Showing papers on "Zeatin published in 2018"

Photoelectrochemical apta-biosensor for zeatin detection based on graphene quantum dots improved photoactivity of graphite-like carbon nitride and streptavidin induced signal inhibition

Abstract: Zeatin is a kind of typical cytokinin, which plays important roles in regulating the proliferation and differentiation of plant cells, and widely distributed in various plant tissues. Herein, a simple and sensitive photoelectrochemical method was developed for zeatin detection using graphite-like carbon nitride (g-C3N4) as photoactive material, graphene quantum dots as photoactivity improvement reagent, gold nanoparticles as immobilization substrate for DNA probe, DNA biotin labeled aptamer as zeatin recognition and streptavidin capture element, and streptavidin as PEC response inhibitor factor. In the presence of zeatin, DNA aptamer can bind with zeatin to form conjugate, which make aptamer releasing from electrode surface and decrease the capture amount of streptavidin, causing the increase of the PEC response. Under optimal conditions, the biosensor showed wide linear range from 0.1 to 100 nM and low detection limit of 0.031 nM (3σ). The developed method also presented good selectivity, even discriminating zeatin analogues. In addition, the proposed detection strategy can be further applied to detect zeatin in complex biological matrix, revealing great potential in practical applicability. This work might provide an excellent platform for zeatin or other phytohormone detection using aptamer as recognition reagent.

Exogenous Hormonal Application Regulates the Occurrence of Wheat Tillers by Changing Endogenous Hormones.

Abstract: Plant hormones play important roles in regulating the occurrence of crop tillers. However, little is known about the relationships and the underlying mechanisms between endogenous hormones and the occurrence of wheat tillers induced by exogenous hormones. In this study, two winter wheat cultivars, Xinong 979 and Xiaoyan 22, were used to investigate the effects of the exogenous application of indole-3-acetic acid (IAA) and zeatin (Z) on the occurrence of wheat tillers and investigate underlying mechanisms regulating the occurrence of tillers. The results showed that the application of IAA inhibited the occurrence of tillers, and external Z application promoted the occurrence rate of tillers under low nitrogen conditions. Further analysis of the results showed that exogenous IAA completely inhibited the growth of tiller buds, while exogenous Z significantly promoted the growth rate of tiller buds in low nitrogen conditions. Endogenous hormones exhibit important functions in regulating the growth of tiller buds, which contents were affected by exogenous hormones. Furthermore, according to the principal component analysis and correlation analysis, the growth of tiller buds was significantly positively correlated with the content of endogenous Z, whereas it was significantly negatively correlated with the ratios of endogenous IAA to endogenous Z (IAA:Z) and endogenous abscisic acid (ABA) to endogenous Z (ABA:Z). Moreover, no significant correlation was observed between the growth of the tiller buds and the endogenous IAA, endogenous gibberellins (GAs), and endogenous ABA content. These results suggested that Z played key roles in regulating the tiller occurrence, and exogenous hormones regulated the growth of wheat tiller buds via affecting the Z contents, thus regulating the occurrence of wheat tiller.

Thidiazuron-induced somatic embryogenesis and changes of antioxidant properties in tissue cultures of half-high blueberry plants

Abstract: An efficient protocol of somatic embryogenesis (SE) has been developed for the first time in four half-high blueberry (Vaccinium corymbosum L. × V. angustifolium Ait.) cultivars. Thidiazuron (TDZ), a plant growth regulator with potential activities for shoot regeneration and shoot proliferation, was found most effective for somatic embryo formation when added to a nutrient medium at high concentration (9 µM). Although TDZ was also best for embryo germination at low concentration (2.3 µM), it was followed by zeatin at 4.6 µM for the same. Plantlets developed from SE were removed from the nutrient medium and transferred on a peat: perlite medium where 100% survival rate was acquired following the acclimatization process in a greenhouse. The concentrations of total phenolic and flavonoid contents were higher in greenhouse-grown conventionally cutting-propagated donor mother plants than those of respective SE plants for ‘St. Cloud’, ‘Patriot’ and ‘Northblue’ but not for ‘Chippewa’. The effect of propagation method and/or the older age of donor mother plants were clearly visible exclusively as the 15-year-old donor plants showed higher level of 2,2-diphenyl-1-picrylhydrazyl scavenging activity than the eight-weeks-old SE plants in all four cultivars.

