Roghayeh Fathi; Mehdi Mohebodini; Esmaeil Chamani
Abstract
Introduction: Transformed hairy roots obtained by infection of plants with Agrobacterium rhizogenes strains are used as a tissue culture tool for secondary metabolite production since hairy roots are genetically and biologically stable and they are able to produce metabolite within a short time. Chicory ...
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Introduction: Transformed hairy roots obtained by infection of plants with Agrobacterium rhizogenes strains are used as a tissue culture tool for secondary metabolite production since hairy roots are genetically and biologically stable and they are able to produce metabolite within a short time. Chicory (Cichorium intybus L.) is a medicinal plant from Asteraceae. This plant contains many important metabolites include chicoric asid, inulin, scoline, coumarin and flavonoids.
Materials and Methods: The seeds were surface-sterilized by rinsing with 2% benomil for 30 min and later sterilized with 5% sodium hypochlorite for 20 min and subsequently with 70% ethanol for 90 s. the wounded leaf of 20 and 28-day-old seedlings inoculated with Agrobacterium rhizogenes 11325. Bacterial colonies were cultured on liquid LB medium for 1 day. The explants were submerged in the solution for 15 min and were shaken gently. After that, the explants were drained on sterile filter paper and then transferred to MS solid medium. The explants were incubated at (25 ± 2) ◦C under dark condition for three different co-cultivation period (24, 48 and 72 hours). In the next step, the explants were transferred to MS solid medium supplemented with 500 mg/L cefotaxime. The explants were incubated at (25 ± 2) ◦C under a 16-h photoperiod condition for four weeks. At the end of incubation time, the percentage of hairy root induction, root number and root length were investigated. Genomic DNA of Cichorium intybus L. hairy root was extracted following the method of CTAB and subjected to PCR analysis. Non-transformed root DNA was used as a negative control and pRi11325 plasmid DNA was used as positive control. The pair of primers specific to the rolB fragment sequences was: 5-ATGGATCCCAAATTGCTATTCCCCACGA -3 and 5-TAGGCTTCTTTCATTCGGTTTACTGCAGC-3. The amplification protocol for the rolB was a 5 min melting at 94 ◦C followed by 35 cycles of a 5 min melting at 94 ◦C, a 45 s annealing at 55 ◦C and a 1 min elongation at 72 ◦C, and final elongation at 72 ◦C for 7 min. PCR products were electrophoretically separated on 0.8 % agarose gels (w/v) and stained with gel red. To determine the best media composition for growth of hairy roots, approximately 100 mg FW roots were cultured in basal liquid MS, solid MS and liquid 1/2MS medium supplemented with 500 mg/L cefotaxime on a rotary shaker (90 rpm) at 25 ◦C in dark for 5 weeks, and increase in fresh weight and dry weight was recorded at 5 week later. Three replicates were made for each experimental set. Non-transformed roots were cultured on same mediums.
Results and Discussion: Each of the two different explants – leaf and petiole showed different levels of hairy root induction in different co-culture time. Hairy root induction was observed 7 days after co-cultivation. There were low adventitious roots formed from control explants. 72 hours co-cultivation period was found to be more efficient in inducing hairy root phenotype in both explant types. The infection of explants by A. rhizogenes is a process that implies a succession of events, including the transfer and integration of T-DNA, into the host plant genome. Therefore, the time of contact between A. rhizogenes and the explants determines the transformation efficiency. A maximum of transformation frequency (53.33%) was observed in leaf explants in 72 hours co-cultivation followed by 33.33% in petiole explants in 72 hours co-cultivation. In leaf explants maximum root number (8.5 roots per explant) and maximum length of root (9.16 cm) induced from 20-day-old seedling in 72 hours co-culture. In petiole explants maximum root number (3.66 roots per explant) and maximum root length (11.46 cm) induced from 28-day-old seedling in 72 hours co-culture. The “hairy root” phenotype characterized by high branching and fast growth was previously reported in “reactive species” like Datura innoxia but not for “difficult species”. The high phenolic content of woody plant species could be responsible for the reduced hairy root phenotype as reported for gingko or pine. Three different culture media (solid MS, liquid MS and 1/2 MS media) were tested to determine the best suitable media for the maximum Biomass of hairy roots line.
