Maryam Zare Hasan Abadi; Ali Ganjeali; Mehrdad Lahouti; Nasrin Moshtaghi
Abstract
Introduction: Agrobacterium rhizogenes hairy roots induction is used for secondary metabolite production in plants. A. rhizogenes is a genus of gram-negative soil bacteria belonging to the Rhizobiaceae family that causes hairy roots at the site of infection. Hairy roots have various advantages, including ...
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Introduction: Agrobacterium rhizogenes hairy roots induction is used for secondary metabolite production in plants. A. rhizogenes is a genus of gram-negative soil bacteria belonging to the Rhizobiaceae family that causes hairy roots at the site of infection. Hairy roots have various advantages, including high growth rate, more genetic stability than the callus and suspension cultures, growing well on hormone-free media that have been reported effective for producing high levels of secondary metabolites. Basil (Ocimum basilicum) is a popular herb with important economical applications in food, cosmetic, and pharmaceutical industry. It is a digestive stimulant with anticarcinogenic, antibacterial, and anticonvulsant properties. The main phenolics reported in basil plants are in the classes of phenolic acids and flavonoids, some of which have human health benefits. This study was designed to develop hairy root culture from O. basilicum using different of A. rhizogenes strains for the production of total phenols and introduce the best strain of A. rhizogenes to induce hairy root and growth and production of total phenol.
Materials and Methods: Different A. rhizogenes strains (ATCC-15834, A4, MSU, and R1000) were studied to investigate their effects for the transformation and production of secondary metabolites in O. basilicum. Therefore, shoot and leaf explants and nodes of the seedlings were used for Agrobacterium-mediated transformation. These explants were inoculated with four A. rhizogenes strains and transferred to ½ MS medium. About four weeks after cultivation with A. rhizogenes, hairy roots were excised from the seedlings and subcultured to fresh medium MS liquid culture containing 500 mg/l cefotaxime. After 60 days of inoculation, various parameters, including dry weight, infection percentage, number of hairy roots per explant, and total phenol contents were measured. The growth rate and phenolic contents of the transformed hairy roots were compared with normal ones. Total genomic DNA was isolated from non-transgenic and transgenic hairy root lines using the Cetyl Trimethyl Ammonium Bromide (CTAB) method. Isolated genomic DNA was used to detect the rolC gene through polymerase chain reaction (PCR) analysis. The PCR using specific primers for rolC of T-DNA and virD2 was used to confirm the nature of resulted transgenic hairy roots.
Results and Discussion: Selecting efficient A. rhizogenes strains, as well as the type of explants, are crucial factors for hairy root induction. All used A. rhizogenes strains were able to produce hairy roots. Hairy roots appeared on the nodes at the point of injection, but were not forming on the shoot and leaf explants. So, the choice of the plant material is crucial for successful transformation with A. rhizogenes and usually, transformation of young tissues gives the best results. The transgenic status of the hairy roots was confirmed using PCR with rolC and virD specific forward and reverse primers. All lines showed the presence of 612 bp rolC amplified products, indicating the integration of T-DNA of A. rhizogenes and O. basilicum. Hairy roots could synthesize phenolic compounds, which was significantly higher in hairy roots than non-transformed control. Four hairy root lines were independently evaluated for their content and these lines showed variation in total phenolic contents, with the highest amount (312 mgGAE/ g DW) in hairy roots induced by ATCC-15834 strain and the lowest amount (113.2 mgGAE/ g DW) in hairy roots induced by R1000 strain. The results showed that the strain ATCC-15834 caused the highest infection percentage (68.1%) along with the highest number of hairy roots (4.8) per explant and root length (1.8 cm). The growth rate and phenolics production were investigated in each hairy root of O. basilicum from infection by four different A. rhizogenes strains. The highest growth rate (103.2 mg DW) and production of total phenol (312 mg/g DW) were found in ATCC-15834. The growth rate of transformed hairy roots was more than that of normal ones.Total phenol contents in all hairy roots were also increased significantly compared with non-transformed control plants (4.6 times in hairy roots induced by A. rhizogenes strain ATCC-15834). ATCC-15834 has been reported as the most widely used A. rhizogenes strain owing to its strong induction ability, and the variation in hairy root induction could be due to disparity in the virulence of different A. rhizogenes strains.
Conclusion: The hairy roots of O. basilicum had shown promising results in terms of significant yield of phenolic contents and had the potential for being scaled-up further for phenol production. It could be concluded that A. rhizogenes strains had different abilities in hairy roots induction. Therefore, the selection of an effective A. rhizogenes strain for the production of transformed root cultures is important, highly dependent on the plant species, and must be determined in future experiments.
