فاطمه کیخا آخر

Among abiotic stresses, Salinity has been increasing over the time for many reasons like using of chemical fertilizers, global warming and rising sea levels. In deed salinity stress and high concentration of ions are of the most determinative factors that simultaneously affect genetical, biochemical and physiological processes of a plant. Some of plants known as halophytes have developed mechanisms that help them to avoid or tolerate the saline conditions. With the aim of understanding the probable mechanisms of saline tolerance in Aeluropus littoralis (as a halophyte) and the saline susceptibility of Oriza sativa var. IR64 (as a glycophyte), In the present study, the similarity of Cu/Zn Superoxide Dismutase (Cu/Zn SOD) gene sequences, the differential biochemical indexes such as aromatic and instability indices of them were evaluated in silico, based on amino acid composition of their Cu/Zn SOD. the gene expression pattern and fluctuation of Cu/Zn SOD as a ROS scavenger (in root and shoot) was also assessed and compared in a span of exposer time to stress and different salt concentratio

The seeds of A. littoralis and O. sativa var IR64 were prepared from Center for Research of Agricultural Science and Natural Resources (CRASN) which were sown in sand as a primary culture medium, after surface sterilization. After 21 days, the seedlings that had grown uniformly were transferred to continuously aerated hydroponic pots containing Yoshida nutrient solution. we established a stress span containing short term (6 hours), mid-term (24 and 48 hours) and long term (6 and 11 days) exposure to salinity stress. the gene expression pattern and fluctuation of Cu/Zn SOD as a ROS scavenger in Aeluropus, was evaluated at the sampling points of 6 h/100 mM, 6 h/200 mM, 6 h/300 mM, 48 h/300 mM, 144 h/300 mM and 264 h/300 mM. the elite sampling timepoint of rice, were also 6 h/30 mM, 6 h/60 mM, 6 h/100 mM, 48 hours/100mM, 144 hours/100 mM and 264 hours/100 mM. To study the structure of genes, genomic DNAs of A. littoralis and Oryza sativa L. Var IR64 were isolated from their leaf. Amplification of Cu/Zn SOD genes was performed with specific primers designed based on NCBI sequences. Amplification of their the maximum length fragments was carried out using a hot start, high fidelity DNA polymerase. The extracted fragments were cloned using pTZ57R vector in Escherichia coli (DH5α strain) and sequenced paired-end. The results were analyzed using NCBI database, and the biochemical features related to their proteins, such as isoelectric points, calculated weight, instability and aromatic indices, were analyzed Insilco using CLC Genomics Workbench 12 and Geneious Prime 2019.

Comparison of the real time quantitative PCR of Cu/Zn SOD transcriptome in roots showed that Aeluropus increase the expression of these genes faster and keeps their expression in long-term while rice loses them significantly. The increased expression of Cu/Zn SOD in the roots of Aeluropus maintained but its expression in rice down regulated. rice did not show any statistically significant changes in compare to control samples and persisted in this manner until the end of investigation. But in Aeluropus, the expression showed another increase after 264 hours of being in 300mM. In the shoots of Aeluropus, the level of Cu/Zn SOD expression increased gradually and reached its highest level after 48 hours commencement of 300 mM salinity stress. While shoots of IR64 Rice did not show any statistically different expression except a very late response at 144th hour of being in 100 mM salinity stress that was only about 40 percent higher than its counterpart control samples. Analysis of amino acid sequences' indices showed that Cu/Zn SOD of Aeluropus would have a superior stability and higher aromatic value in contrast to IR64 counterpart. These indices originate from the arrangement of amino acids which they themselves are the result of nucleotide arrangement in genome.

The early and highly expression of Aeluropus Cu/Zn SOD gene studied in our experiment and their permanency and lasting of their activity in high concentration of salinity makes the sequence of it and its regulatory elements as a prospective candidate for future applied studies and transforming and improving salinity tolerance in many Poacea crops.

Cell suspension culture and elicitation have been playing an important role in the synthesis of active secondary metabolites. Rosmarinic acid (RA) is one of the active compounds found in lavender essential oil that stands out due to its antioxidant and anti-inflammatory characteristics.

