Seyyed Mehran Alavi; Asad Masoumiasl; Naser Zare; Rasul Asghari Zakaria; Parisa Sheikhzade Mosaddegh
Abstract
Introduction: The main habitat of Chavil, Ferulago angulata, in Iran is Zagros area. This plant has a rejuvenating effect and is used to treat digestive diseases and intestinal worms. Because the different explants show different amounts of callogenesis under the effect of different growth regulators, ...
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Introduction: The main habitat of Chavil, Ferulago angulata, in Iran is Zagros area. This plant has a rejuvenating effect and is used to treat digestive diseases and intestinal worms. Because the different explants show different amounts of callogenesis under the effect of different growth regulators, selection of an optimal explant and suitable plant growth regulators combination has a significant effect on the production of callus and their suspension culture. There is no reports on Ferulago angulata callogenesis and its cell suspension culture. Therefore, this study was designed and implemented to optimize callus production and cell suspension culture in this important medicinal plant.
Materials and Methods: Seeds of Chavil were collected from four different habitats in Kohgilooyeh and BoyerAhmad Province in Southwest of Iran include Abenahr, Guayoune, Vezg and Sisakhat. Seedlings obtained from embryo culture were used to prepare the explants. Various explants (leaf, root and stem) were cultured on MS medium supplemented with different concentrations (0, 0.5, 1 and 2 mgl-1) of NAA and BAP. Callus traits were evaluated and from the best culture medium, the best explants and the best PGRs composition for callogenesis of each ecotype were used to cell suspension culture. In order to study the growth rate of cells in suspension culture and plotting the curve of cells growth, two cell density indices and packed cell volume index were evaluated. To determine the cell density index, every 3 days, 10 ml of cell suspension were transferred to the graded falcon and centrifuged at 5000 g for 5 minutes, and the percentage of sediment cells was calculated as the total volume. To determine the packed cell volume index, also every 3 days, 10 ml of culture medium containing cells were transferred to the graded falcon and stored for 30 minutes to precipitate cells and cell masses. Finally, the cell volume was recorded and was calculated as percentage of the total collected medium.
Results and Discussion: According to the callogenesis percentage, the best ecotype is Abenahr and best explant is leaf explant. The highest level of NAA is 2 mgl-1, and the best level of BAP is 2 mgl-1, which causes 100 callogenesis percentage. The best medium for cell suspension culture is MS medium containing 2 mgl-1 NAA and 0.5 mgl-1 BAP for callus was obtained from leaf explant of Abenahr ecotype. Along with these plant growth regulators, 2,4-D was used in combination with BAP to form suspension culture. The results also showed that 2 mgl-1 2,4-D plus 0.5 mgl-1 BAP were useful in producing suspensions. The difference between 2,4-D +BAP and NAA + BAP combinations more cell volumes were observed, and cell suspension was created at a faster rate and in less time, which is an advantage in research work. Growth rate of cell suspension originated from the leaf explant was higher than root explant. In terms of culturing cell suspension, the Abenahr ecotype was favorable compared to other ecotypes. During cell suppression culture of Cyperus aromaticus by applying different levels of NAA, cell growth was increased up to 3 weeks after application, and then decreased. By applying 2,4-D, cell growth also increased until the third week, and after the third week, cell growth declined, which was very low growth rate compared with the NAA. In cell suspension culture of sugar beet, using 2,4-D was much more effective than NAA on all explants. In the present study, 2,4-D was also more effective than NAA for cell suspension culture of Chavil.
Conclusion: In general, the Abenahr was the best ecotype among of investigated. The explants in both callus culture and the suspension culture, and the best combination of plant growth regulator in both culture was 2 mgl-1 NAA plus 0.5 mgl-1 BAP.
Asad Masoumiasl; Ameneh Aryiaeineghad; Masud Dehdeari
Abstract
Introduction: German (Matricaria chamomilla L.) and Shirazi (Matricaria recutita L.) chamomiles are the most important medicinal plants of the Astraceae family which are used in the pharmaceutical, health, food and cosmetics industries. Production of this plant has been undertaken in Iran mainly in Isfahan, ...
