Enzymatic Activity and Its Relationships with the Total Phenolic Content and Color Change in the High Hydrostatic Pressure-Assisted Curing of Vanilla Bean (Vanilla planifolia).

Diverse enzymatic reactions taking place after the killing of green vanilla beans are involved in the flavor and color development of the cured beans. The effects of high hydrostatic pressure (HHP) at 50-400 MPa/5 min and blanching as vanilla killing methods were evaluated on the total phenolic content (TPC), polyphenoloxidase (PPO), and peroxidase (POD) activity and the color change at different curing cycles of sweating-drying (C0-C20) of vanilla beans. The rate constants describing the above parameters during the curing cycles were also obtained. The TPC increased from C1 to C6 compared with the untreated green beans after which it started to decrease. The 400 MPa samples showed the highest rate of phenolic increase. Immediately after the killing (C0), the highest increase in PPO activity was observed at 50 MPa (46%), whereas for POD it was at 400 MPa (25%). Both enzymes showed the maximum activity at C1, after which the activity started to decrease. As expected, the L* color parameter decreased during the entire curing for all treatments. An inverse relationship between the rate of TPC decrease and enzymatic activity loss was found, but the relationship with L* was unclear. HHP appears to be an alternative vanilla killing method; nevertheless, more studies are needed to establish its clear advantages over blanching.

MicroRNA528, a hub regulator modulating ROS homeostasis via targeting of a diverse set of genes encoding copper-containing proteins in monocots.

Plant microRNAs (miRNAs) regulate vital cellular processes, including responses to extreme temperatures with which reactive oxygen species (ROS) are often closely associated. In the present study, it was found that aberrant temperatures caused extensive changes in abundance to numerous miRNAs in banana fruit, especially the copper (Cu)-associated miRNAs. Among them, miR528 was significantly downregulated under cold stress and it was found to target genes encoding polyphenol oxidase (PPO), different from those identified in rice and maize. Expression of PPO genes was upregulated by > 100-fold in cold conditions, leading to ROS surge and subsequent peel browning of banana fruit. Extensive comparative genomic analyses revealed that the monocot-specific miR528 can potentially target a large collection of genes encoding Cu-containing proteins. Most of them are actively involved in cellular ROS metabolism, including not only ROS generating oxidases, but also ROS scavenging enzymes. It also was demonstrated that miR528 has evolved a distinct preference of target genes in different monocots, with its target site varying in position among/within gene families, implying a highly dynamic process of target gene diversification. Its broad capacity to target genes encoding Cu-containing protein implicates miR528 as a key regulator for modulating the cellular ROS homeostasis in monocots.

Inactivation and structural changes of polyphenol oxidase in quince (Cydonia oblonga Miller) juice subjected to ultrasonic treatment.

BACKGROUND: Polyphenol oxidase (PPO) is considered a problem in the food industry because it starts browning reactions during fruit and vegetable processing. Ultrasonic treatment is a technology used to inactivate the enzyme; however, the mechanism behind PPO inactivation is still unclear. For this reason, the inactivation, aggregation, and structural changes in PPO from quince juice subjected to ultrasonic treatments were investigated. Different intensities and times of ultrasonic treatment were used. Changes in the activity, aggregation, conformation, and structure of PPO were investigated through different structural analyses. RESULTS: Compared to untreated juice, the PPO activity in treated juice was reduced to 35% at a high ultrasonic intensity of 400 W for 20 min. The structure of PPO determined from particle size distribution (PSD) analysis showed that ultrasound treatment caused initial dissociation and subsequent aggregation leading to structural modification. The spectra of circular dichroism (CD) analysis of ultrasonic treated PPO protein showed a significant loss of α-helix, and reorganization of secondary structure. Fluorescence analysis showed a significant increase in fluorescence intensity of PPO after ultrasound treatment with evident blue shift, revealing disruption in the tertiary structure. CONCLUSION: In summary, ultrasonic treatment triggered protein aggregation, distortion of tertiary structure, and loss of α-helix conformation of secondary structure causing inactivation of the PPO enzyme. Hence, ultrasound processing at high intensity and duration could cause the inactivation of the PPO enzyme by inducing aggregation and structural modifications. © 2019 Society of Chemical Industry.

