Chalcone synthesis and hydroxylation of flavonoids in 3′-position with enzyme preparations from flowers of Dianthus caryophyllus L. (carnation)

Chalcone synthase activity was demonstrated in enzyme preparations from flowers of defined genotypes of Dianthus caryophyllus L. (carnation). In the absence of chalcone isomerase activity, which could be completely excluded by genetic methods, the first product formed from malonyl-CoA and 4-coumaroyl-CoA proved to be naringenin chalcone, followed by formation of naringenin as a result of chemical cyclization. In the presence of chalcone isomerase activity, however, naringenin was the only product of the synthase reaction. In vitro, both 4-coumaryl-CoA and caffeoyl-CoA were found to be used as substrates for the condensation reaction with respective pH optima of 8.0 and 7.0. The results of chemogenetic and enzymatic studies, however, showed that in vivo only 4-coumaroyl-CoA serves as substrate for the formation of the flavonoid skeleton. In confirmation of these results, an NADPH-dependent microsomal 3-hydroxylase activity could be demonstrated, catalyzing hydroxylation of naringenin and dihydrokaempferol in 3-position. Furthermore, a strict correlation was found between 3-hydroxylase activity and the gene r which is known to control the formation of 3, 4-hydroxylated flavonoid compounds.     

Is a cysteine proteinase inhibitor involved in the regulation of petal wilting in senescing carnation (Dianthus caryophyllus L.) flowers?

Senescence of carnation petals is accompanied by autocatalytic ethylene production and wilting of the petals; the former is caused by the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase genes and the latter is related to the expression of a cysteine proteinase (CPase) gene. CPase is probably responsible for the degradation of proteins, leading to the decomposition of cell components and resultant cell death during the senescence of petals. The carnation plant also has a gene for the CPase inhibitor (DC-CPIn) that is expressed abundantly in petals at the full opening stage of flowers. In the present study, DC-CPIn cDNA was cloned and expressed in E. coli. The recombinant DC-CPIn protein completely inhibited the activities of a proteinase (CPase) extracted from carnation petals and papain. Northern blot analysis showed that the mRNA for CPase (DC-CP1) accumulated in large amounts, whereas that for DC-CPIn disappeared, corresponding to the onset of petal wilting in flowers undergoing natural senescence and exogenous ethylene-induced senescence. Based on these findings, a role of DC-CPIn in the regulation of petal wilting is suggested; DC-CPIn acts as a suppressor of petal wilting, which probably functions to fine-tune petal wilting in contrast to coarse tuning, the up-regulation of CPase activity by gene expression.     

Cryopreservation of in vitro-grown shoot tips of 'Troyer' citrange [Poncirus trifoliata (L.) Raf. × Citrus sinensis (L.) Osbeck.] by encapsulation-dehydration

. In vitro-grown shoot tips excised from preconditioned stock shoots of 'Troyer' citrange were successfully cryopreserved by encapsulation-dehydration. Optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 17.1% water content. The sucrose concentration in the preconditioning medium significantly influenced the growth and dry matter percentage of the stock shoots as well as subsequent survival of the cryopreserved shoot tips. Maximal growth of stock shoots was obtained in sucrose concentrations in the range of 0.15 M to 0.29 M, while the dry matter percentage increased as sucrose concentration increased up to 0.44 M. The survival of cryopreserved shoot tips increased from 40% to approximately 80% as the sucrose concentration for stock shoots increased from 0.09 M to 0.22 M or 0.29 M. The benzyladenine concentration in the post-culture medium significantly affected the survival and regrowth of the cryopreserved shoot tips. Survival of the shoot tips was lowest when they were post-cultured on benzyladenine-free medium. However, high benzyladenine concentrations (3-4 µM) induced callus formation. Optimal recovery was obtained in post-culture medium containing 2 µM benzyladenine and 0.05 µM !-naphthalene acetic acid. The extraction of shoot tips from alginate beads greatly improved the regrowth of cryopreserved shoot tips.     

