نشریه تحقیقات ژنتیک و اصلاح گیاهان مرتعی و جنگلی ایران
سال بیست و دوم شماره 2 (پیاپی 44، تابستان 1393)

Monoterpenes are one of the major groups of terpenes which are present in black cumin (Nigella sativa L.) as limonene، carvacrol، thymoquinone etc. One of the key genes in monoterpene biosynthesis is geranyl diphosphate synthase (GPPS) that produces a monoterpene precursor. The aim of this study was to investigate the expression of the genes in black cumin. RNA was extracted، cDNA was synthesized and a partial sequence of GPPS gene was isolated using degenerate primers and sequenced. Moreover، RNA was extracted from different tissues of the species and semi-quantitative RT-PCR using specific primers of GPPS gene and GAPDH as housekeeping gene in order to study the pattern of GPPS gene expression. Results showed that transcript expression of geranyl diphosphate synthase in stem and leaf is significantly higher than that of root، capsule and seed.

Pistacia atlantica subsp. mutica is one of the most valuable forest species in Iran that has been damaged by uncontrolled exploitation of human being. Therefore، understanding the genetic diversity of different populations of the species، an important step can be taken towards development and restoration of habitat with high value of the species. In order to study the genetic diversity among 10 populations of the species، containing 59 genotypes via 16 ISSR primers، in general estimated 158 alleles of which 100% of alleles were polymorphic. Number of amplified alleles ranged from 3 to 17 with a mean value of 9. 78 alleles for each primer. Polymorphic information content (PIC) varied from 0. 16 (primer UBC866) to 0. 38 (primer UBC884). Marker index criterion ranged from 0. 48 (primer UBC866) to 5. 95 (primer UBC840). Cluster analysis could not completely separate the samples and showed lack of association between molecular diversity and geographic diversity of the studied populations. Principal coordinant analysis also confirmed the results. Kushk population indicated the highest value of polymorphic alleles (72. 15 %) and unbiased expected heterozygosity (0. 257) and Shannon’s Index (0. 364). While، the lowest value of polymorphic alleles (10. 13 %) and unbiased expected heterozygosity (0. 056) and Shannon’s Index (0. 061) observed on out Zagros population. Analysis of molecular variance (AMOVA) showed that a larger proportion of genetic variation (82%) belonged to within the populations، while only a small proportion (18%) observed among the studied populations.

Nucleotide sequences of internal transcribed spacer (ITS) of nuclear ribosomal DNA were used to infer the phylogeny and molecular taxonomy status of Betula species. Three remnant populations of Betula from Hyrcanian forests and one population from Zagros forest were selected. Leaf DNA were extracted and ITS regions were amplified on the selected samples. Entire lengths of the ITS1-5. 8S-ITS2 region were 610-611 bp for all of the studied Betula samples and 611 bp for Iranian samples. Maximum similarity in nucleotide composition and minimum genetic distance of Iranian Betula were observed with Betula pendula from subgenus of Betula. According to character based method، Iranian Betula was distinguished from other Betula species due to having unique nucleotide at position 209. Four secondary structure ITS2 type were recognized for all of the studied Betula species. Construction of the phylogenetic trees of ITS data supported monophyly of the genus Betula and divided into three major groups with more than 73% bootstrap support. The first Group comprises four minor groups by which Iranian Betual with B. pendula was located in Clade I.

Hawthorn (Crataegus sp.) is one of the plants، which suffers of low seed germination rate. Purposes of this research was evaluation of optimizing a medium for propagation of hawthorn and comparison of callus flavonoid، to that of leaf and fruit of the species. Media including MS، N6 and B5 supplemented by various plant growth regulators were used. The study was carried out based on a completely randomized design with 3 replicates. Flavonoid content was determined using a colorimetric (Aluminum Chloride) method. Results showed that mercuric chloride %0. 02 for 3 min، ethanol %70 for 2 min and NaOCl for 15 min were the best treatments for sterilization. N6 medium supplemented with 7 mg/L BA + 1. 5 mg/L NAA was the best medium for callus induction. The most regeneration was observed in B5 medium supplemented with 0. 49 mg/L BA + 0. 49 mg/L Kin and 0. 1 mg/l IAA and MS medium supplemented with 1 mg/L BA + 0. 5 mg/L IAA was the best for multiplication. Rooting was observed only in MS medium supplemented with 1 mg/L IBA with low frequency. The results indicated that flavonoid content of leaf was more than fruit and flavonoid content of callus was very low.