Effect of adsorbent and acidulants on enzymatic browning of sugarcane juice.

Enzymatic browning is a major factor affecting the quality of sugarcane juice, mainly due to the activities of polyphenol oxidase (PPO) and peroxidase (POD). Effect of bentonite (0-1%, w/v) on the activities of these enzymes, when employed alone and also in combination with acidulants, was determined. Bentonite alone could reduce the activities of PPO and POD enzymes to 160 and 24.2 u/mL, respectively. The PPO and POD activity was completely inhibited below pH 4.1 when ascorbic acid was used alone or in combination with bentonite. However, PPO and POD activity was inhibited to 60 and 51 u/mL, respectively, at pH 3.7 when citric acid was used individually and to 112 and 15.36 u/mL, respectively, when employed along with bentonite. In addition, color changes at 4 and 10 °C were measured during the storage of sugarcane juice.

Effect of ultrasonic processing on the changes in activity, aggregation and the secondary and tertiary structure of polyphenol oxidase in oriental sweet melon (Cucumis melo var. makuwa Makino).

BACKGROUND: Polyphenol oxidase (PPO) mainly contributes to the browning reaction of fruits and vegetables and causes serious damage to the quality of sweet melon products. However, traditional methods to inactivate browning may induce more unexpected risks than ultrasonic processing. Meanwhile, there are no reports on the effect of ultrasound on PPO directly purified from sweet melon. RESULTS: The PPO in the original juice was less inactivated than the purified form when treated with ultrasound. As for purified PPO, superior to thermal treatment, less heat was needed to inactivate the PPO with ultrasonic treatment. At intensity lower than 200 W, ultrasound did not significantly affect the structure and activity of PPO (P > 0.05), and latent PPO was activated. At intensity higher than 200 W, ultrasound inactivated PPO, induced aggregation and dissociation of PPO particles and significantly decreased the α-helix structure content. CONCLUSION: Low-frequency high-intensity ultrasound caused an inactivation effect and conformational changes of purified PPO from oriental sweet melons. Changes in the PPO structure induced by ultrasound eventually inactivated the enzyme. Ultrasound may be a potential method to inactivate PPO in oriental sweet melons. © 2016 Society of Chemical Industry.

The effect of ultraviolet treatment on enzymatic activity and total phenolic content of minimally processed potato slices.

In this work, potato slices were exposed to different doses of UV-C irradiation (i.e. 2.28, 6.84, 11.41, and 13.68 kJ m-2) with or without pretreatment [i.e. ascorbic acid and calcium chloride (AACCl) dip] and stored at 4 ± 1 °C. Changes in enzymatic activities of polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia lyase (PAL), as well as total phenolic content (TPC) were investigated after 0, 3, 7 and 10 days of storage. Results showed that untreated and UV-C treated potato slices at 13.68 kJ m-2 dosage level showed significantly higher PPO, POD and PAL activities. Conversely, untreated potato slices showed the lowest TPC during storage period. Potato slices subjected to AACCl dip plus UV-C at 6.84 kJ m-2 produced lower PPO, POD and PAL activities, as well as maintained a high TPC during storage.

A PPO Promoter from Betalain-Producing Red Swiss Chard, Directs Petiole- and Root-Preferential Expression of Foreign Gene in Anthocyanins-Producing Plants.

