Search results for: Murashige and Skoog

Authors: Okafor C. Uche, Agbo P. Ejiofor, Okezie C. Eziuche

Abstract:

This study provides a regeneration protocol for Treculia africana Decne (an endangered plant) through embryo culture. Mature zygotic embryos of T. africana were excised from the seeds aseptically and cultured on varied strengths (full, half and quarter) of Murashige and Skoog (MS) basal medium supplemented. All treatments experienced 100±0.00 percent sprouting except for half and quarter strengths. Plantlets in MS full strength had the highest fresh weight, leaf area, and longest shoot length when compared to other treatments. All explants in full, half, quarter strengths and control had the same number of leaves and sprout rate. Between the treatments, there was a significant difference (P>0.05) in their effect on the length of shoot and root, number of adventitious root, leaf area, and fresh weight. Full strength had the highest mean value in all the above-mentioned parameters and differed significantly (P>0.05) from others except in shoot length, number of adventitious roots, and root length where it did not differ (P<0.05) from half strength. The result of this study indicates that full strength MS basal medium offers a better option for the optimum growth for Treculia africana regeneration in vitro.

Keywords: Medium strengths, Murashige and Skoog, Treculia africana, zygotic embryos.

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Authors: Masoume Amirkhani, Kambiz Mashayekhi, Maurizio Lambardi

Abstract:

Agropyron cristatum L. Gaertn. is a native grass of semiarid region in Iran which is quit resistant to cool and drought climate and withstand heavy grazing. This species has close phylogenetic relationship with Triticum and Hordeum. In this research, the effect of seven different concentrations of growth regulator 2,4-D on callus production and somatic embryogenesis of A. cristatum was investigated on Murashige and Skoog medium. The results showed that the rate of callus, embryo and neomorph were highest in 1 mg L-1 2,4-D. Callus production was increased in 1 mg L-1 2,4-D but dramatically decreased at 5.5 and 9 mg L-1 2,4-D. The somatic embryos were observed at 1 and 4 mg L-1 2,4-D but matured embryos and plantlet were only occurred at 1 mg L-1 2,4-D. There were significant differences between 1 mg L-1 2,4-D and other treatments for producing globular and torpedo embryos, plantlet, rooted callus and number of roots (p<0.05) and there was not any callus production and embryogenesis in control treatment without growth regulator.

Keywords: 2, 4-D, callus production, somatic embryogenesis, Agropyron cristatum.

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Authors: Iriawati, R. R. Esyanti, W. Natalia, N. Zahya

Abstract:

In vitro plant regeneration has been successfully obtained from basal shoot explant of Vetiveria zizanioides through indirect organogenesis. The explant was cultured in Murashige & Skoog’s (MS) media supplemented with 2,4-D, IAA, and kinetin in various concentrations. Callus was well induced in media supplemented with 2 ppm 2,4-D, 1 ppm IAA, and 1 ppm kinetin. This callus was then transferred to MS media supplemented with 1 - 5 ppm of BAP for shoot regeneration. The media supplemented with 3 ppm BAP was a suitable medium for shoot induction, as well as for shoot multiplication. Rooting was well developed in shoot following transferred to half MS media containing 0.2 ppm IBA. Plantlet was then transferred to husk charcoal for acclimatization, and almost all (90%) of plantlets were survived during acclimatization.

Keywords: Callus, plantlet regeneration, shoot induction, Vetiveria zizanioides.

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Authors: Sopalun K., Thammasiri K., Ishikawa K.

Abstract:

The effects of chitosan, a biodegradable polymer, were studied in Grammatophyllum speciosum protocorm-like bodies (PLBs) in vitro culture. The chitosan concentration of 0, 5, 10, 15, 20, 25, 50 or 100 mg/l were supplemented in half-strength Murashige and Skoog (1/2 MS) liquid or on agar media containing 2% (w/v) sucrose. The results showed that liquid medium supplemented with 15 mg/l chitosan showed the highest relative growth rate (7-fold increase) of PLBs. On 1/2 MS agar medium supplemented with 25 mg/l chitosan gave the highest relative growth rate (4-fold increase). The relative growth rate of G. speciosum PLBs on agar medium was significantly lower than that in liquid medium. Moreover, chitosan, supplemented to agar medium promoted shoot formation but not rooting. However, supplementation at too high a level, such as 100 mg/l can inhibit growth and kill PLBs.