This research was conducted to investigate the production efficiency of RA in Lavandula angustifolia suspension culture through elicitation with methyl jasmonate (MeJA) and yeast extract (YE).

Cell suspension culture established in B5 liquid media supplemented with different combinations of Plant Growth Regulators (PGRs). The influence of different PGRs treatments on cell growth and accumulation of RA were analyzed. Then the effect of concentration and time course of elicitation with YE (0.1, 0.5, and 1 g/l after 1, 3 and, 6 days of elicitation) and MeJA (50, 100 and 200 µM after 1, 2 and 3 days of elicitation) separately and in combination with each other on cell growth and intracellular and extracellular content of RA were investigated.

HPLC analysis showed that the highest intracellular RA content (17.03 mg/g dry weight) was observed 24 hours after the addition of 50 µM MeJA in combination with 1 g/l YE, which was approximately 33% higher than that found in leaves. Furthermore, it was 9 and 11 times higher than cultures treated with MeJA and YE alone, respectively. In addition, both elicitors significantly affected the extracellular quantity of RA than control.

Our results documented that the application of elicitors increased biomass and RA accumulation in L. angustifolia suspension cells.

 Flower color is one of the most significant characteristics in ornamental plant breeding. New varieties of various plants in relation to their flower color have been obtained by monitoring the expression levels of genes involved or regulating the flavonoid and anthocyanin biosynthesis pathway. Flavonoids possess significant and diverse biological functions. They are the major pigments for flowers, fruits, seeds, and leaves. They are natural products that contain a C6-C3-C6 carbon framework and are synthesized by a branched pathway that yields both colored and colorless compounds. The gene encoding chalcone isomerase (CHI) is among the genes and enzymes identified in the flavonoid pathway. This enzyme catalyzes the isomerization of naringenin chalcone into the corresponding flavanone. CHI enzyme belongs to the family of isomerases, specifically the class of intramolecular lyases. Chalcone isomerase has a core 2-layer alpha/beta structure and has attracted much attention recently due to its role in stress response and pigment production. One of the most effective methods of genetic engineering is the reduction of flower pigments by suppression of required enzymes for their biosynthesis. RNA interference (RNAi) has provided the tool for the investigation of genes involved in the production of flower color. Silencing of any gene in the anthocyanin biosynthetic pathway can result in reduced or inhibited anthocyanin production. RNAi technology is an effective gene silencing method and a powerful tool for studying gene function and development of new traits by transformation of viral RNA or hairpin RNA (hpRNA) constructs into plants. The processing of dsRNA into 21-23-nt small interfering RNAs (siRNAs), and the mediators of RNAi, triggers cognate mRNA degradation. The hpRNAi methodology simply requires a transgene construct containing an inversely-repeated sequence of the target gene flanked with a promoter and terminator which effectively function in plants.
 
 In this research, with the design and construction of chiRNAi, the transformation of the RNAi construct was carried out of Petunia plants. Potted plants of P. hybrida were grown under standard greenhouse conditions (16-17°C night temperature and 21-24°C day temperature and photoperiod 16/8 (light/dark)). The RNAi construct including the 530 bp cds of the chalcone isomerase (chi) gene and 741 bp of pdk gene as intron between chi sense and antisense were used for transient RNAi-induced silencing. The pBI121-chi530 plasmids were introduced into A. tumefaciens strain LBA4404 by electroporation method. Colonies of A. tumefaciens carrying the desired plasmid were screened by PCR with specific primers for chi gene. RNAi construct co-cultured with petunia’s leave. Samples was kept in dark condition for 3 days and then transferred to branch induction media. Samples were investigated for phenotypical changes and chi gene expression by qRT-PCR.
 Transgenic lines showed a reduced number of pigments and a faded flower color. So that, in purple petunia, was shown 5 phenotypical groups. These groups was indicated different levels of chi gene silencing. In pink petunia was seen two groups of phenotypical changes. In these plants, chi-RNAi construct was reduced pigment production and so, these plants had faded colors in petals. Also, the chi gene expression was reduced in all transgenic lines. Generally, the results of this research showed that RNAi can be used as an efficient method for gene silencing. The application of gene silencing can indicate the gene’s function in biosynthesis pathways of various components such as anthocyanins. In addition, the chalcone isomerase gene was identified as one of the effective genes in anthocyanin biosynthesis pathway in Petunia plants that could be involved in the production of color in these plants; hence, chi gene silencing resulted in clear phenotypic alterations in this plant.
 
