Stages in the Initiation of Root and Shoot Organogenesis in Cultured Leaf Explants of Nicotiana tabacum cv. Xanthi nc.

Reciprocal transfers of Nicotiana tabacum cv. Xanthi nc. leafexplants were made daily between root inducing medium (RIM)and shoot inducing medium (SIM), SIM and a basal medium containingno growth regulators (BM), and RIM and BM. It was found thatthe explants became determined for shoot production after 6d, while roots were produced after only 1 d on RIM before transferto BM. The competence of the explant to produce roots was greatlyreduced by culture on BM prior to culture on RIM. There wasfar less reduction in shoot numbers with preculture on BM. Explantswere found to be only weakly canalized for both caulogenesisand rhizogenesis for the first 2 d after determination. Thereafterthey became strongly canalized. Transfers were also made fromBM to SIM and back to BM, which revealed that the explants becamecompetent for caulogenesis in the absence of cytokinins priorto determination. The period for which SIM is required can bereduced to only 1 d. Key words: Nicotiana tabacum, in vitro, organogenesis, competence, determination     

Feruloylputrescine and Caffeoylputrescine Are Not Involved in Growth and Floral Bud Formation of Stem Explants from Nicotiana tabacum L. var Xanthi nc

The role of feruloylputrescine (FP) and of caffeoylputrescine (CP) was investigated in an explant system of stem explants from day-neutral Nicotiana tabacum L. var Xanthi nc. Previously, a correlation between cortical callus formation and increase in FP content, as well as between in vitro flower formation and increase in CP content had been shown. During the explant growth in vitro, the increase of both FP and CP was inhibited by 4-fluor-(1-amino-2-phenylethyl)phosphonic acid and 2-amino-indene-2-phosphonic acid, both inhibitors of phenylalanine ammonia-lyase (EC 4.3.1.5). dl-α-difluoromethylarginine, an inhibitor of arginine decarboxylase (ADC, EC 4.1.1.19), prevented only the increase in FP, while dl-α-difluoromethylornithine, an inhibitor of ornithine decarboxylase (EC 4.1.1.17), reduced only that of CP. Increase in dry weight and the formation of cortical callus and of floral buds of explants were not affected by any of the inhibitors. We conclude, in contrast to earlier hypotheses, that FP and CP do not trigger growth and differentiation in the explants. It seems more likely that FP and CP increase in response to auxin and cytokinin in the media.     

Occurrence and involvement of adenylate cyclase activity in the first step of tobacco mosaic virus infection of Nicotiana tabacum cv Xanthi nc leaves

Adenylate cyclase activity and its involvement in a physiopathological process (virus infection) were observed in a higher plant, Nicotiana tabacum cv Xanthi nc. The enzyme was characterized in leaves by measuring the conversion of [α-³²P]ATP into cyclic [α-³²P]AMP using a cell membrane preparation. The basal enzyme activity was 1–2 pmol/min per mg protein, was linear with time and protein concentration, and had a temperature optimum between 20 and 25% C. The K_m for ATP was 2 mM in the presence or absence of stimulators. GTP (10⁻⁷ M) increased both basal and sodium fluoride-stimulated activities. During the hypersensitive reaction which follows tobacco mosaic virus (TMV) infection, we detected in the first 10 min a 40–80% increase in the basal activity. These results indicate that cAMP could play an important role by mediating the viral and plant host-cell interaction. The rapid pulse-release of cAMP leads us to propose that this nucleotide may, as in animal tissues, represent a secondary messenger in higher plants.     

An Ethylene Biosynthesis-Inducing Endoxylanase Elicits Electrolyte Leakage and Necrosis in Nicotiana tabacum cv Xanthi Leaves

We have previously demonstrated that a protein purified from xylan-induced culture filtrates of Trichoderma viride contains β-1,4-endoxylanase activity and induces ethylene biosynthesis in tobacco (Nicotiana tabacum cv Xanthi) leaf discs. When the ethylene biosynthesis-inducing xylanase (EIX) was applied to cut petioles of detached tobacco leaves, it induced ethylene biosynthesis within 1 hour and extensive electrolyte leakage and necrosis were observed in tobacco leaf tissue within 5 hours. Ethylene-pretreatment (120 microliters per liter ethylene for 14 hours) of tobacco leaves enhanced ethylene biosynthesis in response to EIX by more than threefold and accelerated development of cellular leakage and necrosis. In intact plants, similar symptoms could be induced in leaves that were distant from the point of the enzyme application. The evidence suggests that EIX is translocated via the vascular system and elicits plant responses similar to those observed in a hypersensitive response.     

