Identification of 2,4-D-responsive proteins in embryogenic callus of Valencia sweet orange (<Emphasis Type="Italic">Citrus sinensis</Emphasis> Osbeck) following osmotic stress

The compound 2,4-Dicholorophenoxyacetic acid (2,4-D) is an important growth regulator which is used in the majority of embryogenic cell and tissue culture systems. However, 2,4-D also appears to have a negative effect on growth and development of plant tissues and organs cultured in vitro. For example, 2,4-D exerts inhibition on in vitro somatic embryo initiation and/or development of most citrus species. To understand the molecular mechanism by which 2,4-D inhibits somatic embryogenesis (SE), proteomic changes of Valencia sweet orange (Citrus sinensis) embryogenic callus induced by treatments with a high concentration of 2,4-D (6 mg l⁻¹) was investigated. Nine 2,4-D-responsive proteins were identified, of which eight were up-regulated and one was down-regulated. Interestingly, three of the eight up-regulated proteins were osmotic stress-associated, suggesting that 2,4-D induced osmotic stress in Valencia embryogenic callus. This speculation was supported by results from our physiological studies: 2,4-D treated callus cells exhibited increased cytoplasm concentration with a significant reduction in relative water content (RWC) and an obvious increase in levels of two osmolytes (proline and soluble sugar). Taken together, our results suggested that 2,4-D could inhibit somatic embryo initiation by, at least in part, inducing osmotic stress to citrus callus cells.     

Maturation of somatic embryos in conifers: Morphogenesis,physiology, biochemistry,and molecular biology

Summary In the past 15 years tremendons progress has been made towards the development of systems for the induction and development of somatic embryos of coniferous species. Since the first report in 1985, several species have been induced to produce somatic embryos. This has been rendered possible by the development of rational media and improvement of culture conditions, which have resulted in increased embryo quality and higher conversion frequency. Understanding the physiological and biochemical events occurring during in vivo embryogenesis has been fundamental in the design of new protocols for improving the somatic embryogenic process. Specifically, the inclusions of abscisic acid (ABA) and osmotic agents, such as polyethylene glycol (PEG), have been shown to be necessary for the functional development of somatic embryos. In the past few years, physiological and biochemical investigations have been useful in increasing our knowledge on the mode of action of ABA and PEG during embryo development. In comparison with the flowering plants, our understanding on the molecular mechanisms regulating the embryogenic process in coniferous species is still very limited. The application of new molecular techniques is therefore fundamental towards this end. The emphasis of this review is on recent information dealing with the maturation of conifer somatic embryos.     

Influence of auxins on somatic embryogenesis and alkaloid accumulation in Leucojum aestivum callus

In vitro cultures of Leucojum aestivum are considered as an alternative for the production of galanthamine, which is used for the symptomatic treatment of Alzheimer’s disease. We studied the effects of auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), 3,6-dichloro-o-anisic acid (dicamba) at concentrations of 25 and 50 µM on the induction of embryogenic callus and its capacity to induce somatic embryogenesis and alkaloid accumulation. The embryogenic response of the explants was from 30% for 25 µM of dicamba to 100% for picloram (for both 25 and 50 µM). 2,4-D (50 µM) stimulated greater callus proliferation and somatic embryo induction as compared to the other auxins. Polyethylene glycol (PEG) stimulated somatic embryo maturation. Callus grown on media containing 50 µM of auxins produced fewer phenolic compounds as compared with callus grown on media containing 25 µM of auxins. GC-MS analyses showed seven alkaloids in the in vivo bulbs and two to four in callus culture. Galanthamine was detected in callus cultivated with 2,4-D (25, 50 µM), picloram (25 µM), and dicamba (50 µM). Other alkaloids, trisphaeridine, tazettine, and 11-hydroxyvittatine were accumulated only in callus growing on medium with picloram (50 µM).     