A 1670 bp 5'-flanking region of the polyphenol oxidase (PPO) gene was isolated from red Swiss chard, a betalain-producing plant. This region, named promoter BvcPPOP, and its 5'-truncated versions were fused with the GUS gene and introduced into Arabidopsis, an anthocyanins-producing plant. GUS histochemical staining and quantitative analysis of transgenic plants at the vegetative and reproductive stages showed that BvcPPOP could direct GUS gene expression in vegetative organs with root- and petiole-preference, but not in reproductive organs including inflorescences shoot, inflorescences leaf, flower, pod and seed. This promoter was regulated by developmental stages in its driving strength, but not in expression pattern. It was also regulated by the abiotic stressors tested, positively by salicylic acid (SA) and methyl jasmonate (MeJA) but negatively by abscisic acid (ABA), gibberellin (GA), NaCl and OH(-). Its four 5'-truncated versions varied in the driving strength, but not obviously in expression pattern, and even the shortest version (-225 to +22) retained the root- and petiole- preference. This promoter is, to our knowledge, the first PPO promoter cloned and functionally elucidated from the betalain-producing plant, and thus provides not only a useful tool for expressing gene(s) of agricultural interest in vegetative organs, but also a clue to clarify the function of metabolism-specific PPO in betalain biosynthesis.

Purification and characterization of polyphenol oxidase from waste potato peel by aqueous two-phase extraction.

Potato peel from food industrial waste is a good source of polyphenol oxidase (PPO). This work illustrates the application of an aqueous two-phase system (ATPS) for the extraction and purification of PPO from potato peel. ATPS was composed of polyethylene glycol (PEG) and potassium phosphate buffer. Effect of different process parameters, namely, PEG, potassium phosphate buffer, NaCl concentration, and pH of the system, on partition coefficient, purification factor, and yield of PPO enzyme were evaluated. Response surface methodology (RSM) was utilized as a statistical tool for the optimization of ATPS. Optimized experimental conditions were found to be PEG1500 17.62% (w/w), potassium phosphate buffer 15.11% (w/w), and NaCl 2.08 mM at pH 7. At optimized condition, maximum partition coefficient, purification factor, and yield were found to be 3.7, 4.5, and 77.8%, respectively. After partial purification of PPO from ATPS, further purification was done by gel chromatography where its purity was increased up to 12.6-fold. The purified PPO enzyme was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by Km value 3.3 mM, and Vmax value 3333 U/mL, and enzyme stable ranges for temperature and pH of PPO were determined. These results revealed that ATPS would be an attractive option for obtaining purified PPO from waste potato peel.

Biosynthesis of Piceatannol from Resveratrol in Grapevine Can Be Mediated by Cresolase-Dependent Ortho-Hydroxylation Activity of Polyphenol Oxidase.

Piceatannol is a naturally occurring hydroxylated analogue of the stilbene phytoalexin resveratrol that can be found in grape fruit and derived products. Piceatannol has aroused great interest as it has been shown to surpass some human health-beneficial properties of resveratrol including antioxidant activity, several pharmacological activities and also bioavailability. The plant biosynthetic pathway of piceatannol is still poorly understood, which is a bottleneck for the development of both plant defence and bioproduction strategies. Cell cultures of Vitis vinifera cv. Gamay, when elicited with dimethyl-ß-cyclodextrin (MBCD) and methyl jasmonate (MeJA), lead to large increases in the accumulation of resveratrol, and after 120 h of elicitation, piceatannol is also detected due to the regiospecific hydroxylation of resveratrol. Therefore, an ortho-hydroxylase must participate in the biosynthesis of piceatannol. Herein, three possible types of resveratrol hydroxylation enzymatic reactions have been tested, specifically, a reaction catalyzed by an NADPH-dependent cytochrome, P450 hydroxylase, a 2-oxoglutarate-dependent dioxygenase and ortho-hydroxylation, similar to polyphenol oxidase (PPO) cresolase activity. Compared with P450 hydoxylase and the dioxygenase activities, PPO displayed the highest specific activity detected either in the crude extract, the particulate or the soluble fraction obtained from cell cultures elicited with MBCD and MeJA for 120 h. The overall yield of PPO activity present in the crude extract (107.42 EU) was distributed mostly in the soluble fraction (66.15 EU) rather than in the particulate fraction (3.71 EU). Thus, partial purification of the soluble fraction by precipitation with ammonium sulphate, dialysis and ion exchange chromatography was carried out. The soluble fraction precipitated with 80% ammonium sulphate and the chromatographic fractions also showed high levels of PPO activity, and the presence of the PPO protein was confirmed by Western blot and LC-MS/MS. In addition, a kinetic characterization of the cresolase activity of partially purified PPO was carried out for the resveratrol substrate, including Vmax and Km parameters. The Km value was 118.35 ± 49.84 µM, and the Vmax value was 2.18 ± 0.46 µmol min-1 mg-1.

