Novel polymorphic nuclear microsatellites in Cupressus sempervirens L.

We report the characterization and optimization of nine polymorphic nuclear microsatellites from Cupressus sempervirens L. using an enriched library method. A total of 24 individuals from four different populations were used to estimate genetic diversity parameters. High level of allelic diversity was found with a number of alleles ranging from two to 13. These markers will prove very useful in screening diversity at different geographical scales and in monitoring gene flow in cypress orchards.     

Palynological study of Cupressus sempervirens L. var. pyramidalis and var. horizontalis

ABSTRACT

Cupressus sempervirens L. var. pyramidalis and C. sempervirens var. horizontalis were examined for their pollen morphometry and ultrastructure to verify whether, in addition to phenotype, pollen could also show characteristics useful to discriminate the two infraspecific taxa. C. sempervirens var. pyramidalis compared to C. sempervirens var. horizontalis has mainly larger, subcircular pollen with a higher percentage of apolar and germinating grains, and a wall with thicker intine; the cytoplasm has a greater number of callose-containing vesicles. C. sempervirens var. horizontalis has pollen distributed in five classes of shape. In addition it is smaller and shows a higher percentage of ruptured grains, and a lower percentage of germination in vitro. The cytoplasm contains vesicles rich in callose and also a higher amount of osmophilic bodies. Pollen grains in both varietas contain only one cell at dispersal. The reported differences in pollen morphometry, structure and biology, together with the phenotypic characteristics of the tree, support the validity of two infraspecific taxa.     

Diterpene composition of Cupressus dupreziana and Cupressus sempervirens

The heartwood of Cupressus dupreziana and C. sempervirens were shown to contain similar mixtures of diterpenes.     

Morphological diversity of some old accessions of Cupressus sempervirens L. in Iran

The aim of this research was to evaluate some old accessions of Cupressus sempervirens L. in Iran and identify any possible relationships exist among them based on phenotypical characteristics. A total of 17 Cupressus accessions mostly grown in situ, from Kerman and Yazd provinces were used. Twenty characteristics (11 quantitative and 9 qualitative) were measured for evaluating the chosen samples. Results from simple correlation analysis showed significant positive and negative correlations for certain important characteristics. Seed length and width were in significant correlation with cone width. There was a close relationship between seed length and width with cone width. Also seed length, seed width and number of total seeds were in significant correlation with cone circumference. The number of seeds per cone was in positive correlation with scale number per cone and number of seed per scale. Factor analysis was used to determine the effective characteristics and the number of main factors. For each factor, a loading value of more than 0.65 was judged as being significant. Effective characteristics were categorized into seven main factors that contributed to 85.5 % of the overall variance. Cone and seed characteristics were defined mainly by the first factor contributing to 25.62 % of the total variance. Cluster analysis divided the trees into two main clusters each consisting of genotypes with lanceolate leaves and ovate, respectively, exception to the thirty sub cluster of the first main cluster that had ovate leaves. The oldest accessions such as Y.A, Y.M, Y.Ch, Y.T₁, Y.T₂ and K.J1 were located in the first cluster. Most of the accessions, especially the oldest ones, in main first cluster had the open growth habit or semi-open, so that growth habit was semi-closed in second main cluster.     

地中海柏挥发油抗氧化、抑肿瘤及其化学表征

为研究地中海柏(Cupressus sempervirens L.)挥发油(CEO)的化学表征,寻找抗氧化、抑肿瘤的物质基础.利用分光光度法研究了CEO总抗氧化能力,及对活性氧自由基、DPPH·的清除作用和对肝组织脂质过氧化产物(MDA)形成的抑制作用;用GC-MS法结合人工解析鉴定化学成分,并用归一化法测定组分相对含量;用MTY与SRB法检测CEO对人鼻咽癌细胞(CNE)的抑制作用,采用形态学观察以及流式细胞术(FCM)检测了CEO体外抑肿瘤的效应.结果发现CEO能很好地清除自由基,显著抑制肝组织MDA的产生;在体外能抑制CNE细胞的增殖;初步推断CEO中占总量82.91%的烯类可能与抗氧化、抗肿瘤的活性有关.     