Artemisia genus from Asteraceae family has 34 species in Iran with valuable medicinal compounds. The most important use of Artemisia has been the treatment of malaria in traditional Chinese medicine. In addition to artemisinin، it produces also other secondary metabolites such as polyphenols، terpenes، phytosterols and alkaloids. MEP (2-C-methyl-D-erythritol-4-phosphate pathway) pathway is a chloroplastic pathway by which monoterpenes are produced. It exists only in photosynthesis plants and some bacteria. In this study، monoterpene compounds produced by MEP pathway in three kinds of tissues including leaf، bud and flower were compared with the expression of three genes in the pathway (DXS، DXR، HDR). Monoterpene compounds were measured by GC/MS and genes expression was evaluated by Real-Time PCR. Six Monoterpene compounds including α-pinene، Camphene، β-Myrcene، 1،8-Cineole، Artemisia ketone and Camphor detected by GC/MS analysis on leaf، bud and flower extracts، with 60. 8، 54 and 78. 76 percent of extract، respectively. Artemisia ketone and Camphor had higher levels compared to other compounds. DXS expression in leaves was three times higher than that of buds and flowers. DXR showed the highest expression in flowers. Expression of HDR in leaves or buds was three times higher than that of flowers. Comparing the results of gene expression and GC/MS suggested that DXR plays more important role in producing monoterpene compounds and might be a candidate gene for genetic engineering.

A major part of drought tolerance mechanisms of Haloxylon aphyllum depends on the depth and number of lateral roots by which the required water is absorbed from more soil volume. Therefore، this research was conducted to evaluate H. aphyllum root morphologic variation and its response to water stress. Progenies of four genotypes taken from a plant population of the species were used in the experiment. Seedlings were exposed to four levels of water stress. At the end of growing period، several root characteristics، such as hairy root density، root length، and root volume were recorded. Genotypes were significantly different for the studied characteristics. The characteristics were highly affected by the water stress treatments. The root characters also varied in responding to the water stress treatments. Root increment at all levels of water stress was also noticeable. In other words، facing water stress، the plant species increased its roots to reach more water. Effects of water deficit on root volume were also noticeable، in such a way that the first level of water stress showed the most recorded root volume. This implies، facing water deficit، Haloxylon roots are increased to cover more soil volume to intake more water.

E. maculata is a fast growing species، adapted to climate conditions of Iran، which is important in agro-forestry، industrial and medical aspects. Proliferation of E. maculata was carried out using photoautotrophic and conventional micropropagation methods as well as comparing efficiency of the two methods. Terminal buds of young plants were used as explants. Explants were placed on MS medium with ½ nitrate and various growth regulators with different concentrations for conventional micropropagation and in Magenta (G7) containers with different number of filters for gas exchange with external and internal environments، containing 70 ml of half-strength، sugar and agar free MS medium، supplemented with vermiculite in photoautotrophic method. After one month، MS medium with ½ nitrate containing IBA (0. 01 mgl-1)، BAP (0. 3 mgl-1)، 2ip (0. 5 mgl-1) for shoot production (with average number of 2. 52)، IBA (0. 01 mgl-1)، BAP (0. 1 mgl-1)، Kinetin (0. 2 mgl-1) for shoot height growth (with average of 2. 07 cm) and MS medium with ½ nitrate containing IBA (1 mgl-1) for root production (with average number of 1. 62) were the best media. In photoautotrophic method، results of study on growth indexes showed that there were not any significant differences between the studied treatments but treatment with 4 filters had better growth conditions. According to observation، photoautotrophic method had better growth characteristics than the conventional micropropagation of E. maculata. The micropropagated plantlets were transferred to greenhouse and field successfully.

