نشریه تحقیقات ژنتیک و اصلاح گیاهان مرتعی و جنگلی ایران
سال بیست و چهارم شماره 1 (پیاپی 47، بهار 1395)

Effects of different concentrations of plant growth regulators and culture media on shoot proliferation and vitrification of Prunus mahaleb L. via in vitro culture was studied using single node explants in twoseparate experiments. Shoots from single node explants were grown on 26 treatments (first experiment) of MS medium supplemented with different concentrations of cytokinins (BAP, TDZ, KIN, 2iP, and Zeatin) in combination with NAA and on 16 treatments (second experiment) of MS, 1/2 MS, WPM and DKW media supplemented with different combinations of CaCl2 and NH4NO3 and cytokinins of BAP, Zeatin, and PG. Experiments were based on completely randomized design with five replicates and each replication included 5 explants. Means comparison after 2 subcultures in the first experiment showed that the highest shoot length (3.14 cm) were obtained on 0.1 mg l-1 NAA in cytokinin -free medium, the highest number of shoot (14.67 shoot numbers per explants) on 0.2 mg l-1 Zeatin plus 0.1 mg l-1 NAA, and the lowest vitrification on 0.1 mg l-1 KIN, 0.05 and 0.1 mg l-1 Zeatin plus 0.1 mg l-1 NAA, respectively. In the second experiment, the highest shoot length (2.88 cm) were observed on DKW medium with 0.2 mg l-1 Zeatin plus 40 mg l-1 PG and the highest number of shoot (19 shoot number per explants) on MS medium with 880 mg l-1 CaCl2 and 825 mg l-1 NH4NO3. The present research by accessing to the highest proliferation (19 shoot numbers per explants) and decreasing vitrification of Prunus mahaleb L. provided a possibility of doing other research projects for protocol commercialization.

Alfalfa (Medicago sativa L.) is one of the most important forage crops because of high quality, growing extensively in farms and rangelands of Iran. Genetic variability is one of the most aspects for crop improvement. The experiments were carried out to determine genetic variability among 17 alfalfa populations based on ISSR and cytogenetic markers. All of the tested ISSR primers showed 44 visible bands. Three primers had the most number of bands while another primers showed the least band numbers. Mean percentage of polymorphism in the primers was 90.18 percent. Polymorphic information content (PIC) and marker index (MI) average were 0.36 and 1.88 respectively. Cluster analysis classified the populations genotype into four clusters. In order to investigate karyotipic variation between the populations, the populations were studied for several karyotipic characteristics. Including long arm, short arm and total length of the chromosomes, long arm to short arm and short arm to long arm ratios and total form percentage (TF%). All of the genotypes had 32 chromosomes. Analysis of variance of mitotic data in completely randomized design, showed highly significant differences between the studied genotypes for all of the studied traits (p

Fennel (Foeniculum vulgare) is one of the most important medicinal plants with many usages in medical, food and cosmetic industries. Development method of two early and medium maturity synthetic cultivars of fennel with the aim of drought tolerance is discussed. The synthetic cultivars were assessed along with their elite parents at two separate environments, with normal irrigation and drought stress conditions. Results showed on the two environmental conditions, there were significant differences among the genotypes. Mean comparison of genotypes showed that in non-stress conditions, the most seed yield belonged to parental medium maturity genotypes of Meshkinshahr and Fozveh; and in drought stress conditions belonged to early and medium maturity synthetic cultivars. Independent mean comparison among the synthetic cultivars and the parents in each growth conditions showed that; in stress conditions according to the elite parents the synthetic cultivars had higher seed and essential oil yields. Finally, for enhancement of drought tolerance of fennel species, breeding method of synthetic cultivar is recommended, so that in environments with variable climate, especially water shortage, the mentioned cultivars can produce satisfactory yield.

Most salinity problems causes by NaCl distribution in dry lands, beaches and water resources. This research was carried out to estimate salinity resistance of three Eucalyptus species: Eucalyptus largiflorens¡ E. sideroxylon and E. wandoo. Five NaCl levels (0, 50, 100, 150 and 200 mM) of salinity were used in three replications by a factorial experiment model based on a completely randomized design. Sampling were made from terminal leaves of the selected trees. Different characteristics such as pigments, total chlorophyle, chlorophyle a and b, carotene, soluble sugar and prolin, glycine betaine, growth parameters such as biomass, leaf area, relative water content, water saturation difference, specific leaf area and leaf water content per unit leaf area were recorded. Increased salt stress lead to increment of prolin level, soluble sugar, withering, falling, dried leaves and decrement of pigments and growth parameters in the species. Between the studied species, E. largiflorens showed the most salt stress tolerance, although for prolin, soluble sugar, pigments, glycine betaine, R/S, RWC, wet and dry root and shoot weight, had the highest values and for the withering, falling and dried leaves had the lowest values.

