Metabolic and genes expression analyses involved in proline metabolism of two rose species under drought stress.

An investigation was conducted to assess proline anabolism and catabolism pathway genes under drought stress. Treatments were irrigation in three levels (25, 50 and 100% field capacity) at 1, 3, 6 and 12 d and two rose species (Rosa canina L. and Rosa damascene Mill.). The results showed that the potential for proline accumulation in R. damascena was higher than R. canina under drought. Simultaneous with proline accumulation, expression of P5CS and P5CR genes increased from 1 to 12 d under 50% FC whereas their expression had an increasing trends from 1 to 6 d at 25% FC and expression of both genes decreased at 12 d in both species. The highest accumulation of proline was observed under 25% FC at 12 d, but expression of genes involved in proline synthesis main pathway decreased on this day. Furthermore, expression of genes (PDH and P5CDH) involved in proline catabolism pathway decreased in 50% FC from 1 to 12 d while their expression remarkably decreased from 1 to 6 d and increased at 12 d under 25% FC. These findings showed that under conditions of 50 and 25% FC, arginine accumulation resulted in the increased expression of the ARG gene, which led to ornithine production. Furthermore, ornithine accumulation increased OAT expression. Therefore, it seems that OAT-induced P5C is transported from the mitochondria to the cytosol and reduced to proline by the P5CR.

Regulation of stomatal aperture in response to drought stress mediating with polyamines, nitric oxide synthase and hydrogen peroxide in Rosa canina L.

To assess the role of genes involved in polyamines synthesis, nitric oxide synthase (NOS), copper amine oxidase activity (CuAO) and hydrogen peroxide (H2O2) in regulation of stomatal aperture to drought stress in Rosa canina L., a study was performed at three irrigating levels (25%, 50%, and 100% field capacity) with three replications at 1, 3, 6 and 12 days. The results showed that putrescine (Put) accumulation occurred under both 50% and 25% FC at 1 d. Furthermore, the role of the Put direct biosynthesis pathway ornithine decarboxylase (ODC) was more effective under 50% FC whereas in the 25% FC the Put indirect production pathway (agmatine iminohydrolase (AIH), N-carbamoyl putrescine amidohydrolase (CPA) and arginine decarboxylase (ADC)) was more effective. HPLC results showed that the accumulation of spermidine (Spd) and spermine (Spm) is consistent with the expression of S-adenosyl methionine decarboxylase (SAMDC), spermidine synthase (SPDS) and spermine synthase (SPMS) genes. Spd accumulation under both 50% and 25% FC occurred on the 3 d and then decreased in the other days. Spm content showed an increasing trend from 6 d under 50% FC and from 3 d under 25% FC. Our results suggest that among the measured polyamines, Put oxidation through CuAO activity increased resulted in an increase in H2O2 production. The H2O2 accumulation also as a secondary messenger led to enhance in NOS gene expression. Increase in NOS gene expression can act as a signal resulting in stomatal closure.

Role of genes and metabolites involved in polyamines synthesis pathways and nitric oxide synthase in stomatal closure on Rosa damascena Mill. under drought stress.

In order to evaluate the genes involved in polyamines synthesis pathway and the role of nitric oxide synthase (NOS) and H2O2 in stomatal closure under drought stress, a research conducted with three irrigation levels (100, 50 and 25% field capacity) at 1, 3, 6 and 12 days on Rosa damascena Mill. HPLC and qPCR results showed that putrescine (Put) accumulation occurred at first day in both 50 and 25% of field capacity and then decreased the other days. Furthermore, Put accumulation in the indirect pathway (ADC, AIH and CPA) was more effective related to the direct pathway (ODC) under severe stress. Increased expression of genes involved in production of spermidine (Spd) and spermine (Spm) i.e., SAMDC, SPDS and SPMS correlated with the highest accumulation of Spd and Spm under 50% FC at 6 d and 25% FC at 12 d, respectively. Moreover, results showed that Put reduction simultaneously accumulated H2O2, which subsequently increased NOS expression suggesting a key signal for stomatal closure.

Applying the AOGCM-AR5 models to the assessments of land suitability for walnut cultivation in response to climate change: A case study of Iran.

