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Trichoderma is an important genus of symbiotic fungi, commonly used around the world as biocontrol agents and as biofertilizer. Although their beneficial effects are well known and are successfully exploited in sustainable agriculture practices, the biochemical mechanisms of plant growth-promoting actions of Trichoderma and their anti-pathogen characteristics are not well understood. This study biochemically surveyed 22 strains of Trichoderma and shows that Trichoderma produces cytokinins (CKs), which has not been reported to date. The phytohormone profiles ranged from 5.34 to 379.99 pmol CKs released to 10 mL of the growth medium and comprised riboside and nucleotide derivatives of cis-zeatin (cZ) and isopentenyladenine (iP), suggesting that fungal CKs originate from a tRNA degradation pathway. We reveal a connection between the levels of free base cZ produced by Trichoderma and the inhibition rate against the pathogen Fusarium graminearum among the tested strains. Furthermore, we analyzed CK profiles of Arabidopsis plants cultured in vitro in the presence of Trichoderma strains. The inoculated plants showed increased levels of cZ-type (cZR, cZROG) and iP-type (iP, iPR) CKs—the forms which dominated CK profiles of all the fungal in vitro cultures tested in this study. The increase in the levels of cZ derivatives was accompanied by a significant reduction in plant trans-zeatin (tZ)-type CKs (tZR, tZNT, tZOG, tZ7G, tZ9G) in Arabidopsis when co-cultured with the fungus. Our work suggests that CKs produced by plant symbiotic Trichoderma strains can be used for plant growth stimulation, may impact the colonization strategy of symbiotic fungi, and include alterations to the host plant phytohormones for enhanced plant resistance against pathogens. [ABSTRACT FROM AUTHOR]

A highly productive and robust adventitious organogenesis protocol is key as an initial step when lemon in vitro mutagenesis or genetic transformation are addressed. Many molecules play a major role on plant regeneration, and in this study, the addition of the main naturally-occurring polyamines (spermine, spermidine, and putrescine) and their effect on endogenous cytokinins and on ethylene production have been analyzed in explants of the lemon (Citrus limon) cultivar 'Verna 51'.The three polyamines were added to the organogenesis media, and the regeneration rate and percentage were recorded. Changes produced in the endogenous cytokinins along with ethylene production were studied. Polyamines were shown to play an important role in the adventitious shoot regeneration of 'Verna 51'. However, this effect would not be related to ethylene production since changes were not detected. Comparing the three studied polyamines, it is possible to conclude that, within the studied doses, spermine (0.2 mM) and spermidine (0.3 and 0.6 mM) are more useful to induce organogenesis than putrescine in 'Verna 51'. In the most regenerative treatments, lower concentrations of cytokinins were found, which could indicate a correlation between the addition of polyamines to the culture media, the adventitious regeneration, and the metabolism of cytokinins. Key message: Polyamines are able to modulate de novo plant regeneration through changes produced in the endogenous cytokinins while do not appear to interfere directly in the ethylene production in lemon tissues. [ABSTRACT FROM AUTHOR]

Cytokinins (CKs) are well‐known as a class of phytohormones capable of delaying senescence in detached leaves. However, CKs are often treated as a monolithic group of compounds even though dozens of CK species are present in plants with varied degrees of reported biological activity. One specific type of CK, isopentenyladenine base (iP), has been demonstrated as having roles in delaying leaf senescence, inhibition of root growth, and promoting shoot regeneration. However, its N‐glucosides isopentenyladenine‐7‐ and ‐9‐glucoside (iP7G, iP9G) have remained understudied and thought of as inactive cytokinins for several decades, despite their relatively high concentrations in plants such as the model species Arabidopsis thaliana. Here we show that iP and one of its glucosides, iP9G, are capable of delaying senescence in leaves, though the glucosides having little to no activity in other bioassays. Additionally, we performed the first transcriptomic study of iP‐delayed cotyledon senescence which shows that iP is capable of upregulating photosynthetic genes and downregulating catabolic genes in detached cotyledons. Transcriptomic analysis also shows iP9G has limited effects on gene expression, but that the few affected genes are CK‐related and are similar to those seen from iP effects during senescence as seen for the type‐A response regulator ARR6. These findings suggest that iP9G functions as an active CK during senescence. [ABSTRACT FROM AUTHOR]

Summary: The diversity of cytokinin (CK) metabolites suggests their interconversions are the predominant regulatory mechanism of CK action. Nevertheless, little is known about their directionality and kinetics in planta.CK metabolite levels were measured in 2‐wk‐old Arabidopsis thaliana plants at several time points up to 100 min following exogenous application of selected CKs. The data were then evaluated qualitatively and by mathematical modeling.Apart from elevated levels of trans‐zeatin (tZ) metabolites upon application of N6‐(Δ2‐isopentenyl)adenine (iP), we observed no conversions between the individual CK‐types – iP, tZ, dihydrozeatin (DHZ) and cis‐zeatin (cZ). In particular, there was no sign of isomerization between tZ and cZ families. Also, no increase of DHZ‐type CKs was observed after application of tZ, suggesting low baseline activity of zeatin reductase. Among N‐glucosides, those of iP were not converted back to iP while tZ N‐glucosides were cleaved to tZ bases, thus affecting the whole metabolic spectrum.We present the first large‐scale study of short‐term CK metabolism kinetics and show that tZ N7‐ and N9‐glucosides are metabolized in vivo. We thus refute the generally accepted hypothesis that N‐glucosylation irreversibly inactivates CKs. The subsequently constructed mathematical model provides estimates of the metabolic conversion rates. [ABSTRACT FROM AUTHOR]

Cytokinins (CKs) encompass a group of phytohormones, known to orchestrate many critical processes in plant development. Excluding Archaea, CKs are pervasive among all kingdoms, but much less is reported about their metabolism beyond plants. Recent evidence from mammalian tissues indicates the presence of six additional CK forms beyond the previously identified, single mammalian CK, N6‐isopentenyladenosine (i6A). There is limited understanding of CK biosynthesis pathways in mammalian systems; therefore, human cervical cancer (HeLa) cells were used to further characterize CK processing by tracking the interconversion of CKs into their various structural derivatives in mammalian cells in a time‐course study. Through high‐performance liquid chromatography‐positive electrospray ionization‐tandem mass spectrometry (HPLC‐(+ESI)‐MS/MS), we document changes in the functional profiles of endogenous CKs in a human cell line following metabolism by HeLa cell cultures. The nucleotide CK fraction (iPRP) was found exclusively within the cell pellet (0.34 pmol/106 cells), and the active free base (FB) form (iP) and riboside fraction (iPR) were found in greater abundance extracellularly (1.67 and 0.10 nmol/L respectively). For further confirmation, we demonstrate that HeLa cells metabolize an exogenously supplied CK, N6‐benzyladenosine (BAR). In the HeLa culture supernatant, a 12‐fold decrease in BAR concentration was observed within the first 24 hours of incubation accompanied by a fivefold increase in the FB form, N6‐benzyladenine (BA). These findings support the hypothesis that HeLa cells have the enzymatic pathways required for the metabolism of both endogenous and exogenous CKs. [ABSTRACT FROM AUTHOR]

The impact of exogenous cytokinins (kinetin, zeatin, 6-benzylaminopurine, and N6-2-isopentenyladenine) on the pattern of Dryopteris filix-mas (L.) Schott spore germination, gametophyte growth and morphology in vitro have been studied. It was found that all studied cytokinins significantly retarded spore germination, inhibited gametophyte growth, caused deformations and decrease in the thallus size, and suppressed the development of reproductive structures and sporophyte growth at the concentration of 10–5 М. The reduction of the hormone concentration to 10–8 М stimulated the gametophyte development, induced cell divisions, particularly in the apical zone, due to which some of thalli were deformed, promoted the production of rhizoids, affected the formation of antheridia and archegonia, and slowed the sporophyte development. [ABSTRACT FROM AUTHOR]

