Somatic embryogenesis and plant regeneration in carob (Ceratonia siliqua L.)

Summary Somatic embryos of carob (Ceratonia siliqua L.) were induced from cotyledonary segments excised from immature seeds when cultured on Murashige and Skoog media supplemented with several combinations of 6-benzylaminopurine (BA) and indole-3-butyric acid (IBA). The best frequencies of induction (33.8%) were obtained when 4.4 μM BA and 0.5 μM IBA were used. Shoots were also sporadically formed in the same media. When IBA was replaced by other auxins in the induction media, only α-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) could induce somatic embryogenesis, although at lower rates than IBA. 2,4-Dichlorophenoxyacetic acid and 4-amino-3,5,6-trichloropicolinic acid were completely ineffective. Besides culture media composition, the developmental stage of the explants at the time of culture showed a strong influence on somatic embryogenesis induction, with cotyledons from stage II pods providing the highest levels of induction. By contrast, the genotype of the explant did not determine a significant role in the induction process. Attempts to achieve somatic embryo germination were mostly unsuccessful, since only shoot development was observed; the highest frequencies of development occurred on media containing only gibberellic acid (3.0 μM). For plant regeneration, the developed shoots were further rooted on IBA-supplemented media, and the plantlets obtained were transferred to soil, where c. 88% of them survived. Histological observations showed the presence of morphologically normal and abnormal somatic embryos, the latter displaying an abnormal pattern of vascular bundles. Ultrastructural analysis showed that the cells of the globular embryos had a dense cytoplasm, whereas those not involved in somatic embryo formation showed signs of senescence. Histological studies were also used to distinguish between somatic embryos and shoots originated in the same media.     

Genetic diversity revealed by EST-SSR markers in carob tree (Ceratonia siliqua L.)

Carob is a caesalpinoid tree species widespread in the Mediterranean. In this paper, carob EST-SSR (Expressed Sequence Tags-Simple Sequence Repeat) markers were used to assess the level of genetic diversity among 71 cultivars and accessions collected in three Mediterranean countries (Italy, Malta and Spain). Starting from 20 microsatellite loci, we identified a set of 9 polymorphic EST-SSRs, with a number of alleles per locus ranging from 2 to 7, and a number of genotypes from 3 to 13. The nine polymorphic EST-SSRs discriminated most of the analyzed genotypes with the exceptions of some cultivated clones having similar phenotypic features and probably belonging to cultivar-populations, and clarified some cases of homonymy.A neighbor joining dendrogram generated three main clusters which did not show a correlation with the distribution areas of the analyzed genotypes. A Bayesian analysis evidenced a high degree of admixture among the gene pools from the three countries. The low level of diversification among geographical areas reveals that the asexual propagation of selected clones played a major role in the diffusion of the species in the western Mediterranean. The work described in this paper represents the first report of carob genotyping achieved through microsatellite markers.     

Studies in the Turkish Carob (Ceratonia siliqua L.)

Summary The total amount of adenine nucleotides was not changed when non-growing cells of a chlorophyll-free, carotenoid-containing mutant of Chlorella vulgaris Beijerinck (211–11 h/20) were transferred from darkness into blue light (<550 nm, 300 W cm^-2). However the level of ATP increased significantly immediately after the onset of blue illumination, while that of both ADP and AMP decreased (Table 1). Induction period, intensity dependency and wave-length dependency of the light-induced change in ATP-concentration (Figs. 1–3) resemble those of the long-known enhancement of respiration by blue light. The observed increase in ATP does not support the often proposed idea of enhanced carbohydrate breakdown being the result of a reduced ATP-level, but rather seems to arise from enhanced substrate degradation.

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Abkürzungen TCE Trichloressigsäure - gZ gepacktes Zellvolumen     

Flavonoid content in leaf extracts of the fig (Ficus carica L.), carob (Ceratonia siliqua L.) and pistachio (Pistacia lentiscus L.)