Due to higher temperatures and lower water availability, climate change is likely to have a major impact on walnut production in the near future. Climate change will alter the land suitability for walnut cultivation around the world, especially in arid and semi-arid regions like Iran. Here, land suitability for the cultivation of walnut (Juglans regia L.) in Iran was determined using the GIS for present and future conditions (2020-2049) with an approach to climate change. Accordingly, data from 375 synoptic stations throughout Iran were gathered for climatic factors including average, minimum and maximum temperatures, relative humidity and chilling requirement. Also, ASTER sensors (Advanced Spaceborne Thermal Emission and Reflection Radiometer) and their data provided this research with cells that make a precision of 150 m (5 s), and the data were used for gauging geological parameters such as altitude and land slope. The electrical conductivity (EC) of soil and water were informed by the data bank of the Iranian Water Resources Management. The results of temperature simulations for the future (2020-2049) were analyzed by 21 AOGCM-AR5 models under the RCP4.5 emission scenario. In the first phase of evaluations, the maps of land suitability were constructed for present conditions by considering a network of the above-mentioned parameters. By combining these layers of information, the final map of land suitability was illustrated for walnut cultivation. In the second phase, the NEX-GDDP was used in order to determine land suitability for the future (2020-2049). The results showed that Iran currently has 582844 km2 of land suitable for walnut cultivation. However, the future will see less suitable lands: the current area will be reduced by 6.19%, from 582844 km2 to 546710 km2. In general, the northern, northwestern and western margins of Iran are currently suitable for walnut cultivation. By approximation, these lands will also be major areas for prospective cultivations of walnut in the future (2020-2049), even though their current stretch will be reduced.

Isopentenyl Transferase (IPT) Gene Transfer to Perennial Ryegrass Through Sonication-Assisted Agrobacterium-Mediated Transformation (SAAT), Vacuum and Heat Treatment.

The successful introduction of isopentenyl transferase (IPT) gene into perennial ryegrass, cultivars Numan and Grassland using Agrobacterium tumefaciens via three explants (callus, seed and meristem tip) under three individual experiment was evaluated. In the first experiment, the calli were inoculated with LBA4404 Agrobacterium strain under vacuum, heat and in combination of both at 42 °C for 5 min followed by vacuum treatment (390 mm Hg pressure) for 15 min. Sonication-assisted Agrobacterium-mediated transformation (SAAT) was applied for seed and meristem tip transformation of perennial ryegrass for the first time. Results showed positive effects of heat treatment on transformation efficiency during Agro-infection in both cultivars. However, heat shock treatment was more effective in 'Grassland' than 'Numan' (14.2% vs 9.2%). In addition, high transformation efficiency of about 46.65% and 29.15% was observed using meristem tip explants of 'Grassland' and 'Numan' based on IPT and RD29A positive PCR results, respectively. Seed transformation efficiency in 'Grassland' and 'Numan' under SAAT method reached to 37.5% and 16.65%, respectively. Results of these experiments revealed that LBA4404 strain was more efficient than GV3101 in transformation of both perennial ryegrass cultivars. The DNA-blot analysis confirmed that a single T-DNA copy of the IPT gene was integrated into the genomic DNA of the positive transgenic T0 plants which obtained from callus and meristem tip explants of 'Grassland' after heat and SAAT treatment, respectively. Because monocots are not the host of Agrobacterium tumefaciens, this novel protocol can be used in further experiments on genetic transformation of perennial ryegrass cultivars.

Temporal characterization of 2-phenylethanol in strongly and weakly scented genotypes of damask rose.

The molecular and physiological properties of 2-phenylethanol (2-PE) in the strongly scented genotype (SSG) and a weakly scented genotype (WSG) of damask rose at six floral developmental stages were investigated. The chemical compositions of volatile emissions were determined by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analysis of the floral headspace. In both genotypes, the relative percentage of 2-PE increased more in SSG than WSG, as flowers developed. In the petals of damask rose the relative transcript levels of phenyl acetaldehyde reductase (PAR) were higher at stages 3 and 4 in SSG and WSG, respectively. Also, the expression pattern of PAR indicated a significant difference between two genotypes during flower developmental stages. In this study, enzymatic activity leading to the synthesis of 2-PE from the phenyl acetaldehyde (PAld) moderately increased during flower development up to stage 5 in SSG. However, high level of PAR enzymatic activity was observed in stage 3 of WSG. These results indicated that the pattern activity of PAR was different in two used genotypes of damask rose. For SSG, PAR activities were low in early stage of flower development and then gradually increased reaching its highest value at full bloom stage. In WSG, no significant change in enzyme activity was seen after stage 3.