The purpose of the present work was to compare calluses of European cranberry (Oxycoccus pal-ustris Pers.) and American cranberry (O. macrocarpus (Ait.) Pers.) by their ability to accumulate biomass and phenolic compounds in the presence of different cytokinins in nutrient medium. In European cranberry, callusogenesis appeared to be more intensive upon the addition of kinetin (Kin) to the medium, while accumulation of phenolic compounds and biomass was most pronounced on the medium with isopentenyladenine (iP). In American cranberry, callusogenesis appeared to be more intensive upon the addition of iP to the medium and in respect to accumulation of biomass and phenolic compounds (in the onset of the cultivation), somewhat higher values were obtained upon addition of benzylaminopurin (BAP). Maximum of phenolic compounds accumulation was observed upon the transition of callus cultures to the stationary growth phase, during the fifth week of the passage on average, except on the medium with BAP. The content of soluble phenolic compounds in European cranberry calluses did not exceed 11 mg/g fr wt, while that in American cranberry calluses did not exceed 17 mg/g fr wt. Cranberry callus cultures under long-term cultivation displayed a decrease in the level of all groups of phenolic compounds, including flavonoids to a higher extent. For European cranberry calluses after 6–10 passages, stabilization of the secondary synthesis was noted, first of all on the medium with iP. In general, more stable accumulation of phenolic compounds in calluses of both cranberry species was observed in the presence of Kin. [ABSTRACT FROM AUTHOR]

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In the present study, an alternate method for germplasm storage in the form of artificial seeds was standardized via nodal explants excised from in vitro proliferated shoots. The explants were encapsulated using sodium alginate and calcium chloride as gelling matrix. For development of root along with shoot, excised nodal segments were pretreated with ½ MS medium along with 20 μM IBA for 24 h and encapsulation was carried thereafter. Combination of 3% sodium alginate augmented with 100 mM CaCl2.2H2O was found appropriate for the formation of clear and uniform beads and subsequent conversion of encapsulated nodal segments into plantlets. Maximum (66%) encapsulated nodal segments were converted into plantlets on MS medium supplemented with 7.5 μM BA and 0.5 μM NAA after eight weeks. Regeneration frequency of auxin-pretreated encapsulated and non-encapsulated nodal segments (stored at 4 ºC) was evaluated at different storage time (0 to 6 weeks). After four weeks of storage, encapsulated propagules exhibited highest conversion response on the optimized medium after eight weeks of culture. Plantlets were hardened and established with success in ex vitro conditions. Conversion of synthetic seeds into plantlets was observed when these were directly sown in autoclaved SoilriteTM (Keltech Energies, Bangalore, India). [ABSTRACT FROM AUTHOR]

Ethylene signaling plays a major role in the regulation of plant growth, but its cross-talk with other phytohormones is still poorly understood. Here, we investigated whether or not a defect in ethylene signaling, particularly in the ETHYLENE INSENSITIVE3 (EIN3) transcription factor, alters plant growth and influences the contents of other phytohormones. With this aim, a hormonal profiling approach using ultrahigh performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS) was used to unravel organ-specific responses (in roots, leaves and fruits) in the ein3-1 mutant and wild-type A. thaliana plants exposed to contrasting phosphate (Pi) availability. A defect in ethylene signaling in the ein3-1 mutant increased the biomass of roots, leaves and fruits, both at 0.5 mM and 1 mM Pi, thus indicating the growth-inhibitory role of ethylene in all tested organs. The hormonal profiling in roots revealed a cross-talk between ethylene signaling and other phytohormones, as indicated by increases in the contents of auxin, gibberellins and the stress-related hormones, abscisic acid, salicylic acid and jasmonic acid. The ein3-1 mutant also showed increased cytokinin contents in leaves. Reduced Pi availability (from 1 mM to 0.5 mM Pi) affected fruit growth, but not root and leaf growth, thus indicating mild Pi deficiency. It is concluded that ethylene signaling plays a major role in the modulation of plant growth in A. thaliana and that the ein3-1 mutant is not only altered in ethylene signaling but in the contents of several phytohormones in an organ-specific manner, thus indicating a hormonal cross-talk. [ABSTRACT FROM AUTHOR]

The explant origin and the factors in the culture medium are responsible for the response to somatic embryogenesis in most plant species, including palms. Acrocomia aculeata is a neotropical palm currently undergoing domestication due to its sustainability and profitability as an alternative oil crop. The attainment of productive cultivars of this palm may be speed up by somatic embryogenesis. Thus, this study sought to refine an existing somatic embryogenesis cloning protocol for A. aculeata and to investigate the possible genetic influence on the in vitro propagation response, using zygotic embryos from 19 matrices or open-pollinated families. The embryos were inoculated in modified induction media. The following characteristics were evaluated in this phase: swollen embryos, percentage of zygotic embryos that formed callus, embryogenic calli and oxidation. Afterwards the resulting pro embryogenic masses were subsequently cultivated in modified multiplication, maturation and gemination media. In those phases the open-pollinated families’ response was measured by the growth of their embryogenic mass and the quantity of regenerated and germinated somatic embryos. The refined micropropagation protocol was successful in establishing embryogenic lines and maintaining their cyclic multiplication for a long period; all without the loss of their potential to maturate and regenerate somatic embryos. In general, the multiplication of pro embryogenic masses was effective even in the lowest dose of Picloram (18 μM). The addition of activated charcoal in the medium favored the maturation of somatic embryos. Plantlets regeneration, although defective in some lineages, was promoted by GA 3 (0.55 μM). The zygotic embryos’ responsiveness to somatic embryogenesis varied according to their matrices, indicating that if micropropagation is to be used as a mass propagation technique for the species, the explant́s genotype must be considered. [ABSTRACT FROM AUTHOR]

Isoprenoid cytokinins play a number of crucial roles in the regulation of plant growth and development. To study cytokinin receptor properties in plants, we designed and prepared fluorescent derivatives of 6-[(3-methylbut-2-en-1-yl)amino]purine ( N 6 -isopentenyladenine, iP) with several fluorescent labels attached to the C2 or N9 atom of the purine moiety via a 2- or 6-carbon linker. The fluorescent labels included dansyl (DS), fluorescein (FC), 7-nitrobenzofurazan (NBD), rhodamine B (RhoB), coumarin (Cou), 7-(diethylamino)coumarin (DEAC) and cyanine 5 dye (Cy5). All prepared compounds were screened for affinity for the Arabidopsis thaliana cytokinin receptor (CRE1/AHK4). Although the attachment of the fluorescent labels to iP via the linkers mostly disrupted binding to the receptor, several fluorescent derivatives interacted well. For this reason, three derivatives, two rhodamine B and one 4-chloro-7-nitrobenzofurazan labeled iP were tested for their interaction with CRE1/AHK4 and Zea mays cytokinin receptors in detail. We further showed that the three derivatives were able to activate transcription of cytokinin response regulator ARR5 in Arabidopsis seedlings. The activity of fluorescently labeled cytokinins was compared with corresponding 6-dimethylaminopurine fluorescently labeled negative controls. Selected rhodamine B C2-labeled compounds 17 , 18 and 4-chloro-7-nitrobenzofurazan N9-labeled compound 28 and their respective negative controls ( 19 , 20 and 29 , respectively) were used for in planta staining experiments in Arabidopsis thaliana cell suspension culture using live cell confocal microscopy. [ABSTRACT FROM AUTHOR]