Photosynthetic Capacities and Growth Characteristics of Nicotiana tabacum (cv. Xanthi) Cell Suspension Cultures

Photoheterotrophic growth of cell suspensions of Nicotiana tabacum L. (cv. Xanthi) in organic culture medium enriched in sucrose (30 g per liter) showed a classical sigmoid growth curve. The cells developed functional chloroplast structures during the exponential growth phase, when their chlorophyll content increased steadily. A limited drop (30%) in the chlorophyll amount and structural changes of the plastids (starch accumulation) were observed during the lag phase. The measurements of photosynthetic capacities (O₂ evolution and CO₂ fixation) during the growth cycle revealed changes in the photosynthetic ratio (O₂/CO₂), which was near 1 during the lag and stationary phases and near 2 during exponential growth. During exponential growth there was also a rapid NO₃^? uptake. Analysis of label distribution among the products of ¹⁴CO₂ fixation showed that both CO₂ assimilation pathways, linked to the ribulose-biphosphate carboxylase (the autotrophic pathway) and to phosphoenolpyruvate carboxylase (the non-autotrophic pathway) were operative with an important increase of the capacity of the latter during the exponential growth phase. Maximum rate of oxygen evolution, either endogenous or with p-benzoquinone as Hill reagent, as well as the increased CO₂ Fixation capacity via the non-autotrophic pathway during the exponential phase were concomitant with a high cyanide inhibited O₂ uptake.     

Ethylene Biosynthesis-Inducing Endoxylanase Is Translocated through the Xylem of Nicotiana tabacum cv Xanthi Plants

Ethylene biosynthesis-inducing xylanase (EIX) from the fungus Trichoderma viride elicits enhanced ethylene production and tissue necrosis in whole tobacco (Nicotiana tabacum cv Xanthi) plants at sites far removed from the point of EIX application when applied through a cut petiole. Symptoms develop in a specific pattern, which appears to be determined by the interconnections of the tobacco xylem. Based on results of tissue printing experiments, EIX enters the xylem of the stem from the point of application and rapidly moves up and down the stem, resulting in localized foliar symptoms on the treated side of the plant above and below the point of EIX application. The observation that a fungal protein that elicits plant defense responses can be translocated through the xylem suggests that plants respond to pathogen-derived extracellular proteins in tissues distant from the invading pathogen.     

Timing of morphological and histological development in premeiotic anthers of Nicotiana tabacum cv. Xanthi (Solanaceae)

The tobacco stamen has been the object of many developmental studies, and the organ has more recently become a model for molecular genetic studies of anther differentiation. However, the spatial and temporal details of cellular differentiation of early anther development have never been thoroughly characterized. In the present study, the age of 15 tobacco flowers from plants grown under constant light and temperature was estimated using growth analysis. Prior to tissue fixation for light microscopy, moulds of stamen and anther primordia were made with a dental impression polymer so morphological and histological observations could be made on each tissue sample. Flower ages spanned an 8-d interval during which petal and stamen initiation occurred, and sporogenous cells reached the leptonema stage of meiosis. The initial development of the tetrasporangiate anther shape largely preceded periclinal division of archesporial initials. Anatomically, periclinal divisions in the hypodermal ∗∗∗(l₂) layer were observed before archesporial initials began to divide. These data indicate differences in the cellular basis of tobacco anther development compared to earlier clonal analyses of Datura. The pattern of mitotic cell division associated with microsporangial development suggested modal peaks in division over time. The ability to estimate developmental time in the tobacco anther has implications for future studies directed at understanding mechanisms of anther evolution via heterochrony.     