Somatic embryogenesis from stem thin cell layers of Dendrobium aqueum

Abstract: An efficient in vitro regeneration protocol through somatic embryogenesis was established from stem transverse thin cell layers (tTCLs) of Dendrobium aqueum Lindley, an imperiled orchid. This study outlines the induction and successive maturation stages of D. aqueum somatic embryos (SEs). The tTCLs (~ 0.5 mm thick) cultured on halfstrength Murashige and Skoog (MS) medium containing cytokinins and auxins, either individually or in combination, produced embryogenic callus (EC). Treatment with 0.5 mg dm-3 zeatin induced EC in 41.42 % of tTCLs. As many as 42.66 globular SEs per tTCL were formed in the presence of 1.5 mg dm-3N6-(2-isopentyl) adenine (2iP) but only on 10.33 % of explants. The combined treatment of 2iP (1.5 mg dm-3) and 0.5 mg dm-3 6-benzyladenine resulted in 34 globular SEs on 14.7 % of tTCLs whereas the combination of 2iP and 1.0 mg dm-3 indole-3-butyric acid (IBA) induced 7.4 globular SEs on 52.33 % of tTCLs. Supplementation of activated charcoal, amino acids, and antioxidants alleviated browning at all the concentrations tested, but the EC response declined. The addition of 0.5 mg dm-3 polyvinylpyrrolidone to 1.5 mg dm-3 2iP and 1.0 mg dm-3 IBA produced 24 SEs on 19.89 % of tTCLs suggesting that the EC and SEs can be effectively induced by individual cytokinins whereas the synergistic treatments with other compounds can only enhance the induction of EC. Histological observations of EC showed the formation of globular SEs from sub-epidermal regions. Successive developmental stages of globular SEs and the intermediate stage of protocorm like bodies until the formation of plantlets were observed. The plantlets obtained through SEs showed no morphological variations, and inter simple sequence repeat profiles also confirmed the genetic fidelity of in vitro-derived progeny with high monomorphism (97.78 %). In conclusion, the use of stem tTCLs is an effective method to produce SEs through indirect somatic embryogenesis in D. aqueum.

γ-Irradiation of Barley Seeds and Its Effect on the Phytohormonal Status of Seedlings

Abstract: We investigated the effect of γ-irradiation (4–50 Gy) of barley seeds (Hordeum vulgare L., cv. Nur) on the content of endogenous phytohormones–stimulators of plant growth and development: indol-3-acetic acid (IAA), indolyl-3-butyric acid (IBA), zeatin and abscisic acid (ABA). The ratio (IAA + IBA + zeatin)/ABA from the third to the seventh day of germination has been measured. It was shown that the changes in the content of phytohormones as a function of the radiation dose were nonlinear. In the dose range of 4–20 Gy, phytohormones balance was changed due to increased content of growth stimulators and decreased ABA content. Using a dose of 50 Gy led primarily to a decrease in the content of growth stimulators and an increase in ABA content, and the ratio (IAA + IBA + zeatin)/ABA shifted toward ABA content.

Plant regeneration via direct somatic embryogenesis from leaf explants of Tolumnia Louise Elmore ‘Elsa’

Abstract: Tolumnia genus (equitant Oncidium) is a group of small orchids with vivid flower color. Thousands of hybrids have been registered on Royal Horticulture Society and showed great potential for ornamental plant market. The aim of this study is to establish an efficient method for in vitro propagation. Leaf explants taken from in vitro-grown plants were used to induce direct somatic embryogenesis on a modified 1/2 MS medium supplemented with five kinds of cytokinins, 2iP, BA, kinetin, TDZ and zeatin at 0.3, 1 and 3 mg l−1 in darkness. TDZ at 3 mg l−1 gave the highest percentage of explants with somatic globular embryos after 90 days of culture. It was found that 2,4-D and light regime highly retarded direct somatic embryogenesis and showed 95–100% of explant browning. Histological observations revealed that the leaf cells divided into meristematic cells firstly, followed by somatic proembryos, and then somatic globular embryos. Eventually, somatic embryos developed a bipolar structure with the shoot apical meristem and the root meristem. Scanning electron microscopy observations showed that the direct somatic embryogenesis from leaf explants was asynchronously. The somatic embryos were found on the leaf tip, the adaxial surface and also the mesophyll through a cleft, and it reflected the heterogeneity of the explant. The 90-day-old globular embryos were detached from the parent explants and transferred onto a hormone-free 1/2 MS medium in light condition for about 1 month to obtain 1-cm-height plantlets. After another 3 months for growth, the plantlets were potted with Sphagnum moss and were acclimatized in a shaded greenhouse. After 1 month of culture, the survival rate was 100%. In this report, a protocol for efficient regenerating a Tolumnia orchid, Louise Elmore ‘Elsa’, was established via direct somatic embryogenesis and might reveal an alternative approach for mass propagation of Tolumnia genus in orchid industry.