Effects of NaCl and iso-osmotic PEG stress on growth,osmolytes accumulation and antioxidant defense in cultured sugarcane cells

In order to discriminate between the ionic and osmotic components of salt stress, sugarcane (Saccharum officinarum L. cv. Co 86032) calli were cultured on media containing NaCl or polyethylene glycol (PEG) 8000 that exerted the same osmotic pressure (−0.7 MPa). PEG stress exposure for 15 days led to significant growth reduction and loss in water content than salt stressed and control tissues. Osmotic adjustment (OA) was observed in callus tissues grown on salt, but was not evident in callus grown on PEG. Oxidative damage to membranes, estimated in terms of accumulation of thiobarbituric acid reactive substances-TBARS and electrolytic leakage was significantly higher in both the stressed calli than the control however, the extent of damage was more in the PEG stressed calli. The stressed callus tissues showed inhibition of ascorbate peroxidase activity, while catalase activity was increased. These results indicate sensitivity of cells to PEG-mediated stress than salt stress and differences in their OA to these two stress conditions. The sensitivity to the osmotic stress indicate that expression of the stress tolerance response requires the coordinated action of different tissues in a plant and hence was not expressed at the cellular level.     

Effects of Different Levels of Water Stress on Leaf Water Potential, Stomatal Resistance, Protein and Chlorophyll Content and Certain Anti-oxidative Enzymes in Tomato Plants

A greenhouse experlment was performed In order to Investigate the effects of dlfferent levels of water stress on leaf water potentlal （ψw）, stomatal resistance （rs）, protein content and chlorophyll （Chl） content of tomato plants （Lycoperslcon esculentum Mill. cv. Nlkita）. Water stress was Induced by addlng polyethylene glycol （PEG 6 000） to the nutrlent solution to reduce the osmotlc potential （ψs）. We Investlgated the behavlor of antl-oxldant enzymes, such as catalase （CAT） and superoxide dlsmutase （SOD）, durlng the development of water stress. Moderate and severe water stress （i.e. ψs= -0.51 and -1.22 MPa, respectlvely） caused a decrease In ψw for all treated （water-stressed） plants compared with control plants, wlth the reductlon belng more pronounced for severely stressed plants. In addltion, rs was slgnlflcantly affected by the Induced water stress and a decrease in leaf soluble protelns and Chl content was observed. Whereas CAT actlvlty remained constant, SOD actlvlty was increased in water-stressed plants compared wlth unstressed plants. These results Indicate the possible role of SOD as an anti-oxidant protector system for plants under water stress condltlons. Moreover, It suggests the possibllity of using this enzyme as an addltional screening crlterlon for detecting water stress in plants.     

大蒜体细胞胚胎发生过程中抗氧化酶活性变化及某些生理特征

以大蒜的发芽叶基(鳞茎)为外植体诱导体细胞胚胎发生,研究大蒜体胚发生过程中SOD、POD和CAT 3种抗氧化酶的活性以及可溶性糖和可溶性蛋白质含量变化.结果表明:在大蒜体胚发生过程中,SOD、POD和CAT活性变化与胚性愈伤组织的诱导及体胚的发育密切相关,POD对体胚的诱导起主导作用,而SOD和CAT在体胚的发育和成熟中起主导作用.可溶性糖和可溶性蛋白质累积与大蒜体细胞胚胎发生密切相关.     

花楸体细胞胚发生过程中抗氧化酶活性的变化

花楸体细胞胚发生过程中,胚性愈伤组织可溶性蛋白含量高于其他类型的愈伤组织,非胚性愈伤组织中超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性均高于其他类型的愈伤组织;SOD、POD活性均在胚性细胞向球形胚转化时下降,球形胚向心形胚发育时下降,心形胚向鱼雷形胚和鱼雷形胚向子叶形胚发育时再升高;CAT活性变化规律与SOD和POD活性变化不同,从胚性细胞到鱼雷形胚的3个发育时间内表现为下降-升高-下降的趋势,鱼雷形胚向子叶胚发育时略有回升。据此认为,SOD酶活性降低似可作为花楸胚性细胞分化以及胚胎早期发育的一个判断指标。     