Organogenesis and somatic embryogenesis in Cupressus sempervirens

Adventitious buds of Cupressus sempervirens L., were formed on excised mature embryos cultured for 10 days on half-strength Quoirin and Lepoivre medium (1/2QP) with 10 M N⁶-benzyladenine. For shoot development, embryos were transferred to 1/2QP without growth regulators. Axillary shoot formation and rooting occurred spontaneously as adventitious shoots aged and transfer intervals were increased. Embryogenic tissue was obtained from immature embryos on induction media consisting of von Arnold and Eriksson (AE) or Gupta and Durzan (DCR) salts with 10 or 20 M 2,4-dichlorophenoxyacetic acid. Cultures were maintained on DCR with 5 M -naphthaleneacetic acid and 5 M BA.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - ABA absicic acid - AC activated charcoal - NAA -naphthaleneacetic acid - QP Quoirin & Lepoivre (1977) - AE von Arnold & Eriksson (1981) - DCR Gupta & Durzan (1985)     

Flora Palinologica Italiana,Sezione Aeropalinologica —S 103:Cupressus sempervirens L

Summary This contribution to the ?Palynological Italian Flora, Aeropalynological Section? deals withCupressus sempervirens L. (Mediterranean cypress). Palynological card regards fresh and acetolyzed pollen.      

A developmental variant of indentation in the scales of female cones of Cupressus sempervirens L.

A developmental variant, in which the contact zone between female cone scales is ectopically expressed in many female cones of Cupressus sempervirens , is common in trees growing in Israel. There are two phenotypes: type A with grooves emerging from the contact zone between two scales, and type B with a blind groove in the inner area of a scale. Some scales show a combination of both. These structures are formed constantly, but in different proportions in individual cones, trees and populations. Within the cone, more ectopic indentations are found in the basal scales. The female cones of other members of the Cupressaceae examined, namely Cupressus macrocarpa, C. arizonica and Thuja orientalis , did not have ectopic indentations.     

Nuclear and cell fusion cause polyploidy in the megagametophyte of common cypress, Cupressus sempervirens L.

In common cypress, Cupressus sempervirens L., the megagametophyte persists in mature seeds as a polyploid endosperm containing cells with even and odd series of DNA contents: 1C, 2C, 3C, 4C, 5C etc., where C is the amount of DNA in the haploid genome. In this study, cytometrical, histological and cytochemical investigations were performed in order to determine the behavior of megagametophyte nuclei during the reproductive cycle. Unexpected nuclear alterations due to a continuous process of nuclear fusion were observed in the megagametophyte, leading to polyploidization and consequently to intense food-reserve synthesis. During the free nuclear stage, the megagametophyte exhibited only sporadic nuclear fusion and limited food-reserve production. When cellularization took place, multinucleated compartments were observed in which nuclei fused, producing odd and even series of DNA contents as proved by flow-cytometric analysis. This polyploidization process considerably increased after fertilization and during embryo development, and was accompanied by increased food-reserve synthesis. During these later stages, fusion mainly involved nuclei of contiguous cells and was preceded by the disintegration of their adjacent walls. Mitoses with incomplete phragmoplast differentiation were also observed to yield polyploid nuclei. Finally, in mature seeds the endosperm still exhibited multinucleate cells and fusion nuclei, and contained high amounts of storage products. The results are interpreted as an alteration of DNA contents in the megagametophyte cells in relation to specific metabolic activity during seed development. Received: 2 September 1998 / Accepted: 31 December 1998     

Photosynthetic changes that occur during aging of cypress (Cupressus sempervirens L.) needles

2-years-old cypress needles (A2) were physiologically most active with regard to net photosynthetic (P _N) and electron transport rates. Variable to maximum fluorescence (F_v/F_m) ratios of dark-adapted needles were higher in A2 needles than in current year (A1) or senescent (A4) needles. Lower F_v/F_m values in these stages seemed to be caused not by photoinhibition but by a low photochemical capacity as suggested from the chlorophyll (Chl) a/b ratios. In isolated thylakoids, lower rates of whole chain and photosystem 2 (PS2) activities were observed in A4 needles, while higher rates were observed in A2 needles. A similar trend was noticed for contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC) and total soluble proteins. The artificial exogenous electron donor Mn²⁺ failed to restore the loss of PS2 activity in 3-year-old (A3) and A4 needles, while diphenylcarbazide and NH₂OH significantly restored the loss of PS2 activity. The marked loss of PS2 activity in A4 needles was primarily the result of the loss of 33, 28–25, 23, and 17 kDa polypeptides. A marked loss of RuBPC activity in A4 needles is mainly due to the loss of 15 (SSU) and 55 (LSU) kDa polypeptides.     

