Local expression of the ipt gene in transgenic tobacco (Nicotiana tabacum L. cv. SR1) axillary buds establishes a role for cytokinins in tuberization and sink formation

The developmental characteristics of a transgenic tobacco line (BIK62) expressing the ipt cytokinin-biosynthetic gene under the control of a tagged promoter were analysed. In situ hybridization and cytokinin immunocytochemistry revealed that the ipt gene was mainly expressed in the axillary buds after the floral transition. The ipt-expressing axillary buds presented morphological alterations such as short and narrow scale-leaflets, and swollen internodes filled with starch grains, giving rise to short and tuberized lateral branches. In addition, the modification of the endogenous cytokinin balance in the axillary meristems resulted in a fast rate of leaf initiation and cytokinins accumulated mostly in the lateral zones of the reactivated axillary meristems, suggesting a role in leaf organogenesis. Cell cycle analysis revealed that the reactivated axillary meristems were characterized by predominant S+G2 nuclei. Terminal internodes displayed low levels of hexose and sucrose concomitant with starch accumulation. Extracellular invertases (EC 3.1.26) were also present in higher amounts in the tuberizing internodes compared to the axillary buds of wild-type tobacco. These results underline the role of cytokinins in cell cycle regulation and in the creation of a sink--source effect. They also provide new information about cytokinin involvement in the process of tuberization and their overproduction in axillary buds giving rise to tuberized lateral branches in a naturally non-tuberizing species.     

Apical reactions to gibberellic acid application according to the genotype inSilene armeria

Two selected strains ofSilene armeria L. were used: S_1-2 (GA-line, not induced to flowering by GA₃ in SD of 8 h) and S_2-1 (GA+ line which reacts to GA₃ with flowering in non-inductive photoperiod). Moreover S_1-2 and S_2-1 differ in their critical daylength 14.5 and 8.0 h, respectively. Changes in the mitotic index and DNA content of cells in the various zones of the apical meristem during GA₃ treatment were described. At the start of the experiment, the functioning of the apex was characterized by a predominance of G₁ phase in the two strains. Therefore S_2-1 differed by a higher mitotic activity specially in the central zone. In S_1-2, GA₃ acted mainly on the rib-meristem cells, which resulted in stem elongation. Lack of response in the cells of the central zone explains why GA₃ fails to induce flowering in this strain. In S_2-1, GA₃ acted mainly on the central zone where a gradual increase in mitotic acitivity and in the percentages of nuclei at the (S + G₂) level was recorded. This stimulation brought the meristems into the prefloral stage. The differences between the two strains are discussed according to the status of control meristems. The reactions induced by GA₃ were compared with the pattern of changes during induction, in LD. This work was supported by grants from the “Centre National de la Recherche Scientifique” (UA 572).     

High frequency plant regeneration from Eucalyptus globulus Labill. hypocotyls: Ontogenesis and ploidy level of the regenerants

Up to 70% regenerating hypocotyls provided with 5 to 20 buds were obtained on MS medium containing 0.01 or 1 mg l^-1 NAA and 0.2 mg l^-1 BA or 0.2 mg l^-1 BA and 0.2 mg l^-1 TDZ. The ability to regenerate buds was correlated with the presence of oil glands at a stage in germination when oil secretion was not yet occurring. The regeneration of shoot meristems took place from the cells involved in the differentiation of these glands. Such glands could also appear during callus redifferentiation, giving rise to indirect regeneration. Rooting of the regenerants was efficient using a two-step procedure of induction under darkness in the presence of 3 mg l^-1 IBA, followed by root development on medium devoid of growth regulators under a 16-h photoperiod, the medium being solidified with Gelrite. Regenerated plants showed no phenotypic alterations. Nuclear DNA contents in mother-plant material and regenerants were analysed using flow cytometry. There was no evidence of polyploidy in any of the samples, indicating the absence of polyploidisation during cell differentiation and under in vitro conditions. No regeneration was obtained from leaf or stem explants from micropropagated plantlets. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mitotic Activities and Levels of Nuclear DNA in the Apical Meristem of Silene armeria (strain SI.2) Following Application of Gibberellin A3