Simin Irankhah; Ali Ganjeali; Mehrdad Lahouti; Mansour Mashreghi
Abstract
Introduction: Fenugreek (Trigonellafoenum-graecum L.) is a traditional medicinal plant belonging to the legume family Fabaceae. Diverse groups of microorganisms are symbiotic with Fenugreek roots system. This integration leads to significant increases in the development and production by increasing nitrogen ...
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Introduction: Fenugreek (Trigonellafoenum-graecum L.) is a traditional medicinal plant belonging to the legume family Fabaceae. Diverse groups of microorganisms are symbiotic with Fenugreek roots system. This integration leads to significant increases in the development and production by increasing nitrogen fixation, phytohormones production, siderophores and phosphate solubilization. Plant growth-promoting bacteria increase plant growth byimproving nutrientuptake and phytohormones production. In addition, the beneficial effect of these bacteria could be due totheirinteractionwithArbuscularMycorrhizal fungi(VAM). Drought is one of the major limiting factors for crop production in many parts of the world including Iran. Symbiotic microorganisms can enhance plant tolerance to drought. This experiment was carried out to investigate the effect of Vesicular ArbuscularMycorrhiza (VAM) and Plant Growth Promoting Rhizobacteria (PGPR) on morphological and biochemical characteristics of Fenugreek in drought stress conditions.
Materials and Methods: The experiment was carried out in completely random design with 3 replications.There were four treatments including inoculation with Pseudomonas putida, inoculation with Glomusintraradices, combined association of Pseudomonas putida and Glomusintraradices and untreated as a check under drought stress (40% of field capacity) and non-stress conditions (80% of field capacity). In this experiment fiveseeds were sowninplastic pots. Before sowing, seeds were inoculated with microorganisms. In order to inoculation ofseed with Mycorrhizal fungi, for each kilogram of soil, 100 grams of powder containing 10 to 15 thousand spores of fungal soil (produced by the biotech company Toos) was added to three centimeters of soil in the pot. For seed inoculation with Plant Growth Promoting Rhizobacteria, the growth curve of the bacteria was drawn at first and then the best time for the growth of bacteria was determined. The bacteria at the best time and at a dilution of 0.5 McFarland was added to the seed.Pots were placed in a growth chamber (with a temperature of 25 ° C and 16 hours of light and 8 hours of darkness photoperiod).After ten days of planting, the water treatment was applied and continued until the end of the experiment.
Results and Discussion:The results showed that the use of co -inoculation treatment ofP.putidaandG.intraradicesand application of G.intraradices aloneat non-stress conditions did not make a significant difference on the amount of plant biomass. In drought conditions, application of G.intraradice alone had significant difference (P≤0.05) compare with control (no inoculation microorganisms) for biomass.In stress conditions, P.putida inoculation and also in combination with G.intraradicesincreased biomass in compare with control (no inoculation of micro-organisms), but this increasing was not statistically significant.VAMare important ecological symbiotic with roots are important component of the ecosystem and affect the absorption of minerals through the roots. The results of the present study showed that the amount of phosphorus in all of treatments was increased and the greatest increase was related to G.intraradicetreatment.The results also showed that drought stress increased the leaf soluble proteins in compared with non-stress condition. Increasing the concentration of soluble proteins under drought stress can be related to increased protein synthesis that maybe related to adaptation and reprogramming under new situation and itprotect the cells against stress. The results showed that the use of co -inoculation treatment ofP.putidaand G.intraradices, as well as each individual treatment, increased the amount of soluble proteins in leaves.In the case of the Diosgenin percentage, drought stress reduced the amount of Diosgenin percentage. Underdrought stress conditions, use of co -inoculation treatment ofP.putidaand G.intraradicesand application of G.intraradicewithout presence of bacteria made a significant increase in plant Diosgenin concentration.
Conclusion: Theresults revealed that seed treatment with Pseudomonas putida and Glomusintraradices increased the biomass, protein content, phosphorus uptake and diosgenin percent in Fenugreek under drought stress condition. Since Diosgenin is very important medicinal compound, inoculation of fenugreek with these microorganisms can be a way to increase the Diosgenin production.
Masumeh Modarres; Mehrdad Lahouti; Ali Ganjeali; Javad Asili
Abstract
Salvia leriifolia (Lamiaceae) is endemic of Khorasan and Semnan province and an endangered plant. Poor seed germination of this plant has a serious problem with the high production. The first step to improve this precious plant is to produce sterile plantlets in order to prepare explants of appropriate ...