In vitro Plant Regeneration of Melia azedarach L.: Shoot Organogenesis from Leaf Explants

In vitro regeneration of Melia azedarach L. was studied. Shoots were regenerated from calli initiated from leaflets of in vitro growing plants. The best medium for establishment of cultures was Murashige and Skoog (MS) medium with 4.44 μM benzylaminopurine (BAP) + 0.46 μM kinetin (KIN) + 16.29 μM adenine sulphate (ADE). Regenerated shoots were multiplied in MS + 0.44 μM BAP + 0.37 μM KIN + 3.26 μM ADE. Maximal rooting of 89 % was achieved by culture of regenerated shoots in MS + 12.26 μM indole-3-butyric acid for 3 d and subsequently in MS lacking growth regulators for 27 d. Rooted shoots were acclimatized and successfully transferred to soil. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi

Plants offer an alternative inexpensive and convenient technology for large scale production of recombinant proteins especially recombinant antibodies (plantibodies). In this paper, we describe the expression of a model single chain antibody fragment (B6scFv) in transgenic tobacco. Four different gene constructs of B6scFv with different target signals for expression in different compartments of a tobacco plant cell with and without endoplasmic reticulum (ER) retention signal were used. Agrobacterium mediated plant transformation of B6scFv gene was performed with tobacco leaf explants and the gene in regenerated plants was detected using histochemical GUS assay and PCR. The expression of B6scFv gene was detected by western blotting and the recombinant protein was purified from putative transgenic tobacco plants using metal affinity chromatography. The expression level of recombinant protein was determined by indirect enzyme-linked immunosorbent assay. The highest accumulation of protein was found up to 3.28 % of the total soluble protein (TSP) in plants expressing B6scFv 1003 targeted to the ER, and subsequently expression of 2.9 % of TSP in plants expressing B6scFv 1004 (with target to apoplast with ER retention signal). In contrast, lower expression of 0.78 and 0.58 % of TSP was found in plants expressing antibody fragment in cytosol and apoplast, without ER retention signal. The described method/system could be used in the future for diverse applications including expression of other recombinant molecules in plants for immunomodulation, obtaining pathogen resistance against plant pathogens, altering metabolic pathways and also for the expression of different antibodies of therapeutic and diagnostic uses.     

K-Nutrition, Growth Bud Formation, and Amine and Hydroxycinnamic Acid Amide Contents in Leaf Explants of Nicotiana tabacum Variety Xanthi n.c. Cultivated in Vitro

The effects of K-nutrition on growth (increase of fresh weight), bud formation (time of emergence, number of buds), and amine and hydroxycinnamic acid amide contents in foliar explants of Nicotiana tabacum cv Xanthi n.c. cultivated in vitro were examined. In K-deficient medium and in high K medium growth and bud formation were markedly inhibited. Marked changes of amine content (a diamine, putrescine; a phenolic amine, phenethylamine) were observed after a few days of culture. No apparent relationship was found between these amines and growth or bud differentiation. In contrast, changes in hydroxycinnamic acid levels were shown to correlate well with growth and bud formation. The greatest stimulation of budding and growth was correlated with the greatest accumulation of these amides. The highest contents of hydroxycinnamic acid amides were found during the first 15 days in culture when intensive cell division took place. Then they declined sharply after 26 days in culture as the rate of cell division decreased and differentiation occurred.     

Alterations in Nicotiana tabacum L. cv Xanthi Cell Membrane Function following Treatment with an Ethylene Biosynthesis-Inducing Endoxylanase

An ethylene biosynthesis-inducing xylanase (EIX) produced by the fungus Trichoderma viride elicited enhanced ethylene biosynthesis and leakage of potassium and other cellular components when applied to leaf disks of tobacco (Nicotiana tabacum L. cv Xanthi). Suspension-cultured cells of Xanthi tobacco responded to EIX by rapid efflux of potassium, uptake of calcium, alkalization of the medium, inhibition of ethylene biosynthesis, and increased leakage of cellular components. EIX-treated cell suspensions released 1-aminocyclopropane-1-carboxylate (ACC) into the surrounding medium, resulting in a reduction of cellular pools of ACC. The responses of both cell suspensions and leaf disks were inhibited (50-80%) by the preincubation of the tissues with the calcium channel blocker La³⁺. High concentrations of EGTA inhibited the alkalization of the medium by cell suspensions responding to EIX, but EGTA alone caused extensive loss of K⁺ and ACC and inhibited ethylene biosynthesis by tobacco cells. Alterations in membrane function appear to be important in the mode of action of EIX in Xanthi cells.     