Cryopreservation of in vitro-grown shoot tips of raspberry (Rubus idaeus L.) by encapsulation–vitrification and encapsulation–dehydration

The first efficient cryopreservation procedure for in vitro-grown shoot tips of raspberry (Rubus idaeus L.) has been developed based on encapsulation–vitrification (EnVi) and encapsulation–dehydration (EnDe). EnVi resulted in higher survival (85%) and regrowth (75%) of cryopreserved shoot tips than EnDe (65 and 50%, respectively). In both cryogenic procedures, shoots regenerated from cryopreserved shoot tips without intermediary callus formation. Histological studies showed that a much larger number of meristematic cells survived following EnVi than EnDe. The EnVi procedure was applied to seven raspberry genotypes with an average survival and regrowth of 71 and 68%, respectively. Regenerated plants showed normal morphology. Results here indicate EnVi as a simple and efficient method for long-term preservation of R. idaeus germplasm.     

Induction of direct shoot organogenesis and in vitro flowering from shoot tip explants of cucumber (Cucumis sativus L. cv. ‘Green long’)

In vitro flowering is an alternative breeding tool for generating hybrid Cucumis spp. as it is able to overcome limitations caused by interspecific incompatibility. The present study describes an efficient method for induction of multiple shoots and in vitro flowering from shoot tip explants of cucumber (Cucumis sativus L.). Shoot tip explants were excised from 7-day-old seedlings and cultured on Murashige and Skoog (MS) medium fortified with different concentrations of 6-benzylaminopurine (BAP; 0.5–2.5 mg/L) alone or in combination with 0.5 mg/L kinetin (KIN). The highest frequency (93.1%) of multiple shoot formation with maximum number of shoots (15.2 shoots/explant) was achieved on MS medium supplemented with 1.0 mg/L BAP. For in vitro flowering, shoots were cultured on MS medium supplemented with 0.5 mg/L BAP and different concentrations of sucrose. Flowering occurred on about 95% of in vitro shoots cultured on MS medium fortified with 6% (w/v) sucrose and 0.5 mg/L BAP after 15 d. For rooting, shoots (>2 cm) were cultured on MS medium augmented with various concentrations of indole-3-butyric acid (IBA; 0.5–2.5 mg/L) alone or in combination with 0.5 mg/L KIN. Among the combinations tested, supplementation with IBA (1.5 mg/L) and KIN (0.5 mg/L) induced maximum rooting rates (95.4%) with 7.8 roots/shoot. Rooted plantlets were successfully transferred into plastic cups containing a mixture of soil and sand (1:1), established in the greenhouse, and subsequently acclimatized in the field. The in vitro flowering reported in this study may facilitate rapid hybridization in Cucumis species and offers a model system for studying the physiological mechanisms involved in flowering.     

Virus infection reduces shoot proliferation of in vitro stock cultures and ability of cryopreserved shoot tips to regenerate into normal shoots in ‘Gala’ apple (Malus × domestica)

Plant cryopreservation has provide secure back-ups of germplasm collections of vegetatively propagated crops. Often, recovery levels vary among laboratories when the same cryogenic procedures are used for the same genotypes. The present study investigated the effects of Apple stem grooving virus (ASGV) on shoot proliferation of in vitro stock cultures and recovery of cryopreserved shoot tips of ‘Gala’ apple. Results showed that virus infection reduced shoot proliferation of in vitro stock cultures and cell ability to regenerate normal shoots in cryopreserved shoot tips. Virus infection increased total soluble protein, total soluble sugar and free proline levels and altered endogenous levels of indoleacetic acid (IAA) and zeatin riboside (ZR), but induced severe cell membrane damage and caused alternation in mitochondria shape of the in vitro stock shoots. The altered levels of IAA and ZR were most likely to be responsible for the reduced shoot proliferation of in vitro stock culture. Cell damage and alternations in mitochondria shape in ASGV-infected shoot tips were most likely responsible for the reduced cell ability to regenerate normal shoots following cryopreservation. To the best of our knowledge, this is the first study on effects of virus infection on recovery of cryopreserved shoot tips. Results reported here emphasize that healthy in vitro stock cultures should be used for cryopreservation.     