Conclusions: Genetically transformed hairy roots obtained by infection of plants with Agrobacterium rhizogenes are suitable source for production of bioactive molecules due to their genetic stability and generally show fast growth in culture media free of growth hormones. This study describes the protocol for hairy root induction which could further be useful for the production of secondary metabolites and biomass.
Mehdi Mohebodini; Zahra Azimzadeh; Esmaeil Chamani; Malihe Erfani
Abstract
Introduction: Lily (L. ledebourii) is the rarestspeciesof thegenusLilium, and grows in Caucasus region. Iranis one of the important distribution areas of this endangered species. It is important as an ornamental plant due to its large and attractive white flowers that are equal to those of commercial ...
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Introduction: Lily (L. ledebourii) is the rarestspeciesof thegenusLilium, and grows in Caucasus region. Iranis one of the important distribution areas of this endangered species. It is important as an ornamental plant due to its large and attractive white flowers that are equal to those of commercial lilies in terms of beauty.The two main constraints on growing this plant are a low multiplication rate and the high cost of bulb production. Five to ten flowers commonly appear on each plant, even specimens with up to 15 flowers have been observed. Plant tissue culture techniques are widely used in plant propagation and using these methods can effectively provide micro-propagation of this plant in large scale. High percentage ofregeneration is necessary for plant protection, using in the breeding programs and gene transfer to this plant. Therefore, the effect of plant growth regulators and abiotic stress (ultrasound) werestudied on the bulblet production and root induction of Lilium ledebourii.
Materials and Methods: The experiment was factorial based on completely randomized design with four replicattions and was carried out in tissue culture lab of University of MohagheghArdabili in 2015. For this purpose, segmentsof scale explant was treated with ultrasound and cultured on MS medium supplemented with different concentrations of NAA and BA alone and/or in combination with each other. In this experiment, different concentrations of NAA (0, 0.01, 0.1 and 1 mgl-1) and BA (0, 0.01, 0.1 and 1 mgl-1) and different Ultrasound exposure duration (0, 5, 10, 20 and 30 second) were studied. In order to remove possible contamination from the media, all media were autoclaved for 20 minutes at 121 °C. At the end of the experiment, the number of bulblet, root length, fresh weight of bulblet were recorded. The cultures were kept at 25±2°C under illumination with daylight fluorescent lamps (30 mol m-2s-1) at 16 h photoperiod. Data was subjected for analysis of variance and compare means using SPSS 16.
Results and Discussion: The results showed that ultrasound had negative effect onroot length, so that the highest root length was observed in explants without ultrasound treatment. Result also indicated that ultrasound had positive effect on bulblet production and root induction. A different effect of growth regulators was observed in similar media on the bulblet formation percentage. The 0.1 NAA concentration had a higher efficiency while increasing NAA insignificantly decreased bulblet induction. The highest total weight and number of bulblets obtained by 0.1 mgl-1 NAA. Concentrations of NAA increased rooting percentage. Different concentrations of NAA had also significant effects on some traits. So that, the highest weight of bulblets obtained by 0.01 and 0.1 mgl-1 BA and the highest number of roots obtained in control. Bulblet maximum mean weightwas in30 seconds ofultrasoundtreatment, which hada significantdifference with the control treatment (without ultrasoundtreatment). In the other hand, ultrasound increased the number and weight of bulblets.Mechanical stress and microstreaming by acoustic cavitation might be considered as the most possible cause of the various physiological effects of ultrasound on cells. The enhancement of V-ATPase transport and ATP hydrolysis activities seem to be an ultrasound-induced metabolic response of cells. High-intensity ultrasound is well known to be destructive to biological materials, disrupting the cell membranes and deactivating biological molecules such as enzymes and DNA. Low-intensity ultrasound, on the other hand, has shown a range of sub lethal biological effects that are of potential significance in biotechnology. There are several processes that take place in the presence of cells or enzymes activated by ultrasonic waves. High-energy ultrasonic waves break the cells and denature the enzymes. Low-energy ultrasound can modify cellular metabolisms or facilitate the uptake of nutrient, and make them easily through the cellular walls and membranes. In the case of enzymes, the increase in the mass transfer rate of the reagents to the active site seems to be a most important factor.