花烛体细胞胚胎发生过程中相关生理生化特征研究

以花烛品种Amigo为材料,研究了悬浮培养条件下花烛体细胞胚胎发生过程中相关生理生化特征。结果表明:POD、CAT在胚性愈伤组织阶段维持较高活性,而SOD在体胚发育后期阶段活性较高;可溶性蛋白质含量在胚性愈伤组织阶段出现高峰;可溶性糖含量变化表现为先上升后下降的趋势,而淀粉含量表现为先下降后上升的趋势;SDS-PAGE电泳分析表明,胚性愈伤组织阶段蛋白质表达量高,种类多,并出现多种特异蛋白。分析认为胚性愈伤组织阶段是调控花烛体细胞胚胎发生过程的关键阶段。     

Somatic Embryogenesis in Abies Alba × Abies Alba and Abies Alba × Abies Nordmanniana Hybrids

Maturation and germination of somatic embryos of hybrids A. alba × A. alba and A. alba × A. nordmanniana were followed by protein analysis of single embryogenic -suspensor masses (ESM) and analysis of storage protein accumulation during somatic embryo development. Very important step was one week pre-cultivation of ESM on medium with polyethylene glycol (PEG) and abscisic acid (ABA). Low osmotic potential of maturation medium and addition of ABA supported development of somatic embryo. Also partial drying of somatic embryo during following three weeks was needed for its normal development. In spite of morphologically fully developed, the somatic embryos were not physiologically ready for germination at least in terms of storage protein accumulation.     

渗透胁迫对黑麦幼苗活性氧和抗氧化酶活性的影响

用20%聚乙二醇(PEG 6000)研究了渗透胁迫对黑麦(Secale cereale L.)幼苗活性氧(reactive oxygen species, ROS)和主要抗氧化酶—— 超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)、抗坏血酸过氧化物酶(ascorbate peroxidase, APX)和谷胱甘肽还原酶(glutathione reductase, GR)活性的影响。结果表明, 与对照相比, PEG处理明显提高了叶子和根中丙二醛(malondialdehyde, MDA)的含量、ROS的水平和以上4种抗氧化酶的活性。渗透胁迫下,叶子和根中MDA和ROS水平变化的规律基本相似, 但抗氧化酶活性在2种器官中表现不完全相同, 叶子中CAT的活性在对照和处理中无显著差异, 但在根中差异明显, 表明叶子中SOD、APX和GR在植物应答渗透胁迫中起重要作用, 而根中这4种抗氧化酶都参与植物对胁迫的反应。GR活性随PEG处理变化幅度显著高于其它抗氧化酶, 表明GR在黑麦应答渗透胁迫中所起作用可能强于其它抗氧化酶。     

Physiological responses of two Jerusalem artichoke cultivars to drought stress induced by polyethylene glycol

Physiological responses of two Jerusalem artichoke (Helianthus tuberosus L.) cultivars with different drought sensitivity to drought stress induced by polyethylene glycol (PEG) 6000 were investigated by characterizing water status, membrane lipid peroxidation, key antioxidant enzymes activity, and proline accumulation. It was observed that the drought-tolerant Jerusalem artichoke cv. Xiuyan maintained a relatively higher water status than the drought-sensitive cv. Yulin upon drought treatments. Meanwhile, lower levels of malondialdehyde (MDA) as well as higher levels of free proline occurred in cv. Xiuyan after 36 h drought treatments. Moreover, the activities of catalase (CAT) and superoxide dismutase (SOD) in cv. Xiuyan were higher than cv. Yulin after drought stress. These results indicated that drought sensitivities actually differ between Jerusalem artichoke cv. Xiuyan and cv. Yulin, and the cv. Xiuyan was more tolerant to drought stress caused by polyethylene glycol.     

