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.     

The role of cytokinins in rooting of stem slices cut from apple microcuttings

ABSTRACT

We examined the role of cytokinins in rooting of 1-mm stem slices cut from microcuttings of the apple rootstock ‘Jork 9?s. Various types of cytokinins inhibited the rooting of apple stem slices to different extents. Highest inhibition was obtained with thidiazuron and benzylaminopurine. Remarkably, isopentenyladenine and isopentenyladenosine enhanced rooting at low concentration (at the optimal concentration of 0.1 μM by 53 and 19%, respectively). We also examined the effect of lovastatin and simvastatin. These drugs are putative cytokinin-synthesis inhibitors. Both inhibited rooting and inhibition was partially reversed by simultaneous addition of zeatin. Moreover, in the presence of lovastatin a higher concentration of zeatin had to be applied to achieve inhibition of rooting than in the absence of the drug. This data indicates that these compounds indeed inhibited cytokinin synthesis. One-day pulses with lovastatin strongly blocked rooting when given just after cutting the slices but had no effect after that. Adding zeatin simultaneously reversed inhibition completely. In conclusion, our data confirm that cytokinins may strongly inhibit rooting but they also show that at low concentration, certain cytokinins enhance rooting. Moreover, synthesis of cytokinin is essential during root formation. We hypothesise that cell division initiated by a relatively high endogenous level of cytokinins just after cutting the slices is a necessary, initial step in adventitious root formation.     

Effects of cytokinins and auxins on cassava shoot organogenesis and somatic embryogenesis from somatic embryo explants

Somatic embryos of cassava (Manihot esculenta Crantz) cultivar ‘Nanzhi 188’ were isolated and cut into fragments to be cultured on media with various cytokinins and auxins. Cytokinin induced of cassava shoot organogenesis, while auxin stimulated somatic embryogenesis. The effectiveness on organogenesis was different based on different cytokinins and in combination with auxins. Benzyladenine and thidiazuron stimulated more shoot organogenesis than kinetin and N-isopentenyladenine. This revised version was published online in June 2006 with corrections to the Cover Date.     

The shoot meristem as a site of continuous organogenesis

In higher plants, organ formation occurs throughout life. This remarkable process occurs at a collection of stem cells termed the shoot meristem. The shoot meristem originates during embryogenesis and is later responsible for generating the above-ground portion of the plant. The shoot meristem can be thought of as having two zones, a central zone containing meristematic cells in an undifferentiated state, and a surrounding peripheral zone where cells enter a specific developmental pathway toward a differentiated state. Recent advances have revealed several genes that specifically regulate meristem development inArabidopsis. The function of these genes and their genetic interactions are described.     

Regeneration of cassava plants via shoot organogenesis

A novel regeneration system based on direct shoot organogenesis is described for cassava. Plants could be regenerated at high frequency by inducing shoot primordia on explants derived from cotyledons of cassava somatic embryos. After a passage on elongation medium, the regenerated shoots were easily rooted in hormone-free medium and could be successfully transplanted to soil. Using the shoot-organogenesis-based regeneration method, up to eight transplantable plantlets per explant could be regenerated. The system was optimised first for one cassava cultivar, and then its transferability to three other cultivars was demonstrated. This method widens the scope of in vitro regeneration modes of cassava, and is also compatible with Agrobacterium-mediated transformation. To develop an efficient system for production of somatic embryos for regeneration experiments, conditions for inducing primary and cycling somatic embryos were also studied, and highly efficient plant regeneration via germination of somatic embryos was achieved using maltose instead of sucrose in the culture medium, and combining paclobutrazol with 2,4-dichlorophenoxyacetic acid in the embryo induction medium. Received: 25 January 1997 / Revision received: 10 February 1997 / Accepted: 20 February 1997     

The role of cytokinins on in vitro shoot production in Salix tetrasperma Roxb.: a tree of ecological importance

A valuable tropical tree, Salix tetrasperma Roxb. commonly known as Indian willow has been investigated for its in vitro regeneration potential using nodal explants obtained from a 30-year-old elite tree. Agar-solidified Woody Plant Medium (WPM) containing different concentrations of Plant Growth Regulators (PGRs) was used in the study. Shoot induction response was best on WPM supplemented with 6-benzyladenine (5.0 μM) where 90% explants responded with an average shoot number (4.40 ± 0.50) and shoot length (0.92 ± 0.04) after 6 weeks of culture. However, multiplication and elongation was best recorded when BA (5.0 μM) treated shoot clusters were transferred to WPM containing BA (1.0 μM) + NAA (0.5 μM) where 18.40 ± 0.92 well-grown healthy shoots with an average shoot length of 5.30 ± 0.43 cm were obtained on completion of 12 weeks culture period. In vitro rooting of shoots was best achieved in half-strength WPM containing 0.5 μM IBA. Well-rooted plantlets were successfully hardened off and acclimatized in plastic cups containing sterile Soilrite. These plantlets were then transferred to pots containing normal garden soil followed by transfer to greenhouse and ultimately to field under full sun.     