The total flavonoid content of leaf extracts (70% ethanol) from fig (Ficus carica L.), carob (Ceratonia siliqua L.) and pistachio (Pistacia lentiscus L.) plants were determined by using reverse phase high-performance liquid chromatography (HPLC)-and analyzed by UV/VIS array and electrospray ionization (ESI)-mass spectrometry (MS) detectors. As a base for comparison, flavonoid type and level were also determined in extracts from soybeans and grape seeds. It was found that the major flavonoids in Ficus are quercetin and luteolin, with a total of 631 and 681 mg/kg extract, respectively. In Ceratonia leaves, nine different flavonoids were detected. The major one was myricetin (1486 mg/kg extract), with a similar level in Pistacia (1331 mg/kg extract, myricetin). The present study is the first to report the presence of the isoflavone genistein in the Pistacia leaf, which was discovered to consist of about a third of the genistein level detected in soybean.     

Inhibitors from Carob (Ceratonia siliqua L.) I. Nature of the Interaction With Gibberellic Acid on Shoot Growth

Concentrated whole extracts of the immature fruit of carob and 3 fractions derived from this extract have been shown to inhibit the gibberellic acid induced growth of pea seedlings. The inhibition can be completely reversed by increasing the amount of gibberellic acid. The inhibitors do not reduce the endogenous growth of seedlings but only that induced by gibberellic acid. One of the fractions is a newly separated one not previously reported.     

Ethylene production by carob (Ceratonia siliqua) callus cultures on varying media

Carob callus from hypocotyl segments produced ethylene in different amounts which were related to the composition of the medium and age of the callus. Both light and darkness stimulated high levels of ethylene production. No correlation was found between growth rate and ethylene production under dark conditions. In the light, a significant correlation was found, indicating that ethylene production and growth rate follow one another. Culture medium was the most important factor in controlling the growth rate and ethylene production. The highest values of ethylene production were obtained on media showing highest callus growth rate. These studies seem to indicate that most of the ethylene produced is a by-product of metabolic changes during carob callus development, though, under certain conditions, the initial evolution could regulate growth.     

Production and secretion of {alpha}-galactosidase and endo-{beta}-mannanase by carob (Ceratonia siliqua L.) endosperm protoplasts

Viable protoplasts were isolated for the first time from maturecarob (Ceratonia siliqua L.) endosperm tissue. After 5 d ofincubation 75% of the protoplasts were viable. During incubationthey underwent vacuolation and produced the carob endospermhydrolases, agalactosidase and endo-ß-mannanase, whichwere secreted in the incubation medium. The secretion of bothenzymes were under Ca²⁺ control. Many characteristics of -galactosidaseand endo-ß-mannanase production by protoplasts werethe same as those of whole endosperms: their production didnot require any hormonal signal and was inhibited in the presenceof ABA or the leachate from the carob endosperm/seed coat. Moderatewater stress (—2.0 MPa) neither affected the activityof these hydrolases nor their secretion by endosperm protoplast.However, when the osmoticum of protoplast incubation mediumwas higher, the production and secretion of both hydrolaseswere reduced. Comparison of the hydrolases activities in theincubation media of leached carob endosperms, which were incubatedunder normal and water stress (—1.5 MPa) conditions, withthe activities of the protoplast-secreted hydrolases indicatedthat (i) carob endosperm cell wall acts as a barrier for thesecreted enzymes and (ii) that water stress reduces the cellwall porosity of the carob endosperm cells, and thus the releaseof the secreted -galactosidase and endo-ß-mannanaseis inhibited. The isolation of carob endosperm protoplasts offersa potent experimental system for the study of aspects of endospermcell physiology, such as enzyme secretion Key words: Abscisic acid, carob endosperm, Ceratonia siliqua L, endo-ß-mannanase, -galactosidase, leachate, protoplasts, water stress     

Effect of N-nutrition and irrigation on fruit production of carob (Ceratonia siliqua)

A combined fertilization and irrigation experiment was initiated in order to see whether such treatment could improve the productivity of carob ( Ceratonia siliqua L. cv. Mulata) on Mediterranean marginal lands. Mature carob trees (20–30 years old) were submitted to 3 different irrigation levels (100%;, 50%; and 0%;) based on daily standard evaporation values, measured with a class A pan. For each water level either 21 or 63 kg N ha⁻¹ was applied as ammonium nitrate. The results reported here refer to a two-year experiment, and show that the productivity of the trees could be improved due to a positive interaction between water and N application. In addition, they suggest that also under the normal dry conditions of these Mediterranean systems nitrogen application is effective, even without irrigation.     

