Effect of CPPU on in vitro axillary shoot proliferation and adventitious shoot regeneration in kiwifruit

Abstract

The effect of CPPU, N‐(2‐chloro‐4‐pyridyl)‐N‐phenylurea, on the development of axillary buds and on adventitious shoot regeneration was investigated in mature leaves of in vitro‐cultured shoots of Actinidia chinensis Planch (Sel. K190) and Actinidia deliciosa A. Chev. cultivars Hayward and Tomuri. In the multiplication phase, 2.2 mM CPPU induced proliferation rates comparable to 4.4 mM benzyadenine (BA) both in Hayward and in Tomuri, while a higher CPPU concentration reduced proliferation. In A. chinensis, significant differences in multiplication rates were not detected between BA and CPPU. However, shoots developed on CPPU appeared hyperhydric, and had very short internodes, reduced leaf laminas, higher water, carotenoid and phenol contents and considerably lower chlorophyll level in comparison with the BA‐grown shoots. On the other hand, in adventitious shoot regeneration CPPU was more effective than zeatin (ZEA) and BA in A. deliciousa cultivars and the best morphogenic response was obtained with the lowest concentration (10 mM) in cv Hayward, while 16 mM was the most efficient treatment in cv Tomuri. In A. chinensis, CPPU was as efficient as ZEA when the highest concentration was used.     

Stable transformation of Mesembryanthemum crystallinum (L.) with Agrobacterium rhizogenes harboring the green fluorescent protein targeted to the endoplasmic reticulum

Stable transformation of Mesembryanthemum crystallinum L. (common ice plant) with a green fluorescent protein (GFP) construct targeted to the endoplasmic reticulum was obtained. Seven and fourteen days after germination seedlings were infected with Agrobacterium rhizogenes strain ARqua1 either by direct coating of the cut radicles with bacteria growing on solid medium or by immersion of the cut surface in bacterial suspension at different optical densities. Both methods of infection resulted in production of GFP-positive roots with a frequency ranging from 6 to 20% according to the age of the explants and the application procedure. The green fluorescing roots displayed the typical hairy root phenotype and were easily maintained in liquid medium without growth regulators for over 2 years. Stable expression of the transgene in the roots was confirmed by polymerase chain reaction (PCR), immunoblotting and the capacity of roots to grow and produce callus on kanamycin-enriched medium. Nineteen endogenous cytokinins were determined in transgenic and non-transformed roots. The results revealed significantly lower levels of the free bases of isopentenyladenine, dihydrozeatin, cis- and trans-zeatin, as well as a conspicuous decline in concentrations of the corresponding nucleosides and most nucleotides in transgenic roots compared to the wild type. Comparison of the cytokinin profiles in transgenic and non-transformed roots suggested that transformation by A. rhizogenes disturbed cytokinin metabolism during the early steps of biosynthesis. Calli obtained from transformed roots were GFP-positive and remained non-regenerative or displayed high rhizogenic potential depending on the auxin/cytokinin ratio in the medium. Calli and callus-derived roots showed a strong GFP signal for over 2 years.     

Enhanced oxidative stress in the ethylene-insensitive (ein3-1) mutant of Arabidopsis thaliana exposed to salt stress

