulation of gene expression by viral suppressors. Since viral suppressors of viruses differentially regulate small RNAs (siRNA and miRNA), viral pathogenicity and symptom development in the host cannot apply universally. In present study, we demonstrated the differential effects of presence of two viruses (Tomato leaf curl virus and Cucumber mosaic virus) in organogenesis of two separate hosts (tomato and tobacco). Presence of ToLCV in the tomato explants severely affected the tomato organogenesis leading to sharp decrease in the survival percentage and severe developmental defects. On the other hand presence of CMV in the tobacco explants had not shown much effect in tobacco organogenesis as well as survival percentage. This study has not only highlighted our basic understanding of host-virus interactions in terms of viral suppressors and symptom development, but also provide an alternative way of maintaining and reviving viral cultures in vitro.

Tiba inhibition of in vitro organogenesis in excised tobacco leaf explants

Abstract Triiodobenzoic acid (TIBA), an anti-auxin, was found to inhibit both shoot and root formation in cultured excised leaf explants of tobacco (Nicotiana tabacum L.). The shoot formation (SF) medium used required only exogenous cytokinin (N6-benzyladenine) and the root formation (RF) medium required both auxin (indole-3-butyric acid) and cytokinin (kinetin). By transferring the explants from SF or RF media to SF or RF media with TIBA (4.0×10−5 M), respectively or vice versa, at different times in culture, it was found that TIBA inhibition was at the time of meristemoid formation and after determination of organogenesis. This indicates that TIBA interfered with endogenous auxin involvement in organized cell division.

Aminoglycoside antibiotics affect the in vitro morphogenic response of chrysanthemum and tobacco

Broadly the success of genetic transformation of plants requires non-chimeric selection of transformed tissues and its subsequent regeneration. With rare exceptions, most plant transformation protocols still heavily utilize antibiotics for the selection of transgenic cells containing an antibiotic-degrading selectable marker gene. The morphogenic capacity of in vitro chrysanthemum and tobacco stem and leaf explants change with the addition of aminoglycoside antibiotics (AAs). Of 6 antibiotics tested, phytotoxicity occurred at 10-25 and 50-100 pgml-I in chrysanthemum and tobacco explants, respectively, depending on the size of the explant and the timing of application. The presence of light or darkness also had a significant effect. The use of transverse thin cell layers (tTCLs) in conjunction with high initial AA selection levels supported the greatest regeneration of transgenic material (adventitious shoots or callus) and the lowest number of escapes. Flow cytometric analyses demonstrate that regeneration can be predicted in both species, depending on the ploidy level of the callus. Endoreduplication was not observed in chrysanthemum, even at high AA levels, but occurred (8C or more) in tobacco callus, even at low AA concentrations (5-10 pgml-1). The higher the AA level, the greater the DNA degradation and the lower the 2C and 4C values.

Efficient recovery of transgenic plants through organogenesis and embryogenesis using a cryptic promoter to drive marker gene expression

Our previous studies have shown that tCUP, a cryptic promoter from tobacco, functions in all living plant cell types in a wide range of plant species. This led us to investigate if an enhanced derivative, EntCUPΔ, could be used to drive the neomycin phosphotransferase II (nptII) gene and select for kanamycin resistance in crop species that regenerate by organogenesis or embryogenesis. Tobacco (leaves), cauliflower (hypocotyls) and alfalfa (leaves, petioles, stems) explants were co-cultivated with Agrobacterium containing either EntCUPΔ-nptII-nos or 35S-nptII-nos to compare the efficiency of selection for kanamycin resistance. The infected alfalfa explants were placed in somatic embryo induction media, whereas tobacco and cauliflower explants were placed in shoot induction media with kanamycin at concentrations that normally inhibit regeneration. Transgenic plants were recovered from all of the explants with both selectable marker gene constructs. The transformation efficiencies using tCUPΔ-nptII-nos were comparable to or higher than those using 35S-nptII-nos in all three species tested. This study demonstrated that promoters which are not associated with expressed plant genes can be used as alternatives for the expression of selectable marker genes in a broad range of tissues and species for the generation of transgenic plants.

