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Hairy root culture is a promising approach to improve production of plant secondary metabolites. The genes, which are located in T-DNA of a root-inducing plasmid, regulate auxin sensitivity of hairy roots. Therefore, this study was aimed to improve the growth and rosmarinic acid production of Lavandula angustifolia hairy roots. Lateral branches of hairy roots were transferred to ½ MS and ½ B5 liquid media. To assess auxin sensitivity, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) with four different concentrations (0, 0.1, 0.5 and 1 mg/l) were also applied. The growth of hairy roots in ½ MS medium was two-fold higher than in ½ B5 medium. In addition, both auxins were found to significantly improve the growth of hairy roots whereas non-transformed roots stopped growing in the presence of the auxins. The highest dry weight and rosmarinic acid production of hairy roots were obtained from ½ MS medium supplemented with IBA irrespective of its concentration. As a result, the hairy roots grown in ½ MS medium supplemented with IBA produced the maximum amount of rosmarinic acid (7.98 mg/g dry weight of hairy roots). This first report of rosmarinic acid production in L. angustifolia hairy roots provides new insights into the auxin sensitivity of L. angustifolia hairy roots.

Pyrus communis is a difficult-to-root species, and its optimal rooting depends on applying an accurate concentration of various chemicals. Since statistical modeling can be applied to estimate the approximate range of factors needed for the success of in vitro plant propagation, we tried to optimize the rooting medium using response surface methodology. Therefore, sucrose and polyvinyl alcohol were selected as independent variables in the central composite design of response surface methodology with α = 1. Based on the pre-test, it was found that the independent variables for the root formation were 2 to 3% sucrose and 0.5 to 1.5 g L−1 polyvinyl alcohol in studied cultivars. The statistical analysis results showed that the quadratic models were significant, with high R2 values > 0.9 for rooting rate response. The optimized values for independent variables in rooting of the studied cultivars included 2% sucrose, 1.5 g L−1 polyvinyl alcohol in ‘Williams’; 2.85% sucrose, 1.47 g L−1 polyvinyl alcohol in ‘Natanzi’; and 3% sucrose, 0.99 g L−1 polyvinyl alcohol in ‘Ghosi’. A separate validation experiment detected that the experimental values of the studied responses are in close agreement with predicted values in all cultivars. For the first time, the methodology was developed using a central composite design to optimize the rooting in P. communis.

Carbon source plays an important role in the induction of embryogenic callus (EC). Therefore it is necessary to determine the effective carbohydrate for improving this process. In this study, different carbohydrates as sucrose, maltose, and sorbitol with different levels (0–30 g/L) were tested in the callus induction of Pyrus communis. At first, in solid culture, calli were divided in relation to their morphological appearance in EC and non-EC. EC was white or yellow with the granular aspect. The morphological study revealed the highest frequency of EC (28.5%) obtained at 30 g/L sucrose. Biochemical analyses showed EC exhibited both soluble sugar and protein high contents, while the starch was at the lowest level. As well as flow cytometry showed EC were genetically similar to donor explants. With transferring the EC into the liquid culture, then, subculturing calli along with 2 ml cell suspension on solid medium led to the globular embryo.

Feverfew (Tanacetum parthenium L. Schultz Bip.) has a lot of secondary metabolites, such as phenols, essential oils and sesquiterpene lactones (parthenolide) which have high value in chemot...

In order to understand the effects of ultraviolet-B (UV-B) radiation and salinity stress on visual quality and morphological parameters, one-year-old seedlings of rosemary (Rosmarinus officinalis L.) were grown under three UV-B levels (0, 4.32, 6.05 kJ m-2 d-1) and four salinity regimes (Control, 50, 100 and 150 mM NaCl) under greenhouse conditions. Results showed that, enhanced UV-B radiation increased visual quality, growth index, plant biomass, shoot/root ratio, number of branches and leaves, leaf fresh and dry weight, leaf area index, specific leaf area, leaf thickness. UV-B radiation had no significant effect on root length, however, shoot height, length of axillary shoots, length of inter node, leaf area, leaf length and width and concentration of chlorophyll b were negatively impacted by UV-B radiation. On the other hand, salinity caused significant decrease in plant biomass, root length, shoot height, shoot/root ratio, length of axillary shoots, length of inter node, number of branches and leaf area, leaf area index, leaf length and width, leaf fresh and dry weight, specific leaf area and concentration of Photosynthetic pigments. But, number leaves and leaf thickness significantly increased under NaCl salinity treatments. Visual quality and growth index were hardly affected by increasing salinity until 100 mM, but declined clearly at 150 mM NaCl salinity. Interaction between UV-B irradiation and salinity showed that, pre-treatment with UV-B irradiation alleviated harmful effects of NaCl and improved visual quality rosemary plants.