 In general the concentration of the target mRNA in a particular tissue could be a factor that influences silencing efficiency. At very low levels of gene expression, small amounts of the silencing target, mRNA, could be completely degraded by the RNA-induced silencing complex (RISC), whereas the presence of higher amounts of the target mRNA may result in incomplete silencing, allowing some residual functional mRNA to be translated into the corresponding protein. This research demonstrated the hpRNA construct has been successfully established for floral tissues of P. hybrida. The hpRNA construct was developed for chi-RNAi silencing of one of the key genes in the anthocyanin biosynthetic pathway in Petunia flowers. The silencing of the chi gene is a prototype for the modification of the anthocyanin biosynthetic pathway in Petunia through gene suppression. This strategy could also be useful for rapid functional analysis of other genes involved in flower development.

have been bred with altered flower color using genetic engineering approaches. One of the most effective applications is the reduction of flower pigments by suppression of involved enzymes in their biosynthesis pathways. RNA interference (RNAi) has provided an effective tool for the knock down of genes involved in the production of flower pigments. In this study, a chi-RNAi construct was designed for chalcone isomerase (chi) gene to transform Petunia plants. Transgenic lines in one phenotype showed 5.6 fold reduction in chi expression in comparison to the control. Chalcone and naringenin were also extracted and quantified. A 24% reduction in naringenin content was obvious in all transgenic lines. Generally, the results of this research showed that RNAi technology can be used as an efficient method for silencing the flower pigments in petunia. In addition, the chalcone isomerase gene was identified as one of the effective genes in anthocyanin biosynthesis pathway in Petunia plants which is involved in the production of color in these plants; hence, chi gene silencing resulted in clear phenotypic alterations in this plant.

There is an imbalance between increase rate in demand for agricultural products and the growth rate of agricultural production. Much of this production deficit is attributed to abiotic stresses. These stresses reduce the yield of crops by more than 50%. Obviously, it worth studying any idea which may lead to reducing the damages of them. In the present research, the transcription level of Catalase in root and shoot of Oryza sativa var. IR64 and Aeluropus littoralis using qtr.PCR and the Aromatic and Instability indices based on amino acid composition were evaluated. The samples were taken at short-term, mid-term and long-term stress span. Analysis of the results showed significant differences in the both gene expression and studied biochemical aspects. The expression of catalase gene in Aeluropus roots was periodic and showed a twice increase and then a decrease however in roots of rice there was just a rise in its expression. In the all sampling of the rice shoots, CAT gene expression levels were either lower or without any significant different in contrast to the control samples. Meanwhile, the rates of expression in most of stressed Aeluropus shoots were significantly higher than control samples. Comparison of the biochemical indices showed that Aeluropus has a relative superiority over rice in terms of amino acid sequences. Based on the evaluated indices, the differences of response to salinity stress in the studied plants could be attributed to the differences in the promoter and nucleotide sequences of their genes.

Algae are an enormous biological group, forming 50% of photosynthetic organisms. In addition to having chlorophyll for the absorption of light photons, algae are rich in red, orange, and yellow carotenoids, which mainly protect cells against harmful radiation and free radicals. Moreover, these organisms have phycobiliproteins (red and blue pigments), which are involved in capturing and passing light energy to chlorophylls during photosynthesis and have a wide range of antioxidant properties. Algae also play a key role in substituting artificial colorants with natural colorants due to the adverse side-effects of chemical colorants, especially since natural colors are commonly used by individuals and various industries. Recently, algal pigments have been widely used in medical, nutraceutical, cosmeceutical, and pharmaceutical industries owing to their antioxidant, antidiabetic, anti-obesity, anti-inflammatory, antiaging, antimalarial, and neuroprotective properties. The growing demand for algal bioproducts highlights the importance of evaluating the trends influential factors in their production. The current review study provided an introduction to algal pigment classification, distribution, function, application, and biological production. In addition, we have discussed crucial biochemical pathways, enzymes, and gene/biotechnological modifications, such as transformation and expression regulation, which noticeably affect the metabolism of their sink and source.