Influence of growth regulators and explants on shoot regeneration in carnation.

Abstract: The influence of growth regulators, explants and their interactions on in vitro shoot bud formation from callus was studied in Dianthus caryophyllus L. The leaf and internode explants were cultured on Murashige and Skoog (MS) medium containing different concentrations of growth regulators. The highest callus induction was observed with 2 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) and 1 mg/l benzyl adenine (Ba). out of twenty seven shoot regeneration media tested, only 2 mg/l thidiazuron (tDZ) and zeatin alone or in combination with naphthalene acetic acid (Naa) and/or indole acetic acid (iaa) could differentiate calli. The highest average number of shoots was observed with 2 mg/l tDZ and 1 mg/l iaa. Significant differences were observed in calli producing shoots and number of shoots per callus in the explants of leaf and internode. The shoots were elongated and multiplied on MS medium supplemented with 1 mg/l Ba and solidified with 1% agar. The shoots were rooted and hardened with 76% survival success in pots after six weeks of transfer to the pots.

Effect of cytokinins on shoots proliferation and rosmarinic and salvianolic acid B production in shoot culture of Dracocephalum forrestii W. W. Smith

Abstract: The current study estimates the effect of different cytokinins on shoot proliferation and biosynthesis of caffeic acid derivatives in Dracocephalum forrestii in vitro culture. The shoots were grown on Murashige and Skoog (MS) agar medium with 1 µM indole-3-acetic acid (IAA) and different content of 6-benzyloaminopurine (BAP), zeatin, kinetin (1, 2, 4, 8, 18 µM) or thidiazuron (TDZ) (0.1, 0.2, 0.5, 1, 2 µM). The highest multiplication rate (about seven shoots and/or buds per explant) was obtained after 4 weeks of culture on MS medium with 1 µM IAA and 8 or 16 µM BAP. Optimal biomass of plant material was also received on the same media. The identity of the compounds present in the hydromethanolic extracts from D. forrestii shoots grown on cytokinin-supplemented media was confirmed using UPLC–PDA–ESI–MS method. The analysis revealed the presence of nine metabolites recognized as caffeic acid derivatives. The content of the predominant phenolic acids in the extracts, i.e. rosmarinic acid (RA) and salvianolic acid B (SAB), was determined with UHPLC. The highest yield of RA was found in shoots cultivated in the medium containing 1 µM IAA and 2 µM BAP (18.7 mg/g DW). The highest level of SAB (5.3–5.9 mg/g DW) was identified in multiple shoots grown in the presence of 1 µM IAA and 0.5–1 µM TDZ or 2 µM BAP.

The trans and cis zeatin isomers play different roles in regulating growth inhibition induced by high nitrate concentrations in maize

Abstract: Abscisic acid (ABA), auxins, and cytokinins (CKs) are known to be closely linked to nitrogen signaling. In particular, CKs control the effects of nitrate availability on plant growth. Our group has shown that treatment with high nitrate concentrations limits root growth and leaf development in maize, and conditions the development of younger roots and leaves. CKs also affect source-sink relationships in plants. Based on these results, we hypothesized that CKs regulate the source-sink relationship in maize via a mechanism involving complex crosstalk with the main auxin indole-3-acetic acid (IAA) and ABA. To evaluate this hypothesis, various CK metabolites, IAA, and ABA were quantified in the roots and in source and sink leaves of maize plants treated with high and normal nitrate concentrations. The data obtained suggest that the cis and trans isomers of zeatin play completely distinct roles in maize growth regulation by a complex crosstalk with IAA and ABA. We demonstrate that while trans-zeatin (tZ) and isopentenyladenine (iP) regulate nitrate uptake and thus control final leaf sizes, cis-zeatin (cZ) regulates source and sink strength, and thus controls leaf development. The implications of these findings relating to the roles of ABA and IAA in plants’ responses to varying nitrate concentrations are also discussed.

Shoot Induction from Axillary Shoot Tip Explants of Fig (Ficus carica) cv. Japanese BTM 6.