Exogenous Nitric Oxide (as Sodium Nitroprusside) Ameliorates Polyethylene Glycol-Induced Osmotic Stress in Hydroponically Grown Maize Roots

The present study was designed to examine whether exogenous sodium nitroprusside (SNP) supplementation has any ameliorating action against PEG-induced osmotic stress in Zea mays cv. FRB-73 roots. Twenty percent or 40 % polyethylene glycol (PEG6000; ?0.5 MPa and ?1.76 MPa, respectively) treatment alone or in combination with 150 and 300 μM SNP was applied to hydroponically grown maize roots for 72 h. Although only catalase (CAT) activity increased when maize roots were exposed to PEG-induced osmotic stress, induction of this antioxidant enzyme was inadequate to detoxify the extreme levels of reactive oxygen species, as evidenced by growth, water content, superoxide anion radical (O ₂ ^?? ), hydroxyl radical (OH^?) scavenging activity, and TBARS content. However, supplementation of PEG-exposed specimens with SNP significantly alleviated stress-induced damage through effective water management and enhancement of antioxidant defense markers including the enzymatic/non-enzymatic systems. Exogenously applied SNP under stress resulted in the up-regulation of glutathione peroxidase (GPX), glutathione S-transferase (GST), ascorbate peroxidase (APX), glutathione reductase (GR), total ascorbate, and glutathione contents involved in ascorbate–glutathione cycle. On the other hand, growth rate, osmotic potential, CAT, APX, GR, and GPX increased in maize roots exposed to both concentrations of SNP alone, but activities of monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase decreased. Based on the above results, an exogenous supply of both 150 and 300 μM SNP to maize roots was protective for PEG-induced toxicity. The present study provides new insights into the mechanisms of SNP (NO donor) amelioration of PEG-induced osmotic stress damages in hydroponically grown maize roots.     

Regeneration from intact and sectioned immature embryos of barley (Hordeum vulgare L.): the scutellum exhibits an apico-basal gradient of embryogenic capacity

Immature zygotic embryos from spring barley cv. Dissa were used to induce somatic embryogenenesis. Up to 158 germinated somatic embryos could be recovered per plated zygotic embryo. Critical factors for obtaining a high yield of regenerants were the size of the explant, the level of 2,4-D used for callus induction and the careful division of callus at each subculture. Use of microsections of immature embryos as explants revealed a pronounced gradient of callus formation and embryogenic response across the scutellum. Sections from the scutellar tissue at the coleoptilar end of the embryo gave the most callus and were highly embryogenic. The regeneration response of sectioned explants was comparable to that recovered from intact embryos of similar size.     

Enhanced Maturation and Desiccation Tolerance of White Spruce [Picea glauca (Moench) Voss] Somatic Embryos: Effects of a Non-plasmolysing Water Stress and Abscisic Acid

A non-plasmolysing moisture stress effected by polyethyleneglycol (PEG) was beneficial when applied to maturing white spruce(Picea glauca) somatic embryos for the following reasons. Anosmotic treatment of 5.0–7.5% PEG stimulated a threefoldincrease in the maturation frequency. The osmotically treatedsomatic embryos displayed higher dry weights and lower moisturecontents than the controls, indicating a greater accumulationof storage reserves. Moisture contents of mature, osmotically-treated,hydrated somatic embryos were 40–45%, in contrast to 57%for the non-osmotically treated controls. Desiccation was achievedby placing the somatic embryos in a range of relative-humidityenvironments. No clear trend for the effect of PEG on survivalof desiccated somatic embryos was observed; mean survival valuesranged from 34 to 62% when somatic embryos from all osmotictreatments were desiccated for 14 d at 81% relative humidity.Following this desiccation treatment, somatic embryos from allosmotic concentrations had moisture contents of 26–31%,similar to the 32% recorded for unimbibed zygotic embryos. Afterimbibition, moisture contents for these zygotic and somaticembryos were in the order of 60%. Somatic embryos matured withPEG remained quiescent during desiccation due to their low initialmoisture contents, and gave rise to plantlets of normal appearance.Gradual desiccation of the somatic embryos directly followingmaturation with abscisic acid (ABA) was crucial to survivalduring desiccation. A plasmolysing water stress effected bysucrose at osmotic potentials similar to PEG was detrimentalto somatic embryo maturation, thereby emphasizing the importanceof the choice of osmoticum. Desiccation, maturation, osmotic potential, Picea glauca, polyethylene glycol, somatic embryo, water stress, white spruce     

Proteins related with embryogenic potential in callus and cell suspensions of sugarcane (Saccharum sp.)