Direct shoot organogenesis and plant regeneration in safflower

Summary  Adventitious shoot buds were induced directly on the adaxial surface of the cotyledons of eight safflower cultivars after 14 d of culture initiation on Murashige and Skoog's (MS) medium supplemented with various levels of 6-benzylaminopurine (BA). Maximum shoot organogenesis of 54.4% with 10.2 shoots per responding cotyledon was obtained with 8.87 μM BA in the cv. S-144. Regenerated shoots were classified into three groups on the basis of their morphological features and were found to be correlated with the levels of BA. The highest number of normal shoots was obtained from 2.2 μM BA treatment. The regenerated shoots of group I (normal shoots) were rooted on half-strength MS medium supplemented with 5.3 μM α-naphthaleneacetic acid, 3% sucrose and 0.8% bacto-agar. Rooted plantlets were successfully transferred to soil and appeared morphologically normal. Histological studies revealed that shoot buds originated adventitiously from subepidermal cells.     

The role of cytokinins in chloroplast lamellar development

The accumulation of chlorophyll, production of two specific lamellar chlorophyll-protein complexes, onset of O(2) evolution, and detection of P700 were examined in intact Jack bean (Canavalia ensiformis [L.] D.C.) leaves treated with 10(-5)m kinetin or benzyladenine and allowed to green under low (30-35%) and high (80-85%) relative humidity. In contrast to reports of the promotion of chlorophyll accumulation by cytokinin treatment in excised tissue or cotyledons, intact greening leaves showed neither promotion of chlorophyll accumulation nor alteration in formation of the lamellar chlorophyll-protein complexes or development of photosynthetic function. Furthermore, cytokinin was ineffective in relieving the consequences of low relative humidity water stress on chlorophyll accumulation and on the formation of at least one lamellar chlorophyll-protein.     

The influence of ethylene and ethylene modulators on shoot organogenesis in tomato

The influence of ethylene and ethylene modulators on the in vitro organogenesis of tomato was studied using a highly regenerating accession of the wild tomato Solanum pennellii and an F1 plant resulting from a cross between Solanum pennellii and Solanum lycopersicum cv. Anl27, which is known to have a low regeneration frequency. Four ethylene-modulating compounds, each at four levels, were used, namely: cobalt chloride (CoCl₂), which inhibits the production of ethylene; AgNO₃ (SN), which inhibits ethylene action; and Ethephon and the precursor 1-aminocyclopropane-1-carboxylic acid (ACC), which both promote ethylene synthesis. Leaf explants of each genotype were incubated on shoot induction medium supplemented with each of these compounds at 0, 10 or 15 days following bud induction. The results obtained in our assays indicate that ethylene has a significant influence on tomato organogenesis. Concentrations of ethylene lower than the optimum (according to genotype) at the beginning of the culture may decrease the percentage of explants with buds (B), produce a delay in their appearance, or indeed inhibit bud formation. This was observed in S. pennellii and the F1 explants cultured on media with SN (5.8–58.0 μM) as well as in the F1 explants cultured on medium with 21.0 μM CoCl₂. The percentage of explants with shoots (R) and the mean number of shoots per explant with shoots (PR) also diminished in media that contained SN. Shoots isolated from these explants were less developed compared to those isolated from control explants. On the other hand, ethylene supplementation may contribute to enhancing shoot development. The number of isolable shoots from S. pennellii explants doubled in media with ACC (9.8–98.0 μM). Shoots isolated from explants treated with ethylene releasing compounds showed a higher number of nodes when ACC and Ethephon were added at 10 days (in F1 explants) or at 15 days (in S. pennellii) after the beginning of culture. Thus, the importance of studying not only the concentration but also the timing of the application of regulators when developing regeneration protocols has been made manifest. An excess of ethylene supplementation may produce an inhibitory effect, as was observed when using Ethephon (17.2–69.0 μM). These results show the involvement of ethylene in tomato organogenesis and lead us to believe that ethylene supplementation may contribute to enhancing regeneration and shoot development in tomato.     

Effects of salt formulations, carbon sources, cytokinins, and auxin on shoot organogenesis from cotyledons of Pinus pinea L.