Abstract: Fig, or Ficus carica, is a fruit tree from the Moraceae family and is widely grown in tropical and subtropical regions of the world. Fig plants are mainly propagated through grafting, air layering, and hardwood cutting whereby these methods were found to be less efficient. Plant tissue culture is efficient method to propagate plants, particularly to produce true-to-type platelets for mass multiplication. The aim of this study is to induce multiple shoot formation on Ficus carica cv. Japanese BTM 6 through identifying and optimising the concentrations of 6-Benzylaminopurine (BAP) and Zeatin suited for shoot formation. The axillary shoot tip explants were cultured in MS media supplemented with different concentrations of BAP and Zeatin (0, 0.5, 1.0, 1.5 and 2.0 mg/L) to determine the optimal concentration for the formation of multiple shoots. Number of shoots per explants and the differences in shoot height of explants were calculated after 8 and 12 weeks of culture respectively. Of all the treatments of BAP, MS media containing with 2 mg/L BAP marked the highest number of shoots per explant with the average value of 1.67 ± 0.33 while 1.5 and 2 mg/L of BAP produced the highest differences in shoot height with 0.51 ± 0.08 cm and 0.51 ± 0.07 cm after 12 weeks respectively. Murashige and Skoog (MS) media supplemented with 2 mg/L Zeatin showed the highest production of multiple shoots and differences in shoot height with the average of 0.83 ± 0.219 and 0.32 ± 0.04 cm respectively among all the different treatments of Zeatin. In this study, BAP performed better in shoot induction and elongation as compared to Zeatin for the cultivar Japanese BTM 6.

In vitro regeneration of adventitious buds from leaf explants and their subsequent cryopreservation in highbush blueberry

Abstract: We report a new cryopreservation method for highbush blueberry (Vaccinium corymbosum) using small leaf squares-bearing adventitious buds. Leaf explants were cultured on adventitious bud regeneration medium composed of Woody Plant Medium (WPM) supplemented with 20 µM zeatin. After 21 days of adventitious bud regeneration, small leaf squares (SLSs, 2 × 3 mm), each bearing multiple adventitious buds, were cut from the leaf explant, precultured on WPM containing 0.3 M sucrose for 24 h and were treated for 30 min with a loading solution composed of WPM containing 1.0 M sucrose and 2 M glycerol, followed by exposure to plant vitrification solution 2 at 0 °C for 40 min. Each of dehydrated SLS was then transferred onto an aluminum foil with small holes and PVS2 was dropped until it covered the SLS, prior to a direct immersion in liquid nitrogen. Cryopreserved SLSs were re-warmed in WPM containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for recovery. With this procedure, more than 23 adventitious buds were produced in each leaf explant, and 100% of SLSs were able to survive and resume shoot regrowth, with more than six shoots per SLS obtained following cryopreservation in three highbush blueberry cultivars. In ‘Misty’, the morphology of plantlets regenerated from cryopreserved SLSs was identical to that of the in vitro-derived ones. No polymorphic bands were detected using inter-simple sequence repeat markers and random amplified polymorphic DNA in plantlets of ‘Misty’ recovered from cryopreservation. The use of SLSs-bearing adventitious buds for cryopreservation reported in the present study eliminates shoot tip excision. This cryopreservation method can be considered an efficient cryopreservation of plant shoot tips, and has potential applications to other plant species.

Transcriptome analysis unravels spatiotemporal modulation of phytohormone-pathway expression underlying gibberellin-induced parthenocarpic fruit set in San Pedro-type fig (Ficus carica L.).

Abstract: Gibberellin (GA) treatments can induce parthenocarpy in the main crop of San Pedro-type figs, the native non-parthenocarpic fruit, however, the underlying mechanism is still largely unclear. In our study, GA3 was applied to San Pedro-type fig main crop at anthesis. Sharply increased GA3 content was detected in both female flowers and receptacle, along with significantly decreased indole-3-acetic acid (IAA), zeatin and abscisic acid (ABA) levels in female flowers, and increased zeatin peak intensity and earlier ABA peak in receptacles. Transcriptome comparison between control and treatment groups identified more differentially expressed genes (DEGs) in receptacles than in female flowers 2 and 4 days after treatment (DAT); 10 DAT, the number of DEGs became similar in the two tissues. Synchronized changing trends of phytohormone-associated DEGs were observed in female flowers and receptacles with fruit development. Modulation of ethylene and GA signaling and auxin metabolism by exogenous GA3 occurred mainly 2 DAT, whereas changes in auxin, cytokinin and ABA signaling occurred mainly 10 DAT. Auxin-, ethylene- and ABA-metabolism and response pathways were largely regulated in the two tissues, mostly 2 and 10 DAT. The major components altering fig phytohormone metabolic and response patterns included downregulated GA2ox, BAS1, NCED and ACO, and upregulated ABA 8′-h and AUX/IAA. Thus GA-induced parthenocarpy in fig is co-modulated by the female flowers and receptacle, and repression of ABA and ethylene biosynthesis and GA catabolism might be the main forces deflecting abscission and producing fig parthenocarpy.