Mentha longifolia L. is well-known to be one of the most pervasive wild-growing species of the Lamiaceae family, which has extensive beneficial properties in the fields of pharmacology and biological products. In the present study, the correlation between Inter-simple sequence repeat (ISSR) markers and morpho-chemical parameters of twenty different M. longifolia accessions (MLACs) were assessed. The geographic information system (GIS) has been employed to interpret the original habitat of the accessions in Iran. ISSR analysis indicated a remarkable difference in the studied accessions, segregated them into three main groups, constructed by an unweighted pair-group method with arithmetic (UPGMA) and principal coordinate analysis (PCoA). A total of 89 bands were generated by 12 ISSR primers, among which 82 (91.97 %) of them were polymorphic. The cluster analysis based on agro-morphological data scattered MLACs into two main groups. The essential oils (EOs) were analyzed through GC/FID/MS, and four chemotypes were characterized according to the major constituents. Pulegone ranged from 0.17 to 69.50 % was the main oil constituent with the highest content. Also, HPLC-PDA was employed to identify and to quantify the phenolic compounds in the MeOH extracts of MLACs. Heatmap cluster based on phenolic compounds produced three main categories of accessions. The components identified in the extracts were rosmarinic acid, rutin, vanillic acid, ferulic acid, chlorogenic acid, caffeic acid, 3,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, and p-coumaric acid, which among them rosmarinic acid (RA) varied from 39.16 to 261.55 mg/100 g (DW) as a predominant constituent. Subsequently, multiple regression analyses between ISSR fragments and morpho-chemical data illustrated considerable relationships in the plant materials. The high variation and correlation observed in metabolic and phenotypic traits of MLACs establish an adequate source to conduct reserves conservation programs.

In the current work, we present an efficient collocation method based on the polynomial–Sinc combined with the Legendre–Gauss quadrature rule for numerically solving a class of distributed order fractional differential equations. This method uses Lagrange approximation at Sinc points. The main purpose of this work is to generalize polynomial–Sinc method to the fractional calculus. The convergence of our method is discussed. Finally, some test problems are used to verify the accuracy and applicability of the presented method.

Nanoelicitors are biological and non-biological factors that can affect the synthesis of secondary metabolites in medicinal plants. Feverfew is a valuable medicinal plant containing effective and important anti-cancer compounds (essential oil and parthenolide). This study was conducted to investigate the effects of zinc oxide nanoparticles (ZnONPs) on yield, metabolites content, and zinc and iron absorption of Feverfew. After seed preparation, seedling production, soil physicochemical properties analysis, ZnONPs treatments, maintenance and harvesting at full flowering stage, morphological traits and yield components were measured. The essential oil was extracted by Clevenger and parthenolide was identified by UPLC-MRM-MS. The content of Zn and Fe were measured by OES-ICP. The effects of ZnONPs were significant on all morpho-phytochemical traits. An increased biological yield (dry weight) was observed at 2000 ppm ZnONPs (32.54 g/day) compared to control (28.09 g/day). The highest (0.9% V/W) and lowest (0.56% V/W) content of essential oil were related to 1000 ppm ZnONPs and control. The content of parthenolide decreased at different levels of ZnONPs. The lowest (36.83 mg/kg DW) and highest (266.02 mg/kg DW) rates of Zn absorption were observed in control and 2000 ppm ZnONPs. Nanoparticles at all concentrations increased the biological yield, essential oil content, and Zn absorption. None of the ZnONPs concentrations improved the flower yield. Along with increasing ZnONPs and Zn uptake, parthenolide levels decreased. Also, it was determined that there was an antagonistic effect between Zn and Fe absorption.