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Introduction: German (Matricaria chamomilla L.) and Shirazi (Matricaria recutita L.) chamomiles are the most important medicinal plants of the Astraceae family which are used in the pharmaceutical, health, food and cosmetics industries. Production of this plant has been undertaken in Iran mainly in Isfahan, Kohgiluyeh and Boyerahmad, Golestan and Hamedan provinces. In vitro propagation of plants have higher potential to produce qualified natural products, restoring and preserving of endangered plants, induction of somaclonal variation, industrial reproduction, valuable secondary metabolites and increased active ingredients. Researchers reported successful micropropagation system for five chamomile varieties on MS medium contained 0.01 mg/l NAA and 2.5 mg/l kinetin. The aim of this study was to investigate the effects of stem (with and without node), leaf and cotyledon explants and different plant growth regulators on direct regeneration of German and Shirazi chamomiles. Although the Shirazi chamomile is native to Iran, but German chamomile is Iran non-indigenous cultivar. By our knowledge, there have been no comparison reports about responses of these cultivars to tissue culture.
Materials and Methods: This research was performed in the central laboratory of Agriculture Faculty in Yasouj University. Seeds were provided from Pakan-Bazr institute, Isfahan. Chamomile seeds were disinfected by ethanol (70%) for 5-10 min and sodium hypocholorite 3% for 5-12 min and then washed for several times by distilled water. Then, seeds were sown on MS medium for germination. After 2-3 weeks, seedlings were grown and then planted in MS medium supplemented with hormonal combinations of NAA at two levels (0.1 and 0.5 mg/l), kinetin at three levels (2, 2.5 and 3 mg/l) and Zeatin, BAP and 2ip at three levels (0.5, 1 and 1.5 mg/l). The experiment was performed in a completely randomized design with four replications. Factors included explants, cultivars and hormonal combinations. In this experiment, traits such as stem induction percentage, stem length, stem fresh and dry weight root induction percent, root length and stem fresh and dry weight were measured. Statistical analysis was performed using SAS software (version 9.1). In order to test the normality and perform mean comparisons, Minitab 14 and MSTAT-C software was used and excel software was used for drawing diagrams.
Results and Discussion: For all traits except stem induction percent and root dry weight, triple interaction of explants, hormonal combinations and cultivar were significant at 1% level. Triple mean comparisons for cultivar, explant and hormonal combinations shows that the highest mean for stem height, stem fresh weight, stem dry weight, root induction percent, root height and root fresh weight was obtained in Shirazi Chamomile cultivar with cotyledon explants in hormonal combinations of 0.1 mg/l NAA and 1.5 mg/l 2ip. The results showed that the best explants in both chamomiles for direct regeneration were stem (whit node) and cotyledon with 78.75% and 75% regenerations, respectively. Also the best genotype and hormone combination were Shirazi chamomile and MS medium supplemented with 1.5mg/l 2ip with 0.1mg/l NAA. To determine the best hormonal combination for root regeneration from direct regeneration, the stems were embedded in medium contained different concentrations of IBA. The first signs of rooting production were observed after 5-7 days. After completing the roots formation (4 weeks after transferring), the rooting percentage, root length, fresh and dry weight of roots were measured. Based upon variance analysis, effect of triple interactions of IBA, explants and cultivar on all traits were not significant, but the effect of IBA for all traits was significant at 1% level. According to the results of mean comparisons for effects of IBA on root traits in direct regeneration of chamomile, the highest percentage of root regeneration (73.75 %), the highest root length (6.60 cm), root fresh weight (174.167 mg) and the highest root dry weight (16.425 mg) were obtained from medium contained 0.5 mg/l IBA. Root differentiation was influenced by auxin (0.5 mg/l IBA) or spontaneously. Regenerated plantlets were transferred to pots contained sterilized soil (3:1:1 mixture of soil: sand: leaf compost). For plant adaptation to natural conditions, glassy caps were used. After adaptation, the caps were removed and the plants were transferred into a growth chamber. Previous studies reported that MS medium supplemented with 0.2 to 1 mg/l of BA and 2 mg/l of NAA induced adventitious bud formation and shoot development in leaf explants of Roman Chamomile. A higher number of adventitious buds were observed at the proximal end of the explants. Plantlets were rooted on MS medium supplemented with 0.1 mg/l of IBA and successfully weaned in vivo.
Conclusion: Based on the results of this research, chamomile showed relevant response to direct regeneration.
