Ana Elisa Valdés

Levels and immunolocalization of endogenous cytokinins in thidiazuron-induced shoot organogenesis in carnation.

We evaluated the capacity of the plant growth regulator thidiazuron (TDZ), a substituted phenylurea with high cytokinin-like activity, to promote organogenesis in petals and leaves of several carnation cultivars (Dianthus spp.), combined with 1-naphthaleneacetic acid (NAA). The involvement of the endogenous auxin indole-3-acetic acid (IAA) and purine-type cytokinins was also studied. Shoot differentiation was found to depend on the explant, cultivar and balance of growth regulators. TDZ alone (0.5 and 5.0 micromol/L) as well as synergistically with NAA (0.5 and 5.0 micromol/L) promoted shoot organogenesis in petals, and was more active than N6-benzyladenine. In petals of the White Sim cultivar, TDZ induced cell proliferation in a concentration-dependent manner and, on day 7 of culture, the proportion of meristematic regions in those petals allowed the prediction of shoot regeneration capacity after 30 days of culture. Immunolocalization of CK ribosides, N6-(delta2-isopentenyl)adenosine, zeatin riboside (ZR) and dihydrozeatin riboside (DHZR), in organogenic petals showed them to be highly concentrated in the tips of bud primordia and in the regions with proliferation capacity. All of them may play a role in cell proliferation, and possibly in differentiation, during the organogenic process. After seven days of culture of White Sim petals, NAA may account for the changes found in the levels of IAA and DHZR, whereas TDZ may be responsible for the remarkable increases in N6-(delta2-isopentenyl)adenine (iP) and ZR. ZR is induced by low TDZ concentrations (0.0-0.005 micromol/L), whereas iP, that correlates with massive cell proliferation and the onset of shoot differentiation, is associated with high TDZ levels (0.5 micromol/L). In addition to the changes observed in quantification and in situ localization of endogenous phytohormones during TDZ-induced shoot organogenesis, we propose that TDZ also promotes growth directly, through its own biological activity. To our knowledge, this study is the first to evaluate the effect of TDZ on endogenous phytohormones in an organogenic process.

Relationships between hormonal contents and the organogenic response in Pinus pinea cotyledons

Isolated cotyledons from Pinus pinea L. germinated embryos cultured in vitro showed shoot organogenesis in response to treatment with 4.4 µM N 6 -benzyladenine (BA). In late germination phases, the extent of the response onset decreased and adventitious buds were redistributed to the basal zone of the cotyledons. To understand these two events, analyses of indole-3-acetic acid (IAA) and several cytokinins have been carried out in apical and basal portions of cotyledons taken at four different culture periods of the embryos on germination medium (0, 2, 4 or 6 d). The highest endogenous content of all hormones, except N 6 -isopentenyladenosine, was found in the most responsive explants, those that were taken from 0-d-old cultured embryos. Apical portions of cotyledons taken from 0 to 4-d-old cultured embryos showed a reduction in the content of IAA and several cytokinins, but basal portions did not show the same pattern. BA dose-response curves were made to explain the loss of caulogenic potential with the age of the explants. The results suggested the combination of two factors, changes in the endogenous hormonal content in original explants and loss of their sensitivity to exogenously applied BA, as the cause for the reduction of the ability of Pinus pinea cotyledons to form adventitious buds.

Could plant hormones be the basis of maturation indices in Pinus radiata

Abstract Maturation and phase change in woody plants are developmental processes at present poorly understood, especially from a physiological point of view. Maturation indices, such as phytohormones, could be used to understand more about the events that accompany the increase in the developmental state and to choose optimal conditions in order to improve forest programs and make them more profitable. In order to determine this putative index, the contents in abscisic acid-like substances (ABA-like), indole-3-acetic acid (IAA) and several cytokinins (Cks) were analysed in terminal and axillary buds of Pinus radiata trees with different developmental states to compare their phytohormonal status. An increase in the content of some zeatin-type (Z-type) Cks, and a decrease in isopentenyladenine-type (iP-type) Cks levels throughout maturation were shown both in terminal and axillary buds. This fact allowed us to consider the ratio iP-type/Z-type Cks, which decreased in parallel with the increasing developmental state of the tree, as a maturation index. According to the results, ABA-like compounds and IAA contents could not be considered as a maturation index in Pinus radiata , even though similar patterns were in fact found between the two kinds of buds.

Alterations in endogenous levels of cytokinins following grafting of Pinus radiata support ratio of cytokinins as an index of ageing and vigour

Markers for ageing or maturation in woody plants provides the possibility of identifying genotypes with a prolonged juvenile phase, or to evaluate reinvigoration procedures, such as grafting of adult material. This study focuses specifically on cytokinins (Cks) as markers, due to their role in the transition process from juvenile to adult states. Previous reports from our group disclosed that the ratio of isopentenyladenine-type (iP-type) to zeatin-type (Z-type) Cks decreases during tree maturation, a maturation and ageing index being likely in Pinus radiata. Based on these results, Cks were tested in terminal buds of adult and grafted adult P. radiata material to corroborate the importance of the aforementioned balance as an index in the reinvigoration process of adult trees associated with grafting procedures. Results revealed the reversion of this index pattern parallel to the reactivation of some juvenile traits, namely, increasing values of the ratio throughout the reinvigoration process. According to previous results, however, the opposite was the case during the reverse process. This fact allowed us to validate the iP-type/Z-type Ck ratio as an ageing and vigour index.

Developmental stage as a possible factor affecting cytokinin content and cytokinin dehydrogenase activity in Pinus sylvestris

In the present study cytokinin dehydrogenase (CKX) activity was for the first time found in a conifer species, Pinus sylvestris. The activities were correlated with the endogenous cytokinin contents. Several enzyme substrates and two different electron acceptors were used to search for the enzyme activity in the extract from seeds, seedlings and plantlets. The highest specific activity was found in one-year-old plantlets with isopentenyladenine as the substrate and 2,6-dichlorophenolindophenol as the electron acceptor, at pH 8. An enhancement in the CKX specific activity corresponded to increasing contents of cytokinins, mainly isopentenyladenine and isopentenyladenosine, indicating that the enzyme activity is affected by the endogenous supply of cytokinins. CKX affinity for the ribosylated form of isopentenyladenine was dependent on the developmental stage, being higher in seeds than in seedlings, and not detectable in plantlets. The results are indicative of the presence of different isoenzymes throughout the development.