This chapter includes the methods for tissue and cell culture of saffron. Saffron is a triploid plant, and therefore vegetative reproduction is a common way for its propagation. In traditional culture, few cormlets are produced each year and thus the production rate is low. Tissue culture methods such as micropropagation, somatic embryogenesis, organogenesis, and in vitro cormogenesis are necessary for mass and free pathogen reproduction of this plant. All of these methods are discussed in the preliminary sections of this chapter. Tissue culture techniques can be very effective and useful methods for genetic modification and to produce new varieties of saffron. Protoplast culture to produce somatic hybrids and stigma-like structure culture to produce stigmas and their compounds that are used in saffron tissue culture and also related research is included at the end of this chapter.

In this research, agro-morphological variation among twenty genotypes of Mentha longifolia L. was assessed in 2 years based on agricultural production under water stress condition. The experiment was arranged in split–split-plot design with water stress treatment in two irrigation levels of 100% (as control) and 60% field capacity in three replications. The result of variance analysis indicated that there was a significant variation (P ≤ 0.01) in traits among the genotypes and in response to the water deficit condition. Different traits were measured such as plant height, yield, date of flowering, inflorescence length, stem diameter, fresh and dry weight of leaf, essential oil content, and essential oil yield. Results showed that traits such as yield and essential oil yield had considerable variation, varying from 374.17 to 899.33 g m−2 and 17.7–391.6 kg ha−1 on the analysis in response to water stress condition, respectively. Cluster analysis using the unweighted pair group method with arithmetic means grouped the studied twenty genotypes into two major clusters based on agro-morphological traits which all the studied genotypes were known as yield ecotype. Five genotypes including Hormozagan, Khuzestan, Kerman-1, West Azarbaijan, and Ghazvin had the most significant biomass and essential oil performance and resistance under water stress condition that can be introduced for domestication breeding programs and cultivation.

The purpose of this investigation is to achieve the procedure of somatic embryogenesis (SE) as an efficient propagation way in four pear cultivars: Natanzi, Ghosi, Williams and Dar Gazi, by focusing on two strategies: One is the application of competent explants, and the other one is stress employment. The results were variable because of cultivars behavior was highly genotype specific. However, the results identified that the highest frequency of embryogenic callus induction was obtained from the endosperm and immature cotyledonary segment explants of P. communis cultivars on Murashige and Skoog medium (MS) containing 8.05 μM 1-naphthaleneacetic acid (NAA), 13.32 μM N6-benzyladenine (BA) supplemented with 250 mg/L glutamine as a reduced nitrogen source and 30 g/L (w/v) sucrose in Ghosi (75.46%). The optimized media were different for each cultivar in the induction phase, namely, Natanzi and Ghosi in MS, Williams in Schenk and Hildebrandt (SH) and Dar Gazi in Nitsch and Nitsch (NN). Then, it was observed that when embryogenic calli were transferred to the media containing 1% (w/v) Polyethylene glycol (PEG), calli of some cultivars further differed into embryos while other cultivars produced in chilling at 4 °C. Also, maturation of embryos occurred at 4 °C for 4 months. Then passages using MS media caused germination of embryos and plantlets were produced. To sum up, the greatest frequency of embryogenic callus (75.46%) occurred in Ghosi, and the highest embryo maturation and germination (77.06% and 37.66%, respectively in mentioned cultivars) were observed in Natanzi.

Glycyrrhiza glabra (licorice) is a medicinal plant with valuable specialised metabolites such as triterpene sweetener glycyrrhizin. Salinity stress is the main environmental stress limiting plant growth and development. The effects of six levels of NaCl (0, 100, 200, 400, 600, and 800 mM) on growth, osmolyte content, oxidative stress markers, antioxidant enzyme activities, K+/Na+ ratio, glycyrrhizin content, and gene expression of glycyrrhizin biosynthesis (bAS, CYP88D6, and CYP72A154) were investigated in licorice rhizomes of two populations. The results showed that the salt stress progressively reduced the growth parameters and increased the proline concentrations in the rhizomes. K+/Na+ ratio showed a significant decrease under salinity as compared to the controls. Salt stress resulted in oxidative stress on the rhizomes, as indicated by increased lipid peroxidation and hydrogen peroxide concentrations and elevated the activities of antioxidant enzymes (i.e., ascorbate peroxidase and superoxide dismutase). The glycyrrhizin content increased only under 100 and 200 mM NaCl treatments. The same trend was observed in the expression of bAS, CYP88D6, and CYP72A154 genes in Fars population. Fars population was found to have more glycyrrhizin content than Khorasan population. But, growth, glycyrrhizin content, and biosynthesis genes of glycyrrhizin showed more reduction in Khorasan population as compared to those of Fars population. The results indicate that the application of 100 mM NaCl up-regulated the expression of key genes involved in the biosynthesis of triterpenoid saponins and directly enhanced the production of glycyrrhizin. Accordingly, G. glabra can be introduced as a halophyte plant.