Conclusions: The results showedthatthebulblet production at first stages and a little root formation in tissue culture is useful for fast bulblet inductionandthenrooting. Finally, it seems that ultrasound in combination with plant growth regulators have the potential to produce the highest average number of bulblets in the scale explant.
Esmaeil Chamani; Marziyeh Ghamari; Mahdi Mohoboldini; Alireza Ghanbari; Hamid Reza Heydari
Abstract
Introduction: Crown imperial (Fritillariaimperialis L.) is an ornamental and medicinal plant native to mountainous regions of Iran. This plant genetic resources is in danger of extinction, because of grazing livestock and pest outbreaks. However, due to slow reproduction in natural conditions and traditional ...
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Introduction: Crown imperial (Fritillariaimperialis L.) is an ornamental and medicinal plant native to mountainous regions of Iran. This plant genetic resources is in danger of extinction, because of grazing livestock and pest outbreaks. However, due to slow reproduction in natural conditions and traditional multiplication methods such as scaling and Bulb division, many species of this genus are endangered. Using of biotechnology, namely in vitro plant propagation, is a solution to the problems of reproduction of rare and endangered plant species with difficult propagation and mass production of valuable genotypes. Therefore, micropropagation of F. imperialis through in vitro regeneration is essential for conservation and commercial production.
Material and Methods: The bulbs of F. imperialis in dormancy stage obtained from Ilam mountainous regions in Iran and theywere placed in wet vermiculite at 4 °C for 4-6 weeks. Then, Bulbs were surface-sterilized with 70% ethanol for 60s followed by immersion in 5% (v/v) NaOCl solution for 20min with gentle agitation, and they rinsed three times in sterile double distilled water. Explants prepared from the lower third of scales with basal plate and were placed in MS basal medium supplemented with different concentrations of NAA and 2,4-D for callus induction. Test tubes with bulb segments were maintained within 25±2°C in growth chamber at 16 hours light period by the illumination from white florescent tube light and 8 hours dark. After two months callus were transferred to MS basal medium without PGRs. Then, callus excised to 0.5 cm pieces and were transferred to MS basal medium supplemented with NAA in 0, 0.3 and 1 mg/l concentration.Three types of cytokinins with different concentrations were arranged in three seperated experiments. Thefirst experiment medium contained NAA with BA (0, 0.3, 0.5 and 1 mg/l), the second experiment NAA combined with 0, 0.1, 0.3 and 0.5 mg/l TDZ and the third experiment MS basal medium included NAA with Kin (0, 0.5, 1 and 1.5 mg/l). After three months, percentage of callogenesis, diameter of calli, percentage of regeneration, number of leaves and roots and length of leaves and roots were measured. This experiment were carried out in completely randomized design with 4 replications.