Redbud (Cercis spp.)، has a great importance in urban forestry and landscape architect، due to its ornamental values. But its use is hampered by some unfavorable traits that can be improved by genetic engineering. In vitro grown of cotyledons and stem explants of redbud were cultured on MS basal medium supplemented with different concentrations of growth regulators for callus induction. EHA105 and AGL1 strains of Agrobacterium tumefaciens were transformed with pCAMBIA2301 binary vector، harboring nptII and gus genes conferring kanamycin (Kan) resistance and reporter enzymatic protein of β-glucuronidase activity، respectively، using calcium chloride (CaCl2) and Tris. Calli and zygotic embryo cells were infected with different densities of Agrobacterium strains under vacuum and normal conditions. Addition of 22. 4 and 9. 1 μM of 2،4-Dicholophenoxy acetic acid (2،4-D) to medium resulted in the highest 92 and 97% callus induction in cotyledon and stem explants، respectively. Optimum concentration of Kan for selecting of transformed shoot and callus was also detected. The results showed that the highest transformation level can be obtained by OD600 = 0. 8 of AGL1 strain in vacuum condition. Histochemical analysis of reporter gene، and polymerase chain reaction (PCR) analysis confirmed transfering of the transgene from agrobacterium to embryo-derived plantlets and callus induced from regenerated shoots. The Agrobacterium tumefaciens-transformation procedure described here for the first time may pave an efficient way for generating transgenic C. griffithii trees using zygotic embryos as explants.

Stinging Nettle (Urtica dioica) is an important medicinal dioecious plant that is widely distributed around the north of Iran. Reproduction of the species is sexual and asexual. In this study، ten AFLP primer combinations were used to evaluate genetic diversity and population structure of five nettle populations collected from Mazandaran province. From 798 bands، 722 bands (90. 5%) were polymorphic. Average polymorphic information content (PIC)، 0. 23، genetic similarity range based on Jaccard''s similarity coefficient were between 0. 44 to 0. 14. Clustering based on UPGMA separated the studied populations into four groups. Analysis of molecular variance indicated high variance within the populations. Wide variation، high rates of Fst and number of migration (Nm) suggested that populations are not isolated implying gene flow between the populations. The results showed that Nettle plant with wide diversity selected the Caspian region as suitable place to reside.

Ferula gummosa B. an endangered and highly valuable medicinal plant belonging to the Apiaceae family is an endemic plant of Iran، on which mass production is exposed to serious problems. Recently hairy root research for production of important secondary metabolites has received a lot of attention. We studied the possibility of hairy root induction، on F. gummosis B. using four strains of Agrobacterium rhizogenes (15834، A4، 1724،2659) by infecting different explants (hypocotyle، cotyledons، leaf، 10-14 day old embryo and callus). Effects of three levels of hormone free MS medium (1، ½، 1/4) on hairy root production were examined. Results showed that different kinds of Agrobacterium rhizogenes and explants were affected by hairy root induction. Fourteen days after infection، only، 10-14 day old embryos produced hairy root by strain 15834. MS1/2 medium was effective for root emergence. In comparison with testifier، successful genetic transformations containing T-DNA، were confirmed by PCR.

Withania coagulans is a multi-purpose medicinal plant. Distribution of the species in Iran is limited and located only in the southeastern sections of Sistan and Baluchestan province. Genetic diversity of 20 accessions of W. coagulans from different natural habitats of Sistan and Baluchestan province was characterized by 18 morphologic characters. Recorded data were analyzed using multivariate analyses. ANOVA revealed high significanct differences between the accessions based on all of the measured characteristics. The most diversity was related to number of branch (60. 82%) and the least was related to root length (13. 28%). Among the accessions، ‘Pigol’ and ‘Damin’ showed the highest values for most of the studied traits. Principal components analysis indicated that the first three principal components with eigenvalues more than 1 explained 86. 62% of the variability amongst the accessions. Cluster analysis based on Euclidian distance، divided the accessions into three major groups. Considering the grouping، there was no relationship between genetic diversity and geographical distance of the accessions. The differential grouping indicated that a factor other than geographical distribution may be responsible for genetic divergence. The results suggested that there is a considerable genetic variation among W. coagulans accessions. Knowledge of genetic diversity in W. coagulans provides different levels of information which is important in the management of germplasm resources and breeding programs.