Petunia (Petunia hybrida) is one of the important plants in horticultural industry. This plant is being used as a model ornamental plant and is one of the most important plants in floriculture market. Flavonoids are the main pigment in this plant. So, genetic engineering with the goal of color alteration in petunia is focused on flavonoids. To gain a global perspective on genes differentially expressed in petunia’s flowers pigment pathway, we investigated the expression of chalcone isomerase (chi) as an essential gene in biosynthesis pathway of pigment production in different types of flower color of petunia and various stages of flowering in this plant. Also, we measured the concentration of total flavonoids, anthocyanins and naringenin to evaluate the probably relationship between the expression profile of chi gene and the concentration of mentioned pigments. The results indicated that chalcone isomerase expression had different profile in different petal color of P. hybrida. So that, the most chi expression observed in red petunia flowers. Naringenin concentrations was the most value in this color flower. In comparison of flowering stages, stage 1, had the most expression. In other words, when the flowers fully closed (bud stage), chi expression and concentration was in the highest value. Our results showed that chi is a key gene in pigment biosynthesis pathway so that in absence of this gene, pigment pathway will be stopped. Identification of effective genes in different pathways of secondary metabolite production will assist in accurate selection of genes for genetically modification of pathways and production of various metabolites.

Begonia is one of the most important ornamental plants around the world. Begonia species are grown for their attractive leaves and flowers in indoor and outdoor conditions. The tissue culture technique is an alternative method for the propagation of begonia species. It can overcome the difficulties of vegetative propagation and lead to high-quality and uniform planting material under disease-free conditions irrespective of the season and weather. In begonia tissue culture, different species showed diverse responses to the medium composition, especially plant growth regulators. Also, lack of information on large scale micropropagation of begonia in Iran highlights research on optimization of begonia species propagation on in vitro culture condition. Thus, in this study the effect of plant growth regulators on shoot regeneration and proliferation of three begonia species (B. elatior‌, B. soli-mutata, and B. tiger), the impact of Auxin and sucrose on rooting of B. elatior‌and diferent pot substrate on acclimation were survived.

In this experiment, petioles of three begonia species (B. soli-mutata, B. elatior, and B. tiger) were used as explant sources. So explants were washed under tab water for 30 mins and sterilized with 1.5% sodium hypochlorite for 15 mins. After sterilization, thin Cell Layers (TCL) with 2 mm thickness were prepared from petiols and cultured in MS medium with different concentrations (0.2, 1, 2 mg/L) of kinetin (kin) or thidiazuron (TDZ) in combination with NAA (0, 0.2 mg/L). Next, the effect of auxin type (1 mg/L of IAA, IBA, and NAA) and sucrose concentration (30 and 40 g/l) on rooting of B. elatior plantlets were determined on in vitro condition. For plantlet acclimation, sand, CocoPeat perlite mixture (1:1 ratio), and Peat moss as pot substrate were evaluated.

The results showed the shoot regeneration response of begonia species to the plant growth regulators of the medium, and there was a significant difference between them in all of the species adding the Auxin to the medium increased shoot regeneration. In B. elatior and B. tiger lacking of NAA to the medium caused no shoot regeneration. Maximum shoot regeneration (100%) was achieved in B. soli-mutata species in medium containing 1 or 2 mg/L of kin and 0.2 mg/l NAA, in B. elatior at 1 or 2 mg/L of TDZ in combination with 0.2 mg/l NAA and in B. tiger at 2 mg/L of both plant growth regulator (TDZ and Kin) and 0.2 mg/l NAA.