Summary Previous results have shown that some proteins secreted in the culture medium are involved with the formation of embryogenic cells and can modify somatic embryo differentiation. Undifferentiated cell suspensions grown in the presence of 13 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and obtained from embryogenic and non-embryogenic callus were used to study these events in sugarcane plants (cv.PR-62258). The cell suspension growth curves were determined and soluble proteins were extracted from embryogenic and non-embryogenic callus and culture medium from cell suspensions. In embryogenic callus we detected 1.43 times more protein than in non-embryogenic callus and the electrophoretic protein patterns show specific polypeptides for both callus types. In embryogenic callus we detected a cluster of four polypeptides in the range of 38–44 kDa and another polypeptide of 23 kDa that were not observed in non-embryogenic callus. In nonembryogenic callus there is a 35-kDa polypeptide that was not detected in embryogenic callus. In the case of extracellular proteins, the medium from embryogenic cell suspensions contained four polypeptides of 41, 38, 34 and 28 kDa that were slightly detected in the medium from non-embryogenic cell cultures; we also detected a band at 15 kDa that could not be observed in the medium from non-embryogenic cell suspensions. These results suggest that the development of embryogenic callus and cell suspensions is related to the type and amount of intracellular proteins in the callus cells and to the secreted proteins from these cells into the medium.     

外源甲基乙二醛对干旱胁迫下板栗幼苗的影响

甲基乙二醛(MG)是一种在植物中具有多种功能的新型信号分子.为探究MG对板栗幼苗干旱胁迫的影响,以两年生'黄棚'板栗幼苗为试材,通过聚乙二醇(PEG)模拟干旱胁迫并进行MG及其清除剂N-乙酰半胱氨酸(NAC)处理,分析板栗幼苗叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(...     

Embryo induction and regeneration from root explants of Medicago truncatula after osmotic pre-treatment

Embryo induction and regeneration from suspension culture of two Medicago truncatula cvs. (cv. R 108 1 and cv. Jemalong) have been studied. The influence of osmotic pre-treatment (1 M solution of sucrose for 48 h and 72 h) of roots as an initial explant, on embryogenic efficiency of the suspension culture was assessed. In comparison to the control, the level of abscisic acid (ABA) increased significantly after osmotic stress. The increased ABA level did not correlate with the induction of embryogenesis neither with the improved embryogenic potential of cv. R 108 1. The shortest regeneration period and the highest percent of conversion to plants were found in cv. R 108 1 after 72-h pre-treatment of roots. The efficiency of somatic embryo conversion was less after 48-h pre-treatment and much less for the untreated control. Osmotic stress did not positively affect the process of embryogenesis from root explants of cv. Jemalong, confirming its cultivar dependence. A single cell suspension fraction was produced in both Medicago trunacatula cvs. during the somatic embryo maturation stage. A higher embryogenic potential than the initial suspension culture was established only for the cell suspension originating from 72-h pre-treated roots of cv. R 108 1. The data confirms that the process of somatic embryo induction and embryo conversion from root explants of cv. R 108 1 could be promoted by osmotic stress pre-treatment.     

Abscisic acid-regulated responses of aba2-1 under osmotic stress: the abscisic acid-inducible antioxidant defence system and reactive oxygen species production