Results and Discussion: In the first experiment application of NAA and BA on in-vitro multiplication of F. imperialis were evaluated. Highest callogenesis and formation (100 %) was observed in mediums contained 0.3 mg/l NAA + 1 mg/l BA, 0.6 mg/l NAA + (0.3, 0.5 and 1 mg/l) BA. Also, callogenesis was obtained in medium contained 0.5 mg/l BA without NAA. This result showed that only in medium supplemented with 1 mg/l BA provided highest (100%) callogenesis, when NAA concentrations were low. However, high levels of NAA (0.6 mg/l) in all concentrations of BA were obtained maximum callogenesis. We concluded that NAA is essential for callogenesis and enhancing its levels can increase callogenesis. Also, application of low levels of BA (0.4 µM) in callogenesis mediums of Cynodon dactylon contained Auxins resulted in increment of embryogenetic calli formation. In the other hand, presence of BA is essential for plantlet regeneration, however NAA is not necessary. Plantlet regeneration was obtained in PGRs free medium. Statistical analysis of results showed that different concentrations of BA and NAA had significant effects on percentage of callogenesis, diameter of calli, percentage of regeneration, length of leaves and roots (P
Hassan Hasani Jifroudi; Mehdi Mohebodini; Behrooz Esmaielpour; Esmaeil Chamani
Abstract
Introduction: Fenugreek (Trigonella foenum- graecum) is a medicinal plant extensively distributed in most regions of the world. Fenugreek is an annual plant from the family of papilionaceae, leguminosae. Fenugreek leaves and seeds have been used extensively to prepare extracts and powders for medicinal ...
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Introduction: Fenugreek (Trigonella foenum- graecum) is a medicinal plant extensively distributed in most regions of the world. Fenugreek is an annual plant from the family of papilionaceae, leguminosae. Fenugreek leaves and seeds have been used extensively to prepare extracts and powders for medicinal uses. Its root, leaf and seed contain a number of important medicinal compounds such as polysaccharide, galactomannan, different saponins such as diosgenin, yamogenin, mucilage, volatile oil and alkaloids such as choline and trigonelline. Plant tissue culture is fundamental to most aspects of biotechnology of plants. Establishment of an efficient callus induction and direct regeneration protocol is an essential prerequisite in harnessing the advantage of cell and tissue culture for genetic improvement. For the successful application of the tissue culture technique in plant breeding, callus induction and plant regeneration potential of each plant must be determined. The present study was performed in order to determine the optimum concentration of plant growth regulators (IBA + TDZ) for producing of in vitro plantlet using cotyledon and hypocotyl as an explant for two different Iranian genotypes (Ardestani and Neyshabouri).
Materials and Methods: In this investigation, Ardestani and Neyshabouri genotypes were used for callus induction and direct shoot regeneration. The medium used in this investigation was MS (Murashige and Skoog) basal medium. Then seeds were germinated on MS medium. For callus induction and direct shoot regeneration, cotyledon and hypocotyl explants were excised from 8-day-old sterile seedlings and cultured on MS medium containing various concentrations of IBA and TDZ. In this experiment, two combinations (TDZ + IBA) were used. In the first composition, IBA had four levels (0, 0.1, 0.3, 0.5 mg l-1) and TDZ had five levels (0, 0.2, 0.4, 0.6, 0.8 mg l-1) and in the second composition, IBA had four levels (0, 0.05, 0.1, 0.15 mg l-1) and TDZ had seven levels (0, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 mg l-1).The experimental designs were factorial based on completely randomized design with four replications. Cultures were incubated at 25° C ± 2 with a 16/8 hour (day/night) photoperiod and an irradiance of 1500 LUX using Sylvania cool white fluorescent tubes. The percentage of callus induction, direct shoot regeneration and average weight of callus were calculated for cotyledon and hypocotyl explants. All Data were analyzed using SPSS16, and mean comparisons were performed with duncan’s multiple range test (P < 0.05).