Strain S1.2 of Silene armeria was grown under an 8h-photoperiodand treated with GA₃ every day for 20 days. This growth substancecaused stem elongation, but no flowering in this long-day plant.Changes in the mitotic index and DNA content of cells in thevarious zones of the apical meristem before, during and afterGA₃ treatment were described. Mitotic activity and increasein the proportion of nuclei at the 4C level (S+G₂ phase of thecell cycle) were strongly stimulated in the rib-meristem, andto a lesser extent in the lateral zone, but not in the axialzone. This stimulation of apical activity reached a peak aftertwo GA₃ treatments and then declined gradually, so that after20 days the activity in GA₃-treated meristems was lower thanthat in untreated controls; at this point most cells were inthe G₁ phase. When the GA₃ treatment was discontinued, there was a gradualincrease in the mitotic activity which ultimately reached thesame level as that in controls. Stem elongation ceased and leavesformed aerial rosettes. It is concluded that in vegetative plants of strain S1.2 ofSilene armeria GA₃ acts mainly on the rib-meristem cells whichresults in stem elongation. Lack of response in the axial cellsexplains why GA₃ fails to induce flowering in this strain ofSilene armeria. (Received June 18, 1983; Accepted August 3, 1983)     

Photoperiod and gibberellic acid – control of the cell cycle in the meristem of Silene armeria and its effects on flowering

Plants of Silene armeria L., strain S_2.1, a quantitative long-day (LD) species which is known to react to GA₃ by flowering after attaining, the'intermediate stage', were induced by two LD or by two GA₃ applications. Changes in the mitotic index and DNA content (microdensitometric estimation) of cells in the axial zone, lateral zone and rib meristem of the shoot apex were observed during the first 48 h of each treatment. Similar mitotic activation occurred in response to LD or GA₃ after a 6-8 h lag period. This was preceded by a decrease in the proportion of nuclei with a 2C DNA content, indicating that in this species the control point for the shortening of the cell cycle was essentially in G₁. A second mitotic peak was observed 16 h later in photoinduced meristems, resulting in more pronounced cellular synchronization. These further events were not seen in GA₃-treated plants where only the meristematic activity was slightly, but reproducibly higher than in the control. Thus, two successive synchronizations of cell division are a typical feature of LD induction. The data are discussed with regard to the competence of shoot apical cells to be reactivated. The essential changes for the transition to flowering depend on these differential patterns of cell reactivation.     

Densitometric analysis of nuclear DNA content in lentil roots grown in space

Lentil seedlings were grown for 28 h in space, on board Spacelab (IML 1 Mission) and growth of the primary root was analysed. The length of cortical cells was less in near weightlessness than on the 1 g centrifuge (flight control) and mitotic index was lower but there was no apparent perturbation in the mitosis. To further investigate which phase of cell cycle was modified, densitometric analysis of nuclear DNA content in cortical cells was carried out by the mean of an image processing system (SAMBA). In microgravity there was a decrease in DNA synthesis and a promotion of the arrest in the G2 phase of cell cycle. These results, and other ones obtained elsewhere on a slowly rotating clinostat, led us to think that in microgravity the perturbation of the gravisensing cells and/or the absence of convection could account for the modification of cell growth registered in the primary root.     

Ectopic expression of Arabidopsis CYCD2 and CYCD3 in tobacco has distinct effects on the structural organization of the shoot apical meristem