To better understand the role of ethylene signaling in plant stress tolerance, salt-induced changes in gene expression levels of ethylene biosynthesis, perception and signaling genes were measured in Arabidopsis thaliana plants exposed to 15 days of salinity. Among the genes analyzed, EIN3 showed the highest expression level increase under salt stress, suggesting a key role for this ethylene-signaling component in response to salt stress. Therefore, we analyzed the salt stress response over 15 days (by adding 100 mM NaCl to the nutrient solution) in the ein3-1 mutant compared to the wild-type (Col-0) in terms of growth, oxidative stress markers (lipid peroxidation, foliar pigments and low-molecular-weight antioxidants) and levels of growth- and stress-related phytohormones (including cytokinins, auxins, gibberellins, abscisic acid, jasmonic acid and salicylic acid). The ein3-1 mutant grew similarly to wild-type plants both under control and salt stress conditions, which was associated with a differential time course evolution in the levels of the cytokinins zeatin and zeatin riboside, and the auxin indole-3-acetic acid between the ein3-1 mutant and the wild-type. Despite showing no signs of physiological deterioration under salt stress (in terms of rosette biomass, leaf water and pigment contents, and PSII efficiency) the ein3-1 mutant showed enhanced lipid peroxidation under salt stress, as indicated by 2.4-fold increase in both malondialdehyde and jasmonic acid contents compared to the wild-type. We conclude that, at moderate doses of salinity, partial insensitivity to ethylene might be compensated by changes in endogenous levels of other phytohormones and lipid peroxidation-derived signals in the ein3-1 mutant exposed to salt stress, but at the same time, this mutant shows higher oxidative stress under salinity than the wild-type.     

Influence of temperature increase on evaportranspiration rate and cytokinin content in wheat seedlings

The content of cytokinins especially zeatin nucleotide decreased in shoots as a result of temperature increase. Simultaneously the cytokinins accumulated in roots. The changes in cytokinins distribution were followed by a decline of evapotranspiration after its initial temperature-induced uprising. This revised version was published online in July 2006 with corrections to the Cover Date.     

Spatial and Temporal Distribution of Zeatin and Zeatin Riboside during the Period of the Egg-Cell Activity in the Ovaries of Amphimictic and Apomictic Plants

Spatial and temporal distribution of zeatin and zeatin riboside was studied in the ovaries of amphimictic (Triticum aestivum L.) and apomictic (Taraxacum officinale Web.) species during the period from the quiescence release of the egg cell to its first division. This period was divided into three stages: (1) no signs of division of the central cell, (2) division of the central cell and no signs of division of the egg, and (3) division of the egg. Cytokinin content was determined in ovary and its four sections (basal, medial, apical, and ovule) using an original modification of ELISA. Apomicts and amphimicts were shown to be similar in the pattern of changes in the cytokinin content in basal and medial zones but differed substantially in that of apical section and ovule.     

Somatic embryogenesis from leaf cultures of potato

An efficient procedure has been developed for inducing somatic embryogenesis from leaf cultures of potato cv. Jyothi. Leaf sections were initially cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) + benzyladenine (BA) and -naphthaleneacetic acid (NAA) + BA supplemented Murashige and Skoog (MS) media. Nodular embryogenic callus developed from the cut ends of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. The explants with primary callus were subsequently moved onto MS media containing zeatin and/or gibberellic acid (GA₃) and BA. Treatment with zeatin (22.8 M) and BA (10.0 M) resulted in the induction of the highest number of somatic embryos directly from meristematic centres produced on the nodular tissue. Embryo induction and maturation took place on this medium. The cotyledonary stage embryos developed into complete plantlets on hormone-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis in leaf cultures of potato which has not been reported previously.     

Identification and quantitation by GC-MS of zeatin and zeatin riboside in xylem sap from rootstock and scion of grafted apple trees

Zeatin and zeatin riboside were identified by full-scan gas chromatography-mass spectrometry (GC-MS) in xylem sap of clonal apple rootstocks (M.27, M.9 and MM.106). These rootstocks exhibit a wide range of control over tree size when grafted to a common scion. The concentrations of zeatin and zeatin riboside were measured by GC-MS selected ion monitoring (SIM) in shoot xylem sap and root pressure exudate obtained from these rootstocks and from trees of Fiesta scion grafted onto the rootstocks. Zeatin was the predominant cytokinin in xylem sap from the dwarfing rootstocks, M.27 and M.9, while zeatin riboside was the predominant cytokinin in xylem sap from the more invigorating rootstock MM.106. Cytokinin concentrations (ng ml^–1) in root pressure exudate and shoot xylem sap, (i.e. from above the graft union in composite trees), increased with increasing vigour of the rootstock, irrespective of whether the plants were non-grafted rootstocks, or were composite plants of Fiesta scion grafted onto the rootstocks. Cytokinin content (ng shoot^–1) of shoot sap differed with rootstock; the more invigorating (MM.106) had greater amounts of cytokinins than the more dwarfing (M.9 and M.27) rootstocks. These results are discussed in relation to possible influences of roots on the growth of shoots via cytokinin supplies in the xylem sap.     