Biodegradation of organic micropollutants by anoxic denitrification: Roles of extracellular polymeric substance adsorption, enzyme catalysis, and reactive oxygen species oxidation.

The control of organic micropollutants (OMPs) in water environments have received significant attention. Denitrification was reported to exhibit good efficiency to remove OMPs, and the mechanisms involved in are too intricate to be well illustrated. In this study, we selected nitrobenzene [NB] and bisphenol A [BPA] as model pollutants and aimed to unravel the mechanisms of Paracoccus Denitrificans in the removal of OMPs, with a specific emphasis on aerobic behavior during denitrification processes. We demonstrated the formation of extracellular superoxide radicals, i.e., extracellular •O2-, using a chemiluminescence probe and found that extracellular polymeric substance adsorption, extracellular •O2-, and microbial assimilation contributed approximately 40 %, 10 %, and 50 % to OMPs removal, respectively. Transcriptome analysis further revealed the high expression and enrichment of several pathways, such as drug metabolism-other enzymes, of which a typical aerobic enzyme of polyphenol oxidase [PPO] participates in the degradation of NB and BPA. Importantly, all the immediate products showed a significant decrease in toxicity during the aerobic activity-related OMPs degradation process based on the proposed degradation pathways. This study demonstrates the formation of extracellular •O2- and the mechanisms of extracellular •O2-- and PPO-mediated OMPs biodegradation, and offers new insights into OMPs control in widely-used denitrification treatment processes.

Evaluating the efficacy of microbial antagonists in inducing resistance, promoting growth, and providing biological control against powdery mildew in wheat.

This study evaluates the biocontrol efficacy of three bacterial strains (Pseudomonas fluorescens DTPF-3, Bacillus amyloliquefaciens DTBA-11, and Bacillus subtilis DTBS-5) and two fungal strains (Trichoderma harzianum Pusa-5SD and Aspergillus niger An-27) antagonists, along with their combinations at varying doses (5.0, 7.5, and 10.0 g/kg of seeds), against wheat powdery mildew. The most effective dose (10 g/kg seeds) was further analyzed for its impact on induced resistance and plant growth promotion under greenhouse conditions. The study measured defense enzyme activities, biochemical changes, and post-infection plant growth metrics. All tested microbial antagonists at 10 g/kg significantly reduced PM severity, with B. subtilis strain DTBS-5 outperforming others in reducing PM severity and achieving the highest biocontrol efficacy. It was followed by B. amyloliquefaciens strain DTBA-11 and P. fluorescens strain DTPF-3, with the fungal antagonists showing no significant effect. Wheat crops treated with B. subtilis strain DTBS-5 exhibited substantial increases in defense-related enzyme activities and biochemicals, suggesting an induced resistance mechanism. The study found a 45% increase in peroxidase (POD) activity, a 50% increase in catalase (CAT) activity, a 30% increase in phenolic content, and a 25% increase in soluble protein content in the wheat plants treated with microbial antagonists. The study highlights the effectiveness of microbial antagonists, particularly B. subtilis strain DTBS-5, in managing wheat PM through biocontrol, induced resistance, and enhanced plant growth, offering a sustainable alternative to chemical treatments.