Adventitious shoots were induced on cotyledons of Pinus pinea. Among seven salt formulations, three carbon sources (sucrose, glucose and fructose), and two cytokinins [6-benzyladenine (BA) and thidiazuron (TDZ)] with and without [-naphthaleneacetic acid (NAA)], cotyledons grown on 1/2 MS medium containing sucrose and 30 M BA produced the highest frequency of shoot organogenesis (>90%) and mean number of shoots/explant (>40% of cotyledons produced over 20 shoots/cotyledon). As for the site of shoot organogenesis along the cotyledonary explant, the highest number of shoots per explant was observed along the basal segment of the cotyledon (distal to the hypocotyl) over all cytokinin and auxin treatments tested. Shoots were elongated on a growth regulator-free medium containing activated charcoal.     

Phenocritical times in the process of in vitro shoot organogenesis

Shoot organogenesis occurs when leaf explants of Convolvulus arvensis are cultured on Murashige and Skoog salts, sucrose, vitamins, and 0.05 mg/liter IAA with 7.0 mg/liter 2-isopentenyl adenine. Under the influence of this shoot inducing medium (SIM), the explants become competent for the organogenic effects of SIM and eventually become determined for shoot formation. The induction process includes five separate transient sensitivities to inhibitors. Such stage-specific inhibitions reflect phenocritical times in development rather than general metabolic toxicities. The phenocopying agents are tri-iodobenzoic acid (TIBA), sorbitol, ribose, ammonium ion, and acetylsalicylic acid. The process of in vitro shoot organogenesis from leaf explants is now seen to include a series of discrete steps which precede morphological differentiation. An initial dedifferentiation process results in the formation of competent callus tissue along the cut edges of the explant. Under the influence of the phytohormone balance in SIM, shoot organogenic induction proceeds. This process involves a time which is sensitive to inhibition by salicylates followed by a time sensitive to TIBA which is followed in turn by a time sensitive to sorbitol and culminates in cells or groups of cells determined for shoot formation. This process also includes a time sensitive to inhibition by ribose, although its place in the order of events is not yet firmly assigned. There is also a sensitivity to ammonium ion (or lack of nitrate) at or near the time the explant becomes determined for shoot production.     

Direct shoot organogenesis and plant regeneration in Fortunella crassifolia

An efficient in vitro regeneration system in kumquats (Fortunella crassifolia Swingle) was established. Explant types and orientations, concentrations and combinations of plant growth regulators were evaluated for their influences on efficiency of plant regeneration. It was found that the optimum explant and its orientation was epicotyl planted vertically with upper part upward, and a shoot regeneration frequency of 1.48 shoots per explant was obtained on Murashige and Skoog (1962; MS) medium supplemented with 22.19 μM 6-benzyladenine (BA). A rooting percentage as high as 74 % was obtained on 1/2 MS supplemented with 0.54 μM 1-naphthaleneacetic acid (NAA), 9.29 μM kinetin (KN), and 0.5 g dm⁻³ activated charcoal.     

The role of angiogenic growth factors in organogenesis

Angiogenic growth factors are a class of molecules which exert a fundamental role in the process of blood vessel formation. Besides vasculogenic and angiogenic properties, these compounds mediate a complex series of patterning activities during organogenesis. Angiogenic factors cooperate in the growth and development of embryo tissues in a cross-talk between endothelial cells and tissue cells. It is well established that many tissue-derived factors are involved in blood vessel formation, but there is now emerging evidence that angiogenic factors and endothelial cells themselves represent a crucial source of instructive signals to non-vascular tissue cells during organ development. Thus, angiogenic factors and endothelial cell signalling are currently believed to provide fundamental cues for cell fate specification, embryo patterning, organ differentiation and postnatal tissue remodelling. This review article will summarize some of the recent advances in our understanding of the role of angiogenic factors and endothelial cells as effectors in organ formation.     

Agrobacterium-mediated transformation of white clover using direct shoot organogenesis

Summary White clover (Trifolium repens L.) plants from the cultivars Grasslands Huia and Grasslands Tahora have been transformed using Agrobacterium-mediated T-DNA transfer. Transgenic plants regenerated directly from cells of the cotyledonary axil. To transform white clover, shoot tips from 3 day old seedlings were co-cultivated with A. tumefaciens strain LBA4404 carrying the plasmid vector pPE64. This vector contains the neomycin phosphotransferase II gene (nptII) and -glucuronidase reporter gene (gus) both under the control of the CaMV 35S promoter. Kanamycin-resistant plants regenerated within 42 days after transfer onto selective media. Integration of the nptII and gus genes into the white clover genome was confirmed using Southern blotting, and histochemical analysis indicated that the gus gene was expressed in a variety of tissues. In reciprocal crosses between a primary transformant and a non-transformed plant the introduced gus gene segregated as a single dominant Mendelian trait.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - MS Murashige and Skoog - GUS -glucuronidase - X-GLUc 5-bromo-4-chloro-3-indolyl--D-glucuronide - MUG methylumbelliferyl--D-glucuronide - CaMV Cauliflower Mosaic Virus - NPTII neomycin phosphotransferase II - OCS octopine synthase - 4-MU 4-methyl umbelliferone     