Biosynthesis of S-associated proteins following self- and cross-pollinations in Brassica campestris L. var. ‘T. 15’

Summary Homozygote plants for the (S) self-incompatibility gene have been produced in Brassica campestris L. var. T 15. Stigmas from plants designated S ₁ S ₁, S ₂ S ₂ and S ₄ S ₄ were extracted and their protein separated on an isoelectric focusing mini-gel. Differences were observed between proteins from stigmas of the three S-homozygous groups: S-genotype specific proteins were determined for S ₁ S ₁ and S ₂ S ₂ stigmas that were absent in the self-compatible S ₄ S ₄ stigmas. Carbon dioxide (CO₂), which is known to block the self-incompatibility reaction in Brassica, was applied to [³⁵S]-methionine unpollinated, self- and cross-pollinated stigmas to observe the effect of external CO₂ on the synthesis of these S-associated proteins. The results indicate that pollination triggers a dramatic reduction in protein synthesis in general and in the synthesis of S₂-associated protein after self-pollination in particular.     

Combining ability and maternal effects in Brassica campestris L. var. ‘yellow sarson’

Summary A diallel analysis of combining ability, including maternal effects, genotype X environment interaction and the progress under selection, is reported in three selected crosses of Brassica campestris L. var. yellow sarson, involving 15 types, including 10 four-valved and 5 two-valved types from different parts of India. Twelve characters, including oil content, were studied in the f₁ generation.The investigation has revealed only marginal superiority of f₁'s over the parents for most of the characters related to yield. There was no relation between heterozygosity and stability of performance over environments for yield or its components or for oil content. Substantial maternal effects were observed which also interacted with environments. Creation of variation for primary and secondary branches would be essential for changing yield level in yellow sarson. The presence of limited additive variation available for selection for yield components should be augmented by biparental mating the early segregating generations to break linkages, and was demonstrated by the recombinants obtained when this method was adopted.The magnitude of genotype — environment interactions in this study, as compared with the total genetic components for yield, oil content, number of siliquae on main axis and presence of large reciprocal variances in relation to general and specific combining ability variances for practically all characters, and the large interaction of , narrowed down the expected effectiveness of selection.Biparental mating in the three best crosses yielded three new recombinants outyielding the best check T 10 by the margins of 14%, 39% and 15%, respectively, in the yield trial. These recombinants had more primary branches and secondary branches, larger main axes and more siliquae with an increased number of seeds per siliqua, than any of the F₁'s in this study.     

Histocytological analysis of yam (Dioscorea alata) shoot tips cryopreserved by encapsulation–dehydration.

In this work, we performed qualitative and quantitative observations of the cytological changes occurring in cells of yam (Dioscorea alata) in vitro shoot tips cryopreserved using the encapsulation–dehydration (E-D) technique. Shoot tip osmoprotection for 24 h in 1.25 M sucrose medium induced drastic changes in cellular cytological features, including high plasmolysis in all three cellular areas studied, the external cell layer (L1), one to three (L1–3) and seven to nine (L7–9) cell layers from the surface of the meristematic dome, pyknotic nuclei in meristematic area cells and disappearance of nucleoli. Nucleus size decreased significantly in all cellular areas studied. Nucleocytoplasmic ratio decreased significantly in L1–3 and L7–9 cells. Nuclear protein content increased, particularly in L1 and L1–3 cells. After physical dehydration, plasma membrane of numerous basal part cells was broken and intracellular soluble protein leakage was observed. Nucleus area and nucleocytoplasmic ratio decreased significantly in L7–9 cells. One week after cryopreservation, shoot tips showed regrowth and living cells had recovered their original morphology. In all cellular areas studied, nuclei had retrieved their original staining and nucleoli were visible. Original nucleus area values were recovered in L1–3 and L1 cells. The nucleocytoplasmic ratio retrieved its initial value in L1 cells but remained at levels observed after osmoprotection for L1–3 and L7–9 cells. The nuclear protein content had retrieved its original level. This investigation provided new insights in changes occurring in D. alata apices throughout an E-D protocol.     