Clonal propagation plays a critical integral role in the growth and success of a global multi-billion dollar horticulture industry through a constant supply of healthy stock plants. The supply chain depends on continuously improving the micropropagation process, thus, understanding the physiology of in vitro plants remains a core component. We evaluated the influence of exogenously applied cytokinins (CKs, N 6 -benzyladenine = BA, isopentenyladenine = iP, meta -topolin =  m T, 6-(3-hydroxybenzylamino)-9-(tetrahydropyran-2-yl)purine =  m TTHP) in Murashige and Skoog (MS)-supplemented media on organogenic response and accumulation of endogenous CK and indole-3-acetic acid (IAA) metabolites. The highest shoot proliferation (30 shoots/explant) was obtained with 20 μM  m T treatment. However, the best quality regenerants were produced in 10 μM  m T treatment. Rooting of Amelanchier alnifolia in vitro plantlets was observed at the lowest CK concentrations, with the highest root proliferation (3 roots/explant) in 1 μM m TTHP regenerants. Similar to the organogenic response, high levels of endogenous bioactive CK metabolites (free bases, ribosides, and nucleotides) were detected in m T and m TTHP-derived regenerants. The level of O -glucosides was also comparatively high in these cultures. All CK-treated plants had high levels of endogenous free IAA compared to the control. This may suggest an influence of CKs on biosynthesis of IAA. [ABSTRACT FROM AUTHOR]

Cytokinins (CKs) play a crucial role in promoting axillary bud outgrowth and targeting the control of CK metabolism can be used to enhance branching in plants. CK levels are maintained mainly by CK biosynthesis (isopentenyl transferase, IPT) and degradation (dehydrogenase, CKX) genes in plants. A systematic study of the IPT and CKX gene families in apple, however, has not been conducted. In the present study, 12 MdIPTs and 12 MdCKXs were identified in the apple genome. Systematic phylogenetic, structural, and synteny analyses were performed. Expression analysis of these genes in different tissues was also assessed. MdIPT and MdCKX genes exhibit distinct expression patterns in different tissues. The response of MdIPT , MdCKX , and MdPIN1 genes to various treatments (6-BA, decapitation and Lovastatin, an inhibitor of CKs synthesis) that impact branching were also investigated. Results indicated that most of the MdIPT and MdCKX , and MdPIN1 genes were upregulated by 6-BA and decapitation treatment, but inhibited by Lovastatin, a compound that effectively suppresses axillary bud outgrowth induced by decapitation. These findings suggest that cytokinin biosynthesis is required for the activation of bud break and the export of auxin from buds in apple tree with intact primary shoot apex or decapitated apple tree. MdCKX8 and MdCKX10 , however, exhibited little response to decapitation, but were significantly up-regulated by 6-BA and Lovastatin, a finding that warrants further investigation in order to understand their function in bud-outgrowth. [ABSTRACT FROM AUTHOR]

The influence of a cytokinin, N6-(2-Isopentenyl) adenine (2iP), on growth of Enhalus acoroides was examined in controlled laboratory experiments. Seeds of E. acoroides were collected from Haad Chao Mai National Park, Trang Province, Southern Thailand, on the Andaman Sea side. The seeds were cultured in sterile MS liquid medium containing 25, 50, 75, and 100 µM 2iP in glass culture bottles. The cultures were stored under 25±2˚C in axenic conditions for 64 days. The results showed that 2iP inhibits the initiation of E. acoroides root. The highest average root length was 19.10±1.13 mm in controlled MS medium. The treatment with increasing 2iP concentration showed positive effects on hypocotyl and leaf developments. The highest average hypocotyl length, 1.60±0.00 cm, was found in seedlings from 50 µM 2iP medium. The hypocotyl diameter recorded in controlled MS medium was not significantly different from those in 50 and 100 µM 2iP media. The highest average leaf length was 5.36±0.67 cm in 100 µM 2iP medium. This culture knowledge is a step forward to the in vitro seagrass propagation and restoration studies. [ABSTRACT FROM AUTHOR]

Salt stress responses in salt-sensitive Arabidopsis thaliana (2–150 mM NaCl) and the closely related salt-tolerant Thellungiella salsuginea ( Eutrema halophila, 150–350 mM NaCl) were compared to identify hormonal and transcriptomic changes associated with enhanced stress tolerance. Phytohormone levels, expression of selected genes, membrane stability, and Na + and K + concentrations were measured in shoot apices, leaves, and roots. Thellungiella exhibited higher salt stress tolerance associated with elevated basal levels of abscisic acid and jasmonic acid, and lower levels of active cytokinins (excluding cis -zeatin) in shoot apices. Analysis of the dynamics of the early salt stress response (15 min to 24 h) revealed that the halophyte response was faster and stronger. Very mild stress, in our hydropony arrangement 2–25 mM NaCl, affected the transcription of genes involved in cytokinin metabolism ( AtIPTs , AtCKXs ). Mild stress induced in Arabidopsis (50 mM) stress responses only in shoot apices, while in Thellungiella (150 mM) across the whole plant. Arabidopsis exhibited in hydropony evidence of severe stress above 75 mM NaCl and died in 150 mM, whereas the halophyte only became severely stressed above 225 mM. The responses of individual phytohormones (cytokinins, auxin, abscisic acid, jasmonic acid, salicylic acid and their metabolites) to salinity are discussed. [ABSTRACT FROM AUTHOR]

Little information is currently available concerning the mechanisms controlling palm seed germination. We compared the anatomical and physiological aspects of seeds of two neotropical palm species showing different levels of dormancy. The seeds of Attalea vitrivir and Butia capitata were evaluated for the endogenous contents of hormones (ABA, GAs, CKs, BRs, IAA, JA, SA and the ethylene precursor ACC) in their cotyledonary petiole and operculum (structures involved in germination control), the force necessary to displace the operculum, endo-β-mannanase activities, and embryo cell elongation. The analyses were carried out on with intact dry and imbibed seeds as well as with seeds with the operculum mechanically removed, 2, 5 and 10 days after sowing. The germinabilities of the intact seeds of A. vitrivir and B. capitata were 68% and 3%, respectively; the removal of the operculum increased germination to more than 90% in both species. Reductions of ABA and increases in GAs contents coincided with cell elongation, although there is no evidence that hormonal balance and endo-β-mannanase activity are involved in operculum weakening. The ratio between the embryo length and the force required for operculum displacement (EL/OF) was found to be 1.9 times greater in A. vitrivir than in B. capitata , which means that very small elongations in each cell would be sufficient to promote germination, resulting in a lower level of dormancy in the former species. EL/OF and cell growth control are therefore important for defining dormancy level in palm seeds. [ABSTRACT FROM AUTHOR]

Postharvest needle abscission in balsam fir varies considerably depending on the season and may be dependent on endogenous hormones of roots or shoots. Samples were obtained from three trees each month from April 2013 to November 2013 and evaluated for retention. In addition, root and needle tissues were analyzed for abscisic acid, cytokinins, auxins, and gibberellins and their conjugates. There was a 3.5-fold increase in time until needle abscission commencement and 1.75-fold increase in time until complete abscission in autumn compared to spring. There were several changes to root and needle hormones with respect to harvest time, but fewer hormones were linked to postharvest needle retention. Among all hormones analyzed, both isopentenyladenine and isopentenyladenosine in roots were strongly linked to needle retention. When isopentenyladenine was present in roots, needle abscission took 114% longer to begin and 58% longer to finish. Isopentenyladenosine in roots was the strongest hormonal predictor of needle retention ( R = 79%). Dihydrozeatin riboside ( R = 37%), trans-abscisic acid ( R = 50%), and isopentenyladenosine ( R = 59%) in the needles were also strong predictors of needle retention. The relative effectiveness of certain hormones in predicting needle retention and speculation pertaining to the roles these hormones could play are discussed. [ABSTRACT FROM AUTHOR]