The occurrence of abscisic acid in inhibitors B1 and C from immature fruit of Ceratonia siliqua L. (carob) and in commercial carob syrup

Summary The presence of abscisic acid in the inhibitors B₁ and C from immature carob fruit, whole and minus seed, has been established by thin-layer and gas chromatography and by combined gas chromatography-mass spectrometry. Abscisic acid has been identified in commercial carob syrup by the same means. Most, if not all, of the growth inhibitory activity in these fractions is accounted for as abscisic acid by quantitative gas chromatography as the methyl ester. Trimethylsilylation of abscisic acid with bis (trimethylsilyl) acetamide in pyridine gives two isomeric tris(trimethylsilyl) derivatives.     

Seasonal variations of leaf water potential and growth in fertigated carob-trees (Ceratonia siliqua L.)

Variations of predawn and midday leaf water potential and relative growth rates were studied in mature carob trees (Ceratonia siliqua L. cv Mulata) submitted to a fertigation experiment. Three levels of irrigation were tested: 0%, 50% and 100%, based on daily standard evaporation values. For each irrigation level two nitrogen amounts were applied –21 and 63 kg N ha^-1 year^-1 as ammonium nitrate. The experiment was run between July 91 and August 1993. Measurements of leaf water potential and absolute branch length increments were made at monthly intervals, during the entire experimental period or during seasonal growth, respectively. Leaf water potential was related to soil volumetric water content, maximum and minimum air temperature and daily evaporation. Predawn leaf water potentials were always higher than –1.1 MPa. Midday leaf water potential values presented very large seasonal variations and very low values independent of treatments. The low leaf water potentials observed for the fertigated trees during summer, suggest that this parameter may be related not only to the evaporative demand but also to growth investment. The amount of fertigation was positively correlated with vegetative growth increment and fruit production. Practical implications for irrigation schedules of leaf water potential patterns together with drought adaptation mechanisms of carob tree are discussed.     

Protoplast isolation and culture from carob (Ceratonia siliqua) hypocotyls: ability of regenerated protoplasts to produce mannose-containing polysaccharides

The aim of this study was to isolate protoplasts from carob (Ceratonia siliqua L.) embryonic tissues with the ability to regenerate cell walls, divide and synthesize galactomannan, a valuable polysaccharide for industry. Protoplasts isolated from carob hypocotyl hooks regenerated cell walls within 24 h. The first divisions of the regenerated cells were observed after 2 days of culture. The highest percentage that successfully divided was achieved when the seedlings were grown under diffuse light, the hypocotyl hooks were plasmolysed for 1 h before incubation in the protoplast isolation solution and the protoplasts were cultured under diffuse light. After 9 days of culture, cell clusters, consisting of eight cells, had been produced, which underwent further mitotic divisions and which were expected to lead to callus formation. Polysaccharide and oligosaccharide synthesis during protoplast regeneration was studied by radiolabelling with exogenous d ‐[U‐¹⁴C]glucose, d ‐[U‐¹⁴C]mannose or d ‐[2‐³H]mannose, which gave rise to uniform, moderately specific and highly specific labelling, respectively. As revealed by the radioactivity distribution in cell wall monosaccharides, the regenerants deposited new wall polymers that differed markedly from those being synthesized by the hypocotyls from which the protoplasts had been isolated. The regenerants deposited large amounts of callose and smaller amounts of galactose‐, arabinose‐ and mannose‐containing polymers. The latter included glucuronomannan, as demonstrated by a new method involving partial acid hydrolysis followed by β‐glucuronidase (EC 3.2.1.31) digestion. The regenerating protoplasts also released soluble extracellular carbohydrates: polysaccharides which appeared to be mainly acidic arabinogalactans, and oligosaccharides which were mainly neutral and contained glucose, galactose and mannose. We conclude that regenerating carob protoplasts are a useful system for studying carbohydrate secretion, including mannose‐rich poly‐ and oligosaccharides.     