Keywords: Chitosan, Grammatophyllum speciosum, Growth stimulator

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Authors: Pratibha Gupta, Satyawati Sharma, Sanjay Saxena

Abstract:

Stevia rebaudiana Bertoni (natural sweetener) belongs to Asteraceae family and can be used as substitute of artificial sweeteners for diabetic patients. Conventionally, it is cultivated by seeds or stem cutting, but seed viability rate is poor. A protocol for callus induction and multiplication was developed to produce large no. of calli in short period. Surface sterilized nodal, leaf and root explants were cultured on Murashige and Skoog (MS) medium with different concentrations of plant hormone like, IBA, kinetin, NAA, 2,4-D, and NAA in combination with 2,4-D. 100% callusing was observed from leaf explants cultured on combination of NAA and 2,4-D after three weeks while with 2,4-D, only 10% callusing was observed. Calli obtained from leaf and root explants were shiny green while with nodal explants it was hard and brown. The present findings deal with induction of callusing in Stevia to achieve the rapid callus multiplication for study of steviol glycosides in callus culture.

Keywords: 2, 4-D, Callusing, NAA, Stevia, Steviol glycosides

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Authors: Chockpisit Thepsithar, Nongnuch Euawong, Nukul Jonghomkajorn

Abstract:

The in vitro culture procedure of purple nutsedge (Cyperus rotundus L.) for multiple shoot induction and tuber formation was established. Multiple shoots were significantly induced from a single shoot of about 0.5 – 0.8 cm long, on Murashige and Skoog (MS) medium supplemented with 4.44 μM 6- benzyladinine (BA) alone or in combination with 2.85 μM 1- indoleacetic acid (IAA), providing 17.6 and 15.3 shoots per explant with 31.2 and 27.5 leaves per explant, respectively, within 6 weeks of culturing. Moreover, MS medium supplemented with 4.44 μM BA and 2.85 μM IAA was suitable for tuber induction, obtaining 5.9 tubers with 3.4 rhizomes per explant. In combination with ancymidol and higher concentration of sucrose, 11.1 μM BA and 60 g/L sucrose or 11.1 μM BA, 7.8 μM ancymidol and 60 g/L sucrose induced 3.5 tubers with 1.6 rhizomes or 3.5 tubers without rhizome, respectively. However, MS medium containing 3.9 or 7.8 μM ancymidol in combination with either 60 or 80 g/L sucrose enchanced significant root formation at 20.9 – 23.6 roots per explant.

Keywords: Purple nutsedge, Cyperus rotundus, multiple shoot induction, tuber formation

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Authors: Shyama Ranjani Weerakoon

Abstract:

Direct and indirect somatic embryogenesis (SE) from petiole and leaf explants of Scaevola aemula R. Br. cv. 'Purple Fanfare' was achieved. High frequency of somatic embryos was obtained directly from petiole and leaf explants using an inductive plant growth regulator signal thidiazuron (TDZ). Petiole explants were more responsive to SE than leaves. Plants derived from somatic embryos of petiole explants germinated more readily into plants. SE occurred more efficiently in half-strength Murashige and Skoog (MS) medium than in full-strength MS medium. Non-embryogenic callus induced by 2, 4-dichlorophenoxyacetic acid was used to investigate the feasibility of obtaining SE with TDZ as a secondary inductive plant growth regulator (PGR) signal. Non-embryogenic callus of S. aemula was able to convert into an “embryogenic competent mode" with PGR signal. Protocol developed for induction of direct and indirect somatic embryogenesis in S. aemula can improve the large scale propagation system of the plant in future.

Keywords: Petiole and leaf explants, Scaevola aemula, Somaticembryogenesis

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Authors: Chockpisit Thepsithar, Aree Thongpukdee, Apichya Daorat

Abstract:

The alternative technique for sterilization of culture medium to replace autoclaving was carried out. For sterilization of culture medium without autoclaving, some commercial pure essential oils, bergamot oil, betel oil, cinnamon oil, lavender oil and turmeric oil, were tested alone or in combinations with some disinfectants, 10% povidone-iodine and 2% iodine + 2.4% potassium iodide. Each essential oil or combination was added to 25-mL Murashige and Skoog (MS) medium before medium was solidified in a 120-mL container, kept for 2 weeks before evaluating sterile conditions. Treated media, supplemented with essential oils, were compared to control medium, autoclaved at 121 degree Celsius for 15 min. In vitro sterile conditions were found 20 – 100% from these treated media compared to 100% sterile condition from autoclaved medium. Treated media obtained 100% sterile conditions were chosen for culturing chrysanthemum shoots. It was found that 10% povidoneiodine in combination with cinnamon oil (3:1) and 2% iodine + 2.4% potassium iodide in combination with lavender oil (1:3) at the concentration of 36 3L/25 mL medium provided the promising growth of shoot explants.