For rooting B. elatior, MS medium containing 1 mg/L IAA plus 30 g/L sucrose was the best and favorite acclimation was acheived in cocopeat perlite mixture and peat moss pot substrate.

Background and objectives Gerbera is one of the most important ornamental plants that used as cut flower and potted plant. The common methods of propagation does not have performance for supplying the global demand of this ornamental plant. So the most common method for commercial propagation of gerbera is micropropagation.
Material and methods In this study, effect of different hormonal treatments and cultivars on micropropagation and rooting of gerbera shoot tip explant was evaluated. Shoot tip explants were first washed with running tap water for 30 min then they were surface sterilized by dipping in 1.5% sodium hypochlorite solution for 5 min and rinsed with sterile distilled water, followed by immersing in 0.1 % mercuric chloride solution for 5 min. After Sterilization with mercuric chloride solution, capitulum was washed with sterile distilled water. Subsequent washing was done with sterile distilled water three times. Sterilization with sodium hypochlorite solution and mercuric chloride and final rinse with sterile distilled water were done under laminar air flow hood. In the proliferation phase, MS medium containing 1 mg/l BA or KIN in combination with 0.1 mg/l IAA or hormone-free medium, 3% sucrose, 8 g/l agar was used. For rooting of propagated plantlets, ½ MS medium containing 1 mg/l IBA, IAA, 2IP or ½ MS hormone-free medium, 3% sucrose, 8 g/l agar was used. After 4 weeks different parameters such as number of plantlet, height of plantlet, number of main root, root length, number of secondary root were measured. Cultivars that was used in this experiment are as follows: 1: ‘Aventura’, 2: ‘Barones’, 3: ‘Mayfair’, 4: ‘Cacharelle’, 5: ‘Real’, 6: ‘Sorbet’, 7: ‘Dalma’, 8: ‘Goldy’, 9: ‘Panama’, 10: ‘Lancaster’, 11: ‘Dune’, 12: ‘Bellavue’, 13: ‘Blinddate’, 14: ‘Applause’, 15: ‘Sunway’.
Results and discussion The results showed that in all studied cultivars, the medium containing BA lead to producing the highest number of plantlet. So using the medium containing BA in propagation phase is recommended for obtaining the highest number of plantlet. Also the results showed that the rooting of gerbera plantlets was affected by cultivar and suitable hormonal composition for rooting of each cultivar was different. However, the appropriate acclimation of produced plantlets is so important. Although the length and number of roots in ½ MS medium was at the average of other treatments, but propagated plantlets from this medium, had a suitable acclimation capacity. Moreover, additional cost for utilization of rooting hormones was reduced and therefore commercial production cost decreased. So application of this medium for rooting of different gerbera cultivars is recommended. Finally acclimation of rooted plantlet was done in cocopeat and perlite medium, with 95% success.