Competence and determination in the process of in vitro shoot organogenesis

Leaf explants of Convolvulus arvensis produce shoots when cultured on Murashige and Skoog salts, sucrose, vitamins and 0.05 mg/liter IAA plus 7.0 mg/liter 2-isopentenyl adenine. Shoot-inducing, root-inducing, or callus-inducing medium (SIM, RIM, or CIM) will cause small amounts of callus to form at the cut edges of the explant. This first-formed callus is developmentally interchangeable: SIM induces shoots in callus formed on CIM or SIM with equal effect and efficiency. Once induction begins in competent callus, the callus is no longer interchangeable. Under the continued influence of SIM, cells, or groups of cells become determined for shoot formation. This determination is strongly canalized for shoot formation: subsequent transfer to root-inducing medium does not affect the formation of shoots by the explant. The control of organogenesis by the auxin/cytokinin balance must occur between the time the tissue becomes competent and the time it is determined for shoot (or root) development. It is not known whether this control is a single or multiple phenomenon.     

Inorganic nitrogen requirements during shoot organogenesis in tobacco leaf discs

The role of nitrate, ammonium, and culture medium pH on shoot organogenesis in Nicotiana tabacum zz100 leaf discs was examined. The nitrogen composition of a basal liquid shoot induction medium (SIM) containing 39.4 mM and 20.6 mM was altered whilst maintaining the overall ionic balance with Na(+) and Cl(-) ions. Omission of total nitrogen and nitrate, but not ammonium, from SIM prevented the initiation and formation of shoots. When nitrate was used as the sole source of nitrogen, a high frequency of explants initiated and produced leafy shoots. However, the numbers of shoots produced were significantly fewer than the control SIM. Buffering nitrate-only media with the organic acid 2[N-morpholino]ethanesulphonic acid (MES) could not compensate for the omission of ammonium. Ammonium used as the sole source of nitrogen appeared to have a negative effect on explant growth and morphogenesis, with a significant lowering of media pH. Buffering ammonium-only media with MES stabilized pH and allowed a low frequency of explants to initiate shoot meristems. However, no further differentiation into leafy shoots was observed. The amount of available nitrogen appears to be less important than the ratio between nitrate and ammonium. Shoot formation was achieved with a wide range of ratios, but media containing 40 mM nitrate and 20 mM ammonium (70:30) produced the greatest number of shoots per explant. Results from this study indicate a synergistic effect between ammonium and nitrate on shoot organogenesis independent of culture medium pH.     

Ethylene and shoot regeneration: hookless1 modulates de novo shoot organogenesis in Arabidopsis thaliana

We have investigated the role of ethylene in shoot regeneration from cotyledon explants of Arabidopsis thaliana. We examined the ethylene sensitivity of five ecotypes representing both poor and prolific shoot regenerators and identified Dijon-G, a poor regenerator, as an ecotype with dramatically enhanced ethylene sensitivity. However, inhibiting ethylene action with silver nitrate generally reduced shoot organogenesis in ecotypes capable of regeneration. In ecotype Col-0, we found that ethylene-insensitive mutants (etr1-1, ein2-1, ein4, ein7) exhibited reduced shoot regeneration rates, whereas constitutive ethylene response mutants (ctr1-1, ctr1-12) increased the proportion of explants producing shoots. Our experiments with ethylene over-production mutants (eto1, eto2 and eto3) indicate that the ethylene biosynthesis inhibitor gene, ETO1, can act as an inhibitor of shoot regeneration. Pharmacological elevation of ethylene levels was also found to significantly increase the proportion of explants regenerating shoots. We determined that the hookless1 (hls1-1) mutant, a suppressor of the ethylene response phenotypes of ctr1 and eto1 mutants, is capable of dramatically enhancing shoot organogenesis. The effects of ACC and loss of HLS1 function on shoot organogenesis were found to be largely additive.     

The role of cytokinins in root development of pea seedlings

Endogenous cytokinin-like activity was detected in pea seedlings usingthe soybean callus bioassay. Higher levels of activity were found in two-day-oldseedling roots and in the root free zone of four-day-old seedlings compared tothe lateral root zone of four-day-old seedlings. By day six, the levels ofendogenous cytokinin-like activity was greatly reduced in both the lateral rootzone and root free zone. Decapitation of the root tip disrupted the spatialorganization of the root. Lateral roots were subsequently found along the entirelength of the root rather than in a discreet lateral root zone. Application of10^–3 M dihydrozeatin to decapitated root tipsovercame the effect of root tip removal and restored the situation to what isnormally found in intact roots. There was little mobility of dihydrozeatin inthe root, with applied ³H-DHZ not moving from the root free zone,even 24 h after application.