Keywords: Sterilizing agents, essential oils, disinfectants, MS medium, in vitro culture, chrysanthemum, sterilization of medium without autoclaving

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Authors: Kullanart Obsuwan, Suluck Namchote, Natdhera Sanmanee, Kamolchanok Panishkan, Sirichai Dharmvanij

Abstract:

Humic acids (HAs) have been shown to activate some ion uptakes along with stimulating the lateral roots at effective concentration of micronutrients. However, the effects of HA on ion adsorption by plant roots are not easily explainable due to the varieties of HAs that differ from origins. Therefore, this study was aimed to investigate the effect of various concentrations of HA obtained from the compost derived from mix manures and some agricultural wastes on the growth of eggplant seedlings (Solanum melongena L. cv. Chao Praya) in tissue cultures at low nutrient level. Egg plant seeds were surfaced sterilized and germinated in ½ Murashige and Skoog medium (MS) without HA added or in ¼ MS supplemented with 0, 25, 50, 75 and 100 ppm of HAs. Then, they were cultured for 4 weeks under the controlled environment. The results showed that seedlings grown on ¼MS supplemented with HAs at the concentration of 25 and 50 ppm had the average plant heights (2.49 and 2.28 cm, respectively) higher than the other treatments. Both treatments also significantly showed the maximum average fresh and dry weights (p<0.05). Also the later yielded the highest average number of leaves and the longest average root length (p<0.05). However, there was no statistically different in the number of roots among treatments (p>0.05). This suggested that HAs at the concentration of 25 and 50 ppm could improve the growth of egg plant seedlings in tissue cultures at low nutrient level (¼ MS).

Keywords: growth, seedling, humic acid, fresh weght, dry weight, tissue culture

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Authors: Anuar, N., Markom, M., Khairedin, S., Johari, N. A.

Abstract:

Quercetin and (+)-catechin are metabolites present in Phyllanthus niruri plant, have potential in medicinal uses as anticancer and antioxidant agents. Studies on production of quercetin and (+)-catechin from P. niruri callus culture via in vitro technique were carried out and the results were compared to the intact plant. P. niruri explants were cultured on Murashige and Skoog (MS) solidified media supplemented with several phytohormone combinations for one month. The metabolites were extracted from P. niruri callus and intact plant by using carbon dioxide supercritical fluid extraction (SFE) with ethanol as modifier and solvent extraction techniques. The extracts were analyzed by means of HPLC method. Results showed that P. niruri callus culture was successfully established. The highest content of quercetin (1.72%) was found from P. niruri callus grown in media supplemented with 0.8mg/L kinetin and 0.2mg/L 2,4-dicholophenoxyacetic acid (2,4-D), which was 1.2 fold higher than intact plant. Meanwhile, the highest amounts of (+)-catechin (0.63%) was found from P. niruri callus grown in media with addition of 0.2mg/L 1-naphthalene acetic acid (NAA) and 0.8mg/L 2,4-D. The SFE condition in this study showed better extraction efficiency when higher contents of selected metabolites were found in all SFE extracts compared to the common solvent extracts.

Keywords: Callus culture, Phyllanthus niruri, secondary metabolite, supercritical fluid extraction.