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Salvia leriifolia (Lamiaceae) is endemic of Khorasan and Semnan province and an endangered plant. Poor seed germination of this plant has a serious problem with the high production. The first step to improve this precious plant is to produce sterile plantlets in order to prepare explants of appropriate vigor. In this study,in vitro culture of Salvia leriifolia zygotic embryo was performed through a factorial experiment in the form of completely random design including culture medium, BAP and NAA. The results showed that, MS and 1/2MS media appeared to be more efficient than B5 medium and significant differences were observed. Efficient concentrations of BAP and NAA were 1mgL-1 and had a significant effect in growth and development of embryos. The seedlings were obtained 10 days after planting. Based on these results, the best choice for quick access to strong seedlings, seedling growth and development of embryos is MS and 1/2MS media supplemented with 1mgL-1 BAP and NAA.
Maryam Sadat Araghi Shahri; Mehrdad Lahouti; Fereshte Ghasemzadeh; Hamid Ejtehadi
Abstract
Antimony is a toxic heavy metal for plants, animals and human being. Considering its effects on plants growth, different concentrations on watermelon growth and development were studied. The experiment was established on a factorial experiment based on a completely randomized design with 3 replications. ...
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Antimony is a toxic heavy metal for plants, animals and human being. Considering its effects on plants growth, different concentrations on watermelon growth and development were studied. The experiment was established on a factorial experiment based on a completely randomized design with 3 replications. The seedlings were grown hydroponically in nutrient solution containing Sb-EDTA (0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6 and 8 mg/L). Samples were harvested after four weeks to determine biochemical and morphological parameters. With increasing Sb concentration in culture medium, resulted in significantly decreased chlorophyll content and some other growth parameters in treated plants (p
Azadeh Saffar Yazdi; Mehrdad Lahouti; Ali Ganjeali
Abstract
Selenium is a metalloid that it is antioxidant characteristics is proved for humans, animals and plants. However, some plants are known as hyper accumulators for selenium and sometimes seems to be useful for growth of some plants. In this paper, we have studied the effect of different selenium concentrations ...
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Selenium is a metalloid that it is antioxidant characteristics is proved for humans, animals and plants. However, some plants are known as hyper accumulators for selenium and sometimes seems to be useful for growth of some plants. In this paper, we have studied the effect of different selenium concentrations on growth and morpho-physiological characteristics of spinach. Seeds of spinach were germinated in germinator and seedlings were transferred to hydroponic cultures. The seedlings were grown in Hogland´s solution with different selenium concentrations (in form of sodium selenite) of 0,1,2,4,6, and 10 mgL-1 SeO3-2. The experiment was conducted based on a completely randomized design with four replications. Four weeks after treatments, morphophysiological characteristics including plant biomass, length of shoot and root, number of leaves, amount of chlorophyll a and b were measured. The results showed that application of different selenium concentrations had significant effect on morphological and physiological characteristics of root and shoot consisting wet and dry weight, root and shoot length and amount of chlorophyll. In this experiment, increasing selenium concentrations (except in 1 mgL-1 SeO3-2 concentration) decreased amounts of all mentioned characters in comparison with control. Morphological symptoms of selenium toxicity on spinach was chlorosis on young leaves and significant reduction in root and shoot growth.
Zahra Khakshoor Moghadam; Mehrdad Lahouti; Ali Ganjeali
Abstract
Abstract
In order to study the effect of different water potential on Anethum graveolens two experiments were conducted for germination and vegetative stages. This study was carried out as a factorial experiment based on completely randomized design with three replications. In the first experiment, ...
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Abstract
In order to study the effect of different water potential on Anethum graveolens two experiments were conducted for germination and vegetative stages. This study was carried out as a factorial experiment based on completely randomized design with three replications. In the first experiment, response of dill seeds germination to levels of drought stress including to drought potentials zero(control), -1.5, -2, -2.5 and -3 bar that were obtained by polyethylene glycol 6000 were investigated. In the second experiment, similar to first, influence of drought stress on dill morphophysiological parameters were investigated. The results showed that effect of drought stress on germination characteristics including to germination percentage, germination rate, radicle and plumule length , radicle and plumule dry weight , plumule to radicle dry weight ratio was significant (p≤ 0.01). Mean camparison of treatments showed that with increasing drought stress, all parameters decreased. Effect of drought stress on amount of proline and soluble carbohydrates in shoot and root was significant (p≤ 0.01). Mean camparison of treatments showed that with increasing stress, amount of proline and soluble carbohydrates in shoot and root, shoot to root proline and soluble carbohydrates ratio increased. Shoot to root proline and soluble carbohydrates ratio were not influenced by drought stress (p≤0.05).
Keywords: PEG, Morphological parameters, Proline, Soluble carbohydrate