Influence of cold pretreatment on shoot regeneration from callus in date palm (Phoenix dactylifera L.) cv. ‘Barhee’

Mass propagation of date palm through indirect somatic embryogenesis or organogenesis has attracted the interest of commercial producers. But, this technique still faces some problems that hindered the production of date palm plantlets in vitro. Tissue browning is one of the serious problems that reduce callus growth and shoot regeneration. So the objective of the present study is to investigate the effect of cold pretreatment on callus growth, shoot regeneration, and polyphenol oxidase (PPO) activity during the callus culture. Results showed that a high survival rate of callus cultures (100%) were obtained when cultures were incubated in low temperature (cold treatment) for 45 and 75?days. On the other hand, total amount on phenolic compounds was also reduced to 0.47 and 0.53?mg GAE/g after same period of incubation (45 and 75?days respectively) at low temperature. In additional, our results showed that the highest frequency of shoot formation (66.67 and 73.34, %) and the highest shoot numbers (7.8 and 8.6 shoots/100?mg) were obtained from callus treated with low temperature for 45 and 75?days, respectively.     

Effects of 4-CPPU on in vitro multiplication and sustainable generation of Hibiscus rosa-sinensis L. ‘White Butterfly’

There are more than nine thousand cultivars of Hibiscus rosa-sinensis L., with a series of flowers with shapes, colors and new cultivars continues as generated through both traditional and modern breeding techniques. In this study, advanced biotech methods of in vitro culture have been used to identify a technique for the efficient mass multiplication of H. rosa-sinensis ‘White Butterfly’, using phenyl urea, N-(2-Chloro-4-pyridyl)-N′-phenylurea (4-CPPU). For the first time, the effects of 4-CPPU for stimulating axillary shoot proliferation and multiple shoot regenerations from nodal explants were evaluated, and the optimal nutrient media deduced. From the diverse concentrations as 0.1, 0.5, 2.5, 5.0 & 10.0 µM of 4-CPPU, the highest frequency of shoots was recorded at 2.5 µM supplied in Murashige and Skoog (MS, pH-5.8) medium. After eight-weeks of culture, on an average of 6.7 shoot were obtained on this media with shoot heights of 4.2 cm from each explant. With the involvement of 0.5 µM-IBA (indole-3-butyric acid) in MS medium the regenerated shoots were rooted and followed by successful acclimation to ex vitro conditions. The ploidy consistency among the micro-plants was analyzed using flow cytometry and compared with ex vitro grown plants. No differences in the ploidy levels were observed among the 4-CPPU induced plants, when compared with the donor plants.     

Culture-independent analysis of an endophytic core microbiome in two species of wheat: Triticum aestivum L. (cv. ‘Hondia’) and the first report of microbiota in Triticum spelta L. (cv. ‘Rokosz’)

The main goal of the study was to determine the structure of endophytic bacteria inhabiting different parts (endosperm, germ, roots, coleoptiles, and leaves) of two wheat species, Triticum aestivum L. (cv. ‘Hondia’) and Triticum spelta L. (cv. ‘Rokosz’), in order to provide new knowledge about the stability and/or changeability of the core microbiome in different plant organs. The endophytic core microbiome is associated with plants throughout their whole life cycle; however, plant organs can determine the actual endophytic community. Therefore, next generation sequencing with MiSeq Illumina technology was applied to identify the endophytic microbiome of T. aestivum and T. spelta. Bioinformatic analyses were performed with the use of the DADA2(1.8) package and R software (3.5.1).It was demonstrated that wheat, which is an important crop plant, was associated with beneficial endophytic bacteria inside the endosperms, germs, roots, leaves, and coleoptiles. Importantly, for the first time, biodiversity was recognized in the coleoptiles of the investigated wheat species. Flavobacterium, Pseudomonas and Janthinobacterium were shown to be common genera for both tested wheat cultivars. Among them, Pseudomonas was found to be the only endophytic genus accompanying both wheat species from the endosperm stage to the development of the leaf. Paenibacillus was recognized as a core genus for the ‘Hondia’ cv., whereas Pedobacter and Duganella constituted the core microbiome in the ‘Rokosz’ cv. In addition, the first insight into the unique and yet unrecognized endophytic microbiome of T. spelta is presented.     