Evaluation of genetic diversity and relationship among traits are an essential prerequisite for direct and indirect selection in plant breeding. This experiment was carried out to evaluate the relationship between morphological and agronomic traits and to classify 56 sainfoin accessions using multivariate statistical analysis methods during 2010-2011. Results of estimated correlation coefficients showed that dry matter yield had significant positive correlation with plant height، number of stem per plant and length of panicle. Whereas، there was a negative significant correlation between dry matter yield and leaf percent and leaf to stem ratio. On the basis of stepwise regression، number of stem per plant and plant height explained the majority of dry matter yield variation and leaf percent solely could justify 64% of forage palatability variation. Cluster analysis could separate Iranian and exotic sainfoin accessions on the base of morphological traits. Also accessions of similar geographic origin were mainly clustered in the same groups. Moreover، principle components analysis confirmed the result of cluster analysis. Finally results indicated that there was high variation for studied traits among Iranian and exotic sainfoin genotypes which can be useful to select suitable parents to produce synthetic varieties in breeding programs.

In order to investigate variation in qualitative and quantitative traits، 19 accessions of Elymus hispidus were studied based on a randomised complete block design (RCBD) at Hosseinabad station، Shiraz، Iran. Data were collected and analyzed three years on dry matter (DM) yield، plant height، stem number، leaf to stem ratio (LSR)، as well as five quality traits as: dry matter digestibility (DMD)، water soluble carbohydrate (WSC)، crude protein (CP)، acid detergent fiber (ADF) and total ash. Significant differences were observed between the studied genotypes for all of the traits except for LSR، CP and ash. Khoshyeelagh (Gorgan)، Patava (Yasuj)، Sabzkoh (Chaharmahal) and Mimand (Yasuj) genotypes with average values of 4034، 3068، 2942 and 2450 kg/ha had higher annual DM yield. Khoshyeelagh and Mimand genotypes had hugher quality and recognized as the best genotypes for both yield and quality for cultivation in Fars province، Iran. Results of correlation showed positive correlation between DM yield with plant height and stem number، DMD was positively with WSC and negatively correlated with ADF. Relationships among CP and total ash were positively significant. Using principal components analysis، the first four components determined 76% of the total variation. Five quality traits (DMD، WSC، CP، ADF and ash) were determinant in the first component. DM yield and stem number were important traits in the second components. The genotypes were grouped into 4 clusters based on Ward cluster analysis method.

Chamomile، a herbaceous and perennial plant from Asteracaea and Anthemis is the second largest genus in the family. Knowledge of genetic diversity and differentiation related genes، genotypes and knowledge of how to operate، is essential for breeding programs and may play an important role in genetics، biochemistry and plant breeding. Therefore، genetic diversity of 90 genotype of Anthemis (one population of A. pseudocotula، two populations of A. haussknechtti and six populations of A. triumfetti) were studied by electrophoretic pattern of total proteins and investigated to determine extent of genetic diversity. On the basis of SDS-PAGE، 24 reproducible bands were observed. A. haussknechtti and A. triumfetti species had highest number of bands (23) and A. pseudocutula had minimum number of bands (17). Percentage of polymorphism was 49/07% and specific band was observed. Longest distance was observed between A. haussknechtti (Ivan) and A. triumfetti (Ramian). Analysis of molecular variance showed that genetic differentiation between and within the populations were 57% and 43% respectively. Correlation between the protein data، latitude and longitude showed significant differences (R2=/03049 and P=0/010).