Study of cytokinin transport from shoots to roots of wheat plants is informed by a novel method of differential localization of free cytokinin bases or their ribosylated forms by means of their specific fixation

Abstract: The aim of the present report was to demonstrate how a novel approach for immunohistochemical localization of cytokinins in the leaf and particularly in the phloem may complement to the study of their long-distance transport. Different procedures of fixation were used to conjugate either cytokinin bases or their ribosides to proteins of cytoplasm to enable visualization and differential localization of these cytokinins in the leaf cells of wheat plants. In parallel to immunolocalization of cytokinins in the leaf cells, we immunoassayed distribution of free bases of cytokinins, their nucleotides and ribosides between roots and shoots of wheat plants as well as their presence in phloem sap after incubation of leaves in a solution supplemented with either trans-zeatin or isopentenyladenine. The obtained data show ribosylation of the zeatin applied to the leaves and its elevated level in the phloem sap supported by in vivo localization showing the presence of ribosylated forms of zeatin in leaf vessels. This suggests that conversion of zeatin to its riboside is important for the shoot-to-root transport of zeatin-type cytokinins in wheat. Exogenous isopentenyladenine was not modified, but diffused from the leaves as free base. These metabolic differences may not be universal and may depend on the plant species and age. Although the measurements of cytokinins in the phloem sap and root tissue is the most defining for determining cytokinin transport, study of immunolocalization of either free cytokinin bases or their ribosylated forms may be a valuable source of information for predicting their transport in the phloem and to the roots.

Transcriptome Profiles Reveal the Crucial Roles of Auxin and Cytokinin in the "Shoot Branching" of Cremastra appendiculata.

Abstract: Cremastra appendiculata has become endangered due to reproductive difficulties. Specifically, vegetative reproduction is almost its only way to reproduce, and, under natural conditions, it cannot grow branches, resulting in an extremely low reproductive coefficient (reproductive percentage). Here, we performed RNA-Seq and a differentially expressed gene (DEG) analysis of the three stages of lateral bud development in C. appendiculata after decapitation—dormancy (D2), transition (TD2), and emergence (TG2)—and the annual axillary bud natural break (G1) to gain insight into the molecular regulatory network of shoot branching in this plant. Additionally, we applied the auxin transport inhibitors N-1-naphthylphthalamic acid (NPA) and 2,3,5-triiodibenzoic acid (TIBA) to a treated pseudobulb string of C. appendiculata to verify the conclusions obtained by the transcriptome. RNA-Seq provided a wealth of valuable information. Successive pairwise comparative transcriptome analyses revealed 5988 genes as DEGs. GO (Gene Ontology) and KEGG (Kyoto encyclopedia of genes and genomes) analyses of DEGs showed significant enrichments in phytohormone biosynthesis and metabolism, regulation of hormone levels, and a hormone-mediated signaling pathway. qRT-PCR validation showed a highly significant correlation (p < 0.01) with the RNA-Seq generated data. High-performance liquid chromatography (HPLC) and qRT-PCR results showed that, after decapitation, the NPA- and TIBA-induced lateral buds germinated due to rapidly decreasing auxin levels, caused by upregulation of the dioxygenase for auxin oxidation gene (DAO). Decreased auxin levels promoted the expression of isopentenyl transferase (IPT) and cytochrome P450 monooxygenase, family 735, subfamily A (CYP735A) genes and inhibited two carotenoid cleavage dioxygenases (CCD7 and CCD8). Zeatin levels significantly increased after the treatments. The increased cytokinin levels promoted the expression of WUSCHEL (WUS) and inhibited expression of BRANCHED1 (BRC1) in the cytokinin signal transduction pathway and initiated lateral bud outgrowth. Our data suggest that our theories concerning the regulation of shoot branching and apical dominance is really similar to those observed in annual plants. Auxin inhibits bud outgrowth and tends to inhibit cytokinin levels. The pseudobulb in the plant behaves in a similar manner to that of a shoot above the ground.

Natural plant hormones cytokinins increase stress resistance and longevity of Caenorhabditis elegans.

Abstract: Cytokinins are phytohormones that are involved in many processes in plants, including growth, differentiation and leaf senescence. However, they also have various activities in animals. For example, kinetin and trans-zeatin can reduce levels of several aging markers in human fibroblasts. Kinetin can also protect mice against oxidative and glyoxidative stress, and prolong fruit flies’ lifespan. Additionally, several cytokinins are currently used in cosmetics. To extend knowledge of the breadth of cytokinins’ activities, we examined effects of natural cytokinin bases on the model nematode Caenorhabditis elegans. We found that kinetin, para-topolin and meta-topolin prolonged the lifespan of C. elegans. Kinetin also protected the organism against oxidative and heat stress. Furthermore, our results suggest that presence of reactive oxygen species, but not DAF-16 (the main effector of the insulin/insulin-like growth factor signaling pathway), is required for the beneficial effects of kinetin. Ultra-high performance liquid chromatography-tandem mass spectrometric analysis showed that kinetin is unlikely to occur naturally in C. elegans, but the worm efficiently absorbs and metabolizes it into kinetin riboside and kinetin riboside-5′-monophosphate.