Properties of a Trypsin Inhibitor from Job’s-tears (Coix lacryma-jobi L. var. Ma-yuen Stapf)

A heat stable trypsin inhibitor was found in the bran of soft-shelled job’s-tears (Coix lacryma-jobi L. var. Ma-yuen Stapf) seeds. This inhibitor seemed to be a simple protein, and the molecular weight was about 12,000. Similar to other heat stable trypsin inhibitors, this inhibitor also contained many cysteine or cystine residues in the molecule. This inhibitor inhibited bovine trypsin at the molar ratio of 1 to 2, showing that it was double-headed. Its activity was stable against the change of pH at the range of 3 to 11 and high temperature of 100°C under certain conditions. However, the degree of heat stability of the inhibitory activity depended highly upon the kind of the solution in which this inhibitor was dissolved.     

Shoot regeneration and cryopreservation of shoot tips of apple (Malus) by encapsulation–dehydration

Here, we report an efficient and widely applicable method for cryopreservation of Malus shoot tips by encapsulation–dehydration using adventitious shoots. Shoots were induced from leaf segments cultured on a shoot induction medium containing 2–3 mg L^?1 thidiazuron, depending on genotype, and 0.5 mg L^?1 indole-3-butyric acid. Shoot tips (3 mm in length) containing six leaf primordia excised from 11-wk-old adventitious shoots were encapsulated and precultured with 0.5 M sucrose for 5 d, followed by air-drying for 6 h prior to direct immersion in liquid nitrogen. With our protocol, we obtained a mean organogenesis rate of 100%, a mean of 4.5 adventitious shoots per explant (leaf segment), and a mean shoot recovery of 57.0% from cryopreserved shoot tips in four Malus species. Inter-simple sequence repeat (ISSR) analysis did not reveal any polymorphic bands in regenerants recovered from either leaf segments or cryopreserved shoot tips of ‘Gala’. To the best of our knowledge, this is the first report on cryopreservation of Malus shoot tips using adventitious shoots derived from leaf segments and is the most widely applicable protocol so far reported for cryopreservation of Malus. Establishment of this protocol provides an alternative means for cryopreservation of Malus.     

Potential role of cytokinin–auxin synergism, antioxidant enzymes activities and appraisal of genetic stability in Dianthus caryophyllus L.—an important cut flower crop

The regeneration potential, antioxidative enzyme activities, and genetic stability among micropropagated plantlets of Dianthus caryophyllus L. were evaluated. Multiple adventitious shoots were induced from leaf explants on Murashige and Skoog medium incorporated with various combinations and concentrations of plant growth regulators (PGRs). The highest leaf explant response (90%), number of shoots per explant (15.30?±?1.19), and shoot length (6.75?±?0.63 cm) was recorded in response to a combination of 2.5 μM 6-benzyladenine and 0.5 μM α-naphthaleneacetic acid (NAA) after 8 wks culture. Subsequent subculturing for five passages, on a medium with the same composition of PGRs, induced the highest shoot number (42.50?±?1.44), with an average shoot length of 8.06 cm after the fourth subculture. Different concentrations of indole-3-butyric acid (IBA) were tested to determine the optimum conditions for ex vitro rooting of microshoots. The best result was accomplished with a pulse treatment of IBA (100 μM) applied to the basal end of the microshoot for 30 min, followed by transfer to plastic cups containing soilrite, and eventually established in natural soil with an 85% survival rate. The determination of activities of antioxidative enzymes (superoxide dismutase, ascorbate peroxidase, catalase, and glutathione reductase) revealed involvement of these enzymes in shoot differentiation and development. All of these activities were interlinked with each other and played significant roles in the scavenging of toxic free radicals. Intersimple sequence repeat DNA analysis was carried out using five primers. The amplification products were monomorphic in micropropagated plants, similar to those of the mother plant. No polymorphisms were detected revealing the genetic integrity of the micropropagated plants.     