Seasonal changes in some photosynthetic properties of Ceratonia siliqua (carob tree) leaves under natural conditions

Rates of photosynthesis and leaf conductance of the leaves of carob trees ( Ceratonia siliqua L.) growing in natural conditions were measured during the course of the seasons to define the effects of the main climatic factors limiting growth in the region: temperature during the winter and water in the summer. The highest photosynthetic rates were measured in spring and autumn and could reach 25 μmol m₋₂ s₋₁ with optimal temperature and available water. Due to lower temperatures (4 to 6°C in the night) these values were frequently around 15 μmol m₋₂ s₋₁ during winter, but the strongest depression was due to prolonged drought in summer. However a reduction in photosynthesis rate down to 5 μmol m₋₂ s₋₁ occurred only after depletion of all the available water in the soil layer up to a depth of 50 cm. In the end of the summer, leaf conductance and water potential were in the order of 20 mmol m₋₂ s₋₁ and −3 MPa respectively. Compared to other trees that make up the Mediterranean sclerophyll forest, the photosynthetic activity of carob is high, and the tree tolerates a considerable depletion of soil water.     

Uptake of ammonium and nitrate by carob (Ceratonia siliqua) as affected by root temperature and inhibitors

Seedlings of carob ( Ceratonia siliqua L. cv. Mulata) were grown in nutrient solution culture for 5 weeks, with or without nitrogen at different root temperatures (10, 16, 22, 30, 35 or 40deg;C) and with the air temperature kept between 20 and 24°C. The nitrogen was given as either ammonium or nitrate. At all root temperatures studied, nitrogen-depleted plants developed higher net uptake rates for nitrogen than plants grown in the presence of nitrogen. Temperature affected the kinetic parameters of nitrate uptake more than those of ammonium uptake. With increasing root temperature, the K_m of ammonium uptake decreased, but to a lesser extent than the K_m for nitrate. The increase in V_max of ammonium uptake with temperature was also less noticeable than that for nitrate uptake. Ammonium and nitrate uptakes were inhibited in a similar way by respiratory or protein synthesis inhibitors. It may be noted that ammonium uptake in the presence of inhibitors at 40°C was higher than uptake at 10°C without inhibitors. Some similarities between the transport mechanisms for nitrate and ammonium are underlined in the present work. Components of both transport systems displayed saturation kinetics and depended on protein synthesis and energy. The following components of nitrate uptake were distinguished: (a) a passive net influx into the apparent free space; (b) a constitutive active uptake and (c) active uptake dependent on protein synthesis. We may similarly define three ammonium uptake systems: (a) a passive influx into the apparent free space; (b) passive diffusion uptake at high temperature and (c) active uptake dependent on protein synthesis. The possible role of the ratio between mechanism (c) and mechanism (b) as determinant of ammonium sensitivity is discussed.     

A phi Layer in Roots of Ceratonia siliqua L

The central cylinder of the primary root of the carob tree (Ceratonia siliqua) is encircled by a layer of cells with wall thickenings, known as a phi (φ) cell layer. The development of the φ layer and the chemical composition of the cell wall thickenings have been studied in roots of C. siliqua. The results reveal the presence of condensed tannins in the mature phi thickenings and that the development of the φ layer is asynchronous: at 0–1 cm from the root tip φ thickenings appear before endodermis differentiation at the sites opposite phloem, at 1–4 cm new φ thickenings are developed at the sites opposite xylem, at 4–7 cm the φ layer consists of two layers of cells and it completely encloses the central cylinder.     

Lenticel hypertrophy in shoot cultures of Ceratonia siliqua L.