Impact of Agronomic Treatments on the Enzymatic Browning of Eggplants (Solanum melongena L.).

Enzymatic browning could negatively affect the sensory and nutritional properties of eggplants post-harvest. Polyphenols, polyphenol oxidase (PPO), and reactive oxygen species (ROS) are three material conditions involved in enzymatic browning. This paper seeks to evaluate the effect of fertilization techniques and grafting on the activity of PPO and colorimetric parameters in cultivated eggplants. Fertilization alone significantly increased the PPO activity in all eggplant fleshes (p ≤ 0.05), whereas the grafting technique combined with fertilization decreased the PPO activity in most of the samples significantly (p ≤ 0.05). Moreover, there was a significant positive correlation between the PPO activity and the a* values of the eggplants. The a* values in grafted eggplants were significantly different from each other (p ≤ 0.05), showing that grafting the fertilized eggplants could be effective in controlling the enzymatic browning. The eggplant slices exposed to air for 60 min at room temperature showed a significant increase (p ≤ 0.05) in PPO activity, browning index (BI), total color difference (ΔE), and a*, b*, and c* values. Thus, it is necessary to minimize the exposure time of the slices to air at room temperature, even if combining fertilization techniques with grafting could delay the enzymatic browning in fresh-cut eggplants.

Comparison of Inhibitory Effects of Cinnamic Acid, ß-Cyclodextrin, L-Cysteine, and Ascorbic Acid on Soluble and Membrane-Bound Polyphenol Oxidase in Peach Fruit.

There has been considerable interest in controlling polyphenol oxidase (PPO) activity to prevent enzymatic browning in foods. However, studies on inhibitions of different forms of PPO are very limited. Thus, this study focuses on the effects of cinnamic acid, ß-cyclodextrin, L-cysteine, and ascorbic acid on soluble PPO (sPPO) and membrane-bound PPO (mPPO) in peach fruit. The activity of partially purified sPPO was 3.17 times higher than that of mPPO. However, mPPO was shown to be more stable than sPPO in the presence of inhibitors with different concentrations (i.e., 1, 3, 5 mM); activation of mPPO was found by 5 mM L-cysteine. Both sPPO and mPPO inhibitions were PPO substrate-dependent. Ascorbic acid showed the highest inhibitory effect on both sPPO and mPPO with all studied inhibitors and substrates. The inhibition of 1 mM ascorbic acid on sPPO and mPPO reached 95.42 ± 0.07% and 65.60 ± 1.16%, respectively. ß-Cyclodextrin had a direct inhibitory effect only on sPPO, while the other three inhibitors had direct effects on both sPPO and mPPO. Cinnamic acid exhibited a non-competitive inhibition on sPPO and mPPO, with L-cysteine showing the same, though on sPPO. The inhibition of studied inhibitors on sPPO and mPPO is highly related to the substrate environment, type, and concentration of inhibitors. This study provides a basis for the further prevention of peach fruit browning from the perspective of different enzyme forms.

Multispectroscopic and computational simulation insights into the inhibition mechanism of epigallocatechin-3-gallate on polyphenol oxidase.

Polyphenol oxidase (PPO)-mediated enzymatic browning occurs in fruit, vegetables and aquatic products and causes huge economic losses every year. In this study, epigallocatechin-3-gallate (EGCG) displayed high affinity for and efficient inhibitory capacity against PPO. To explore the inhibition mechanism, multispectroscopic methods and computational simulations were implemented. Initially, EGCG inhibited PPO activity reversibly in a mixed-type manner. Then, the conformation and secondary structure changes of PPO after binding with EGCG were discovered by fluorescence emission spectra and circular dichroism. Molecular docking and dynamic simulation results revealed that EGCG could tightly bind with the binuclear copper domain of PPO through hydrophobic stacking and hydrogen bonds. Moreover, EGCG might act as a linker to interact with different PPO molecules at another binding site. Transmission electron microscopy observation suggested that EGCG induced the aggregation of PPO. Therefore, the inhibition mechanism of EGCG on PPO included competition for catalytic centers and induced aggregation.