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Authors: Wittaya Deein, Chockpisit Thepsithar, Aree Thongpukdee

Abstract:

Plant tissue culture is an important in vitro technology applied for agricultural and industrial production. A sterile condition of culture medium is one of the main aspects. The alternative technique for medium sterilization to replace autoclaving was carried out. For sterilization of plant tissue culture medium without autoclaving, ten commercial pure essential oils and 5 disinfectants were tested. Each essential oil or disinfectant was added to a 20-mL Murashige and Skoog (MS) medium before medium was solidified in a 120-mL container, kept for 2 weeks before evaluating sterile conditions. Treated media, supplemented with essential oils or disinfectants, were compared to control medium, autoclaved at 121 degree Celsius for 15 min. Sterile conditions of MS medium were found 100% from betel oil or clove oil (18 mL/20 mL medium), cinnamon oil (36 mL/20 mL medium), lavender oil or holy basil oil (108 mL/20 mL medium), and lemon oil or tea tree oil or turmeric oil (252 mL/20 mL medium), compared to 100% sterile condition from autoclaved medium. For disinfectants, 2% iodine + 2.4% potassium iodide, 2% merbromine solution, 10% povidone-iodine, 6% sodium hypochlorite or 0.1% thimerosal at 36 mL/20 mL medium provided 100% sterile conditions. Furthermore, growth of new shoots from chrysanthemum node explants on treated media (fresh weight, shoot length, root length and number of node) were also reported and discussed in the comparison of those on autoclaved medium.

Keywords: Chrysanthemum, disinfectants, essential oils, MS medium, sterilizing agents, sterilization of medium without autoclaving.

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Authors: T. Punjansing, M. Nakkuntod, S. Homchan, P. Inthima, A. Kongbangkerd

Abstract:

The influence of organic supplements on growth and multiplication efficiency of Phaius tankervilleae var. alba seedlings was investigated. 12 week-old seedlings were cultured on half-strength semi-solid Murashige and Skoog (MS) medium supplemented with 30 g/L sucrose, 8 g/L agar and various concentrations of coconut water (0, 50, 100, 150 and 200 mL/L) combined with potato extract (0, 25 and 50 g/L) and the pH was adjusted to 5.8 prior to autoclaving. The cultures were then kept under constant photoperiod (16 h light: 8 h dark) at 25 ± 2 °C for 12 weeks. The highest number of shoots (3.0 shoots/explant) was obtained when cultured on the medium added with 50 ml/L coconut water and 50 g/L potato extract whereas the highest number of leaves (5.9 leaves/explant) and roots (6.1 roots/explant) could receive on the medium supplemented with 150 ml/L coconut water and 50 g/L potato extract. with 150 ml/L coconut water and 50 g/L potato extract. Additionally, plantlets of P. tankervilleae var. alba were transferred to grow into seven different substrates i.e. soil, sand, coconut husk chip, soil-sand mix (1: 1), soil-coconut husk chip mix (1: 1), sand-coconut husk chip mix (1: 1) and soil-sand-coconut husk chip mix (1: 1: 1) for four weeks. The results found that acclimatized plants showed 100% of survivals when sand, coconut husk chip and sand-coconut husk chip mix are used as substrates. The number of leaves induced by sand-coconut husk chip mix was significantly higher than that planted in other substrates (P > 0.05). Meanwhile, no significant difference in new shoot formation among these substrates was observed (P < 0.05). This precursory developing protocol was likely to be applied for more large scale of plant production as well as conservation of germplasm of this orchid species.

Keywords: Acclimatization, coconut water, orchid, Phaius tankervilleae var. alba., potato extract.

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Authors: A. B. A. Ahmed, Amar, D. I., R. M. Taha

Abstract:

This research aims to investigate callus induction, somatic embryogenesis and indirect plant regeneration of Crassula ovata (Mill.) Druce – the famous ornamental plant. Experiment no.1: Callus induction was obtained from leaf and stem explants on Murashige and Skoog (MS) medium supplemented with various plant growth regulators (PGRs). Effects of different PGRs, plant regeneration and subsequent plantlet conversion were also assessed. Indirect plant regeneration was achieved from the callus of stem explants by the addition of 1.5 mg/L Kinetin (KN) alone. Best shoot induction was achieved (6.5 shoots/per explant) after 60 days. For successful rooting, regenerated plantlets were sub-cultured on the same MS media supplemented with 1.5 mg/L KN alone. The rooted plantlets were acclimatized and the survival rate was 90%. Experiment no.2: Results revealed that 0.5 mg/L 2,4-D alone and in combination with 1.0 mg/L 6-Benzyladenine (BA) gave 89.8% callus from the stem explants as compared to leaf explants. Callus proliferation and somatic embryo formation were also evaluated by ‘Double Staining Method’ and different stages of somatic embryogenesis were revealed by scanning electron microscope. Full Strength MS medium produced the highest number (49.6%) of cotyledonary stage somatic embryos (SEs). Mature cotyledonary stage SEs developed into plantlets after 12 weeks of culture. Wellrooted plantlets were successfully acclimatized at the survival rate of 85%. Indirectly regenerated plants did not show any detectable variation in morphological and growth characteristics when compared with the donor plant.