Exposure of Petunia Seedlings to Ethylene Decreased Apical Dominance by Reducing the Ratio of Auxin to Cytokinin

Seedlings of Petunia x hybrida Orchid treated with the ethylene-releasing compound ethephon at 0.9, 1.7, and 3.5 mM evolved ethylene at a higher rate as the concentration of ethephon increased. Regardless of the concentration of ethephon applied, ethylene evolution peaked 6 to 8 h following application. Evidence that ethephon application decreased apical dominance included an increase in the number of new nodes on the main stem and a sustained increase in the length of new and existing lateral shoots compared to the control (no ethephon). Plants treated with 3.5 mM ethephon developed mild chlorosis, whereas a concentration of 1.7 mM ethephon decreased apical dominance without phytotoxic effects. The auxin/cytokinin ratio decreased in the apical shoot section as early as 1 h after ethephon treatment. In contrast, a decrease in the ratio in the subapical shoot section was not detected until 24 h after ethephon application. Reduction in auxin/cytokinin ratio was a result of a decrease in indole-3-acetic acid (IAA) and an increase of zeatin riboside (ZR), but not isopentenyladenosine (iPA). These results suggest that exposing Orchid petunia seedlings to ethylene via ethephon lowers the auxin/cytokinin ratio, thereby promoting the outgrowth of lateral shoots.     

The uptake and metabolism of 3H-benzylaminopurine in tobacco (Nicotiana tabacum L.) and cucumber (Cucumis sativus L.) explants

The uptake and metabolism of ³H-benzylaminopurine(³H-BAP) were studied in explanted stem pith andleaves of tobacco and in the hypocotyls and cotyledonsof cucumber. The explants were kept for 2, 5, 8 and 20h on MS medium with 0.8 mg.l^–1 2,4-D,0.5 mg.l^–1 BAP and 13.2 mg.l^–1 aspartic acid(induction medium) with or without ³H-BAP and¹⁴C-sucrose. The highest uptake of ³H-BAPwas observed in tobacco leaves and cucumbercotyledons. The major metabolite in both species was³H-benzylaminopurine riboside (³H-BAPR). Thehighest level was found in explanted cucumbercotyledons after 20 h in culture, the lowest inexplanted tobacco stem pith. Intensive 7-glucosylationof ³H-BAP was observed in explanted tobaccoleaves after 20 h in culture, where the levels of7-glucoside of ³H-BAP and of free ³H-BAPwere equal. To study the morphogenic effect of growth regulators(BAP and 2,4-D), the explants were subcultured aftershort-term induction (20 h) to MS medium without anygrowth regulators. In most cases, incubation of 20 hon induction medium was sufficient to induce therespective morphogenesis. Cucumber hypocotyl andtobacco stem pith explants formed a callus on theirbasal end. Root formation was observed on explantedcucumber cotyledons and shoot formation on tobaccoleaves. Long-term culture (3 weeks) of tobacco leaveson induction medium led to the formation of callus andglobules. The microscopic analysis of globulesindicated the presence of meristematic and tracheidalcells.     

Cytokinins and Water Stress

It is almost impossible to find a single process in plant life that is not affected by both stress and hormones directly or indirectly. This minireview is focused on the interactions between water stress and cytokinins (CKs). The attention was paid mainly to changes in endogenous CK content and composition under water stress, involvement of CK in plant responses to water stress mainly in stomatal regulation of gas exchange, water relations of transgenic plants with elevated CK content, and possibilities to ameliorate the negative effects of water stress by application of exogenous CKs.