Photoreactivation of nitrate reductase production in Nicotiana tabacum var. Xanthi.

Ultraviolet (254 nm) irradiation of liquid-cultured tobacco cells inhibited the production of nitrate reductase; subsequent illumination with white light allowed a partial restoration of the synthesis of the enzyme (photoreactivation). Ultraviolet irradiation of these same cells also inhibited their ability to incorporate labeled uridine and labeled amino acids. Subsequent illumination with white light gave a partial restoration of the ability of the cells to incorporate uridine while a similar post-ultraviolet-irradiation treatment failed to restore the amino acid incorporation. The system in tobacco known to repair ultraviolet-damaged viral RNA thus does not seem to repair ultraviolet damage to the protein-synthesizing system of the cell. The photoreactivation of nitrate reductase production is best explained by the action of a DNA photorepairing system.     

Shoot Regeneration from Cultured Leaf Explants of Lycium barbarumand Agrobacterium-Mediated Transformation1

A method for fast plant regeneration via organogenesis directly from Lycium barbarumleaf explants has been developed. The key factor for shoot regeneration was the presence of benzyladenine (BA) in the medium. NAA could only induce root formation and explant callusing. Murashige and Skoog (MS) medium supplemented with 2 mg/l BA and 0.5 mg/l NAA is the most efficient condition for shoot formation, with up to 92.6% shoot regeneration and no callus formation. All adventitious shoots cultured on MS medium supplemented with 1 mg/l IAA formed an extensive root system. Regenerated plants were morphologically normal and were also proved to be diploid (2n = 24). Using the optimized regeneration system, the genetic transformation of L. barbarumwas carried out mediated by Agrobacterium tumefaciensEHA101(pIG121Hm). 11.8% leaf explants produced kanamycin-resistant shoots after infection by A. tumefaciens.The putative transgenic nature of plants was confirmed by GUS assay and PCR analysis. Expression of the nptIIgene in the regenerated plants was also detected by observing the callus formation by leaf pieces on MS medium containing 0.2 mg/l 2,4-D and 0–100 mg/l kanamycin.     

In Vitro Clonal Propogation of Coleus forskohlii via Direct Shoot Organogenesis from Selected Leaf Explants

Present study provides an easy and efficient protocol for large scale clonal propagation of Coleus forskohlii, a threatened medicinal plant of commercial importance. Basal leaf lamina excised from upper three nodes of shoot was used as explant and its size, position, orientation and season of collection were initially optimized to select the most responsive explant condition. Enhanced shoot production and proliferation has been achieved on medium containing 2 μM BA + 0.1 μM NAA wherein, a highest number of 35 shoots/explant were produced. The regenerated shoots of varied length (3–5 cm) were transferred to root induction medium comprising of IBA, NAA and IAA (1–5 μM) in half-strength MS medium to determine the most suitable shoot length for proper root induction. Rooted plantlets were acclimatized in field conditions after proper hardening. Histological analysis was also carried out to confirm the nature of origin of shoot buds from leaf explants.     

Sensitivity to an Ethylene Biosynthesis-Inducing Endoxylanase in Nicotiana tabacum L. cv Xanthi Is Controlled by a Single Dominant Gene

The ethylene biosynthesis-inducing xylanase (EIX) is known to be a potent elicitor of ethylene biosynthesis and other responses when applied to leaf tissue of Nicotiana tabacum L. cv Xanthi. In contrast, leaf tissue of the tobacco cultivar Hicks was insensitive to EIX at concentrations 100-fold higher than was needed to elicit responses from Xanthi. Cell-suspension cultures of Xanthi and Hicks showed similar differences in sensitivity to EIX. Equivalent levels of ethylene production were elicited in leaf discs of both cultivars after treatment with CuSO4. The F1 and Xanthi backcross progeny of Hicks and Xanthi crosses were all sensitive to EIX, whereas the F2 and Hicks backcross progeny segregated for sensitivity to EIX. Individual plants from the F2 and Hicks backcross that were insensitive to EIX produced only insensitive progeny when they were self-pollinated. Progeny from sensitive plants either segregated for sensitivity to EIX or produced all sensitive progeny (an F2 plant). Sensitivity to EIX is controlled by a single dominant gene, based on chi-square analysis of segregation ratios.