An RP-HPLC-PDA method for the simultaneous analysis of 9 compounds deriving from the phytohormones class was developed and optimized, namely indoleacetic acid (IAA), indolebutyric acid (IBA), phenyleacetic acid (PAA), naphtyleacetic acid (NAA), trans -zeatin (TZ), kinetin (KA), isopentenyladenine (IA), 6-benzylaminopurine (6-BA) and abscisic acid (ABA). Validation of the method was performed on the SFE-CO 2 extract made out of the mixture of Baltic algae. The regression coefficients for plant hormones were in the range from 0.997 to 0.999. The LOD and LOQ were on the levels from 0.05–0.29 and 0.15–0.88 mg/L, respectively. Developed method was used for the separation and determination plant hormones in extracts obtained by SFE-CO 2 (supercritical fluid extraction) made out of the mixture of Baltic algae, Cladophora glomerata and Spirulina sp. In the extract of Baltic seaweed 2 of tested compounds were present in the concentration of 154,45 ± 20,63 μg/g for TZ and 362,47 ± 13,00 μg/g for PAA, whereas in Cladophora glomerata extract contained PAA and IAA in the concentration of 229,30 ± 7,90 μg/g and 23,91 ± 0,80 μg/g, respectively (all values per g of extract). The differences in the hormones levels may occur due to the different scale of extract preparation (laboratory or industrial) and other factors like the place of algae collection, year of collection or the way of biomass preparation. [ABSTRACT FROM AUTHOR]

In order to evaluate whether brassinosteroids (BS) and green light regulate the transcription of plastid genes in a cross-talk with cytokinins (CKs), transcription rates of 12 plastid genes ( ndhF , rrn23 , rpoB , psaA , psaB , rrn16 , psbA , psbD , psbK , rbcL , atpB , and trnE / trnY ) as well as the accumulation of transcripts of some photoreceptors ( PHYA , CRY2 , CRY1A , and CRY1B ) and signaling ( SERK and CAS ) genes were followed in detached etiolated barley leaves exposed to darkness, green or white light ±1 μm 24- epi brassinolide (EBL). EBL in the dark was shown to up-regulate the transcription of 12 plastid genes, while green light activated 10 genes and the EBL combined with the green light affected the transcription of only two genes ( psaB and rpoB ). Green light inhibited the expression of photoreceptor genes, except for CRY1A. Under the green light, EBL practically did not affect the expression of CRY1A , CAS and SERK genes, but it reduced the influence of white light on the accumulation of CAS , CRY1A , CRY1B , and SERK gene transcripts. The total content of BS in the dark and under white light remained largely unchanged, while under green light the total content of BRs (brassinolide, castasterone, and 6-deoxocastasterone) and HBRs (28-homobrassinolide, 28-homocastasterone, and 6-deoxo-28-homocastasterone) increased. The EBL-dependent up-regulation of plastome transcription in the dark was accompanied by a significant decrease in CK deactivation by O-glucosylation. However, no significant effect on the content of active CKs was detected. EBL combined with green light moderately increased the contents of trans -zeatin and isopentenyladenine, but had a negative effect on cis -zeatin. The most significant promotive effect of EBL on active CK bases was observed in white light. The data obtained suggest the involvement of CKs in the BS- and light-dependent transcription regulation of plastid genes. [ABSTRACT FROM AUTHOR]

In the current study, we evaluated the effect of α-naphthaleneacetic acid (NAA) individually or in combination with different cytokinins (CKs) including benzyladenine (BA), meta -topolin ( m T) and isopentenyladenine (iP) on organogenesis, auxin and CK content in Eucomis autumnalis subspecies autumnalis (EA) and Eucomis zambesiaca (EZ). These species were used as model plants due to their ornamental and medicinal properties. Three leaf explants were inoculated in screw-cap jars containing 30 mL Murashige and Skoog (MS) media supplemented with 5 μM NAA alone or in combination with 5 μM CK (BA, m T or iP). After 10 weeks (EA) or 15 weeks (EZ), parameters including shoot and root growth as well as plant fresh weight were recorded. For analysis of auxin and CK content, whole plantlets were harvested, pooled and freeze-dried for the different treatments. In both species, shoot and root proliferation as well as plant biomass were generally higher when NAA was combined with the individual CK than in NAA or CK treatment. The highest concentration of indole-3-acetic acid (IAA, 619 pmol g −1 DW) and 2-oxindole-3-acetic acid (OxIAA, 2381 pmol g −1 DW) were observed in EA-treated with NAA alone while m T treatment (without NAA) had the most abundant indole-3-acetyl- l -aspartic acid (IAAsp, 904 and 582 pmol g −1 DW for EA and EZ, respectively) in both species. A significant concentration of total endogenous CK accumulated in both Eucomis regenerants from m T and m T + NAA when compared to the other treatments. The majority of the detected CKs were of the aromatic CK-type, mainly free bases. The potential physiological roles of these quantified phytohormones in relation to the observed morphological responses are discussed. [ABSTRACT FROM AUTHOR]

The objective of the present investigation was to evaluate the effect of the genotype and culture age on appearance of somaclonal variation in olive plants regenerated via somatic embryogenesis. For this purpose, plants were produced from 8 embryogenic lines maintained by repetitive subcultures in ECO medium for different time periods. Four and a half years after acclimatization, plants were phenotypically evaluated by morphological and biometric analysis of vegetative and reproductive characters and compared with control plants derived from seeds. The results obtained revealed the existence of somaclonal variation in olive plants regenerated via somatic embryogenesis. Fourteen variant phenotypes were identified affecting plant vegetative and reproductive development. Biometric analysis also showed the existence of intraclonal variation in quantitative traits related to plant architecture. The genotype was a fundamental factor in the appearance of somaclonal variation, with some lines especially susceptible to change. Higher variation was in general observed in plants derived from aged lines, although this effect was strongly influenced by the embryogenic line. Variants exhibiting shortening of the juvenile period obtained in the present investigation can constitute a very interesting material to be included in olive breeding programs nowadays in progress. [ABSTRACT FROM AUTHOR]

The red alga Gracilaria caudata J. Agardh (Gracilariales) is commercially exploited in Brazil as a source of raw material for agar production, and its cultivation is needed for sustainable biomass production to avoid overexploitation of natural populations. The objective of the present study was to evaluate the effects of aromatic and isoprenoid-derived cytokinins under two photon flux densities (PFDs) on growth rates, formation of lateral branches, pigment content (chlorophyll a and phycobiliproteins), and total soluble proteins in G. caudata. Aromatic cytokinins (benzylaminopurine, BA, and kinetin, K) and isoprenoid cytokinins (2-isopentenyladenine, 2iP, cis-zeatin, and trans-zeatin) were tested at 50 and 100 μmol photons m s, temperature of 23 ± 1 °C, and light:dark cycle of 14:10 h. Cytokinins were added to the ASP 12-NTA synthetic medium (salinity 30 PSU, pH 8.0) in concentrations ranging from 0.0 to 50.0 μM. Growth rates of G. caudata were not affected by addition of isoprenoid and aromatic cytokinins. Zeatins ( cis and trans forms) and BA at the concentrations of 0.5 and/or 5.0 μM stimulated the branch formation at 100 μmol photons m s. In general, concentrations of isoprenoid cytokinins showed negative correlation with total soluble proteins and pigment contents at both PFDs. On the other hand, aromatic cytokinins did not affect protein contents, and K inhibited the pigment contents in both PFD levels. In conclusion, our results indicate that the isoprenoid and aromatic cytokinins and PFD levels did not affect the growth rates of G. caudata, but influence the branching and contents of pigments and total soluble proteins. Moreover, aromatic cytokinins and zeatins stimulated branch formation, which is a morphogenetic process useful for micropropagation of G. caudata, and could improve its cultivation in the Brazilian coast. [ABSTRACT FROM AUTHOR]