Keywords: Callus induction, Crassula ovata, Double Staining, Indirect plant regeneration, Somatic embryogenesis.

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Authors: R. Razavizadeh, F. Adabavazeh, M. Rezaee Chermahini

Abstract:

Salinity is one of the most widespread agricultural problems in arid and semi-arid areas that limits the plant growth and crop productivity. In this study, the salt stress effects on protein, reducing sugar, proline contents and antioxidant enzymes activities of Carum copticum L. under in vitro conditions were studied. Seeds of C. copticum were cultured in Murashige and Skoog (MS) medium containing 0, 25, 50, 100 and 150 mM NaCl and calli were cultured in MS medium containing 1 μM 2, 4-dichlorophenoxyacetic acid, 4 μM benzyl amino purine and different levels of NaCl (0, 25, 50, 100 and 150 mM). After NaCl treatment for 28 days, the proline and reducing sugar contents of shoots, roots and calli increased significantly in relation to the severity of the salt stress. The highest amount of proline and carbohydrate were observed at 150 and 100 mM NaCl, respectively. The reducing sugar accumulation in shoots was the highest as compared to roots, whereas, proline contents did not show any significant difference in roots and shoots under salt stress. The results showed significant reduction of protein contents in seedlings and calli. Based on these results, proteins extracted from the shoots, roots and calli of C. copticum treated with 150 mM NaCl showed the lowest contents. The positive relationships were observed between activity of antioxidant enzymes and the increase in stress levels. Catalase, ascorbate peroxidase and superoxide dismutase activity increased significantly under salt concentrations in comparison to the control. These results suggest that the accumulation of proline and sugars, and activation of antioxidant enzymes play adaptive roles in the adaptation of seedlings and callus of C. copticum to saline conditions.

Keywords: Antioxidant enzymes, Carum copticum, organic solutes, salt stress.

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Authors: Santosh Kumar, Anand Prakash, Kanak Sinha, Anita K Verma

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Vernonia divergens Benth., commonly known as “Insulin Plant” (Fam: Asteraceae) is a potent sugar killer. Locally the leaves of the plant, boiled in water are successfully administered to a large number of diabetic patients. The present study evaluates the putative anti-diabetic ingredients, isolated from the in vivo and in vitro grown plantlets of V. divergens for their antimicrobial and anticancer activities. Sterilized explants of nodal segments were cultured on MS (Musashige and Skoog, 1962) medium in presence of different combinations of hormones. Multiple shoots along with bunch of roots were regenerated at 1mg l-1 BAP and 0.5 mg l-1 NAA. Micro-plantlets were separated and sub-cultured on the double strength (2X) of the above combination of hormones leading to increased length of roots and shoots. These plantlets were successfully transferred to soil and survived well in nature. The ethanol extract of plantlets from both in vivo & in vitro sources were prepared in soxhlet extractor and then concentrated to dryness under reduced pressure in rotary evaporator. Thus obtainedconcentrated extracts showed significant inhibitory activity against gram negative bacteria like Escherichia coli and Pseudomonas aeruginosa but no inhibition was found against gram positive bacteria. Further, these ethanol extracts were screened for in vitro percentage cytotoxicity at different time periods (24 h, 48 h and 72 h) of different dilutions. The in vivo plant extract inhibited the growth of EAC mouse cell lines in the range of 65, 66, 78, and 88% at 100, 50, 25 & 12.5μg mL-1 but at 72 h of treatment. In case of the extract of in vitro origin, the inhibition was found against EAC cell lines even at 48h. During spectrophotometric scanning, the extracts exhibited different maxima (ʎ) - four peaks in in vitro extracts as against single in in vivo preparation suggesting the possible change in the nature of ingredients during micropropagation through tissue culture techniques.

Keywords: Anti-cancer, Anti-microbial, EAC mouse cell, Tissue culture, Vernonia divergens.

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