Application of ISSR markers to analyze molecular relationships in Iranian jasmine (Jasminum spp.) accessions.

There are many species of jasmines in different regions of Iran in natural or cultivated form, and there is no information about their genetic status. Therefore, inter-simple sequence repeat (ISSR) analysis was used to evaluate genetic variations of the 53 accessions representing eight species of Jasminum collected from different regions of Iran. A total of 21 ISSR primers were used which generated 981 bands of different sizes. Mean percentage of polymorphic bands was 90.64 %. Maximum resolving power, polymorphic information content average, and marker index values were 21.55, 0.35, and 14.42 for primers of 3, 4, and 3 respectively. The unweighted pair group method with arithmetic mean dendrogram based on Jaccard's coefficients indicated that 53 accessions were divided into two major clusters. The first major cluster was divided into two subclusters; the subcluster A included Jasminum grandiflorum L., J. officinale L., and J. azoricum L. and the subcluster B consisted of three forms of J. sambac L. (single, semi-double, and double flowers). The second major cluster was divided into two subclusters; the first subcluster (C) included J. humile L., J. primulinum Hemsl., J. nudiflorum Lindl. and the second subcluster (D) consisted of J. fruticans L. At the species level, the highest percentage of polymorphism (34.05 %), numbers of effective alleles (1.16), Shannon index (0.151), and Nei's genetic diversity (0.098) were observed in J. officinale. The lowest values of percentage polymorphism (0.011), number of effective alleles (1.009), Shannon index (0.007), and Nei's genetic diversity (0.005) were obtained for J. nudiflorum. Based on pairwise population matrix of Nei's unbiased genetic identity, the highest identity (0.85) was found between J.officinale and J. azoricum and the lowest identity (0.69) was between J. grandiflorum and J. perimulinum. Based on analysis of molecular variance, the amount of genetic variations among the eight populations was 83 %. This study demonstrated that the ISSR is an useful tool in jasmine genomic diversity studies and to detect their relationships.

Seismomorphogenesis: a novel approach to acclimatization of tissue culture regenerated plants.

Plantlets under in vitro conditions transferred to ex vivo conditions are exposed to biotic and abiotic stresses. Furthermore, in vitro regenerated plants are typically frail and sometimes difficult to handle subsequently increasing their risk to damage and disease; hence acclimatization of these plantlets is the most important step in tissue culture techniques. An experiment was conducted under in vitro conditions to study the effects of shaking duration (twice daily at 6:00 a.m. and 9:00 p.m. for 2, 4, 8, and 16 min at 250 rpm for 14 days) on Sansevieria trifasciata L. as a model plant. Results showed that shaking improved handling, total plant height, and leaf characteristics of the model plant. Forty-eight hours after 14 days of shaking treatments with increasing shaking time, leaf length decreased but proline content of leaf increased. However, 6 months after starting the experiment different results were observed. In explants that received 16 min of shaking treatment, leaf length and area and photosynthesis rate were increased compared with control plantlets. Six months after starting the experiment, control plantlets had 12.5 % mortality; however, no mortality was observed in other treated explants. The results demonstrated that shaking improved the explants' root length and number and as a simple, cost-effective, and non-chemical novel approach may be substituted for other prevalent acclimatization techniques used for tissue culture regenerated plantlets. Further studies with sensitive plants are needed to establish this hypothesis.

Micropropagation of Araucaria excelsa R. Br. var. glauca Carrière from orthotropic stem explants.

The objectives of the present work were in vitro propagation of Araucaria excelsa R. Br. var. glauca Carrière (Norfolk Island pine) with focus on the evaluation of the mean number of shoots per explant (MNS/E) and mean length of shoots per explants (MLS/E) produced by different parts of the orthotropic stem of A. excelsa R. Br. var. glauca in response to plant growth regulators. Norfolk Island pine axillary meristems responded very well to the 2-iso-pentenyl adenine (2iP) and thidiazuron (TDZ) levels. Explants taken from stem upper segments in the media containing 2iP had a higher MNS/E (3.47) and MLS/E (6.27 mm) in comparison to those taken from stem lower segments, which were 0.71 and 0.51 mm, respectively. Using 0.045 µM TDZ in the MS medium not only resulted in 4.60 MNS/E with 7.08 mm MLS/E but proliferated shoots showed a good performance as well. Investigating the best position of stem explant on mother plant as well as the best concentrations of growth regulators were performed which were useful for efficient micropropagation of this plant. Thirty three percent of explants were rooted in the MS medium containing 3 % sucrose, supplemented with 7.5 µM of both NAA and IBA for 2 weeks before transferring to a half strength MS medium without any growth regulator. Plantlets obtained were acclimatized and transferred to the greenhouse with less than 20 % mortality. This procedure considered the first successful report for regeneration and acclimatization of A. excelsa R. Br. var. glauca plantlet through main stem explants.