Cytological basis for a tetraspory in Cupressus sempervirens L. megagametogenesis and its implications in genetic studies

 The processes of megasporogenesis and early megagametogenesis were cytologically investigated in Cupressus sempervirens L. in order to elucidate, at the cellular level, the origin of the megagametophyte. After pollination, sporogenous tissue developed in the chalazal region of the nucellus, but only one megaspore mother cell differentiated and divided meiotically without cell-wall formation. This led to the development of a cell with four nuclei which directly functioned as a megaspore. The C. sempervirens megagametophyte is thus tetrasporic, in contrast to the majority of conifers where the megagametophyte is monosporic. The consequenses of this observation are discussed from a genetics point of view. Received: 15 August 1997 / Accepted: 19 September 1997     

Essential‐Oil Composition of the Tunisian Endemic Cypress (Cupressus sempervirens L. var. numidica Trab.)

The essential oils isolated from leaves, wood, and cones of the Tunisian endemic cypress Cupressus sempervirens L. var. numidica Trab. collected from three natural populations were characterized by GC‐FID and GC/MS analyses. In the wood, leaf, and cone oils, 38, 35, and 26 constituents, representing 94.4, 97.8, and 98.5% of the total oil composition, respectively, were identified. Monoterpenes constituted the major fraction of the oils from all organs and for all populations. The oils were found to be of an α‐pinene (64.2%)/δ‐car‐3‐ene (11.1%) chemotype with considerable contents of α‐humulene (3.4%) in the leaf oil, cedrol (2.8%) in the wood oil, and sabinene (3.2%) in the cone oil, respectively. α‐Pinene, δ‐car‐3‐ene, limonene, carvacrol methyl ether, α‐humulene, and α‐amorphene were the main components that differentiated the oils of the three organs in the cypress of Makthar.     

Enhancing planting stock quality of Italian cypress (Cupressus sempervirens L.) by pre-cultivation in mini-plugs

Native species are important in ecological restoration of degraded forest ecosystems provided that their ecological engineering potential is exploited. Their failure to get established is usually due to the use of low-quality planting stock and adverse site conditions. Mini-plug transplants are a relatively new and promising production system in the forest nursery area. Objectives of our study were the evaluation of seedling production of Italian cypress (Cupressus sempervirens) in mini-plug containers, the study of the effect of mini-plug density and substrate on planting stock quality, the comparison of the performance between mini-plug seedlings and the standard planting stock produced by Greek nurseries and the evaluation of physiological and morphological variables as predictive indicators of Italian cypress field performance. Our results showed that cypress seedlings could be produced using the mini-plug technique, resulting in higher quality seedlings than the standard stock type. Pre-cultivation of cypress seedlings under favorable conditions for a period of 5 weeks using peat and high mini-plug densities (1800–3500 mini-plugs m^?2) could be recommended. Grading of seedlings by using both easily measured morphological variables (root length, leaf area, root and shoot dry weight) and physiological tests, such as shoot electrolyte leakage, may improve survival and transplanting success. It is concluded that mini-plugs can serve ecological restoration of degraded forest ecosystems more efficiently than standard planting stock.     

Taxonomic differentiation of Cupressus sempervirens and C. atlantica based on morphometric evidence

We investigated the interspecific morphological relationships of two related Mediterranean Cupressus taxa and the possible geographic differentiation of Eastern Mediterranean C. sempervirens using morphometric analysis. A total of 446 individuals, representing eight C. atlantica (Morocco) and 10 C. sempervirens (Greece, Turkey and Lebanon) populations were examined. The 17 morphological characters of cone, seed and shoot with leaves were measured and analysed statistically. Significant morphological differences between C. atlantica and C. sempervirens were detected for the majority of analysed characters. For C. sempervirens, significant differences were also detected and these were related to its three regions of origin, the Greek Islands, Taurus Mts and Lebanon Mts. Multivariate analyses did not indicate a clear geographic pattern of differentiation in C. sempervirens populations. A relatively low level of phenotypic differentiation was detected between populations originating from the Greek Islands and Taurus Mts. We also observed a moderate multivariate differentiation between the Lebanese and remaining C. sempervirens populations. Phenotypic differentiation detected in the Asian part of the C. sempervirens range reflects patterns of differentiation similar to those described for other taxa occurring in the Taurus and Lebanon Mts.     