Electrochemical aptasensors for zeatin detection based on MoS2 nanosheets and enzymatic signal amplification.

Abstract: A simple and sensitive electrochemical aptasensor was constructed for zeatin detection, where MoS2 nanosheets were used as the immobilization matrix for gold nanoparticles (AuNPs), and AuNPs were employed as the immobilization matrix to probe DNA. After the aptamer DNA and assist DNA hybridized with probe DNA, Y-type DNA can be formed with two biotins at the terminals of aptamer DNA. Then, avidin modified alkaline phosphatase (Avidin-ALP) can be further modified on the electrode surface through the biotin and avidin interaction. Under the catalytic effect of ALP, p-nitrophenylphosphate disodium (PNPP) can be hydrolyzed to produce p-nitrophenol (PNP). However, in the presence of zeatin, the formed Y-type DNA can be destroyed due to the formation of the zeatin–aptamer conjugate, which further reduces the amount of PNP and leads to the decrease of the oxidation signal of PNP. Under the optimum conditions, the change of the oxidation peak current of PNP was inversely proportional to the logarithm value of zeatin concentration in the range of 50 pM–50 nM. The detection limit was calculated to be 16.6 pM. This electrochemical method also showed good detection selectivity and stability. The potential applicability of this method was proved by detecting zeatin in real samples.

The effect of endogenous hormones on plant morphology and fruit quality of tomato under difference between day and night temperature

Abstract: The difference between day and night temperature (DIF) was reported to influence plant morphology and fruit quality, but the mechanism was poorly known. Therefore, controlled-environment experiments were carried out to investigate the mechanism of DIF influenced plant morphology and fruit quality attributes of tomato during fruit stage. Five day/night temperature regimes 16/34, 19/31, 25/25, 31/19 and 34/16°C with respective DIFs of –18, –12, 0, +12 and +18 at a common 25°C mean daily temperature were used. The results showed that gibberellin 3, indoleacetic acid and zeatin content of stem tip were enhanced significantly by positive DIF and inhibited by negative DIF, while abscisic acid was not significantly influenced by DIF. Plant height, stem diameter, fruit diameter and leaf area were enhanced significantly by positive DIF regimes and inhibited by negative DIF regimes. The soluble sugars, vitamin C and soluble protein content increased under positive DIF while decreased under negative DIF. Both plant morphology and fruit quality of tomato were significantly related to endogenous hormones.

Detection of cytokinins and auxin in plant tissues using histochemistry and immunocytochemistry.

Abstract: We report a new method for histochemical localization of cytokinins (CKs) in plant tissues based on bromophenol blue/silver nitrate staining. The method was validated by immunohistochemistry using anti-trans-zeatin riboside antibody. Indole-3-acetic acid (auxin, IAA) was localized by anti-IAA antibody in plant tissues as a proof for IAA histolocalization. We used root sections, because they are major sites of CKs synthesis, and insect galls of Piptadenia gonoacantha that accumulate IAA. Immunostaining confirmed the presence of zeatin and sites of accumulation of IAA indicated by histochemistry. The colors developed by histochemical reactions in free-hand sections of plant tissues were similar to those obtained by thin layer chromatography (TLC), which reinforced the reactive sites of zeatin. The histochemical method for detecting CKs is useful for galls and roots, whereas IAA detection is more efficient for gall tissues. Therefore, galls constitute a useful model for validating histochemical techniques due to their rapid cell cycles and relatively high accumulation of plant hormones.

Regeneration of highland papaya (Vasconcellea pubescens) from anther culture

Abstract: Premise of the Study Vasconcellea pubescens is an important Caricaceae species cultivated in several countries of South America. The objective of this study was to investigate different media compositions and plant growth regulators to induce plant regeneration. Methods Anthers were cultured in Murashige and Skoog medium with varying concentrations of naphthalene acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) plus a cytokinin (N-(2-chloro-4-pyridyl)-N'-phenylurea). The effect of the basal medium supplemented with auxins and cytokinins on shoot regeneration from the induced calli was also evaluated. Addition of maltose to the basal medium was also tested. Results The combination of 0.54 μM NAA and 22.66 μM 2,4-D induced the highest rate of calli formation. Regeneration via organogenesis was obtained in Murashige and Skoog and Woody Plant Medium supplemented with maltose and containing 8.88 μM 6-benzylaminopurine, 5.71 μM indoleacetic acid, and 2.28 μM zeatin. Discussion The plant regeneration protocol reported here permits the development of haploid and double haploid plants that can be useful for propagation purposes, allow a better molecular understanding of the species, and facilitate the production of new cultivars.

