M. Sayadi Nejad; S.M. Sadeghi
Abstract
Introduction: Zamiifolia is a perennial ornamental plant and is one of the most important medicinal plants of the Aracea family. The origin of this evergreen, low-anticipated plant is East Africa. Zamiifolia spreads through the leaves and split rhizomes, which is very time-consuming. The traditional ...
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Introduction: Zamiifolia is a perennial ornamental plant and is one of the most important medicinal plants of the Aracea family. The origin of this evergreen, low-anticipated plant is East Africa. Zamiifolia spreads through the leaves and split rhizomes, which is very time-consuming. The traditional Zamiifolia proliferation method have been done by dividing rhizomes and leaf cuttings, but the production efficiency is low due to the slow growth of the plant, tubers and roots. In addition, due to the warm and humid environment, reproduction is limited to summer season. Due to the traditional reproductive problems in this plant, tissue culture or microbial culture is the best way to replicate rapidly and to achieve a large number of plants with the same genetic structure, as well as the elimination of diseases in the short term and reducing the costs. The aim of this study was to compare different microorganisms in terms of calogenesis and regeneration, as well as to determine the optimum culture medium for Zamiifolia tissue culture.
Materials and Methods: In this study, the explants prepared for the first experiment, including rhizome and petiole and the explants for the second experiment were the leaeaves and shoots. In the first experiment, rhizome and petiole were cultured in three replications in ½ MS medium containing BA (0, 2, 4 mg / L) and 2,4-D (0,1,2 mg / L) in combination with vitamins, 30 g/l sucrose, 5 g / L agar and adjusted to 5.8 PH. The cultivars were cultured for the callus induction under temperature of 27-25 ° C and light conditions of 16 hours light and 8 hours darkness. After 5 weeks, the percentage of callus and fresh callus weight were measured. The callus generated from rhizome and petiole in three replicates on ½ MS medium containing BA (0, 1, 2 mg / L) and 2,4-D (0, 0.5, 1 mg / l) for shoots and after the observation of branches and leaf buds were grafted on to ½ MS medium containing BA (0, 1, 2 mg / L) and NAA (0, 0.5, 1 mg / L) for rooting. Traits such as time to shoot elongation were recorded at regeneration stage, and after 5 weeks, shoot length and the number of leaves were measured. The time to rootstock was also recorded. In the second experiment leaf and shoot explants were cultured in ½ MS medium containing BA (0, 2, 4 mg / l) and NAA (0, 0.5, 1 mg / L) in combination with vitamins, 30 g/l sucrose, 5 g / L agar and PH adjusted to 5.8. The cultivars were cultured for the callus induction under temperature 27-25 °C and light conditions of 16 hours light and 8 hours darkness. The time to reach the callus was recorded and after 5 weeks, the percentage of callus and fresh callus weight were measured. The calli generated from the leaves and shoots were cultures on ½ MS medium containing BA (0, 1, 2 m g / L) and NAA (0, 0.5, 1 mg / L) for shoots and after observation of branch and leaves were transplanted to the ½ MS medium containing BA (0, 1, 2 mg / L) and NAA (0, 1, 2 mg /L) for rooting. The traits such as time to shoot elongation were recorded at the regeneration stage, After 5 weeks, the shoot length and the number of leaves were measured.The time to rootstock was also recorded.
Results and Discussion: The results of the first experiment showed that the effect of the rhizome and petiole type on the callus formation was significant at 1% level. So that the rhizome showed greater ability to callogenesis. The results of the second experiment showed that the effect of the type leaf and shoot on the callus formation was significant at 1% level. So that the leaf showed greater ability to callogenesis. The highest percentage of callosing (94.5%), the shortest time to reach the callus (14 days) and the highest callus weight (1.1 g) in culture medium with 2 mg / l BA and 1 mg / l hormones NAA was observed in leaf samples from the second experiment. The best treatment in the shoot elongation stage, which included the shortest time to shoot formation (10.5 days), the longest shoot length (4.10 cm), and the highest leaf number (8 leaves) in the leaf extract with hormonal concentrations of 2 mg / 0 mg / L NAA was observed from the second experiment. In the rooting stage, the best treatment for petiole extracts with hormonal concentrations was 1 mg / l BA and 0.5 mg / l NAA with 14 days to rooting from the first experiment.