Black cumin (Nigella sativa) is an important medicinal plant belongs to the Ranunculaceae which produce large diversity of secondary metabolites including industrial and medicinal monoterpenes/ triterpenes. In the present work, relative gene expression of genes involved in biosynthetic pathway of terpenes including a monoterpene synthase (MTS), geranyldiphosphate synthase (GDS), β- amyrin synthase (βAS) and squaleneepoxidase(SQE) were investigated in black cumin plants treated with methyl jasmonate. The plants were grown in a greenhouse sprayed with 0.1 mM methyl jasmonate. Samples were collected at 0, 4, 8, 24, 48 and 72 hours after applying the treatments. RT-PCR was performed by gene-specific primers after RNA extraction and cDNA synthesis. Results of semi quantitative RT-PCR showed that the expression pattern of GDS gene differed with the results of other genes. The highest gene expression of GDS was observed at 4 h and its expression remained at the same level till 8 h then dropped at next time periods. The expression patterns of monoterpene synthase, β-amyrin synthase and squaleneepoxidase showed rising trend. Gene expression analysis of the genes at transcript level using semi-quantitative PCR in black cumin leaves treated by methyl jasmonate revealedinduced-geneexpression which might show the roles of terpenes in defense mechanisms and signal transduction. Consequently, methyl jasmonate treatment can be considered to induce the terpene biosynthetic pathway and to study the enzymes/ metabolites associated with terpenoid metabolism.

Woody vegetative coverage of Zagros forests is mainly composed by oaks. Information on natural patterns of genetic variation is of fundamental practical importance for sustainable forest conservation and management. In the present study, to assess the genetic differentiation and population diversity of eight Quercus brantii populations with 104 genotypes from Ilam forests, genomic DNA was extracted and then amplified fragments from different primers were visualized and scored on 1.2% agarose gel. Of 22 screened ISSR primers, 15 primers produced repeatable and scorable bands. Dice and Jaccard similarity matrix were analysed. Then the data were molecularly analyzed using molecular analysis software, GenAlEx. The selected primers amplified 189 bands, from which all were polymorphic. The number of amplified fragments varied from 7 to 20 with an average of 12.6 bands per primer, and with size range of 100 to 1800 bp. Analysis of molecular variance (AMOVA) showed that major proportion of total genetic variation (79%) was attributed to within populations, and the remaining 21% was resulted from among population variation. High level of genetic variation was observed within Q. brantii natural populations from Ilam forests.

Castor (Ricinus communis L.) is one of the most ancient medicinal oil crops in the world. It has been vastly distributed in different parts of Iran. Association analysis based on 16 combinations of ISSR primers was applied in order to identify ISSR markers associated with genes controlling 26 morphological traits in 12 populations of castor. The genotypes were evaluated for morphological traits in randomized complete block design with three replications in experimental field of Agricultural Research Center of West-Azerbaijan during 2012. Locus UBC-859 was the most effective marker to assessment the diversity in populations of castor. Based on 116 loci produced by ISSR primers, the studied genotypes divided into 6 subpopulations. Using mixed linear model (MLM), 26 loci were identified to be significantly associated with 17 morphological traits. R2 values ranged from 12.4% (UBC857-5) to 28.1% (UBC844-2). Most of the identified markers were associated specifically with one trait. UBC812-8 was associated with 8 traits. Common markers between some of the studied traits can be due to linkage or pleiotropic effects. The common markers lead to increase the efficiency of marker-assisted selection in plant breeding programs via simultaneously selection for several traits.

Genus Berberis include 660 species and only one of them, Berberis vulgaris is cultivated as a horticultural species. Berberis vulgaris is propagated via cutting and souker that is very time consuming. Using tissue culture method may product larg number of plants in a short period of time. In this research, effects of plant growth regulating hormones on micropropagation of Berberis vulgaris were studied. Axillary buds were sterillized as explants. The explants were transferred to different medium after sterilization stages including mercuric chloride, 70% ethanol and sodium hypochlorite supplemented with different concentrations of BAP, IAA, Kin and GA3 growth regulating hormones. The cultures were kept in a growth room at 25±2 ˚C in 16/8 h light and dark periods. Results showed that 3% NaOCl for 15 minutes was the best sterilization treatment. Also ANOVA results indicated that MS medium supplemented with 1 mg/l BAP was the best medium for regeneration. The most number of shoots (6) was observed in MS supplemented with 2 mg/l BAP 0.01 mg/l IBA. Therefore, BAP was the best hormone for regeneration of Berberis vulgaris.The best medium for root initiation was MS containing 0.5 mg/l IBA. The regenerated plant were succesfuly transferred to Jify pots and then to soil.

RNA extraction from hard wood tissues, especially in coniferous family trees, is very hard because they have high concentrations of phenolic compounds, polysaccharides, secondary metabolites and lignin. Moreover, up to now, no commercial kit is made for the mentioned purpose. In this study, the problems were resolved and a rapid, simple, and economic protocol is presented for RNA extraction from bark tissue of Cedrus deodara and Abies grandis, which belong to conifer family. Ratios of optical density of 260/280 nm and 260/230 nm were respectively obtained as 1.88 and 1.1 for Cedrus deodara and 1.8 and 0.9 for Abies grandis. Modifications for optimization of RNA extraction were preheating treatment of extraction buffer, and decreasing centrifuge rounds to eliminate DNA contaminations and DEPC. Synthesis of cDNA was performed using extracted RNA. RT-PCR successfully produced expected bands. Therefore, it seems that the presented protocol is suitable for RNA extraction from woody tissues.