Results and Discussion: According to our results, explants cultured on MS basal medium without plant growth regulators (control) produced no callus. However, after two weeks, callus formed in both of Ardestani and Neyshabouri genotypes from cotyledon and hypocotyl explants in the presence of IBA + TDZ plant growth regulators in most of the combinations. In hypocotyl explants of Neyshabouri genotype, the highest callus induction was obtained from the medium containing 0.15 mg l-1 IBA + 0.45 mg l-1 TDZ (96.87%). Various important factors such as genotype, source of explants and plant growth regulators significantly influence direct regeneration. Direct regeneration was obtained from hypocotyl explants for Neyshabouri genotype in combination IBA + TDZ. The highest percentage of direct shoot regeneration was observed in MS medium containing 0.05 mg l-1 IBA + 0.35 mg l-1 TDZ in hypocotyl explants of Neyshabouri genotype (37.5%). Direct shoot regeneration requires plant cells to undergo dedifferentiation which is known to be affected by not only exogenous plant growth regulators but also endogenous content of the hormones. Different tissues may have different levels of endogenous hormones and, therefore, the type of explant source would have a critical impact on the regeneration success. In our study, when cotyledon and hypocotyl explants were compared, it was clear that hypocotyl explants were much more productive for direct shoot regeneration than cotyledon explants.
Conclusions: Callus induction and direct shoot regeneration are as in vitro tissue culture methods. Plant growth regulators and types of explant and genotype are the most important factors for callus induction and direct shoot regeneration phases. Therefore, optimization of these factors is essential to establish a high frequency of callus induction, direct shoot regeneration and gene transferring to this plant. According to the results of this investigation, it is recommended to apply plant growth regulators that were used in this study for other landraces of fenugreek cultured in Iran and select the best genotypes in response to tissue culture conditions for using in future studies.
Homeyra Hadizadeh; Mehdi Mohebodini; Behrooz Esmaeilpour; Esmaeil Chamani
Abstract
Introduction: Chicory (Cichorium intybus L.) belongs to Asteraceae family is commonly known as witloof chicory. The leaves and the roots of this medicinal plant are edible and commonly used as salad. Some varieties are also cultivated as coffee substitute after roasting the roots. All parts of the plant ...
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Introduction: Chicory (Cichorium intybus L.) belongs to Asteraceae family is commonly known as witloof chicory. The leaves and the roots of this medicinal plant are edible and commonly used as salad. Some varieties are also cultivated as coffee substitute after roasting the roots. All parts of the plant contain these volatile oils, with the majority of the toxic components concentrated in the plant's root. In folk medicine, the plant is used for the treatment of diarrhea, spleen enlargement, fever, and vomiting. Antihepatotoxic activity on damaged rat’s liver sections and anti-bacterial activity of this crop has been recently reported. In vitro regeneration from leaf explants with various hormonal combinations has been reported previously. Moreover, in vitro regeneration of Chicory from cotyledon explants using different combinations of plant growth regulators has been studied. Also, a protocol for the regeneration of plantlets from leaf and petiole explants of witloof chicory has been developed. The aim of the present investigation was optimization of callus induction and shoot regeneration from leaf and petiole tissues of Chicory (Esfahan genotype).
Materials and Methods: In this investigation, Esfahan genotype was used for callus induction and direct shoot regeneration. Seeds were first washed with running tap water for 30 min then seeds were surface sterilized by dipping in 70% ethanol for 90 s and rinsed with sterile distilled water, followed by immersing in 5% sodium hypochlorite solution for 25 min and thereafter rinsed for 30 min with sterile distilled water. The basal medium used in this investigation was MS. For shoot regeneration, leaf and petiole explants (5 mm segments) were excised from 4-week-old sterile seedlings and cultured on MS medium containing different combinations of NAA / BA and KIN / BA in two separate experiments. Experiments were performed factorial based on completely randomized design. Cultures were incubated at 25° C ± 2 with a 16/8 hour (day/night) photoperiod and an irradiance of 1500 LUX using Sylvania cool white fluorescent tubes. The percentage of callus induction, shoot regeneration and the number of regenerated shoots were calculated for the leaf and petiole explants. Data was subjected for analysis of variance and means were compared in 5% level with Duncan’s multiple range tests.