We investigated the interaction among abscisic acid (ABA), reactive oxygen species (ROS) and antioxidant defence system in the transduction of osmotic stress signalling using Arabidopsis thaliana WT (Columbia ecotype, WT) and an ABA-deficient mutant (aba2-1). For this, 50 μm ABA and osmotic stress, induced with 40% (w/v) polyethylene glycol (PEG8000; -0.7 MPa), were applied to WT and aba2-1 for 6, 12 or 24 h. Time course analysis was undertaken for determination of total/isoenzyme activity of the antioxidant enzymes, superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), NADPH oxidase (NOX; EC 1.6.3.1) activity; scavenging activity of the hydroxyl radical (OH˙), hydrogen peroxide (H(2) O(2) ); endogenous ABA and malondialdehyde (MDA). The highest H(2) O(2) and MDA content was found in PEG-treated groups of both genotypes, but with more in aba2-1. ABA treatment under stress reduced the accumulation of H(2) O(2) and MDA, while it promoted activity of SOD, CAT and APX. APX activity was higher than CAT activity in ABA-treated WT and aba2-1, indicating a protective role of APX rather than CAT during osmotic stress-induced oxidative damage. Treatment with ABA also significantly induced increased NOX activity. Oxidative damage was lower in ABA-treated seedlings of both genotypes, which was associated with greater activity of SOD (Mn-SOD1 and 2 and Fe-SOD isoenzymes), CAT and APX in these seedlings after 24 h of stress. These results suggest that osmotic stress effects were overcome by ABA treatment because of increased SOD, CAT, APX and NOX.     

Effect of hydropriming on Trigonella foenum callus growth,biochemical traits and phytochemical components under PEG treatment

The induction of secondary metabolites under osmotic stress is well documented. However, cell death is probably due to osmotic stress. This work tries to study the synergetic effect of hydropriming and polyethylene glycol (PEG) on enhancing the secondary metabolites production in fenugreek callus cultures without facing cell death. PEG initiates the stress and the hydropriming increase the plant cell response against the stress. Fenugreek calli were initiated from hypocotyl of two groups of seeds, the first was hydroprimed overnight before germination, the second remained dry. Three months old calli of the two groups were subcultured on media containing two different concentration of PEG (5, 10%). The calli growth, biochemical analysis, secondary metabolism keys, and secondary metabolites were determined after 4 weeks. PE induced oxidative stress, which increased the membrane lipid peroxidation and decreased cell viability and growth. Hydropriming enhanced the activity of antioxidant enzymes, regulating the reactive oxygen species level, accumulating the osmolytes and secondary products. Therefore the primed callus can tolerate the osmotic stress initiated with PEG. Consequently, cell biomass increased and not affected by PEG treatment. On the other hand, the calli from non-primed seeds have a significant decrease in fresh weight, and dry weight under the higher PEG treatment. The hydropriming protected the growth of the cells under PEG treatment with a high content of secondary metabolites and high antioxidant machinery. The synergetic effect of hydropriming and PEG can be used as a simple and low-cost way to produce valuable compounds in commercial industrial bioreactors.

     

Normalizing sweet orange (C. sinensis (L.) osbeck) somatic embryogenesis with semi-permeable membranes

Summary Development of citrus somatic embryos initiated from embryogenic callus generally results in abnormal morphogenesis of somatic embryos. To normalize development, glycerol-induced globular-stage somatic embryos of sweet orange [C. sinensis (L.) Osbeck cv. ‘Hamlin’] were cultured on 6000–8000 MW cutoff cellulose acetate, >400 000 MW cutoff cellulose acetate, nitrocellulose, polyvinylidene fluoride (PVDF), cellulose filter paper, or positive or neutral charged nylon membranes. Only the two cellulose acetate membranes resulted in the development of normal, two-cotyledon, bipolar, heart-shaped embryos, and no aberrant teratoma-like structures. Heart-shaped embryos developed and germinated normally on Murashige and Tucker basal medium with 0.5% sucrose and 1 μM gibberellic acid. Culture of embryogenic callus directly onto cellulose membranes also resulted in the development of normal heart-shaped embryos, indicating that glycerol induction of globular-stage embryos is not necessary. Heart-shaped embryos were not observed when the osmotic potential of the medium was increased by the addition of 2.5–15% polyethylene glycol; neither were they observed when the matric potential of the medium was increased by increasing the gelling agent concentrations of agar and Gelrite from 0.8% to 3% and 0.15% to 0.9%, respectively.