Lily (Lilium spp.) is a genus of herbaceous flowering plants, which consisted of many beautiful ornamental species with large prominent flowers. Most species are native to the Northern hemisphere temperate, though their range extends into the Northern subtropics. Some specific hybrids of Lilium spp. have been developed as cut flower in controlled conditions and in some cases can be grown as pot plant. Propagation rate of lily in natural clonal propagation methods is very low and one year produces of 1-2 bulblets per bulb scale. There is also possibility of disease transmission; so that, tissue culture techniques has provided an efficient method for its micropropagation.
In this study, two separate experiments under In vitro conditions the bulblet regeneration from thin cell layer (TCL) explants of Lilium spp. was investigated. In the first experiment, after two months the effect of TCL explants with 1, 3 and 5 mm thickness on MS medium containing 1 mg/l BA, 2ip and kin in combination with 0.5 mg/l NAA on regeneration parameters were assayed. In the second experiment to determine the effect of cultivar and cytokinin types, 3mm thickness TCL explants of five cultivars (Robina, Donato, Nymph, Lessoto and Roxana) were tested on MS medium containing different plant growth regulator (PGR) compounds including BA, kin, 2ip and TDZ at concentration of 1 mg/l in combination with 0.5 mg/l NAA. The regeneration parameters were assayed after four months. In all experiments, the medium was adjusted to pH 5.8 and autoclaved at 121°C for 15 min. All cultures were incubated at 25 ± 2°C with a 16 h photoperiod under cool white flourescent lights (30 µmol/m2).
According to the first experiment results, plant growth regulator of BA in all of surveyed parameters except root number was better than other PGRs and explants with 3 mm thickness was the best in all of parameters. The interaction of PGR and explants was significant, however maximum bulblet regeneration was observed in TCL explants with 3 mm thickness in all of PGR treatments (100%). While 1 mm thickness TCL in 2ip and 1 and 5 mm thickness TCL in Kin had the least regeneration percentage. Results revealed that the interaction of explants and medium is a key factor for suitable establishment, regeneration and growth of TCLs. Bulb dormancy is one of the limiting factors in regeneration of bulbous crop species. It seems under In vitro condition explants size and PGR combination of media especially cytokinin affected on breaking of dormancy. Maximum number of leaves and dry weight of bulblets in medium containing BA was significantly higher compared with other treatments. Most of studies confirmed the positive effect of BA on regeneration of lily. The function of cytokinin in plant promoted cell division and differentiation, which lead to growth and maintaining cells in meristematic status.
Result of second experiment showed that cultivar was one of the effective factors on regeneration trait. Oriental lily cultivar "Roxana" had the highest number of roots, bulblets, dry weight and length of plantlets and "Nymph" cultivar showed the lowest percentage of regeneration, dry weight, length of plantlets and rooting obtained. In all of cultivars BA induced more organogenesis percentage and plantlet dry weight, while TDZ induced more rooting percentage.The interaction of cultivar and PGR treatments on percentage of regenerated bulblets and rooting were significant. "Nymph" cultivar had minimum percentage of regeneration and rooting in medium containing TDZ and Kin. Furthermore, "Roxana" cultivar in medium containing BA showed the best dry weight comparison to other treatments.
Conclusion Lily has widely used in the floral industry as a cut flower or potted plant. In recent years, tissue culture was developed as reliable and highly effective method to overcome its limitations of vegetative propagation. The most advantage of this method is high multiplication rate and disease free propagation. In this study, bulblet regeneration of lilium Spp. from TCL explants under in vitro condition was considered as a highly efficient procedure for its micropropagation. With optimization of TCL system some parameters such as exogenously applied plant growth regulators, cultivar, explants types were investigated. Favorable conditions for bulblet regeneration were achieved with 3 mm thickness TCLs in MS medium containing 1 mg/l BA with 0.5 mg/l NAA. This protocol can be used for rapid micropropagation of many cultivars.