Transgenic tobacco lines expressing Arath-CYCD2 or Arath-CYCD3 genes under a cauliflower mosaic virus 35S promoter are modified in the timing of their development, but not in the phenotype of their vegetative organs. They display an increased rate of leaf initiation, which is shown to be associated with distinct changes in the structural organization of their shoot apical meristem (SAM). Constitutive expression of Arath-CYCD2 leads to a progressive modification of the SAM structural organization with predominant periclinal divisions in the L3 layer and to the loss of the classical cytophysiological zonation, the central zone being reduced to the central cells of the L1 and L2 layers. These changes reveal a particular sensitivity of the corpus cells (L3) to Arath-CYCD2 over-expression and suggest a role for CYCD2 in controlling the planes of cell division in these cells. The SAM structural modifications in the Arath-CYCD3 over-expressing lines are less drastic; only an increased cell number together with a reduced cell size, particularly in the L1 layer, characterizes the peripheral zones. This could be related to the shortening of the G1-phase duration that renders cell growth incomplete between successive mitoses. Cell proliferation continues beyond the SAM in the developing internodes and confers a delayed senescence to Arath-CYCD3 over-expressing juvenile tissues. In addition, the ploidy levels of mature stem tissues in both types of transgenic lines are unaffected, suggesting that the studied G1 to S cell-cycle genes have no effect on the extent of endoreduplication in tobacco stem tissues.     

Plant regeneration fromin vitro leaf culture of severalGerbera species

Efficient bud regeneration was obtained from a clone ofGerbera hybrida Bol. L. leaf explants cultured on modified Murashige and Skoog medium supplemented with 10 µM benzyladenine and 2.5 µM naphthalenacetic acid. The morphogenic potential varied with the developmental stage of the leaves. Up to 90% of excised developing leaves formed 3 to 5 shoots per explant. Bud regeneration was not obtained on the same medium with fully expanded leaves. Addition of 0.05 µM or 0.5 µM thidiazuron to the above medium significantly promoted regeneration from mature leaves but was ineffective for similar explants of a recalcitrant clone. The two wild speciesG. viridifolia Schultz Bip. andG. piloselloides L. Cass. also displayed specific multiplication habits and regeneration abilities. Bud regeneration occurred from callus. Chromosome counts and DNA flow cytometry indicated that all the regenerants were typically diploid, as were the various tissues of the mother plants. Afterin vitro rooting and acclimatization, no phenotypic variations were detected among the regenerants during both their vegetative and reproductive phases.     

Dynamics of Cytokinins in Apical Shoot Meristems of a Day-Neutral Tobacco during Floral Transition and Flower Formation

This study considered cytokinin distribution in tobacco (Nicotiana tabacum L.) shoot apices in distinct phases of development using immunocytochemistry and quantitative tandem mass spectrometry. In contrast to vegetative apices and flower buds, we detected no free cytokinin bases (zeatin, dihydrozeatin, or isopentenyladenine) in prefloral transition apices. We also observed a 3-fold decrease in the content of cytokinin ribosides (zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine) during this transition phase. The group concluded that organ formation (e.g. leaves and flowers) is characterized by enhanced cytokinin content, in contrast to the very low endogenous cytokinin levels found in prefloral transition apices, which showed no organogenesis. The immunocytochemical analyses revealed a differing intracellular localization of the cytokinin bases. Dihydrozeatin and isopentenyladenine were mainly cytoplasmic and perinuclear, whereas zeatin showed a clear-cut nuclear labeling. To our knowledge, this is the first time that this phenomenon has been reported. Cytokinins do not seem to act as positive effectors in the prefloral transition phase in tobacco shoot apices. Furthermore, the differences in distribution at the cellular level may be indicative of a specific physiological role of zeatin in nuclear processes.     

The pleiotropic effects induced by therolC gene in transgenic plants are caused by expression restricted to protophloem and companion cells

Expression of therolC gene fromAgrobacterium rhizogenes causes morphological and developmental alterations in transgenic plants. The histological alterations underlying the macroscopic changes and the cellular localization of the site of expression of therolC gene have shown that: (i) the expression of therolC gene is developmentally regulated, (ii) in vegetative transgenic plants, the expression of therolC gene under the control of its own promoter is restricted to companion and protophloem cells, (iii) the site of action of the product(s) of the activity of the rolC enzyme is distinct from its site of expression, (iv) precise localization of the rolC peptide has been achieved by immunocytochemistry but not by the histochemical GUS assay. These results imply that the sites of action and expression of therolC gene in trangenic plants are physically separated. Thus the product(s) of the activity of the rolC enzyme must be a factor capable of being transported. Current models forrolC gene action are discussed taking into account the reported results.