Application of in vitro plant biotechnology in berry crops depends on the availability of efficient regeneration protocols that are specific to the genotype background and to the correct combination of exogenous hormones (auxin and cytokinin) added to the medium. Strawberry regeneration protocols are available for different cultivars, while more limited information are available for blueberry cultivars. In this study, we show that the best regeneration efficiency for the leaves of strawberry cultivar Calypso were obtained culturing in a medium supplemented with thidiazuron (TDZ) 0.5 mg L −1 and 2,4-dichlorophenoxyacetic acid (2,4-D) 0.02 mg L −1 . The best regeneration efficiency for cultivar Sveva leaves was obtained culturing in a medium supplemented with N6-benzyladenine (BA) 3 mg L −1 and indole-3-butyric acid (IBA) 0.2 mg L −1 . In blueberry cultivar Duke, shoot proliferation trials were carried out comparing the effects of 2-isopentenyladenine (2iP) and TDZ; furthermore, experiments of blueberry direct and indirect organogenesis were made, using TDZ as alternatives to zeatin, the most common and expensive hormone used for blueberry in vitro regeneration. Different regeneration responses were observed by using TDZ alone or combined with 2iP. The addition of both 0.2 mg L −1 or 0.5 mg L −1 of TDZ in the medium led to improved callus formation. The addition of 15 mg L −1 of 2iP in the same medium promoted blueberry stem elongation, while inhibiting callus growth. Zeatin was most efficient in direct regeneration of shoots while 0.5 mg L −1 TDZ induced a highest number of shoots by indirect organogenesis. [ABSTRACT FROM AUTHOR]

Cytokinins (CKs) are an important group of phytohormones. Their tightly regulated and balanced levels are essential for proper cell division and plant organ development. Here we report precise quantification of CK metabolites and other phytohormones in maize reproductive organs in the course of pollination and kernel maturation. A novel enzymatic activity dependent on NADP + converting trans- zeatin ( t Z) to 6-(3-methylpyrrol-1-yl)purine (MPP) was detected. MPP shows weak anticytokinin properties and inhibition of CK dehydrogenases due to their ability to bind to an active site in the opposite orientation than substrates. Although the physiological significance of t Z side-chain cyclization is not anticipated as the MPP occurrence in maize tissue is very low, properties of the novel CK metabolite indicate its potential for utilization in plant in vitro tissue culture. Furthermore, feeding experiments with different isoprenoid CKs revealed distinct preferences in glycosylation of t Z and cis- zeatin ( c Z). While t Z is preferentially glucosylated at the N9 position, c Z forms mainly O- glucosides. Since O- glucosides, in contrast to N9- glucosides, are resistant to irreversible cleavage catalyzed by CK dehydrogenases, the observed preference of maize CK glycosyltransferases to O- glycosylate zeatin in the cis- position might be a reason why c Z derivatives are over-accumulated in different maize tissues and organs. [ABSTRACT FROM AUTHOR]

Tree peony ( Paeonia sect. Moutan ) is a famous ornamental crop worldwide that is difficult to propagate. The present study provides efficient protocols for the micropropagation of three economically important tree peony cultivars ( Paeonia suffruticosa ‘Jin Pao Hong’, P. suffruticosa ‘Wu Long Peng Sheng’, and P. × lemoinei ‘High Noon’). For optimal shoot multiplication, different concentrations of GA 3 were added into woody plant medium (WPM) (double strength Ca 2+ ) containing 1.0 mg l −1 BAP; 0.5 mg l −1 GA 3 yielded the highest multiplication rate with more than 4 shoots per explant for the three cultivars. For better rooting, different polyamines (putrescine, spermine, or spermidine) were added individually to half-strength MS medium containing 1.0 mg l −1 IBA. 5.0, 1.0, and 1.0 mg l −1 putrescine produced the optimal rooting for ‘Jin Pao Hong’ (100%), ‘Wu Long Peng Sheng’ (100%), and ‘High Noon’ (80%), respectively; each rooted shoot had 4–6 roots measuring 4–6 cm in length. Poor survival rates during acclimatization were obtained for ‘Jin Pao Hong’ (52.6%), ‘Wu Long Peng Sheng’ (40.1%), and ‘High Noon’ (64.0%) when the plantlets were grown in uninoculated vermiculite/peat/perlite sterilized substrate; in contrast, ‘High Noon’ plantlets inoculated with Glomus mosseae showed a high survival rate (83.4%) and vigorous growth. The protocols reported in this study can be used for rapid and large-scale propagation of the three tree peony cultivars. Furthermore, this study raises interesting possibilities for stimulating the in vitro rooting of tree peony cultivars through polyamine application and for improving the ex vitro establishment of tree peony plantlets through arbuscular mycorrhizal fungi inoculation. [ABSTRACT FROM AUTHOR]

In vitro regeneration via somatic embryogenesis is at the base of application of biotechnological tools to olive ( Olea europaea L.) genetic improvement. The development of elite plant material utilizing these techniques requires a considerable period of time, but little is known about the real influence of the duration of the maintenance phase on somatic embryogenesis of olive. The objective of this investigation was to evaluate the effect of culture age on the efficiency of the different phases of the somatic embryogenesis process and the quality of the regenerated plants. With this purpose, 10 independent embryogenic lines of two different ages (2 and 8 years) were monitorized throughout the somatic embryogenesis process. Behavior of the regenerated plants was compared with that of seedlings over the subsequent micropropagation, rooting and acclimatization steps. The results showed alteration of the proliferation pattern with time in culture. Efficiency of the maturation phase significantly decreased in aged lines and a decline in embryos quality was observed as revealed by the reduced germination capacity. Both effects gave rise to a significant decrease in the regeneration potential as duration of the proliferation phase increased. Plants obtained from aged cultures were smaller than those derived from young cultures although more shoots developed per germinated somatic embryo. During the elongation phase, shoots derived from younger cultures were more vigorous, giving rise to longer axillary shoots. No significant differences were found in the rest of the parameters studied in the elongation, rooting and acclimatization phases. Behavior of somatic plants during these steps was similar to that of seedlings, thus revealing the high quality of plants regenerated following this somatic embryogenesis protocol. Events involved in loss of morphogenic potential in olive appear to be different than those described in conifer species. [ABSTRACT FROM AUTHOR]

‘Hass’ avocado ( Persea americana Mill.) is characterized by distinct periods of excessive flower and fruit drop. Determining the cause(s) of these abscission events is critical for the development of strategies to increase fruit set and yield. Therefore, the objectives of this research were to determine pollination and fertilization rates of abscising flowers and to quantify the viability of developing ovules (seeds) and hormone concentrations of abscising versus persisting fruit through early and June drop of ‘Hass’ avocado. Experiments were conducted during sequential off- and on-crop years. Pollination, pollen germination, pollen tube growth and ovule viability of abscising flowers were determined by microscopic analysis. During early and June drop, cellular deterioration of developing ovules of abscising and persisting fruit was visualized with Evan’s blue stain. Abscisic acid (ABA), indole-3-acetic acid (IAA), and isopentenyladenine (IPA) concentrations were determined by radio-immuno assay (RIA) during the on-crop year only. On average ≥ 70% of abscising ‘Hass’ flowers were pollinated but due to the failure of the pollen grains to germinate and produce pollen tubes, fertilization never occurred and the ovule deteriorated. Fruit abscission during early and June drop was also due primarily to a lack of fertilization. Fruit abscission was associated initially with deterioration of the nucellus within the developing ovule in early drop, and subsequently with deterioration of the integument (seed coat) in June drop; both occurred more frequently in abscising than persisting fruit. Abscising fruit had greater ABA concentrations during early drop, June drop and fall fruit drop compared to persisting fruit; IAA and IPA were greater in abscising fruit during June drop, but not early drop or fall fruit drop. Only 25% of the total number of fruit that abscised per crop year were fertilized. Taken together, these results provide strong evidence that the majority of flowers and fruit of ‘Hass’ avocado abscise due to a lack of pollen germination and subsequent fertilization. In addition, the results suggest that ABA accumulation is related to ovule (seed) abortion and reduced fruit growth in abscising fruit. [ABSTRACT FROM AUTHOR]