Photoinhibition and Recovery of Photosynthesis in Canker-susceptible and Resistant Needles of Cypress (Cupressus sempervirens L.)

The aim of the present investigation was to test the hypothesis that the cypress canker caused by a fungus (Seiridium cardinale) infection induced effects on photosynthesis which could be related to photoinhibition and the process of recovery in susceptible and resistant needles. Photoinhibition of photosynthesis and recovery was studied in canker‐infected susceptible and resistant needles of cypress (Cupressus sempervirens L.) under controlled conditions (irradiation of detached needles to approximately 1900 μmol/m²/s). The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll (Chl) fluorescence (F_v/F_m) and electron transport measurements. The potential efficiency of photosystem (PS) II, F_v/F_m declined, and F_o increased significantly in canker‐susceptible needles, while F_o did not change in resistant needles. In isolated thylakoids, high light (HL) decreased the rate of whole chain and PS II activity markedly more in susceptible than in resistant needles. A smaller reduction of PS I activity was noticed only in susceptible needles. Upon subsequent dark incubation, fast recovery was noticed in both needle types and reached maximum rates of PS II efficiency similar to those noticed in non‐photoinhibited needles. The artificial exogenous electron donors such as diphenyl carbazide (DPC), NH₂OH and Mn²⁺ failed to restore the HL induced loss of PS II activity in susceptible needles, while DPC and NH₂OH significantly restored it in resistant needles. The results suggest that HL inactivates the donor side of PS II in resistant and the acceptor side of PS II in susceptible needles. The results on the quantification of the PS II reaction centre protein D1 and 33 kDa protein of water‐splitting complex following HL exposure showed pronounced differences between susceptible and resistant needles. The marked loss of PS II activity in HL‐irradiated needles was due to the marked loss of D1 protein in susceptible and 33 kDa protein in resistant needles, respectively.     

The Annual Rhythm of Activity of the Lateral Meristems (Cambium and Phellogen) in Cupressus sempervirens L

The annual rhythm of cambial activity in Cupressus sempervirensseems to be endogenous and fits well the Mediterranean climaticrhythm. Under outdoor conditions the cambium begins its activitytowards the autumn when temperatures drop and water is lesslimiting. The cambium continues to be active during the mildMediterranean winter and enters dormancy towards the beginningof the dry summer. A constant supply of water is not enoughto avoid cambial dormancy but may result in earlier onset anda prolonged period of activity. Relationships between xylemand phloem production, between shoot and root, and between cambialand phellogen activity are discussed. Cupressus sempervirens L., cypress, cambium, phellogen, annual rhythm, wood production     

Somatic plant regeneration from selected common cypress (Cupressus sempervirens L.) clones resistant to the bark canker disease

Regeneration of nodular cultures involves the production of new individuals from organogenic nodules. Despite the existence of well-established protocols for bromeliads species, many gaps in understanding the mechanisms of this route still remain, particularly the regeneration of new shoots, a stage little explored. Therefore, this work aimed to monitor the morpho and histodifferentiation processes of shoot regeneration from nodular cultures of Billbergia zebrina. To accomplish this, nodular cultures previously obtained from nodal segments of B. zebrina were transferred to regeneration induction medium consisting of semi-solid MS medium supplemented with 1.0 µM of naphthalene acetic acid and 2.0 µM of benzylaminopurine, respectively. Cultures were maintained in a growth chamber over 8 weeks, and samples were collected weekly for analysis under light, transmission electron, and scanning electron microscopy. Cells in the shoot formation region presented characteristics of mitotically active cells, such as the presence of numerous plasmodesmata and conspicuous microtubules. New shoots maintained vascular connection to the node. Cells binding the nodule vascular bundle toward the shoot showed degeneration of organelles, which, in turn, originated vascular neoelements. The dome presented a depressed or prominent surface, depending on shoot development. Rounded stomata were conspicuous in the leaf primordia and generally higher in number as compared to the epidermal surface. This study elucidated the nodular culture in vitro system in aspects associated to the regeneration of shoots and it is anticipated that such data will lead to the optimization of present nodular cultures based protocols with the aim of conserving endangered bromeliad species.