Gerbera is one of the most important ornamental plants in the world. The importance of Gerbera is due to its beauty, diversity and economically aspects. Traditional propagation methods such as crown division and cutting methods are not suitable for obtaining disease free plants and rapid multiplication. These methods also do not have the capacity to fulfill global demands. Therefore, obtaining efficient protocol for micropropagation of this ornamental plant is necessary.
In this study the effect of various factors on in vitro regeneration, proliferation, rooting and acclimation of gerbera capitulum explants were analyzed in four separate experiments. Capitulum explants were first washed with running tap water for 30 min then surface sterilized by dipping in 1.5% sodium hypochlorite solution for 15 min and rinsed with sterile distilled water, followed by immersing in 0.1 % mercuric chloride solution for 10 min. To remove mercuric chloride residue, capitulum was rinsed with sterile distilled water. Subsequent washing was done with sterile distilled water for three times. Sterilization steps were done under laminar air flow hood. For regeneration, eight genotypes of gerbera capitulum explants (Famous, Sunway, Red Pearl, Pink Snow, Popov, Balance, Dune, Eagle)were cultured on solid MS medium containing several cytokynins, BA, TDZ, 2IP or KIN (4 mg/l) in combination with IAA (0.2 mg/l). In proliferation stage, the effect of different concentrations of BA was evaluated on proliferation rate of Sunway regenerated explants. In the rooting stage, Sunway genotype plantlets were cultured on ½ MS medium containing NAA, IBA or IAA (1 mg/l) or ½ MS medium without any hormones. The pH of the medium was adjusted to 5.7-5.8 prior to autoclaving (15 min at 121 oC and 1.5 kg.cm-2 pressure). The cultures were incubated in a growth chamber at 25±2 oC with a 16-h photoperiod (2500-3000 Lux) provided by cool-white fluorescent lamps. For acclimation of rooted plantlets, different substrates used as follow: 1- perlite, 2- perlite: Cocopeat, 3- Cocopeat: peat moss, 4- Cocopeat: peat moss; treated with fungicide.
After 30 days, the response of explants was evaluated for each experiment. Data preparation was done in the Excel program and data analysis was done using JMP-8 software. Mean comparison of the treatments was done by Tukey test and finally the charts were drawn using the Excel program.
The results of regeneration stage showed that application of MS media containing kinetin or 2IP did not make an appropriate response to capitulum explants and no regeneration was observed in this condition. The medium containing 4 mg/l BA and 0.2 mg/l IAA indicated the highest percentage of regeneration in all genotypes. The highest regeneration was observed in Sunway genotype with an average of 21.96%. On the other hand no regeneration was observed in Eagle genotype. In terms of the number of regenerated plantlet, the highest number (61.2) was attributed to the Sunway genotype while no plantlet was recorded for Eagle genotype. No significant differences were also observed between Pink Snow and Dune genotypes. For the proliferation stage, only Sunway genotype was utilized due to its vigorous growth in comparison to other genotypes. In this stage, the highest (6 regenerated plantlets) and the lowest (1 regenerated plantlet) regeneration rate were observed in MS medium containing 2 mg/l BA and hormone-free medium, respectively. Hormone-free ½ MS medium and ½ MS medium containing 1 mg/l IAA or IBA, indicated the highest rooting rate (100% rooting) while medium containing 1 mg/l NAA showed 55% rooting rate. It seems that the application of NAA in the medium composition had the lowestimpact on the rooting of regenerated plantlets. At the end of the experiment, the highest (90.42%) and the lowest (47.5%) acclimation rate was obtained in peat moss cocopeat fungicide medium and perlite medium, respectively.
Generally, for shoot induction of gerbera through capitulum culture, application of MS medium containing 4 mg/l BA and 0.2 mg/l IAA is recommended. It is also concluded that for proliferation stage, the MS medium containing 2 mg/l BA showed the highest rate of regeneration. Using of Hormone-free ½ MS medium is economically affordable. Finally for acclimation of the plantlets, application of peat moss cocopeat fungicide medium is recommended.

Breeding and production of new varieties of ornamental plants as well as their propagation and development is an expanding industry. The application of ornamental plants in landscape and increasing the demand for new varieties has led the breeders to plan for production of new varieties with attractive colors, excessive perfumes, more durability and resistance to biotic and abiotic stresses. Generally, diversity is a basic principle in any breeding program and plant breeding relies on variety, selection, evaluation and production of new genotypes. One way to increase diversity is mutation induction at in vitro conditions. Application of this method can help to increase diversity and enhance the efficiency of breeding programs and improve or achieve useful traits which have been lost in the plants evolution process. This article reviews the new research outcomes for increasing the diversity via induced mutation of ornamental plants combined with modern methods such as plant tissue culture and molecular markers. According to this study, the application of mutagens at in vitro conditions, using the molecular markers for selection of mutant plants in the early stages and increasing the rate of their propagation for enhancing the efficiency of breeding of ornamental plants is suggested.