RAPD fingerprint to appraise the genetic fidelity of in vitro propagated Araucaria excelsa R. Br. var. glauca plantlets.

Randomly amplified polymorphic DNA (RAPD) was used as a tool to assess the genetic fidelity of in vitro propagated Araucaria excelsa R. Br. var. glauca with explants taken from orthotropic stem along with their related mother plants after treatment with kinetin, 2iP, BA (0.02-0.26 mg/l) and TDZ (0.001-1 mg/l) to produce axillary shoots. TDZ and kinetin induced more shoot and higher length per explant. Results showed a total of 1,676 fragments were generated with 12 RAPD primers in micropropagated plants and their donor mother plants. The number of loci ranged from 6 in OPB 12-18 in OPY 07 with a size ranging from 250 bp in OPH 19-3500 bp in OPH 11. Cluster analysis of RAPD data using UPGMA (unweighted pair group method with arithmetic average) revealed more than 92% genetic similarities between tissue cultured plants and their corresponding mother plant measured by the Jaccard's similarity coefficient. Similarity matrix and PCoA (two dimensional principal coordinate analysis) resulted in the same affinity. Primers had shown 36% polymorphism. However, careful monitoring of tissue culture derived plants might be needed to determine that rooted shoots are adventitious in origin.

Micropropagation of Lilium ledebourii (Baker) Boiss as affected by plant growth regulator, sucrose concentration, harvesting season and cold treatments

A protocol for the micropropagation in different harvesting time of Lilium ledebourii (Baker) Boiss, an endangered rare species endemic to Iran has been developed. In vitro scale culture of this species, using bulbs from three harvesting seasons (spring, summer and winter), was attempted. Among the various treatments tested, the Murashige and Skoog (MS) medium supplemented with 0.1 mg l-1 naphthaleneacetic acid (NAA) + 0.1 mg l-1 benzyladenin (BA) and 6% sucrose in all harvesting seasons proved to be superior to others. The best results for fresh weight of bulblets, rooting parameters and the survival rate after transplantation to greenhouse were obtained from early winter-harvested bulbs. Summer-harvested bulbs had the highest number of bulblets per explant. The bulblets at the end of the culture period were given cold treatment at 4ºC for 2-8 weeks at a 2-weeks interval and then transplanted to a potting mixture of sand, leaf mold and peat moss (1:1:1 v/v). The best emergence rate (90%) was achieved at 8 weeks cold treatment for winter harvested bulbs.

Microsatellite analysis of Damask rose (Rosa damascena Mill.) accessions from various regions in Iran reveals multiple genotypes.

BACKGROUND: Damask roses (Rosa damascena Mill.) are mainly used for essential oil production. Previous studies have indicated that all production material in Bulgaria and Turkey consists of only one genotype. Nine polymorphic microsatellite markers were used to analyze the genetic diversity of 40 accessions of R. damascena collected across major and minor rose oil production areas in Iran. RESULTS: All microsatellite markers showed a high level of polymorphism (5-15 alleles per microsatellite marker, with an average of 9.11 alleles per locus). Cluster analysis of genetic similarities revealed that these microsatellites identified a total of nine different genotypes. The genotype from Isfahan province, which is the major production area, was by far the most common genotype (27/40 accessions). It was identical to the Bulgarian genotype. Other genotypes (each represented by 1-4 accessions) were collected from minor production areas in several provinces, notably in the mountainous Northwest of Iran. CONCLUSION: This is the first study that uncovered genetic diversity within Damask rose. Our results will guide new collection activities to establish larger collections and manage the Iranian Damask rose genetic resources. The genotypes identified here may be directly useful for breeding.