Background: Transient and stable transformation of host plants are the common techniques to produce transgenic plants. However, the main drawback of stable transformation is the fact that it takes quite a long time to produce a transgenic line. While, transient gene expression is a quick method to produce recombinant proteins in plants. Objective: The main goal of the present study was to evaluate efficient agroinfiltration as an efficient and rapid method for production of recombinant antigen of FMDV. Materials and Methods: Tobacco leaves were transformed via agroinfiltration using a needle-free syringe. Presence of the gene cassette was verified by polymerase chain reaction (PCR). Expression of the foreign gene was evaluated using Real Time PCR, protein dot blot and enzyme-linked immunosorbent assay (ELISA). Results: PCR analysis confirmed successful transformation of plant leaves. Expression of foreign protein was confirmed at both transcription and translation levels. Results of Real Time PCR assay indicated that the foreign gene was transcribed in transformed leaves. ELISA results showed that the foreign gene was expressed in the transformed leaves in high level. Conclusion: Here, the efficacy of agroinfiltration for transient expression of FMDV coat protein in tobacco was illustrated. Accordingly, transient agroinfilteration expedites the process of recombinant antigens expression in plant tissues.

Mentha longifolia L. is one of the most important aromatic and perennial herbs of the Lamiaceae family, having potential sources of essential oils and compounds with interesting pharmacological and therapeutic properties. In this study, the variability of morphological parameters, essential oil composition, and phenolic compounds such as rosmarinic acid and caffeic acid content were assessed among twenty different accessions of Iranian horsemint at full flowering stage. The average of essential oil content illustrated a high variability, ranging from 0.38% to 4.33% (w/w), and the phytomass amounts varied from 471.5 g/m2 to 860.66 g/m2. Also, the clustering of morphological traits of the studied accessions generated two main groups. The characterization of the essential oils by GC-MS/FID analyses revealed a total of forty-two constituents in the horsemint accessions. Although all the accessions were classified as oxygenated monoterpenes-rich volatile oils (42.38–94.7%), they were divided into seven main chemotypes by cluster and principal component analysis (PCA). The major constituents in the essential oil of the twenty accessions were piperitenone oxide (0.27–75.39%) > pulegone (0.17–69.49%) > trans-piperitone epoxide (0.3–54.39%) > menthone (0.5–48.12%) > 1,8-cineole (1.27–44.7%) > piperitenone (0.35–36.71%) > (Z)-β-ocimene (14.11–21.27%) > menthol (0.13–20.98%), respectively. The rosmarinic acid content of the methanolic extracts using HPLC analysis showed significant quantitative variability, ranging from 36.95 mg/100 g to 302.97 mg/100 g (based on the dry matter). Since all the accessions were subjected to similar soil and climate conditions, it was evident the priority of genetic effects on ecological factors. The high intraspecific variability in Iranian horsemint directs a holistic approach to gain novel and homogeneous genetic materials, required for the breeding programs and pharmaceutical industries.

The plant vacuolar Na + /H + antiporter plays an important role in salt tolerance. A vacuolar Na + /H + cDNA ( LfNHX1 ) gene was cloned from Leptochloa fusca (L.) Kunth, a halophyte forage grass, by cDNA and RACE methods using degenerate primers. The results show that the LfNHX sequence contains 2452 nucleotides and an open-reading frame of1620 nucleotides that encode a protein of 540 amino acids with a deduced molecular mass of 59.8 kDa. The deduced amino acid contains the consensus amiloride-binding domain. The effects of NaCl stress on the Na + / H + gene expression were analysed using Real Time PCR. The analysis showed that the expression level of LfNHX1 increases in response to different levels of NaCl, indicating that V-Na + /H + plays an important role in salt tolerance in this plant.