Numerous white surface proliferations appeared in cultures of Ceratonia siliqua L. grown three to four weeks on medium containing 0.5 mg l^-1 BA and 0.1 mg l^-1 IBA. It was histologically confirmed that these proliferations were hypertrophied lenticels. Proliferations appeared first at the basal shoot internode and gradually spread acropetally, covering eventually the whole shoot except the uppermost internodes. Increase of BA concentration in the medium increased both the number of hypertrophied lenticels per shoot and the shoot multiplication index.Abbreviations BA 6-benzylamino-purine - IBA indole-3-butyric acid Dubravka Bojovi-Cveti deceased July 8, 1991.     

Responsiveness of Carob (Ceratonia siliqua L.) Plants to Arbuscular Mycorrhizal Symbiosis Under Different Phosphate Fertilization Levels

This experiment was carried out in pots in a greenhouse to evaluate the effects of arbuscular mycorrhizal fungi (Funneliformis mosseae, Rhizophagus intraradices and Rhizophagus fasciculatus) on carob plant performance under different levels of phosphate fertilization. Non-mycorrhizal (NMyc) and mycorrhizal (Myc) carob plants were subjected to three levels of phosphate fertilization, L1 (0 mg P kg⁻¹ soil), L2 (25 mg P kg⁻¹ soil) and L3 (100 mg P kg⁻¹ soil). Results showed that under L1 and L2 P-fertilization levels, arbuscular mycorrhizal symbiosis significantly improved growth and biomass production of carob plants. Moreover, mineral nutrient (P, K, Na and Ca) acquisition, photosynthetic activity (F_v/F_m), stomatal conductance, total chlorophyll content, and soluble sugar accumulation were also strongly improved in Myc plants in comparison with NMyc ones. Under L1 P-fertilization level, Myc plants showed strongly increased acid phosphatase activity in roots and in the rhizospheric soil than NMyc plants. Furthermore, Myc plants maintained high membrane integrity (over 80%) and low hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents, associated with increased activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (G-POD), and catalase (CAT) compared to NMyc plants. However, high phosphorus input (L3) negatively affected root colonization and mycorrhizal plant performance. Thus, carob plants associated with Funneliformis mosseae performed best under phosphorus deficiency and were the least sensitive to the variations of phosphorus input levels.

     

Microgeographic genome size differentiation of the carob tree, Ceratonia siliqua, at 'Evolution Canyon', Israel

BACKGROUND AND AIMS: We tested whether the local differences in genome size recorded earlier in the wild barley, Hordeum spontaneum, at 'Evolution Canyon', Mount Carmel, Israel, can also be found in other organisms. As a model species for our test we chose the evergreen carob tree, Ceratonia siliqua. METHODS: Genome size was measured by means of DAPI flow cytometry. KEY RESULTS: In adults, significantly more DNA was recorded in trees growing on the more illuminated, warmer, drier, microclimatically more fluctuating 'African' south-facing slope than in trees on the opposite, less illuminated, cooler and more humid, 'European' north-facing slope in spite of an interslope distance of only 100 m at the canyon bottom and 400 m at the top. The amount of DNA was significantly negatively correlated with leaf length and tree circumference. In seedlings, interslope differences in the amount of genome DNA were not found. In addition, the first cases of triploidy and tetraploidy were found in C. siliqua. CONCLUSIONS: The data on C. siliqua at 'Evolution Canyon' showed that local variability in the C-value exists in this species and that ecological stress might be a strong evolutionary driving force in shaping the amount of DNA.     

Characterization of rhizobia isolated from Carob tree (Ceratonia siliqua)

Bacteria were isolated from root nodules of Carob tree (Ceratonia siliqua). We studied their cultural, physiological and biochemical characteristics. Strains showed some variability concerning tolerance to NaCl and KCl, and none grew at pHs below 7. The mean temperature for growth was 30°C and some strains could grow at 45°C. These isolates could use a great variety of carbohydrates as a sole carbon source (hexoses, pentos, disaccharides, trisaccharides, polysaccharides and organic acids). According to their cultural and physiological features and their in vitro infectivity on their original host, we suggest that these isolates may belong to the genus Rhizobium. Moreover, with regard to their average generation time, carbohydrate utilization, antibiotic resistance and other biochemical properties, they may be related to fast-growing rhizobia until further characterization.