Cytokinins are plant hormones with biological functions ranging from coordination of plant growth and development to the regulation of senescence. A series of 2-chloro- N 6 -(halogenobenzylamino)purine ribosides was prepared and tested for cytokinin activity in detached wheat leaf senescence, tobacco callus and Amaranthus bioassays. The synthetic compounds showed significant activity, especially in delaying senescence in detached wheat leaves. They were also tested in bacterial receptor bioassays using both monocot and dicot members of the cytokinin receptor family. Most of the derivatives did not trigger cytokinin signaling via the AHK3 and AHK4 receptors from Arabidopsis thaliana in the bacterial assay, but some of them specifically activated the ZmHK1 receptor from Zea mays and were also more active than the aromatic cytokinin BAP in an ARR5::GUS cytokinin bioassay using transgenic Arabidopsis plants. Whole transcript expression analysis was performed using an Arabidopsis model to gather information about the reprogramming of gene transcription when senescent leaves were treated with selected C2-substituted aromatic cytokinin ribosides. Genome-wide expression profiling revealed that the synthetic halogenated derivatives induced the expression of genes related to cytokinin signaling and metabolism. They also prompted both up- and down-regulation of a unique combination of genes coding for components of the photosystem II (PSII) reaction center, light-harvesting complex II (LHCII), and the oxygen-evolving complex, as well as several stress factors responsible for regulating photosynthesis and chlorophyll degradation. Chlorophyll content and fluorescence analyses demonstrated that treatment with the halogenated derivatives increased the efficiency of PSII photochemistry and the abundance of LHCII relative to DMSO- and BAP-treated controls. These findings demonstrate that it is possible to manipulate and fine-tune leaf longevity using synthetic aromatic cytokinin analogs. [ABSTRACT FROM AUTHOR]

Cytokinin, auxin and gibberellin contents in resting and wound-responding potato tubers have not been fully determined and coordinated with wound-healing processes. Using a well-defined wound-healing model system, hormone content and expression of genes associated with hormone turnover were determined in tubers following wounding. Changes in hormone content were coordinated with: (I) formation and completion of the wound closing layer (0–5/6 days), and (II) initiation of phellogen and wound periderm formation (∼7 days). Quantifiable amounts of biologically active cytokinins (Z, DZ and IP) were not detected in resting or wound-responding tubers. However, the precursor IPA and catabolic product c -ZOG were found in small amounts in resting and wound-responding tubers. Wound-induced activation of cytokinin biosynthesis was suggested by an increase in t -ZR and c -ZR content at 0.5 days and large increases in IPA and c -ZR content by 3 days and throughout 7 days after wounding suggesting roles in II, but little or no role in I. Expression of key genes involved in cytokinin metabolism followed similar profiles with transcripts decreasing through 3 days and then increasing at 5–7 days after wounding. Both free IAA and IAA-Asp were present in resting tubers. While IAA-Asp was no longer present by 3 days after wounding, IAA content nearly doubled by 5 days and was more than 4-fold greater at 7 days compared to that in resting tuber (0 day) suggesting roles in II, but little or no role in I. Gibberellins were not present in quantifiable amounts in resting or wound-responding tubers. These results suggest that bio-active cytokinins are wound-induced, but their residency is temporal and highly regulated. The transient presence of active cytokinins and corresponding increases in IAA content strongly suggest their involvement in the regulation of wound periderm development. The absence of gibberellins indicates that they are not a regulatory component of wound-healing processes. [ABSTRACT FROM AUTHOR]

Isopentenyl diphosphate isomerase (IPI) catalyzes the isomerization between the common terpene precursor substances isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) during the terpenoid biosynthesis process. In this study, tissue expression analysis revealed that the expression level of the Salvia miltiorrhiza IPI1 gene (SmIPI1) was higher in the leaves than in the roots and stems. Furthermore, color complementation and RNA interference methods were used to verify the function of the SmIPI1 gene from two aspects. A recombinant SmIPI1 plasmid was successfully constructed and transferred into engineered E. coli for validating the function of SmIPI1 through the color difference in comparison to the control group; the observed color difference indicated that SmIPI1 served in promoting the accumulation of lycopene. Transformant hairy root lines with RNA interference of SmIPI1 were successfully constructed mediated by Agrobacterium rhizogenes ACCC 10060. RNA interference hairy roots had a severe phenotype characterized by withering, deformity or even death. The mRNA expression level of SmIPI1 in the RSi3 root line was only 8.4% of that of the wild type. Furthermore the tanshinone content was too low to be detected in the RNA interference lines. These results suggest that SmIPI1 plays a critical role in terpenoid metabolic pathways. Addition of an exogenous SmIPI1 gene promoted metabolic flow toward the biosynthesis of carotenoids in E. coli, and SmIPI1 interference in S. miltiorrhiza hairy roots may cause interruption of the 2-C-methyl-D-erythritol-4-phosphate metabolic pathway. [ABSTRACT FROM AUTHOR]

Product inhibition is the main problem of high-strength rice vinegar fermentation by Acetobacter pasteurianus. An optimized semi-continuous operation mode was proposed with a modified starting-up protocol which can improve the average acetification rate 35 % higher than original one. First, we revealed PQQ-ALDH is the rate-limiting enzyme responsible for ethanol oxidation route, whose apparent K of PQQ-ADH and PQQ-ALDH is 1.89 and 7.80 mM, respectively. Further work elucidated that PQQ-ALDH follows ordered Bi-Bi mechanism which indicated that Co Q can improve the reaction. Among five Co Q precursors, supplementation of 20 mM isopentenyl alcohol got the best result, which improved strain acid resistance ability that accelerates biomass accumulation. A two-stage starting-up protocol was developed, which reduced the duration of the starting-up process from 45 to 34 h. An optimized semi-continuous operation mode was proposed whose whole operation time reduced from 116 to 98 h and average acetification rate has been raised to 1.77 g/Lh, while the original is 1.31 g/Lh. The average activity of ALDH is 43 % higher than original process, while ADH is 19 %, which shows a good correlation with acetification rate. [ABSTRACT FROM AUTHOR]

Background: Cytokinins are known to play an important role in fruit set and early fruit growth, but their involvement in later stages of fruit development is less well understood. Recent reports of greatly increased cytokinin concentrations in the flesh of ripening kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang & A.R. Ferguson) and grapes (Vitis vinifera L.) have suggested that these hormones are implicated in the control of ripening-related processes. Results: A similar pattern of isopentenyladenine (iP) accumulation was observed in the ripening fruit of several grapevine cultivars, strawberry (Fragaria ananassa Duch.) and tomato (Solanum lycopersicum Mill.), suggesting a common, ripening-related role for this cytokinin. Significant differences in maximal iP concentrations between grapevine cultivars and between fruit species might reflect varying degrees of relevance or functional adaptations of this hormone in the ripening process. Grapevine orthologues of five Arabidopsis (Arabidopsis thaliana L.) gene families involved in cytokinin metabolism and signalling were identified and analysed for their expression in developing grape berries and a range of other grapevine tissues. Members of each gene family were characterised by distinct expression profiles during berry development and in different grapevine organs, suggesting a complex regulation of cellular cytokinin activities throughout the plant. The post-veraison-specific expression of a set of biosynthesis, activation, perception and signalling genes together with a lack of expression of degradation-related genes during the ripening phase were indicative of a local control of berry iP concentrations leading to the observed accumulation of iP in ripening grapes. Conclusions: The transcriptional analysis of grapevine genes involved in cytokinin production, degradation and response has provided a possible explanation for the ripening-associated accumulation of iP in grapes and other fruit. The pre- and post-veraison-specific expression of different members from each of five gene families suggests a highly complex and finely-tuned regulation of cytokinin concentrations and response to different cytokinin species at particular stages of fruit development. The same complexity and specialisation is also reflected in the distinct expression profiles of cytokinin-related genes in other grapevine organs. [ABSTRACT FROM AUTHOR]