Results and Discussion: Explants cultured on medium containing either no plant growth regulators (control) or cytokines alone produced no callus. However, after 2 weeks, other concentrations of NAA and BA indicated callus formation from leaf and petiole explants in all hormone combinations. In leaf explants, the highest callus induction were obtained in the medium containing 0.3 mg l-1 NAA with 1 mg l-1 KIN and 0.3 mg l-1 NAA with 1.5 mg l-1 KIN (81.25%). Leaf and petiole explants cultured on medium containing no plant growth regulators (control treatment) and medium containing NAA produced no shoots. The combination of 0.3 mg l-1 NAA and 0.1 mg l-1 BA was the best treatment tested. This treatment produced 2.7 shoots per explant at 71% shoot regeneration frequency in leaf explant and 2.73 shoots per explant at 73% shoot regeneration frequency in petiole explants. The results also showed that the highest percentage of regeneration and the highest number of regenerated shoots were obtained in the medium containing 0.1 mg l-1 NAA and 1 mg l-1 KIN in leaf explants (65.6% regeneration and 1.37 shoots per explant, respectively). The highest number of regenerated shoots was obtained in the medium containing 0.3 mg l-1 NAA and 0.5 mg l-1 KIN in petiole explants (40.6% regeneration and 0.5 shoot per explants, respectively. Shoot regeneration requires plant cells to undergo dedifferentiation which is known to be affected by not only exogenous plant growth regulators but also endogenous content of the hormones. Different tissues may have different levels of endogenous hormones and, therefore, the type of explant source would have a critical impact on the regeneration success. In our study, when leaf and petiole explants were compared, it was clear that leaf explants were much more productive for regeneration than petiole explants.
Conclusion: Callus induction and shoot regeneration are in vitro tissue culture methods. Plant growth regulators and types of explant are the most important factors for callus induction and shoot regeneration phases. Therefore, optimization of these factors is essential to establish a high frequency of callus induction, shoot regeneration and gene transfer to this plant.
Mehdi Mohebodini; Mokhtar Jalali Javaran; Hooshang Alizadeh; Fereydoon Mahboodi; Hossein Khosravi
Abstract
Abstract
Lettuce (Lactuca sativa L.) is a major leafy vegetable and belongs to the Asteraceae family (Compositae). The genetic manipulation of lettuce requires a reliable and efficient regeneration method. Since lettuce is a suitable bioreactor for production of recombinant proteins especially edible ...
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Abstract
Lettuce (Lactuca sativa L.) is a major leafy vegetable and belongs to the Asteraceae family (Compositae). The genetic manipulation of lettuce requires a reliable and efficient regeneration method. Since lettuce is a suitable bioreactor for production of recombinant proteins especially edible vaccines. This study was conducted to identify an appropriate method for lettuce gene transformation. At first, the effects of explants age and seven different combinations of plant growth regulators on callus induction and direct shoot regeneration of lettuce were examined. The experiment was factorial based on a completely randomized design. The sensitivity of lettuce cotyledons to hygromycin was assayed by culturing the cotyledons without co-cultivation with Agrobacterium tumefaciens on selection medium containing different concentrations of hygromycin. For gene transformation, Agrobacterium tumefaciens (LBA4404 with pCAMBIA1304) was used and transgenic plants were analyzed with PCR method. The highest percentage of callus induction was obtained using 0.54 µM NAA and 0.44 µM BA on 7 days old cotyledon explants. The highest number of direct shoot regenerations was also obtained using M1 (0.54 µM NAA and 0.44 µM BA) and M7 (0.1 µM NAA and 0.44 µM BA) media. Hygromycin with a concentration of 15 mg.l-1, completely inhibited the regeneration from untransformed explants and it was therefore used in selection medium. Optimal transformation conditions were obtained by co-culturing cotyledon explants with Agrobacterium tumefaciens in MS medium without any plant growth regulators for 72 hours. Primary analysis of regenerated plants in DNA level was conducted by specific primers for hph gene and transgenic plants were screened for further studies.
Keywords: Lettuce, Genetic manipulation, Tissue culture, Direct shoot regeneration, hph selectable gene