Smirnovia iranica Sabeti is an endemic shrub species in Fabaceae family. It is found in sand dunes of central parts of Iran. It is very important for soil conservation, forage, landscape and medicinal values. RAPD is a useful genetic marker to determine polymorphism and relationship among plant species. In order to evaluate genetic diversity within and between Smirnovia iranica populations by 11 RAPD primers, 20 samples were collected from 4 sites of sand dune of Kashan. According to diversity indices, Dice similarity, cluster analysis and band information a total of 72 bands were amplified and 61 of the bands were polymorphic. Cluster analysis revealed a high amount of diversity among the studied samples. The most similar samples with 0.97 similarity were found in site 4, while the least similarity (0.65) was obtained for samples from different sites. Cluster analysis classified the samples in five groups. Two groups had only one sample. However, the other samples were distributed in other three groups one of which contained all samples of site 4. There was relatively fair coordination between genetic divergence and geographical origins.

The present study optimized micropropagation protocol for Ferula assa- foetida L. The effect of cytokinins (BAP, 2ip and Kinetin), auxins (IBA, NAA and Picloram) and hormones combination was assayed in different concentrations (0 - 2.5 mg/l) on MS medium. Results showed that BAP, promoted the highest shoot elongation and shoot numbers. Concentration of 1 mg/l BAP enhanced three shoots per explants and compared to control and 66 percent shooting was increased. Effect of 1.5 mg/l BAP produced the highest shoot. MS media containing 1mg/l BAP and the 0.01 mg/l of IBA was the best treatment to micropropagate ferula and tuber production. Hormone combination enhanced 3.96 cm plant height, compared with control (2.28 cm) at the rate of 57.6 percent increase. Number of lateral shoots with an average of 3 per explants grew three fold higher than control. The study proved that root like tuber in compared to long roots had higher ability to shoot production. Endogenous auxin in ferula is the most important reason for the existence of root elongation in vivo and in vitro.

Satureja edmondiis an important medicinal plant species which is one of the rare endemic plant species of Iran. Since seeds of the species are not found enough in the nature and the available seeds hardly germinate as well, micropropagation and mass reproduction is required in order to prevent the extinction of the species. Hence, the green branches of the species were cultured on MS medium (with half contents of the KNO3 and NH4NO3) to increase the number of branches. Then, the branches were transferred to 1/2 MS and DKW media with different levels of BAP, 2iP and IBA hormones. Then the branches were transferred to DKW medium containing NAA and IBA. Most of the branch productions were occurred on the DKW media, containing 0.5 mgl-1BAP and the best hormone for rooting was 0.5 mgl-1IBA. Regenerated plantlets were established in greenhouse and then grown under field conditions.

Fennel (Foeniculum vulgare Mill.) is an important, well-known aromatic and medicinal herb. In order to evaluate karyotipic variation and relationship between karyotipic traits of 16 Iranian Foeniculum vulgare accessions, an experiment was conducted based on a completely randomized design with five replications at biotechnology laboratory of Payame Noor University of Kermanshah, Iran. Using squash method several kariotipic characters were measured. All of the 16 fennel accessions had 22 chromosomes. Analysis of variance showed significant differences among the accessions for the studied traits. There was significant variation among the accessions for total chromosome form. There were significant differences between the groups for the karyotypic traits. Two satellites were observed in the accessions 14726 (Markazi), 8615 (Ardabil), 29667 (Ghazvin). One satellite was also observed in the other studied accessions. Based on Stebbins table, all of the accessions, except 18650 (Ardabil) located in 1A class while Ardabil accession located in 2A class. Hormozgan (24482) accession had minimum asymmetrical index while Ardabil accession had the greatest amount of Asymmetrical index value. Cluster analysis classified the accessions into 4 groups. Principle components analysis revealed that 99 percent of variance was explained by the first two principal components.

In order to study symbiosis effects of Sinorhizobium sp. on sainfoin (Onobrichis vicifolia) growth, 6 strains of Sinorhizobium sp. were collected from sainfoin fields in (Ardabil, Shahrekord, Samirom, Karaj, Khorramabad). The strains were inoculated on four sainfoin ecotypes originated from Ghazvin, Kermanshah, Aligoodarz and Fridonshahr, Iran. A factorial experiment on the basis of completely randomized design with four replications was conducted at Research Institute of Forests and Rangelands of Iran during 2012. The strains were propagated with YAM solution and then were inoculated on seeds of the sainfoin ecotypes. Seeds were sown in pots and treated with Hoagland solution (without N) in glasshouse and outdoor for 90 days. On the harvest date, data were collected on root and stem length, fresh and dry weight of roots, stems and node number of root. Data were analyzed and means were classified using Duncan method. Results showed significant effects of ecotypes, Sinoryzobium sp. and their interaction effects (P