Conclusion: In this study, explants and various concentrations of growth regulators had significant effect on the response to callus induction in Zamiifolia. In the first experiment, the rhizome and in the second experiment the leaf showed a better reaction to callus induction. According to this research, it can be suggested that the treatments applied in both experiments should be applied on all four leaves, petiole, rhizome and shoot samples, and the best culture type and the best culture medium for the cultivation of Zamiifolia plant tissue should be determined in subsequent studies.
M. Aghdaei; S.H. Nemati; L. Samiei; A. Sharifi
Abstract
Introduction: Pepino (Solanum muricatum Aiton) is a diploid herbaceous plant belongs to the Solanaceae family, which is growing in subtropical zone, originates from Andes in South America. It is commercially grown for its fruit, which is appreciated not only for food but also for its appearance, in South ...
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Introduction: Pepino (Solanum muricatum Aiton) is a diploid herbaceous plant belongs to the Solanaceae family, which is growing in subtropical zone, originates from Andes in South America. It is commercially grown for its fruit, which is appreciated not only for food but also for its appearance, in South American countries, including Bolivia, Colombia, Ecuador and Peru, as well as in countries such as New Zealand and Australia. Pepino is propagated by seed, cutting, and tissue culture methods. Most pepino cultivars are sexually fertile and produce viable seeds, but their seeds have poor germination and high level of heterozygosis causing to highly variable plants. Both mentioned negative aspects have limited the mass production of this plant through seed. In this case, stem cutting is used as the most common way of propagating pepino led to transmission of viral diseases and increasing propagation costs as two main limiting factors of pepino propagation. So, micropropagation systems are a promising tool to produce disease-free clonal plant material with low costs. Therefore, the present study was aimed to assess the effect of different media and plant growth regulators on micropropagation traits of pepino.
Materials and Methods: Three separate experiments were carried out in institute of plant sciences of Ferdowsi University of Mashhad in 2016. Pepino seeds were bought from company of Plant World Seed, UK, were cultivated on MS medium. Grown plants were used as source of providing explants. Four mediums, including MS, ½ MS, SH and B5 were used to determine the best culture medium for shoot regeneration of pepino using single node explant. A factorial experiment was conducted based on a completely randomized design. Some growth properties such as number of shoots, shoot length, number of roots, root length, leaf number and leaf length were evaluated after two and four weeks. In proliferation experiment, MS medium was compared with MS supplemented with different concentrations of BA (0.5, 1 and 2 mg L-1) and Kin (0.5, 1 and 2 mg L-1) applied as combined treatments, and also BA used alone at concentrations of 2, 4 and 6 mg L-1 that was conducted based on a completely randomized design. For rooting of explants, an experiment was conducted based on a completely randomized design containing of two concentrations of IBA (at 0.3 and 0.6 mg L-1) and three concentrations of NAA (at 0.3, 0.6 and 0.9 mg L-1) in MS medium. Some growth properties including root number and length, root density and root quality were evaluated after four weeks
Results and Discussion: Results indicated that micropropagation rate of pepino was affected by culture medium type. The highest shoot length, number of root, root length and leaf number were obtained in MS medium, although statistically there was no significant difference between MS and ½ MS media. The highest number of shoots and leaf length were observed in MS medium, which led to a significant difference with other media (½ MS, SH and B5). Overall, Based on obtained results MS medium was the best culture medium for micropropagation of pepino using single node. In the proliferation experiment, the highest shoot and leaf number and plant color were obtained with using 2 mg L-1 BA + 1 mg L-1 Kin, whereas the highest shoot length and leaf length were observed in the 1 mg L-1 BA + 2 mg L-1 Kin and 1 mg L-1 BA+1 mg L-1 Kin treatments, respectively. Increasing in concentration of BA up to 2 mg L-1 in combination with Kin had a positive effect on shoot proliferation, while applying BA at concentration 2, 4 and 6 mg L-1 alone led to decrease in proliferation. Results obtained from rooting experiment showed that the highest root number, root density and root quality were obtained using IBA at the concentration of 0.6 mg L-1, whereas the highest root length was observed by applying IBA at concentration of 0.3 mg L-1, which led to a significant difference with other treatments. Furthermore, results indicated that the effect of IBA on rooting of pepino microshoots was more than NAA.
Conclusion: Generally, the best results were obtained by MS medium, 2 mg L-1 BA with 1 mg L-1 Kin for shoot proliferation, and IBA at concentration of 0.6 mg L-1 for the rooting of pepino nodal segments.
Esmaeil Chamani; Marzyeh Ghamari; Mahdi Mohoboldini; Alireza Ghanbari; Hamidreza 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
H. Hadizadeh; M. Mohebodini; B. Esmaeilpoor; E. 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.