Leaves, one year old stems and buds were used as explants. WPM, B5, MS, half and one-fourth strength MS with 2,4-?D, NAA, IBA, IAA and picloram 2, 5 and 10 mg/l were used for callus induction. Investigation into the seasonal effect on callus regeneration and explants browning was carried out in mid-April until September. Results showed that washing with sterile distilled water for one hour after surface sterilization was the best treatment for browning inhibition of explants than other treatments. Leaf explants have best callus formation. MS with 8 g/l agar had strong effect on callus formation. Culture for callus formation in May and June found to be higher than other months. 2,4-D, NAA and 10 mg/l picloram significantly enhanced callus formation. Leaf and bud explants sampling in April to May did not show any endophyte contamination but leaf explants in June until September, specially the one year old stems, showed intensive contamination. Key words: Seasonal effect, Browning inhibitor, Berberis vulgaris D. O. I. 10.3329/ptcb.v21i2.10239 Plant Tissue Cult. & Biotech. 21(2): 161-168, 2011 (December)

The synthesis of chitinases is one of the strategies evolved in higher plants to resist stresses. To investigate the role of chitinases in the interaction between chickpea (Cicer arietinum L.) and pathogens, the expression of two classes of chitinase, I and III, was monitored in infected and non-infected plants with different disease resistance (resistant MCC10 and sensitive MCC724 genotypes). The expression levels of these genes in chickpea were studied at different times after inoculation with Fusarium oxysporum f. sp. ciceri isolate 85. Semi-quantitative reverse transcription PCR was used to study the differential induction of the chitinase genes. Expression levels of chitinase I in non-infected and infected MCC724 plants were higher than those in MCC10. Since chitinase III may play an effective role in plant defense, its expression levels were quantified by reverse transcription PCR. Chitinase III was induced in MCC724 within 6 h and in MCC10 in 48 h after inoculation. However, the expression levels of chitinase III in MCC10 non-infected plants were significantly higher than in MCC724. The results showed that chitinase III can be effective to enhance resistance to F. oxysporum f. sp. ciceri isolate 85.

Eukaryotic NHX transporters are trans-membrane proteins which are vital for cellular homeostasis. Plants vacuolar NHX antiporters have been suggested to be involved in sequestering Na+ into vacuoles, thus preventing toxic effects of Na+ in the cytoplasm. Primary and secondary structure analysis of NHX antiporter of Leptochloa fusca suggested that this antiporter is a hydrophobic protein containing a significant proportion of alpha helixes. In this study, a three-dimensional structures of NHX antiporter predicted by in silico 3D homology modeling studies. The resulting model was refined by energy minimization, subjected to the quality assessment from both geometric and energetic aspects and was found to be of reasonable quality. Validation of 3D structure was done by plotting Ramachandran plot and calculation of QMEAN score. This predicted information will help in better understanding of mechanisms underlying to salt tolerance in monocot plants and use of this information in protein engineering to improve plants to high salinity conditions.

This study surveyed a rapid, efficient and reproducible protocol for in vitro shoot regeneration and rooting by different explants and different concentration of BAP. Due to optimization of shoot regeneration, two media including of MS and modified MS (MS salts with double concentration of CaC21, and B5 vitamins), different explants such as decapitated embryo axes, epicotyls and cotyledonary nodes and different concentrations of BAP hormone (1, 1.5, 2, 2.5, 3 and 4 mg L(-1)) in two genotypes (Gachsaran and Flip. 92-12 L) were used. The results showed that modified MS is a suitable medium for in vitro shoot regeneration of lentil. High levels of BAP caused increasing of shoot regeneration in lentil genotypes. Three milligram per liter of BAP induced the highest level of shoot regeneration. In addition, decapitated embryo explants were the best explants for highest shoot regeneration (5.8) (p0.05). However, increasing of hormone concentration from 2 to 3 and 4 mg L(-1) caused decreasing in the number of shoots, so 2 mg L(-1) of BAP was best. For rooting, the in vitro-in vivo method of rooting was better than only in vitro method. The shoots regenerated in 2 mg L(-1) BAP had higher rooting percentage than the shoots were regenerated in 3 and 4 mg L(-1) BAP. These results indicate on the inhibitory effect of high concentration of BAP on root induction. But the genotype didn't have any significant effect on rooting percentage and length of roots.