Methyl jasmonate upregulates biosynthetic gene expression, oxidation and conjugation of polyamines, and inhibits shoot formation in tobacco thin layers

The effect of methyl jasmonate (MJ) on de novo shoot formation and polyamine metabolism was investigated in thin layer explants of tobacco (Nicotiana tabacum L. cv. Samsun). A relatively low concentration of MJ (0.1 microM) enhanced explant fresh weight, but had no effect on the final number of shoots per explant while higher concentrations (1 and 10 microM) significantly inhibited organogenesis. The histological study revealed that, with increasing concentrations of MJ, the formation of meristemoids and shoot domes declined and the incidence of cell hypertrophy increased. In explants cultured with 0.1, 1 or 10 microM MJ, the endogenous levels of free putrescine, spermidine and spermine generally declined compared with controls, after 7 and 15 d. Perchloric acid-soluble conjugated polyamines accumulated dramatically during culture, but much more so in the presence of MJ than in controls. Acid-insoluble conjugated spermidine alone increased in response to the elicitor. Activities of the putrescine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) in the soluble fraction of MJ-treated explants displayed up to 3-fold increases relative to control explants. However, the most relevant increases in these enzyme activities occurred in the particulate fraction. The activity of S:-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), an enzyme involved in spermidine and spermine biosynthesis, was also stimulated by exposure to MJ. Northern analyses revealed MJ-induced, generally dose-dependent, increases in the mRNA levels of all three enzymes. Diamine oxidase (DAO, EC 1.4.3.6) activity was stimulated by MJ mainly in the cell wall fraction. The upregulation of polyamine metabolism is discussed in relation to the morphogenic behaviour of MJ-treated explants.

Methyl jasmonate upregulates biosynthetic gene expression, oxidation and conjugation of polyamines, and inhibits shoot formation in tobacco thin layers

The effect of methyl jasmonate (MJ) on de novo shoot formation and polyamine metabolism was investigated in thin layer explants of tobacco (Nicotiana tabacum L. cv. Samsun). A relatively low concentration of MJ (0.1 μM) enhanced explant fresh weight, but had no effect on the final number of shoots per explant while higher concentrations (1 and 10 μM) significantly inhibited organogenesis. The histological study revealed that, with increasing concentrations of MJ, the formation of meristemoids and shoot domes declined and the incidence of cell hypertrophy increased. In explants cultured with 0.1, 1 or 10 μM MJ, the endogenous levels of free putrescine, spermidine and spermine generally declined compared with controls, after 7 and 15 d. Perchloric acid‐soluble conjugated polyamines accumulated dramatically during culture, but much more so in the presence of MJ than in controls. Acid‐insoluble conjugated spermidine alone increased in response to the elicitor. Activities of the putrescine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) in the soluble fraction of MJ‐treated explants displayed up to 3‐fold increases relative to control explants. However, the most relevant increases in these enzyme activities occurred in the particulate fraction. The activity of S‐adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), an enzyme involved in spermidine and spermine biosynthesis, was also stimulated by exposure to MJ. Northern analyses revealed MJ‐induced, generally dose‐dependent, increases in the mRNA levels of all three enzymes. Diamine oxidase (DAO, EC 1.4.3.6) activity was stimulated by MJ mainly in the cell wall fraction. The upregulation of polyamine metabolism is discussed in relation to the morphogenic behaviour of MJ‐treated explants.

2-Deoxyglucose resistance: a novel selection marker for plant transformation

A novel selection marker for plant transformation alternative to antibiotic and herbicide resistance is described. The selective agent applied is 2-deoxyglucose (2-DOG) which in the cytosol of plant cells is phosphorylated by hexokinase yielding 2-DOG-6-phosphate (2-DOG-6-P). 2-DOG-6-P exerts toxic effects on overall cellular metabolism leading to cell death. We observed that constitutive expression of the yeast DOG R1 gene encoding a 2-DOG-6-P phosphatase resulted in resistance towards 2-DOG in transgenic tobacco plants. This finding was exploited to develop a selection system during transformation of tobacco and potato plants. The lowest concentration of 2-DOG leading to nearly complete inhibition of regeneration of wild-type explants was found to range between 400 and 600 mg/l 2-DOG for tobacco, potato and tomato plants. After Agrobacterium tumefaciens-mediated transformation cells expressing the DOG R1 gene were selected by resistance to 2-DOG. More than 50% of tobacco explants formed shoots and on average 50% of these shoots harboured the DOG R1 gene. Similar results were obtained for potato cv. Solara. The acceptability of the resistance gene derived from baker's yeast, the unobjectionable toxicological data of 2-DOG as well as the normal phenotype of DOG R1-expressing plants support the use of this selection system in crop plant transformation.