Calendula (Calendula officinalis) is relatively cold tolerant plant، but in some years plant seriously injured due to harsh winter. In order to evaluate freezing tolerance of calendula an experiment was carried out at college of Agriculture، Ferdowsi University of Mashhadin a factorial-completely randomized design with three replications andplants of two sowing dates (summer and autumn) were exposed to12 temperatures (0، -2، -4، -6، -8، -10، -12، 14-، 16-، -18، -20 and -22oC). Seeds of calendula plants were sown in summer (summer plant) and autumn (autumn plant) in the bed and in six to eight-leaf stages were transplanted to the pots. After the cold acclimation in natural condition، freezing stress was applied with using a thermogradient freezer. To employ stability of cytoplasmic membrane، percentage of electrolyte leakage (EL%) was measured after freezing. Also survival percentage (Su%) and regrowth of calendula plants determined after three weeks recovery. Leaves EL% in autumn plants wassignificantly more than summer plants and autumn plants havehigher Su%، but plant height، number of lateral branches، numbers of reproductive traits، total dry matter، vegetative and reproductive dry matter in summer plants were more than autumn plants. However، there were no difference between calendula plants for LT50el in both autumn and summer plants،but there was significant difference between them for LT50su and total dry matter، and LT50su and reduced dry matter temperature50 (RDMT50) for summer plants were -18. 6 oC and -11. 3 oC and for autumn plants were -19. 4 oC and -13. 7 oC، respectively.

Freezing stress affected some vital processes of plant, and causing disturbance on the plant growth. In order to study the effect of low temperatures on freezing tolerance of chickpea (Cicer arietinum L.) a factorial experiment with 4 replications carried out using 3 chickpea genotypes (MCC496، MCC763 and MCC798) and 7 freezing temperatures (-4, -6, -8, -10, -12, -14 and -160C). Explants were cultured on MS medium with 2mg. l-1 BAP and 0. 125 mg. l-1 IBA. For acclimation, explants were transfered to 4ºC for 12 days. Then explants were freezed in thermo- gradient freezer. The percentage of survival and injury were determined after 3 weeks. Result showed that the effect of different freezing temperatures were significant on survival and injury percentage (P≤0. 05). By temperature decreasing, survival percentage decreased, wherease injury percentage increased but between 3 genotype differences were not significant. LT50 (injury percentage) in genotypes MCC763 and MCC798 were -15 ºC and in genotype MCC496 was -15/5 ºC. Also, LT50 (survival percentage) in 3 genotypes MCC496, MCC763 and MCC798, were -13, -12 and -12/5 ºC. This study suggests that in vitro screening of chickpea genotypes for cold tolerance is satisfactory and can be replaced by selection in the field, mainly due to its high repeatability.

In order to evaluation of freezing tolerance in viola (Viola gracilis L.) under controlled conditions, a trial carried out in completely randomized design with six replications at college of agriculture, Ferdowsi University of Mashhad, Iran, during 2008-2009. Seeds were planted in bed and seedlings were acclimated during natural conditions in autumn and at 5-7 leaves stage plants transferred to the thermo-gradient freezer to apply 12 freezing temperatures (0, -2, -4, -6, -8, -10, -12, -14, -16, -18, -20 and -22°C). Leaf cell’s membrane integrity was measured through electrolyte leakage (EL) after the freezing and survival percentage and plant recovery (e.g. plant height and dry weight, number of branches and flowers and flower’s diameter) were determined after three weeks of plant re-growth in the cold frame. As temperature decreased, EL% increased significantly and reached to the maximum in -20oC. Survival percentage did not affected by temperatures between 0oC to -18oC but all plants killed in -22oC. LT50el and LT50su were -20.0°C and -19.4°C, respectively. Decreasing the temperature lower than -18 oC reduced plant dry matter severely and RDMT50 was -19.2°C.

The aim of this experiment was the study of freezing tolerance of Bellis perennis under controlled conditions and were arranged in a completely randomized design with three replications. Plants after sowing and grow in the bed, at the middle of autumn (after hardening in natural conditions), in the 7-8 leaf stage put on the thermogradiant freezer with the 12 freezing temperatures (0,-2,-4,-6,-8,-10,-12,-14,-16,-18,-20,-22 oC). Cell membrane stability was measured through electrolyte leakage (EL) and survival percentage and regrowth of the plants after 3 weeks in cold frame were measured, by counting the number of plants and determining their proportional with the number of plants before freezing and measuring the dry matter, number of flowers and fully developed leaves. With decreasing the temperature EL increased significantly (P