Measurement of selected trace elements in Olea europaea L. cv. ‘Sigoise’

BackgroundOlive-trees (Olea europaea L.) are the dominant rustic trees cultivated in the Mediterranean agricultural zones. Major and micronutrients play an indispensable role in their plant physiological functions although; the effect of trace elements on metabolic processes has not been sufficiently investigated, especially in olive-trees.MethodsIn the current study, we have used X-ray fluorescence (XRF) spectrometry to determine selected major and trace elements (Br, Cu, Fe, K, Mn, P, Rb and Zn) in the main olive cultivar cultivated in Algeria, cv.‘Sigoise’. Certified reference materials viz. IAEA-336 (Lichen) and NIST-1646a (Estuarine sediment) were evaluated simultaneously with the soil and plant samples for quality control of the analytical method.ResultsThe results show that Fe and Mn concentrations were superior in leaves than fruits. However large amounts of K, Cu and Rb were accumulated in the olive-fruits. The contents of all chemical elements were above the threshold limits for possible plant nutrient deficiencies, except for P whose concentration was in borderline requirement of olive trees. High values of a translocation factor index were found for K, Cu and Rb (TFs > 4). Principal component analysis (PCA) indicated that K was highly related with olives-fruits, suggesting that the fruit was the principal organ of K storage. Furthermore, dietary element intake through consuming olives was also estimated and compared to recommended daily intakes (RDIs) and daily permissible limits (DPLs). The estimations of chemical element intakes were below the DPLs set by WHO/FAO guidelines for human nutrition.ConclusionThe present work indicates that the concentrations of macro- and microelements (Cu, Fe, K, Mn and Zn) were above the threshold limits for possible plant deficiencies except for P, and this cultivar can easily accumulate high amount of K in their organs (predominance in olives). These findings will be used to achieve efficient fertilization for O. europaea orchards.     

Culturalizing politics,hyper-politicizing ‘culture’: ‘White’ vs. ‘Black Turks’ and the making of authoritarian populism in Turkey

This paper focuses on the uses and abuses of two terms, ‘White’ and ‘Black Turk’, which have been significant in the ways modern Turkish society and national identity have been defined and contested in recent decades. Initially emerging in social analysis in the 1990s, ‘White Turk’ was a metaphor for and critique of the class culture, subjectivity and worldviews of the ‘new middle classes’ in a period of rapid integration to neoliberalism and globalized capitalism. Over time, both White and Black Turks have come to be used as part of a politics of identity and a politics of authenticity to characterize who are seen as the ‘authentic self’ and inauthentic others of national identity and to assert different visions for the future of Turkish society. White Turk has been adopted as an identity by outspoken members of the media and business elite, whereas its binary opposite, Black Turk, has been appropriated by Islamist politicians of the Justice and Development Party (AKP) as a metaphor to characterize the marginalization and purported oppression of their conservative Muslim constituency. As White and Black Turks were adopted as self-proclaimed identities, they provided a basis for a culturalist depiction of Turkish society, contributing over time to an increasingly divisive politics. Even though the AKP initially used the reference to White and Black Turks to appeal to specific demands for inclusion, as it increased its grip on power, it also (hyper)politicized the terms to articulate nativist claims to authenticity. In recent years, this nativist populism has been used to justify increasing authoritarianism and to delegitimize belonging and political participation of those deemed inauthentic others of the body politics.     

Expression of Rice (Oryza sativa L. var. Nipponbare) α-Galactosidase Genes in Escherichia coli and Characterization

Two putative α-galactosidase genes from rice (Oryza sativa L. var. Nipponbare) belonging to glycoside hydrolase family 27 were cloned and expressed in Escherichia coli. These enzymes showed α-galactosidase activity and were purified by Ni Sepharose column chromatography. Two purified recombinant α-galactosidases (α-galactosidase II and III; α-Gal II and III) showed a single protein band on SDS–PAGE with molecular mass of 42 kDa. These two enzymes cleaved not only α-D-galactosyl residues from the non-reducing end of substrates such as melibiose, raffinose, and stachyose, but also liberated the galactosyl residues attached to the O-6 position of the mannosyl residue at the reducing-ends of mannobiose and mannotriose. In addition, these enzymes clipped the galactosyl residues attached to the inner-mannosyl residues of mannopentaose. Thus, α-Gal II catalyzes efficient degalactosylation of galactomannans, such as guar gum and locust bean gum.