There is a continuous search for new chemical entities to expand the collection of suitable compounds to increase the efficiency of micropropagation protocols. Two cytokinin (CK) analogues, 2-chloro-6-(3-methoxyphenyl)aminopurine (INCYDE) and CK antagonist 6-(2-hydroxy-3-methylbenzylamino)purine (PI-55) were used as a tool to elucidate the auxin-CK crosstalk under in vitro conditions in the medicinally important plant, Eucomis autumnalis subspecies autumnalis . These compounds were tested at 0.01, 0.1 and 10 μM alone as well as in combination with benzyladenine (BA) and naphthaleneacetic acid (NAA). The organogenesis, phytohormone content, phytochemical and antioxidant response in 10 week-old- in vitro regenerated E . autumnalis subspecies autumnalis was evaluated. INCYDE generally favoured shoot regeneration while the effect of PI-55 was more evident in root proliferation. Overall, INCYDE promoted the accumulation of higher concentrations and varieties of endogenous CK relative to the PI-55 treatments. In contrast, higher concentration of indole-3-acetic acid and 2-oxindole-3-acetic acid were generally observed in PI-55-supplemented cultures when compared to plantlets derived from INCYDE. Both CK analogues (individually and in-conjunction with exogenously applied PGRs) significantly influenced the phytochemicals and consequently the antioxidant potential of the in vitro regenerants. These results provided insight on how to alleviate root inhibition, a problem which causes considerable loss of several elite species during micropropagation. [ABSTRACT FROM AUTHOR]

Within the cultivated tomato germplasm, sun , ovate and fs8.1 are the three predominant QTLs controlling fruit elongation. Although SUN and OVATE have been cloned, their role in plant growth and development are not well understood. To compare and contrast the effects of the three QTLs in a homogeneous background, we developed near isogenic lines (NILs) in the wild species Solanum pimpinellifolium LA1589 background. We carried out detailed morphological characterization of reproductive and vegetative organs in the single, double and triple NILs and determined the epistatic interactions of the three loci affecting fruit shape. The phenotypic evaluations demonstrated that the three loci regulate unique aspects of ovary and fruit elongation and in different temporal manners. The strongest effect on organ shape was caused by sun . In addition to fruit shape, sun also affected leaf and sepal elongation and stem thickness. The synergistic interaction between sun and ovate or fs8.1 suggested that the pathways involving SUN , OVATE and the gene(s) underlying fs8.1 may converge at a common node. The results of an extensive profiling analysis suggested that the degree of fruit elongation was not related to the accumulation of any of the classical hormones. [ABSTRACT FROM AUTHOR]

A simple and rapid method was developed for the determination of three free cytokinins, namely, N6-(Δ2-isopentenyl)adenine, zeatin, and dihydrozeatin, in plants using TurboFlow on-line cleanup liquid chromatography combined with hybrid quadrupole-Orbitrap high-resolution mass spectrometry. The samples were extracted using acetonitrile, and then the extract was purified on a C18-p column, in which the sample matrix was removed and the analytes were retained. Subsequently, the analytes were eluted from the extraction column onto the analytical column (Hypersil Gold C18 column) prior to chromatographic separation and hybrid Q-Orbitrap detection using the targeted-MS2 scan mode. The linearity was satisfactory with a correlation coefficient of >0.999 at concentrations ranging from 5-5000 pg/mL. The limits of quantification for the analytes ranged from 4.2-5.2 pg/mL. The intra- and inter-day average recoveries of analytes fortified at three levels ranged from 85.4-108.2%, and the intra- and inter-day relative standard deviations ranged from 4.04-8.57%. The method was successfully applied for the determination of free cytokinins in different tissue samples of Oryza sativa and Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

Artemisinin, produced in very low amounts in Artemisia annua L. plants, is one of the most efffective drugs in treating malaria. In this study, the effect of exogenous applications of methyl jasmonate (MeJA) and 2-isopentenyladenine (2-iP) in artemisinin production and the main genes of its biosynthesis pathway within a 1-week time period were investigated. Both MeJA and 2-iP increased artemisinin content, but no correlation was found between gene expression and its content. In plants treated with 2-iP, despite increased gene expression following elicitor application, downregulation of gene expression was also observed, which might be attributed to the negative effects of rising artemisinin levels in gene expression. In plants treated with MeJA, the steady increase in artemisinin content was not explained by the few changes observed in gene expression, suggesting that some other mechanism may increase artemisinin. Our results indicate that the negative feedback mechanism of artemisinin is likely an obstacle for the selection of biotechnological strategies to increase artemisinin content. his may be overcome by studying the role of trichome in artemisinin production as a step toward achieving high yields of this valuable component. [ABSTRACT FROM AUTHOR]

It has been found that application of exogenous cytokinins at a specific time induced flower induction inHylocereus undatus(Haw.) Britt. & Rose. The aim of the present paper is to explore the role of cytokinins in flower induction in the cacti. Since endogenous cytokinins have never been analyzed in any cactus species, the initial step was to analyze endogenous cytokinins the year-round, and especially during the flowering period, when flower bud induction occurs. We found that the areoles contained all the cytokinin groups that are found in other plants, namely, active, non-active, metabolites, and storage forms. An important finding was that the concentrations of the active forms oftrans-zeatin and isopentenyladenine increased several-fold prior to the formation of flower buds in plants grown outdoors under natural Israeli conditions.Endogenous cytokinins were analyzed further inH. undatus, flowering of which was induced with various temperature and long-day regimes. Cytokinins were also analyzed in two closely related clones, one of which flowers continuously through the warm season while the other flowers in two waves. In all tested cases, the active forms of the endogenous cytokinins peaked before the peaks of flowering occurred. The cytokinin dynamics of all these treatments supported the hypothesis that active cytokinins are involved in regulation of flowering in this vine-cactus species. [ABSTRACT FROM PUBLISHER]

Cytokinins are involved in key developmental processes in rice (Oryza sativa), including the regulation of cell proliferation and grain yield. However, the in vivo action of histidine kinases (OsHks), putative cytokinin receptors, in rice cytokinin signaling remains elusive. This study examined the function and characteristics of OsHk3, 4 and 6 in rice. OsHk6 was highly sensitive to isopentenyladenine (iP) and was capable of restoring cytokinin-dependent ARR6 reporter expression in the ahk2 ahk3 Arabidopsis mutant upon treatment with 1 nM iP. OsHk4 recognized trans-zeatin (tZ) and iP, while OsHk3 scarcely induced cytokinin signaling activity. OsHk4 and OsHk6 mediated the canonical two-component signaling cascade of Arabidopsis to induce phosphorylation of ARR2. OsHk4 and OsHk6 were highly expressed in spikelets, suggesting that tZ and iP might play key roles in grain development. OsHk6 formed a self-interacting homomer in rice protoplasts, although the trans-phosphorylation activity between subunits was much lower than the intra-molecular trans-phosphorylation activity. This indicates that the action mechanism of OsHks is evolutionarily diverged from bacterial histidine kinases. Ectopic expression of OsHk6 in rice calli promoted green pigmentation and subsequent shoot induction, further supporting an OsHk6 in planta function as a cytokinin receptor. From the results of this study, OsHks are homomeric cytokinin receptors with distinctive cytokinin preferences in rice. [ABSTRACT FROM PUBLISHER]

Abstract: The discovery of cytokinin receptors of Arabidopsis thaliana ten years ago was a milestone in plant hormone research. Since then, research has yielded insights into the biochemical properties and functions of these sensor histidine kinases. Their affinities to both trans-zeatin and isopentenyladenine are in the low nM range. Cytokinin ribosides, cis-zeatin and thidiazuron were established as compounds with genuine cytokinin activity and the first cytokinin antagonists were identified. Numerous functions of cytokinin receptors in plant development, as well as in the plant''s responses to the environment, have been elucidated and are summarized. Finally, we address the question how the receptors have evolved during plant evolution. [Copyright &y& Elsevier]