Determination of Post-Harvest Biochemical Composition, Enzymatic Activities, and Oxidative Browning in 14 Apple Cultivars.

Phenolic compounds in fruit provide human health benefits, and they contribute to color, taste, and the preservation of post-harvest fruit quality. Phenolic compounds also serve as modifiers of enzymatic activity, whether inhibition or stimulation. Polyphenol oxidases (PPO) and peroxidases (POD) use phenolic compounds as substrates in oxidative browning. Apple browning leads to flesh color, taste, texture, and flavor degradation, representing a drawback for the variety and its' market appraisal. This study was conducted to investigate the process of browning in 14 apple cultivars throughout post-harvest at three-time points: immediately (T0), one hour (T1), and 24 h (T2) after apples were cut in half. Color parameters L* (lightness), a* (red/green), b* (yellow/blue) were measured, and chroma (ΔC*) and color (ΔE) were calculated to quantify differences between T0₋T1 and T1₋T2 on the fruit surface. Enzymatic activity (PPO, POD) and phenolic composition were also quantified for each cultivar. 'Granny Smith' and 'Cripps Pink' browned minimally. In contrast, 'Fiesta' and 'Mondial Gala' browned severely, reporting high enzymatic activity and quantified phenolic concentration (QPC). Phenolic compound polymerization appears to play a significant role in enzymatic inhibition. 'Topaz' does not fit the high QPC, PPO, and browning formula, suggesting alternative pathways that contribute to apple browning.

Ultrasound-homogenization-assisted extraction of polyphenols from coconut mesocarp: Optimization study.

Coconut pericarp (shell fiber (mesocarp) and shell (endocarp)), the main by-product of coconut production, is often discarded and causing serious environmental pollution. To make better use of coconut pericarp, the extraction process of polyphenols from coconut mesocarp (CM) carefully studied by screening seven solvent systems, optimizing the assisted ultrasonic process by response surface methodology, and comparing the four processes of Ultrasound-Assisted Extraction (UAE), Homogenization-Assisted Extraction (HAE), Homogenization-Ultrasound-Assisted Extraction (HUAE), and Ultrasound-Homogenization-Assisted Extraction (UHAE). The UAE and HAE are considered to be the main methods for efficient extraction of natural active ingredients. The former effectively destroys the cell wall structure and promotes the intermolecular diffusion based on the cavitation, thermal and mechanical effect of ultrasonic, while the latter breaks the material based on strong shear force between the rotor and stator. Their combinations (HUAE and UHAE) enhance the damage to the cell wall of raw materials and improve the extraction efficiency by the synergistic effect. The results showed that using 60% acetone (V : V) as extraction solvent, solid-liquid ratio of 1:5 g mL-1, ultrasonic temperature of 80 â„ƒ, ultrasonic time of 80 min, ultrasonic power of 225 W, and then homogenizing at 10,000 rpm for 10 min, the total flavonoid content of CM reached the maximum value of 551.99 ± 12.69 mg Rutin g-1 dry weight (dw), while the total phenolic content reached the maximum value of 289.48 ± 4.41 mg GAE g-1 dw at 10,000 rpm for 5 min, which may be related to the oxidative degradation of polyphenols caused by the increase of polyphenol oxidase with the extension of homogenization time. This study provides a technical guarantee for the further utilization of phenolic substances in CM.

Inhibitory mechanism of salicylic acid on polyphenol oxidase: A cooperation between acidification and binding effects.