MaMADS2 repression in banana fruits modifies hormone synthesis and signalling pathways prior to climacteric stage

Abstract: While the role of ethylene in fruit ripening has been widely studied, the contributions of additional plant hormones are less clear. Here we examined the interactions between the transcription factor MaMADS2-box which plays a major role in banana fruit ripening and hormonal regulation. Specifically, we used MaMADS2 repressed lines in transcriptome and hormonal analyses throughout ripening and assessed hormone and gene expression perturbations as compared to wild-type (WT) control fruit. Our analyses revealed major differences in hormones levels and in expression of hormone synthesis and signaling genes mediated by MaMADS2 especially in preclimacteric pulp. Genes encoding ethylene biosynthesis enzymes had lower expression in the pulp of the repressed lines, consistent with reduced ethylene production. Generally, the expression of other hormone (auxin, gibberellins, abscisic acid, jasmonic acid and salicylic acid) response pathway genes were down regulated in the WT pulp prior to ripening, but remained high in MaMADS2 repressed lines. Hormone levels of abscisic acid were also higher, however, active gibberellin levels were lower and auxin levels were similar with MaMADS2 repression as compared to WT. Although abscisic level was higher in MaMADS2 repression, exogenous abscisic acid shortened the time to ethylene production and increased MaMADS2 mRNA accumulation in WT. Exogenous ethylene did not influence abscisic acid level. CRE - a cytokinin receptor, increased its expression during maturation in WT and was lower especially at prebreaker in the repressed line and zeatin level was lower at mature green of the repressed line in comparison to WT. In addition to previously reported effects of MaMADS2 on ethylene, this transcription factor also influences other plant hormones, particularly at the pre-climacteric stage. The cytokinin pathway may play a previously unanticipated role via MaMADS2 in banana ripening. Finally, abscisic acid enhances MaMADS2 expression to promote ripening, but the transcription factor in turn auto inhibits ABA synthesis and signaling. Together, these results demonstrate a complex interaction of plant hormones and banana fruit ripening mediated by MaMADS2.

Evaluation of the antioxidant potential of in vitro propagated hyssop (Hyssopus officinalis L.) with different plant growth regulators

Abstract: An efficient method for the micro propagation of hyssop (Hyssopus officinalis L.) has been developed. The plants were cultured on Murashige and Skoog's medium supplemented with 6-benzyl-aminopurine, thidiazuron, zeatin and indole-3butyric acid after four weeks of culture. Maximum numbers of shoots, shoot length, fresh weight and dry weight were established at 1.0 mg/L 6-benzyl-aminopurine and 0.1 mg/L indole-3-butyric acid. For induction of root, uniform micro shoots were excised and transferred to the rooting medium (half strength Murashige and Skoog medium macro and microelements) supplemented with 0.1 mg/L indole-3-butyric acid and 20 g/L sucrose. Indole-3-butyric acid increased culture rooting, number of roots and root length more efficiently at 0.1 mg/L after four weeks of culture. The multiple plants were successfully ex vitro adapted with 90% survival. The described protocol allows the establishment of numerous micro propagated plants of H. officinalis. The used growth regulators for hyssop micro propagation influenced plant antioxidant system. Antioxidant defence of micropropagated H. officinalis determined by the activities of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and guaiacol peroxidase) resulted in higher shoot formation and increasing of shoot number per explant.

Multiple shoot bud induction and plant regeneration studies of Pongamia pinnata.

Abstract: Pongamia pinnata is a legume plant which has great potential to be used as a biofuel feedstock. Conventional propagation of P. pinnata was found to be inefficient for mass propagation. Employing plant tissue culture techniques for micropropagation and further plant improvement of P. pinnata will be the right path to fulfill future challenges in biofuel production. This study aimed to establish a plant regeneration system for potential micropropagation and genetic manipulation of P. pinnata in future. In vitro nodal explants were used and Woody Plant Medium (WPM) containing 30 µM 6-benzylaminopurine (BAP) and 1 mM phloroglucinol (PG) was able to induce higher frequency of multiple shoot buds compared to other media investigated in this study. For shoot regeneration study, WPM containing 15 µM of zeatin and 1 mM PG was able to induce longer shoots while rooting of the regenerated shoots was enhanced by WPM supplemented with indole-3-butyric acid (IBA) in combination with silver thiosulphate (STS). A simple and effective acclimatisation protocol was established with very high survival frequency of regenerated plantlets. Root nodulation of the successfully acclimatised plants was also observed. In short, multiple shoot buds were successfully induced, regenerated and rooted in vitro. The rooted plantlets were successfully acclimatised and grown healthily. It was concluded that a successful plant regeneration protocol of P. pinnata was achieved for potential application in micropropagation and genetic manipulation.