Our paper presents aspects regarding in vitro culture initiation in cranberry (Vaccinium macrocarpon), cultivar 'Pilgrim', in vitro multiplication and ex vitro acclimation. In order to establish the optimal explant for initiation, several types of explants: single node fragments without leaves, single node fragments with leaves, two-node fragments without leaves, two-node fragments with leaves, multiple node fragments (3-4 nodes with leaves) and 1-1.5 cm long shoot tips were tested on media of the same composition (modified Woody Plant Medium). After testing the explant types, three variants of nutritive media were tested: WPM with three concentrations of 2-Isopentenyladenine: 5, 10 and 20 mg/l in order to induce the proliferation of axillary buds in the initiation phase. In the multiplication phase, several types of culture vessels were tested, as well as the optimal number of inoculalvessei and the influence of growth regulators upon multiplication rate. Among the variants of nutritive media that were tested, modified WPM containing either 1 mg/l zeatin or 5 mg/l 2-Ip presented reasonable proliferation rates and relatively well-developed shoots. In the in vitro layering experiment, 2-Ip at 10 mg/l generated tens of thin shoots/explant. As gelling agents, starch and Plant Agar were tested. Ex vitro acclimation was carried out in Jiffy7 pellets in either covered or uncovered plastic trays and various survival rates (17-92 %) were obtained depending on explant types and culture conditions. [ABSTRACT FROM AUTHOR]

A protocol is described for rapid and large scale propagation of an endangered, important Sikkim Himalayan rhododendron (R. maddeni Hook. f.) by in vitro culture of cotyledonary nodes from 15 days old seedlings. Several cytokinin types were evaluated for their effect on shoot multiplication from cotyledonary nodes. Maximum numbers of shoot (12.00 ±0.58) were observed on the AM containing 7 mg/l 2iP, 0.1 mg/l IAA after eight weeks of culture. Incorporation of 0.1 mg/l IAA in the medium during the first subculture after establishment and initiation of shoot buds significantly improved the shoot elongation. Regenerated shoots were separated and rooted on same strength AM medium supplemented with 0.2 mg/l of IBA alone for three weeks. Well-developed complete plantlets were transferred on to specially made plastic cup containing soilrite. The rooted plantlets were hardened and successfully established in greenhouse, the plants were transferred to field site at Pangthang arboretum of the Institute and the 'Rare & Threatened Plant Conservation Park' of Zoological Park, Gangtok, Sikkim. [ABSTRACT FROM AUTHOR]

Aconitum chasmanthum Stapf ex Holmes, a highly valued medicinal plant, is a critically endangered plant species with restricted global distribution. Because there is no published report on the in vitro micropropagation of A. chasmanthum, the present study was undertaken to contribute to the development of an efficient micropropagation protocol for its conservation. Seeds collected from the wild showed enhanced germination after being given a chilling treatment (−4 °C and −20 °C) for different durations (10, 20, 30 and 40 days). Seeds given a chilling treatment of −4 °C for 10 days showed enhanced germination rates of 47.59 ± 0.53% with a mean germination time of 10.78 ± 0.21 days compared to seeds kept at room temperature when grown in an MS basal medium. Nodes, leaves and stems, taken from 20–40-day-old seedlings, were used as an explant for micropropagation. An MS medium supplemented with different concentrations of cytokinins (BAP, Kn), auxins (2,4-D, NAA), and an additive adenine sulphate were tested for callusing, direct shoot regeneration and rooting. Only nodal explants responded and showed direct multiple shoot regeneration with 7 ± 0.36 shoots with an elongation of 5.51 ± 0.26 cm in the MS medium supplemented with BAP 0.5 mg/L, and with a response time (RT) of 10.41 ± 0.51 days and a percentage culture response of 77.77 ± 2.77%. Rhizome formation was observed after 8 weeks, with the highest culture response of 36.66 ± 3.33% in the MS basal media with an RT of 43.75 ± 0.50 days. These rhizomes showed a 60% germination rate within 2 weeks and developed into plantlets. The present in vitro regeneration protocol could be used for the large-scale propagation and conservation of A. chasmanthum. [ABSTRACT FROM AUTHOR]

The roots and stems of dicotyledonous plants thicken by the cell proliferation in the cambium. Cambial proliferation changes in response to environmental factors; however, the molecular mechanisms that regulate cambial activity are largely unknown. The quadruple Arabidopsis thaliana mutant atipt1;3;5;7, in which 4 genes encoding cytokinin biosynthetic isopentenyltransferases are disrupted by T-DNA insertion, was unable to form cambium and showed reduced thickening of the root and stem. The atipt3 single mutant, which has moderately decreased levels of cytokinins, exhibited decreased root thickening without any other recognizable morphological changes. Addition of exogenously supplied cytokinins to atiptl;3;5;7 reactivated the cambium in a dosedependent manner. When an atiptl;3;5;7 shoot scion was grafted onto WT root stock, both the root and shoot grew normally and trans-zeatin-type(tZ-type)cytokinins in the shoot were restored to WI levels, but isopentenyladenine-type cytokinins in the shoot remained unchanged. Conversely, when a WI shoot was grafted onto an atipt1;3;5;7 root, both the root and shoot grew normally and isopentenyladenine-type cytokinins in the root were restored to WT levels, but tZ-type cytokinins were only partially restored. Collectively, it can be concluded that cytokinins are important regulators of cambium development and that production of cytokinins in either the root or shoot is sufficient for normal development of both the root and shoot. [ABSTRACT FROM AUTHOR]

The scope of this paper is to present the in vitro propagation of the highbush blueberry cultivar Blue Crop, a cultivar that has important difficulties regarding in vitro propagation as well as propagation by cuttings. As basal medium, Woody Plant Medium (WPM, after Lloyd & McCown) was used. As growth regulators, 2-Isopentenyladenine, Zeatin, Thidiazuron and raw coconut water obtained from ripe fruit were used. The iron source is also very important, FeNaEDDHA (Sequestrene 138) providing far better results than FeNaEDTA. FeNaEDDHA can either be added as iron supplement to the prepackaged media or it can be used as the only iron source in the media prepared from stock solutions of macroelements and microelements. The pH recommended for the media used for bluecrop in vitro propagation is 5. Also, some peculiarities of the in vitro propagation of cultivar Blue Crop are presented, in comparison with other cultivars. [ABSTRACT FROM AUTHOR]

With the aim to contribute to elucidation of the role of phytohormones in plant responses to stresses the endogenous contents of abscisic acid (ABA) and cytokinins (CK) were followed in French bean, maize, sugar beet, and tobacco during water stress and subsequent rehydration. The effects of pre-treatments with exogenous ABA or benzyladenine (BA) before imposition of water stress were also evaluated. The content of ABA increased by water stress, and with the exception of bean plants increased content of ABA remained also after rehydration. In all plant species the ABA content was further increased by ABA pre-treatment, but in bean and maize it decreased by BA pre-treatment. The highest total content of CK was observed in bean and the lowest in maize during water stress. In their spectrum, the storage CK were dominant in bean, and inactive CK in tobacco while in sugar beet and maize all groups were present in comparable amounts. In all plant species, the contents of CK increased during water stress and with exception of bean they decreased back after rehydration. ABA pre-treatment further increased contents of CK in water-stressed bean and tobacco. BA pre-treatment increased contents of CK in sugar beet and tobacco after rehydration. [ABSTRACT FROM AUTHOR]

We describe a new enzymatic reaction method for the preparation of the radioisotope-labeled cytokinins isopentenyladenine (iP), trans-zeatin (tZ), and their ribosides. The method is based on the three enzyme activities of an adenylate isopentenyltransferase (IPT; EC 2.5.1.27) from Arabidopsis thaliana, an alkaline phosphatase (EC 3.1.3.1) from calf intestine, and a purine-nucleoside phosphorylase (EC 2.4.2.1) from Escherichia coli. The A. thaliana IPT, AtIPT7, utilized both dimethylallyldiphosphate and 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate as isoprenoid donors. The dual specificity of the substrates enabled us to produce iP-type and tZ-type cytokinins separately in the same system simply by switching the substrates. Our method affords a much higher yield of the labeled products than the chemical reaction methods previously used. These labeled compounds will be useful tools for cytokinin research, such as receptor–ligand assays and cell metabolism studies. [ABSTRACT FROM AUTHOR]