Salicylic acid is generally considered to combine with polyphenol oxidase (PPO) to inhibit activity and enzymatic browning, while its acidification effect on PPO activity was usually neglected. In this study, the inhibitory mechanism of salicylic acid on PPO was examined from acidification and binding effects by altering the buffer conditions. As the buffer concentration increased, contribution of acidification decreased while the binding effect became more predominant. Salicylic acid exhibited competitive inhibition on PPO, inducing the changes in secondary structure with a reduction in α-helix. Molecular docking results showed that salicylic acid interacted with residues HIS61, HIS85, HIS259, HIS263 and VAL283 through hydrogen bond and hydrophobic interaction. Furthermore, acidic pH enhanced the binding of salicylic acid to PPO with lower binding energy, additional hydrogen bond and electrostatic interactions. Therefore, both acidification and binding effects were important for salicylic acid on PPO inhibition and enzymatic browning control in fruit and vegetables.

Determination of Biochemical Composition in Peach (Prunus persica L. Batsch) Accessions Characterized by Different Flesh Color and Textural Typologies.

The rising interest in beneficial health properties of polyphenol compounds in fruit initiated this investigation about biochemical composition in peach mesocarp/exocarp. Biochemical evaluation of phenolic compounds and ascorbic acid were quantified through high-performance liquid chromatography (HPLC) in relation to three flesh colors (white, yellow and red) and four flesh typologies (melting, non-melting, slow softening and stony hard) within six commercial cultivars and eight breeding selections of peach/nectarine in 2007. While in 2008, quality and sensorial analyses were conducted on only three commercial cultivars ('Big Top', 'Springcrest' and 'Ghiaccio 1'). The red flesh selection demonstrated the highest levels of phenolic compounds (in mesocarp/exocarp) and ascorbic acid. Total phenolic concentration was approximately three-fold higher in the exocarp than the mesocarp across all accessions. Breeding selections generally reported higher levels of phenolics than commercial cultivars. Flesh textural typologies justified firmness differences at harvest, but minimally addressed variations in quality and phenolic compounds. Flesh pigmentation explained variation in the biochemical composition, with the red flesh accession characterized by an abundancy of phenolic compounds and a high potential for elevated antioxidant activity. Sensorial analyses ranked the cultivar with high soluble solids concentration:titratable acidity (SSC:TA) and reduced firmness the highest overall. Red flesh is a highly desirable trait for breeding programs aiming to improve consumption of peaches selected for nutraceutical properties.

Synergistic Effect of NaCl Pretreatment and PVP on Browning Suppression and Callus Induction from Petal Explants of Paeonia Lactiflora Pall. 'Festival Maxima'.

Browning is prevalent in tissue cultures of Paeonia lactiflora Pall. (herbaceous peony), and severely affects and restricts the growth and differentiation of the explants. In this study, dipping excised explants in a sodium chloride (NaCl) solution as a pretreatment, adding polyvinyl pyrrolidone (PVP) to the culture medium, storing planted explants at 4 °C for 24 h, and transferring planted explants to a new medium after 24 h were considered as browning-suppression methods in tissue cultures of herbaceous peony 'Festival Maxima'. The treated petal explants were cultured in a culture room with a 16-hour photoperiod, 25 °C temperature, and 80% relative humidity in darkness for 4 to 8 weeks. The results demonstrated that dipping excised explants in a 0.5 g·L-1 NaCl solution, adding 0.5 g·L-1 PVP to the medium, storing planted explants at 4 °C for 24 h, and transferring planted explants to the same fresh medium after 24 h could effectively inhibit browning. Adding PVP to the medium led to the greatest browning suppression percentage of 95%. Storing planted explants at 4 °C for 24 h reduced the effectiveness of other treatments in suppressing browning. After 8 weeks, dipping excised explants in a NaCl solution resulted in the highest callus induction percentage of 75%, while storing explants at 4 °C for 24 h suppressed callus formation. It was observed in all treatments that decreases in browning was accompanied with higher levels of phenols and lower activities of phenylalanine ammonia-lyase (PAL) and polyphenoloxidase (PPO). Overall, the results suggest that dipping in a NaCl solution was effective in alleviating the browning issues of herbaceous peony tissue cultures, and had positive synergistic effects with PVP on browning suppression and callus induction.

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.