Micropropagation of cranberry (Vaccinium macrocarpon) through shoot tip cultures - short communication.

Abstract: Sedlak J, Paprstein F, 2011 Micropropagation of cranberry (Vaccinium macrocarpon) through shoot tip cultures – Short communication Hort Sci (Prague), 38: 159–162 The goal of this study was to determine an efficient micropropagation system for cranberry (Vaccinium macrocarpon Ait) Cranberry cultivar Howes was successfully established in vitro using mercuric chloride in a concentration of 015% as a sterilization solution Anderson’s rhododendron medium (AN), half-strength Murashige and Skoog medium (halfMS) and McCown woody plant medium (WPM) containing the cytokinin zeatin in concentrations 05, 1 or 2 mg/l were tested Generally, the highest multiplication rate (27) was obtained for cv Howes on AN medium with the concentration 1 mg/l of zeatin The effects of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and naphtalen acetic acid (NAA) on root induction were tested in WPM medium The percentage of rooting was 100% for NAA, 86% for IBA and 81% for IAA From the tested variants, auxin NAA also promoted the highest development of good quality roots (more than 5) per shoot without callus formation

Application of a System for Targeting Fe-Dependent Superoxide Dismutase and Choline Oxidase Enzymes to Chloroplast as a Strategy for Effective Plant Resistance to Abiotic Stresses

Abstract: The plastids involved in photosynthesis and other essential metabolic processes are the cellular compartments most susceptible to abiotic stress factors with adverse effects in plants. In order to protect the chloroplast compartment, the plants of the Belyi Naliv tomato cultivar and the Darenka potato cultivar were transformed with genes for Fe-dependent superoxide dismutase and choline oxidase with the use of the method for plant genetic engineering. These target genes were equipped with signal sequences directing the proteins into the plastid compartment. The experimental results have demonstrated the optimization of the agrobacterium-mediated transformation method for the analyzed tomato and potato cultivars. The naturally growing leaves and stems of the mature plants proved to be the most effective explants to obtain the regenerants. The highest regeneration frequency in the explants was reached with the use of a medium containing 5 mg L–1 BAP, 0.1 mg L–1 NAA, and 1 mg L–1 zeatin. Augmentin and timentin were equally efficient among the antibiotics tested for the inhibition of the agrobacterium; the concentration of 200 mg L–1 in the culture medium was sufficient to eliminate the agrobacterium. In addition, they had no inhibitory effects on the shoot regeneration process. However, the cefotaxime concentration required to inhibit the agrobacterium should be 500 mg L–1 and higher. Moreover, this antibiotic at the concentration of 100 mg L–1 was proven to inhibit the regenerant growth.

Germination Studies of Critically Endangered Medicinal Angiosperm Plant Species Meconopsis Aculeta Royle Endemic to Kashmir Himalaya, India: A Multipurpose Species

Abstract: The critically endangered and perennial alpine endemic angiosperm, Meconopsis aculeta Royle inhabit such habitats in the Kashmir Himalaya that are characterized by short growing season and heavy snow cover for about 3-4 months during winter season. The seeds of this species under natural conditions experience a long period of pre-chilling during winter prior to their germination in following spring season. Taking cue from such a requirement, present study investigated the effect of exogenous application of growth hormones (BAP, NAA Zeatin, Kinetin and TDZ) on germination percentage of deep-dormant seeds of Meconopsis aculeta, under alternate light/dark regimes. Treatment of seeds with different doses of hormonal combinations had a pronounced stimulatory effect on the total germination percentage. In fact, highest germination percentage (78%) was recorded on MS basal supplemented with combinations of Zeatin (4 mg/l) and NAA (0.5 mg/l) within 30 days. The seeds cultured on MS basal medium has no significant influence on germination percentage in Meconopsis aculeta. Among various treatments highest in vitro shoot regeneration was achieved on MS medium supplemented with combination of Zeatin (2 mg/l) and IAA (0.1 mg/l) with 3.41 ± 1.00 cm mean length of shoots with a culture response of 40% within 28 days. In vitro raised shoots regenerated roots on MS medium supplemented with combination of Zeatin (2 mg/l) and IAA (0.1 mg/l) with 14.65 ± 1.23 mean number of roots and 2.76 ± 1.21 cm mean length of roots within 42 days.